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Time‐Resolved Hierarchical Modeling Highlights Metabolites Influencing Productivity and Cell Death in Chinese Hamster Ovary Cells | 3a6d5fd2-59e7-4e7d-86a9-c5edc2eb4f6c | 11894446 | Biochemistry[mh] | Introduction Biopharmaceuticals, or biologics, are pharmaceuticals derived from biological sources. Biopharmaceuticals include vaccines, monoclonal antibodies (mAbs), and other recombinant proteins. Mammalian cells are the leading expression systems in the biologics industry , comprising 2/3 of newly approved drugs from 2018 to 2022, of which Chinese hamster ovary (CHO) cells accounted for 89% . CHO cells are good production systems, reaching high densities in chemically defined media and suspension cultures . CHO cells can also make human‐compatible post‐translational modifications like protein folding and glycosylation and are less susceptible to human viruses than other cells . The rising demand for biologics necessitates higher production. Currently, protein titers around 10 g/L are possible in prevailing fed‐batch cultures . However, continuous cultures are emerging due to advances in upstream bioprocessing. Continuous cultures cycle waste with perfusion feeding systems, improving the idle reactor time and viable cell density (VCD), in turn enhancing the volumetric productivity (g/L/day). Productivity can also be improved on a cell‐specific (qP) level via cell line engineering or optimized culture conditions . Twenty million cells/mL and 50 pg/cell/day have been achieved in fed‐batch cultures. However, VCDs cannot increase indefinitely, and higher VCDs may not lead to higher titers if qP is adversely affected . Thus, optimizing not only VCD but also qP is crucial to increasing volumetric productivity further. Cell proliferation is governed by two complementary processes: cell division, measured by the specific growth rate ( µ g , 1/day), and cell death, measured by the specific death rate ( µ d , 1/day) . These rates inform how VCD evolves: increasing in the exponential phase ( µ g > µ d ), peaking in the stationary phase ( µ g ≈ µ d ), and declining in the death phase ( µ g < µ d ) . Most CHO studies target the exponential and stationary phases. Fewer examine the death phase , indicating a need to study CHO metabolism late in production further. Cell death is detrimental to product quality and titer. Dying cells release enzymes that degrade the recombinant proteins and alter the glycan structures. Cellular debris also creates impurities, complicating downstream bioprocessing . µ d increases over time, accelerating from the stationary phase, likely due to toxic byproducts, not nutrient exhaustion . Removing waste metabolites via perfusion could improve viability and productivity. Lactate and ammonia, the primary waste metabolites from glucose and amino acids, respectively, can be limited with optimized feeding strategies , but other toxic metabolites must also be considered to enhance culture productivity . Multidimensional omics techniques aid in investigating biological systems. Metabolomics, in particular, links snapshots of cellular physiology to important phenotypes like high productivity or low cell death . Chemometric techniques help interpret metabolomics data. Principal component analysis (PCA) identifies overall trends and outliers, while orthogonal partial least squares (OPLS) relates metabolites to process variables, or distinguishes between sample classes . These techniques have been applied to CHO metabolic growth phases , cell densities , and media compositions , identifying targets for cell line and medium development, such as metabolites that stifle growth or induce apoptosis . Even under identical growth conditions, CHO cells show clonal variation, leading to growth and productivity differences between production batches that affect the titer and critical quality attributes . However, the underlying biology is underexplored. The obstacle here is time. Metabolomics samples from the same batch are not independent over time, creating artificially high metabolite‐process variable correlations. PCA struggles when the greatest variation is time‐related . Various (O)PLS‐based techniques have been used with time series, each with its strengths and drawbacks: PLS on batch‐level models can provide metabolic correlations per time point , but monotonic or cumulative response variables are necessarily time‐dependent . OPLS‐discriminant analysis (DA) can compare time points pair‐wise , but the differences might not relate to a process variable of interest. Multiway PLS‐DA can compare groups of cell lines for a single time point or overall, but may not capture changes in the response variable over time. To overcome the time dependence, in this study, we devised a hierarchical method based on OPLS regression to analyze metabolites at each time point separately. We compared our method against conventional, global OPLS models that consider the metabolites at all time points simultaneously. Seeking metabolites linked to productivity and cell death, we analyzed the extracellular metabolome of CHO cells with varying growth and productivity properties. The new method pinpointed several metabolites with consistent relations to qP and/or µ d in the stationary and death phases that the global method missed. Many metabolites were biologically relevant to CHO cell death and/or productivity, demonstrating the new method's effectiveness in analyzing time‐dependent bioprocess omics data.
Materials and Methods 2.1 Cell Line and Culture Conditions We grew eleven clones of the same CHO‐DG44 parental cell line (Sartorius, Germany) as fed‐batch cultures in 0.25 L automated parallel Ambr 250 High Throughput bioreactors (Sartorius, Germany) as previously described . Each clone had distinct growth and productivity properties, but all expressed the same IgG1 mAb. The reactors ran for 14 days (one was halted on day 12 after contamination). Samples were automatically drawn daily by an Ambr 250 liquid handler (Sartorius, Germany). 2.2 Extracellular Metabolomics Analysis We conducted untargeted metabolomics analysis of extracellular metabolites using tandem liquid and gas chromatography‐mass spectrometry (LC‐MS/MS, GC‐MS), and nuclear magnetic resonance (NMR) spectroscopy. The details sample preparation, quantitation, and data pre‐processing. We removed metabolites with relative standard deviations over 30% in the quality control samples, signal‐to‐noise ratios below 3, and duplicates across LC, GC, and/or NMR. In all, 109 unique metabolites were annotated. 2.3 Bioprocess Variables Cell culture variables (Figure ) were measured with a BioProfile FLEX2 (Nova Biomedical, USA), as was the titer using an Octet R8 system (Sartorius, Germany). After removing outliers, we calculated two process variables): the specific death rate ( µ d , 1/day) and the specific productivity (qP, pg/cell/day). For qP, the titer data were smoothed using a generic logistic function, and its derivative was normalized to the VCD. µ d and the specific growth rate ( µ g , 1/day) were calculated as described previously . We assigned cell growth phases as follows: exponential: µ g > µ d (≈ days 1–6); stationary: µ g ≈ µ d (≈ days 7–8); death: µ g < µ d , (≈ days 9–14). 2.4 Multivariate Data Analysis (MVDA) We performed MVDA using SIMCA, version 18.0.0 (Sartorius, Sweden). All data were mean‐centered and scaled to unit variance (UV) unless noted otherwise. PCA provided a data overview and outlier detection. OPLS regression linked the relative metabolite levels ( X ) to µ d or qP as the response ( y ) variable. SIMCA automatically estimated the number of model components from the coefficient of prediction ( Q 2 ) using cross‐validation. Models exceeding two components were manually reduced to 1 predictive + 1 orthogonal to avoid the risk of overfitting . To find the timeframe where the metabolite concentrations correlated with a response ( µ d or qP), we created one model per day and response, yielding 14 µ d and 14 qP OPLS models. Similar to other studies , we used Q 2 > 0.25 as the threshold for acceptable models, retaining only contiguous days for cohesion, meaning days 8–14 for qP and 7–14 for µ d , covering the stationary and death phases. We assessed the metabolites’ relationship to the bioprocess variables with two methods. In the global method, we created a single OPLS model for each response ( µ d or qP) using samples from the retained days. In the hierarchical method, we used the 8 µ d ‐ and 7 qP‐retained OPLS models and calculated the metabolites’ correlation‐scaled loadings ( p (corr)) . The p (corr) values were then used as input in OPLS effect projection (EP) models without mean‐centering. OPLS‐EP used p (corr) as X data against a y = 1 column vector to identify common trends between time points. We compared which metabolites were highlighted as significant in the global and hierarchical methods. Significance was estimated using p (corr) values from the two global OPLS models and the two OPLS‐EP models. p (corr) is functionally equivalent to the Pearson correlation coefficient (PCC or Mr) with significance based on Student's two‐tailed t‐distribution: (1) t = r n − 2 1 − r 2 where t is the t ‐statistic and n is the number of independent samples. The global method set the number of batches as n since samples from different days were dependent. If n were the number of samples, nearly all metabolites would be significant. The hierarchical method set the number of days as n since all batches were condensed to one value . The global method's p(corr) describes the metabolite‐response correlation, while the hierarchical method's p(corr) describes consistently sized correlations between time points. To avoid falsely flagging consistent but negligibly correlated metabolites as significant, we set the mean p(corr) threshold to 0.30 (Figure ). The contains pathway overrepresentation analysis of the significant metabolites.
Cell Line and Culture Conditions We grew eleven clones of the same CHO‐DG44 parental cell line (Sartorius, Germany) as fed‐batch cultures in 0.25 L automated parallel Ambr 250 High Throughput bioreactors (Sartorius, Germany) as previously described . Each clone had distinct growth and productivity properties, but all expressed the same IgG1 mAb. The reactors ran for 14 days (one was halted on day 12 after contamination). Samples were automatically drawn daily by an Ambr 250 liquid handler (Sartorius, Germany).
Extracellular Metabolomics Analysis We conducted untargeted metabolomics analysis of extracellular metabolites using tandem liquid and gas chromatography‐mass spectrometry (LC‐MS/MS, GC‐MS), and nuclear magnetic resonance (NMR) spectroscopy. The details sample preparation, quantitation, and data pre‐processing. We removed metabolites with relative standard deviations over 30% in the quality control samples, signal‐to‐noise ratios below 3, and duplicates across LC, GC, and/or NMR. In all, 109 unique metabolites were annotated.
Bioprocess Variables Cell culture variables (Figure ) were measured with a BioProfile FLEX2 (Nova Biomedical, USA), as was the titer using an Octet R8 system (Sartorius, Germany). After removing outliers, we calculated two process variables): the specific death rate ( µ d , 1/day) and the specific productivity (qP, pg/cell/day). For qP, the titer data were smoothed using a generic logistic function, and its derivative was normalized to the VCD. µ d and the specific growth rate ( µ g , 1/day) were calculated as described previously . We assigned cell growth phases as follows: exponential: µ g > µ d (≈ days 1–6); stationary: µ g ≈ µ d (≈ days 7–8); death: µ g < µ d , (≈ days 9–14).
Multivariate Data Analysis (MVDA) We performed MVDA using SIMCA, version 18.0.0 (Sartorius, Sweden). All data were mean‐centered and scaled to unit variance (UV) unless noted otherwise. PCA provided a data overview and outlier detection. OPLS regression linked the relative metabolite levels ( X ) to µ d or qP as the response ( y ) variable. SIMCA automatically estimated the number of model components from the coefficient of prediction ( Q 2 ) using cross‐validation. Models exceeding two components were manually reduced to 1 predictive + 1 orthogonal to avoid the risk of overfitting . To find the timeframe where the metabolite concentrations correlated with a response ( µ d or qP), we created one model per day and response, yielding 14 µ d and 14 qP OPLS models. Similar to other studies , we used Q 2 > 0.25 as the threshold for acceptable models, retaining only contiguous days for cohesion, meaning days 8–14 for qP and 7–14 for µ d , covering the stationary and death phases. We assessed the metabolites’ relationship to the bioprocess variables with two methods. In the global method, we created a single OPLS model for each response ( µ d or qP) using samples from the retained days. In the hierarchical method, we used the 8 µ d ‐ and 7 qP‐retained OPLS models and calculated the metabolites’ correlation‐scaled loadings ( p (corr)) . The p (corr) values were then used as input in OPLS effect projection (EP) models without mean‐centering. OPLS‐EP used p (corr) as X data against a y = 1 column vector to identify common trends between time points. We compared which metabolites were highlighted as significant in the global and hierarchical methods. Significance was estimated using p (corr) values from the two global OPLS models and the two OPLS‐EP models. p (corr) is functionally equivalent to the Pearson correlation coefficient (PCC or Mr) with significance based on Student's two‐tailed t‐distribution: (1) t = r n − 2 1 − r 2 where t is the t ‐statistic and n is the number of independent samples. The global method set the number of batches as n since samples from different days were dependent. If n were the number of samples, nearly all metabolites would be significant. The hierarchical method set the number of days as n since all batches were condensed to one value . The global method's p(corr) describes the metabolite‐response correlation, while the hierarchical method's p(corr) describes consistently sized correlations between time points. To avoid falsely flagging consistent but negligibly correlated metabolites as significant, we set the mean p(corr) threshold to 0.30 (Figure ). The contains pathway overrepresentation analysis of the significant metabolites.
Results and Discussion 3.1 Benchmarking Hierarchical Modeling with OPLS‐EP Against Global OPLS Modeling The method we present here rests on a hierarchical approach to model bioprocess data by Alinaghi et al. . Their approach captures the relationship between metabolites and response variables with separate OPLS regression models for each time point, then utilizes the metabolites’ p(corr) values, which summarize each batch into one value per time point, visualizing them in a PCA model . However, PCA models show overall differences, such as which metabolic correlations change over time. In this study, we focused on similarities instead: metabolites with consistent, strong relationships to a response at each time point. Thus, we replaced the PCA model with an OPLS‐EP model (Figure ). OPLS‐EP was originally developed for dependent samples where within‐group variation can exceed between‐group variation. OPLS‐EP captures the difference X after — X before as X effect against a y = 1 column vector, highlighting variables that change in the same direction for most observations . Here, we used p(corr) as a stand‐in for X effect , revealing metabolites that correlated consistently with a response variable across most days without the time dependence. We benchmarked integrating hierarchical modeling with OPLS‐EP against conventional, global OPLS with all time points simultaneously. We created one model per method (global/hierarchical) and response (qP/ µ d ), covering the stationary and death phases. We assessed the metabolite‐response correlations’ significance using shared and unique structures (SUS)‐plots . Table lists all correlations and trajectories over time. For both methods (Figure ), most metabolites were associated negatively with qP and positively with µ d , whether we considered all metabolites or only significant ones. Most metabolites also had the same correlation sign (±) in both methods for a given response. However, the hierarchical method generally showed higher p(corr) values (closer to ± 1) and more significant metabolites, meaning more metabolites to investigate further compared with the global method. Interestingly, the responses were inversely related: metabolites correlated with µ d were generally anti‐correlated with qP, and vice versa. As living cells synthesize mAbs, factors benefiting productivity should not lead to cell death simultaneously. A tradeoff between cell growth and productivity has been reported before . This inverse relationship was clearer in the global models (Figure ) as both µ d and qP co‐varied with time. Without time as a confounder, the metabolites were more spread out in the hierarchical models (Figure ). Thus, the hierarchical approach more accurately reflected the metabolite‐process variable relationship. In total, 109 metabolites were detected. For 66, the outcome was consistent across methods: 26 correlated significantly with the same response(s) in both methods, while 40 did not correlate significantly with any response. The method affected the remaining 43 metabolites, correlating significantly with qP and/or µ d only in one method. The hierarchical method had more unique metabolites: 30 versus 8 for qP; and 32 versus 6 for µ d , as shown by the hierarchical method's significant metabolites (Figure ) appearing both near the corners (significant) and the center (non‐significant) of the global SUS‐plot (Figure ). Conversely, the global method's significant metabolites (Figure ) mostly appear in the hierarchical SUS‐plot's corners (Figure ). Most unique metabolites correlated moderately with the response(s) ( r ≈ ±0.5) in the other method, though some were biologically negligible ( r < ±0.3). The unique metabolites were interesting as they differentiated the methods, highlighting separate metabolites depending on their relationship with the response variables over time. We illustrate the methods’ differences using four sample metabolites representing different categories: significant in both methods, only in the global, only in the hierarchical, or in neither. Guanosine monophosphate (Figure ) was highlighted as significantly correlated across all days and on each day separately. N‐acetylserine (Figure ) correlated positively across all days. However, by splitting the data by time point, the correlation switched signs from negative (day 8) to positive (day 11, 14) Thus, while significant in the global approach, N‐acetylserine was nonsignificant in the hierarchical method due to day‐to‐day inconsistency. Oxidized glutathione (GSSG) (Figure ) was nonsignificant for µ d across all days, but splitting the data by day revealed a consistent correlation over time: moderate (day 8, 11) but decreasing (day 14). The consistent day‐to‐day sign made GSSG significant for µ d in the hierarchical method. γ‐glutamylalanine (Figure ) was not significant, being weakly correlated both overall and on each day. Each category (Figure ) contained multiple metabolites, so the methods targeted different metabolites rather than just having sharper cutoffs. The global method strictly pinpointed metabolites with overall correlations, which the hierarchical method did not do if the sign (±) was inconsistent over time. Conversely, hierarchical modeling pinpointed consistent metabolites, even if the size varied so the overall correlation was weak. Thus, the hierarchical method found metabolites whose consistent response relationships were obscured by time in the global models. 3.2 Metabolites Significantly Correlated with qP and µ d Are Enriched in Similar Pathways After comparing the methods, we explored whether certain sets of the hierarchical models’ significantly correlated metabolites were enriched in specific pathways relative to all known compounds in CHO‐related pathways for days 7–14 ( µ d , Figure ) and 8–14 (qP, Figure ). Table lists pathways per metabolite. Because most metabolites correlated with both responses, their enrichment profiles overlapped, suggesting that many pathways are related to both responses: Both responses: ATP‐binding cassette (ABC) transporters; the tricarboxylic acid (TCA) cycle; pantothenate and coenzyme A (CoA) biosynthesis; beta‐alanine metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; glycine, serine, and threonine metabolism; and glutathione metabolism. Unique to µ d : ferroptosis (cell death from accumulated iron and lipid peroxides). Unique to qP: aminoacyl‐tRNA biosynthesis, glycerophospholipid metabolism, D‐amino acid metabolism, and pyruvate metabolism. In previous studies, the TCA cycle was enriched for qP . Pantothenate and CoA metabolism was notable in the stationary phase, as was pyruvate, glyoxylate, and dicarboxylate metabolism in the death phase . Glycerophospholipid metabolism has been linked to growth limitation . Glutathione metabolism is tied to the oxidative stress response and to productivity . While ABC transporters and aminoacyl‐tRNA biosynthesis were significant, they are not specific and could reflect any individual amino acid. We explored the response‐amino acid correlations further. Many amino acids were present in the feed and/or medium (Table ), affecting their levels over time and limiting how they reflected CHO metabolic changes. Most amino acids were detected by NMR, which had three daily samples, but we only modeled with one to work with the LC/GC‐MS data. The NMR data showed that each bolus raised the amino acid levels (Figure ) more than the cells consumed between feeds, for a net increase over time, giving spurious correlations. Only glutamine (Figure ) did not display this behavior, potentially due to its early depletion . While beyond this study's scope, studying production and consumption rates instead of concentrations might better characterize how amino acids impact productivity and cell death. For now, we suggest users monitor feed metabolites online or draw multiple samples per feed interval in fed‐batch and continuous cultures to ensure that observed concentration changes stem from cellular metabolism, not feeding. 3.3 Metabolites Unique to Hierarchical Modelling Are Biologically Relevant The pathway analysis included all significant metabolites from the hierarchical method, but some were also significant in the global method. Excluding amino acids, 28 metabolites were unique to the hierarchical method (Table ). Next, we examined their biological relevance to the responses. Carbohydrate metabolism is crucial for cell growth and productivity. CHO cells preferably convert glucose to lactate via aerobic glycolysis, even with ample oxygen. Lactate reduces cell growth and productivity and can induce apoptosis . We found that lactate anti‐correlated significantly with qP. If glucose is depleted, CHO cells consume lactate instead , converting lactate to pyruvate and acetyl‐CoA. Acetyl‐CoA can be made into acetate, which we observed was significantly correlated with µ d and anti‐correlated with qP, accruing extracellularly in the stationary phase. Still, acetyl‐CoA mainly enters the TCA cycle and oxidative phosphorylation, which are highly active in productive CHO cells . TCA intermediates can escape into the cytosol , so replenishing them upholds TCA cycle activity. We detected several TCA intermediates. Cis‐ and trans‐aconitate and citrate/isocitrate (indistinguishable) were significantly correlated with qP and anti‐correlated with µ d . In one study, supplementation with succinate, malate, and α‐ketoglutarate improved qP by up to 35% without viability loss . Succinate addition yielded positive growth into the stationary phase and the highest qP among all tested intermediates . Citrate, cis‐aconitate, fumarate, malate, and succinate correlated with qP and anti‐correlated with VCD in the stationary phase. Adding citrate in the exponential phase reduced peak VCD but increased qP . Citrate in the feed decreased the VCD earlier than in controls, possibly due to apoptosis . TCA intermediates are replenished using amino acids from the medium and cellular biosynthesis , first with glutamine, then other amino acids . Excessive or catabolized amino acids can create growth‐inhibiting derivatives that may accumulate extracellularly . Ammonia, the primary byproduct, negatively affects productivity and cell growth . Other derivatives include 4‐hydroxyphenyllactate, a growth inhibitor derived from phenylalanine and tryptophan , which we found was significantly correlated with µ d and anti‐correlated with qP. N‐formylmethionine, a cysteine and methionine product, accumulates in the exponential and stationary phases . Here, N‐formylmethionine declined in the death phase, significantly correlating with qP and anti‐correlating with µ d . Formate, a product of serine, glycine, and threonine, negatively impacts growth . In our study, formate was significantly correlated with qP and anti‐correlated with µ d . Fatty acid production keeps pace with cellular demand during growth but remains active even during nongrowth, causing build‐up over time . Glycerol‐3‐phosphocholine and phosphocholine decrease inside cells and accumulate outside in the exponential phase, correlating with apoptosis . In our study, glycerol‐3‐phosphocholine was significantly correlated with µ d . Glycerol‐3‐phosphocholine and phosphocholine were anti‐correlated with qP and accumulated in the death phase. Phospholipid build‐up may indicate poor control of lipid metabolism and membrane composition . Conversely, intracellular plasma membrane precursors decrease over time , so lipid metabolism and cell growth limitations are tightly linked. Energy production is vital in mAb synthesis as cells need ATP to form peptide bonds . High mAb producers have elevated levels of electron carriers in oxidative phosphorylation . However, this process creates reactive oxygen species, which can be detoxified by reduced glutathione (GSH) through GSH's oxidation to GSSG. GSH synthesis is upregulated in productive CHO cells , while GSSG correlates with caspase activity and can induce apoptosis in fresh cultures . Curiously, GSH depletion leads to reduced cholesterol synthesis. Since high cholesterol synthesis enhances productivity by improving secretion capacity, GSH's productivity effects may be tied to cholesterol . In our study, cholesterol was significantly correlated with µ d and anti‐correlated with qP. To our knowledge, several metabolites exclusively significant in the hierarchical models have not been linked to productivity or cell death in CHO cells before, making them interesting for future research. Some metabolites have ties in other cell lines, though. For example, 3‐methoxytyrosine was significantly correlated with qP, anti‐correlated with µ d , and accumulated in the death phase. In humans, pathogenic 3‐methoxytyrosine levels can lead to oxidative stress and possibly cell death . γ ‐glutamyltryptophan inhibits cell growth in tumor cell lines . In our study, γ ‐glutamyltryptophan significantly correlated with qP and anti‐correlated with µ d in the death phase. Succinyladenosine was significantly correlated with µ d , anti‐correlated with qP, and accumulated over time in our study. In adipocytes, succinyladenosine was significantly enriched after apoptosis . In summary, most metabolites highlighted only by the hierarchical method were biologically relevant to productivity and/or cell death in CHO cells. Some correlated metabolites have, to our knowledge, not been reported in CHO cells before. While several metabolites were already known from other studies, they often concern the exponential or stationary phases, and either productivity or cell death. We report correlations for the death phase and both responses for most metabolites. Although we have metabolite levels for the entire batch (14 days), we only obtained functional class models in the stationary and death phases. This might stem from insufficient time for the cells to impact the extracellular medium in the exponential phase . Alternatively, µ d and qP may not have been relevant initially; for instance, µ d remained low throughout the exponential phase.
Benchmarking Hierarchical Modeling with OPLS‐EP Against Global OPLS Modeling The method we present here rests on a hierarchical approach to model bioprocess data by Alinaghi et al. . Their approach captures the relationship between metabolites and response variables with separate OPLS regression models for each time point, then utilizes the metabolites’ p(corr) values, which summarize each batch into one value per time point, visualizing them in a PCA model . However, PCA models show overall differences, such as which metabolic correlations change over time. In this study, we focused on similarities instead: metabolites with consistent, strong relationships to a response at each time point. Thus, we replaced the PCA model with an OPLS‐EP model (Figure ). OPLS‐EP was originally developed for dependent samples where within‐group variation can exceed between‐group variation. OPLS‐EP captures the difference X after — X before as X effect against a y = 1 column vector, highlighting variables that change in the same direction for most observations . Here, we used p(corr) as a stand‐in for X effect , revealing metabolites that correlated consistently with a response variable across most days without the time dependence. We benchmarked integrating hierarchical modeling with OPLS‐EP against conventional, global OPLS with all time points simultaneously. We created one model per method (global/hierarchical) and response (qP/ µ d ), covering the stationary and death phases. We assessed the metabolite‐response correlations’ significance using shared and unique structures (SUS)‐plots . Table lists all correlations and trajectories over time. For both methods (Figure ), most metabolites were associated negatively with qP and positively with µ d , whether we considered all metabolites or only significant ones. Most metabolites also had the same correlation sign (±) in both methods for a given response. However, the hierarchical method generally showed higher p(corr) values (closer to ± 1) and more significant metabolites, meaning more metabolites to investigate further compared with the global method. Interestingly, the responses were inversely related: metabolites correlated with µ d were generally anti‐correlated with qP, and vice versa. As living cells synthesize mAbs, factors benefiting productivity should not lead to cell death simultaneously. A tradeoff between cell growth and productivity has been reported before . This inverse relationship was clearer in the global models (Figure ) as both µ d and qP co‐varied with time. Without time as a confounder, the metabolites were more spread out in the hierarchical models (Figure ). Thus, the hierarchical approach more accurately reflected the metabolite‐process variable relationship. In total, 109 metabolites were detected. For 66, the outcome was consistent across methods: 26 correlated significantly with the same response(s) in both methods, while 40 did not correlate significantly with any response. The method affected the remaining 43 metabolites, correlating significantly with qP and/or µ d only in one method. The hierarchical method had more unique metabolites: 30 versus 8 for qP; and 32 versus 6 for µ d , as shown by the hierarchical method's significant metabolites (Figure ) appearing both near the corners (significant) and the center (non‐significant) of the global SUS‐plot (Figure ). Conversely, the global method's significant metabolites (Figure ) mostly appear in the hierarchical SUS‐plot's corners (Figure ). Most unique metabolites correlated moderately with the response(s) ( r ≈ ±0.5) in the other method, though some were biologically negligible ( r < ±0.3). The unique metabolites were interesting as they differentiated the methods, highlighting separate metabolites depending on their relationship with the response variables over time. We illustrate the methods’ differences using four sample metabolites representing different categories: significant in both methods, only in the global, only in the hierarchical, or in neither. Guanosine monophosphate (Figure ) was highlighted as significantly correlated across all days and on each day separately. N‐acetylserine (Figure ) correlated positively across all days. However, by splitting the data by time point, the correlation switched signs from negative (day 8) to positive (day 11, 14) Thus, while significant in the global approach, N‐acetylserine was nonsignificant in the hierarchical method due to day‐to‐day inconsistency. Oxidized glutathione (GSSG) (Figure ) was nonsignificant for µ d across all days, but splitting the data by day revealed a consistent correlation over time: moderate (day 8, 11) but decreasing (day 14). The consistent day‐to‐day sign made GSSG significant for µ d in the hierarchical method. γ‐glutamylalanine (Figure ) was not significant, being weakly correlated both overall and on each day. Each category (Figure ) contained multiple metabolites, so the methods targeted different metabolites rather than just having sharper cutoffs. The global method strictly pinpointed metabolites with overall correlations, which the hierarchical method did not do if the sign (±) was inconsistent over time. Conversely, hierarchical modeling pinpointed consistent metabolites, even if the size varied so the overall correlation was weak. Thus, the hierarchical method found metabolites whose consistent response relationships were obscured by time in the global models.
Metabolites Significantly Correlated with qP and µ d Are Enriched in Similar Pathways After comparing the methods, we explored whether certain sets of the hierarchical models’ significantly correlated metabolites were enriched in specific pathways relative to all known compounds in CHO‐related pathways for days 7–14 ( µ d , Figure ) and 8–14 (qP, Figure ). Table lists pathways per metabolite. Because most metabolites correlated with both responses, their enrichment profiles overlapped, suggesting that many pathways are related to both responses: Both responses: ATP‐binding cassette (ABC) transporters; the tricarboxylic acid (TCA) cycle; pantothenate and coenzyme A (CoA) biosynthesis; beta‐alanine metabolism; alanine, aspartate, and glutamate metabolism; glyoxylate and dicarboxylate metabolism; glycine, serine, and threonine metabolism; and glutathione metabolism. Unique to µ d : ferroptosis (cell death from accumulated iron and lipid peroxides). Unique to qP: aminoacyl‐tRNA biosynthesis, glycerophospholipid metabolism, D‐amino acid metabolism, and pyruvate metabolism. In previous studies, the TCA cycle was enriched for qP . Pantothenate and CoA metabolism was notable in the stationary phase, as was pyruvate, glyoxylate, and dicarboxylate metabolism in the death phase . Glycerophospholipid metabolism has been linked to growth limitation . Glutathione metabolism is tied to the oxidative stress response and to productivity . While ABC transporters and aminoacyl‐tRNA biosynthesis were significant, they are not specific and could reflect any individual amino acid. We explored the response‐amino acid correlations further. Many amino acids were present in the feed and/or medium (Table ), affecting their levels over time and limiting how they reflected CHO metabolic changes. Most amino acids were detected by NMR, which had three daily samples, but we only modeled with one to work with the LC/GC‐MS data. The NMR data showed that each bolus raised the amino acid levels (Figure ) more than the cells consumed between feeds, for a net increase over time, giving spurious correlations. Only glutamine (Figure ) did not display this behavior, potentially due to its early depletion . While beyond this study's scope, studying production and consumption rates instead of concentrations might better characterize how amino acids impact productivity and cell death. For now, we suggest users monitor feed metabolites online or draw multiple samples per feed interval in fed‐batch and continuous cultures to ensure that observed concentration changes stem from cellular metabolism, not feeding.
Metabolites Unique to Hierarchical Modelling Are Biologically Relevant The pathway analysis included all significant metabolites from the hierarchical method, but some were also significant in the global method. Excluding amino acids, 28 metabolites were unique to the hierarchical method (Table ). Next, we examined their biological relevance to the responses. Carbohydrate metabolism is crucial for cell growth and productivity. CHO cells preferably convert glucose to lactate via aerobic glycolysis, even with ample oxygen. Lactate reduces cell growth and productivity and can induce apoptosis . We found that lactate anti‐correlated significantly with qP. If glucose is depleted, CHO cells consume lactate instead , converting lactate to pyruvate and acetyl‐CoA. Acetyl‐CoA can be made into acetate, which we observed was significantly correlated with µ d and anti‐correlated with qP, accruing extracellularly in the stationary phase. Still, acetyl‐CoA mainly enters the TCA cycle and oxidative phosphorylation, which are highly active in productive CHO cells . TCA intermediates can escape into the cytosol , so replenishing them upholds TCA cycle activity. We detected several TCA intermediates. Cis‐ and trans‐aconitate and citrate/isocitrate (indistinguishable) were significantly correlated with qP and anti‐correlated with µ d . In one study, supplementation with succinate, malate, and α‐ketoglutarate improved qP by up to 35% without viability loss . Succinate addition yielded positive growth into the stationary phase and the highest qP among all tested intermediates . Citrate, cis‐aconitate, fumarate, malate, and succinate correlated with qP and anti‐correlated with VCD in the stationary phase. Adding citrate in the exponential phase reduced peak VCD but increased qP . Citrate in the feed decreased the VCD earlier than in controls, possibly due to apoptosis . TCA intermediates are replenished using amino acids from the medium and cellular biosynthesis , first with glutamine, then other amino acids . Excessive or catabolized amino acids can create growth‐inhibiting derivatives that may accumulate extracellularly . Ammonia, the primary byproduct, negatively affects productivity and cell growth . Other derivatives include 4‐hydroxyphenyllactate, a growth inhibitor derived from phenylalanine and tryptophan , which we found was significantly correlated with µ d and anti‐correlated with qP. N‐formylmethionine, a cysteine and methionine product, accumulates in the exponential and stationary phases . Here, N‐formylmethionine declined in the death phase, significantly correlating with qP and anti‐correlating with µ d . Formate, a product of serine, glycine, and threonine, negatively impacts growth . In our study, formate was significantly correlated with qP and anti‐correlated with µ d . Fatty acid production keeps pace with cellular demand during growth but remains active even during nongrowth, causing build‐up over time . Glycerol‐3‐phosphocholine and phosphocholine decrease inside cells and accumulate outside in the exponential phase, correlating with apoptosis . In our study, glycerol‐3‐phosphocholine was significantly correlated with µ d . Glycerol‐3‐phosphocholine and phosphocholine were anti‐correlated with qP and accumulated in the death phase. Phospholipid build‐up may indicate poor control of lipid metabolism and membrane composition . Conversely, intracellular plasma membrane precursors decrease over time , so lipid metabolism and cell growth limitations are tightly linked. Energy production is vital in mAb synthesis as cells need ATP to form peptide bonds . High mAb producers have elevated levels of electron carriers in oxidative phosphorylation . However, this process creates reactive oxygen species, which can be detoxified by reduced glutathione (GSH) through GSH's oxidation to GSSG. GSH synthesis is upregulated in productive CHO cells , while GSSG correlates with caspase activity and can induce apoptosis in fresh cultures . Curiously, GSH depletion leads to reduced cholesterol synthesis. Since high cholesterol synthesis enhances productivity by improving secretion capacity, GSH's productivity effects may be tied to cholesterol . In our study, cholesterol was significantly correlated with µ d and anti‐correlated with qP. To our knowledge, several metabolites exclusively significant in the hierarchical models have not been linked to productivity or cell death in CHO cells before, making them interesting for future research. Some metabolites have ties in other cell lines, though. For example, 3‐methoxytyrosine was significantly correlated with qP, anti‐correlated with µ d , and accumulated in the death phase. In humans, pathogenic 3‐methoxytyrosine levels can lead to oxidative stress and possibly cell death . γ ‐glutamyltryptophan inhibits cell growth in tumor cell lines . In our study, γ ‐glutamyltryptophan significantly correlated with qP and anti‐correlated with µ d in the death phase. Succinyladenosine was significantly correlated with µ d , anti‐correlated with qP, and accumulated over time in our study. In adipocytes, succinyladenosine was significantly enriched after apoptosis . In summary, most metabolites highlighted only by the hierarchical method were biologically relevant to productivity and/or cell death in CHO cells. Some correlated metabolites have, to our knowledge, not been reported in CHO cells before. While several metabolites were already known from other studies, they often concern the exponential or stationary phases, and either productivity or cell death. We report correlations for the death phase and both responses for most metabolites. Although we have metabolite levels for the entire batch (14 days), we only obtained functional class models in the stationary and death phases. This might stem from insufficient time for the cells to impact the extracellular medium in the exponential phase . Alternatively, µ d and qP may not have been relevant initially; for instance, µ d remained low throughout the exponential phase.
Conclusions Bioprocess time series data are difficult to analyze. Both metabolic profiles and process variables are time‐dependent, confounding how the metabolites relate to process variables. Traditional correlation‐based statistical methods cannot resolve this time dependence. Here, we adopted a novel hierarchical method to model time‐dependent data at each time point separately based on p(corr), combining the results in an OPLS‐EP model. We applied this method to the extracellular metabolome of 11 CHO clones with different growth and productivity properties over 14 days, seeking metabolites with similar correlations with µ d and/or qP between time points. The hierarchical method pinpointed many metabolites consistently related to µ d and/or qP that conventional, global OPLS modeling missed. The new approach also avoided flagging metabolites with inconsistent relations as significant, unlike the global method. µ d and qP were inversely related: most metabolites were positively associated with one and negatively with the other, reflecting the relation between cell viability and mAb production. The key pathways concerned carbohydrate metabolism (TCA cycle, pyruvate, glyoxylate, pantothenate). Amino acid metabolism was highlighted as important too, but the correlations were spurious, mainly caused by amino acids’ being in the feed, necessitating caution when analyzing changes in amino acid levels in fed‐batch cultures. Our study focused on the extracellular metabolome, which admittedly has limited relevance to intracellular processes. Likewise, intracellular metabolomics cannot adequately describe extracellular metabolic dynamics. Most studies explore either intra‐ or extracellular metabolomics, so integrating both sides could provide a more holistic understanding of cellular metabolism during bioprocess production. Importantly, the metabolites in our study that only the hierarchical method highlighted were biologically relevant. Some were described previously as relevant for cell death or productivity, but others have not been reported in CHO literature before, showing the new method's benefits. We hope our findings contribute to a better understanding of biopharmaceutical production and to improving bioprocesses for cheaper and safer medicines.
Andreas Eriksson : Data curation (lead); investigation (lead); methodology (equal); visualization (lead); writing–original draft (lead); writing–review and editing (equal). Anne Richelle : Data curation (supporting); formal analysis (equal); writing–review and editing (equal). Johan Trygg : Funding acquisition (equal); writing–review and editing (supporting). Steffi Scholze : Investigation (supporting); resources (lead); writing–review and editing (supporting). Shanti Pijeaud : Formal analysis (equal); writing–review and editing (supporting). Henrik Antti : Funding acquisition (equal); supervision (equal); writing–review and editing (supporting). Christoph Zehe : Conceptualization (equal); writing–review and editing (supporting). Izabella Surowiec : Conceptualization (equal); project administration (lead); supervision (equal); writing–review and editing (equal). Pär Jonsson : Formal analysis (equal); methodology (equal); supervision (equal); writing–review and editing (equal).
The authors declare no conflicts of interest.
Supporting Information
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Fracture resistances of heat-treated nickel-titanium files used for minimally invasive instrumentation | 606a1bce-375c-480e-a56a-7dc95110b507 | 11761776 | Dentistry[mh] | Recently, the concept of minimal invasiveness has been introduced in dentistry and it has been applied in various practical dentistry, including restorative dentistry and implant surgery . Minimally invasive endodontic treatment has been also introduced to clinical dentistry to increase the preservation of tooth material for better prognosis of endodontically treated teeth (ETT) . Various kinds of access cavity designs, such as conservative and truss access, have been proposed to preserve coronal tooth structure . The fracture resistance of an ETT was increased by preparing the conservative endodontic access cavity . Minimally invasive access was reported to maintain coronal dentin and offer increased fracture resistance of ETT . In these minimally invasive approaches to access cavity preparation, while the smaller size from the occlusal surface is the main approach, different access directions and locations were attempted depending on cases (mainly, caries location) in anterior teeth and premolar . During the root canal preparation procedure after access cavity preparation, instrumentation methods and/or instruments themselves have been reported as to be related to the reduced resistance to mechanical failure of the ETT . The potential risk of root dentin crack or vertical root fracture (VRF) from endodontic treatment due to excessive canal preparation has been reported . A certain size of an instrument under certain canal conditions may have a higher potential risk of root dentin damages . To reduce this kind of unfavorable situation, martensite dominant nickel-titanium (NiTi) files were developed through various heat treatments and geometric alterations . These heat-treated files have been reported to have several advantages, such as maintaining the center of the canal well to prevent it and causing less transportation or development due to their lower stiffness and higher ductility than the files made of conventional NiTi alloy . Meanwhile, the preservation of peri-cervical dentin (PCD) has been suggested as an important anatomical structure for the long-term prognosis of the ETT . Preservation of PCD has been reported to reduce VRF in the tooth and prolong the prognosis after root canal treatment . According to the minimal invasiveness concept, the usage of reduced-tapered NiTi files and conservative endodontic access cavity preparation has gained popularity. Recently, some NiTi file systems in the form of smaller tapers and thinner shafts were introduced to preserve PCDs and consequently have a better prognosis for the ETT. It was suggested that minimally invasive root canal preparation performed using NiTi files with a smaller taper may preserve the root dentin tissue compared to that of larger tapered NiTi files and increase the fracture resistance of the ETT . TruNatomy system (Dentsply Sirona, Ballaigues, Switzerland) is designed to preserve PCDs and increase file flexibility by reducing the diameter of file shaft from approximately 1.2 mm to 0.8 mm, followed by a post-manufacturing heat treatment. TruNatomy file has a square cross-section with an off-center design, a variable taper and 4% taper at apical few millimeters . Another instrument system aimed at minimal invasive root canal shaping, EndoRoad (Maruchi, Wonju, Korea) made of MT-wire (by memory-triple heat treatment technology as named by the manufacturer) with surface treatment was recently introduced . EndoRoad file system has a triangular cross-sectional shape with increased ductility achieved by using the heat-treated alloy (MT-wire) at body temperature . Typically, files with thinner shafts and/or smaller cross-sectional areas have lower torsional strength and higher fatigue resistances, as reported by geometric optimization studies . This relationship is primarily attributed to the reduction in metal mass, which decreases rigidity and enhances flexibility. However, these studies have predominantly focused on general file designs, and there is a notable lack of research specifically addressing files designed for minimally invasive root canal procedures. Files that underwent specialized heat treatments and surface modifications are especially noteworthy, as these processes have the potential to significantly modify their mechanical properties, such as torsional strength and cyclic fatigue resistance. The purpose of this study was to compare the torsional fracture resistance, bending stiffness, and cyclic fatigue resistance of heat-treated NiTi files for minimally invasive instrumentation with a common heat-treated NiTi instrument system (ProTaper Gold; Dentsply Sirona). The null hypothesis was that all of these heat-treated NiTi files have the same mechanical properties.
Two file systems with a minimally invasive concept were used in this study: TruNatomy (TN; Prime, #26/.04v) and EndoRoad (ER; #25/.06v). They were compared with ProTaper Gold (PG; F2, #25/.08v; Dentsply Sirona), which is one of the popular systems as contemporary heat-treated file systems . All three file systems have a shaft length of 25 mm and a similar tip size of the International Organization for Standardization (ISO) #25. Before the test, all instruments were inspected under a dental operating microscope (M320; Leica Microsystems, Wetzlar, Germany) at ×10 magnification to confirm that there was no deformation of the files. Table provides the manufacturers’ descriptions for each of these files. Sample size calculations were conducted using G*Power 3.1 software (Heinrich-Heine-Universität, Düsseldorf, Germany). With an α level of 5%, a β level of 20%, and the chosen effect size, the required total sample size was determined to be 12. Therefore, each group in this study consisted of 15 files. In this study, torsional resistances, bending stiffness, and cyclic fatigue resistance were compared using customized devices (AEndoS and EndoC; DMJ System, Busan, Korea) (Fig. ). AEndoS was used for the torsional test and bending stiffness measurement test with dedicated jigs. EndoC was used for cyclic fatigue resistance with a simulated canal. All the experiments were carried out in a heat-controlled environment similar to the intraoral temperature (37℃). The temperature was controlled using a thermal sensor and heat generator attached to the test devices. Torsional resistance test For the torsional resistance test, the torsional strength and distortion angle were measured using the AEndoS (Fig. A and B) ( n = 15). The file tip of 3 mm was fixed between copper plates and driven clockwise at 60 rpm until fracture occurred . While the files rotated, the torsional load (Ncm) and distortion angle (°) were recorded at a speed of 50 Hz. The toughness until fracture was numerically calculated from the area under the plot presenting distortion angle (X axis) and torsional load (Y axis) using Origin v6.0 Professional (Microcal Software Inc, Northampton, USA) . Bending stiffness test The bending stiffness was measured also with the AEndoS (Fig. A and C) following the American National Standard/American Dental Association specification no. 28 and ISO specification 3630-1:2008 . The file tip of 3 mm files was fixed between two resin blocks and bent at 45° to their long axis. After bending the file 45°, the angle between the location of the bent file and the initial location was defined as the residual angle ( n = 15). To measure the residual angle, the distance between the initial location of the file and the location after bending was measured using a micro-caliper. The residual angle was calculated using the Pythagorean method. Cyclic fatigue resistance test Cyclic fatigue resistance was evaluated in another custom-made device, EndoC (Fig. D). Cyclic fatigue resistance was tested by pecking and rotating instruments in an artificial canal with a 7.8 mm radius and a 45° angle of curvature until fracture (Fig. E). The instruments from each group ( n = 15) were rotated in a pecking motion using an endodontic motor and handpiece (X-smart Plus; Dentsply Sirona) at speeds of 600 revolutions per minute (rpm), 500 rpm, and 300 rpm for ER, TN, and PG, respectively. The pecking distance comprised a 4 mm increment in each direction every 0.5 s, with a 50 ms dwell time. The moment the instrument was broken was recorded using a chronometer. The number of cycles to failure (NCF) was calculated by applying the rotation speed and time (seconds) to fracture. Scanning electron microscope (SEM) observation After the torsional and cyclic fatigue tests, the fractured files underwent examination via scanning electron microscopy (SEM). Before microscopic observation, all the files were subjected to a cleaning process with absolute alcohol and immersion in an ultrasonic bath to eliminate any debris. Subsequently, the files were left to air dry at room temperature and attached onto metal plates using double-sided adhesive carbon tape. The mounted samples were then placed inside the SEM (JSM-7200 F: JEOL, Tokyo, Japan). Finally, four fractured fragments from each group were observed under the SEM in various magnification ranges to assess the topographic features of the surface. Differential scanning calorimetry Differential scanning calorimetry (DSC) analysis was performed to determine phase transformation temperatures. Specimens from three groups were analyzed (DSC Q2000 V24.4 Build 116; TA Instruments, New Castle, USA) to obtain the features of austenite peak (A p ) temperature during heating and cooling curves. To determine various temperature points, including the austenite starting temperature (A s ), austenite finishing temperature (A f ), R-phase starting temperature (R s ), R-phase finishing temperature (R f ), martensite starting temperature (M s ), and martensite finishing temperature (M f ), a specific method was employed. Referring to earlier our laboratory tests, the maximum temperature during calorimetric heating was determined to be 80 °C, which was higher than the temperature required to achieve a fully austenite state for each of the studied specimens . Statistics analysis The normality of the data was assessed using Levene test. In cases of normal distribution, for data evaluation and comparison among the file groups, one-way analysis of variance and Duncan’s post-hoc comparison were conducted using statistic program SPSS (version 25.0; SPSS Inc., Chicago, IL). Statistical significance was set at 95%.
For the torsional resistance test, the torsional strength and distortion angle were measured using the AEndoS (Fig. A and B) ( n = 15). The file tip of 3 mm was fixed between copper plates and driven clockwise at 60 rpm until fracture occurred . While the files rotated, the torsional load (Ncm) and distortion angle (°) were recorded at a speed of 50 Hz. The toughness until fracture was numerically calculated from the area under the plot presenting distortion angle (X axis) and torsional load (Y axis) using Origin v6.0 Professional (Microcal Software Inc, Northampton, USA) .
The bending stiffness was measured also with the AEndoS (Fig. A and C) following the American National Standard/American Dental Association specification no. 28 and ISO specification 3630-1:2008 . The file tip of 3 mm files was fixed between two resin blocks and bent at 45° to their long axis. After bending the file 45°, the angle between the location of the bent file and the initial location was defined as the residual angle ( n = 15). To measure the residual angle, the distance between the initial location of the file and the location after bending was measured using a micro-caliper. The residual angle was calculated using the Pythagorean method.
Cyclic fatigue resistance was evaluated in another custom-made device, EndoC (Fig. D). Cyclic fatigue resistance was tested by pecking and rotating instruments in an artificial canal with a 7.8 mm radius and a 45° angle of curvature until fracture (Fig. E). The instruments from each group ( n = 15) were rotated in a pecking motion using an endodontic motor and handpiece (X-smart Plus; Dentsply Sirona) at speeds of 600 revolutions per minute (rpm), 500 rpm, and 300 rpm for ER, TN, and PG, respectively. The pecking distance comprised a 4 mm increment in each direction every 0.5 s, with a 50 ms dwell time. The moment the instrument was broken was recorded using a chronometer. The number of cycles to failure (NCF) was calculated by applying the rotation speed and time (seconds) to fracture.
After the torsional and cyclic fatigue tests, the fractured files underwent examination via scanning electron microscopy (SEM). Before microscopic observation, all the files were subjected to a cleaning process with absolute alcohol and immersion in an ultrasonic bath to eliminate any debris. Subsequently, the files were left to air dry at room temperature and attached onto metal plates using double-sided adhesive carbon tape. The mounted samples were then placed inside the SEM (JSM-7200 F: JEOL, Tokyo, Japan). Finally, four fractured fragments from each group were observed under the SEM in various magnification ranges to assess the topographic features of the surface.
Differential scanning calorimetry (DSC) analysis was performed to determine phase transformation temperatures. Specimens from three groups were analyzed (DSC Q2000 V24.4 Build 116; TA Instruments, New Castle, USA) to obtain the features of austenite peak (A p ) temperature during heating and cooling curves. To determine various temperature points, including the austenite starting temperature (A s ), austenite finishing temperature (A f ), R-phase starting temperature (R s ), R-phase finishing temperature (R f ), martensite starting temperature (M s ), and martensite finishing temperature (M f ), a specific method was employed. Referring to earlier our laboratory tests, the maximum temperature during calorimetric heating was determined to be 80 °C, which was higher than the temperature required to achieve a fully austenite state for each of the studied specimens .
The normality of the data was assessed using Levene test. In cases of normal distribution, for data evaluation and comparison among the file groups, one-way analysis of variance and Duncan’s post-hoc comparison were conducted using statistic program SPSS (version 25.0; SPSS Inc., Chicago, IL). Statistical significance was set at 95%.
Using descriptive statistics, the average and standard deviation results from the three tests are shown in Table . PG showed the highest ultimate torsional strength, followed by the ER and TN ( p < 0.05). ER showed a significantly greater distortion angle than TN and PG. ER showed the highest toughness, followed by PG and TN, in the order ( p < 0.05). ER and TN showed significantly lower bending stiffness than PG ( p < 0.05). ER and TN had similar bending stiffness. ER showed higher residual angles than PG and TN ( p < 0.05). ER showed the highest cyclic fatigue resistance, followed by PG and TN ( p < 0.05). SEM examination of the fractured fragments after the torsional resistance tests showed typical features of torsional fractures, such as concentric abrasion marks and fibrous dimples from the torsional center (Fig. ). In the lateral aspects, while the PG and TN showed distinct milling grooves and curved and slippery was observed. However, ER group did not present the milling groove and slippery. SEM examination of the cyclic fatigue fracture fragments showed typical features of fatigue fractures, such as crack initiation area(s), fatigue zone, and rolled over fast fracture area (Fig. ). The results of the DSC analysis exhibited a distinct difference in phase transformation temperatures for all groups (Fig. ). DSC analysis showed that PG and ER showed a peak of austenite (A p ) at temperatures higher than body temperature, 42 °C and 40 °C respectively. TN showed A p at 25 °C, in a relatively low temperature.
The primary goal of endodontic treatment to save a natural tooth is the removal of bacterial infection from the root canal system. After root canal treatment, the restoration of the tooth function and prevention of reinfection are essential. ETT should be reinforced by restorative treatments such as intra-coronal or full veneer restorations, to ensure proper coronal sealing . The durability and prognosis of the ETT should be considered when selecting materials and methods for these post-endodontic restoration procedures . Minimally invasive endodontic procedures primarily consist of access cavity preparation and root canal instrumentation . Many studies have investigated access cavity designs to increase mechanical resistance under occlusal loading using in-vitro tests and finite element analysis . However, there is a lack of sufficient studies examining the effects of different file systems and apical root canal preparation on extending the mechanical durability of ETT . This study compared the mechanical properties of NiTi files recently developed with the concept of minimal invasiveness. These files may have different mechanical properties due to their thinner and smaller size compared to conventional and traditional NiTi files, aimed at preserving the root dentin’s PCD during instrumentation . Silva et al. reported that minimal endodontic access cavities may increase the remaining root dentin and, consequently, reduce the fracture of ETT. Thus, manufacturers have developed files with relatively thin shafts, with the maximum flute diameter (MFD) reduced to 0.8 mm instead of usual 1.2 mm for most generic variable tapered files . While these small-sized instruments generally exhibit high fatigue resistance and flexibility, they may have lower torsional strength . In conclusion, each file used in the experiment exhibited differences in physical properties based on heat treatment and cross-sectional area. Therefore, the null hypothesis was rejected. In this study, PG was used for comparison, although it does not adhere to the minimally invasive concept, serving as a comparison/control group due to its common size and composition of heat-treated alloy (Gold wire). Including PG, TN and ER were evaluated for the mechanical properties of torsional resistance, bending stiffness, and flexural fatigue resistance. Torsional resistance assesses the ability to withstand distortion or fracture when a file becomes stuck in the root canal, particularly focusing on ductility that can affected by heat treatment . This is an important aspect for the thin file systems because they usually have lower torsional strength and are at risk of unwinding or distortion in tight canal conditions . Bending stiffness measures flexibility, while cycle fatigue resistance evaluates the ability to resist fractures from repeated bends in curved root canals . In this study, PG showed the higher ultimate torsional strength than ER and TN ( p < 0.05). This difference can be attributed to the larger cross-section of PG, which usually has higher torsional resistance compared to files with smaller cross-Sects. . Meanwhile, ER showed a significantly greater distortion angle than TN and PG, leading to higher toughness, attributable to its heat treatment. From the perspective of toughness—a composite parameter of torsional resistance—this increase in flexibility through heat treatment is particularly significant, as it enables the file to better endure torsional stress . Thus, clinicians using heat-treated files may have a better chance of identifying distorted file before fractures occur, thereby improving both safety and performance during clinical procedures. As found in the bending stiffness test, ER has a lower bending stiffness and larger residual angles compared to PG and TN. These characteristics contributed to ER showing the highest cyclic fatigue resistance than others. This outcome could be attributed to ER’s thin geometry and the properties of heat-treated NiTi alloy. However, TN showed lower fatigue resistance than ER and similar resistance to PG, contrary to expectations based on their geometries and alloy characteristics. The lower fatigue resistance of TN could be attributed to surface irregularities, particularly machining grooves. In comparison to ER, TN’s surfaces have numerous machining grooves that could initiate and propagate fatigue fractures more readily than ER’s smoother surfaces resulting from heat treatment and surface coating . In terms of bending stiffness, PG showed higher stiffness than ER and TN. In other words, ER showed the most flexible properties with low bending stiffness. Therefore, the heat-treated ER exhibits remarkable flexibility even at room temperature, owing to its dominant martensitic property. This is the most important characteristic of the ER file and has not been previously reported. According to the manufacturer (Maruchi), ER was designed to possess soft properties while increasing fracture resistance through patented heat treatment and surface treatment (MT wire technology) . Reddy et al. reported that the cyclic fatigue resistance of TN was significantly higher compared to PG, which contrasts with the results of this study. However, Elnaghy et al. reported that the HyFlex CM instruments (Coltene-Whaledent, Allstetten, Switzerland) had greater fatigue resistance than Vortex Blue (DENTSPLY Tulsa Dental Specialties, Johnson City, TN), TN, and FlexMaster. Although Elnaghy et al. did not compare TN with the PG, similar to this study, TN did not demonstrate the highest fatigue resistance when compared to the HyFlex CM and Vortex Blue. These findings of lower fatigue resistance in TN might be attributed to its lower A p temperature (25 °C), which is around room temperature and lower than body temperature. As discussed above considering the test condition, temperature is an important factor to make differences on the in-vitro fatigue resistance tests. Considering the body temperature, it is important to determine the A p temperature of the file alloys. If the file alloy has a lower A p temperature than the body temperature, it may not gain any specific advantage from heat treatment. Thus, DSC evaluation is important for assessing the thermal properties of the file alloys. The effect of phase transition temperature on torsional strength is relatively minimal. This may be primarily due to experimental limitations, as the influence of temperature was only applied to the apical 3 mm of the file during the torsional test. As a result, the full impact of temperature on the entire length of the file could not be accurately assessed. The localized temperature effect on the apical end may not reflect the overall behavior of the file, and further studies with more comprehensive temperature control along the entire length of the file could provide a better understanding of its influence on torsional strength. In this study, for the torsional resistance test, a rotation speed of 60 rpm was applied, despite the ISO and American Dental Association (ADA) suggesting a speed of 2 rpm . Ha et al. reported that the torsional resistances of the rotary instruments were not affected by the rotational speed. As they described, the ISO and ADA methods are designed to test the mechanical performance of stainless-steel hand files, for which a speed of 2 rpm is reasonable. However, NiTi rotary instruments typically have a much higher recommended rotational speed. The instruments used in this study for the cyclic fatigue resistance tests were rotated in a pecking motion using an endodontic motor at speeds of 600 rpm, 500 rpm, and 300 rpm for ER, TN, and PG, respectively, following the manufacturer’s instructions. Li et al. evaluated cyclic fatigue resistances of rotary files depending on rpm and found that the time to failure significantly decreased as the rotational speeds were increased, although the cycles until fracture were not significantly affected. Therefore, comparing the NCF in this study to assess fatigue resistance would be an appropriate criterion. Minimally invasive approaches often result in small access cavities, which may lead to a curved or inclined access of the file to the root canals. This situation may increase the difficulty of maintaining straight-line access, and result in higher stress on files due to curvature or angulation, thereby heightened the risk of file fracture or deformation. In such clinical situations, clinicians may select heat-treated files to reduce the risk of file fracture . This study has some limitations because the physical properties of files are influenced not only by heat treatment but also by geometry, such as thickness and cross-sectional shape. Therefore, it is unclear whether the results of this in-vitro experiment were sorely due to heat treatment or geometry. To understand the changes in physical properties resulting from heat treatment, different heat treatments applied to the files with the same geometry could be performed. Thus, further investigation may consider examining the differences in physical properties resulting from various heat treatment applied to files with identical geometries. In summary, the file used for minimally invasive root canal preparation is small in size and heat-treated, leading to excellent fatigue fracture resistance. However, it is essential to select the file appropriately based on the specific anatomical conditions of the root canal system and the tooth, such as canal curvature, calcification levels, and access cavity designs. Clinicians should balance the preservation of tooth structure with the mechanical performance requirements of the file to ensure optimal treatment outcomes.
This study compared the torsional fracture resistance, bending stiffness, and cyclic fatigue resistances of heat-treated NiTi files (TruNatomy and EndoRoad) designed for minimally invasive instrumentation with a common heat-treated NiTi instrument system (ProTaper Gold). Among the tested heat-treated instruments, ProTaper Gold exhibited the highest torsional strength and resistance compared to others. EndoRoad was the most flexible instrument with lower bending stiffness. EndoRoad also showed higher torsional resistance than TruNatomy as a minimally invasive instrument.
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Managing women‐specific bleeding in inherited bleeding disorders: A multidisciplinary approach | bd3ad107-5bed-48d1-9fdb-1991d60df6fe | 8246991 | Gynaecology[mh] | INTRODUCTION Women and Girls Bleeding Disorders (WBD) face specific challenges associated with reproduction and the menstrual cycle. Multidisciplinary management is important to preserve quality of life and social participation. Here, we present two case examples to discuss the management of WBD from different perspectives of involved healthcare professionals, including the aspect of sexuality, and the patients' perspective. Our aim is to support appropriate multidisciplinary care for WBD in haemophilia treatment centres (HTC). CASE 1 An 11‐year‐old girl with severe type 2A von Willebrand disease (VWD), with von Willebrand Factor (VWF) antigen 40 IU/dl, VWF activity 5 IU/dl and factor VIII 53 IU/dl consults her paediatric haematologist. Her medical history includes an uncomplicated adenotonsillectomy at the age of 4 years covered with VWF concentrate. Recurrent epistaxis has been controlled with cauterization once and occasional use of Desmopressin and Tranexamic acid. She is an active girl who participates in sports (swimming), about to go to secondary school and to have her first menstruation. 2.1 Question for the multidisciplinary team Over 80% of women with VWD experience heavy menstrual bleeding , : how should this girl and her family be prepared for the menarche? 2.1.1 Nurse perspective Haemophilia treatment centres nurses have an important role in HTC multidisciplinary team providing education, being accessible for patient's questions and uncertainties/worries and making sure that the right team members are contacted in case of a specific problem of concern. In educating, it should be taken into mind to talk with both parents and the girl in ‘understandable’ language (no jargon) and to adjust the mode of education to the patient's age and culture. Education of WBD should include information on the normal menstrual cycle and on symptoms of iron deficiency. Next to information on pads and tampons, information about use of menstrual cups—how to measure blood loss and if ‘period poverty’ is an issue, the need for (psycho)social support can be explored in their families. The Pictorial Blood Loss Assessment Chart (PBAC) can be used to measure (ab)normal menstruation; a PBAC score >100 corresponds to abnormal blood loss. Families can be made aware of mobile applications (apps) that track periods and blood loss, for example www.sisterhoodapp.com , if appropriate in the local setting. Also other practical issues should be discussed, such as information about sports participation and the need to have pads available at school. Basic sexual education, including information on how to manage bleedings, should also be part of the conversation. Adolescent girls should be prepared for transition to adult care, for example starting with an appointment by an adult nurse at age 14–15 years. In case of heavy menstrual bleeding (HMB), the girl should have medication available to treat her coagulation defect (such as Tranexamic acid, Desmopressin and/or clotting factor concentrate). If severe menstrual pain occurs they should know what kinds of painkillers are allowed; non‐steroidal anti‐inflammatory drugs (NSAIDs) preferably not to avoid additional platelet dysfunction, cyclo‐oxygenase‐2 (COX‐2) selective NSAIDs are allowed since these drugs comprise platelet function to a lesser extent. The aim of hormonal treatment should be explained mainly as a treatment of HMB rather than as an contraconceptive although both effects should be discussed. Haemoglobin and ferritin levels should be checked regularly in case of HMB. Women and Girls Bleeding Disorders suffering from HMB are likely to experience psychosocial issues which can affect academic and social pursuits. Offering peer support can help to maintain their self‐esteem, as well as extending management beyond the hospital, teaching and organizing home treatment for those who require regular factor replacement therapy and liaising with school or place of work when required. If bleeding problems interfere with social participation, a social worker should be involved. 2.1.2 Psychologist perspective Based on the clinical experiences, menarche may be a traumatic experience affecting quality of life and even provoke a post‐traumatic stress syndrome (PTSS). HMB has negative impact on the quality of life in WBD, impairing their daily activities and social life. , The health‐related quality of life (HRQoL) in 13 years old girls with a bleeding disorder is lower compared to their healthy peers. In contrast, there is no apparent difference in psychosocial functioning and HRQoL between young adult women with a bleeding disorder compared to peers. However, women with bleeding disorders participating in a large survey stated that they had mainly problems on reproduction and sexual relations. They also felt more fatigued. Psychosocial support is important to prevent further deterioration which may hamper the social development of these girls. To check whether reference to a psychologist is indicated, standard screening with patient‐reported outcome measurement instruments, such as HRQoL questionnaires, can be performed ahead of an outpatient clinical visit. Ideally, a psychologist, or at least a social worker, is part of the multidisciplinary team to discuss WBD who experience serious psychosocial issues. In case of PTSS eye movement desensitization reprocessing treatment by a psychologist is indicated. When problems like fatigue, acceptance, shame or fear interfere with normal social functioning, treatment with cognitive behaviour therapy may be indicated. 2.1.3 Gynaecologist perspective Menstrual disorders are common in adolescent girls. Menstrual periods can be heavy and irregular, usually secondary to anovulatory cycles caused by immaturity of the hypothalamic‐pituitary‐ovarian axis. Adolescent girls with inherited bleeding disorders are at a higher risk of HMB; often starting from menarche with a significant health implications such as anaemia, hospitalization and need for transfusion of blood or blood products. HMB has a negative impact on quality of life, can be a source of distress for adolescent girls and may affect their school attendance and performance as well as their social life. In case of suspected heavy menstrual bleeding (HMB), assessment of menstrual blood loss using the PBAC score is useful. Alternatively, narrative assessment should be adopted; excessive bleeding is defined as prolonged (>7 days), and/or the need to change a pad every 2 h or more. Careful history taking is important. Pelvic examination and ultrasound, if appropriate, may reveal an additional reason for HMB. The goals of HMB treatment are to reduce morbidity, prevent iron deficiency and improve quality of life through reducing menstrual blood loss and attainment of a predictable pattern of menstruation. Treatment options for HMB depend on age, menstrual pattern, the need for contraception and patients tolerability and acceptability of available treatment options. Management of HMB in adolescents is usually medical and there are several haemostatic and hormonal options that can be used singly or in combination (Table ). However, there are specific considerations when using these agents in adolescent girls that must be incorporated into multidisciplinary and shared decision‐making with the girls and their family. This is ideally provided in a joint clinic, where the experts can agree on optimal use of various treatments and assess the girls in one visit. WBD value provision of care in such models with a high level of satisfaction and better compliance to treatment. 2.1.4 Patients' perspective A 42‐year‐old woman, suffering from severe VWD, shared her personal experience with the menarche. This was very traumatic. She did not receive any education or information in advance and there was no proper treatment. She had to stay in the hospital for weeks due to uncontrolled bleeding. She strongly feels that educating patients and parents on menstrual bleeding and on the psychosocial effects may prevent such negative healthcare experiences. She also emphasizes unrestricted use of factor concentrates to treat HMB, if needed, since this made a huge difference for her. Medical doctors in HTCs should be aware that HMB may be very stressful and anticipate better, in order to prevent a traumatic experience with lifelong impact. Question for the multidisciplinary team Over 80% of women with VWD experience heavy menstrual bleeding , : how should this girl and her family be prepared for the menarche? 2.1.1 Nurse perspective Haemophilia treatment centres nurses have an important role in HTC multidisciplinary team providing education, being accessible for patient's questions and uncertainties/worries and making sure that the right team members are contacted in case of a specific problem of concern. In educating, it should be taken into mind to talk with both parents and the girl in ‘understandable’ language (no jargon) and to adjust the mode of education to the patient's age and culture. Education of WBD should include information on the normal menstrual cycle and on symptoms of iron deficiency. Next to information on pads and tampons, information about use of menstrual cups—how to measure blood loss and if ‘period poverty’ is an issue, the need for (psycho)social support can be explored in their families. The Pictorial Blood Loss Assessment Chart (PBAC) can be used to measure (ab)normal menstruation; a PBAC score >100 corresponds to abnormal blood loss. Families can be made aware of mobile applications (apps) that track periods and blood loss, for example www.sisterhoodapp.com , if appropriate in the local setting. Also other practical issues should be discussed, such as information about sports participation and the need to have pads available at school. Basic sexual education, including information on how to manage bleedings, should also be part of the conversation. Adolescent girls should be prepared for transition to adult care, for example starting with an appointment by an adult nurse at age 14–15 years. In case of heavy menstrual bleeding (HMB), the girl should have medication available to treat her coagulation defect (such as Tranexamic acid, Desmopressin and/or clotting factor concentrate). If severe menstrual pain occurs they should know what kinds of painkillers are allowed; non‐steroidal anti‐inflammatory drugs (NSAIDs) preferably not to avoid additional platelet dysfunction, cyclo‐oxygenase‐2 (COX‐2) selective NSAIDs are allowed since these drugs comprise platelet function to a lesser extent. The aim of hormonal treatment should be explained mainly as a treatment of HMB rather than as an contraconceptive although both effects should be discussed. Haemoglobin and ferritin levels should be checked regularly in case of HMB. Women and Girls Bleeding Disorders suffering from HMB are likely to experience psychosocial issues which can affect academic and social pursuits. Offering peer support can help to maintain their self‐esteem, as well as extending management beyond the hospital, teaching and organizing home treatment for those who require regular factor replacement therapy and liaising with school or place of work when required. If bleeding problems interfere with social participation, a social worker should be involved. 2.1.2 Psychologist perspective Based on the clinical experiences, menarche may be a traumatic experience affecting quality of life and even provoke a post‐traumatic stress syndrome (PTSS). HMB has negative impact on the quality of life in WBD, impairing their daily activities and social life. , The health‐related quality of life (HRQoL) in 13 years old girls with a bleeding disorder is lower compared to their healthy peers. In contrast, there is no apparent difference in psychosocial functioning and HRQoL between young adult women with a bleeding disorder compared to peers. However, women with bleeding disorders participating in a large survey stated that they had mainly problems on reproduction and sexual relations. They also felt more fatigued. Psychosocial support is important to prevent further deterioration which may hamper the social development of these girls. To check whether reference to a psychologist is indicated, standard screening with patient‐reported outcome measurement instruments, such as HRQoL questionnaires, can be performed ahead of an outpatient clinical visit. Ideally, a psychologist, or at least a social worker, is part of the multidisciplinary team to discuss WBD who experience serious psychosocial issues. In case of PTSS eye movement desensitization reprocessing treatment by a psychologist is indicated. When problems like fatigue, acceptance, shame or fear interfere with normal social functioning, treatment with cognitive behaviour therapy may be indicated. 2.1.3 Gynaecologist perspective Menstrual disorders are common in adolescent girls. Menstrual periods can be heavy and irregular, usually secondary to anovulatory cycles caused by immaturity of the hypothalamic‐pituitary‐ovarian axis. Adolescent girls with inherited bleeding disorders are at a higher risk of HMB; often starting from menarche with a significant health implications such as anaemia, hospitalization and need for transfusion of blood or blood products. HMB has a negative impact on quality of life, can be a source of distress for adolescent girls and may affect their school attendance and performance as well as their social life. In case of suspected heavy menstrual bleeding (HMB), assessment of menstrual blood loss using the PBAC score is useful. Alternatively, narrative assessment should be adopted; excessive bleeding is defined as prolonged (>7 days), and/or the need to change a pad every 2 h or more. Careful history taking is important. Pelvic examination and ultrasound, if appropriate, may reveal an additional reason for HMB. The goals of HMB treatment are to reduce morbidity, prevent iron deficiency and improve quality of life through reducing menstrual blood loss and attainment of a predictable pattern of menstruation. Treatment options for HMB depend on age, menstrual pattern, the need for contraception and patients tolerability and acceptability of available treatment options. Management of HMB in adolescents is usually medical and there are several haemostatic and hormonal options that can be used singly or in combination (Table ). However, there are specific considerations when using these agents in adolescent girls that must be incorporated into multidisciplinary and shared decision‐making with the girls and their family. This is ideally provided in a joint clinic, where the experts can agree on optimal use of various treatments and assess the girls in one visit. WBD value provision of care in such models with a high level of satisfaction and better compliance to treatment. 2.1.4 Patients' perspective A 42‐year‐old woman, suffering from severe VWD, shared her personal experience with the menarche. This was very traumatic. She did not receive any education or information in advance and there was no proper treatment. She had to stay in the hospital for weeks due to uncontrolled bleeding. She strongly feels that educating patients and parents on menstrual bleeding and on the psychosocial effects may prevent such negative healthcare experiences. She also emphasizes unrestricted use of factor concentrates to treat HMB, if needed, since this made a huge difference for her. Medical doctors in HTCs should be aware that HMB may be very stressful and anticipate better, in order to prevent a traumatic experience with lifelong impact. Nurse perspective Haemophilia treatment centres nurses have an important role in HTC multidisciplinary team providing education, being accessible for patient's questions and uncertainties/worries and making sure that the right team members are contacted in case of a specific problem of concern. In educating, it should be taken into mind to talk with both parents and the girl in ‘understandable’ language (no jargon) and to adjust the mode of education to the patient's age and culture. Education of WBD should include information on the normal menstrual cycle and on symptoms of iron deficiency. Next to information on pads and tampons, information about use of menstrual cups—how to measure blood loss and if ‘period poverty’ is an issue, the need for (psycho)social support can be explored in their families. The Pictorial Blood Loss Assessment Chart (PBAC) can be used to measure (ab)normal menstruation; a PBAC score >100 corresponds to abnormal blood loss. Families can be made aware of mobile applications (apps) that track periods and blood loss, for example www.sisterhoodapp.com , if appropriate in the local setting. Also other practical issues should be discussed, such as information about sports participation and the need to have pads available at school. Basic sexual education, including information on how to manage bleedings, should also be part of the conversation. Adolescent girls should be prepared for transition to adult care, for example starting with an appointment by an adult nurse at age 14–15 years. In case of heavy menstrual bleeding (HMB), the girl should have medication available to treat her coagulation defect (such as Tranexamic acid, Desmopressin and/or clotting factor concentrate). If severe menstrual pain occurs they should know what kinds of painkillers are allowed; non‐steroidal anti‐inflammatory drugs (NSAIDs) preferably not to avoid additional platelet dysfunction, cyclo‐oxygenase‐2 (COX‐2) selective NSAIDs are allowed since these drugs comprise platelet function to a lesser extent. The aim of hormonal treatment should be explained mainly as a treatment of HMB rather than as an contraconceptive although both effects should be discussed. Haemoglobin and ferritin levels should be checked regularly in case of HMB. Women and Girls Bleeding Disorders suffering from HMB are likely to experience psychosocial issues which can affect academic and social pursuits. Offering peer support can help to maintain their self‐esteem, as well as extending management beyond the hospital, teaching and organizing home treatment for those who require regular factor replacement therapy and liaising with school or place of work when required. If bleeding problems interfere with social participation, a social worker should be involved. Psychologist perspective Based on the clinical experiences, menarche may be a traumatic experience affecting quality of life and even provoke a post‐traumatic stress syndrome (PTSS). HMB has negative impact on the quality of life in WBD, impairing their daily activities and social life. , The health‐related quality of life (HRQoL) in 13 years old girls with a bleeding disorder is lower compared to their healthy peers. In contrast, there is no apparent difference in psychosocial functioning and HRQoL between young adult women with a bleeding disorder compared to peers. However, women with bleeding disorders participating in a large survey stated that they had mainly problems on reproduction and sexual relations. They also felt more fatigued. Psychosocial support is important to prevent further deterioration which may hamper the social development of these girls. To check whether reference to a psychologist is indicated, standard screening with patient‐reported outcome measurement instruments, such as HRQoL questionnaires, can be performed ahead of an outpatient clinical visit. Ideally, a psychologist, or at least a social worker, is part of the multidisciplinary team to discuss WBD who experience serious psychosocial issues. In case of PTSS eye movement desensitization reprocessing treatment by a psychologist is indicated. When problems like fatigue, acceptance, shame or fear interfere with normal social functioning, treatment with cognitive behaviour therapy may be indicated. Gynaecologist perspective Menstrual disorders are common in adolescent girls. Menstrual periods can be heavy and irregular, usually secondary to anovulatory cycles caused by immaturity of the hypothalamic‐pituitary‐ovarian axis. Adolescent girls with inherited bleeding disorders are at a higher risk of HMB; often starting from menarche with a significant health implications such as anaemia, hospitalization and need for transfusion of blood or blood products. HMB has a negative impact on quality of life, can be a source of distress for adolescent girls and may affect their school attendance and performance as well as their social life. In case of suspected heavy menstrual bleeding (HMB), assessment of menstrual blood loss using the PBAC score is useful. Alternatively, narrative assessment should be adopted; excessive bleeding is defined as prolonged (>7 days), and/or the need to change a pad every 2 h or more. Careful history taking is important. Pelvic examination and ultrasound, if appropriate, may reveal an additional reason for HMB. The goals of HMB treatment are to reduce morbidity, prevent iron deficiency and improve quality of life through reducing menstrual blood loss and attainment of a predictable pattern of menstruation. Treatment options for HMB depend on age, menstrual pattern, the need for contraception and patients tolerability and acceptability of available treatment options. Management of HMB in adolescents is usually medical and there are several haemostatic and hormonal options that can be used singly or in combination (Table ). However, there are specific considerations when using these agents in adolescent girls that must be incorporated into multidisciplinary and shared decision‐making with the girls and their family. This is ideally provided in a joint clinic, where the experts can agree on optimal use of various treatments and assess the girls in one visit. WBD value provision of care in such models with a high level of satisfaction and better compliance to treatment. Patients' perspective A 42‐year‐old woman, suffering from severe VWD, shared her personal experience with the menarche. This was very traumatic. She did not receive any education or information in advance and there was no proper treatment. She had to stay in the hospital for weeks due to uncontrolled bleeding. She strongly feels that educating patients and parents on menstrual bleeding and on the psychosocial effects may prevent such negative healthcare experiences. She also emphasizes unrestricted use of factor concentrates to treat HMB, if needed, since this made a huge difference for her. Medical doctors in HTCs should be aware that HMB may be very stressful and anticipate better, in order to prevent a traumatic experience with lifelong impact. CASE 2 A 36‐year‐old carrier of severe haemophilia B, who has mild haemophilia herself (factor IX of 15 IU/dl), desires to get pregnant. The gene defect is unknown. Her father died from HIV, and there is no contact with other family members except for a younger sister who has never been tested either. The medical history includes HMB, bleeding after sexarche and an ovulation bleed. She was three times treated with clotting factor and uses a combined oral contraceptive pill to control menstrual bleeding and suppress ovulation. Difficulties with penetration, which arose after the negative experience at her sexarche, hamper conception. 3.1 Question for the multidisciplinary team The 25% of WBD report that they experience a severe impact on reproductive decision‐making. How should this woman be guided in conceiving, pregnancy and childbirth? 3.1.1 Psychosexual therapist perspective Good sex education is very important, underscored by the fact that girls who are more sexually autonomous have more pleasant and less unwanted sexual experiences. , In contrast to common opinion, adolescents who start their sexual careers well prepared tend to have their sexual debut at a later age than those who do not. Health professionals who feel reluctant to talk about sex with their patients may be pleased to learn that the majority of patients are grateful that their healthcare provider initiates a conversation about sex. A good heuristic is to first explain why you ask questions about sexuality (because sexual problems in people with this condition are common), then ask permission to talk about it (in young girls ask permission from the parents too), and be specific in your questioning using clear, non‐obscuring language. More than 25% of young women are familiar with pain during intercourse. This pain is mostly caused by a lack of adequate sexual arousal in combination with a high pelvic floor muscle tone. Even though systematic data are lacking, it is likely that fear of bleeding, like any other fear, impairs sexual arousal and therefore increases the risk of bleeding. Difficulties with sexual arousal and with penetrative sex require a multidisciplinary approach, taking into account all relevant biopsychosocial variables. In this particular case, an important question is as follows: Is there fear of pain or fear of bleeding? Does she have symptoms of PTSS related to excessive bleeding at coital debut, which impair sexual response? Are there other negative sexual experiences? Fear (of any kind) not only impairs sexual arousal but also increases the likelihood of having an increased pelvic floor muscle tone. This patient's difficulties with vaginal penetration suggest that this is the case for her too. In that case, physical therapy may be indicated, but only in conjunction with treatment of the fear of bleeding or the PTSS, otherwise physical therapy is likely to be ineffective. When complaints are severe or physical therapy is unhelpful, referral to a psychosexual therapist is in order. Discussing the role of sexual arousal and vaginal lubrication in the promotion of successful and pleasurable vaginal intercourse is important. To encourage the experience of sexual pleasure, it helps to relax her body and her pelvic floor, to pay attention to arousing and pleasurable bodily sensations, to eliminate all pressures to perform, and to not focus on intercourse as the only and final goal of sexual interactions. Encouragement to use lubricants to enable unaroused vaginal penetration is likely to exacerbate the sexual difficulties. Because she wants to become pregnant, she may be inclined to continuing attempts to engage in intercourse. A much more successful and healthy strategy is to separate the sexual problem from the wish to conceive, by instructing her and her partner to deliver sperm to the uterus on fertile days using a small syringe, while encouraging them to engage in arousing and pleasurable non‐penetrative sexual activities. 3.1.2 Geneticist perspective Haemophilia follows an X linked recessive inheritance pattern (Figure ). Affected men have a mutation in the factor 8 or factor 9 gene, which are both located on the X chromosome. Because men do not have a second X chromosome with a normal copy of the factor 8 or 9 gene (but a Y chromosome instead), they will not produce enough factor VIII or IX for normal coagulation and have symptoms of haemophilia. Daughters of male haemophilia patients are obligate carriers of haemophilia. Daughters of haemophilia carriers have a 50% chance of inheriting the X chromosome with the factor 8 or 9 mutation and are therefore potential carriers. Haemophilia carriers (HCs) should be identified and genetically counselled, preferably before they become pregnant, to be able to choose from all available reproductive options. Normal clotting factor levels in HCs do not exclude carriership of the familial haemophilia mutation. Carriership can be detected with direct DNA analysis if the familial mutation is known. In case the familial mutation is unknown, as in the presented case, DNA analysis to identify the familial mutation should preferably be done in a male family member affected with haemophilia or, if they are not available, an obligate carrier. Last choice is to primarily test a potential carrier, because of the (small) chance of failure to detect the culprit mutation in carriers due to technical reasons. There are several reproductive options for haemophilia carriers: no children or adoption, oocyte donation, preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PND). Preimplantation genetic diagnosis is an in vitro fertilization (IVF) treatment in which one single cell of an embryo is tested for gender or a known familial mutation. When using PGD for haemophilia, only female or unaffected male embryos are placed into the uterus to create a pregnancy. It is a reliable, but costly and time‐consuming method to prevent having an affected child, with high emotional burden and a 25% chance of an ongoing pregnancy per IVF cycle in general. Before or after birth, the absence of the familial mutation should be confirmed. Prenatal diagnosis with the goal to terminate pregnancy in case of an affected boy is different from PND with the aim to guide obstetric management. For PND with the aim to terminate pregnancy, in case of an affected boy, a Y‐PCR test in maternal blood can be done at around 9 weeks of gestation to determine the sex of the foetus. In case of a boy, chorionic villus biopsy is done around 11 weeks of gestation. There is a small chance of pregnancy loss with this procedure. In PND to guide obstetric recommendations, ultrasound for gender determination is done around 16 weeks gestation. In case of a boy, amniocentesis is performed around 30–32 weeks of gestation. In case of an affected boy or when parents choose no PND, an atraumatic delivery is indicated. In the near future, non‐invasive prenatal diagnosis (NIPD) with genetic analysis on cell‐free foetal DNA isolated from maternal blood will hopefully become available to enable early, reliable and safe prenatal diagnosis. 3.1.3 Haematologist perspective The 30% of haemophilia carriers have clotting factor levels below 40 IU/dl and about a third experiences abnormal bleeding, although the bleeding tendency is not 1:1 related to the clotting factor level. , HMB and post‐partum haemorrhages are the most frequent bleeding episodes seen associated with lower quality of life and iron deficiency anaemia. When a carrier, who is on hormonal treatment because of heavy menstruation, has a wish to become pregnant she should be prepared for the possibility of HMB once this medication has been stopped. Tranexamic acid must be at home and the haemoglobin level and ferritin level should be checked before stopping hormonal treatment and repeated when heavy blood loss exists. Adequate levels of haemoglobin and ferritin should be maintained with oral and/or iv iron‐ ensuring they are adequate before pregnancy. It should also be checked whether DNA testing has been performed and the possibilities of PND and the modern treatment of haemophilia discussed, preferably in a multidisciplinary setting. Since haemophilia treatment has evolved greatly the past years, it is important to discuss the current treatment options and perspectives of having haemophilia, to prevent decisions being made solely based on negative family experiences with older haemophilia patients. This information has to be tailored according to the personal and family history of severe/moderate or mild haemophilia and repeated before each pregnancy. Clotting factor correction is required for invasive procedures like PND, PGD and chorion villus collection in case of a decreased FVIII or FIX level. During pregnancy, a physiologic increase in factor VIII levels occurs and levels should therefore be checked repeatedly in carriers of haemophilia A. However, in haemophilia B carriers, like the presented case, a rise in factor IX hardly occurs. As there is a risk for maternal and neonatal bleeding, a proper pregnancy and delivery plan should be made in time, preferable before 32 weeks of gestation in case preterm delivery occurs, and communicated with the patient and other members of the multidisciplinary team. This plan should include discussing the need of clotting factor correction to prevent post‐partum bleeding and measures to prevent bleeding in (possibly) affected male neonates, as well the type of painkillers and use of Tranexamic acid post‐partum. In contrast to haemophilia A carriers, where desmopressin can be used in addition to factor replacement, unsatisfactory factor IX levels for delivery in haemophilia B carriers can only be corrected with factor IX concentrate. Instrumental delivery must be avoided to decrease maternal and neonatal (intracranial) bleeding risks and is contraindicated in case of a (possibly) affected boy. In moderate/severe haemophilia, invasive neonatal surveillance procedures are also contraindicated. Regional block anaesthesia is possible if factor levels are >50 IU/dl. When clotting factor levels are <50 IU/dl at 32–34 weeks of gestation, clotting factor correction is given to secure haemostasis during and after delivery. This should be repeated aiming for a through level >50 IU/dl for 3−5 days after vaginal delivery and 7–10 days in case of caesarean section. The risk of intracranial bleeding in the neonate with haemophilia is 3.6% in countries with good quality of care. This is 40–80 times higher than non‐affected neonates and is related to the way of delivery and awareness of carriership in mothers. , A multidisciplinary delivery plan is key to both mother and child. 3.1.4 Patients' perspective Anxiety occurs that prior traumatic experiences with HMB may return when contraceptives are stopped. Sexual problems are not easily discussed and it is much valued if healthcare providers offer an opening to start this conversation. WBD have to cope with feelings of guild towards their possible offspring which may inherit a severe disease they carry. Negative family experiences play an important role and may add to the anxiety, even causing her to decide not to have children if she is not well informed about the current health care perspectives. Support and guidance from the multidisciplinary team, starting in the phase of conception and onwards is required, especially in this case, since normal penetration in an issue. Taken together, many insecurities and uncertainties cause a lot of worry and difficulties in reproductive decision‐making. A safe environment to openly discuss these issues with members the multidisciplinary team is imperative. Question for the multidisciplinary team The 25% of WBD report that they experience a severe impact on reproductive decision‐making. How should this woman be guided in conceiving, pregnancy and childbirth? 3.1.1 Psychosexual therapist perspective Good sex education is very important, underscored by the fact that girls who are more sexually autonomous have more pleasant and less unwanted sexual experiences. , In contrast to common opinion, adolescents who start their sexual careers well prepared tend to have their sexual debut at a later age than those who do not. Health professionals who feel reluctant to talk about sex with their patients may be pleased to learn that the majority of patients are grateful that their healthcare provider initiates a conversation about sex. A good heuristic is to first explain why you ask questions about sexuality (because sexual problems in people with this condition are common), then ask permission to talk about it (in young girls ask permission from the parents too), and be specific in your questioning using clear, non‐obscuring language. More than 25% of young women are familiar with pain during intercourse. This pain is mostly caused by a lack of adequate sexual arousal in combination with a high pelvic floor muscle tone. Even though systematic data are lacking, it is likely that fear of bleeding, like any other fear, impairs sexual arousal and therefore increases the risk of bleeding. Difficulties with sexual arousal and with penetrative sex require a multidisciplinary approach, taking into account all relevant biopsychosocial variables. In this particular case, an important question is as follows: Is there fear of pain or fear of bleeding? Does she have symptoms of PTSS related to excessive bleeding at coital debut, which impair sexual response? Are there other negative sexual experiences? Fear (of any kind) not only impairs sexual arousal but also increases the likelihood of having an increased pelvic floor muscle tone. This patient's difficulties with vaginal penetration suggest that this is the case for her too. In that case, physical therapy may be indicated, but only in conjunction with treatment of the fear of bleeding or the PTSS, otherwise physical therapy is likely to be ineffective. When complaints are severe or physical therapy is unhelpful, referral to a psychosexual therapist is in order. Discussing the role of sexual arousal and vaginal lubrication in the promotion of successful and pleasurable vaginal intercourse is important. To encourage the experience of sexual pleasure, it helps to relax her body and her pelvic floor, to pay attention to arousing and pleasurable bodily sensations, to eliminate all pressures to perform, and to not focus on intercourse as the only and final goal of sexual interactions. Encouragement to use lubricants to enable unaroused vaginal penetration is likely to exacerbate the sexual difficulties. Because she wants to become pregnant, she may be inclined to continuing attempts to engage in intercourse. A much more successful and healthy strategy is to separate the sexual problem from the wish to conceive, by instructing her and her partner to deliver sperm to the uterus on fertile days using a small syringe, while encouraging them to engage in arousing and pleasurable non‐penetrative sexual activities. 3.1.2 Geneticist perspective Haemophilia follows an X linked recessive inheritance pattern (Figure ). Affected men have a mutation in the factor 8 or factor 9 gene, which are both located on the X chromosome. Because men do not have a second X chromosome with a normal copy of the factor 8 or 9 gene (but a Y chromosome instead), they will not produce enough factor VIII or IX for normal coagulation and have symptoms of haemophilia. Daughters of male haemophilia patients are obligate carriers of haemophilia. Daughters of haemophilia carriers have a 50% chance of inheriting the X chromosome with the factor 8 or 9 mutation and are therefore potential carriers. Haemophilia carriers (HCs) should be identified and genetically counselled, preferably before they become pregnant, to be able to choose from all available reproductive options. Normal clotting factor levels in HCs do not exclude carriership of the familial haemophilia mutation. Carriership can be detected with direct DNA analysis if the familial mutation is known. In case the familial mutation is unknown, as in the presented case, DNA analysis to identify the familial mutation should preferably be done in a male family member affected with haemophilia or, if they are not available, an obligate carrier. Last choice is to primarily test a potential carrier, because of the (small) chance of failure to detect the culprit mutation in carriers due to technical reasons. There are several reproductive options for haemophilia carriers: no children or adoption, oocyte donation, preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PND). Preimplantation genetic diagnosis is an in vitro fertilization (IVF) treatment in which one single cell of an embryo is tested for gender or a known familial mutation. When using PGD for haemophilia, only female or unaffected male embryos are placed into the uterus to create a pregnancy. It is a reliable, but costly and time‐consuming method to prevent having an affected child, with high emotional burden and a 25% chance of an ongoing pregnancy per IVF cycle in general. Before or after birth, the absence of the familial mutation should be confirmed. Prenatal diagnosis with the goal to terminate pregnancy in case of an affected boy is different from PND with the aim to guide obstetric management. For PND with the aim to terminate pregnancy, in case of an affected boy, a Y‐PCR test in maternal blood can be done at around 9 weeks of gestation to determine the sex of the foetus. In case of a boy, chorionic villus biopsy is done around 11 weeks of gestation. There is a small chance of pregnancy loss with this procedure. In PND to guide obstetric recommendations, ultrasound for gender determination is done around 16 weeks gestation. In case of a boy, amniocentesis is performed around 30–32 weeks of gestation. In case of an affected boy or when parents choose no PND, an atraumatic delivery is indicated. In the near future, non‐invasive prenatal diagnosis (NIPD) with genetic analysis on cell‐free foetal DNA isolated from maternal blood will hopefully become available to enable early, reliable and safe prenatal diagnosis. 3.1.3 Haematologist perspective The 30% of haemophilia carriers have clotting factor levels below 40 IU/dl and about a third experiences abnormal bleeding, although the bleeding tendency is not 1:1 related to the clotting factor level. , HMB and post‐partum haemorrhages are the most frequent bleeding episodes seen associated with lower quality of life and iron deficiency anaemia. When a carrier, who is on hormonal treatment because of heavy menstruation, has a wish to become pregnant she should be prepared for the possibility of HMB once this medication has been stopped. Tranexamic acid must be at home and the haemoglobin level and ferritin level should be checked before stopping hormonal treatment and repeated when heavy blood loss exists. Adequate levels of haemoglobin and ferritin should be maintained with oral and/or iv iron‐ ensuring they are adequate before pregnancy. It should also be checked whether DNA testing has been performed and the possibilities of PND and the modern treatment of haemophilia discussed, preferably in a multidisciplinary setting. Since haemophilia treatment has evolved greatly the past years, it is important to discuss the current treatment options and perspectives of having haemophilia, to prevent decisions being made solely based on negative family experiences with older haemophilia patients. This information has to be tailored according to the personal and family history of severe/moderate or mild haemophilia and repeated before each pregnancy. Clotting factor correction is required for invasive procedures like PND, PGD and chorion villus collection in case of a decreased FVIII or FIX level. During pregnancy, a physiologic increase in factor VIII levels occurs and levels should therefore be checked repeatedly in carriers of haemophilia A. However, in haemophilia B carriers, like the presented case, a rise in factor IX hardly occurs. As there is a risk for maternal and neonatal bleeding, a proper pregnancy and delivery plan should be made in time, preferable before 32 weeks of gestation in case preterm delivery occurs, and communicated with the patient and other members of the multidisciplinary team. This plan should include discussing the need of clotting factor correction to prevent post‐partum bleeding and measures to prevent bleeding in (possibly) affected male neonates, as well the type of painkillers and use of Tranexamic acid post‐partum. In contrast to haemophilia A carriers, where desmopressin can be used in addition to factor replacement, unsatisfactory factor IX levels for delivery in haemophilia B carriers can only be corrected with factor IX concentrate. Instrumental delivery must be avoided to decrease maternal and neonatal (intracranial) bleeding risks and is contraindicated in case of a (possibly) affected boy. In moderate/severe haemophilia, invasive neonatal surveillance procedures are also contraindicated. Regional block anaesthesia is possible if factor levels are >50 IU/dl. When clotting factor levels are <50 IU/dl at 32–34 weeks of gestation, clotting factor correction is given to secure haemostasis during and after delivery. This should be repeated aiming for a through level >50 IU/dl for 3−5 days after vaginal delivery and 7–10 days in case of caesarean section. The risk of intracranial bleeding in the neonate with haemophilia is 3.6% in countries with good quality of care. This is 40–80 times higher than non‐affected neonates and is related to the way of delivery and awareness of carriership in mothers. , A multidisciplinary delivery plan is key to both mother and child. 3.1.4 Patients' perspective Anxiety occurs that prior traumatic experiences with HMB may return when contraceptives are stopped. Sexual problems are not easily discussed and it is much valued if healthcare providers offer an opening to start this conversation. WBD have to cope with feelings of guild towards their possible offspring which may inherit a severe disease they carry. Negative family experiences play an important role and may add to the anxiety, even causing her to decide not to have children if she is not well informed about the current health care perspectives. Support and guidance from the multidisciplinary team, starting in the phase of conception and onwards is required, especially in this case, since normal penetration in an issue. Taken together, many insecurities and uncertainties cause a lot of worry and difficulties in reproductive decision‐making. A safe environment to openly discuss these issues with members the multidisciplinary team is imperative. Psychosexual therapist perspective Good sex education is very important, underscored by the fact that girls who are more sexually autonomous have more pleasant and less unwanted sexual experiences. , In contrast to common opinion, adolescents who start their sexual careers well prepared tend to have their sexual debut at a later age than those who do not. Health professionals who feel reluctant to talk about sex with their patients may be pleased to learn that the majority of patients are grateful that their healthcare provider initiates a conversation about sex. A good heuristic is to first explain why you ask questions about sexuality (because sexual problems in people with this condition are common), then ask permission to talk about it (in young girls ask permission from the parents too), and be specific in your questioning using clear, non‐obscuring language. More than 25% of young women are familiar with pain during intercourse. This pain is mostly caused by a lack of adequate sexual arousal in combination with a high pelvic floor muscle tone. Even though systematic data are lacking, it is likely that fear of bleeding, like any other fear, impairs sexual arousal and therefore increases the risk of bleeding. Difficulties with sexual arousal and with penetrative sex require a multidisciplinary approach, taking into account all relevant biopsychosocial variables. In this particular case, an important question is as follows: Is there fear of pain or fear of bleeding? Does she have symptoms of PTSS related to excessive bleeding at coital debut, which impair sexual response? Are there other negative sexual experiences? Fear (of any kind) not only impairs sexual arousal but also increases the likelihood of having an increased pelvic floor muscle tone. This patient's difficulties with vaginal penetration suggest that this is the case for her too. In that case, physical therapy may be indicated, but only in conjunction with treatment of the fear of bleeding or the PTSS, otherwise physical therapy is likely to be ineffective. When complaints are severe or physical therapy is unhelpful, referral to a psychosexual therapist is in order. Discussing the role of sexual arousal and vaginal lubrication in the promotion of successful and pleasurable vaginal intercourse is important. To encourage the experience of sexual pleasure, it helps to relax her body and her pelvic floor, to pay attention to arousing and pleasurable bodily sensations, to eliminate all pressures to perform, and to not focus on intercourse as the only and final goal of sexual interactions. Encouragement to use lubricants to enable unaroused vaginal penetration is likely to exacerbate the sexual difficulties. Because she wants to become pregnant, she may be inclined to continuing attempts to engage in intercourse. A much more successful and healthy strategy is to separate the sexual problem from the wish to conceive, by instructing her and her partner to deliver sperm to the uterus on fertile days using a small syringe, while encouraging them to engage in arousing and pleasurable non‐penetrative sexual activities. Geneticist perspective Haemophilia follows an X linked recessive inheritance pattern (Figure ). Affected men have a mutation in the factor 8 or factor 9 gene, which are both located on the X chromosome. Because men do not have a second X chromosome with a normal copy of the factor 8 or 9 gene (but a Y chromosome instead), they will not produce enough factor VIII or IX for normal coagulation and have symptoms of haemophilia. Daughters of male haemophilia patients are obligate carriers of haemophilia. Daughters of haemophilia carriers have a 50% chance of inheriting the X chromosome with the factor 8 or 9 mutation and are therefore potential carriers. Haemophilia carriers (HCs) should be identified and genetically counselled, preferably before they become pregnant, to be able to choose from all available reproductive options. Normal clotting factor levels in HCs do not exclude carriership of the familial haemophilia mutation. Carriership can be detected with direct DNA analysis if the familial mutation is known. In case the familial mutation is unknown, as in the presented case, DNA analysis to identify the familial mutation should preferably be done in a male family member affected with haemophilia or, if they are not available, an obligate carrier. Last choice is to primarily test a potential carrier, because of the (small) chance of failure to detect the culprit mutation in carriers due to technical reasons. There are several reproductive options for haemophilia carriers: no children or adoption, oocyte donation, preimplantation genetic diagnosis (PGD) and prenatal diagnosis (PND). Preimplantation genetic diagnosis is an in vitro fertilization (IVF) treatment in which one single cell of an embryo is tested for gender or a known familial mutation. When using PGD for haemophilia, only female or unaffected male embryos are placed into the uterus to create a pregnancy. It is a reliable, but costly and time‐consuming method to prevent having an affected child, with high emotional burden and a 25% chance of an ongoing pregnancy per IVF cycle in general. Before or after birth, the absence of the familial mutation should be confirmed. Prenatal diagnosis with the goal to terminate pregnancy in case of an affected boy is different from PND with the aim to guide obstetric management. For PND with the aim to terminate pregnancy, in case of an affected boy, a Y‐PCR test in maternal blood can be done at around 9 weeks of gestation to determine the sex of the foetus. In case of a boy, chorionic villus biopsy is done around 11 weeks of gestation. There is a small chance of pregnancy loss with this procedure. In PND to guide obstetric recommendations, ultrasound for gender determination is done around 16 weeks gestation. In case of a boy, amniocentesis is performed around 30–32 weeks of gestation. In case of an affected boy or when parents choose no PND, an atraumatic delivery is indicated. In the near future, non‐invasive prenatal diagnosis (NIPD) with genetic analysis on cell‐free foetal DNA isolated from maternal blood will hopefully become available to enable early, reliable and safe prenatal diagnosis. Haematologist perspective The 30% of haemophilia carriers have clotting factor levels below 40 IU/dl and about a third experiences abnormal bleeding, although the bleeding tendency is not 1:1 related to the clotting factor level. , HMB and post‐partum haemorrhages are the most frequent bleeding episodes seen associated with lower quality of life and iron deficiency anaemia. When a carrier, who is on hormonal treatment because of heavy menstruation, has a wish to become pregnant she should be prepared for the possibility of HMB once this medication has been stopped. Tranexamic acid must be at home and the haemoglobin level and ferritin level should be checked before stopping hormonal treatment and repeated when heavy blood loss exists. Adequate levels of haemoglobin and ferritin should be maintained with oral and/or iv iron‐ ensuring they are adequate before pregnancy. It should also be checked whether DNA testing has been performed and the possibilities of PND and the modern treatment of haemophilia discussed, preferably in a multidisciplinary setting. Since haemophilia treatment has evolved greatly the past years, it is important to discuss the current treatment options and perspectives of having haemophilia, to prevent decisions being made solely based on negative family experiences with older haemophilia patients. This information has to be tailored according to the personal and family history of severe/moderate or mild haemophilia and repeated before each pregnancy. Clotting factor correction is required for invasive procedures like PND, PGD and chorion villus collection in case of a decreased FVIII or FIX level. During pregnancy, a physiologic increase in factor VIII levels occurs and levels should therefore be checked repeatedly in carriers of haemophilia A. However, in haemophilia B carriers, like the presented case, a rise in factor IX hardly occurs. As there is a risk for maternal and neonatal bleeding, a proper pregnancy and delivery plan should be made in time, preferable before 32 weeks of gestation in case preterm delivery occurs, and communicated with the patient and other members of the multidisciplinary team. This plan should include discussing the need of clotting factor correction to prevent post‐partum bleeding and measures to prevent bleeding in (possibly) affected male neonates, as well the type of painkillers and use of Tranexamic acid post‐partum. In contrast to haemophilia A carriers, where desmopressin can be used in addition to factor replacement, unsatisfactory factor IX levels for delivery in haemophilia B carriers can only be corrected with factor IX concentrate. Instrumental delivery must be avoided to decrease maternal and neonatal (intracranial) bleeding risks and is contraindicated in case of a (possibly) affected boy. In moderate/severe haemophilia, invasive neonatal surveillance procedures are also contraindicated. Regional block anaesthesia is possible if factor levels are >50 IU/dl. When clotting factor levels are <50 IU/dl at 32–34 weeks of gestation, clotting factor correction is given to secure haemostasis during and after delivery. This should be repeated aiming for a through level >50 IU/dl for 3−5 days after vaginal delivery and 7–10 days in case of caesarean section. The risk of intracranial bleeding in the neonate with haemophilia is 3.6% in countries with good quality of care. This is 40–80 times higher than non‐affected neonates and is related to the way of delivery and awareness of carriership in mothers. , A multidisciplinary delivery plan is key to both mother and child. Patients' perspective Anxiety occurs that prior traumatic experiences with HMB may return when contraceptives are stopped. Sexual problems are not easily discussed and it is much valued if healthcare providers offer an opening to start this conversation. WBD have to cope with feelings of guild towards their possible offspring which may inherit a severe disease they carry. Negative family experiences play an important role and may add to the anxiety, even causing her to decide not to have children if she is not well informed about the current health care perspectives. Support and guidance from the multidisciplinary team, starting in the phase of conception and onwards is required, especially in this case, since normal penetration in an issue. Taken together, many insecurities and uncertainties cause a lot of worry and difficulties in reproductive decision‐making. A safe environment to openly discuss these issues with members the multidisciplinary team is imperative. CONCLUSION Woman and girls with bleeding disorders may experience heavy menstruation from menarche onwards. This has physical, psychosocial and psychosexual impact which requires a patient‐centred multidisciplinary approach. If a woman with an inherited bleeding disorder desires to become pregnant, preconception counselling is essential, to discuss genetic diagnosis, modern treatment for the bleeding disorder in question and possible choices to prevent having an affected child. Maternal bleeding risks during conception, delivery and the post‐partum period should be anticipated and severe bleeding prevented by making personalized multidisciplinary plans. Adequate management and good education is best provided in HTCs by an experienced multidisciplinary team. Dr. van Galen has received unrestricted research grants from CSL Behring and Bayer in the past and speakers fee from Takeda. Dr. d'Oiron has served as a consultant for Baxalta/shire, Bayer, CSL Behring, LFB, NovoNordisk, Octapharma, Pfizer, Roche and Sobi, Spark and was invited speaker for Baxalta/shire, Bayer, CSL Behring, LFB, NovoNordisk, Octapharma, Pfizer, Roche and Sobi, Spark. |
Lost in the plot: missing visual elements in Kaplan-Meier plots of phase III
oncology trials | 6183fe54-3c2c-43ba-869b-73ac01659abe | 11224967 | Internal Medicine[mh] | Kaplan-Meier (KM) curves are the most commonly used visual presentation of time-to-event outcomes in oncology; these plots rely on standard visual features for interpretability. , Missing visual elements (MVE) in KM curves may distort data, mislead readers, and prevent secondary analyses. , For example, in a recent study using survival curve reconstructions, Das et al excluded 66 of 405 phase III trials because of missing data in the KM plot. MVE may also prevent the assessment of key trial assumptions, such as proportional hazards or lack of informative censoring. Despite published guidelines, the quality of KM curves in contemporary trials remains unclear. , Thus, this study was conducted to evaluate KM plots of published phase III oncology trials. Phase III oncology trials were screened from the ClinicalTrials.gov registry using previously reported search criteria. Institutional review board approval was not required. The study objective was to evaluate the incidence of any MVE in KM plots, defined as (1) an incomplete range for survival probability, or (2) missing number at risk . , Surrogate endpoints were defined based on previously reported criteria. Because surrogate endpoints are influenced by the interval of disease assessments and potentially impacted by informative censoring, KM plots of surrogate endpoints were assessed for complete interpretability, which was defined by the following: (1) there were no MVE, (2) the number of patients censored (or number of events, from which the number censored could be derived) was reported over time, and (3) the number at risk interval corresponded to the assessment interval . If the assessment interval changed over time, the number at risk interval was considered corresponding if the intervals overlapped for at least half of the KM plot. Number at risk intervals that were more frequent than the assessment intervals were also considered corresponding, so long as the number at risk was reported at each assessment time point. Trends were examined by ordinary least squares linear regression. Structural causal models were created for each trial characteristic to identify confounder variables . Multivariable logistic regressions calculated adjusted odds ratios (aOR). All tests were 2-sided. Significance was set at 0.05, and CIs were calculated at 95%. Analyses were performed using SAS v9 (Cary, NC) and plots were created using Prism v9 (GraphPad, La Jolla, CA). Of 1877 screened trials, 1036 were phase III interventional randomized trials; of these, 395 were excluded (lack of manuscript, N = 251; lack of KM curve, N = 144), leaving a total of 641 trials enrolling 518 235 patients eligible, with publications from 2002 to 2020. Among these, 116 trials (18%) had MVE in KM curves . Specifically, 19 trials (3%) excluded the possible range(s) of survival probabilities, and 103 trials (16%) did not report the number at risk. MVE in surrogate endpoint KM plots and overall survival KM plots were found in 15% of trials (87/574; y -axis exclusions: 11/574; missing number at risk: 82/574) and 15% of trials (78/513; y -axis exclusions: 5/513; missing number at risk: 75/514), respectively. MVE decreased over time (m = −4.44; 95% CI: −5.38 to −3.51, P < 0.0001) from 45% (9/20, 2002-2007) to 34% (39/115, 2008-2011) to 18% (49/265, 2012-2015) to 8% (18/235, 2016-2019; P < 0.0001; ). High-impact journals publishing a plurality of phase III trials appeared to have lower rates of MVE: MVE incidence for trials published in The Lancet and Lancet Oncology was 4% (2/53) and 1% (1/109), respectively. MVE seemed to be associated with certain factors . On adjusted analysis, trials studying metastatic solid tumors, trials with industry funding, more recently published trials, and larger trials were associated with lower odds of MVE . The association of enrollment and MVE persisted when evaluating trials with enrollments exceeding 200 patients as well as trials with enrollments exceeding 100 patients, suggesting this overall association is attributable to a few small trials . Only 3% of trials (15/574) displayed surrogate endpoint KM plots with complete interpretability. The number of censored patients over time was present in 9% of trials (50/574), and the disease assessment interval (when reported) corresponded with the number at risk interval in 27% of trials (139/507). All trials with completely interpretable surrogate endpoint KM plots were published in either a Lancet group journal ( N = 13) or the New England Journal of Medicine ( N = 2). Our study demonstrates a modest prevalence of MVE in KM plots of phase III oncology trials. The rate of MVE overall has reassuringly declined over time. Trial-level factors, including publication journal, enrollment, and sponsor, appear associated with lower rates of MVE, which may be related to methodological rigor and quality editorial review. However, only 3% of phase III trials published completely interpretable surrogate endpoint KM plots. Trials evaluating surrogate endpoints should report the number at risk plus the number of censored patients over time. The number at risk interval should correspond to the interval of disease assessment for full interpretability of the study’s findings and assessment of key assumptions, such as informative censoring. , Future trials should consider the novel strategy of displaying 95% CIs for the difference in outcomes, which may better facilitate visual comparative inferences compared to plotting the curves alone. 95% CIs for group-specific outcomes are not recommended for convenience samples typical in randomized trials. Limitations of this study include the source database (ClinicalTrials.gov), which may not be representative of global trials. KM curves in the supplement section of manuscripts were not assessed, and this may have reduced the detection of MVE. In summary, there is a modest and decreasing prevalence of MVE in the KM plots of phase III oncology trials. However, the vast majority of surrogate endpoint KM plots were not fully interpretable. Improved adherence to quality guidelines for KM plots, particularly for trials evaluating surrogate endpoints, is needed to improve the interpretability, transparency, and reproducibility of phase III oncology research. oyae067_suppl_Supplementary_Material |
Clinical and economic effects of the transformation from an open to a laparoscopic center for colorectal surgery | 7da751bc-4eb8-42aa-a1d7-b5359b00ca55 | 11732922 | Laparoscopy[mh] | Since the late 1990s, minimally invasive surgery has been used in the surgical treatment of colorectal carcinoma . The advantages in the perioperative course compared to the conventional open approach, such as the reduction of postoperative pain, the reduction of postoperative intestinal atony, earlier mobilization and consequently shorter hospital stays, are evident. The oncological equivalence of laparoscopy with open surgery, especially in the case of colon and rectal carcinoma, has already been proven by several trials [ – ] Nevertheless, only about 30–40% of patients with colorectal carcinoma in Germany are treated laparoscopically . The data is based on the recordings of health companies. The reasons for this are manifold. Still not all procedures for colorectal cancer are performed in colorectal cancer centers. On the other hand, oncological equivalence is still being discussed in some groups . Above all, however, the open procedure is the established procedure in many hospitals and a switch to laparoscopic care is not carried out for several reasons, which, in addition to the perioperative quality data and economic aspects, primarily focus on patient safety. In this analysis, the transformation process over period of three years (2012 – 2014) from open to laparoscopic surgery is monitored in a center of primary care, during certification as a colorectal cancer center. Special attention is given to perioperative data such as the length of hospital stay, intensive care unit monitoring, complication data, quality of specimen as well as overall and disease-free survival.
To access the transformation effects, the data of 237 patients who underwent surgical treatment with a colorectal cancer in the Westküstenklinikum Heide and Brunsbüttel gGmbH in the period from 01/2012 to 12/2014 were prospectively recorded and retrospectively evaluated. The data was collected as part of the certification process for the colon cancer center (DKG, OnkoZert). After completion of the certification in 2013, the data collection was continued. All patients included had consented to the collection and use of the collected data as part of the inpatient treatment contract. The tumor staging and the surgical procedure corresponded to the quality requirements of the professional societies and the current guidelines. The study was carried out with the approval of the responsible ethics committee (AZ 15-341A). Applicable inclusion criteria were the presence of colorectal cancer and open or laparoscopic oncological curative resection of the tumor. The patient collective was divided into two groups. Group A included the laparoscopically operated patients and group B the patients who were treated conventionally via a midline laparotomy. The allocation to the groups was decided by the surgeon. In this process, patient characteristics, including age, body mass index (BMI), comorbidities, and general health status, were crucial in assessing suitability for different surgical methods. Another relevant consideration was the severity and location of the disease. Cases involving larger or more advanced tumors, high disease stage, or the presence of adhesions require open surgery to allow for thorough exploration and intervention. Finally, patient preference played a role in surgical planning but was not a frequent variable in the selection process. In the further differentiation, a distinction was made between colon and rectal tumors to show differentiated course and quality parameters. In addition to demographic data, general and specific anamnestic parameters were recorded and evaluated. General medical history data included: Chronic obstructive pulmonary disease (COPD), myocardial infarction (MI), diabetes mellitus (DM), arterial hypertension (AHT), coronary artery disease (CHD), wound healing disorders in pre-op (WHD), hyperlipoproteinemia (HLP), prostate diseases (PD) and sleep apnea (SAP). The specific anamnestic data record included pre-existing underlying malignant diseases and previous abdominal operations. Furthermore, peri- and postoperative course data was recorded. The operating time (OT), the length of stay in hospital (LOS), the length of stay in the intensive care unit (ICU), the need for the administration of erythrocyte concentrates (ERY) and complications were recorded. Complications were classified into major and minor complications. Complications requiring surgical intervention were considered major complications. Major complications included anastomotic leaks (AI) and wound healing disorders (WHD). Minor complications included pneumonia (PM) and intestinal atonia (IA). In addition, the Clavien-Dindo score was recorded for all patients. A special focus of this work was on the assessment and comparison of the quality of the surgical specimen. For this purpose, the number of lymph nodes, the distal resection margin and the circumferential resection margin were recorded and evaluated. The operations were performed by a total of three surgeons. Two of the surgeons had many years of experience in open colorectal oncological surgery. In addition, the surgeons were familiar with all common general laparoscopic procedures. The supervision of the implementation of laparoscopic oncologic procedures was monitored by a proven minimally invasive visceral surgeon, who had already performed more than 500 laparoscopic oncological colorectal resections before. The supervision was carried out with the assistance of the procedure with only temporary takeover of surgical steps. All cases were performed under supervision during the period of transformation. The surgical standards of the procedures were performed according to the oncological guidelines at that time. This included central vessel ligation and adequate specimen length. Explicit CME procedures were not performed. Patients were positioned in supine position with both arms attached to the body using a vacuum mattress, regardless of the operation. Only sigmoid resection and deep anterior rectum resection were performed in lithotomy positioning. In addition, pneumatic calf compression cuffs were applied during each operation to minimize thrombotic or thromboembolic events. For right sided and central colon resections the specimen was recovered via a small supraumbilical median laparotomy. The anastomosis was usually created extracorporeally, side-to-side, isoperistaltic using a stapler. The auxiliary incision required for this is closed with a 4/0 serosubmucous suture. Only in the case of the left hemicolectomy is the anastomosis created end-to-end as a single-row hand suture and serosubmucous continuously with suture 4/0. The closure of the meso-slot was not performed. For sigmoid and rectum resection the anastomosis was created using a trans anal circular stapler. To ensure a well-perfused anastomosis, the stapler anvil was only tied in if there was significant bleeding from the marginal arcade. The anastomosis was tested as an air–water test. In the case of anterior resections, a protective ileostomy is created in each case. If rectal extirpation was necessary, the specimen was retrieved transanally. A left-sided omental-plasty was created for both anterior resections and rectal extirpation. Statistics In order to analyze the collected data, SPSS Version 26 from IBM (SPSS Inc., Chicago, IL, USA) was used. Continuous parameters were descriptively characterized by the following information: Median (Mdn), Minimum (Min), Maximum (Max) and Percentage (%). Dichotomous variables were examined for their frequency distribution and their percentage. The chi-square test (χ^2) was used to determine statistical relationships. With a sample size N < 20 and an expected cell count < 5, Fisher's exact test was used. To compare defined endpoints at the rational scale level between two groups, the Mann–Whitney U test (MWU) was performed for unpaired samples. The above tests were two-tailed with a probability of error of p = 0.05. A statistically significant relationship was assumed with a p-value ≤ 0.05. Survival analysis was performed using Kaplan–Meier estimations. Differences between groups were analyzed using the Log-Rank-Test.
In order to analyze the collected data, SPSS Version 26 from IBM (SPSS Inc., Chicago, IL, USA) was used. Continuous parameters were descriptively characterized by the following information: Median (Mdn), Minimum (Min), Maximum (Max) and Percentage (%). Dichotomous variables were examined for their frequency distribution and their percentage. The chi-square test (χ^2) was used to determine statistical relationships. With a sample size N < 20 and an expected cell count < 5, Fisher's exact test was used. To compare defined endpoints at the rational scale level between two groups, the Mann–Whitney U test (MWU) was performed for unpaired samples. The above tests were two-tailed with a probability of error of p = 0.05. A statistically significant relationship was assumed with a p-value ≤ 0.05. Survival analysis was performed using Kaplan–Meier estimations. Differences between groups were analyzed using the Log-Rank-Test.
Demographic data is analyzed in Table . The gender distribution showed a ratio of 100 women (42.2%) to 137 men (57.8%) in the total collective. The median age was 74.16 years, and the average BMI was 27 kg/m 2 . Concerning gender and BMI, there were no significant differences between open operated and laparoscopic operated patients. Regarding the age distribution of the patients in 2014, the conventional operated patients were significantly older than the laparoscopically operated patients ( p = 0.039). Also, the American Society of Anaesthesiologists (ASA) Distribution showed no significant differences between the groups (Table ). In the total collective, 17% of the patients had previously undergone an open abdominal operation and 4% had previous laparoscopic abdominal operation. In comparison, depending on the surgical procedure and the observation period, no significance for the existence of previous operations could be determined. Table shows the distribution of tumor specifications in comparison between patients treated laparoscopically and openly referring to the observation period. In the overall collective and over the entire observation period, most patients had a postop. UICC stage III (mean = 25.6 cases per year; 33.2% of all cases). When comparing the open vs. laparoscopic groups referring to the observation period, no significance could be demonstrated when comparing the distribution pattern according to the tumor stage. Analyzing the perioperative parameters, Table shows the median operation time according to the observation periods. Likewise, in the previous parameters, no significant differences between the two techniques could be found. Further postoperative parameters included in the analysis of the length of hospital stay (LOS), the duration of the need for epidural anesthesia (epidural catheter, PDA) (Table ) and the complications according to the Clavien-Dindo-Score (Table ). For the required length of stay in hospital, a significantly shorter stay was shown for laparoscopically operated patients in 2013 compared to the open operated group ( p = 0.011). In the years 2012 and 2014 no significant difference could be determined but a shorter LOS for the laparoscopic group in 2014 was obvious. The analysis of the postoperative stay on the intensive care unit (ICU) showed a significant shorter time for the laparoscopic group in the years 2013 and 2014. No significance could be observed between the two procedures the length of a PDA. The median in 2012 was 2 days for laparoscopic surgery and 1 day for open surgery. In 2013, the median for both procedures was 2 d. In 2014, 2.5 days were required for laparoscopic surgery vs. 3 days for open procedures. A PDD was applied in 85 patients (72.0%) in the laparoscopic group and in 69 patients (59.8%) in the open group. Complications from open and laparoscopic surgery were evaluated using the Clavien-Dindo-Score (Table ). This analysis revealed no significant advantage for either method. A total of 163 patients (68.7%) could be discharged with a score of 0 (no complication). A total of 74 patients (31.2%) had a Clavien-Dindo score of 1–3. Of these 74 patients, 34 patients underwent laparoscopic surgery, and 40 patients underwent open surgery. The scores 4 and 5 could not be observed over the observation period. The complications were also differentiated to minor and major complications. Major complications were defined as the need for surgical or endoscopic re-intervention and listed in Table . Over the entire observation period and independent of the surgical procedure, 12 patients (7.6%) with colon carcinoma developed an anastomotic leak and 8 patients (5.4%) experienced a complication of impaired wound healing. In patients with rectal carcinoma, the rate of anastomotic leaks was 12.3% (n = 10) and of wound healing disorders 5.4% (n = 4). No significant difference could be shown when comparing the surgical procedures regarding major complications. In 2012, an anastomotic leak was observed in 10.0% (n = 2) rectal cancer patients who underwent open surgery. In 2012, no anastomotic leak was observed in laparoscopically operated rectal cancer patients. The open operated colon carcinoma patients in 2012 were affected by an anastomotic leakage in 6.0% of the cases. In the laparoscopic group, no anastomotic leak was observed in the patients with colon carcinoma in 2012 either. In 2013, the rate of anastomotic leaks in patients with rectal cancer was 21.1% (n = 4) in the laparoscopic group and 11.1% (n = 1) in the open group. Wound healing disorders could be observed in one case this year after open rectal carcinoma resection. In 2014, patients after rectal carcinoma resection were affected by an anastomotic leak in 7.4% (n = 2) of the cases after laparoscopic surgery and in 25.0% (n = 1) of the cases with open surgery. In the same observation year, after resection of a colon carcinoma, 2 anastomotic leaks (11.8%) were recorded in the open operated group and 4 (10.3%) in the laparoscopic operated group. To monitor the surgical quality the number of lymph nodes and the safety margin of the specimens were included in the analysis. The distal safety margin was evaluated for colon carcinomas and the distal and circumferential safety margins for rectal carcinomas. Also, the MERCURY grade and the R- status of rectal resections were analyzed. (Table , ). Concerning all analyzed quality parameters for rectal and colonic resections, no statistically significant differences between both surgical techniques could be demonstrated. To evaluate the economic dimension of both procedures a detailed cost calculation was performed (Table ). This results in additional costs for laparoscopic operations per case due to the additionally required material of 122 euros for colon resection (0.84% of the total costs/case) and 137 euros for rectal resection (0.94% of the total costs/case) with the same revenues according to the DRG regardless of the surgical procedure. These additional costs can be compensated for by shortening the LOS and the ICU stay. In this analysis, the median LOS in the last year of transformation was 11 days for laparoscopically operated patients, while the open operated patients required a median LOS of 14 days. With a median reduction in LOS of 3 days and hospital costs for a peripheral bed of around 360 euros per bed/d (data from 2021, Department of Controlling, WKK Heide), this results in savings of 1,080 euros/case. In addition, over the entire observation period after laparoscopic surgery compared to open surgery, treatment duration in the intensive care unit was reduced by a median of 1.3 days. With costs for an intensive care unit of 1,696 euros/day (data from 2021, department for controlling, WKK Heide), this results in additional savings of 2,205 euros/case, which leads to an effective saving of 3,147 euros/case. Finally, Figs. , and show the Overall survival (OS) and Figs. , and the Disease-free survival (DFS) of the laparoscopic and open group stratified by the operation year 2012 to 2014. The log rank test comparing the Kaplan–Meier-Curves showed no significant differences for the years 2012 and 2013 whereas the curves for the year 2014 show a significant longer OS and DFS for the laparoscopic group.
The transition from open surgery to the adoption of laparoscopic techniques in the treatment of colorectal cancer has manifested as a transformative journey, linked with both clinical and economic dimensions. This study contributes substantially to the ongoing discourse by offering an analysis of the implications associated with this paradigm shift, enhancing the understanding of the multifaceted landscape in colorectal surgery. The recurrent debate about the adoption of laparoscopic surgery in colorectal carcinoma treatment has persisted despite the evident advantages documented since the late 1990s .This study, conducted over a three-year period (2012–2014) at a colorectal cancer-certified primary care center, provides a comprehensive analysis of the transformation process. The primary motivation behind the study initiates from the observation that, despite established benefits such as reduced postoperative pain, shorter hospital stays, and earlier mobilization, only 30–40% of colorectal cancer patients in Germany undergo laparoscopic procedures . This discrepancy is attributed to concerns about oncological equivalence, and concerns about patient safety. The data, collected prospectively and analyzed retrospectively, encompasses 237 patients undergoing colorectal surgery. Notably, the operations were executed by three surgeons, each bringing a distinct skill set — one proficient in minimally invasive surgery and two with extensive experience in open oncological surgery. This degree of expertise underscores the pragmatic approach adopted during the transition period. Key results showed a significant reduction in the need for postoperative intensive care, symbolizing enhanced recovery following laparoscopic procedures. The reduction in the average length of stay from 2.5 days to 1.2 days further substantiates the enhanced postoperative recovery associated with laparoscopic colorectal surgery. Notably, the length of hospital stay following laparoscopic surgery emerges as significantly shorter than the open approach, correlating with existing literature that underscores the efficiency of minimally invasive approaches in colorectal surgery [ – ]. Quality metrics, a cornerstone in surgical assessments, emerge unscathed during the transformation. Therefore, the study examines the number of lymph nodes in surgical specimens, a crucial parameter in oncological outcomes. The consistent high quality of specimens, irrespective of the surgical approach or the observation period, shows the standards maintained throughout the transition. This effect also is seen in literature even in advanced rectal cancer . Moreover, quality parameters such as the Distal and Circumferential Resection Margin do not differ between groups. Also, the R-Status and the MERCURY grade showed no significant differences between the laparoscopic and the open approach ovar all three years. Furthermore, the study focuses on the stability of major complications, with anastomotic leaks exhibiting no significant differences between laparoscopy and open surgery. This commitment to surgical quality stands for a successful transformative process and is a cornerstone for laparoscopic adoption. Even for patients aged 80 and older Chung et al. could demonstrate similar effects showing advantages for the laparoscopic approach and making it the approach of choice . Regarding long-term survival, only the year 2014 shows differences in the OS as well as the DFS. Curves show a significant advantage of the laparoscopic approach. This could be a result of an selection bias, but could also result from the advantages of the laparoscopic technique, which in literature also showed relevant survival benefits resulting from modulated immune response as well as the tumor cell spread . In summary, the laparoscopic approach shows no inferiority in the underlying data, which is in line with the well-known randomized prospective studies addressing this issue . Economic considerations form another important part of the study, addressing concerns related to the financial implications of embracing laparoscopic techniques. Despite the upfront investment costs and higher material expenses associated with laparoscopy, the study introduces a compelling narrative of economic viability. The reduction in intensive care monitoring time and hospital stay not only offsets the initial costs but results in a net economic advantage. The projected savings of around 3150 euros per case for laparoscopic surgery underscore the fiscal prudence inherent in this paradigm shift. These results are in line with comparable international studies also suggesting the cost effectiveness of the laparoscopic approach [ – ]. A cost-effectiveness analysis made in the USA for patients older than 65 years also revealed a cost advantage of $3276 per case and therefore resulting in a comparable saving amount . However, a nuanced interpretation necessitates acknowledgment of certain limitations inherent in the study. The focus on a single center introduces a potential limitation in terms of generalizability. Surgeon experience, albeit briefly mentioned, could potentially influence outcomes, warranting a more in-depth exploration of its impact. Additionally, the three-year observation period, while providing valuable short- and long-term insights, prompts contemplation about the sustainability of the observed benefits over a more extended timeframe. Moreover, the limited number of patients, the retrospective study design and the group allocation by surgeon preference limits the validity of the results. In conclusion, this study offers an analysis of the transformative process from open to laparoscopic colorectal surgery, including both clinical and economic dimensions. The consolidation of positive clinical outcomes, economic advantages, and the constant surgical quality emerges as a powerful narrative favoring for the continued adoption of laparoscopic techniques. As the healthcare landscape navigates the difficulties of evidence-based practices and endeavors for cost-effective healthcare delivery, the study positions laparoscopic colorectal surgery as not just a pragmatic but a strategic choice. The sustained success of this transformative paradigm shifts relies on ongoing training, strict adherence to established guidelines , and the implementation of robust quality monitoring mechanisms, ensuring that the benefits outlined in this study show the feasibility of a save transition process.
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The Effectiveness of Video Animations as a Tool to Improve Health Information Recall for Patients: Systematic Review | dd7250e5-ee7c-49fa-a310-38a3bf9e2453 | 11730234 | Patient Education as Topic[mh] | Accessible, accurate, and understandable health information is crucial in health care, empowering patients to make informed decisions, take appropriate actions, and actively participate in their own health management . It enables patients to understand their conditions, treatments, and health behaviors, leading to improved self-care, better adherence to treatment plans, and overall better health outcomes . Studies have shown that lacking or inaccurate knowledge of health conditions or treatment reduces the ability of the patient to engage in treatment and preventive management behaviors with lower compliance leading to poorer health outcomes, such as physical disabilities and decreased mental health . Information about illness and relevant treatments is traditionally provided through written documents, for example, information pamphlets handed out by a health care professional, or oral information given during a consultation. However, these types of information often lead to poor memorization and comprehension, resulting in potential mistrust and confusion. Furthermore, these types of information were strongly correlated with level of education and health literacy levels . Over the past decade, there has been a growing interest in using innovative tools to improve patient information, such as animation videos, to enhance patients’ knowledge and skills in managing their illnesses. Combining verbal and pictorial information in a coordinated way has been shown to enhance learning outcomes, for individuals with high and low health literacy . While previous research has delved into the use of animated videos and shown promising results in selected health areas , a notable gap exists in the comprehensive examination of animations’ precise influence as a tool for improving the recall of health-related information in general. More knowledge is particularly needed when exploring more technical parameters when using animation videos . Factors such as the type of animation video used, the specific health topics addressed, the duration of the animation’s impact, and other relevant variables need to be further investigated. Therefore, the objective of this systematic review is to investigate the effectiveness of animation videos on health information recall in patients as they engage with the health care system across diverse health care sectors. Protocol and Registration The review protocol was registered at PROSPERO (International Prospective Register of Systematic Reviews; ID: CRD42022380016; November 2022). PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines were followed when reporting the search results . Our original protocol specified the inclusion of studies investigating only patients with musculoskeletal disorders. This criterion was later broadened to include studies examining all types of disorders. Eligibility Criteria Participants This systematic review included studies involving adult participants aged 18 years and older in a health care setting. No restrictions were imposed based on ethnicity, gender, or socioeconomic status. However, studies on patients with personality disorders or conditions known to affect cognitive functionality were excluded. Intervention Animation videos were only considered if they were objective and factual and provided information about a patient’s specific health condition, such as the course of treatment or physiological and anatomical facts. In addition, the animation videos should enhance patients’ understanding of their health condition and how to manage it. The delivery mode of interventions should involve either the use of CD-ROMs, websites, tablet devices, computers, or mobile phones within a health care context. The animation was eligible if it consisted of, for example, cartoons, avatars, “whiteboard” animation, or animated 2D or 3D models. The intervention was only eligible if sufficient details about the animation video were provided, either by providing detailed descriptions, screenshots, or links; authors were contacted for more information if details were scarce. We used the exclusion definition of insufficient details about the intervention when it was unclear if the animation video component was the main component in the intervention or just a bi-part of the intervention as a whole. Furthermore, we used the definition could not contact the author if we were uncertain if the studies included what we defined as animation or not, and did not get any response when trying to contact authors to provide sufficient details about the animation component. All exclusion reasoning is displayed in . Comparator Usual care encompasses conventional information delivered by health care professionals, such as the use of written printed materials or verbal communication. Outcome Health information recall refers to the participant’s ability to remember and recall the information provided by the animation video immediately after receiving it or during a follow-up period. Therefore, all studies that aimed to measure information recall, knowledge gain, or acquisition of information about the specific disorders as a primary or secondary outcome were included. Types of Studies This review included randomized controlled trials (RCTs) that were peer-reviewed. No specific criteria were set for the language used in the studies. If necessary, a translation tool (DeepL) was used to review and assess a study. The time frame was not restricted, as it was anticipated that research related to animation videos would primarily be limited to the last few decades. Search Strategy A 3-step search was performed with the assistance of an experienced research librarian. First, an initial search was conducted and was ongoing from July 2022 to August 2022. This search was limited to PubMed, CINAHL, and Embase using preliminary subject headings and keywords based on experience and knowledge of the field. Articles that appeared appropriate to the aim were viewed. Notes were made of relevant keywords contained in the title, abstract, and index terms in each relevant article. The preliminary subject headings and keywords were revised in accordance with the findings obtained in the initial search. Articles found relevant in the first search were set aside to confirm that the second search identified these. The second search was performed between September 12, 2022, and September 16, 2022, using the revised subject headings and keywords in PubMed, CINAHL, Embase, and Web of Science. The search was divided into blocks consisting of main keywords and additional variables and is shown in . The third and final search was performed under and after the screening process from January 24, 2023, to September 18, 2023, and was conducted using reference lists. Reference lists were manually consulted to identify any additional studies, and finally, a crosscheck was done to confirm that previously identified articles were included in the search. Study Selection A 3-step selection and assessment process were conducted. First, all studies were imported into Covidence, a web-based collaboration software for managing systematic reviews and also to remove duplicates . Second, all titles and abstracts were independently screened twice by 2 reviewers (SH, TSJ, AMS, and MTH). Articles identified for full-text underwent a similar process, being independently screened twice by the 2 reviewers (SH, TSJ, AMS, and MTH). Discrepancies between the reviewers’ decisions in the inclusion and exclusion process were clarified by involving a third reviewer who had not been part of the initial review (SH, TSJ, AMS, and MTH; ). Finally, all studies reviewed in full-text that did not meet the inclusion criteria were excluded. Data Collection Characteristics and outcomes of the studies were extracted using a data collection form developed in Covidence . This was done by 2 separate reviewers (SH, TSJ, AMS, and MTH), and in case of conflicting understandings, the subject was resolved by discussion or handled by the third reviewer. Data were extracted on author and date, setting (health care sector) and country, sample size, mean age of participants, intervention and comparator, follow-up period, and outcome, including type of measurement instrument. Study Quality Assessment The studies that met the eligibility criteria were assessed for their methodological validity using the Revised Cochrane risk-of-bias tool for randomized trials (RoB2) template . A total of 2 independent reviewers (SH, TSJ, AMS, and MTH) appraised the studies, and in cases of disagreement, a third reviewer was consulted. Data Analysis Due to the diverse nature, use of measurement instruments, and the heterogeneity of articles in this field, it was not planned to perform a meta-analysis. Therefore, a narrative synthesis approach was planned. Detailed information on the study characteristics, and methodological quality, as well as a summary of the outcome measures and the results, was obtained. Also, the narrative synthesis summarized the findings according to three pre-identified outcome-related categories (length of the animation videos and time for outcome assessment; animation styles and health-related topics; and comparators and the role of the animation video) inspired by the data extraction template made for this study. We have used P <.05 as an indicator of effect. The review protocol was registered at PROSPERO (International Prospective Register of Systematic Reviews; ID: CRD42022380016; November 2022). PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines were followed when reporting the search results . Our original protocol specified the inclusion of studies investigating only patients with musculoskeletal disorders. This criterion was later broadened to include studies examining all types of disorders. Participants This systematic review included studies involving adult participants aged 18 years and older in a health care setting. No restrictions were imposed based on ethnicity, gender, or socioeconomic status. However, studies on patients with personality disorders or conditions known to affect cognitive functionality were excluded. Intervention Animation videos were only considered if they were objective and factual and provided information about a patient’s specific health condition, such as the course of treatment or physiological and anatomical facts. In addition, the animation videos should enhance patients’ understanding of their health condition and how to manage it. The delivery mode of interventions should involve either the use of CD-ROMs, websites, tablet devices, computers, or mobile phones within a health care context. The animation was eligible if it consisted of, for example, cartoons, avatars, “whiteboard” animation, or animated 2D or 3D models. The intervention was only eligible if sufficient details about the animation video were provided, either by providing detailed descriptions, screenshots, or links; authors were contacted for more information if details were scarce. We used the exclusion definition of insufficient details about the intervention when it was unclear if the animation video component was the main component in the intervention or just a bi-part of the intervention as a whole. Furthermore, we used the definition could not contact the author if we were uncertain if the studies included what we defined as animation or not, and did not get any response when trying to contact authors to provide sufficient details about the animation component. All exclusion reasoning is displayed in . Comparator Usual care encompasses conventional information delivered by health care professionals, such as the use of written printed materials or verbal communication. Outcome Health information recall refers to the participant’s ability to remember and recall the information provided by the animation video immediately after receiving it or during a follow-up period. Therefore, all studies that aimed to measure information recall, knowledge gain, or acquisition of information about the specific disorders as a primary or secondary outcome were included. Types of Studies This review included randomized controlled trials (RCTs) that were peer-reviewed. No specific criteria were set for the language used in the studies. If necessary, a translation tool (DeepL) was used to review and assess a study. The time frame was not restricted, as it was anticipated that research related to animation videos would primarily be limited to the last few decades. This systematic review included studies involving adult participants aged 18 years and older in a health care setting. No restrictions were imposed based on ethnicity, gender, or socioeconomic status. However, studies on patients with personality disorders or conditions known to affect cognitive functionality were excluded. Animation videos were only considered if they were objective and factual and provided information about a patient’s specific health condition, such as the course of treatment or physiological and anatomical facts. In addition, the animation videos should enhance patients’ understanding of their health condition and how to manage it. The delivery mode of interventions should involve either the use of CD-ROMs, websites, tablet devices, computers, or mobile phones within a health care context. The animation was eligible if it consisted of, for example, cartoons, avatars, “whiteboard” animation, or animated 2D or 3D models. The intervention was only eligible if sufficient details about the animation video were provided, either by providing detailed descriptions, screenshots, or links; authors were contacted for more information if details were scarce. We used the exclusion definition of insufficient details about the intervention when it was unclear if the animation video component was the main component in the intervention or just a bi-part of the intervention as a whole. Furthermore, we used the definition could not contact the author if we were uncertain if the studies included what we defined as animation or not, and did not get any response when trying to contact authors to provide sufficient details about the animation component. All exclusion reasoning is displayed in . Usual care encompasses conventional information delivered by health care professionals, such as the use of written printed materials or verbal communication. Health information recall refers to the participant’s ability to remember and recall the information provided by the animation video immediately after receiving it or during a follow-up period. Therefore, all studies that aimed to measure information recall, knowledge gain, or acquisition of information about the specific disorders as a primary or secondary outcome were included. This review included randomized controlled trials (RCTs) that were peer-reviewed. No specific criteria were set for the language used in the studies. If necessary, a translation tool (DeepL) was used to review and assess a study. The time frame was not restricted, as it was anticipated that research related to animation videos would primarily be limited to the last few decades. A 3-step search was performed with the assistance of an experienced research librarian. First, an initial search was conducted and was ongoing from July 2022 to August 2022. This search was limited to PubMed, CINAHL, and Embase using preliminary subject headings and keywords based on experience and knowledge of the field. Articles that appeared appropriate to the aim were viewed. Notes were made of relevant keywords contained in the title, abstract, and index terms in each relevant article. The preliminary subject headings and keywords were revised in accordance with the findings obtained in the initial search. Articles found relevant in the first search were set aside to confirm that the second search identified these. The second search was performed between September 12, 2022, and September 16, 2022, using the revised subject headings and keywords in PubMed, CINAHL, Embase, and Web of Science. The search was divided into blocks consisting of main keywords and additional variables and is shown in . The third and final search was performed under and after the screening process from January 24, 2023, to September 18, 2023, and was conducted using reference lists. Reference lists were manually consulted to identify any additional studies, and finally, a crosscheck was done to confirm that previously identified articles were included in the search. A 3-step selection and assessment process were conducted. First, all studies were imported into Covidence, a web-based collaboration software for managing systematic reviews and also to remove duplicates . Second, all titles and abstracts were independently screened twice by 2 reviewers (SH, TSJ, AMS, and MTH). Articles identified for full-text underwent a similar process, being independently screened twice by the 2 reviewers (SH, TSJ, AMS, and MTH). Discrepancies between the reviewers’ decisions in the inclusion and exclusion process were clarified by involving a third reviewer who had not been part of the initial review (SH, TSJ, AMS, and MTH; ). Finally, all studies reviewed in full-text that did not meet the inclusion criteria were excluded. Characteristics and outcomes of the studies were extracted using a data collection form developed in Covidence . This was done by 2 separate reviewers (SH, TSJ, AMS, and MTH), and in case of conflicting understandings, the subject was resolved by discussion or handled by the third reviewer. Data were extracted on author and date, setting (health care sector) and country, sample size, mean age of participants, intervention and comparator, follow-up period, and outcome, including type of measurement instrument. The studies that met the eligibility criteria were assessed for their methodological validity using the Revised Cochrane risk-of-bias tool for randomized trials (RoB2) template . A total of 2 independent reviewers (SH, TSJ, AMS, and MTH) appraised the studies, and in cases of disagreement, a third reviewer was consulted. Due to the diverse nature, use of measurement instruments, and the heterogeneity of articles in this field, it was not planned to perform a meta-analysis. Therefore, a narrative synthesis approach was planned. Detailed information on the study characteristics, and methodological quality, as well as a summary of the outcome measures and the results, was obtained. Also, the narrative synthesis summarized the findings according to three pre-identified outcome-related categories (length of the animation videos and time for outcome assessment; animation styles and health-related topics; and comparators and the role of the animation video) inspired by the data extraction template made for this study. We have used P <.05 as an indicator of effect. Study Selection A total of 2505 studies were identified, yielding 79% (1985/2505) studies after removal of duplicates. After reviewing the 1985 titles and abstracts, 1825 studies were excluded, resulting in a total of 160 (8.1%) eligible for full-text assessment. After full-text assessment, 145 additional studies were excluded, resulting in a total of 15 included studies . All inconsistencies in data assessment and extraction were resolved by consensus among the reviewers. A total of 2454 patients were included, and details of the study selection are displayed in . Study Characteristics Detailed characteristics and results of the included studies are shown in . All studies were conducted in the last 10 years, with most studies 75% (10/15) published within the past 5 years . Of the 15 studies included the majority 12 (80%) were conducted in high-income countries , and 3 (20%) were conducted in upper- to middle-income countries . Most studies originated from the United States (20%) , Australia (13.3%) , Canada (13.3%) , and France (13.3%) , and one (6.7%) each from Japan , Austria , United Kingdom , Singapore , Brazil , and Türkiye . Length of the Animation Videos and Time for Outcome Assessment The animation videos exhibited a diverse range of durations, spanning from 1 to 15 minutes. Dividing the length into percentiles revealed distinct patterns among the studies. A third (5/15) of the studies used short animation videos lasting 1 to 5 minutes . An equal proportion featured middle-length animation videos ranging from 6 to 8 minutes . In total, 3 studies used longer animation videos, falling within the 13- to 15-minute range . In addition, 2/15 had animation videos with unspecified durations . Most of the studies (12/15) assessed the outcome immediately after the patients were given the intervention or on the day of treatment . Only 3 of 15 studies had follow-up, one after 1 month , one after 6 weeks , and one after 6 and 8 weeks . Animation Styles and Health-Related Topics The studies used a variety of animation styles on different health-related topics. 2D cartoon animated videos were used in 7 studies, on the subjects of diabetes food care ; imaging and inevitable consequences of lower back pain ; living with atrial fibrillation ; anesthesia during surgery ; the etiology, symptoms, and treatments for stress and urgency urinary incontinence ; and preoperative retention undergoing bowel resection and postoperative risk with benign parotid tumor . 3D computer animation was used in 4 studies, with topics regarding; informed consent before operative laparoscopy ; informed content before elective inpatient coronary angiography ; knowledge regarding Mohs surgery ; and age-related cataract surgery . A total of 3 studies used narrated whiteboard animation the subjects presented by this form of animation were opioid safety and proper usage, storage, and disposal ; first-time intravenous fluorescein angiography ; and patients undergoing coronary angiography . Finally, one study used a mixture of 2D and 3D animation on the subject of Mohs surgery . Comparators and the Role of the Animation Video A variety of comparators, commonly referred to as “usual care,” were used across the studies. Notably, the majority of the studies (12/15) evaluated animation videos as a supplementary tool alongside usual care. Among these 12 studies, 6 used usual care that combined written materials with verbal information provided by health care professionals . Out of 4 other studies within this subset described usual care as either a routine verbal consent process or individual face-to-face conversations . A more flexible approach to usual care was noted in 1 study, which incorporated a mix of verbal explanations, drawings, or picture imagery . The remaining study relied solely on written information sheets as usual care . In contrast, the last 3 studies out of the 15 had a different approach, examining animation videos as an alternative, rather than a supplement, to their usual care. In total, 2 of these studies used a combination of verbal and written information as their usual care while the last study only used written materials . Outcomes Health Information Recall The 15 studies used different outcome measurements to assess health information recall. All the studies used self-developed questionnaires, which were based on the information provided during the consultation. The knowledge measurement tools included multiple-choice answers, open-ended questions, and statement questions with true or false and yes or no options. The studies included in the review used varying numbers of questions related to the specific medical subject, with the number of questions ranging from 4 to 16. Positive Effects Using Animation Videos In total, 11 studies reported statistically significant improvements in health information recall when compared with control groups . Notably when looking at animation style, the 3 studies that used whiteboard animation as an educational tool demonstrated a significant positive effect over usual care . A total of 2 out of the 3 studies using 3D animation videos had a positive effect , and 5 of the total 8 studies using 2D cartoon animation including the study using a mixture of 2D and 3D had a positive effect. When looking at the time of outcome assessment 10 out of the 11 studies that found effect was measured immediately after intervention . One of those studies did not have a persistent effect at the 6-week follow-up assessment . Conversely, the study conducted by Dincer and Bahçecik in 2021 observed a positive effect 4 weeks post intervention . Equal Effect of Using Animation Video In total, 3 out of the 15 studies found equal benefits between the use of animation video and usual care . Two of the studies used 2D cartoon animation and 1 used a 3D animation video . In the cases of these 3 studies, the usual care alone achieved the same positive effect as supplementing usual care with the animation video. The studies’ usual care consisted of leaflets and verbal information , a combination of verbal communication, drawing or pictorial imagery, using a more open and flexible information style , and finally a simple information sheet . Negative Effect of Using Animation Video In 1 study , it was observed that usual care achieved significantly greater benefits than the use of animation videos when assessing the accuracy of patients’ responses regarding the correct use of imaging. Notably, the animation video was introduced as an alternative to conventional consent information. The comparators to animation were in this case (1) a 2-page written summary on an A4 paper and (2) an infographic solution combining both imagery and written summary, both these solutions had equal positive effects. Study Quality Assessment An overview of the risk of bias assessment for the included studies is presented in . In general, most studies exhibited “some concerns” related to the risk of bias, with the most prominent issues being observed in domain D2: Bias due to deviations from intended interventions. The primary reason for this was our inability to access trial protocols or get a sufficient description of the protocol, which would have provided crucial insights into the trial context. Without access or information in the protocols, it was difficult to determine if the intended interventions were consistently followed, which could have potentially introduced bias into the results. In some cases, there was a lack of transparency regarding blinding procedures, further contributing to concerns in this domain. Moreover, the domain “D5: Bias in selection of the reported results,” also raised some concerns. These issues were partly due to the unavailability of information on study protocols, making it difficult to determine if the reported results align with a prespecified analysis plan. To a lesser extent, some concerns were identified in the domain “D4: Risk of bias in the measurement of the outcome.” Many studies of interventions in this area face a common challenge of using self-developed outcome measurement tools instead of validated instruments. This is because the outcomes being measured are often highly specific and directly related to the particular disorder targeted by the animation. However, using nonvalidated tools raises questions about the reliability and validity of the outcome measurements. A total of 2505 studies were identified, yielding 79% (1985/2505) studies after removal of duplicates. After reviewing the 1985 titles and abstracts, 1825 studies were excluded, resulting in a total of 160 (8.1%) eligible for full-text assessment. After full-text assessment, 145 additional studies were excluded, resulting in a total of 15 included studies . All inconsistencies in data assessment and extraction were resolved by consensus among the reviewers. A total of 2454 patients were included, and details of the study selection are displayed in . Detailed characteristics and results of the included studies are shown in . All studies were conducted in the last 10 years, with most studies 75% (10/15) published within the past 5 years . Of the 15 studies included the majority 12 (80%) were conducted in high-income countries , and 3 (20%) were conducted in upper- to middle-income countries . Most studies originated from the United States (20%) , Australia (13.3%) , Canada (13.3%) , and France (13.3%) , and one (6.7%) each from Japan , Austria , United Kingdom , Singapore , Brazil , and Türkiye . The animation videos exhibited a diverse range of durations, spanning from 1 to 15 minutes. Dividing the length into percentiles revealed distinct patterns among the studies. A third (5/15) of the studies used short animation videos lasting 1 to 5 minutes . An equal proportion featured middle-length animation videos ranging from 6 to 8 minutes . In total, 3 studies used longer animation videos, falling within the 13- to 15-minute range . In addition, 2/15 had animation videos with unspecified durations . Most of the studies (12/15) assessed the outcome immediately after the patients were given the intervention or on the day of treatment . Only 3 of 15 studies had follow-up, one after 1 month , one after 6 weeks , and one after 6 and 8 weeks . The studies used a variety of animation styles on different health-related topics. 2D cartoon animated videos were used in 7 studies, on the subjects of diabetes food care ; imaging and inevitable consequences of lower back pain ; living with atrial fibrillation ; anesthesia during surgery ; the etiology, symptoms, and treatments for stress and urgency urinary incontinence ; and preoperative retention undergoing bowel resection and postoperative risk with benign parotid tumor . 3D computer animation was used in 4 studies, with topics regarding; informed consent before operative laparoscopy ; informed content before elective inpatient coronary angiography ; knowledge regarding Mohs surgery ; and age-related cataract surgery . A total of 3 studies used narrated whiteboard animation the subjects presented by this form of animation were opioid safety and proper usage, storage, and disposal ; first-time intravenous fluorescein angiography ; and patients undergoing coronary angiography . Finally, one study used a mixture of 2D and 3D animation on the subject of Mohs surgery . A variety of comparators, commonly referred to as “usual care,” were used across the studies. Notably, the majority of the studies (12/15) evaluated animation videos as a supplementary tool alongside usual care. Among these 12 studies, 6 used usual care that combined written materials with verbal information provided by health care professionals . Out of 4 other studies within this subset described usual care as either a routine verbal consent process or individual face-to-face conversations . A more flexible approach to usual care was noted in 1 study, which incorporated a mix of verbal explanations, drawings, or picture imagery . The remaining study relied solely on written information sheets as usual care . In contrast, the last 3 studies out of the 15 had a different approach, examining animation videos as an alternative, rather than a supplement, to their usual care. In total, 2 of these studies used a combination of verbal and written information as their usual care while the last study only used written materials . Health Information Recall The 15 studies used different outcome measurements to assess health information recall. All the studies used self-developed questionnaires, which were based on the information provided during the consultation. The knowledge measurement tools included multiple-choice answers, open-ended questions, and statement questions with true or false and yes or no options. The studies included in the review used varying numbers of questions related to the specific medical subject, with the number of questions ranging from 4 to 16. Positive Effects Using Animation Videos In total, 11 studies reported statistically significant improvements in health information recall when compared with control groups . Notably when looking at animation style, the 3 studies that used whiteboard animation as an educational tool demonstrated a significant positive effect over usual care . A total of 2 out of the 3 studies using 3D animation videos had a positive effect , and 5 of the total 8 studies using 2D cartoon animation including the study using a mixture of 2D and 3D had a positive effect. When looking at the time of outcome assessment 10 out of the 11 studies that found effect was measured immediately after intervention . One of those studies did not have a persistent effect at the 6-week follow-up assessment . Conversely, the study conducted by Dincer and Bahçecik in 2021 observed a positive effect 4 weeks post intervention . Equal Effect of Using Animation Video In total, 3 out of the 15 studies found equal benefits between the use of animation video and usual care . Two of the studies used 2D cartoon animation and 1 used a 3D animation video . In the cases of these 3 studies, the usual care alone achieved the same positive effect as supplementing usual care with the animation video. The studies’ usual care consisted of leaflets and verbal information , a combination of verbal communication, drawing or pictorial imagery, using a more open and flexible information style , and finally a simple information sheet . Negative Effect of Using Animation Video In 1 study , it was observed that usual care achieved significantly greater benefits than the use of animation videos when assessing the accuracy of patients’ responses regarding the correct use of imaging. Notably, the animation video was introduced as an alternative to conventional consent information. The comparators to animation were in this case (1) a 2-page written summary on an A4 paper and (2) an infographic solution combining both imagery and written summary, both these solutions had equal positive effects. The 15 studies used different outcome measurements to assess health information recall. All the studies used self-developed questionnaires, which were based on the information provided during the consultation. The knowledge measurement tools included multiple-choice answers, open-ended questions, and statement questions with true or false and yes or no options. The studies included in the review used varying numbers of questions related to the specific medical subject, with the number of questions ranging from 4 to 16. In total, 11 studies reported statistically significant improvements in health information recall when compared with control groups . Notably when looking at animation style, the 3 studies that used whiteboard animation as an educational tool demonstrated a significant positive effect over usual care . A total of 2 out of the 3 studies using 3D animation videos had a positive effect , and 5 of the total 8 studies using 2D cartoon animation including the study using a mixture of 2D and 3D had a positive effect. When looking at the time of outcome assessment 10 out of the 11 studies that found effect was measured immediately after intervention . One of those studies did not have a persistent effect at the 6-week follow-up assessment . Conversely, the study conducted by Dincer and Bahçecik in 2021 observed a positive effect 4 weeks post intervention . In total, 3 out of the 15 studies found equal benefits between the use of animation video and usual care . Two of the studies used 2D cartoon animation and 1 used a 3D animation video . In the cases of these 3 studies, the usual care alone achieved the same positive effect as supplementing usual care with the animation video. The studies’ usual care consisted of leaflets and verbal information , a combination of verbal communication, drawing or pictorial imagery, using a more open and flexible information style , and finally a simple information sheet . In 1 study , it was observed that usual care achieved significantly greater benefits than the use of animation videos when assessing the accuracy of patients’ responses regarding the correct use of imaging. Notably, the animation video was introduced as an alternative to conventional consent information. The comparators to animation were in this case (1) a 2-page written summary on an A4 paper and (2) an infographic solution combining both imagery and written summary, both these solutions had equal positive effects. An overview of the risk of bias assessment for the included studies is presented in . In general, most studies exhibited “some concerns” related to the risk of bias, with the most prominent issues being observed in domain D2: Bias due to deviations from intended interventions. The primary reason for this was our inability to access trial protocols or get a sufficient description of the protocol, which would have provided crucial insights into the trial context. Without access or information in the protocols, it was difficult to determine if the intended interventions were consistently followed, which could have potentially introduced bias into the results. In some cases, there was a lack of transparency regarding blinding procedures, further contributing to concerns in this domain. Moreover, the domain “D5: Bias in selection of the reported results,” also raised some concerns. These issues were partly due to the unavailability of information on study protocols, making it difficult to determine if the reported results align with a prespecified analysis plan. To a lesser extent, some concerns were identified in the domain “D4: Risk of bias in the measurement of the outcome.” Many studies of interventions in this area face a common challenge of using self-developed outcome measurement tools instead of validated instruments. This is because the outcomes being measured are often highly specific and directly related to the particular disorder targeted by the animation. However, using nonvalidated tools raises questions about the reliability and validity of the outcome measurements. Principal Findings The primary objective of this systematic review was to investigate the effectiveness of animation videos as informative tools in the health care system, particularly in enhancing patient health information recall. The main finding is that 11 of the 15 studies reported a significant positive impact of animation videos on health information recall. The studies predominantly originated from high-income countries and were mostly conducted within the last decade, with a significant concentration (75%) published in the past 5 years. The studies demonstrated a diversity in the length and style of animation videos, ranging from short (1-5 minutes) to longer durations (up to 15 minutes). Different animation styles were used, including 2D cartoon animation, 3D computer animation, and narrated whiteboard animation. This variety underscores the adaptable nature of animation as a medium for conveying health information. Comparison to Previous Work Similar results are found in a recent systematic review investigating the effectiveness of animations as an information medium for patients and the general public; they found that across 30 trials assessing knowledge, 19 of those had greater knowledge compared with a range of comparators. However, the evidence base was highly variable, with mostly small trials . The same tendencies are shown in a systematic review and meta-analysis by Feeley et al , which found an overall weighted effect size of 0.38, which indicates that improvements are modest, but not less reliable across 20 out of 21 included studies . This indicates an overall positive effect in increasing patient understanding when using animation videos across different health care settings. Contrasting our work with the review by Moe-Byrne et al , several distinctions become apparent. First, our review focuses exclusively on animations designed as a tool for patient information, whereas the review by Moe-Byrne et al includes public interventions, such as health promotion campaigns. Second, we considered solely RCTs, while the previous review also encompassed quasi-experimental studies. Finally, their analysis was not limited to health information recall but also examined changes in attitudes and behaviors . Interestingly, Feeley et al found that animations work better with patients than they do with samples from the general population or mixed groups of patients and nonpatients . This finding supports our focus on patient populations and suggests that animations tailored specifically for certain patient groups may be particularly effective, possibly due to increased motivation and relevance of the information presented. Notably, in our systematic review, all studies using whiteboard animation showed a significant positive effect, suggesting its effectiveness as an educational tool . Even though only 3 studies used whiteboard animation, this can indicate that simple forms of animations are efficient in their ability to inform patients. This finding aligns with previous studies investigating the impact of details in animation videos, and their ability to be informing. For example, it was found that realism in animation, such as the level of visual detail, did not significantly affect cognitive learning performance, suggesting different levels of animation details can be equally effective for instructional purposes . Furthermore, a study found that increasing detail can result in overloading the viewer’s visual information processing, resulting in worse performance tests regarding information retention . Most studies assessed recall immediately after intervention , with only a few extending the follow-up period . This raises questions about the long-term efficacy of animations in patient recall since only 3 out of the 15 studies had follow-up, and only one of them found a significant effect at 4 weeks . When comparing these results with the systematic review by Moe-Byrne et al they conclude similar findings, indicating that the effect tends to be most present immediately after intervention , suggesting that while animations are effective in the short term, the sustainability of this effect warrants further investigation. Our systematic review observed a diverse range of animation durations, from 1 to 15 minutes. Interestingly, Feeley et al found that video length was a significant moderate of effectiveness, but only at an α=.06 criterion. Their analysis suggested that longer videos yielded smaller learning effects across the 16 studies that reported video length . This aligns with our observation that a third of our included studies used short animation videos lasting 1 to 5 minutes, all of which demonstrated positive effects. These findings collectively suggest that concise, well-designed animations might be more effective in conveying health information, possibly due to their ability to maintain viewer attention and avoid cognitive overload. Finally, our review found that most studies (80%) were conducted in high-income countries, Feeley et al reported that 9 out of 21 studies were conducted in the United States, with the remaining 12 done in countries Germany, Japan, the Netherlands, Australia, Taiwan, Singapore, and Saudi Arabia. This similarity in geographical distribution underscores the need for more research in diverse global settings, particularly in low- and middle-income countries. Strengths and Limitations For this systematic review, we used different processes to reduce the potential risk of bias. First, it was a strength that the review protocol was registered with PROSPERO, and the PRISMA guidelines were adhered to, ensuring transparency and consistency in our reporting. Our collaboration with an experienced research librarian enabled a thorough 3-step search across multiple databases and manual searches, enhancing the breadth and relevance of the literature covered. The use of dual independent reviewers for screening, selection, data extraction, and risk of bias assessment, along with the systematic use of Covidence software, helped minimize biases and ensured a rigorous selection process. Using the Revised Cochrane risk-of-bias tool for randomized trials added robustness to our methodology, with discrepancies resolved through consultation with a third researcher. This systematic review had several limitations. First, our search strategy may have missed some relevant papers. While we conducted a comprehensive search across multiple databases (PubMed, CINAHL, and Embase) and performed manual searches of reference lists, we did not have access to all potentially relevant databases, such as PsycINFO or CENTRAL. To mitigate this, we worked closely with an experienced research librarian to develop and refine our search strategy. However, future reviews could expand the search to include additional databases and consider using a wider range of search terms to capture all relevant studies. Second, we faced challenges regarding the accessibility and evaluation of the animation video content used in the included studies. Only 8 out of 15 studies provided direct links to the animation videos, while the remaining studies offered only screenshots or written explanations. This limited our ability to fully assess the quality and effectiveness of the animations. Furthermore, we had to exclude several studies from the screening process due to this problem. To address this, we attempted to contact all authors to gain access to the animation videos or sufficient information about the intervention, but with limited success. This limitation may have influenced our results by preventing a comprehensive analysis of the animation characteristics that contribute to effective health information recall. Future studies in this area should focus on elaborating the design of the animation video and its role in the intervention. Third, our decision to include only RCTs in this systematic review may have limited the breadth of evidence we were able to consider. While this choice was made to focus on the highest quality evidence available, it potentially excluded valuable insights from well-designed quasi-experimental studies or other study types. To mitigate this, we ensured a thorough search and screening process for RCTs. However, this limitation may have influenced our results by potentially overestimating the effectiveness of animation videos, as RCTs with positive outcomes are more likely to be published. In addition, we may have missed nuanced findings from real-world implementations that are often captured in quasi-experimental designs. For future reviews in this area, researchers could consider including a wider range of study designs, such as high-quality quasi-experimental studies, while using appropriate tools to assess their risk of bias. This approach could provide a more comprehensive understanding of the effectiveness of animation videos in various real-world health care settings, while still maintaining a focus on methodological rigor. Finally, the final search was conducted in mid-2023 and additional studies may have been published in the interim given the progressive nature of this topic. Future Directions First, our review and Feeley et al suggest that shorter animations may be more effective. Future interventions should focus on creating concise, targeted animations, typically around 5-8 minutes in length, to maintain viewer engagement and minimize cognitive overload. Second, the effectiveness of whiteboard animations in our review suggests that simple, clear animations can be highly effective. Future work should explore how to maximize the impact of these simpler animation styles across various health topics. Third, given the limited long-term follow-up in current studies, future interventions should incorporate strategies to reinforce information over time. This could include providing patients with access to animations for repeated viewing or developing complementary materials that build on the animated content. Our systematic review is a great stepping stone for organizations, institutions, and so on, that want to develop their own animation videos on the subject of health care information. Conclusion In conclusion, this systematic review finds an overall positive impact of animation videos on short-term health information recall. Further studies should investigate the identified research gaps, particularly the long-term efficacy, impact on diverse populations, and the impact of animation style on the effectiveness of health information recall. Furthermore, future studies should focus on including a detailed description of the type of animation used in the intervention, or provide a link, to enable full transparency and usability of the studies. The primary objective of this systematic review was to investigate the effectiveness of animation videos as informative tools in the health care system, particularly in enhancing patient health information recall. The main finding is that 11 of the 15 studies reported a significant positive impact of animation videos on health information recall. The studies predominantly originated from high-income countries and were mostly conducted within the last decade, with a significant concentration (75%) published in the past 5 years. The studies demonstrated a diversity in the length and style of animation videos, ranging from short (1-5 minutes) to longer durations (up to 15 minutes). Different animation styles were used, including 2D cartoon animation, 3D computer animation, and narrated whiteboard animation. This variety underscores the adaptable nature of animation as a medium for conveying health information. Similar results are found in a recent systematic review investigating the effectiveness of animations as an information medium for patients and the general public; they found that across 30 trials assessing knowledge, 19 of those had greater knowledge compared with a range of comparators. However, the evidence base was highly variable, with mostly small trials . The same tendencies are shown in a systematic review and meta-analysis by Feeley et al , which found an overall weighted effect size of 0.38, which indicates that improvements are modest, but not less reliable across 20 out of 21 included studies . This indicates an overall positive effect in increasing patient understanding when using animation videos across different health care settings. Contrasting our work with the review by Moe-Byrne et al , several distinctions become apparent. First, our review focuses exclusively on animations designed as a tool for patient information, whereas the review by Moe-Byrne et al includes public interventions, such as health promotion campaigns. Second, we considered solely RCTs, while the previous review also encompassed quasi-experimental studies. Finally, their analysis was not limited to health information recall but also examined changes in attitudes and behaviors . Interestingly, Feeley et al found that animations work better with patients than they do with samples from the general population or mixed groups of patients and nonpatients . This finding supports our focus on patient populations and suggests that animations tailored specifically for certain patient groups may be particularly effective, possibly due to increased motivation and relevance of the information presented. Notably, in our systematic review, all studies using whiteboard animation showed a significant positive effect, suggesting its effectiveness as an educational tool . Even though only 3 studies used whiteboard animation, this can indicate that simple forms of animations are efficient in their ability to inform patients. This finding aligns with previous studies investigating the impact of details in animation videos, and their ability to be informing. For example, it was found that realism in animation, such as the level of visual detail, did not significantly affect cognitive learning performance, suggesting different levels of animation details can be equally effective for instructional purposes . Furthermore, a study found that increasing detail can result in overloading the viewer’s visual information processing, resulting in worse performance tests regarding information retention . Most studies assessed recall immediately after intervention , with only a few extending the follow-up period . This raises questions about the long-term efficacy of animations in patient recall since only 3 out of the 15 studies had follow-up, and only one of them found a significant effect at 4 weeks . When comparing these results with the systematic review by Moe-Byrne et al they conclude similar findings, indicating that the effect tends to be most present immediately after intervention , suggesting that while animations are effective in the short term, the sustainability of this effect warrants further investigation. Our systematic review observed a diverse range of animation durations, from 1 to 15 minutes. Interestingly, Feeley et al found that video length was a significant moderate of effectiveness, but only at an α=.06 criterion. Their analysis suggested that longer videos yielded smaller learning effects across the 16 studies that reported video length . This aligns with our observation that a third of our included studies used short animation videos lasting 1 to 5 minutes, all of which demonstrated positive effects. These findings collectively suggest that concise, well-designed animations might be more effective in conveying health information, possibly due to their ability to maintain viewer attention and avoid cognitive overload. Finally, our review found that most studies (80%) were conducted in high-income countries, Feeley et al reported that 9 out of 21 studies were conducted in the United States, with the remaining 12 done in countries Germany, Japan, the Netherlands, Australia, Taiwan, Singapore, and Saudi Arabia. This similarity in geographical distribution underscores the need for more research in diverse global settings, particularly in low- and middle-income countries. For this systematic review, we used different processes to reduce the potential risk of bias. First, it was a strength that the review protocol was registered with PROSPERO, and the PRISMA guidelines were adhered to, ensuring transparency and consistency in our reporting. Our collaboration with an experienced research librarian enabled a thorough 3-step search across multiple databases and manual searches, enhancing the breadth and relevance of the literature covered. The use of dual independent reviewers for screening, selection, data extraction, and risk of bias assessment, along with the systematic use of Covidence software, helped minimize biases and ensured a rigorous selection process. Using the Revised Cochrane risk-of-bias tool for randomized trials added robustness to our methodology, with discrepancies resolved through consultation with a third researcher. This systematic review had several limitations. First, our search strategy may have missed some relevant papers. While we conducted a comprehensive search across multiple databases (PubMed, CINAHL, and Embase) and performed manual searches of reference lists, we did not have access to all potentially relevant databases, such as PsycINFO or CENTRAL. To mitigate this, we worked closely with an experienced research librarian to develop and refine our search strategy. However, future reviews could expand the search to include additional databases and consider using a wider range of search terms to capture all relevant studies. Second, we faced challenges regarding the accessibility and evaluation of the animation video content used in the included studies. Only 8 out of 15 studies provided direct links to the animation videos, while the remaining studies offered only screenshots or written explanations. This limited our ability to fully assess the quality and effectiveness of the animations. Furthermore, we had to exclude several studies from the screening process due to this problem. To address this, we attempted to contact all authors to gain access to the animation videos or sufficient information about the intervention, but with limited success. This limitation may have influenced our results by preventing a comprehensive analysis of the animation characteristics that contribute to effective health information recall. Future studies in this area should focus on elaborating the design of the animation video and its role in the intervention. Third, our decision to include only RCTs in this systematic review may have limited the breadth of evidence we were able to consider. While this choice was made to focus on the highest quality evidence available, it potentially excluded valuable insights from well-designed quasi-experimental studies or other study types. To mitigate this, we ensured a thorough search and screening process for RCTs. However, this limitation may have influenced our results by potentially overestimating the effectiveness of animation videos, as RCTs with positive outcomes are more likely to be published. In addition, we may have missed nuanced findings from real-world implementations that are often captured in quasi-experimental designs. For future reviews in this area, researchers could consider including a wider range of study designs, such as high-quality quasi-experimental studies, while using appropriate tools to assess their risk of bias. This approach could provide a more comprehensive understanding of the effectiveness of animation videos in various real-world health care settings, while still maintaining a focus on methodological rigor. Finally, the final search was conducted in mid-2023 and additional studies may have been published in the interim given the progressive nature of this topic. First, our review and Feeley et al suggest that shorter animations may be more effective. Future interventions should focus on creating concise, targeted animations, typically around 5-8 minutes in length, to maintain viewer engagement and minimize cognitive overload. Second, the effectiveness of whiteboard animations in our review suggests that simple, clear animations can be highly effective. Future work should explore how to maximize the impact of these simpler animation styles across various health topics. Third, given the limited long-term follow-up in current studies, future interventions should incorporate strategies to reinforce information over time. This could include providing patients with access to animations for repeated viewing or developing complementary materials that build on the animated content. Our systematic review is a great stepping stone for organizations, institutions, and so on, that want to develop their own animation videos on the subject of health care information. In conclusion, this systematic review finds an overall positive impact of animation videos on short-term health information recall. Further studies should investigate the identified research gaps, particularly the long-term efficacy, impact on diverse populations, and the impact of animation style on the effectiveness of health information recall. Furthermore, future studies should focus on including a detailed description of the type of animation used in the intervention, or provide a link, to enable full transparency and usability of the studies. |
An autopsy case of disseminated | 4c621e6a-e2e1-4844-bd3a-b48e06374681 | 10022292 | Forensic Medicine[mh] | Mucormycosis is an important infection that is associated with high mortality . In recent decades, its incidence has increased in populations with underlying conditions, such as those with malignancies and recipients of bone marrow transplant; among these, pulmonary mucormycosis is the primary cause at initial diagnosis . Among zygomycetes, Cunninghamella spp. are rarely isolated from samples from immunocompromised patients; however, the associated mortality is significantly higher than that associated with other zygomycetes . Only few cases of disseminated pulmonary , cardiovascular , and aortic infections have been reported in immunocompromised patients. Furthermore, in immunocompetent patients, the occurrence of a disseminated Cunninghamella infection is rarer. Therefore, its clinical and pathological features are not fully understood. We experienced a case wherein an immunocompetent patient was diagnosed with disseminated Cunninghamella bertholletiae infection; sputum culture indicated bronchial colonization prior to the diagnosis. Histological findings from their autopsy could reveal the expected invasion sites.
A 67-year-old Japanese man with emphysema visited our hospital every month for bronchodilator medication. He was hospitalized for progressive dyspnea, productive cough, and moderate fever that had developed 3 days prior to admission. He had a smoking habit (33 smoking pack years) and recurrent pneumothoraxes with chest tube drainage management, and a home oxygen therapy (2 L/min). He did not have a history of allergy or immunodeficiency, and did not consume alcohol as a habit. Physical examination revealed an elevated body temperature (37.8℃) and respiratory failure with SpO 2 97% and 3.5 L/min oxygen therapy; he also had bilateral coarse crackles without leg edema. Chest radiography revealed a bilateral consolidation shadow, with emphysema in the right lower lobe and pleural effusion in the left lung (Fig. A). Chest computed tomography indicated consolidation in multiple lung lobes, with left-sided pleural effusion and an adhesive collapsed lung appearance in the right upper-lung field (Fig. B). Temporal manual drainage was performed to relieve the left pleural exudative effusion; the sputum, blood, and pleural effusion all tested negative for a bacterial infection. Although sputum culture had yielded Cunninghamella spp. 6 months prior to the most recent presentation, the infection seemed to have been a respiratory tract colonization because of his good condition. Laboratory findings revealed that the only abnormalities were anemia (hemoglobin: 10.5 g/100 mL), a low albumin level (3.0 g/100 mL), and an elevated C-reaction protein level (14.0 mg/100 mL). Tests for β-d glucan and galactomannan were normal; thus, an Aspergillus infection was ruled out. Although there were no bacterial evidence, tazobactam-piperacillin hydrate (13.5 g/day) was administered empirically; however, it was discontinued on the ninth disease day because renal dysfunction occurred as an adverse event. The bilateral lung consolidation around the emphysema worsened gradually, and repeated sputum cultures yielded fungal agents on the 17 th disease day (Fig. A). Mass spectrometry and polymerase chain reaction (PCR)-based direct sequencing revealed the pathogen to be Cunninghamella bertholletiae (according to the DDBJ/EMBL/GenBank [ http://blast.ncbi.nlm.nih.gov/Blast.cgi ] and MycoBank Database [ http://www.mycobank.org/ ]). Cytological analysis mainly revealed sporangiophores, with all branches swelling up to vesicles producing unicellular sporangioles (Fig. B, C). These findings were consistent with a pulmonary Cunninghamella infection. Although liposomal amphotericin B (5 mg/kg/day) was administered since the 28 th disease day in addition to cefepime (2 g/day), chest radiography and electrocardiography revealed cardiomegaly and atrial fibrillation on the 29 th disease day, respectively. The serum brain natriuretic peptide (BNP) level was also elevated (956 pg/mL). Respiratory dysfunction occurred gradually and the plasma BNP level increased to 2,338 pg/mL. The patient died from multiorgan dysfunction on the 37 th disease day (Fig. C). Macroscopically, a postmortem examination (Fig. A) revealed a cavity region and coagulative necrosis in the right upper lung, a small amount (50 mL) of bilateral pleural effusion, and pericardial fluid (50 mL). Histopathological examination revealed a cluster of fungal hyphae within the arteries of the right cavity wall (Fig. B), subpericardial artery (Fig. C), intramyocardial capillary blood vessels (Fig. D–G), and esophageal subserosa vein. The fungus cultures from the right upper bronchial secretion, pleural, and pericardial fluid were positive for Cunninghamella bertholletiae (Fig. A, B, F). These findings suggested that the colonized Cunninghamella bertholletiae in the upper bronchus had invaded the blood vessels and disseminated to other organs, and myocardial invasion had resulted in the critical damage that led to the death in this case.
This was a rare case of pulmonary Cunninghamella bertholletiae infection that occurred in an immunocompetent patient. Most Cunninghamella infections tend to occur in immunocompromised hosts, including transplantation recipients and patients with hematological malignancies . Recently, Cunninghamella was reported to have accounted for only 7% of all mucormycosis cases and isolated primarily from patients with pulmonary or disseminated disease. The associated mortality was significantly higher than that associated with other Mucorales (71% [23/30] vs. 44% [185/417]; p < 0.001) . However, the pathophysiology has not been fully understood because of its rarity and rapid disease progression in immunocompromised patients. Only two cases of pulmonary Cunninghamella bertholletiae infection have been reported in immunocompetent patients . One of these occurred in a 61-year-old White man with a history of alcoholic binges who had right pleural effusion, pneumothorax, and a right upper lobe cavitary lesion. Cunninghamella bertholletiae was detected in the abscess wall and surrounding lung parenchyma sections without vascular involvement or dissemination. The lung deformity may have promoted the growth of Cunninghamella bertholletiae , similar to in our case . The other case was of a 74-year-old man with exacerbated asthma–chronic obstructive pulmonary disease overlap syndrome, who was diagnosed with allergic bronchopulmonary mycosis; tests detected a prolonged serum-specific immunoglobulin type E antibody against mucormycetes. This indicates the possibility of Cunninghamella bertholletiae having contaminated his organs (such as the respiratory tract) or emphysema . Both cases suggest the colonization of Cunninghamella bertholletiae with pulmonary deformation, as in our case, which is sometimes misdiagnosed as a contamination. Distinguishing between a definite diagnosis of mucormycetes and colonization seems rather difficult. Therefore, the detailed morphological features were investigated in this case. Generally, temperature plays an important role in fungal growth; the optimal growth temperature for Cunninghamella elegans is known to be approximately 35℃, which is lower than the human body temperature or the temperature of the respiratory tract. Conversely, for other Cunninghamella spp. (such as Cunninghamella bertholletiae and Cunninghamella echinulata ), the best growth temperature is higher than that for Cunninghamella elegans , and these species are thought to have thermotolerance . In our case, biological culture examination suggested that the Cunninghamella bertholletiae infection occurred prior to admission to our hospital; the pathogen may have colonized the respiratory tract for a long time and grown gradually to invade the cardiovascular system until the critical stage of death. In immunocompetent patients in particular, Cunninghamella bertholletiae might likely colonize the respiratory tract. To detect mucormycetes at the earliest stage, a novel DNA sequencing method has been suggested . In this present case, the serum Cunninghamella bertholletiae DNA load was measured using quantitative PCR. The copy number on the day of the patient’s death was higher than that on the onset day. This suggests that quantification of Cunninghamella bertholletiae DNA in the serum could be useful for the diagnosis and evaluation of mucormycosis. Because the DNA is not detected in healthy or non-pathological patients, this would be a useful way for distinguishing between definite infection and colonization. In our case, we observed biological and pathological evidence in autopsy tissues from several organs in an immunocompetent patient. This finding is indicative of the common invasion sites of Cunninghamella bertholletiae itself. Fungal hyphae were found within the pulmonary cavity wall, subpericardial artery, intramyocardial capillary blood vessels, and esophageal subserosa veins (Fig. ); this is in line with previous reports [ , , , ]. Pulmonary and cardiovascular deformities are considered common invasive regions ; cardiovascular invasion sometimes follows a critical course in the presence of a Cunninghamella infection, with arrhythmia or acute exacerbation of heart failure, as in our case. Only two cases of cardiovascular invasion with a large mass within the left inferior wall of the ventricular cavity have been reported; one involved hypokinesis and the other involved invasion of the septate hyphae of Cunninghamella spp. into the vascular wall . Both patients in these cases died, and an autopsy was performed. In conclusion, we have presented a rare case of an Cunninghamella bertholletiae infection that occurred in an immunocompetent patient and followed a critical course even under antifungal treatment. Because useful diagnostic markers are lacking, it is difficult to distinguish between colonization and definite diagnosis in order to initiate antifungal treatment at an earlier stage. This pathogen can rapidly progress from colonizing the bronchi to infecting the surrounding organs via vascular invasion even in immunocompetent patients.
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Applicability and usefulness of the Declaration of Helsinki for forensic research with human cadavers and remains | 710a8e48-cfc2-406d-96ce-616d037c3add | 9362629 | Pathology[mh] | Forensic research applies several methods to many different types of research that may involve human subjects, human material obtained from either living persons or dead persons, including the whole body, and personal data. In particular, the use of human cadavers and remains (hereinafter HC&R), or their specimens (i.e., organs, bones, tissues, or biological samples), is essential for advancement in forensic research but may raise ethical concerns . Indeed, as suggested by Jones and Whitaker “the manner in which we respond to the dead, the use we make of their skeletal remains and their tissues, and the ways in which we learn about ourselves by studying them, raises ethical queries that go to the heart of what it means to be human.” Accordingly, forensic doctors and assistants have reported that the dignity of the body is a central issue in everyday forensic professional practice . However, while research on living people is subject to intense ethical scrutiny today , and ethics guidelines have been proposed for anatomical dissection or for research with recently deceased and brain dead cadavers , no ethical standards or guidelines for forensic research involving HC&R are available internationally. This may be, at least in part, the result of a lack of consensus among forensic pathologists regarding the ethical acceptability of diverse uses of autopsy tissues for research . On the other hand, ethical standards are needed both to avoid possible unethical practices and to sustain research in the forensic field. Indeed, in the absence of guidelines, ethical issues such as confidentiality and the use of “non-consented autopsy tissues” were among the main limitations to research reported by US academic forensic pathologists . A cornerstone of biomedical research involving human subjects is the Declaration of Helsinki issued by the World Medical Association (WMA) . The Declaration of Helsinki, the first version of which dates to 1964, was not issued in order to be legally binding, but many national laws, and the Regulation on clinical trials on medicinal products for human use recently adopted by the European Parliament and the Council , require its principles to be followed in all medical research involving human beings. According to the Preamble, the WMA developed the Declaration of Helsinki “as a statement of ethical principles for medical research involving human subjects, including research on identifiable human material and data,” and “encourages others [than physicians] who are involved in medical research involving human subjects to adopt these principles” . These premises fit very well with forensic research that is characterized by both the use of human material and data and the involvement of researchers with non-medical backgrounds. However, deceased individuals are not considered human subjects in some countries, as is the case under US federal regulations, or HC&R may be deemed as res nullius , with little oversight, consequently, of their use . In addition, while almost all the Declaration of Helsinki principles are applicable in forensic research with HC&R, with only a few statements not applicable (e.g., those related to the use of placebo or post-trial provisions), the vast differences between clinical research involving human beings and forensic research involving HC&R stand out. Bearing these aspects in mind, in the following paragraphs we discuss the main problems and benefits of the applicability of the Declaration of Helsinki to forensic research on HC&R, especially in relation to the provisions concerning risks, burdens, and benefits involved with research, ethics committee approval, and informed consent requirements. Our aim is to stimulate discussion within the forensic scientific community regarding this topic. Since laws vary from country to country, we will consider the Declaration of Helsinki in terms of its global value and we will mention national and supranational laws or regulations when needed in order to better contextualize the issues concerning the use of HC&R for forensic research purposes. Protecting people from the risk of being harmed because of their involvement in research is the main concern of research ethics . The Declaration of Helsinki provides clear guidance regarding the need for careful assessment of predictable risks, burdens, and benefits, the implementation of measures to minimize the risk, and continuous monitoring, assessment, and documentation of risks. Of course, in the case of forensic research involving HC&R, the risks and burdens are different from those that may harm a living person. On the other hand, as summarized by Pentz and colleagues, “even though they are not identical, there is direct continuity between the body of a person who has died and the living person” . Therefore, when handling HC&R, particular attention should be paid to those behaviors that may undermine the dignity of the individual to whom the sample belonged. Which ethical limits should not be exceeded in the use of HC&R for forensic research purposes? The answer to this question encompasses scientific, ethical, cultural, religious, and psychological aspects. Moreover, professionals dealing with HC&R should develop procedures to ensure that donor programs and the use of cadaveric material reflect progress in ethical awareness within a geographical context . When dealing with human cadavers, the first focus is not on the body itself, but upon the person to whom the body and the remains belonged ante-mortem and belong post-mortem . The cadaver should be respected as a symbol of the living person , and should be handled in a dignified manner . It was suggested that human remains deserve to be treated with the same respect and dignity . In addition, it should be considered that some individuals or groups may accord a “special status” to the body and its parts , due to different religious beliefs or thoughts . It is, therefore, of paramount importance to assess the ethics of research involving HC&R “with an awareness of and sensitivity to the known values, beliefs and attitudes of those from whom the materials originated” . With specific reference to these aspects, the research protocol should provide not only the scientific background of the research, but also clear information about how to carry it out with regard to the collection, use, storage, and destiny of HC&R. Research lacking sufficient scientific background, or characterized by unnecessary loss or damage of samples, should not be considered to be ethically acceptable. For instance, according to Hostiuc , the use of whole cadavers to study the biomechanics of falls from various heights cannot be considered acceptable considering the existence of alternative methods (e.g., molds). With regard to specimens, careful handling is crucial when they are taken, brought to the laboratory, stored, analyzed, and eventually destroyed. Research involving HC&R may also imply several risks with regard to data protection, especially in the case of identifiability of the body or specimens , and when genetic testing is involved . Therefore, the research protocol should address risks for the deceased individual’s privacy, detailing how confidentiality will be assured, as well as risks for family members or ethnic groups in the case of genetic testing. Policies to manage incidental findings should be included if appropriate. Remarkably, in forensic research involving HC&R, risks and burdens could concern researchers themselves, as became evident in the case of COVID-19 forensic autopsies performed for scientific purposes . As suggested by Sperhake , “every corpse must be considered potentially infectious.” Indeed, biological risks may be present both when carrying out autopsies and handling human remains through materials or soil (e.g., tetanus or diphtheria) . Therefore, the protection of researchers’ health is an issue that also ought to be thoroughly considered: safety in the workplace should be guaranteed, research methods should be evaluated critically in advance and monitored, and individual protective equipment must always be available and used. Dead bodies do not pose serious risks to the public generally and for those handling them if the morgue attendants and pathologists follow recommended biocontainment precautions in autopsy practice and in the transportation of human remains. Safety in managing HC&R should therefore be considered itself an ethical priority both to protect those involved in the research and to avoid misconceptions about body infectivity and unnecessary public worries with regard to HC&R. The biological risk related to HC&R should not be overestimated, and may and should be managed according to existing safety recommendations. Finally, the overall implications of the research should be predicted in terms of potential benefits and application. In many medical disciplines, body donor programs have already provided useful contributions to medical research. The availability of human body parts is of the utmost importance for basic research and for clinical research aimed at improving therapies or developing new treatments. The benefits of forensic research do not only concern advancements in knowledge within the forensic field, such as the improvements made in the assessment of the cause of death by virtopsy . Indeed, even if burdened by an apparently non-therapeutic interest, the value of forensic research for third parties and the entire community has long been established . The advantages of forensic research involving HC&R may include benefits for family members when the research results allow them to adopt preventive strategies, and benefits for society, when the results may help to prevent or treat diseases or avoid accidents. Indeed, according to Byard , preventive pathology extends beyond accidents at any age, also addressing suicides, homicides, and certain heritable and non-heritable diseases. As recently outlined with regard to the COVID-19 pandemic, “the scientific benefit that can be drawn from experience with autopsies and further examination of tissue samples is immeasurable” . With this regard, early in the pandemic, the forensic community contributed to bring new knowledge to the pathophysiology and immunopathology of severe COVID-19 . The prevalence of benefits over risks is one of the main arguments supporting forensic research in the sensitive field of forensic taphonomy. The first human taphonomy research center was created in 1981 at the University of Tennessee (Knoxville, USA), and currently there are several centers in the USA, one in Australia, and one in The Netherlands . These facilities provide “a unique opportunity in the forensic sciences to study human decomposition using cadavers in a controlled research environment” . Indeed, animal proxies do not seem to provide sufficiently accurate data regarding the time of death for a human being . The ethical issues of the use of HC&R in these human decomposition facilities have been extensively addressed, and guidelines and best practices have been proposed . A second major issue of interest concerns the ethical scrutiny of research. While authorization of an ethics committee is usually needed before starting any research on both animal and human subjects worldwide, similar authorization for the use of HC&R for scientific purposes is only mandatory, in specific circumstances, in a few countries. For instance, a protocol approval by an ethics committee is required for body donation programs in Italy , it is also required in Spain before the removal of body material after death when the subject’s wishes are unknown and are impossible to discover , and it is necessary for research projects involving human remains in Norway . On the other hand, according to the Declaration of Helsinki, every protocol “must be submitted for consideration, comment, guidance and approval to the concerned research ethics committee before the study begins” (art. 23) . Even if this provision may be time-consuming and sometimes challenging for forensic researchers, the importance of a formal institutional approval for research projects involving the use of HC&R has been consistently underlined , and has several advantages for forensic research. First, the scrutiny of a research ethics committee helps forensic researchers to align the study protocol with the complex regulations concerning data protection and informed consent established in each country. Auditing by an ethics committee may be fundamental in order to establish whether or not the informed consent process and forms are required and appropriate, and whether broad consent is acceptable in the specific case in question . Second, ethics committee approval may potentially increase the numbers of body donations for forensic research. Indeed, fear or anxiety regarding possible disrespectful behavior towards cadavers was consistently found to be a reason for not donating bodies over time . These concerns may be in part related to media cases regarding unauthorized storage and use of human samples, such as the instance of the Bristol Royal Infirmary and of the Liverpool Alder Hey Children’s Hospital . Scrutiny by the research ethics committee prevents ethically unacceptable studies from being carried out, and represents a guarantee of ethical integrity of the research for both the donor and family members. Finally, a systematic submission of forensic research protocols to the ethics committee for approval may prevent regrettable situations for both biomedical journal editors requiring information on this issue and researchers dealing with the publication of study results. A closely-related key point is the registration protocol in publicly accessible databases. To the best of our knowledge, no study has addressed this issue yet with regard to forensic research involving the use of HC&R. However, trial registration could be fundamental for forensic research for many reasons: the unintended duplication of existing studies could be avoided, results from different studies could be compared, thus expanding scientific knowledge, and the publication of selective results could be prevented. In addition, protocol registration could increase body donations thanks to the publication of the purpose of the research, encouraging the interest of possible donors. An example of a public database is ClinicalTrials.gov ( https://clinicaltrials.gov/ ), which is a registration and results database for clinical studies conducted around the world. At the present time, this register contains only a few forensic protocols involving HC&R. Since a public register for forensic research is lacking, the registration of forensic study protocols in one of the existing clinical trial public registers should be encouraged. The sensitive issue of informed consent to forensic research on HC&R has been addressed by several scholars to the work of whom we refer the readers. An in-depth examination of this issue, including a list of the various cases, goes beyond the objectives of this article. In summary, apart from very few exceptions , research should only be carried out with the free and informed consent of a competent person. This means that a competent person, after having been adequately informed as to the potential use of the body and its parts, would donate his/her body or biological samples for forensic research purposes. Indeed, only through detailed information can the donors freely choose whether or not to give their consent . However, as shown by Bach , scarce ethical guidelines and regulatory oversight have allowed worrying practices with regard to body donation for research: people are often poorly informed or misled as to the risks, and some tissue banks use language that may even be potentially exploitative in their advertisements. In this regard, the Declaration of Helsinki is a very useful reference when developing informative material for donation programs. Indeed, according to the Declaration of Helsinki, “each potential subject must be informed adequately of the aims, methods, sources of funding, any possible conflicts of interest, institutional affiliation of the researcher, the anticipated benefits and potential risks of the study and the discomfort it may entail, post-study provisions and any other relevant aspects of the study” (art. 26) . Applied to forensic research, this means that potential donors and their next of kin should at least be provided with the following information: for what purposes the body can be used, what invasive—to a greater or lesser extent—interventions or destructive actions may be performed (autopsy, sampling of organs, post-mortem biopsies, various research methods, recovery of bones, etc.), how many years the sample can be held in storage by the department, and what fate may be chosen at the end of the retention time (cremation, burial, return to family, etc.). After verifying that all the information has been clearly understood, written informed consent should be acquired for both the body donation and the collection and storage of personal data by the department, and for the use of the results provided by the scientific research. The potential subject should be informed of the right to withdraw consent at any time and with regard to communication of incidental findings to family members. However, body donation for research purposes is neither a common nor an accepted practice worldwide , and in the daily practice of forensic research body donation is even less common. While compliance with the deceased person’s wishes, goals, and values is one of the requirements for a respectful use of HC&R, in the forensic field it is not easy to obtain informed consent before death, which is, in most cases, unexpected. In some situations, not only the wishes but even the very identity of the deceased may be, and may remain, unknown. In these instances, regulations such as the UK Human Tissue Act or the EU Recommendation CM/Rec(2016)6 on research on biological materials of human origin are of little help. Furthermore, the use of biological materials obtained during forensic autopsies is poorly or inadequately regulated by the law in most countries . On the other hand, forensic researchers could rely on article 32 of the Declaration of Helsinki concerning “medical research using identifiable human material or data, such as research on material or data contained in biobanks or similar repositories,” that establishes that in “exceptional situations where consent would be impossible or impracticable to obtain for such research […] the research may be done only after consideration and approval of a research ethics committee” . We argue that this statement could also be applied to the use of both unclaimed bodies, which still represent an important source for forensic research, and of human remains from unknown people. The absence of ad hoc ethical guidelines for forensic research involving HC&R may represent a barrier for research and expose researchers to the risk of ethically questionable practices, especially in countries where this issue is poorly or inconsistently regulated by the law. Bearing in mind the inherent differences in comparison with clinical research involving human beings and the different moral obligations involved, the Declaration of Helsinki is applicable and useful for forensic research involving HC&R. Indeed, the Declaration of Helsinki framework allows researchers to focus on substantial ethical principles and issues, promoting the consistency, integrity, and quality of forensic research. Of course, the Declaration of Helsinki is not a panacea for forensic research. Consensus regarding ethical standards and the adoption of national and supranational laws that clearly regulate the use of human cadavers and remains, including those from autopsies, continued to be of primary importance for the forensic science community. However, the systematic and publicized adoption of the Declaration of Helsinki principles, along with improved visibility and transparency of forensic research through the registration protocol in public databases, could increase public trust in forensic research and, ultimately, increase “good” research in the field. Editors of forensic science journals could play a pivotal role, by uniformly adopting publication policies that set the Declaration of Helsinki as the ethical standard for research involving human biological material. Bodies of deceased persons and human remains are essential in forensic research but ad hoc worldwide-recognized ethical standards for their use are still lacking. A clear ethical reference is needed both to avoid possible unethical practices and to sustain research in the forensic field. Even if moral obligations are different, there is a continuity between the living person and his/her body after death. The Declaration of Helsinki is applicable and useful for forensic research involving human cadavers and remains, promoting the consistency, integrity, and quality of research. Consensus regarding ethical standards and the adoption of national and supranational laws that clearly regulate the topic remains of primary importance for the forensic science community. |
Enhancing the future of simulation-based education in pediatrics | 056c498b-d5ba-4048-a207-45c0b5d1ac06 | 7888690 | Pediatrics[mh] | Simulation-based medical education (SBME) is a form of experiential learning that takes advantage of the use of simulation to create realistic clinical scenarios in a well-controlled environment . By virtue of it, the trainee can improve their technical and non-technical skills through the interaction with other learners and the privilege of making mistakes safely, with corrective feedback from the simulator itself or their mentor . Particularly in pediatrics, simulation has emerged as a powerful tool to enhance 360-degree medical education and has become part of pediatric residency and fellowship training programs . Furthermore, it may represent a method to boost skills even in other more experienced professionals (e.g. neonatologists) or strengthen provider performance throughout real-time consultations with experienced clinicians while handling simulated resuscitation scenarios, using tele-medicine . Nevertheless, considering at least procedural skills and teamwork behaviors, SBME usually requires strictly interactive and face-to-face educational sessions. As senior instructors , we directly experienced that social distancing policy forced to suddenly stop simulation sessions, at least for groups of trainees. Therefore, during the COVID-19 period, SBME switched from shared simulation environments to e-learning platforms, virtual reality 360-degree videos and individual just-in-time training (where a simulation center was located inside the hospital and the ward itself). However, the efforts to avoid a gap in knowledge among professionals in this unique educational period could represent a great springboard for reshaping the future of SBME. Notwithstanding this unforeseen and impressive storm, we ought to take advantage of it. Indeed, if on the one hand we currently do not know how long this unique period will last, we are aware of the added value of simulation in pediatric education. Hence, we should strengthen what we still can achieve face-to-face, while enabling new perspectives for things that we will be forced to do remotely. Indeed, as clinicians and researchers, we are witnessing a great expansion of diagnostic and prognostic tools, thanks to the introduction of newest technologies- e.g. Next Generation Sequencing (NGS) among the others- that are more and more available, even for the study of rare and complex pediatric pathologies, such as multiple sclerosis . Moreover, as instructors, it is time for us to enable a technology enhanced environment in simulation-based learning. For this purpose, we herein provide some tips for reshaping SBME, striving to usher a new era in this essential element of pediatric education.
Multi-sensory augmentation as a key enabling factor for a new generation of pediatric simulation frameworks New technologies should become part and parcel of procedural skills training. This could be the case of Augmented Reality (AR) systems, such as Microsoft HoloLens, Oculus devices or Google Glass . From this standpoint, their use could enable procedural skills teaching as if both mentor and trainee were in the same location, even if they work remotely. As an example, these devices could be used to capture the learner’s first-person view of a simulated emergency/delivery room through mixed reality capture, while the mentor’s hand gestures are captured through motion tracking (such as Leap Motion) and virtually displayed in the trainee’s AR space. These may represent valid solutions to exploit communication through visual channel. However, vision is not the only sense one can use to provide information. Indeed, an alternative solution may be the use of tactile feedback. Touch is one of the most ancestral senses in Nature, which typically requires lower cognitive efforts as compared to audio/visual feedback. Because of this peculiarity, the use of tactile communication may introduce considerable benefits. Roboticists have proposed many solutions to convey a number of different stimuli, such as vibrations, normal and tangential forces, temperature. Many pilot studies investigated the use of this kind of stimuli in medical settings . As an example, vibration-based feedback seems very effective in conveying directional cues, which may be used to naturally guide the arm of the trainee toward given targets. Force stimuli, e.g. through an engineered fabric band which squeezes the arm of the operator, could be used to codify “alert” signals. The very same technology, redesigned to fit at fingertip level, may be controlled to replicate pulsations, which for example could simulate the perception of arterial beats during palpation. Voice coils motors, finally, seem very effective in reproducing the contact with a surface, its roughness, and potentially its temperature with the addition of thermal displays. It would be fascinating to investigate this line of research, especially considering that haptic technologies may be integrated in existing AR/VR settings, opening highways of potential applications in SBME. Exploiting the available resources to improve non-technical skills The learner could enhance their teamwork, leadership and clinical decision-making skills throughout the use of suitable serious games (games used with a pedagogical purpose) . Meanwhile, the mentors could take advantage of the available recorded simulation sessions to assess what has been done before and find what can be improved, even with the help of experienced staff (e.g. counselors). This would result in a “meta-analysis” which could definitely lead to future tailor-made interventions in order to endlessly improve non-technical skill teaching. Moreover, virtual e-learning platforms should be implemented with live webinars, which should be preferred to video-recorded lessons, to facilitate the debate among mentors and learners. From this standpoint, debriefing sessions should be the core of e-learning education and video-assisted ones should be provided (even using previously video-recorded simulation sessions when a simulation session cannot be performed). This could enable the trainees to learn from (other people’s) mistakes. A focus on old and new problems, and keys on how to solve them A great obstacle to these challenging perspectives could be funding resources. Indeed, the newest technologies often require conspicuous capital investments. Especially for rural hospitals, or low- and middle-income countries this could be unaffordable. For this reason, great effort should be put into devising and producing low-cost devices, to avoid the introduction of social disparities. Furthermore, it is worth mentioning that -at the current stage - the usage of such technologies does not require conspicuous investments. Indeed, their cost typically ranges between hundreds to a maximum of few thousand dollars, thanks to the extensive employment for other commercial purposes, such as electronic games. On the other hand, governments and University institutions should guarantee their maximum effort in covering medical education costs, as this lays the foundation for a safer world while potentially sparing dreaded expensive medico-legal issues.
New technologies should become part and parcel of procedural skills training. This could be the case of Augmented Reality (AR) systems, such as Microsoft HoloLens, Oculus devices or Google Glass . From this standpoint, their use could enable procedural skills teaching as if both mentor and trainee were in the same location, even if they work remotely. As an example, these devices could be used to capture the learner’s first-person view of a simulated emergency/delivery room through mixed reality capture, while the mentor’s hand gestures are captured through motion tracking (such as Leap Motion) and virtually displayed in the trainee’s AR space. These may represent valid solutions to exploit communication through visual channel. However, vision is not the only sense one can use to provide information. Indeed, an alternative solution may be the use of tactile feedback. Touch is one of the most ancestral senses in Nature, which typically requires lower cognitive efforts as compared to audio/visual feedback. Because of this peculiarity, the use of tactile communication may introduce considerable benefits. Roboticists have proposed many solutions to convey a number of different stimuli, such as vibrations, normal and tangential forces, temperature. Many pilot studies investigated the use of this kind of stimuli in medical settings . As an example, vibration-based feedback seems very effective in conveying directional cues, which may be used to naturally guide the arm of the trainee toward given targets. Force stimuli, e.g. through an engineered fabric band which squeezes the arm of the operator, could be used to codify “alert” signals. The very same technology, redesigned to fit at fingertip level, may be controlled to replicate pulsations, which for example could simulate the perception of arterial beats during palpation. Voice coils motors, finally, seem very effective in reproducing the contact with a surface, its roughness, and potentially its temperature with the addition of thermal displays. It would be fascinating to investigate this line of research, especially considering that haptic technologies may be integrated in existing AR/VR settings, opening highways of potential applications in SBME.
The learner could enhance their teamwork, leadership and clinical decision-making skills throughout the use of suitable serious games (games used with a pedagogical purpose) . Meanwhile, the mentors could take advantage of the available recorded simulation sessions to assess what has been done before and find what can be improved, even with the help of experienced staff (e.g. counselors). This would result in a “meta-analysis” which could definitely lead to future tailor-made interventions in order to endlessly improve non-technical skill teaching. Moreover, virtual e-learning platforms should be implemented with live webinars, which should be preferred to video-recorded lessons, to facilitate the debate among mentors and learners. From this standpoint, debriefing sessions should be the core of e-learning education and video-assisted ones should be provided (even using previously video-recorded simulation sessions when a simulation session cannot be performed). This could enable the trainees to learn from (other people’s) mistakes.
A great obstacle to these challenging perspectives could be funding resources. Indeed, the newest technologies often require conspicuous capital investments. Especially for rural hospitals, or low- and middle-income countries this could be unaffordable. For this reason, great effort should be put into devising and producing low-cost devices, to avoid the introduction of social disparities. Furthermore, it is worth mentioning that -at the current stage - the usage of such technologies does not require conspicuous investments. Indeed, their cost typically ranges between hundreds to a maximum of few thousand dollars, thanks to the extensive employment for other commercial purposes, such as electronic games. On the other hand, governments and University institutions should guarantee their maximum effort in covering medical education costs, as this lays the foundation for a safer world while potentially sparing dreaded expensive medico-legal issues.
In conclusion, we strongly believe that our mission is to guarantee high quality education, especially in the pediatric setting, notwithstanding the current pandemic. As described above, several opportunities are available to pursue this aim. Ultimately, we suggest keeping in mind an age-old truth: “As for the future, your task is not to foresee it, but to enable it”- Antoine de Saint-Exupéry, Le Petit Prince (1943).
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Decomposition and entomological colonization of charred bodies – a pilot study | b4da8717-8fd6-4951-b2e7-201a06bece52 | 3760664 | Pathology[mh] | Two experiments were performed in a field in the outskirts of Milan, in Northern Italy (45° 20’ N; 09 13’ E), in the winter and summer of 2007. The weather at the site was hot and damp in the summer and cold in the winter with moderate surface winds. Meteorological data were collected from the closest meteorological station located 1.5 km from the studied area and compared with the measurements performed during the sampling. Adult domestic pig ( Sus scrofa ) carcasses were used as models for human cadavers. This animal is considered to be an excellent model for human decomposition and is frequently used in taphonomic experiments, particularly concerning insect/arthropod colonization ( , - ). Four 60-kg pigs were obtained from the Department of Veterinary Medicine (University of Milan); each animal died from causes independent from the experimental project. For each experiment, a pig carcass was burnt on a wooden pyre until it reached the level 2-3 of the Glassman-Crow scale ( ), corresponding to the destruction of the extremities, initial charring of the skin, and a substantial preservation of the corpse. The second pig of similar weight, not burnt, was used as control. In both experiments, the carcasses were maintained at a 50 m distance from each other to avoid reciprocal contamination and a wire mesh (5 cm mesh size) was placed over each carcass to prevent vertebrate depredation. The animals were placed in the same place during the winter and summer experiments. Observation and sample collections were performed after 3, 6, 15, 18, 24, 36, 42, 60, 95, and 120 days in the winter and after 1, 6, 9, 12, 15, 18, 27, 34, and 42 days in the summer. For each observation, carcasses were macroscopically analyzed in order to determine the state of decomposition according to the Goff terrestrial model, which distinguishes fresh, bloated, decay, postdecay, and skeletal stages ( ). In order to perform the morphological evaluation of the exposed pigs, samples for histological analyses (square in shape, 1 cm wide) were taken from the charred skin areas, and fixed in 10% formalin and then paraffin-embedded according to standard histological technique. Four microtome-thick sections were cut from paraffin blocks and stained with standard hematoxylin eosin stain and Trichrome stain. All observations were made using a light microscope equipped with a digital camera and DP software for computer-assisted image acquirement and managing (Wild Heerbrugg, Switzerland). Eight insect pitfall traps containing a saturated NaCl solution and soap were placed at 50 cm all around the carcass. Moreover, entomological samples were collected by hand on the carcasses, under them, and where and when possible in the carrion cavities. Insect identification was performed using specific entomological key and description ( - ) and by comparison with specimens stored in the private collection of one of the authors (SV). Zoological nomenclature followed Minelli et al ( ).
The first part of the study was conducted from February to June. The average temperature in this period was 16.0°C (min 1.0°C, max 32.4°C) and the rainfall was considerable during March (48 mm) and May (152 mm). The second part of the study was conducted from June to August. The average temperature was 25.5°C (min 12.8°C, max 36.0°C). The rainfall during this time was negligible. Macroscopic ( and ), histological, and entomological observations were carried out in order to describe the decomposition processes and insect colonization. The list of the saprophagous and saprophilous insects collected on the carcasses is shown in . During the first two weeks of the winter part of the experiment, after the charring process, no clear external modifications occurred on the carrion, there was no decomposition fluid, and no insect egg depositions. The first insect activity (flight) ( Calliphora vomitoria ) was observed at the day 18, but without egg laying. During the fourth week (day 26), a considerable presence of calliphorid maggots ( C. vomitoria ) and several adults and larvae of other Diptera [ Sphaerocera curvipes (Sphaeroceridae), Themira sp, Sepsis sp (Sepsidae), Gen. spp (Sciaridae)] and Coleoptera (Staphylinidae, mainly Creophilus maxillosus ) was recorded. At the same time, a clear reduction of the tissues in several body regions (head, thorax, and abdomen) was observed. On the day 26, the carrion appeared completely skeletonized, with complete bone disarticulation. The maggot activity ( C. vomitora, Calliphora vicina, Phormia regina, Hydrotaea capensis ) was localized only on the soil and under a few skin fragments, whereas Coleoptera (Silphidae, Staphylinidae, Carabidae, Anthicidae) were widely spread on all the body remains. There was no presence of larvae from the sixth week after exposure (day 42 and the following days). Two months after the exposure (day 60), the bones were clean and only a few remains of burnt skin and muscles were still present. Larvae and adults of coleopterans belonging to different families (Staphylinidae, Carabidae, Trogidae, and Aphodidae) were still recovered. The control pig was exposed at the same time as the burnt pig. No evident morphological modifications were observed in the first 15 days after the exposure. The beginning of the initial putrefactive stage was detected at the end of the second week. In the third week (day 18), the presence of first instar larvae ( C. vicina ), adults of scuttle flies and Coleoptera (Staphylinidae, Carabidae, Trogidae) was recorded. At the same time, a moderate emphysematic phase in the head region and discharge of decomposition fluids from the mouth was observed. In the abdominal region, the beginning of a colliquative phase was observed. With the progression of this phase, breaking of the skin was evident and the maggot activity ( C. vicina, C. vomitoria, Ph. regina, H. capensis, Themira sp) was localized mainly in the bodily area in contact with the soil. The rest of the skin became drier and drier and the maggot mass invaded the whole abdominal and thoracic cavities. In the summer period, in the days immediately after the exposure the burnt carrion showed important morphological modifications, with evident decomposition processes in the head and the presence of adult flies and first instar maggots ( Ph. regina ) concentrated on the head and in the abdominal and thoracic cavities. The entry of the larvae into the body cavities occurred through the skin fissures caused by fire. After one week (day 6), the carrion showed some clear skeletonized areas (head, thorax). After the first week, the rate of skeletonization and the exposure of bones slowed down. Larval activity ( Ph. regina, Lucilia sericata ) was concentrated only under the skin, whereas coleopterans of different families (Staphylinidae, Carabidae, Silphidae, Histeridae, Anthicidae) were largely spread. During the third week (day 18), no maggots were observed on the body. In the fourth week (day 27), soft tissues were almost completely lost, except for large fragments of dry or burnt skin; a decrease in species richness was observed. Several Dermestidae larvae ( Dermestes laniarius , Dermestes frishii ) were present in all the body regions. In the sixth week, all the bones were exposed, with disarticulation of leg bones. The control pig in the summer period was exposed on the same day as the burnt one. The decomposition processes during the next 2 months followed the typical pattern with the bloated, active, and dry decay stage and the beginning of the skeletonization phase. The most important concentration of maggots ( Ph. regina, L. sericata ) was detected in the abdominal area. After 6 weeks, the control pig showed about 40% skeletonization. Coleopterans (Staphylinidae, Carabidae, Silphidae, Histeridae, Anthicidae, Dermestidae) were collected during the whole decomposition period. Histological screening of the charred skin fragments showed that the outer crisp surface was completely destroyed macroscopically; but frequently underneath there was a thin layer of dehydrated skin, which showed moderate preservation of cellular patterns. The dermis, subcutaneous adipose, and muscle tissues were always visible during the whole experiment, both in the winter and summer part ( ).
The entomo-fauna that was collected during the two experiments in both not-burnt and burnt carcasses included a large number of species typical of all colonization waves ( ). The first flies ( Calliphora spp, Ph. regina , L. sericata ) arrived on burnt and not-burnt carcasses at the same time, whereas other species (Diptera and Coleoptera) arrived earlier on burnt carcasses. The colonization of burnt carcasses showed a classic pattern for the first wave insects and anticipation of other waves, with insects usually attributed to different waves arriving at the same time. This was probably a result of the carbonization process, which brought about two main advantages for fly colonization; first, the breaking up of charred soft tissues, with multiple fissures of the skin exposing the viscera, which therefore become immediately available for fly colonization. This may explain why the flies attacked the charred carcass earlier than the control carcass. In addition, the disruption of the skin surface and fast decomposition of the viscera during the first days creates a substratum of tissues in different phases of decomposition and with different water concentrations. This complex environment may explain the arrival of different waves of insects within a short period of time, which in cases of standard decomposition usually arrive sequentially. Moreover, the burning process caused the transformation of several molecules resulting in a wide spectrum of odors (volatile molecules), attractive for different insects. Entomological approach proves to be a reliable method for the time since death determination. PMI estimation is of utmost importance in cases involving charred corpses, where decomposition processes often have different dynamics, and tissue destruction prevents evaluation based on morphological appearance or chemical variation, although some studies on accumulated degree days produced useful results ( , ). Some authors recently calculated a correction factor of the original formula, which adequately takes into account the carbonization process, but at the moment the morphological approach for PMI estimation is experimental, and there are still doubts concerning the standardization of specific carbonization variables (temperature, use of accelerants, etc) ( ). In this study, decomposition of burnt carcasses stopped quite soon, which is in contrast with four stages of decomposition observed by Avila and Goff ( ). Both in winter and summer, charred tissues did not show what could be referred to as actual decomposition; they became more brittle and were affected by progressive crumpling, which lasted during the entire experimental period, reducing the bodily area covered by the soft tissues and exposing the bone surface ( ). Charred corpses were better conserved probably due to the loss of water, with a decrease in bacterial activity, which was confirmed by histological analysis showing that heat retains the tissues, probably through dehydration. In fact, the observed coartation of the dermis and the presence of large gas bubbles in the dermis and picnotic nuclei in the epithelium are reported to be changes caused by heat ( - ). The results are in contrast with data reported by Gruenthal et al ( ), which verified the decomposition rate in charred pig carcasses, and observed that, although the general decomposition trend was similar both in charred and uncharred carcasses, a more advanced pattern was visible in bodily regions highly affected by fire ( ). The differences in the decomposition process found in these two studies may be explained by the lower carbonization degree and a limited charred area in the study by Gruenthal et al ( ). In conclusion, our results showed that in burnt remains, entomological approach can be used for estimation of the minimum PMI in the presence of insects belonging to the first colonization wave (mainly Calliphoridae). Our study does not support the claim that the burnt corpse is hardly colonized by flies ( , ) and indicates that the “classic” insect waves of colonization model reported by several authors cannot be applied to burnt remains. Further research is needed to evaluate the influence of temperature of carbonization, accelerants use, environment, and body size in PMI estimation in charred bodies and fill in the gap in this important field of forensic practice.
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Demonstrating the presence of | 717c27e4-0668-4b2f-9eae-fc702049d71b | 7561240 | Pathology[mh] | Canine monocytic ehrlichiosis (CME) is caused by Ehrlichia canis , an intracellular parasitic bacterium and tick-borne pathogen. Recently, this pathogen has received further attention because it has led to increasing morbidity and mortality in animals . Transmission is mediated by the tick Rhipicephalus sanguineus ( sensu lato ), and, before infection, the bacteria replicate in monocytes and macrophages . Clinical presentation of CME results in acute, chronic or subclinical phases, with several clinical manifestations. The acute phase persists for 2–4 weeks and is characterised by signs in diverse systems, yet the most common are fever, weight loss, anorexia, depression, lymphadenomegaly, splenomegaly and vasculitis . In addition, dogs in this phase show thrombocytopenia as the most common laboratory abnormality . In the subclinical phase, dogs have persistent thrombocytopenia and leukopenia in laboratory analysis; however, during this stage, in some dogs the thrombocytopenia may be mild to non-existent , and they usually do not show clinical signs. The duration of this phase varies from months to years . Additionally, during this phase it is common that the microorganism may not circulate in the blood but is deposited in some target organ, such as the spleen, bone marrow or liver . Furthermore, previous research has shown that E. canis is widely distributed in different organs of infected dogs . Otherwise, in the chronic phase dogs show severe pancytopenia, haemorrhagic diathesis, and general debilitation . Immune system deficiency, stress, co-infections, virulence strain, and geographical region are factors that influence the presentation of this phase in affected dogs . In recent times, diagnosis of the disease has been challenging for practicing veterinarians . Identification of morulae in monocytes in a blood smear is diagnostic of the disease; however, a low frequency of morulae in buffy coat smears has previously been reported, which could be due to the low parasitaemia observed in the natural infection . Besides, other more specific methods are used as diagnostics, including the immunofluorescence antibody test (IFA) and ELISA (enzyme-linked immunosorbent assay), which are both able to detect specific antibodies , as well as other molecular techniques such as the polymerase chain reaction (PCR) . Presently, the Infectious Disease Group of the American College of Veterinary Internal Medicine (ACVIM) requires that dogs diagnosed with this disease show suggestive clinical signs and have positive tests, either by serology and/or by PCR . A complication in the diagnosis comes about in dogs in the subclinical phase of the disease because dogs normally do not show clinical signs. Furthermore, cross-reactivity and a failure to differentiate between current and past infections with ELISA and IFA tests has been reported . On the other hand, both in the subclinical and chronic phases, there is a possibility that parasitaemia is low in the dog , as the bacteria are located in the target organs . Therefore, in these cases, the dogs will be negative in a PCR blood test . Presently, the presence of the DNA of E. canis in several tissues, such as blood, bone marrow, spleen, liver, kidney and lymph nodes has been demonstrated by PCR in experimentally infected dogs . The goal of this study was to evaluate the occurrence of E. canis in different tissues, such as liver, spleen, lymph nodes and bone marrow, in dogs naturally infected with monocytic ehrlichiosis, assuming that a considerable percentage of dogs negative to E. canis by blood PCR will show positive results in biopsies of different tissues. An analysis of the variation in infection by E. canis in four tissues was carried out in two groups of dogs: positive and negative by PCR of blood samples. Animals Fifty-nine dogs obtained from the municipal Anti-Rabies Centre of Juárez were used in this study. Based on the Centre’s internal regulations, animals that were not adopted 8 weeks after their arrival were euthanised. Euthanasia was performed by an overdose of sodium pentobarbital according to national and international animal welfare regulations. In order to increase the possibility that dogs will present the subclinical phase of the disease, the inclusion criteria were that the dogs should have ticks but be clinically healthy; therefore, dogs without ticks or with signs of any disease were excluded. Sample collection Whole blood samples were collected in tubes containing EDTA (Vacutainer BD ® , Mexico City, Mexico) by cephalic venepuncture with prior administration of sodium pentobarbital. The other tissue samples were acquired by biopsies immediately after euthanasia, following the steps of surgical asepsis in order to prevent cross-contamination. In addition, with the same purpose, a change of instruments was made for the biopsy of each tissue, and particular attention was taken to avoid blood or other fluid from the dog coming into contact with the tissue samples. Bone marrow aspirates were obtained with bone marrow aspiration needles (Argon Medical Devices ® , Dallas, TX, USA) from the greater tubercle of the humerus, as described by Raskin & Messickin . Hepatic and splenic biopsies were obtained by celiotomy and with the ligature fracture technique . Finally, prescapular lymph node were biopsied with a biopsy punch (Premier ® , Plymouth Meeting, PA, USA) as previously described . Tissues samples were marked and frozen at − 20 °C for future extraction of DNA and PCR analysis. Biopsies obtained from spleen, liver and lymph node had an average weight of 200 mg (range 150–210 mg). The amount of whole blood obtained was 1.5 ml and the bone marrow biopsy obtained 0.6 ml on average (range 0.4–0.7 ml) DNA extraction For the blood samples, the extraction of genomic DNA from the cellular package of the dogs’ samples was performed using the UltraClean Blood DNA Isolation Kit (MoBio Lab®, Carlsbad, CA, USA), according to the manufacturer’s instructions. The other tissues were handled in a sterile fashion prior to the extraction of DNA. For the extraction of DNA from the biopsies, the protocol was modified with the previous addition of lysis reagents . The tissues were then macerated with the use of a low-velocity drill (Jorvet Lab ® , Loveland, CO, USA) and a dental burn (JOTA Technical®, Rüthi, Switzerland). Once each tissue was macerated, DNA extraction was performed in the same way as for the blood. PCR amplification and analysis Detection of E. canis DNA was achieved with the use of nested PCR molecular test. Initially, to amplify the Ehrlichia spp. 16S rRNA gene, 2 pmol of primers ECC (5′′-AGA ACG AAC GCT GGC GGC CAA GC-3′) and ECB (5′-CGT ATT ACC GCG GCT GCT-3′) were used . In the second PCR, to amplify the E. canis 16S rRNA gene, 2 pmol of primer HE-3(5′-TAT AGG TAC CGT CAT TAT CTT CCC TAT-3′) combined with the reverse primer ECA (5′-CAA TTA TTT ATA GCC TCT GGC TAT AGG AA-3′) were used . Initially, the PCR was performed in a thermocycler (Bio-Rad ® C-1000 Touch, Hercules, CA, USA) starting at 94 °C for 1 min followed by 35 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (hybridisation) and 72 °C for 3 min (extension). This was followed by 94 °C for 5 min and then 40 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (hybridisation), and 72 °C for 1 min (extension), as described previously . Statistical analyses A multivariate logistic regression model was used for the response variable ‘infection’ which was binary (dummy variable) with y = 1 if positive, and y = 0 if negative, depending on two explanatory variables: blood positivity (two levels) and positivity in four separate tissues (four levels). Therefore, the model was: infection = blood + tissue + error. The model analysed separately infection in both groups of dogs. In each group, the model compared infection among the four tissues using statistical tests ‘z’ between pairs of tissues, using a multiple-comparison Scheffe test. Comparison of the proportions of positive and negative results in blood, lymph node, liver and spleen samples were performed using Chi square and Fisher’s exact tests with the FREQ procedure of SAS (9.0). Significance was considered with a P -value of < 0.05. Fifty-nine dogs obtained from the municipal Anti-Rabies Centre of Juárez were used in this study. Based on the Centre’s internal regulations, animals that were not adopted 8 weeks after their arrival were euthanised. Euthanasia was performed by an overdose of sodium pentobarbital according to national and international animal welfare regulations. In order to increase the possibility that dogs will present the subclinical phase of the disease, the inclusion criteria were that the dogs should have ticks but be clinically healthy; therefore, dogs without ticks or with signs of any disease were excluded. Whole blood samples were collected in tubes containing EDTA (Vacutainer BD ® , Mexico City, Mexico) by cephalic venepuncture with prior administration of sodium pentobarbital. The other tissue samples were acquired by biopsies immediately after euthanasia, following the steps of surgical asepsis in order to prevent cross-contamination. In addition, with the same purpose, a change of instruments was made for the biopsy of each tissue, and particular attention was taken to avoid blood or other fluid from the dog coming into contact with the tissue samples. Bone marrow aspirates were obtained with bone marrow aspiration needles (Argon Medical Devices ® , Dallas, TX, USA) from the greater tubercle of the humerus, as described by Raskin & Messickin . Hepatic and splenic biopsies were obtained by celiotomy and with the ligature fracture technique . Finally, prescapular lymph node were biopsied with a biopsy punch (Premier ® , Plymouth Meeting, PA, USA) as previously described . Tissues samples were marked and frozen at − 20 °C for future extraction of DNA and PCR analysis. Biopsies obtained from spleen, liver and lymph node had an average weight of 200 mg (range 150–210 mg). The amount of whole blood obtained was 1.5 ml and the bone marrow biopsy obtained 0.6 ml on average (range 0.4–0.7 ml) For the blood samples, the extraction of genomic DNA from the cellular package of the dogs’ samples was performed using the UltraClean Blood DNA Isolation Kit (MoBio Lab®, Carlsbad, CA, USA), according to the manufacturer’s instructions. The other tissues were handled in a sterile fashion prior to the extraction of DNA. For the extraction of DNA from the biopsies, the protocol was modified with the previous addition of lysis reagents . The tissues were then macerated with the use of a low-velocity drill (Jorvet Lab ® , Loveland, CO, USA) and a dental burn (JOTA Technical®, Rüthi, Switzerland). Once each tissue was macerated, DNA extraction was performed in the same way as for the blood. Detection of E. canis DNA was achieved with the use of nested PCR molecular test. Initially, to amplify the Ehrlichia spp. 16S rRNA gene, 2 pmol of primers ECC (5′′-AGA ACG AAC GCT GGC GGC CAA GC-3′) and ECB (5′-CGT ATT ACC GCG GCT GCT-3′) were used . In the second PCR, to amplify the E. canis 16S rRNA gene, 2 pmol of primer HE-3(5′-TAT AGG TAC CGT CAT TAT CTT CCC TAT-3′) combined with the reverse primer ECA (5′-CAA TTA TTT ATA GCC TCT GGC TAT AGG AA-3′) were used . Initially, the PCR was performed in a thermocycler (Bio-Rad ® C-1000 Touch, Hercules, CA, USA) starting at 94 °C for 1 min followed by 35 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (hybridisation) and 72 °C for 3 min (extension). This was followed by 94 °C for 5 min and then 40 cycles of 94 °C for 1 min (denaturation), 60 °C for 1 min (hybridisation), and 72 °C for 1 min (extension), as described previously . A multivariate logistic regression model was used for the response variable ‘infection’ which was binary (dummy variable) with y = 1 if positive, and y = 0 if negative, depending on two explanatory variables: blood positivity (two levels) and positivity in four separate tissues (four levels). Therefore, the model was: infection = blood + tissue + error. The model analysed separately infection in both groups of dogs. In each group, the model compared infection among the four tissues using statistical tests ‘z’ between pairs of tissues, using a multiple-comparison Scheffe test. Comparison of the proportions of positive and negative results in blood, lymph node, liver and spleen samples were performed using Chi square and Fisher’s exact tests with the FREQ procedure of SAS (9.0). Significance was considered with a P -value of < 0.05. Of the 59 dogs analysed in this study, 28 (47.45%) showed a positive result for E. canis by PCR of blood samples, and 31 (52.55%) were negative. When evaluating the 28 dogs that were positive by PCR of blood samples, it was observed that 16 (57.14%) were also positive by PCR of some of the tissues. Otherwise, when analysing dogs with negative PCR results in blood ( n = 31) and comparing them with the results of PCR in different tissues of the same dogs, it was observed that 19 dogs (61.30%) presented positive results for E. canis in some of the tissues and 12 (38.70%) were negative in all tissues biopsied. The tissue biopsy with the highest number of positive samples was the bone marrow, with 26 (44.60%). Positive results from bone marrow samples occurred in both positive and negative blood samples. For example, 10 dogs (35.71%) that were positive by PCR of blood samples were also positive in PCR of the bone marrow (Table ). Furthermore, it was found that 12 of 19 cases (63.15%) were positive with negative PCR of blood samples (Table ). In half of the negative cases ( n = 6), the results of the PCR of other tissues were negative. Conversely, in two cases, the PCR was positive for all tissues analysed. The tissue with the second highest number of positive results was the spleen, with a prevalence of 42.37% ( n = 25). When analysing PCR-positive blood samples, 16 samples (57.14%) were also positive in PCR of spleen (Table ). In blood PCR-negative dogs, the splenic tissue showed 9 (47.36%) positive PCR results, although there were spleen-only positive samples on two occasions (Table ). Also, on two occasions the PCR was positive for all the tissues analysed. The remaining of the combinations are presented in Tables and . The liver had 22 PCR-positive cases (37.28%) from the total samples evaluated. Of the PCR-positive blood samples, 12 (42.85%) were also positive for the liver tissue (Table ). Similarly, with the spleen, of the 19 PCR-negative blood samples, 10 (52.63%) were positive for the liver tissue. In the negative blood samples, there was one liver-only positive result (Table ). In addition, the PCR was positive in all tissues twice. Finally, the tissue with the fewest positive results in the study was the lymph node, with 5 cases (8.47%). In the PCR-positive blood samples, only 2 cases were positive (10.52%; Table ). On the other hand, the blood samples negative by PCR were positive for lymphatic tissue in 3 cases, representing 15.78%. In none of these three cases was the lymph node the only tissue with positive results (Table ). Considering infection in the four tissues, the infection rate was the same in both negative and positive dogs in PCR of blood samples ( P > 0.05). The infection in tissues of negative dogs was an average rate of 0.23 ± 0.05, and for positive dogs was 0.35 ± 0.04 ( df = 233, P < 0.001). In the present study, of the 59 clinically healthy dogs analysed, 47.45% had a positive result for E. canis with PCR of blood samples. In addition, PCR recognised a higher prevalence of E. canis in different tissues of naturally infected dogs, in those with both positive and negative results by PCR of blood samples. With these results it was demonstrated that some dogs suspected of presenting subclinical ehrlichiosis, presented E. canis DNA in various tissues, even though they had negative PCR results from blood. At the present time, diagnosis by PCR is more useful than serology for the differentiation of concurrent infections and co-infections with diverse Ehrlichia spp. and is used for treatment monitoring . However, in naturally-occurring CME, the diagnostic sensitivity and optimal tissue for PCR testing in the untreated dog or in the post-treatment setting has not yet been clarified . Results obtained at this point demonstrate that in dogs with naturally-occurring CME infection it is feasible to detect E. canis in different tissues, even if they have negative blood tests. Additionally, in the acute phase of infection, E. canis is easily detected in blood, while in the subclinical and chronic phases there is the possibility of false negatives. Therefore, some tissues are more appropriate for sampling, such as the bone marrow and the spleen , an argument that has been corroborated by the present investigation. This study does not suggest performing tissue PCR for routine diagnosis of CME in dogs because performing biopsies in dogs with no clinical signs is impractical. However, sampling tissues may be relevant in understanding the distribution of CME in dogs. Comparative information on the spread and presence of E. canis by PCR analysis in multiple organs is limited, especially in dogs with the natural form of the disease, although some research has been done in experimentally inoculated dogs. For example, it is proven that PCR is effective in detecting E. canis in diverse tissues of dogs with experimental disease . In the same way, it has been described that the spleen is a tissue that can be useful to demonstrate the presence of E. canis DNA by PCR . In addition, the possibility of dogs in the subclinical phase being negative to PCR in blood samples and positive to PCR of splenic aspirates has also been established . Splenic aspirates have previously been performed to detect E. canis DNA by PCR. Previous research has shown that dogs that were blood-positive were also positive to splenic aspirates, compared to those that were negative in blood . These results differ from those obtained in the present investigation, where a prevalence of 42.37% ( n = 25) was obtained. Furthermore, of the 19 blood PCR-negative dogs, nine (47.36%) were positive by PCR in the splenic biopsies. It has been revealed that in the acute phase of disease, splenic aspirates are not superior to blood samples for detection of ehrlichial DNA by PCR. However, splenic aspirates are superior to blood in the evaluation of the response to therapy in experimentally treated dogs, because E. canis DNA could be detected in the spleen after its elimination from the blood . The results of the present study also differ from previous reports in which the number of dogs positive and negative for E. canis by PCR is similar in blood samples and splenic aspirates. The results revealed that DNA of E. canis was isolated in 29 (72.5%) spleen samples and in 30 (75%) whole blood samples; and ehrlichial DNA was not isolated in 11 (27.5%) spleen samples and in 10 (25%) whole blood samples . The difference between the other studies and the present investigation is the spleen tissue analysed. In our study, DNA was obtained through splenic biopsy, whereas in others DNA was obtained from blood through splenic aspirates. In another investigation, it was found that out of 78 dogs with splenic disease, only one was positive for E. canis by PCR in a splenic biopsy . The present study creates the expectation of performing research to establish the most suitable technique to obtain E. canis DNA from the spleen in dogs by comparing splenic aspirates with biopsies, including those taken with minimally invasive techniques, such as ultrasound-guided or laparoscopic methods. Furthermore, another important difference in our study is that the tissue with the highest number of positive samples was the bone marrow, in contrast to a previous report that obtained more positives from aspirates of the spleen . Nevertheless, other studies have demonstrated that other tissues besides the spleen are better in detecting E. canis by PCR. For example, some authors describe results similar to those obtained in the present study and show that E. canis DNA was most often amplified from bone marrow . But, in these cases, there was experimental disease, and PCR was performed using aspirates. On the other hand, in one study on biopsies of dog cadavers, contrary to the results of the present study, none of the bone marrow biopsies was positive for E. canis by PCR . An important limitation of the present study was the absence of blood analysis, especially blood counts. This could have established in a more accurate way the dogs presenting with the subclinical phase of monocytic ehrlichiosis . However, it can be assumed that positive dogs were in this phase, since they were clinically healthy. Ehrlichia canis is widespread throughout the different body systems of infected dogs. In addition, the molecular detection of E. canis DNA has shown that it can be present in different target organs . In the subclinical and chronic phases, E. canis could be ‘hiding’ in splenic macrophages . In this case, the spleen may be the principal reservoir of E. canis , probably because it has an abundance of macrophages. Moreover, some studies propose that it is the last organ to contain the microorganism before its elimination . Therefore, when containing a large number of bacteria, the spleen is considered by some authors as the organ of choice for molecular detection in different phases of the disease . Although in our study E. canis DNA was detected in the spleen, our results differ slightly from this statement, since it was the third most affected organ, surpassed by the bone marrow and liver. However, our results are similar to those of other studies that suggested that the spleen was inferior when compared to other tissues . In conclusion, results of this study could be applicable in some cases where the diagnostic sensitivity of PCR may be suboptimal . In some special cases, it will be necessary to search for E. canis DNA in different organs by molecular methods. In this study we have demonstrated that although infection in organs was 30% lower in dogs negative by PCR on blood samples, a considerable number of dogs ( n = 19 or 61.30%) showing negative results by blood PCR were positive for E. canis in some organs. Dogs with positive blood results were positive in three tissues (liver, bone marrow and spleen) in 48% of cases. At the same time, these three tissues were more positive than the lymph node, which was positive in only 8% of the samples evaluated, and was four times lower than in any of the other three tissues. Dogs with negative results in blood showed 33% detection of E. canis DNA in the spleen, liver and bone marrow; however, the presence of DNA was higher in liver and bone marrow than in the lymph node. Because in some cases DNA was detected in only one of these tissues, it is proposed that biopsies be performed of at least these three. This assertion is stipulated for other rickettsial diseases, such as Anaplasma spp., where blood samples are routinely used for screening, but in persistently infected dogs with intermittent or low-level bacteraemia other tissues might be useful . The results open the possibility of performing similar research aimed at detecting E. canis by PCR of different tissues in treated dogs that continue to show signs or alterations in blood tests, as well as in dogs that show signs suggestive of the disease but have negative results in serological and molecular blood analyses. |
Unveiling the efficacy and safety of Erenumab, a monoclonal antibody targeting calcitonin gene-related peptide (CGRP) receptor, in patients with chronic and episodic migraine: a GRADE-assessed systematic review and meta-analysis of randomized clinical trials with subgroup analysis | 4501d43f-361c-4f6d-8810-08d11190fa71 | 11938773 | Pathologic Processes[mh] | Migraine is a neurological disorder characterized by headaches of moderate to severe intensity, possibly accompanied by an aura. The pain is usually unilateral, pulsatile, and associated with sensitivity to light and sound . Migraine is a highly prevalent and disabling disease. It affects approximately 14% of the global population and caused over 45.1 million years of life lived with disability in 2016 . Despite the high prevalence and burden, the disease pathophysiology remains poorly understood. Besides, the pharmacotherapy of the disease encounters many challenges, with few drugs showing evidence-based efficacy in management and prevention [ – ]. Preventive strategies often involve the use of drugs such as beta-adrenergic blockers (like propranolol), certain antidepressants (such as amitriptyline), and anticonvulsants (like topiramate) . These drugs, however, were not originally developed to treat migraines, and their exact mechanisms of action in preventing migraine attacks remain unclear . As many as 50% of patients report inadequate effectiveness or poor tolerance of these treatments, leading to early cessation of therapy [ – ]. Accordingly, a significant number of individuals struggle to control their migraines with current preventive options, leading to high levels of disability and a significant reduced quality of life . Furthermore, low adherence rates (81% of patients had gaps of > 90 days in their migraine prevention in the first year) and low persistence (20% of patients at 12 months) for oral migraine preventive therapies lead to frequent switching, re-initiation, or complete cessation of preventive therapies across the migraine spectrum. As patients switch between preventive therapies, these discontinuation rates increase . Therefore, novel therapies have recently been proposed for the management of migraine. Erenumab, a calcitonin gene-related peptide (CGRP) receptor blocker, has recently shown promising results in managing migraine [ – ]. CGRP plays an integral role in the complex pathophysiology of migraine. During a migraine attack, CGRP is released from the nerve terminals of the trigeminal nerve, subsequently causing vasodilation and neurogenic inflammation . The role of CGRP in migraine has been even more prominent when administration of CGRP elicited migraine-like symptoms in susceptible individuals . The present study aims to provide class-one evidence regarding the effectiveness and safety of Erenumab in treating migraines, supported by what we believe is the most extensive meta-analysis to date. Unlike the latest meta-analysis that included only observational studies , the pooled results of this study are derived entirely from randomized-controlled trials, providing a more precise insight into the effect of Erenumab in migraine. Besides, our meta-analysis sub-grouped the pooled effect with respect to Erenumab doses, which were not taken into account in other meta-analyses like Fernandez-Bravo-Rodrigo et al. . Moreover, our analysis delved into details not covered in previous relevant studies ; our meta-analysis is the first to include a subgroup analysis based on the history of prior failures with migraine-preventive treatments. Additionally, we conducted two further subgroup analyses: one comparing episodic and chronic migraines and another comparing different doses of Erenumab (70 mg versus 140 mg). We addressed several metrics assessing migraine management efficacy, including MMD (Monthly Migraine Days), MSMD (Monthly Severe Migraine Days), and HIT-6 (Headache Impact Test). Furthermore, we aimed to provide insights into the safety profile of the drug. Additionally, we investigated sources of heterogeneity whenever possible using sensitivity analysis and assessed the quality of evidence using GRADE (Grading of Recommendations, Assessment, Development, and Evaluations).
This systematic review and meta-analysis followed the criteria of the Preferred Reporting Items for Systematic Review and Meta-analysis (PRISMA) statement . The protocol held a registration number CRD42024573300 on PROSPERO. Eligibility criteria The included studies followed the following criteria: randomized controlled trials (RCTs); studies including patients diagnosed with migraine: either chronic migraine defined as ≥ 15 headache days/month plus ≥ 8 migraine days/month for at least three months or episodic migraine defined as 4 to < 15 migraine days per month and < 15 headache days per month for at least three months before screening and during the baseline period of the trial ; the intervention group in the participating RCTs was Erenumab 70 mg or 140 mg, and the comparator was placebo with a minimum follow-up period of 3 months; English-language studies only. Observational studies, case reports, conference abstracts, uncontrolled studies, and studies not written in English were excluded. Search strategy Comprehensive research was conducted from inception until July 2024 in Scopus, PubMed, WOS, Embase, Clinical trials.gov, and Cochrane Central Register of Controlled Trials (CENTRAL) databases. The search strategy comprised specific keywords and Medical Science Heading (MeSH) terms, including the following: "Erenumab," "Erenumab-aooe," "AMG334," "headache*," "migraine*," and "Cephalgia." Study selection data extraction After developing the search strategy, two authors independently performed studies screening using Rayyan online software . We began initially with title-abstract screening, followed by full-text screening. A third reviewer was responsible for resolving conflicts between the two authors in the inclusion process. Four authors have extracted data independently on an online Excel sheet for easier access and communication between authors. The online sheet included study characteristics, population baseline characteristics, and outcome measures data. Study characteristics included study name and year, sample size, design, duration of treatment, population, and the key findings. Population baseline characteristics included sample size, age, gender, history of use or failure of prior migraine-preventive treatment, and duration of migraine. Outcome measures involved: MMD (Monthly Migraine Days) refers to the number of days per month a person experiences migraine headaches and either at least two pain characteristics (unilateral, throbbing, moderate to severe, or aggravated by physical activity) and one non-pain symptom (nausea, vomiting, or both photophobia and phonophobia). MSMD (monthly acute migraine-specific medication treatment days) refers to the number of days in a month that a person requires migraine medication (only migraine-specific medications like triptans or/and ergots) . HIT-6 (Headache Impact Test) assesses the headaches' impact on quality of life. It comprises six questions that evaluate headache-related disability, covering pain severity, daily activity interference, fatigue, cognitive issues, and emotional distress. Scores vary from 36 to 78, with higher scores reflecting a higher impact . MPFID-PI (Migraine Physical Function Impact Diary—Physical Impairment) and MPID-EA (Migraine Physical Function Impact Diary—Everyday Activities) are part of the MPFID, a validated patient-reported outcome tool developed to measure the impact of migraine on physical functioning and daily activities . mMIDAS (modified Migraine Disability Assessment) is a simplified questionnaire that assesses the impact of migraine on the quality of life . Our primary outcomes involved MMD, MSMD, HIT-6, and ≥ 50 reduction from baseline in MMD, and our secondary outcomes comprised MPFID-PI, mMIDAS, and the safety of Erenumab. Quality assessment The included (RCTs) quality was assessed using the Revised Cochrane risk-of-bias tool for randomized trials (ROB 2). Five domains were evaluated during the quality assessment: randomization process bias, bias due to deviations from intended intervention, missing outcome data bias, bias in the measurement of the outcome, and bias in the selection of the reported result. Two reviewers independently assessed each study, and disagreements were resolved by discussion or consultation with a third reviewer. Studies were categorized as having a low risk of bias, some concerns, or a high risk of bias based on the ROB 2 criteria . Statistical analysis Review Manager 5.4 software (RevMan) was used for the statistical analysis of both continuous and dichotomous outcomes . We applied the random effect model employing the DerSimonian-Laird method . The statistical analysis was based on a mean difference (MD) and standard deviation (SD) whenever the outcomes were continuous and on risk ratio (RR) when they were dichotomous, with a confidence interval (CI) of 95% and a statistically significant P -value was considered if it was < 0.05. The heterogeneity of the included studies was evaluated using the Higgins score ( I 2). I-square values ≥ 50% were indicative of high heterogeneity . Adverse events were reported as the number per study arm and pooled as RR. Furthermore, we conducted sensitivity analyses to assess the heterogeneity and robustness of the results whenever possible. One study adopted a crossover design ; therefore, we used the paired analysis method mentioned in the Cochrane Handbook . Quality of evidence The level of certainty of the generated evidence was evaluated using the Grading of Recommendations, Assessment, Development and Evaluation criteria (GRADE) by the GRADEpro Guideline Development Tool (GDT) online tool . GRADE tool assesses the evidence and classifies it into four levels of certainty: very low, low, moderate, and high, taking into consideration the following domains of evaluation: risk of bias, inconsistency, indirect evidence, imprecision, publication bias, and other domains like dose–response effect and plausible confounding. Publication bias The Luis Furuya-Kanamori asymmetry index (LFK index) and the Doi plot were used to evaluate the publication bias. Publication bias is reflected by the existence of asymmetry, whereas its absence is reflected by symmetry. LFK index values of ≤ ± 1, > ± 1 but ≤ ± 2, and > ± 2 indicate no asymmetry, minor asymmetry, and major asymmetry, respectively . Using the MetaXL Version 5.3 add-in for Microsoft Excel, we conducted a publication bias analysis and generated Doi plots for our primary outcomes. Furthermore, the funnel plots using Stata 17.0 software were generated for the primary outcomes.
The included studies followed the following criteria: randomized controlled trials (RCTs); studies including patients diagnosed with migraine: either chronic migraine defined as ≥ 15 headache days/month plus ≥ 8 migraine days/month for at least three months or episodic migraine defined as 4 to < 15 migraine days per month and < 15 headache days per month for at least three months before screening and during the baseline period of the trial ; the intervention group in the participating RCTs was Erenumab 70 mg or 140 mg, and the comparator was placebo with a minimum follow-up period of 3 months; English-language studies only. Observational studies, case reports, conference abstracts, uncontrolled studies, and studies not written in English were excluded.
Comprehensive research was conducted from inception until July 2024 in Scopus, PubMed, WOS, Embase, Clinical trials.gov, and Cochrane Central Register of Controlled Trials (CENTRAL) databases. The search strategy comprised specific keywords and Medical Science Heading (MeSH) terms, including the following: "Erenumab," "Erenumab-aooe," "AMG334," "headache*," "migraine*," and "Cephalgia."
After developing the search strategy, two authors independently performed studies screening using Rayyan online software . We began initially with title-abstract screening, followed by full-text screening. A third reviewer was responsible for resolving conflicts between the two authors in the inclusion process. Four authors have extracted data independently on an online Excel sheet for easier access and communication between authors. The online sheet included study characteristics, population baseline characteristics, and outcome measures data. Study characteristics included study name and year, sample size, design, duration of treatment, population, and the key findings. Population baseline characteristics included sample size, age, gender, history of use or failure of prior migraine-preventive treatment, and duration of migraine. Outcome measures involved: MMD (Monthly Migraine Days) refers to the number of days per month a person experiences migraine headaches and either at least two pain characteristics (unilateral, throbbing, moderate to severe, or aggravated by physical activity) and one non-pain symptom (nausea, vomiting, or both photophobia and phonophobia). MSMD (monthly acute migraine-specific medication treatment days) refers to the number of days in a month that a person requires migraine medication (only migraine-specific medications like triptans or/and ergots) . HIT-6 (Headache Impact Test) assesses the headaches' impact on quality of life. It comprises six questions that evaluate headache-related disability, covering pain severity, daily activity interference, fatigue, cognitive issues, and emotional distress. Scores vary from 36 to 78, with higher scores reflecting a higher impact . MPFID-PI (Migraine Physical Function Impact Diary—Physical Impairment) and MPID-EA (Migraine Physical Function Impact Diary—Everyday Activities) are part of the MPFID, a validated patient-reported outcome tool developed to measure the impact of migraine on physical functioning and daily activities . mMIDAS (modified Migraine Disability Assessment) is a simplified questionnaire that assesses the impact of migraine on the quality of life . Our primary outcomes involved MMD, MSMD, HIT-6, and ≥ 50 reduction from baseline in MMD, and our secondary outcomes comprised MPFID-PI, mMIDAS, and the safety of Erenumab.
The included (RCTs) quality was assessed using the Revised Cochrane risk-of-bias tool for randomized trials (ROB 2). Five domains were evaluated during the quality assessment: randomization process bias, bias due to deviations from intended intervention, missing outcome data bias, bias in the measurement of the outcome, and bias in the selection of the reported result. Two reviewers independently assessed each study, and disagreements were resolved by discussion or consultation with a third reviewer. Studies were categorized as having a low risk of bias, some concerns, or a high risk of bias based on the ROB 2 criteria .
Review Manager 5.4 software (RevMan) was used for the statistical analysis of both continuous and dichotomous outcomes . We applied the random effect model employing the DerSimonian-Laird method . The statistical analysis was based on a mean difference (MD) and standard deviation (SD) whenever the outcomes were continuous and on risk ratio (RR) when they were dichotomous, with a confidence interval (CI) of 95% and a statistically significant P -value was considered if it was < 0.05. The heterogeneity of the included studies was evaluated using the Higgins score ( I 2). I-square values ≥ 50% were indicative of high heterogeneity . Adverse events were reported as the number per study arm and pooled as RR. Furthermore, we conducted sensitivity analyses to assess the heterogeneity and robustness of the results whenever possible. One study adopted a crossover design ; therefore, we used the paired analysis method mentioned in the Cochrane Handbook . Quality of evidence The level of certainty of the generated evidence was evaluated using the Grading of Recommendations, Assessment, Development and Evaluation criteria (GRADE) by the GRADEpro Guideline Development Tool (GDT) online tool . GRADE tool assesses the evidence and classifies it into four levels of certainty: very low, low, moderate, and high, taking into consideration the following domains of evaluation: risk of bias, inconsistency, indirect evidence, imprecision, publication bias, and other domains like dose–response effect and plausible confounding. Publication bias The Luis Furuya-Kanamori asymmetry index (LFK index) and the Doi plot were used to evaluate the publication bias. Publication bias is reflected by the existence of asymmetry, whereas its absence is reflected by symmetry. LFK index values of ≤ ± 1, > ± 1 but ≤ ± 2, and > ± 2 indicate no asymmetry, minor asymmetry, and major asymmetry, respectively . Using the MetaXL Version 5.3 add-in for Microsoft Excel, we conducted a publication bias analysis and generated Doi plots for our primary outcomes. Furthermore, the funnel plots using Stata 17.0 software were generated for the primary outcomes.
The level of certainty of the generated evidence was evaluated using the Grading of Recommendations, Assessment, Development and Evaluation criteria (GRADE) by the GRADEpro Guideline Development Tool (GDT) online tool . GRADE tool assesses the evidence and classifies it into four levels of certainty: very low, low, moderate, and high, taking into consideration the following domains of evaluation: risk of bias, inconsistency, indirect evidence, imprecision, publication bias, and other domains like dose–response effect and plausible confounding.
The Luis Furuya-Kanamori asymmetry index (LFK index) and the Doi plot were used to evaluate the publication bias. Publication bias is reflected by the existence of asymmetry, whereas its absence is reflected by symmetry. LFK index values of ≤ ± 1, > ± 1 but ≤ ± 2, and > ± 2 indicate no asymmetry, minor asymmetry, and major asymmetry, respectively . Using the MetaXL Version 5.3 add-in for Microsoft Excel, we conducted a publication bias analysis and generated Doi plots for our primary outcomes. Furthermore, the funnel plots using Stata 17.0 software were generated for the primary outcomes.
Literature search We identified a total of 4426 records after conducting our search strategy. Using Endnote, we identified 1660 duplicates and removed them. The remaining 2766 records underwent vigorous title/abstract screening, yielding 200 records left for the full-text screening process. Eventually, 21 RCTs were eligible for our qualitative analysis, and 20 ( n = 5212) underwent quantitative analysis. The PRISMA flow diagram is shown in Fig. . Study and population characteristics A total of 21 [ – ] studies were enrolled in our systematic review and meta-analysis. All the included studies were RCTs, with a total number of participants of 5212. Eight studies were either post-hoc analyses or subgroup analyses based on previously conducted RCTs. The STRIVE study provided the largest number of participants among all studies ( n = 955), while Hoon et al. provided the smallest number with 12 patients. Most RCTs included three arms comprising placebo and two doses of Erenumab: 140 mg and 70 mg. The majority of the studies ( n = 16) included patients diagnosed with episodic migraine. Yu et al. (DRAGON) and Tepper et al. studies involved exclusively patients diagnosed with chronic migraine. In addition, Basedau et al. , Takeshiama et al. , and Hirata et al. studies included patients with either chronic or episodic migraine (mixed). The patients included in most studies exhibited a mixture of no prior treatment failures and previous failures of other preventive treatments. The studies that assessed only patients with previous treatment failures are Filippi et. , Reuter et al. (LIBERTY) , Hirata et al. , Ashina et al. , and Goadsby et al. 2019 . The latter three studies provided further details on patient subgroups according to the occurrence of prior treatment failure. The mean age across studies ranged from 24 to 48 years old, with females being the predominant participants in all studies. All study characteristics, including sample size, duration of treatment, and the key findings, are represented in Table . Furthermore, the characteristics of the studies’ population are summarized in Table . Quality assessment The risk of bias assessed by the Cochrane Risk of Bias tool version 2 in all studies is represented in Fig. . All the included RCTs have been assigned low risk in all domains and consequently considered to have an overall low risk of bias. Moreover, one study employed a crossover design and was assessed for additional considerations given its different design. It showed some concerns regarding the overall risk of bias, which could be attributed to the unclear reporting of the washout period and carry-over effect details. The risk of bias summary for the crossover study is shown in Supplementary File: Figure (S1).
We identified a total of 4426 records after conducting our search strategy. Using Endnote, we identified 1660 duplicates and removed them. The remaining 2766 records underwent vigorous title/abstract screening, yielding 200 records left for the full-text screening process. Eventually, 21 RCTs were eligible for our qualitative analysis, and 20 ( n = 5212) underwent quantitative analysis. The PRISMA flow diagram is shown in Fig. .
A total of 21 [ – ] studies were enrolled in our systematic review and meta-analysis. All the included studies were RCTs, with a total number of participants of 5212. Eight studies were either post-hoc analyses or subgroup analyses based on previously conducted RCTs. The STRIVE study provided the largest number of participants among all studies ( n = 955), while Hoon et al. provided the smallest number with 12 patients. Most RCTs included three arms comprising placebo and two doses of Erenumab: 140 mg and 70 mg. The majority of the studies ( n = 16) included patients diagnosed with episodic migraine. Yu et al. (DRAGON) and Tepper et al. studies involved exclusively patients diagnosed with chronic migraine. In addition, Basedau et al. , Takeshiama et al. , and Hirata et al. studies included patients with either chronic or episodic migraine (mixed). The patients included in most studies exhibited a mixture of no prior treatment failures and previous failures of other preventive treatments. The studies that assessed only patients with previous treatment failures are Filippi et. , Reuter et al. (LIBERTY) , Hirata et al. , Ashina et al. , and Goadsby et al. 2019 . The latter three studies provided further details on patient subgroups according to the occurrence of prior treatment failure. The mean age across studies ranged from 24 to 48 years old, with females being the predominant participants in all studies. All study characteristics, including sample size, duration of treatment, and the key findings, are represented in Table . Furthermore, the characteristics of the studies’ population are summarized in Table .
The risk of bias assessed by the Cochrane Risk of Bias tool version 2 in all studies is represented in Fig. . All the included RCTs have been assigned low risk in all domains and consequently considered to have an overall low risk of bias. Moreover, one study employed a crossover design and was assessed for additional considerations given its different design. It showed some concerns regarding the overall risk of bias, which could be attributed to the unclear reporting of the washout period and carry-over effect details. The risk of bias summary for the crossover study is shown in Supplementary File: Figure (S1).
MMD, MSMD, and HIT-6 Erenumab showed a statistically significant reduction in terms of MMD at three months compared to placebo (MD: -1.78, 95% CI: [-2.37 to -1.20], P < 0.00001, as shown in Fig. A), and a significant substantial heterogeneity appeared in the analysis ( p = 0.0007; I^2 = 70%). Similarly, MMD at 4–6 months showed a significant reduction (MD: -1.77, 95% CI: [-2.11 to -1.43], P < 0.00001, as shown in Supplementary File: Figure (S2)). However, no heterogeneity was evident in the analysis ( p = 0.70; I^2 = 0%). Both MSMD at three months and 4–6 months showed statistically significant improvement in favour of Erenumab compared to placebo (MD: -1.36, 95% CI: [-1.92 to -0.81], P < 0.00001, as shown in Fig. B) and (MD: -1.56, 95% CI: [-2.08 to -1.03], P < 0.00001, as shown in Supplementary File: Figure (S2)), respectively. Heterogeneity was significant in both ( p < 0.00001; I^2 = 84%) and ( p = 0.02; I^2 = 69%), respectively. HIT-6 at three months exhibited a statistically significant reduction favouring Erenumab (MD: -2.83, 95% CI: [-3.83 to -1.82], P < 0.00001, as shown in Fig. C), and showed significant heterogeneity ( p = 0.02; I^2 = 65%). ≥ 50%, ≥ 75%, and 100% reduction from baseline in MMD The drug showed statistically significant improvements in all responder rates, where significantly more subjects reached ≥ 50% reduction in MMD at three months (RR: 1.52, 95% CI: [1.31 to 1.76], P < 0.00001, as shown in Fig. A) and at 4–6 months (RR: 2.03, 95% CI: [1.53 to 2.70], P < 0.00001, as shown in Supplementary File: Figure (S2)). Similarly, 75% and ≥ 100% reductions from baseline in MMD at three months were significant compared to placebo (RR: 1.84, 95% CI: [1.43 to 2.35], P < 0.00001, as shown in Fig. B) and (RR: 1.93, 95% CI: [1.11 to 3.36], P = 0.02, as shown in Fig. C), respectively. ≥ 50% responder rate outcome at three months showed significant moderate heterogeneity ( p = 0.04; I^2 = 52%). MPFID-PI, MPID-EA, and mMIDAS at three months Each of MPFID-PI, MPID-EA, and mMIDAS showed a statistically significant reduction favouring Erenumab compared to placebo, (MD: -1.85, 95% CI: [-2.87 to -0.83], P = 0.0004, as shown in Fig. A), (MD: -2.10, 95% CI: [-3.45 to -0.75], P = 0.002, as shown in Fig. B), and (MD: -1.80, 95% CI: [-2.62 to -0.99], P < 0.00001, as shown in Fig. C), respectively.
Erenumab showed a statistically significant reduction in terms of MMD at three months compared to placebo (MD: -1.78, 95% CI: [-2.37 to -1.20], P < 0.00001, as shown in Fig. A), and a significant substantial heterogeneity appeared in the analysis ( p = 0.0007; I^2 = 70%). Similarly, MMD at 4–6 months showed a significant reduction (MD: -1.77, 95% CI: [-2.11 to -1.43], P < 0.00001, as shown in Supplementary File: Figure (S2)). However, no heterogeneity was evident in the analysis ( p = 0.70; I^2 = 0%). Both MSMD at three months and 4–6 months showed statistically significant improvement in favour of Erenumab compared to placebo (MD: -1.36, 95% CI: [-1.92 to -0.81], P < 0.00001, as shown in Fig. B) and (MD: -1.56, 95% CI: [-2.08 to -1.03], P < 0.00001, as shown in Supplementary File: Figure (S2)), respectively. Heterogeneity was significant in both ( p < 0.00001; I^2 = 84%) and ( p = 0.02; I^2 = 69%), respectively. HIT-6 at three months exhibited a statistically significant reduction favouring Erenumab (MD: -2.83, 95% CI: [-3.83 to -1.82], P < 0.00001, as shown in Fig. C), and showed significant heterogeneity ( p = 0.02; I^2 = 65%).
The drug showed statistically significant improvements in all responder rates, where significantly more subjects reached ≥ 50% reduction in MMD at three months (RR: 1.52, 95% CI: [1.31 to 1.76], P < 0.00001, as shown in Fig. A) and at 4–6 months (RR: 2.03, 95% CI: [1.53 to 2.70], P < 0.00001, as shown in Supplementary File: Figure (S2)). Similarly, 75% and ≥ 100% reductions from baseline in MMD at three months were significant compared to placebo (RR: 1.84, 95% CI: [1.43 to 2.35], P < 0.00001, as shown in Fig. B) and (RR: 1.93, 95% CI: [1.11 to 3.36], P = 0.02, as shown in Fig. C), respectively. ≥ 50% responder rate outcome at three months showed significant moderate heterogeneity ( p = 0.04; I^2 = 52%).
Each of MPFID-PI, MPID-EA, and mMIDAS showed a statistically significant reduction favouring Erenumab compared to placebo, (MD: -1.85, 95% CI: [-2.87 to -0.83], P = 0.0004, as shown in Fig. A), (MD: -2.10, 95% CI: [-3.45 to -0.75], P = 0.002, as shown in Fig. B), and (MD: -1.80, 95% CI: [-2.62 to -0.99], P < 0.00001, as shown in Fig. C), respectively.
Sensitivity analysis by removing one study at a time has been conducted to investigate sources of heterogeneity and test the robustness of the analysis in our primary outcomes. In both MMD ( p = 0.0007; I^2 = 70%) and HIT-6 ( p = 0.02; I^2 = 65%) at three months, the substantial heterogeneity was resolved by excluding Filippi et al. 2023 , yielding homogenous results ( p = 0.38; I^2 = 7%) and ( p = 0.90; I^2 = 0%), respectively, as shown in Supplementary File: Figure (S3). Regarding MSMD at three months, heterogeneity ( P < 0.00001; I^2 = 84%) was resolved by excluding both Dodick et al. 2018 and Tepper et al. 2017 ( P = 0.19; I^2 = 35%). Moreover, the heterogeneity in ≥ 50% responder rate outcome at three months ( p = 0.04; I^2 = 52%) was resolved by excluding Sakai et al. ( p = 0.18; I^2 = 33%), as shown in Supplementary File: Figure (S3). No single study exclusion affected the overall significance of any of the study`s outcomes, ensuring robust results.
We conducted subgroup analysis according to multiple criteria not only to investigate sources of heterogeneity but also to provide estimates of treatment effects for clinically relevant subgroups of patients. According to the status of previous failures, in all three primary outcomes, MMD, MSMD, and ≥ 50% reduction in MMD, the test for subgroup differences suggested that there was a statistically significant subgroup difference, p = 0.02, p = 0.010, p = 0.0007, respectively, as shown in Table . The presence of prior failure significantly modified the response of Erenumab compared to placebo. The treatment effect favoured Erenumab over placebo in both subgroups; however, it was significantly higher for the subgroup with prior failures indicating a quantitative subgroup effect. Although there was a significant difference between subgroups, an uneven distribution of trials and participants between subgroups existed. The forest plots are shown in Supplementary File: Figure (S4). In terms of different doses of Erenumab, only the MSMD outcome revealed a statistically significant test for subgroup difference ( p = 0.01) with a quantitative subgroup effect showing a more significant improvement favouring the 140 mg dose over the 70 mg compared to placebo, as shown in Table . The forest plots demonstrating subgroups of different doses are shown in Supplementary File: Figure (S5). Regarding the type of migraine, the test for subgroup differences indicated that there was no statistically significant subgroup difference in either MMD or ≥ 50% reduction in MMD ( p = 0.48) and ( p = 0.69), respectively, as shown in Table . This finding suggested that the type of migraine does not influence the effect of Erenumab compared to placebo. However, a smaller number of trials and participants contributed data to the chronic migraine subgroup (two studies) than to the episodic migraine subgroup (six studies in MMD and five in ≥ 50% reduction in MMD), proposing a possibility that the analysis may not be able to detect subgroup differences. The forest plots of subgrouping according to the type of migraine are shown in Supplementary File: Figure (S6). Publication bias Regarding publication bias, the DOI plot of MMD at 3 months showed minor asymmetry with an LFK index of -1.69, as shown in Fig. , suggesting possible publication bias. MSMD, HIT-6, and ≥ 50% reduction in MMD at 3 months showed major asymmetry in the DOI plots with LFK indices of -3.51, -2.95, and 5.92, respectively, indicating potential publication bias. The DOI plots of MSMD, HIT-6, and ≥ 50% reduction in MMD at 3 months are shown in Supplementary File: Figure (S7). Furthermore, funnel plots for MMD, HIT-6, MSMD, and ≥ 50% reduction in MMD at 3 months are shown in Supplementary File: Figures (S8) and (S9). Safety of erenumab Erenumab showed an acceptable safety profile compared to placebo. The intervention resulted in a significantly higher incidence of only constipation compared to the placebo (RR: 2.53, 95% CI: [1.60 to 4.02], P < 0.0001). Although nasopharyngitis and upper respiratory tract infection were the most reported adverse effects in both the placebo and Erenumab groups, they were not statistically significant. A summary of the adverse events is displayed in Table . All the forest plots demonstrating the adverse effects are represented in Supplementary File: Figures (S10)—(S14). Quality of the evidence The certainty of evidence regarding Erenumab efficacy in the most important and relevant outcomes was assessed using GRADE. All the outcomes at three months, including MMD, MSMD, HIT-6, and ≥ 50% reduction in MMD, were downgraded in two domains: inconsistency and the presence of suspected publication bias. Therefore, this downgrade resulted in a low overall certainty of evidence regarding the aforementioned four outcomes. A summary of the findings and a GRADE evaluation of the outcomes are shown in Table .
Regarding publication bias, the DOI plot of MMD at 3 months showed minor asymmetry with an LFK index of -1.69, as shown in Fig. , suggesting possible publication bias. MSMD, HIT-6, and ≥ 50% reduction in MMD at 3 months showed major asymmetry in the DOI plots with LFK indices of -3.51, -2.95, and 5.92, respectively, indicating potential publication bias. The DOI plots of MSMD, HIT-6, and ≥ 50% reduction in MMD at 3 months are shown in Supplementary File: Figure (S7). Furthermore, funnel plots for MMD, HIT-6, MSMD, and ≥ 50% reduction in MMD at 3 months are shown in Supplementary File: Figures (S8) and (S9).
Erenumab showed an acceptable safety profile compared to placebo. The intervention resulted in a significantly higher incidence of only constipation compared to the placebo (RR: 2.53, 95% CI: [1.60 to 4.02], P < 0.0001). Although nasopharyngitis and upper respiratory tract infection were the most reported adverse effects in both the placebo and Erenumab groups, they were not statistically significant. A summary of the adverse events is displayed in Table . All the forest plots demonstrating the adverse effects are represented in Supplementary File: Figures (S10)—(S14).
The certainty of evidence regarding Erenumab efficacy in the most important and relevant outcomes was assessed using GRADE. All the outcomes at three months, including MMD, MSMD, HIT-6, and ≥ 50% reduction in MMD, were downgraded in two domains: inconsistency and the presence of suspected publication bias. Therefore, this downgrade resulted in a low overall certainty of evidence regarding the aforementioned four outcomes. A summary of the findings and a GRADE evaluation of the outcomes are shown in Table .
In this meta-analysis, we assessed the efficacy and safety of Erenumab in patients with episodic and chronic migraine. Erenumab resulted in a significant reduction in MMD and HIT-6 compared to placebo. However, there was considerable heterogeneity, with [12pt]{minimal}
$${I}^{2}$$ I 2 =70% for MMD and 65% for HIT-6TM, possibly due to several reasons. First and most importantly, the crossover design utilized in the study by Filippi et al. (where patients were randomly assigned to receive either Erenumab or placebo for 12 weeks, then switched to the other treatment for another 12 weeks) raised some concerns that it might contribute to this heterogeneity. Filippi et al. reported they observed that the impact of Erenumab on MMD at week 16 persisted in patients who were randomized to the treatment sequence of Erenumab followed by placebo (who discontinued the drug at week 12). However, there was no evidence that the carry-over effect persisted up to week 24 when data from the second period were included in our analysis. Besides, Jenssen et al. addressed the crossover design in migraine preventive treatment in four RCTs and detected no carry-over effect in migraine patients . However, it is noteworthy that Filippi et al. included only patients who had failed two or more previous migraine preventives, which could be a plausible cause for heterogeneity. This hypothesis was confirmed by the resolution of heterogeneity when conducting a leave-one-out analysis for the study by Filippi et al. . Although there was a significant decline in MSMD with Erenumab, substantial heterogeneity was evident ( [12pt]{minimal}
$${I}^{2}$$ I 2 =84%), which was resolved by excluding Dodick et al. and Tepper et al. Additionally, excluding Sakai et al. resolved the heterogeneity with ≥ 50% response rate outcome. Differences in the eligibility criteria of each study regarding the use of concurrent or prior migraine preventive treatment might account for this heterogeneity. For instance, Tepper et al. prevented migraine preventive drugs during the study and two months before the baseline. Sakai et al. and Dodick et al. allowed using one preventive drug as long as the dose did not differ from two months before. Moreover, Reuter et al. required patients to have failed previous treatment with 2–4 preventive medications. Our subgroup analysis revealed that patients with prior failures to preventive treatments exhibited a higher response to Erenumab compared to those without such a history. This enhanced treatment effect appears to be influenced, in part, by a lower placebo response in this subgroup, particularly concerning migraine frequency-related endpoints such as change in MMD, ≥ 50%, and ≥ 75% response rates [ , , ]. This observation is consistent across multiple studies, where a reduced placebo effect has been linked to lower expectations among patients who have unsuccessfully tried several preventive medications [ , , ]. Moreover, the placebo response in treatment-naive patients tends to be higher, which may dilute the apparent efficacy of active treatments in such populations [ , , , ]. However, the uniform placebo response observed in the MSMD endpoint suggests that the influence of prior treatment failures may vary across different outcome measures . Overall, these findings underscore the importance of considering prior treatment history in the design and interpretation of migraine trials, as including patients with failed prior treatments might reduce placebo effects and provide a better assessment of the treatment's true efficacy. Further research is warranted to explore the mechanisms underlying these differences and to optimize treatment strategies for this challenging patient population. The aforementioned results are consistent with the recommendations of the European Headache Federation guidelines and the position statement of the American Headache Society , which suggest using a CGRP monoclonal antibody in patients who have failed two or more preventive treatments. Besides, subgroup analysis by doses revealed no significant difference between the two doses (70 mg and 140 mg) in primary outcomes, except for MSMD, in which Erenumab 140 mg exhibited significantly higher response than the 70 mg dose. This finding aligns with Gui MM et al., who also reported that the 140 mg dose was more effective than the 70 mg dose specifically for MSMD . Regarding physical function in migraine patients, Yang et al. reported no significant difference between the placebo and Erenumab groups in the change of MPFID-EA and MPFID-PI scores from baseline . In contrast, our analysis showed that Erenumab led to a statistically significant greater reduction in both scores compared to placebo. These findings indicate that Erenumab not only alleviates migraine symptoms but also improves physical function and daily activity performance. In terms of safety, although the most common adverse events were nasopharyngitis and upper respiratory tract infection, there was no statistically significant difference between both groups. Most importantly, Erenumab showed a statistically significant increase in rates of constipation compared to placebo (RR: 2.53, P < 0.0001). These higher rates of constipation could be explained by the crucial role CGRP plays in regulating gastrointestinal motor activity, thus affecting intestinal transit, propulsion, and secretion . Lampl et al. found that both doses of Erenumab (70 mg and 140 mg) demonstrated a safety profile comparable to that of a placebo for patients with episodic or chronic migraine. This finding was consistent across all age groups, including those aged 50 and older, with no increase in adverse events noted in the older age group. In contrast, many standard oral preventive treatments are used cautiously in older adults due to poor safety and tolerability profiles. Therefore, Erenumab emerges as a viable, well-tolerated, and effective alternative for patients of all ages . To the best of our knowledge, this is the largest and most comprehensive meta-analysis evaluating the efficacy and safety of Erenumab for migraine patients. Notably, our meta-analysis is the first to conduct a subgroup analysis based on prior migraine-preventive treatment failure status. We performed two other subgroup analyses according to the type of migraine and doses. Besides, we conducted sensitivity analyses to detect the source of heterogeneity found in primary outcomes. Furthermore, to assess the quality of the evidence, we utilized the GRADE system. Given the small number of studies per outcome, we employed Doi plots and the LFK index to evaluate publication bias. Nevertheless, there are inevitable limitations. First, the small and unequal distribution of sample sizes and studies in the subgroup analysis might cause imbalance and affect the overall analysis, potentially leading to biased or less generalizable results for specific subgroups. Additionally, variability in study protocols posed another challenge, as some studies allowed participants to use a concomitant migraine-preventive treatment during the study, while others prohibited using any. This inconsistency could have impacted the overall assessment of Erenumab’s efficacy, potentially underestimating or overestimating its effects. Due to the unavailability of separate data for patients who used concomitant preventive treatment and those who did not, we could not perform a subgroup analysis. Furthermore, the different definitions of a "migraine day" among studies represented another limitation. Most studies defined a migraine day as any calendar day on which the patient experienced a qualified migraine lasting for ≥ 30 min with either ≥ 2 pain features or ≥ 1 associated non-pain feature. However, four studies [ , , , ] required qualified migraine to have lasted for at least 4 h to be considered a migraine day. Therefore, we recommend that future research focus on carrying out more RCTs comparing patients with migraine who have failed previous preventive treatment with those who have not failed any prior treatment. Such studies could help provide better insights into the enhanced effectiveness of Erenumab in the non-failure group. Furthermore, it would be valuable to conduct studies evaluating the efficacy of Erenumab when administered concomitantly with other migraine-preventive treatments. Additionally, long-duration RCTs investigating the formation of anti-Erenumab antibodies and their potential impact on clinical improvement are necessary.
In conclusion, this meta-analysis demonstrated that Erenumab significantly reduced the frequency of migraine attacks, the number of days on which migraine patients require medication, and physical impairment scores. Erenumab was more effective in patients with prior preventive treatment failures compared to patients with no prior failure. There was no significant difference in Erenumab`s response between episodic and chronic migraine patients. Furthermore, Erenumab administered at a dose of 140 mg showed superior efficacy in MSMD reduction compared to the 70 mg dose, though no significant differences were evident in MMD or ≥ 50% response rates. The safety profile of Erenumab was comparable to that of the placebo, with constipation being the only significant adverse event for Erenumab. Overall, Erenumab is a safe and effective treatment for migraine. However, long-term RCTs comparing Erenumab with other preventive migraine treatments, including further investigations focused on patient groups with a history of prior treatment failures, are required.
Supplementary Material 1: Figure (S1) The risk of bias summary for the crossover study (Filippi et al.). Figure (S2) Comparison of Erenumab vs placebo at 4-6 months in terms of (A) MMD, (B) MSMD, (C) 50% reduction in MMD. Figure (S3) Sensitivity Analysis of primary outcomes at 3 months. (A) MMD by excluding Filippi et al., (B) HIT-6 by excluding Filippi et al., (C) MSMD by excluding Dodick et al. and Tepper et al., (D) 50% reduction in MMD by excluding Sakai et al. Figure (S4) Subgroup analysis based on prior preventive treatment failure status (Prior failure versus No failure). (A) MMD at 3 months, (B) MSMD at 3 months (C) 50% reduction in MMD at 3 months. Figure (S5) Subgroup analysis based on doses (Erenumab 70mg versus Erenumab 140mg). (A) MMD at 3 months, (B) MSMD at 3 months, (C) 50% reduction in MMD at 3 months. Figure (S6) Subgroup analysis based on type of migraine (Episodic versus Chronic). (A) MMD at 3 months, (B) 50% reduction in MMD at 3 months. Figure (S7) Doi plot and LFK index for (A) HIT-6 at 3 months, (B) MSMD at 3 months, (C) 50% reduction in MMD at 3 months. Figure (S8) Funnel plots for (A) MMD 3 months, (B) HIT-6 at 3 months. Figure (S9) Funnel plots for (A) MSMD at 3 months, (B) 50% reduction in MMD at 3 months. Figure (S10) Adverse Events. (A) Any adverse event, (B) Any serious adverse event (C) Any adverse event leading to treatment discontinuation. Figure (S11) Adverse Events (A) Nasopharyngitis (B) Upper respiratory tract infection (C) Constipation. Figure (S12) Adverse Events (A) Nausea, (B) Urinary Tract Infection, (C) Back pain, (D) Influenza. Figure (S13) Adverse Events (A) Injection site pain, (B) Abdominal pain, (C) Vomiting, (D) Diarrhea. Figure (S14) Adverse Events (A) Migraine, (B) Arthralgia, (C) Fatigue, (D) Gastroenteritis.
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Key interventions and outcomes in perioperative care pathways in emergency laparotomy: a systematic review | ba9e6db0-a508-47d6-86f1-27f604fa85d6 | 11892323 | Surgical Procedures, Operative[mh] | Major emergency abdominal surgery is a complex clinical arena serving a heterogenous patient population, with variable physiological status. This high-risk cohort requires time-sensitive, definitive care to potentially mitigate the impact of their physiological and pathological status on post-operative outcomes. The burden of emergency surgery is significant, with reported rates of post-operative morbidity and mortality of 14–47% and 10–20% respectively . There have been considerable efforts made in recent times to try and improve these outcomes through the introduction of structured and standardised care pathways to attenuate the physiological stress of emergency laparotomy and improve post-operative clinical outcomes. Initiatives such as the ELPQuiC (Emergency Laparotomy Quality Improvement Care Bundle) have demonstrated the feasibility of implementing dedicated EmLap pathways into the early peri-operative period in the emergency setting to improve post-operative mortality . Modified Enhanced Recovery after Surgery (ERAS) protocols in the emergency setting have demonstrated improvements in broader clinical outcomes, including reduced length of stay, post-operative complications and improved gastrointestinal functions . These perioperative pathways often comprise several components, which interact to exert their overall effects. As demonstrated by the EPOCH trial, it is the combination of high-fidelity component interventions and overall compliance to the perioperative pathway, that drives overall improvement . Understanding the design and delivery of perioperative pathways in the EmLap setting is essential to evaluate their clinical and cost-effectiveness, and to facilitate broader adoption and implementation. Surgical and perioperative interventions are often poorly reported with a lack of detailed and in-depth intervention reporting . There is growing recognition of the importance of intervention reporting. The Template for Intervention Description and Replication (TIDieR) checklist and guide was developed in 2014 to provide a structure for assessing the completeness of intervention descriptions . The overarching purpose of the TIDieR checklist is to describe interventions in sufficient detail to allow their replication. The use of the TIDieR checklist has led to enhanced and in-depth reporting of complex interventions, which has led to improved implementation across clinical practice and trials . Detailed reporting of the types of interventions delivered across EmLap perioperative pathways, as well as, key aspects of each component, including mode of delivery, frequency, intensity and overall duration, is essential to ensure effective and time sensitive treatment is delivered. Comprehensive reporting of all aspects of perioperative pathways is important in clinical studies to ensure appropriate assessment of clinical effectiveness and onward implementation into clinical practice. Incorrect implementation leads to the initiation of ineffective or lesser treatment. This has implications for the patient, potentially impacting on their clinical outcomes, and on wider healthcare resources. The aim of this systematic review was to identify the current design and make-up of perioperative pathways in the EmLap setting, including identifying component interventions, their associated reported clinical and patient-reported outcomes and to understand their design and reporting in line with the TIDieR checklist.
This systematic review was conducted according to a pre-specified protocol based on guidance from the Centre for Reviews and Dissemination and the Cochrane Handbook and is reported in line with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement . Our protocol was registered with the international, prospective register of systematic reviews, PROSPERO (CRD42021277211). Eligibility criteria All randomised and non-randomised cohort studies reporting outcomes on perioperative care pathways (PCP) in adult patients (≥ 18 years old) undergoing major emergency abdominal surgery were included. Perioperative care pathways were defined as multimodal perioperative care bundles, perioperative protocols, dedicated clinical pathways or ERAS protocols comprising of a number of components. Studies were excluded if they reported on perioperative care protocols/pathways in the trauma or elective setting or did not include clinical outcomes. Search strategy The OVID SP versions of MEDLINE (1950 to 31st December 2023), EMBASE (1980 to 31st December 2023) and the Cochrane Central Register of Controlled Trials were searched using the following search terms ‘emergency surgery’, ‘laparotomy’ ‘enhanced recovery’, ‘fast track’, ‘multimodal’, ‘care bundles’, ‘perioperative protocols’, ‘care pathways’ separated by the Boolean operator ‘AND’. Reference lists of included articles were hand-searched to identify any additional studies. All citations were collated within EndNote X7.8 ® , USA and duplicates were removed. All relevant titles and abstracts were screened by two reviewers (DH and BG). The full text versions of potentially eligible abstracts were retrieved in full. Only studies that fulfilled all eligibility criteria were included. Any conflicts were resolved through discussion. Study quality Methodological quality assessment of included studies was undertaken using the ‘Risk of Bias In Non-Randomised Studies of Intervention’ (ROBINS-I) assessment tool for non-randomised studies and the Cochrane risk of bias tool for randomised controlled trials (RCTs) . Data analysis A narrative description of all perioperative pathways was reported to identify design of the pathway including the delivery and timing of component interventions. To assess the completeness of intervention reporting and its replicability each PCP was assessed against the TIDieR checklist. To assess the consistency of outcome reporting the frequency of each definition and any inconsistencies in definitions across individual studies were reported. Descriptive data were expressed using basic statistics including proportions and averages. All data were entered into Microsoft Excel (Microsoft, Redmond, Washington USA) for analysis.
All randomised and non-randomised cohort studies reporting outcomes on perioperative care pathways (PCP) in adult patients (≥ 18 years old) undergoing major emergency abdominal surgery were included. Perioperative care pathways were defined as multimodal perioperative care bundles, perioperative protocols, dedicated clinical pathways or ERAS protocols comprising of a number of components. Studies were excluded if they reported on perioperative care protocols/pathways in the trauma or elective setting or did not include clinical outcomes.
The OVID SP versions of MEDLINE (1950 to 31st December 2023), EMBASE (1980 to 31st December 2023) and the Cochrane Central Register of Controlled Trials were searched using the following search terms ‘emergency surgery’, ‘laparotomy’ ‘enhanced recovery’, ‘fast track’, ‘multimodal’, ‘care bundles’, ‘perioperative protocols’, ‘care pathways’ separated by the Boolean operator ‘AND’. Reference lists of included articles were hand-searched to identify any additional studies. All citations were collated within EndNote X7.8 ® , USA and duplicates were removed. All relevant titles and abstracts were screened by two reviewers (DH and BG). The full text versions of potentially eligible abstracts were retrieved in full. Only studies that fulfilled all eligibility criteria were included. Any conflicts were resolved through discussion.
Methodological quality assessment of included studies was undertaken using the ‘Risk of Bias In Non-Randomised Studies of Intervention’ (ROBINS-I) assessment tool for non-randomised studies and the Cochrane risk of bias tool for randomised controlled trials (RCTs) .
A narrative description of all perioperative pathways was reported to identify design of the pathway including the delivery and timing of component interventions. To assess the completeness of intervention reporting and its replicability each PCP was assessed against the TIDieR checklist. To assess the consistency of outcome reporting the frequency of each definition and any inconsistencies in definitions across individual studies were reported. Descriptive data were expressed using basic statistics including proportions and averages. All data were entered into Microsoft Excel (Microsoft, Redmond, Washington USA) for analysis.
A total of thirty studies outlining 26 unique pathways in EmLap were included in this review . A total of 10 randomised controlled trials (RCTs), 1 pilot RCT, 4 prospective cohort studies, 1 propensity matched cohort study, 5 retrospective cohort studies, 8 before and after studies and 1 case-control study were included (Table ; Fig. ). Outcomes were reported in 44,055 patients undergoing major emergency abdominal surgery. Care pathways were defined in different ways with 16 studies reporting on emergency ERAS protocols, 7 studies reporting on care bundles, 3 studies reporting on the implementation of a perioperative protocol, 2 studies reporting on protocolised care pathways and 1 study defined its care pathway as intermediate care and 1 study defined it PCP as a quality improvement programme. The earliest reported perioperative pathway was in 2011, with a total of 3 studies predating the introduction of the TIDieR checklist, and 27 studies published following its introduction. Study Bias The majority of RCTs were low overall risk of bias: with 6 RCTs identified to be low risk, 4 RCTs were considered to have some concerns and 1 RCT was considered to be high risk (Fig. a). The majority of 19 non-randomised studies were moderately biased: with 16 identified moderate risk and 3 considered to be seriously biased (Fig. b). Key areas for concern include confounding variables, participant selection, measurement of outcomes and selection of reported results. Peri-operative pathway design Twenty-six unique pathways were identified, with a total of 400 component interventions delivered across all studies. These component interventions were classified into 24 domains (Fig. ) across three distinct time points; pre-, intra- and post-operatively. There was significant overlap with delivery of domain interventions across perioperative timepoints. Six domains; multimodal analgesia, goal-directed fluid therapy, antibiotics, monitoring, thromboembolism prophylaxis and post-operative nausea and vomiting (PONV), were delivered across all three timepoints. Urgent radiology was identified as the only domain intervention delivered exclusively in the pre-operative phase. Risk stratification, timely intervention, prescriptive anaesthetic strategy and prescriptive surgical strategy were domain interventions delivered during the pre- and intra-operative phases of PCPs. There were no exclusive intra-operative domain interventions identified. Five domains were exclusively delivered during post-operative phase; early nutrition, chest physiotherapy, early mobilisation, early removal of drains and discharge/follow up criteria. Three domain interventions were delivered in the pre- and post-operative phases: medical optimisation, review and escalation policies and stress ulceration prophylaxis. Maintaining normothermia was the only domain that was delivered in the intra- and post-operative phases. Twenty-one studies reported on EmLap care pathways with a pre-operative phase, consisting of a median of 6 individual components (Table ). A total of 108 pre-operative component interventions were mapped to 14 broad pre-operative intervention domains. There was significant variation in the coverage of domains delivered in the pre-operative phase, with the sepsis screening/antibiotic prophylaxis domain being the most commonly employed; 14 (66.7%) studies reported component interventions within this domain. Twenty-two studies reported PCPs with an intra-operative phase, consisting of a median of 3 individual components. One hundred and ten intra-operative component interventions were mapped to 12 intra-operative intervention domains (Table ). Commonly covered domains across PCPs intra-operatively were prescriptive surgical strategy ( n = 13, 59.1%), prescriptive anaesthetic strategy ( n = 10, 45.5%), normothermia ( n = 12, 54.5%), goal directed fluid therapy ( n = 10, 45.5%) and analgesia ( n = 14, 63.6%). Twenty-five studies reported PCPs with a post-operative phase, consisting of a median of 8 components (Table ). A total of 191 individual component interventions were identified and mapped to 18 post-operative intervention domains. The most commonly employed domain interventions across PCPs were early nutrition, early mobilisation, early removal of drains and analgesia. PCPs tidier checklist The intervention characteristics of PCPs according to the TIDieR framework are outlined in Table . The majority of studies ( n = 20, 66.6%) did not report the TIDieR framework items, with thirteen studies reporting less than 50% of all items. Three studies reported 90% of the items within the TIDiER framework; reporting on all components of the PCPs intervention, apart from the item on modifications. There was no in-depth detail provided across all PCPs regarding the component intervention delivered, with no data provided on component interventions in specific patient or clinical groups. The PCP designed for use by Burcharth et al. was designed specifically in keeping with the TIDiER framework . The commonest TIDiER item reported across all studies was Item 2: why to describe the rationale, theory, or goal of the elements essential to the intervention. Poorly reported domains included Item 5: Who provided the interventions ( n = 8, 30.8%), Item 7: Where ( n = 7, 26.9%), and Item 9: Tailoring ( n = 5, 19.2%). There was a failure to report Item 10: Modifications across all studies. PCPs reported outcomes Seventeen studies reported on a primary outcome; with 6 studies reporting on post-operative mortality, 3 studies on length of stay (LoS), 3 studies reported on outcomes related to complications, 2 studies reported of composite post-operative outcomes, 1 study reported on compliance, 1 study reported on cost and 1 study reported on gastrointestinal function. A total of 250 individual outcomes were extracted from 30 studies and mapped to 13 overarching categories: mortality, length of stay (LoS), readmission, reoperation, complications, gastrointestinal function, invasive tube removal, analgesic requirements, mobility, cost-effectiveness, compliance rates, post-operative treatment, recovery and function and quality of life (QoL) (Table ). Clinical outcomes such as morbidity, mortality and LoS were most commonly reported. Outcomes relating to analgesic requirement, compliance, mobility, recovery, function and QoL were poorly reported across all studies. Post-operative mortality was the most frequently reported outcome measure across all studies, with 24 (80%) studies reporting this outcome. However, there was significant heterogeneity in the definitions and timing in reporting this outcome measure, with 8 different definitions identified. The most commonly used definition was overall 30-day mortality, with other definitions including in-hospital and risk-adjusted mortality, as well as reporting mortality outcomes at 90 days, 180 days and 1 year post-operatively. Post-operative morbidity was reported by 23 (76.7%) studies in 27 different ways at variable timepoints ranging from 3 to 180 days post-operatively. Seven studies reported specific complications including pulmonary complications, acute kidney injury, ileus, surgical site infection, post-operative bleeding, trocar site hernia, urinary tract infections, septic shock, anastomotic leak, peritonitis and abscess. Two grading systems were identified to grade the severity of complications; the Clavien-Dindo classification and the Post-operative Morbidity Score, across 7 studies. Outcomes for the gastrointestinal function domain were reported across 12 (40%) studies using 8 different definitions. No standardised definition of gastrointestinal function was identified. Patient-reported outcomes such as recovery and function and QoL were poorly reported, with 6 identified outcome measures across 2 (6.6%) studies.
The majority of RCTs were low overall risk of bias: with 6 RCTs identified to be low risk, 4 RCTs were considered to have some concerns and 1 RCT was considered to be high risk (Fig. a). The majority of 19 non-randomised studies were moderately biased: with 16 identified moderate risk and 3 considered to be seriously biased (Fig. b). Key areas for concern include confounding variables, participant selection, measurement of outcomes and selection of reported results.
Twenty-six unique pathways were identified, with a total of 400 component interventions delivered across all studies. These component interventions were classified into 24 domains (Fig. ) across three distinct time points; pre-, intra- and post-operatively. There was significant overlap with delivery of domain interventions across perioperative timepoints. Six domains; multimodal analgesia, goal-directed fluid therapy, antibiotics, monitoring, thromboembolism prophylaxis and post-operative nausea and vomiting (PONV), were delivered across all three timepoints. Urgent radiology was identified as the only domain intervention delivered exclusively in the pre-operative phase. Risk stratification, timely intervention, prescriptive anaesthetic strategy and prescriptive surgical strategy were domain interventions delivered during the pre- and intra-operative phases of PCPs. There were no exclusive intra-operative domain interventions identified. Five domains were exclusively delivered during post-operative phase; early nutrition, chest physiotherapy, early mobilisation, early removal of drains and discharge/follow up criteria. Three domain interventions were delivered in the pre- and post-operative phases: medical optimisation, review and escalation policies and stress ulceration prophylaxis. Maintaining normothermia was the only domain that was delivered in the intra- and post-operative phases. Twenty-one studies reported on EmLap care pathways with a pre-operative phase, consisting of a median of 6 individual components (Table ). A total of 108 pre-operative component interventions were mapped to 14 broad pre-operative intervention domains. There was significant variation in the coverage of domains delivered in the pre-operative phase, with the sepsis screening/antibiotic prophylaxis domain being the most commonly employed; 14 (66.7%) studies reported component interventions within this domain. Twenty-two studies reported PCPs with an intra-operative phase, consisting of a median of 3 individual components. One hundred and ten intra-operative component interventions were mapped to 12 intra-operative intervention domains (Table ). Commonly covered domains across PCPs intra-operatively were prescriptive surgical strategy ( n = 13, 59.1%), prescriptive anaesthetic strategy ( n = 10, 45.5%), normothermia ( n = 12, 54.5%), goal directed fluid therapy ( n = 10, 45.5%) and analgesia ( n = 14, 63.6%). Twenty-five studies reported PCPs with a post-operative phase, consisting of a median of 8 components (Table ). A total of 191 individual component interventions were identified and mapped to 18 post-operative intervention domains. The most commonly employed domain interventions across PCPs were early nutrition, early mobilisation, early removal of drains and analgesia.
The intervention characteristics of PCPs according to the TIDieR framework are outlined in Table . The majority of studies ( n = 20, 66.6%) did not report the TIDieR framework items, with thirteen studies reporting less than 50% of all items. Three studies reported 90% of the items within the TIDiER framework; reporting on all components of the PCPs intervention, apart from the item on modifications. There was no in-depth detail provided across all PCPs regarding the component intervention delivered, with no data provided on component interventions in specific patient or clinical groups. The PCP designed for use by Burcharth et al. was designed specifically in keeping with the TIDiER framework . The commonest TIDiER item reported across all studies was Item 2: why to describe the rationale, theory, or goal of the elements essential to the intervention. Poorly reported domains included Item 5: Who provided the interventions ( n = 8, 30.8%), Item 7: Where ( n = 7, 26.9%), and Item 9: Tailoring ( n = 5, 19.2%). There was a failure to report Item 10: Modifications across all studies.
Seventeen studies reported on a primary outcome; with 6 studies reporting on post-operative mortality, 3 studies on length of stay (LoS), 3 studies reported on outcomes related to complications, 2 studies reported of composite post-operative outcomes, 1 study reported on compliance, 1 study reported on cost and 1 study reported on gastrointestinal function. A total of 250 individual outcomes were extracted from 30 studies and mapped to 13 overarching categories: mortality, length of stay (LoS), readmission, reoperation, complications, gastrointestinal function, invasive tube removal, analgesic requirements, mobility, cost-effectiveness, compliance rates, post-operative treatment, recovery and function and quality of life (QoL) (Table ). Clinical outcomes such as morbidity, mortality and LoS were most commonly reported. Outcomes relating to analgesic requirement, compliance, mobility, recovery, function and QoL were poorly reported across all studies. Post-operative mortality was the most frequently reported outcome measure across all studies, with 24 (80%) studies reporting this outcome. However, there was significant heterogeneity in the definitions and timing in reporting this outcome measure, with 8 different definitions identified. The most commonly used definition was overall 30-day mortality, with other definitions including in-hospital and risk-adjusted mortality, as well as reporting mortality outcomes at 90 days, 180 days and 1 year post-operatively. Post-operative morbidity was reported by 23 (76.7%) studies in 27 different ways at variable timepoints ranging from 3 to 180 days post-operatively. Seven studies reported specific complications including pulmonary complications, acute kidney injury, ileus, surgical site infection, post-operative bleeding, trocar site hernia, urinary tract infections, septic shock, anastomotic leak, peritonitis and abscess. Two grading systems were identified to grade the severity of complications; the Clavien-Dindo classification and the Post-operative Morbidity Score, across 7 studies. Outcomes for the gastrointestinal function domain were reported across 12 (40%) studies using 8 different definitions. No standardised definition of gastrointestinal function was identified. Patient-reported outcomes such as recovery and function and QoL were poorly reported, with 6 identified outcome measures across 2 (6.6%) studies.
We highlight the heterogenous nature of current perioperative pathway design in the EmLap setting, with multiple component interventions delivered in a variable manner. Our review identified 400 individual components mapping to 24 domains, with variable quality intervention and outcome reporting as measured by the TIDiER checklist. The overall lack of intervention description and reporting for EmLap perioperative pathways limits understanding their effectiveness, implementation and generalisability. EmLap perioperative pathways consist of several interacting components, with little understanding of the underlying interaction due to the variable quality evidence base underpinning each component intervention . This leads to significant heterogeneity in the type of interventions employed, with this systematic review identifying 26 unique perioperative pathways. Although the interventions delivered within these pathways mapped to 24 broad overarching domains, the overall delivery and reporting of individual interventions within these domains was heterogenous and inconsistent across different pathways. The TIDieR framework provides a standardised and robust manner to report complex interventions to enable broader adoption and implementation. However, adherence to this framework is variable in EmLap perioperative pathways. There is a significant focus on key aspects of the TIDieR framework including reporting on rationale for implementation and evaluation with reporting of key procedures outlined in 92.3% ( n = 24) and materials required to deliver these procedures in 57.7% ( n = 15). Despite the majority of studies reporting on key procedures and materials, these descriptions were often minimal or lacked sufficient detail, and therefore are unlikely to facilitate broader adoption or implementation. Several key details regarding intervention description and reporting are underreported, including, who delivered the intervention ( n = 8, 30.8%), where the intervention was delivered ( n = 7, 26.9%), tailoring of interventions ( n = 5, 19.2%), modifications ( n = 0, 0%), and planned and actual adherence ( n = 5, 19.2%). These key reporting criteria are often underreported across a range of complex interventions in multiple disease settings, with the focus largely being on the actual intervention delivered. Key detail on the broader reporting standards of intervention delivery are essential for implementation of complex interventions such as a EmLap perioperative pathway, which is often delivered by several key members of the multidisciplinary team, at different timepoints and stages of the pathway, to a broad and heterogenous clinical population. Three studies were identified to have excellent compliance with the TIDieR framework reporting. Vester-Andersen demonstrated variable compliance of 14.3–85.8% to key components of their complex intervention to improve post-operative EmLap care using intermediate care. However, when compared to standard care, the overall compliance to interventions was much higher due to the key reporting and educational components of the TIDieR framework . Using a similar approach, Burcharth et al. were able to demonstrate overall compliance of 83% to 15 component interventions . Boden et al. assessed the feasibility of implementing a complex intervention of intensive physiotherapy aimed at reducing postoperative pneumonia and improving physical recovery . Through the use of the TIDieR framework the authors identified key interventions with poor compliance and implementation in clinical practice and the associated barriers/challenges. These three studies demonstrate the value of the TIDieR framework, using indepth intervention description and reporting in ensuring the delivery of effective and feasible interventions within the EmLap setting. Through robust and standardised reporting of interventions, complex interventions can be appropriately implemented into clinical practice. Transparent reporting is essential for pathway effectiveness research due to the complex nature of developing and implementing clinical pathways, which is further amplified in the emergency setting.This limits healthcare resource wastage through the early identification of undeliverable interventions and ensuring the delivery of concise, high-fidelity, clinically effective interventions within complex clinical settings. This work contributes to the growing evidence-base in perioperative pathways in EmLap by identifying the content of these pathways and by identifying their associated reporting outcomes. However, our work is limited by the overall quality of the existing evidence-base, consisting primarily of moderately biased, non-randomised studies. We only identified ten RCTs for inclusion into this review. The disproportionate number of non-randomised studies is associated with inherent biases including selection bias and outcome reporting. This has a potential impact on determining the clinical effectiveness of the interventions and perioperative pathways reported within these studies. It is also important to note the limitations of the TIDIER checklist, as it has been designed for the generic use of intervention reporting across medicine and surgery leading to broad descriptors and the lack of thresholds to define adequate reporting.
Perioperative pathways in the EmLap setting are complex interventions, with variable design and structure, spanning across the entire perioperative pathway. This review identified 26 unique pathways delivering 400 individual component interventions across 24 domains, with a variety of outcome metrics used to assess their clinical effectiveness. These pathways are multimodal, consisting of multiple component interventions. Currently, they are reported and therefore implemented in a variable manner. Future studies in EmLap perioperative pathways should ensure in depth reporting of the design and delivery of the pathway, including an in-depth description of component interventions, using existing frameworks such at the TIDIER framework. This will help identify component interventions that are valuable, effective and feasible for implementation in the EmLap setting.
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Consensus-Based Recommendations on Priority Activities to Address Acute Kidney Injury in Children | 3593039a-0ed2-444f-9fd1-d54633aa5659 | 9756303 | Internal Medicine[mh] | The pediatric acute kidney injury (AKI) research field has expanded during the past decade with international epidemiologic studies, long-term AKI follow-up studies, validation of AKI prediction models, and continued investigation of novel AKI biomarkers. – The 26th Acute Disease Quality Initiative (ADQI XXVI) convened in Napa, California, in November 2021, the first Pediatric ADQI (pADQI) that specifically addressed the global burden of AKI in the child and young adult population, with focused attention paid to kidney development and AKI, prenatal or neonatal kidney physiology, dialysis devices engineered for children, and the unique aspects of pediatric AKI epidemiology. The pADQI was assembled to mirror the global workforce in pediatrics and used a public and patient-facing approach. The primary aim of the pADQI was to identify the work priorities across the pediatric AKI landscape of care, education, research, and advocacy.
The pADQI consensus meeting followed the established ADQI process, as previously described, to provide expert-based statements and interpretation of current knowledge for use by clinicians according to professional judgment and identify evidence care gaps to establish research priorities. We performed an objective scientific review and distillation of literature through September 2021 of (1) epidemiology, (2) risk assessment and diagnosis, (3) fluid assessment, (4) kidney support and extracorporeal therapies, (5) pathobiology, nutrition, and pharmacology, and (6) education and advocacy. The 6 workgroups were composed of representatives from 5 continents of varying age (median: 43 years; range, 24–74 years), gender (23 male, 23 female) and professional discipline (physicians, nurses, pharmacists, and nutritionists). In addition, a pediatric patient survivor of AKI and an expert in equity and care delivery were included in the pADQI and assisted all workgroups. The in-person meeting was conducted using workgroups and large group in mixed in-person and virtual (72% in person) formats. Work following the in-person meeting was completed virtually. Using a modified Delphi method, each workgroup determined key questions supported by representative literature, or professional opinion when evidence was scarce, and presented their drafts iteratively to develop consensus statements (target of 2–3 per group) (main document), and to articulate a research agenda with a more comprehensive background ( ; and in the ). The consensus statements were categorized as recommendations or suggestions. The work product was finalized and approved by the entire group in 2 follow-up web-based meetings. This summary statement was based on existing evidence in the literature, thus institutional approval was not required. All members of pADQI consented to their inclusion in this article. The statements are presented herein as direct quotations. The age spectrum of patients receiving the care of pediatric specialists ranged from extremely low birthweight neonates to young adults. Throughout this manuscript, the terms child , children , and pediatric reflect the entire age spectrum, unless specifically noted.
Consensus Statement 1A (Recommendation) AKI epidemiology related to short-term outcomes is well-described in critically ill neonates and children from high-income countries; however, further study is needed in other health care contexts, such as low-middle income countries and in non-ICU and ambulatory settings and for socioeconomic and long-term outcomes. Consensus Statement 1B (Suggestion) An understanding of pediatric AKI epidemiology allows us to begin employing strategies to improve primary, secondary, and tertiary AKI prevention in at-risk patient groups. Implementation of standardized criteria to define the presence and severity of AKI has facilitated a description of pediatric AKI epidemiology. – Prospective multicenter studies provide insight into the epidemiology of AKI in critically ill neonates and children. , Other studies describe AKI epidemiology in specific cohorts of children including sepsis, those receiving nephrotoxins, and cardiac surgical populations. , However, residual knowledge gaps remain regarding the accurate description of AKI epidemiology in other health care contexts as the most data come from high-income countries, and few studies have described AKI epidemiology outside critical care settings. Pediatric literature has described AKI and morbidity, mortality, and health resource use in critically ill children with a gradient-response association between AKI severity and the risk of worse outcomes. , , More recently, research has focused on assessing the medium and long-term sequalae of AKI, including longitudinal kidney function and its socioeconomic outcomes. , , A comprehensive description of pediatric AKI epidemiology reflects current practice and best available information ( in the ). Given this, information should be up to date and readily available to clinicians and institutions in real time. A risk prediction study and a randomized clinical trial suggest a causal link between pediatric AKI and a limited scope of short-term outcomes, supporting prevention and mitigation of pediatric AKI and its complications as targets for improving outcomes. Pediatric AKI epidemiologic characteristics can be categorized as pre-event (eg, risk factors, key drivers, and predictors), peri-event (eg, demographic characteristics and etiology), and postevent (eg, outcomes and recovery). Studies of AKI epidemiology should explore the temporal nature of AKI, health contexts (eg, populations and locations) in which AKI has been inadequately described, and outcomes that are focused on patient, family, cost, and performance. Thus, by following epidemiologic trends, clinicians can help focus efforts to enhance public health surveillance, augment field investigations, clinical and preclinical studies, and further policy development to promote child health ( and in the ).
AKI epidemiology related to short-term outcomes is well-described in critically ill neonates and children from high-income countries; however, further study is needed in other health care contexts, such as low-middle income countries and in non-ICU and ambulatory settings and for socioeconomic and long-term outcomes.
An understanding of pediatric AKI epidemiology allows us to begin employing strategies to improve primary, secondary, and tertiary AKI prevention in at-risk patient groups. Implementation of standardized criteria to define the presence and severity of AKI has facilitated a description of pediatric AKI epidemiology. – Prospective multicenter studies provide insight into the epidemiology of AKI in critically ill neonates and children. , Other studies describe AKI epidemiology in specific cohorts of children including sepsis, those receiving nephrotoxins, and cardiac surgical populations. , However, residual knowledge gaps remain regarding the accurate description of AKI epidemiology in other health care contexts as the most data come from high-income countries, and few studies have described AKI epidemiology outside critical care settings. Pediatric literature has described AKI and morbidity, mortality, and health resource use in critically ill children with a gradient-response association between AKI severity and the risk of worse outcomes. , , More recently, research has focused on assessing the medium and long-term sequalae of AKI, including longitudinal kidney function and its socioeconomic outcomes. , , A comprehensive description of pediatric AKI epidemiology reflects current practice and best available information ( in the ). Given this, information should be up to date and readily available to clinicians and institutions in real time. A risk prediction study and a randomized clinical trial suggest a causal link between pediatric AKI and a limited scope of short-term outcomes, supporting prevention and mitigation of pediatric AKI and its complications as targets for improving outcomes. Pediatric AKI epidemiologic characteristics can be categorized as pre-event (eg, risk factors, key drivers, and predictors), peri-event (eg, demographic characteristics and etiology), and postevent (eg, outcomes and recovery). Studies of AKI epidemiology should explore the temporal nature of AKI, health contexts (eg, populations and locations) in which AKI has been inadequately described, and outcomes that are focused on patient, family, cost, and performance. Thus, by following epidemiologic trends, clinicians can help focus efforts to enhance public health surveillance, augment field investigations, clinical and preclinical studies, and further policy development to promote child health ( and in the ).
Consensus Statement 2A (Recommendation) Validated tools which incorporate patient characteristics and exposure and also interface with the local health care environment should be utilized to estimate AKI risk in children, including assessment of objective measures of kidney fitness in at-risk children prior to a predictable or planned intervention. Consensus Statement 2B (Suggestion) Unique AKI phenotypes in children may overlap and change over time. Differentiating AKI phenotype(s) informs prognosis and has the potential to guide therapeutics. Previous studies , – identified baseline patient characteristics and exposures that place children at increased risk for AKI, including but not limited to cancer therapy, cardiopulmonary bypass, extracorporeal membrane oxygenation, major surgical procedures, and a high nephrotoxic medication burden. , , Integrating diagnostic resources may optimize AKI risk assessment for high-risk diagnoses (eg, by using point-of-care testing and automated algorithms) or exposures (eg, by using the electronic medical record) could be updated continuously in real time ( ). Prior to a planned exposure associated with increased AKI risk, ascertainment of kidney fitness using traditional measures of kidney function and damage, including serum creatinine and cystatin C levels, and urine albumin or protein level can be used to direct comprehensive kidney protection and surveillance and prevent AKI development. Preoperative values of candidate biomarkers may have utility in predicting AKI after cardiac surgery in adults and children. – Kidney functional reserve values can predict postoperative AKI in adults. ( ). When available, biomarkers that indicate structural kidney injury may provide further insight into AKI risk and phenotype. For example, structural biomarker concentration elevation in the absence of functional biomarker elevation may indicate early kidney injury that is not readily reversible. Conversely, functional biomarker elevation alone may be indicative of decreased effective circulating volume and direct care toward restoration of volume. The terms functional AKI and structural AKI , to supplant the terms pre-renal AKI and intrinsic AKI were first proposed by the ADQI X Consensus Conference. Single center pediatric study and systematic reviews provide initial validation of these concepts, with structural AKI being associated with worse outcomes than functional AKI alone. – Pediatric AKI is a heterogeneous disease with multiple etiologic factors, biochemical signatures, and clinical manifestations. Expert consensus and emerging data support the view that these elements combined with individual susceptibility contribute to unique, discernible AKI phenotypes in children with AKI ( ). In particular, evidence supports the use of clinical features such as urine output, , severity and duration of creatinine elevation, , fluid overload, response to loop diuretics, , and tubular injury biomarkers, , to refine AKI diagnosis and reveal unique phenotypes. The concept of subclinical AKI is defined by an elevation in a urine tubular injury biomarker without elevation in functional marker. Subclinical AKI is associated with worse outcomes in children without AKI or with functional AKI. , Although these studies examined static 1 or 2 factor-based AKI phenotypes, it is likely that more than 1 of these discernible phenotypes may be present in a child with AKI at a given time and that this unique phenotypic signature is dynamic ( ). A dynamic diagnostic model to address a dynamic phenotype, akin to blood gas assessment in acute lung injury, may be required for AKI ( and in the ).
Validated tools which incorporate patient characteristics and exposure and also interface with the local health care environment should be utilized to estimate AKI risk in children, including assessment of objective measures of kidney fitness in at-risk children prior to a predictable or planned intervention.
Unique AKI phenotypes in children may overlap and change over time. Differentiating AKI phenotype(s) informs prognosis and has the potential to guide therapeutics. Previous studies , – identified baseline patient characteristics and exposures that place children at increased risk for AKI, including but not limited to cancer therapy, cardiopulmonary bypass, extracorporeal membrane oxygenation, major surgical procedures, and a high nephrotoxic medication burden. , , Integrating diagnostic resources may optimize AKI risk assessment for high-risk diagnoses (eg, by using point-of-care testing and automated algorithms) or exposures (eg, by using the electronic medical record) could be updated continuously in real time ( ). Prior to a planned exposure associated with increased AKI risk, ascertainment of kidney fitness using traditional measures of kidney function and damage, including serum creatinine and cystatin C levels, and urine albumin or protein level can be used to direct comprehensive kidney protection and surveillance and prevent AKI development. Preoperative values of candidate biomarkers may have utility in predicting AKI after cardiac surgery in adults and children. – Kidney functional reserve values can predict postoperative AKI in adults. ( ). When available, biomarkers that indicate structural kidney injury may provide further insight into AKI risk and phenotype. For example, structural biomarker concentration elevation in the absence of functional biomarker elevation may indicate early kidney injury that is not readily reversible. Conversely, functional biomarker elevation alone may be indicative of decreased effective circulating volume and direct care toward restoration of volume. The terms functional AKI and structural AKI , to supplant the terms pre-renal AKI and intrinsic AKI were first proposed by the ADQI X Consensus Conference. Single center pediatric study and systematic reviews provide initial validation of these concepts, with structural AKI being associated with worse outcomes than functional AKI alone. – Pediatric AKI is a heterogeneous disease with multiple etiologic factors, biochemical signatures, and clinical manifestations. Expert consensus and emerging data support the view that these elements combined with individual susceptibility contribute to unique, discernible AKI phenotypes in children with AKI ( ). In particular, evidence supports the use of clinical features such as urine output, , severity and duration of creatinine elevation, , fluid overload, response to loop diuretics, , and tubular injury biomarkers, , to refine AKI diagnosis and reveal unique phenotypes. The concept of subclinical AKI is defined by an elevation in a urine tubular injury biomarker without elevation in functional marker. Subclinical AKI is associated with worse outcomes in children without AKI or with functional AKI. , Although these studies examined static 1 or 2 factor-based AKI phenotypes, it is likely that more than 1 of these discernible phenotypes may be present in a child with AKI at a given time and that this unique phenotypic signature is dynamic ( ). A dynamic diagnostic model to address a dynamic phenotype, akin to blood gas assessment in acute lung injury, may be required for AKI ( and in the ).
Consensus Statement 3A (Recommendation) Fluid balance is the difference between total input and output that can be expressed as “daily” and/or “cumulative” over a defined duration of time. Consensus Statement 3B (Suggestion) Fluid overload denotes a pathologic state of positive fluid balance associated with a clinically observable event(s), which may vary by age, case-mix, acuity, and phase of illness. No specific threshold of positive fluid balance alone can define fluid overload across all sick children. As with AKI diagnosis, consistent use of consensus terminology is essential to further our understanding of fluid management and outcomes. Many terms have been used imprecisely to assess the impact of fluid volume on outcomes in sick children, including fluid balance, fluid accumulation, and fluid overload. Fluid overload has been conflated to describe both the degree of positive fluid balance within a patient and its association with adverse outcomes. – Use of the term in this manner introduces bias into research and clinical practice, as it presumes any degree of positive fluid balance is synonymous with fluid overload and is therefore detrimental. Moreover, such use presumes that net fluid removal and targeting negative fluid balance is beneficial. Fluid overload is a distinct pathologic state of positive fluid balance with adverse consequences. Fluid balance is an objective calculation using specific methods. We suggest that the term percent cumulative fluid balance be used to describe the previously referred term, percent fluid overload , to separate the pathologic state from specific calculations. The terms daily fluid balance, cumulative fluid balance, and percent cumulative fluid balance describe fluid status of patients for purpose of clinical care and research ( in the ) more precisely. The 2 methods most used to describe fluid balance are calculations from (1) cumulative fluid inputs and outputs and (2) changes in measured body weight. – , – The weight change method has certain advantages because it accounts for unmeasured insensible losses, does not require an invasive bladder catheter, and is less resource intensive. A weight-change method is preferred in neonates to estimate fluid balance. , – Sick children are a heterogenous cohort of patients, and fluid overload may vary according to patient-specific susceptibilities (eg, by patient age, case-mix, phase, or severity of illness). Thus, no specific threshold can be recommended uniformly to define fluid overload in sick children. Interventions to mitigate or treat fluid overload will need to be patient-specific and consider the pathophysiologic context, the dynamics of the patient’s fluid balance, and the degree to which fluid balance affects the severity of the patient’s fluid overload or volume depletion. in the provides a visual model of this precision approach ( and in the ).
Fluid balance is the difference between total input and output that can be expressed as “daily” and/or “cumulative” over a defined duration of time.
Fluid overload denotes a pathologic state of positive fluid balance associated with a clinically observable event(s), which may vary by age, case-mix, acuity, and phase of illness. No specific threshold of positive fluid balance alone can define fluid overload across all sick children. As with AKI diagnosis, consistent use of consensus terminology is essential to further our understanding of fluid management and outcomes. Many terms have been used imprecisely to assess the impact of fluid volume on outcomes in sick children, including fluid balance, fluid accumulation, and fluid overload. Fluid overload has been conflated to describe both the degree of positive fluid balance within a patient and its association with adverse outcomes. – Use of the term in this manner introduces bias into research and clinical practice, as it presumes any degree of positive fluid balance is synonymous with fluid overload and is therefore detrimental. Moreover, such use presumes that net fluid removal and targeting negative fluid balance is beneficial. Fluid overload is a distinct pathologic state of positive fluid balance with adverse consequences. Fluid balance is an objective calculation using specific methods. We suggest that the term percent cumulative fluid balance be used to describe the previously referred term, percent fluid overload , to separate the pathologic state from specific calculations. The terms daily fluid balance, cumulative fluid balance, and percent cumulative fluid balance describe fluid status of patients for purpose of clinical care and research ( in the ) more precisely. The 2 methods most used to describe fluid balance are calculations from (1) cumulative fluid inputs and outputs and (2) changes in measured body weight. – , – The weight change method has certain advantages because it accounts for unmeasured insensible losses, does not require an invasive bladder catheter, and is less resource intensive. A weight-change method is preferred in neonates to estimate fluid balance. , – Sick children are a heterogenous cohort of patients, and fluid overload may vary according to patient-specific susceptibilities (eg, by patient age, case-mix, phase, or severity of illness). Thus, no specific threshold can be recommended uniformly to define fluid overload in sick children. Interventions to mitigate or treat fluid overload will need to be patient-specific and consider the pathophysiologic context, the dynamics of the patient’s fluid balance, and the degree to which fluid balance affects the severity of the patient’s fluid overload or volume depletion. in the provides a visual model of this precision approach ( and in the ).
Consensus Statement 4A (Recommendation) A dedicated multidisciplinary team made up of kidney health care workers, patients, and families along with institutional investments of personnel, time, materials, and quality assurance/improvement systems are essential to a pediatric acute kidney support therapy (paKST) program. Consensus Statement 4B (Suggestion) Patient-centered goals of care, degree of kidney recovery, physiological stability, fluid balance, and global recovery and rehabilitation priorities inform decisions for de-escalation, liberation, transition, and follow-up to optimize hospital-based and lifelong outcomes. A paKST program requires thoughtful planning and synchronization across the health care enterprise ( in the ). , Program development begins by identification of key stakeholders with a vested interest in paKST delivery and outcomes. A multidisciplinary team of kidney health care workers is responsible for fostering a culture of safety and transparency, navigating organizational barriers, facilitating shared decision-making, providing education, maintaining equipment, developing and maintaining policies, procedures, and guidelines, and managing quality improvement data. , – Patients and families are vital partners, identifying goals and advocating for effective and safe care. paKST device manufacturers and suppliers play an important role in working with pediatric paKST providers to design products that can be used safely and effectively in children, and to respond to the unique needs of a pediatric program. The care delivery model should be based on available resources with representation from multiple disciplines. , , , Multiple blood purification and extracorporeal treatment modalities have been developed ( in the ). Programs must define their standards of high-quality delivery for the procedures they provide. Most quality metrics proposed by the ADQI XXII conference are relevant for children. If a program is unable to provide high-quality care in a particular area, the team must develop criteria and processes for transfer to an institution that provides such care, and should examine the components lacking in their program to see if they can be addressed. The education curriculum encompasses patient and team learning, is tailored to fit specific roles, and includes competency assessments. , , , , Quality assurance or improvement systems should be integrated into clinical practice, recognizing that cost, varied definitions, and lack of agreed-on benchmarks are known barriers to implementation. , – , – , – Previous work by Rewa et al , , and the 22nd ADQI conference , has provided minimum programmatic standards that may be used for paKST. While most children who require paKST for AKI will achieve liberation from dialysis, there is scant data to guide clinicians during kidney recovery. , – Current practices use estimates of the ability to maintain euvolemia and metabolic balance for decisions about timing of paKST liberation, transition, or de-escalation. Inability to execute timely transition from continuous paKST may negatively affect outcomes, especially in light of emerging data on effectiveness of the ICU Liberation Bundle. – After paKST liberation, children are at risk for lifelong morbidity, including chronic kidney disease, but guidance on optimal follow-up practices is lacking ( and in the ). , –
A dedicated multidisciplinary team made up of kidney health care workers, patients, and families along with institutional investments of personnel, time, materials, and quality assurance/improvement systems are essential to a pediatric acute kidney support therapy (paKST) program.
Patient-centered goals of care, degree of kidney recovery, physiological stability, fluid balance, and global recovery and rehabilitation priorities inform decisions for de-escalation, liberation, transition, and follow-up to optimize hospital-based and lifelong outcomes. A paKST program requires thoughtful planning and synchronization across the health care enterprise ( in the ). , Program development begins by identification of key stakeholders with a vested interest in paKST delivery and outcomes. A multidisciplinary team of kidney health care workers is responsible for fostering a culture of safety and transparency, navigating organizational barriers, facilitating shared decision-making, providing education, maintaining equipment, developing and maintaining policies, procedures, and guidelines, and managing quality improvement data. , – Patients and families are vital partners, identifying goals and advocating for effective and safe care. paKST device manufacturers and suppliers play an important role in working with pediatric paKST providers to design products that can be used safely and effectively in children, and to respond to the unique needs of a pediatric program. The care delivery model should be based on available resources with representation from multiple disciplines. , , , Multiple blood purification and extracorporeal treatment modalities have been developed ( in the ). Programs must define their standards of high-quality delivery for the procedures they provide. Most quality metrics proposed by the ADQI XXII conference are relevant for children. If a program is unable to provide high-quality care in a particular area, the team must develop criteria and processes for transfer to an institution that provides such care, and should examine the components lacking in their program to see if they can be addressed. The education curriculum encompasses patient and team learning, is tailored to fit specific roles, and includes competency assessments. , , , , Quality assurance or improvement systems should be integrated into clinical practice, recognizing that cost, varied definitions, and lack of agreed-on benchmarks are known barriers to implementation. , – , – , – Previous work by Rewa et al , , and the 22nd ADQI conference , has provided minimum programmatic standards that may be used for paKST. While most children who require paKST for AKI will achieve liberation from dialysis, there is scant data to guide clinicians during kidney recovery. , – Current practices use estimates of the ability to maintain euvolemia and metabolic balance for decisions about timing of paKST liberation, transition, or de-escalation. Inability to execute timely transition from continuous paKST may negatively affect outcomes, especially in light of emerging data on effectiveness of the ICU Liberation Bundle. – After paKST liberation, children are at risk for lifelong morbidity, including chronic kidney disease, but guidance on optimal follow-up practices is lacking ( and in the ). , –
Consensus Statement 5A (Recommendation) Successful pediatric translational AKI research programs include diverse teams using reverse translational approaches in partnership with clinical and epidemiological findings that prioritize development as a biologic variable. Sufficient support including pediatric specific government and industry funding along with meaningful partnerships among health professionals is necessary to understand and leverage the unique aspects of pediatric AKI to address kidney health and disease across the life course. Consensus Statement 5B (Recommendation) Patient centered outcomes such as functional status, quality of life, and optimal growth and development must drive the targeted nutritional interventions, optimizing short- and long-term nutrition, and incorporate measures of acute and chronic changes of anthropometrics, body composition, physical function, and metabolic control. Engagement of diverse stakeholders across the preclinical and translational research realms is needed to disseminate findings and transform care. Most translational AKI models do not account for the additional complexity of pediatric AKI including the concept of development as a biologic variable (DABV). The association between premature birth and kidney function and maturation remain incompletely understood. Furthermore, the associations between a single AKI episode during kidney development and short-term and long-term outcomes remain unclear. Potential sex differences in AKI have prevented the inclusion of both males and females together in preclinical animal studies. In pediatrics, sex as a biologic variable is further confounded by DABV; hormone levels change throughout pubertal stages, and few clinical studies capture this information. Animal models of AKI that consider sex hormones and sex chromosomes could inform clinical evaluations sensitive to sex as a biologic variable and DABV. The association between AKI and chronic kidney disease has been established, however, there is a paucity of data on the systemic long-term impact of AKI. , The potential associations between AKI with respect to DABV and long-term outcomes are unknown. Opportunities remain to use the unique aspects of pediatric patients, including but not limited to the lack of chronic comorbidities and ability to understand the impact of nephron endowment prior to nephron loss ( ). Malnutrition and protein energy wasting are prevalent and independently associated with mortality in children hospitalized with AKI, especially those requiring paKST, with prevalence ranging from 30% to 55%. – Mortality is a crude end point to assess the importance of nutrition support in children with AKI. AKI survivors are at risk of worsened functional status, chronic ventilation, or dialysis after hospital discharge. Nutrition support and mobilization offer opportunities to improve functional outcomes in survivors through attenuation of muscle loss and promotion of muscle protein synthesis in critically ill children. The outcomes of malnutrition associated with somatic growth, body composition, development, immune function, metabolic derangements, and long-term physical and neurocognitive functioning require more precise measurement tools and targets. Nutritional therapeutic interventions are nuanced and include changes in feeding modality, composition (macronutrient or micronutrient) and volume of nutrition-related fluids which intersects with medical AKI management and metabolic control, which can be further complicated by institution of paKST. Nutritional needs are further affected by DABV. Identifying and measuring appropriate outcome variables represent the first step to define the phenotypic nutritional morbidities in pediatric AKI, and thereby enable evaluation of nutritional intervention efficacy. , We recommend program-based focused nutrition support for children with AKI and transitioning through acute kidney disease. Such a program would use evidence-based nutrition therapy adjusted in response to changes in objectively measured nutrition-related outcomes characterized by mitigating functional decline, a return to metabolic homeostasis, and facilitation of long-term physical and neurocognitive rehabilitative processes. Physiologic derangement in acute illness, with susceptibility factors of DABV render kidney-eliminated and nephrotoxic medication use uniquely complex in sick children. High-value medications can be selected for detailed pharmacokinetic/pharmacodynamic/pharmaco-omics characterization to inform drug disposition, dosing, and monitoring decisions at the bedside for AKI and paKST ( and in the ). ,
Successful pediatric translational AKI research programs include diverse teams using reverse translational approaches in partnership with clinical and epidemiological findings that prioritize development as a biologic variable. Sufficient support including pediatric specific government and industry funding along with meaningful partnerships among health professionals is necessary to understand and leverage the unique aspects of pediatric AKI to address kidney health and disease across the life course.
Patient centered outcomes such as functional status, quality of life, and optimal growth and development must drive the targeted nutritional interventions, optimizing short- and long-term nutrition, and incorporate measures of acute and chronic changes of anthropometrics, body composition, physical function, and metabolic control. Engagement of diverse stakeholders across the preclinical and translational research realms is needed to disseminate findings and transform care. Most translational AKI models do not account for the additional complexity of pediatric AKI including the concept of development as a biologic variable (DABV). The association between premature birth and kidney function and maturation remain incompletely understood. Furthermore, the associations between a single AKI episode during kidney development and short-term and long-term outcomes remain unclear. Potential sex differences in AKI have prevented the inclusion of both males and females together in preclinical animal studies. In pediatrics, sex as a biologic variable is further confounded by DABV; hormone levels change throughout pubertal stages, and few clinical studies capture this information. Animal models of AKI that consider sex hormones and sex chromosomes could inform clinical evaluations sensitive to sex as a biologic variable and DABV. The association between AKI and chronic kidney disease has been established, however, there is a paucity of data on the systemic long-term impact of AKI. , The potential associations between AKI with respect to DABV and long-term outcomes are unknown. Opportunities remain to use the unique aspects of pediatric patients, including but not limited to the lack of chronic comorbidities and ability to understand the impact of nephron endowment prior to nephron loss ( ). Malnutrition and protein energy wasting are prevalent and independently associated with mortality in children hospitalized with AKI, especially those requiring paKST, with prevalence ranging from 30% to 55%. – Mortality is a crude end point to assess the importance of nutrition support in children with AKI. AKI survivors are at risk of worsened functional status, chronic ventilation, or dialysis after hospital discharge. Nutrition support and mobilization offer opportunities to improve functional outcomes in survivors through attenuation of muscle loss and promotion of muscle protein synthesis in critically ill children. The outcomes of malnutrition associated with somatic growth, body composition, development, immune function, metabolic derangements, and long-term physical and neurocognitive functioning require more precise measurement tools and targets. Nutritional therapeutic interventions are nuanced and include changes in feeding modality, composition (macronutrient or micronutrient) and volume of nutrition-related fluids which intersects with medical AKI management and metabolic control, which can be further complicated by institution of paKST. Nutritional needs are further affected by DABV. Identifying and measuring appropriate outcome variables represent the first step to define the phenotypic nutritional morbidities in pediatric AKI, and thereby enable evaluation of nutritional intervention efficacy. , We recommend program-based focused nutrition support for children with AKI and transitioning through acute kidney disease. Such a program would use evidence-based nutrition therapy adjusted in response to changes in objectively measured nutrition-related outcomes characterized by mitigating functional decline, a return to metabolic homeostasis, and facilitation of long-term physical and neurocognitive rehabilitative processes. Physiologic derangement in acute illness, with susceptibility factors of DABV render kidney-eliminated and nephrotoxic medication use uniquely complex in sick children. High-value medications can be selected for detailed pharmacokinetic/pharmacodynamic/pharmaco-omics characterization to inform drug disposition, dosing, and monitoring decisions at the bedside for AKI and paKST ( and in the ). ,
Consensus Statement 6A (Recommendation) Given the adverse immediate and lifelong outcomes for children with AKI, education and advocacy are essential, starting with the patient and family and expanding across health care teams, systems, and communities. Consensus Statement 6B (Suggestion) Customized AKI education and advocacy require a comprehensive, multidisciplinary, and patient/family centered focus, with AKI champions embedded at every level, embracing the complexities of diverse settings and individuals, which need to evolve over the pediatric life span. Education may address the widespread gaps in AKI recognition. Lack of AKI awareness may be associated with disparities in AKI outcomes. Extending education to all stakeholders can reduce the risk of missing AKI or delaying care. Because delayed changes in clinical markers (serum creatinine level, urine output) may delay recognition of kidney injury, education requires awareness of risk factors. Education must be delivered in varied settings, different backgrounds of health care workers, and available resources with a flexible approach adjusted for the scenario and depth of knowledge needed ( ). Education must include recognition of AKI risk, prevention and treatment of volume depletion, careful attention to fluid balance, avoidance or limitation of nephrotoxic medications, and blood pressure monitoring. Checklists, standardized screening and care protocols (care bundles), adapted to and used across varied settings, can enhance identification of patients with or at risk for AKI. Successful education programs also require strong advocacy efforts to engage different constituents, including patients and families, community members, physicians, allied health care professionals, industry affiliates, and governmental and nongovernmental organizations ( in the ). Education must be tailored to all members of the health care team, including patients and family, and should occur on micro (individual learners, training program curriculum, teaching modules, and alert systems for providers) and macro (health care system, communitywide educational programs, and governmental policy) levels. AKI education must be an integral part of the curriculum of foundational and advanced education for all health care professionals, with continuing medical education for all practitioners. Programs should view AKI as a core competency metric. , Specialists in AKI (including educators, researchers, and advocates) must use educational methods for those who access online free open-access medical education, available globally 24 hours per day. Patients and families may be unaware of the AKI episode or of the potential for long-term adverse outcomes. AKI education may help empower them to participate in healthy lifestyle modification and may improve follow-up, which has the potential to improve long-term outcomes. , Adaptable and multifaceted educational tools, available in a variety of primary languages and for varying levels of health literacy, should support comprehensive care from admission through discharge, follow-up and potentially, readmission. Connectivity between patient and family with clinician may be essential in the AKI care continuum. AKI diagnosis remains a significant challenge across areas with varying resource availability (low-income, war zones, and disaster areas), with the lack of appropriate laboratory supplies, adequate medical infrastructure, and personnel. A recent survey emphasized the importance of involving and educating governments about the implications of AKI. Fostering partnerships with industry and the international medical community will also be beneficial. Public-private partnerships and programs may have established platforms that can be used for earlier diagnosis of AKI ( and in the ).
Given the adverse immediate and lifelong outcomes for children with AKI, education and advocacy are essential, starting with the patient and family and expanding across health care teams, systems, and communities.
Customized AKI education and advocacy require a comprehensive, multidisciplinary, and patient/family centered focus, with AKI champions embedded at every level, embracing the complexities of diverse settings and individuals, which need to evolve over the pediatric life span. Education may address the widespread gaps in AKI recognition. Lack of AKI awareness may be associated with disparities in AKI outcomes. Extending education to all stakeholders can reduce the risk of missing AKI or delaying care. Because delayed changes in clinical markers (serum creatinine level, urine output) may delay recognition of kidney injury, education requires awareness of risk factors. Education must be delivered in varied settings, different backgrounds of health care workers, and available resources with a flexible approach adjusted for the scenario and depth of knowledge needed ( ). Education must include recognition of AKI risk, prevention and treatment of volume depletion, careful attention to fluid balance, avoidance or limitation of nephrotoxic medications, and blood pressure monitoring. Checklists, standardized screening and care protocols (care bundles), adapted to and used across varied settings, can enhance identification of patients with or at risk for AKI. Successful education programs also require strong advocacy efforts to engage different constituents, including patients and families, community members, physicians, allied health care professionals, industry affiliates, and governmental and nongovernmental organizations ( in the ). Education must be tailored to all members of the health care team, including patients and family, and should occur on micro (individual learners, training program curriculum, teaching modules, and alert systems for providers) and macro (health care system, communitywide educational programs, and governmental policy) levels. AKI education must be an integral part of the curriculum of foundational and advanced education for all health care professionals, with continuing medical education for all practitioners. Programs should view AKI as a core competency metric. , Specialists in AKI (including educators, researchers, and advocates) must use educational methods for those who access online free open-access medical education, available globally 24 hours per day. Patients and families may be unaware of the AKI episode or of the potential for long-term adverse outcomes. AKI education may help empower them to participate in healthy lifestyle modification and may improve follow-up, which has the potential to improve long-term outcomes. , Adaptable and multifaceted educational tools, available in a variety of primary languages and for varying levels of health literacy, should support comprehensive care from admission through discharge, follow-up and potentially, readmission. Connectivity between patient and family with clinician may be essential in the AKI care continuum. AKI diagnosis remains a significant challenge across areas with varying resource availability (low-income, war zones, and disaster areas), with the lack of appropriate laboratory supplies, adequate medical infrastructure, and personnel. A recent survey emphasized the importance of involving and educating governments about the implications of AKI. Fostering partnerships with industry and the international medical community will also be beneficial. Public-private partnerships and programs may have established platforms that can be used for earlier diagnosis of AKI ( and in the ).
The pADQI consensus statements are expert-derived landscape definitions of the multifaceted and multidisciplinary approach needed to improve prediction, diagnosis, management, and follow-up care for AKI in children. The AKI story requires partnerships between patients and providers, across the medical landscape and across the ages of children. We have provided recommendations for current efforts and identified opportunities to address knowledge gaps ( in the ). Limitations This study has limitations. The recommendations of the pADQI are based on existing evidence and consensus but a systematic review on all individual studies and pieces of data was not performed. We also acknowledge recent studies on AKI epidemiology, biomarkers, fluid accumulation, kidney support therapy, nutrition, pharmacology, and education are in progress or have been completed since the completion of pADQI and we could not account for their findings in our recommendations.
This study has limitations. The recommendations of the pADQI are based on existing evidence and consensus but a systematic review on all individual studies and pieces of data was not performed. We also acknowledge recent studies on AKI epidemiology, biomarkers, fluid accumulation, kidney support therapy, nutrition, pharmacology, and education are in progress or have been completed since the completion of pADQI and we could not account for their findings in our recommendations.
Nearly 2 decades of work in critical care nephrology has delivered a robust foundation on which to frame future AKI education, advocacy, research, and clinical practice. AKI in children is unique and is associated with long-lasting consequences. Improved outcomes in patients will require a comprehensive and cross-discipline approach requiring stakeholders across medicine, community, and government to partner with children and their families.
Supplementary Material eBackground. Group Descriptions eBox. Research Agenda for Pediatric Acute Kidney Injury eFigure 1. An Evolving Approach to AKI Epidemiology eTable 1. Definitions of Fluid Balance eFigure 2. The Spectrum of Fluid Balance eFigure 3. Multipronged Approach to Pediatric Acute Kidney Support Therapy eTable 2. Blood Purification and Extracorporeal Treatment Modalities for Different Pathophysiological Conditions eTable 3. Key Components of Education and Advocacy for Children With Acute Kidney Injury (AKI)</SI_Caption>
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Exploiting the potential of | fc8764c7-b0e4-4c13-90b0-f6426be5125a | 11899226 | Pharmacology[mh] | Patient adherence to a treatment regimen remains one of key challenges within the pharmaceutical community as it significantly influences therapeutic outcomes, especially in the treatment of chronic disorders and diseases (Bassand et al., ). Managing chronic illnesses demands strict adherence to a treatment regimen, which commonly includes multiple daily or weekly doses to maintain constant therapeutic levels of the drug in the human body. This is of particular importance for drugs susceptible to rapid in vivo clearance (Jindal et al., ). Yet, compliance with extended therapeutic regimen typically stands at approximately 50%, even in developed countries (Sabaté, ). The fast-growing field of long-acting depot drug delivery systems for subcutaneous (SC) administration holds the potential to revolutionize the current treatment of chronic conditions (Nkanga et al., ; Dubbelboer & Sjögren, ; Rama & Ribeiro, ). Long-acting depots provide minimized dosing frequency and with that associated reduced side effects, while the SC delivery route enables quick and convenient self-administration. Ultimately, these benefits lead to improved patient adherence and associated therapeutic outcomes with reduced overall healthcare costs (van den Bemt et al., ; Li et al., ). A plethora of different drug delivery technologies have been proposed for the formulation of long-acting depot drug delivery systems up to now (Chaudhary et al., ; Park et al., ; Lou et al., ). Simple oil-based solutions and suspensions were initially introduced. Following this, polymeric microspheres were utilized. Lately, in situ forming drug delivery systems have emerged as an attractive platform where the depot is formed upon injection due to a phase transition trigger such as body biological fluid, enzyme catalysis, physiological temperature, or pH (Jain & Jindal, ). Among them, injectable in situ forming liquid crystalline systems have acquired a high appeal due to remarkable structural features, resulting in unique functionalities (Rahnfeld & Luciani, ; Sharma et al., ). Liquid crystalline mesophases are formed spontaneously by the self-assembly of amphiphilic lipids upon contact with aqueous environment providing a liquid crystalline gel with embedded drug molecules. The sustained drug release is a result of the slow drug diffusion through the network of nanochannels in the mesophases, whereby the channel size can be adjusted by changing the composition or other conditions (Shanmugam & Banerjee, ). Various mesophases can be formed corresponding to molecular shapes, local and global packing constraints, and the average interfacial mean curvature (Mezzenga et al., ). In accordance, lyotropic liquid crystals (LCCs) can be classified into lamellar, hexagonal, and cubic mesophases. For the development of long-acting depot drug delivery systems for SC administration hexagonal and cubic LCCs are of prime interest due to their highly ordered microstructure. Hexagonal mesophases are defined as cylindrical micelles packed in an infinite two-dimensional lattice arrangement of a hexagonal pattern, while cubic mesophases consist of three-dimensional structures formed by a continuous curved lipid bilayer with non-contacting aqueous channels (Chavda et al., ). Hexagonal and cubic LCCs are thermodynamically stable colloidal systems and, owing to their high degree of order, they are less prone to fusion or aggregation as well as drug leakage when compared to other in situ forming drug delivery systems (Allegritti et al., ). In addition, their tunable properties concerning their microstructural organization enable relatively high loading and predictable release of small molecules as well as biomolecules such as proteins and peptides (Clogston & Caffrey, ; Chavda et al., ). In keeping with this, in situ forming LCCs as long-acting depot drug delivery systems are especially relevant for peptides as there is a great interest for the improvement of their pharmacokinetic properties (Tiwari et al., ; Wang et al., ). Namely, despite their high efficacy and low toxicity, their use is limited by their short plasma half-life due to significant metabolism by enzymes in vivo as well as their inherent poor physical and chemical stability. Given their immense therapeutic potential, market prospects, and economic value, there is a high demand for novel drug delivery systems that would effectively address the challenges described above (Gonella et al., ; Yang et al., ). Thymosin alpha 1 (Tα1), an immunomodulatory bioactive peptide with 28 amino acid residues, characterized by a short plasma half-life and with that associated twice-weekly SC administration regimen (Dominari et al., ), was chosen as a model peptide drug in this study. Herein, we thus aimed to exploit the potential of liquid crystalline platform for development of patient-friendly in situ forming system for SC administration, designed to achieve sustained release of the peptide drug Tα1. Accordingly, our study prioritized the following main objectives: (i) optimal rheological properties of nonaqueous precursor formulation for SC injection, (ii) easy and quick in situ phase transition to hexagonal and/or cubic LCCs triggered merely by water absorption, (iii) sustained release kinetics of peptide drug Tα1 that would notably minimize its dosing frequency.
Materials Ethanol (96% v/v) was provided by Pharmachem, Ljubljana, Slovenia. Lipoid ® S-100, soybean lecithin with not less than 94% (w/w) phosphatidylcholine content, was supplied from Lipoid GmbH, Ludwigshafen, Germany. According to the manufacturer’s specification the fatty acids of the two acyl groups of phosphatidylcholine are palmitic (15%), stearic (3%), oleic and isomers (12%), linoleic (62%), and α-linolenic (5%). Glycerol monooleate, type 40 (Peceol), and glycerol monolinoleate (Maisine ® CC) were obtained as a gift sample from Gattefossé SAS, Saint-Priest, France. According to the manufacturer’s specification, the first consists of monoglycerides (32–52%), diglycerides (30–50%) and triglycerides (5–20%) of mainly oleic (C 18:1 ) acid and the latter consist of monoglycerides (32–52%), diglycerides (40–55%) and triglycerides (5–20%) of mainly linoleic (C 18:2 ) and oleic (C 18:1 ) acids. Tα1, a 28 amino acid sequence peptide, was purchased from Pure Peptides UK, Epsom, United Kingdom. Bidistilled water and phosphate buffered solution (PBS), respectively, were used throughout the experiments. All other chemicals and reagents were of analytical grade. For polarized light microscope examination, gelation time measurements, gelation test, and water uptake evaluation PBS with pH = 7.4 was used to mimic the subcutaneous environment at the injection site. As for the in vitro release testing, PBS with pH = 6.8 containing 5% (m/m) of ethanol was used as the release medium for the stability improvement of the peptide drug Tα1. Pseudoternary phase diagram construction In order to determine the concentration ranges of the LCCs formation, four pseudoternary phase diagrams were constructed by a water titration method. Each diagram depicted three phases, with certain phases consisting of different combinations of ingredients. The first phase was composed of either a hydrotropic substance, i.e. ethanol, or a mixture of a hydrotropic substance and an amphiphile, i.e. ethanol and lecithin with a mass ratio of 1/1. The next phase was designated as the lipid phase consisting of glycerol monooleate or glycerol monolinoleate. Finally, the third vertex of the diagram was represented by the hydrophilic phase. Nine dilution lines were constructed for each diagram, and the starting point of each line denoted a precursor formulation. All precursor formulations were thus composed of ethanol or ethanol-lecithin mixture and glycerol monooleate or glycerol monolinoleate with mass ratios ranging from 90/10 to 10/90%. For the titration process, each precursor formulation was slowly titrated with aliquots of the hydrophilic phase and stirred at room temperature for a sufficient time to obtain equilibrium. Subsequently, samples were checked for homogeneity, consistency, and appearance. Homogeneous, highly viscous, and opaque samples were characterized as LCCs. Next, samples visually identified as LCCs were further checked under a polarized light microscope at 25 °C and 37 °C for the presence of liquid crystalline mesophases. Based on the obtained results, eight precursor formulations were then selected for further characterization. The composition of these selected studied precursor formulations is detailed in , while the preparation procedure is outlined in the following section. Sample preparation Unloaded precursor formulations were prepared by mixing appropriate amounts of ethanol or ethanol-lecithin mixture and glycerol monooleate or glycerol monolinoleate for a sufficient time to form a homogeneous system. In the case of Tα1-loaded precursor formulations containing 1.6 mg/g of peptide drug (i.e. dose of reference medicine) (Dominari et al., ), Tα1 was first dissolved in ethanol or ethanol-lecithin mixture. The resulting solution was then mixed with appropriate amount of glycerol monooleate or glycerol monolinoleate for a sufficient time until a homogeneous system was obtained. Both unloaded and Tα1-loaded precursor formulations were prepared at room temperature. Polarized light microscopy Polarized light microscopy (PLM) was used for phase transition analysis of the samples from the pseudoternary phase diagram construction as well as the gelation test. In the former, samples that were identified as LCCs based on macroscopic examination of mixtures formed along dilution lines of the pseudoternary phase diagrams were examined at 25 °C and 37 °C to assess phase transition and presence of liquid crystalline mesophases, respectively. In the latter, phase transition of precursor formulations upon contact with PBS at predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days) was examined at 37 °C. PLM was performed using a CX31-P Upright Microscope (Olympus, Tokyo, Japan). The magnification was 40×. Gelation time measurements Gelation time is the time required for a precursor formulation to convert into an in situ formed gel upon contact with an excess aqueous medium. Within the scope of gelation time measurements, 0.5 mL of each precursor formulation was injected with a 25-gauge needle into 5 mL of PBS preheated at 37 °C. The time upon contact of a liquid transparent precursor formulation with the aqueous medium until complete transformation into an opaque in situ formed gel was recorded as the gelation time (Mei et al., ). For each precursor formulation, the measurement was performed in triplicate. Gelation test The ability to form and maintain an in situ formed gel of a precursor formulation upon contact with excess aqueous medium for a prolonged period of time was evaluated based on the macroscopic appearance of in situ formed gels at predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days). In addition, for better visualization, in situ formed gels in vials immediately after injection were also photographed. 0.5 mL of each precursor formulation was injected with a 25-gauge needle into a 10 mL vial with 5 mL of PBS preheated at 37 °C. To note, for every time point, each precursor formulation was injected into a separate vial. During the whole testing period samples were stored in an orbital shaker-incubator ES-20 (SIA Biosan, Riga, Latvia) set at 50 rpm and 37 °C. At each time point, PBS on the surface of each in situ formed gel was cautiously wiped off. Subsequently, in situ formed gels were observed visually for homogeneity, consistency, and appearance (Ki et al., ; Mei et al., ). In addition, phase transition analysis of in situ formed gels at 37 °C using a polarized light microscope was performed at each time point. Water uptake evaluation To explore the swelling behavior of in situ formed gels, their water uptake was monitored by gravimetric analysis according to a standard protocol (Mei et al., ) at predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days). In keeping with this, the time point at which equilibrium with water was reached and the water maximum absorption, denoted as water capacity, was also determined. 0.5 g of each precursor formulation was injected with a 25-gauge needle into a 10 mL vial with 5 mL of PBS preheated at 37 °C. During the whole testing period samples were stored in an orbital shaker-incubator ES-20 (SIA Biosan, Riga, Latvia) set at 50 rpm and 37 °C. Experiments were performed in triplicate. At each time point, the respective masses were determined and the percentage of water uptake was calculated from , where M vg represents mass of a vial together with in situ formed gel, M v represents mass of a vial alone, M vpp represents mass of a vial together with PBS and precursor formulation, and M vp represents the mass of a vial together with PBS. (1) W % = Mvg − Mv Mvpp − Mvp − 1 × 100 % Differential scanning calorimetry Differential scanning calorimetry (DSC) measurements were carried out for individual compounds (i.e. ethanol, lecithin, glycerol monooleate, glycerol monolinoleate, and bidistilled water) and in situ formed gels after reaching equilibrium with the water. DSC was performed to analyze intermolecular interactions and water state within samples. A DSC 1 differential scanning calorimeter (Mettler Toledo, Greifensee, Switzerland) was used. Approximately 10 mg of the sample was accurately weighed into a small aluminum pan and sealed. An empty sealed pan was used as a reference. Nitrogen with a flow rate of 50 mL/min was used as a purge gas. One cooling and one heating scan were recorded during each analysis. Samples were cooled from 20 °C to −80 °C, kept at −80 °C for 5 min, and heated to 140 °C. The cooling and heating rate was 5 K/min. Rheological measurements The rheological behavior of precursor formulations and in situ formed gels after reaching equilibrium with water was characterized using a Physica MCR 301 rheometer equipped with RheoCompass software (Anton Paar GmbH, Graz, Austria). Rotational tests were conducted at 25 ± 0.1 °C for precursor formulations and at 37 ± 0.1 °C for in situ formed gels after reaching equilibrium with water. Experiments were performed in duplicate. Rotational measurements were carried out to determine the viscosity (η), which was calculated according to , where τ is the shear stress and γ̇ is the shear rate. (2) η = τ / γ ˙ Oscillatory tests were employed to define the storage (elastic; G′) and loss (viscous; G″) moduli of in situ formed gels after reaching equilibrium with water at 37 ± 0,1 °C. They were calculated using and , respectively, where τ is the shear stress, γ is the deformation, and δ is the phase shift angle. (3) G ′ = ( τ / γ ) × cos δ (4) G ″ = ( τ / γ ) × sin δ In addition, complex viscosity (η*) was calculated according to , where τ is the shear stress, γ is the deformation, and ω is the angular frequency. (5) η ∗ = τ / ( γ × ω ) Rotational tests were performed using a cone and plate measuring system CP50-2 (cone diameter 49.961 mm, cone angle 2.001°, sample thickness 0.209 mm). The shear rate ranged from 1 s −1 to 100 s −1 . For the oscillatory tests, the stress sweep measurements were carried out at a constant frequency of 10.0 s −1 to determine the linear viscoelastic region. Afterward, the oscillatory shear measurements were performed as a function of frequency (0.1–100 s −1 ) at a small stress (0.1%) chosen within the linear region to provide the least disturbance of the microstructure. In vitro release testing A membraneless model, which enables direct contact between in situ formed gel and release medium, was applied for in vitro release testing. 1 g of Tα1-loaded precursor formulation containing 1.6 mg/g of peptide drug (i.e. dose of reference medicine) (Dominari et al., ) was injected with a 25-gauge needle directly into 15 mL of release medium preheated at 37 °C. Considering physiological conditions after SC administration and chemical stability of peptide drug Tα1, PBS (pH = 6.8) containing 5% (m/m) of ethanol was selected as the most appropriate release medium for the test completion in 2 weeks at 37 °C. At predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days) 1 mL aliquots of release medium were withdrawn and replaced by an equal volume of fresh preheated receptor medium to keep the volume constant. During the whole testing period samples were stored in an orbital shaker-incubator ES-20 (SIA Biosan, Riga, Latvia) set at 50 rpm and 37 °C. Experiments were performed in quadruplicate. The medium that was taken at each time point was analyzed quantitatively by ultra-high performance liquid chromatography (UHPLC) analysis described below. The cumulative amount of the released peptide drug Tα1 (Q t ) was plotted as a function of time and calculated according to where c t is the peptide drug Tα1 concentration of receptor medium at each sampling time, V rm is the volume of receptor medium, c i is the peptide drug Tα1 concentration at previous sampling times, and V i is the sampling volume. (6) Q t = c t × V rm + ∑ i = 0 t − 1 c i × V i UHPLC analysis An Infinity 1290 ultra-high performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector with a high-sensitivity Max-Light cartridge cell (60 mm) and an EZChrom acquisition system was used. Chromatographic separation was performed on a reversed-phase Synergi Hydro column 150 × 4.6 mm, 4 µm particle size (Phenomenex, Torrance, CA, USA) at 40 °C. The mobile phase consisted of solvent A: 0.1% H 3 PO 4 and solvent B: acetonitrile with a gradient elution of 12.0% to 16.5% solvent B in 12 min at a flow rate of 1 mL/min. The total run time was 14 min. The injection volume was 10 μL, and a detection wavelength of 214 nm was selected. The method was validated in terms of selectivity (no interference at the retention time of the Tα1), linearity ( R 2 = 1.000 in the concentration range between 1 and 100 mg/L), precision (RSD < 5%) and accuracy (10 0 ± 5%). Tα1 was stable in the samples for at least 4 days when 5% ethanol was added to the solution. Circular dichroism spectroscopy Circular dichroism (CD) measurements were performed using a Chirascan CD spectrometer equipped with a Peltier temperature controller (Applied Photophysics Ltd, London, United Kingdom). CD spectra were recorded in a 1-mm quartz cell (Hellma GmbH & Co, Müllheim, Germany) at 37 °C using 1-nm step, 1-nm bandwidth and 1-s sampling. The secondary structure of the peptide drug Tα1 was analyzed by scan measurement in the range from 200 nm to 260 nm. The results are the average of three scans. Data and statistical analysis The data and statistical analysis were performed with GraphPad Prism 10.2.0. All results, unless stated otherwise, were expressed as mean ± standard deviation (SD).
Ethanol (96% v/v) was provided by Pharmachem, Ljubljana, Slovenia. Lipoid ® S-100, soybean lecithin with not less than 94% (w/w) phosphatidylcholine content, was supplied from Lipoid GmbH, Ludwigshafen, Germany. According to the manufacturer’s specification the fatty acids of the two acyl groups of phosphatidylcholine are palmitic (15%), stearic (3%), oleic and isomers (12%), linoleic (62%), and α-linolenic (5%). Glycerol monooleate, type 40 (Peceol), and glycerol monolinoleate (Maisine ® CC) were obtained as a gift sample from Gattefossé SAS, Saint-Priest, France. According to the manufacturer’s specification, the first consists of monoglycerides (32–52%), diglycerides (30–50%) and triglycerides (5–20%) of mainly oleic (C 18:1 ) acid and the latter consist of monoglycerides (32–52%), diglycerides (40–55%) and triglycerides (5–20%) of mainly linoleic (C 18:2 ) and oleic (C 18:1 ) acids. Tα1, a 28 amino acid sequence peptide, was purchased from Pure Peptides UK, Epsom, United Kingdom. Bidistilled water and phosphate buffered solution (PBS), respectively, were used throughout the experiments. All other chemicals and reagents were of analytical grade. For polarized light microscope examination, gelation time measurements, gelation test, and water uptake evaluation PBS with pH = 7.4 was used to mimic the subcutaneous environment at the injection site. As for the in vitro release testing, PBS with pH = 6.8 containing 5% (m/m) of ethanol was used as the release medium for the stability improvement of the peptide drug Tα1.
In order to determine the concentration ranges of the LCCs formation, four pseudoternary phase diagrams were constructed by a water titration method. Each diagram depicted three phases, with certain phases consisting of different combinations of ingredients. The first phase was composed of either a hydrotropic substance, i.e. ethanol, or a mixture of a hydrotropic substance and an amphiphile, i.e. ethanol and lecithin with a mass ratio of 1/1. The next phase was designated as the lipid phase consisting of glycerol monooleate or glycerol monolinoleate. Finally, the third vertex of the diagram was represented by the hydrophilic phase. Nine dilution lines were constructed for each diagram, and the starting point of each line denoted a precursor formulation. All precursor formulations were thus composed of ethanol or ethanol-lecithin mixture and glycerol monooleate or glycerol monolinoleate with mass ratios ranging from 90/10 to 10/90%. For the titration process, each precursor formulation was slowly titrated with aliquots of the hydrophilic phase and stirred at room temperature for a sufficient time to obtain equilibrium. Subsequently, samples were checked for homogeneity, consistency, and appearance. Homogeneous, highly viscous, and opaque samples were characterized as LCCs. Next, samples visually identified as LCCs were further checked under a polarized light microscope at 25 °C and 37 °C for the presence of liquid crystalline mesophases. Based on the obtained results, eight precursor formulations were then selected for further characterization. The composition of these selected studied precursor formulations is detailed in , while the preparation procedure is outlined in the following section.
Unloaded precursor formulations were prepared by mixing appropriate amounts of ethanol or ethanol-lecithin mixture and glycerol monooleate or glycerol monolinoleate for a sufficient time to form a homogeneous system. In the case of Tα1-loaded precursor formulations containing 1.6 mg/g of peptide drug (i.e. dose of reference medicine) (Dominari et al., ), Tα1 was first dissolved in ethanol or ethanol-lecithin mixture. The resulting solution was then mixed with appropriate amount of glycerol monooleate or glycerol monolinoleate for a sufficient time until a homogeneous system was obtained. Both unloaded and Tα1-loaded precursor formulations were prepared at room temperature.
Polarized light microscopy (PLM) was used for phase transition analysis of the samples from the pseudoternary phase diagram construction as well as the gelation test. In the former, samples that were identified as LCCs based on macroscopic examination of mixtures formed along dilution lines of the pseudoternary phase diagrams were examined at 25 °C and 37 °C to assess phase transition and presence of liquid crystalline mesophases, respectively. In the latter, phase transition of precursor formulations upon contact with PBS at predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days) was examined at 37 °C. PLM was performed using a CX31-P Upright Microscope (Olympus, Tokyo, Japan). The magnification was 40×.
Gelation time is the time required for a precursor formulation to convert into an in situ formed gel upon contact with an excess aqueous medium. Within the scope of gelation time measurements, 0.5 mL of each precursor formulation was injected with a 25-gauge needle into 5 mL of PBS preheated at 37 °C. The time upon contact of a liquid transparent precursor formulation with the aqueous medium until complete transformation into an opaque in situ formed gel was recorded as the gelation time (Mei et al., ). For each precursor formulation, the measurement was performed in triplicate.
The ability to form and maintain an in situ formed gel of a precursor formulation upon contact with excess aqueous medium for a prolonged period of time was evaluated based on the macroscopic appearance of in situ formed gels at predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days). In addition, for better visualization, in situ formed gels in vials immediately after injection were also photographed. 0.5 mL of each precursor formulation was injected with a 25-gauge needle into a 10 mL vial with 5 mL of PBS preheated at 37 °C. To note, for every time point, each precursor formulation was injected into a separate vial. During the whole testing period samples were stored in an orbital shaker-incubator ES-20 (SIA Biosan, Riga, Latvia) set at 50 rpm and 37 °C. At each time point, PBS on the surface of each in situ formed gel was cautiously wiped off. Subsequently, in situ formed gels were observed visually for homogeneity, consistency, and appearance (Ki et al., ; Mei et al., ). In addition, phase transition analysis of in situ formed gels at 37 °C using a polarized light microscope was performed at each time point.
To explore the swelling behavior of in situ formed gels, their water uptake was monitored by gravimetric analysis according to a standard protocol (Mei et al., ) at predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days). In keeping with this, the time point at which equilibrium with water was reached and the water maximum absorption, denoted as water capacity, was also determined. 0.5 g of each precursor formulation was injected with a 25-gauge needle into a 10 mL vial with 5 mL of PBS preheated at 37 °C. During the whole testing period samples were stored in an orbital shaker-incubator ES-20 (SIA Biosan, Riga, Latvia) set at 50 rpm and 37 °C. Experiments were performed in triplicate. At each time point, the respective masses were determined and the percentage of water uptake was calculated from , where M vg represents mass of a vial together with in situ formed gel, M v represents mass of a vial alone, M vpp represents mass of a vial together with PBS and precursor formulation, and M vp represents the mass of a vial together with PBS. (1) W % = Mvg − Mv Mvpp − Mvp − 1 × 100 %
Differential scanning calorimetry (DSC) measurements were carried out for individual compounds (i.e. ethanol, lecithin, glycerol monooleate, glycerol monolinoleate, and bidistilled water) and in situ formed gels after reaching equilibrium with the water. DSC was performed to analyze intermolecular interactions and water state within samples. A DSC 1 differential scanning calorimeter (Mettler Toledo, Greifensee, Switzerland) was used. Approximately 10 mg of the sample was accurately weighed into a small aluminum pan and sealed. An empty sealed pan was used as a reference. Nitrogen with a flow rate of 50 mL/min was used as a purge gas. One cooling and one heating scan were recorded during each analysis. Samples were cooled from 20 °C to −80 °C, kept at −80 °C for 5 min, and heated to 140 °C. The cooling and heating rate was 5 K/min.
The rheological behavior of precursor formulations and in situ formed gels after reaching equilibrium with water was characterized using a Physica MCR 301 rheometer equipped with RheoCompass software (Anton Paar GmbH, Graz, Austria). Rotational tests were conducted at 25 ± 0.1 °C for precursor formulations and at 37 ± 0.1 °C for in situ formed gels after reaching equilibrium with water. Experiments were performed in duplicate. Rotational measurements were carried out to determine the viscosity (η), which was calculated according to , where τ is the shear stress and γ̇ is the shear rate. (2) η = τ / γ ˙ Oscillatory tests were employed to define the storage (elastic; G′) and loss (viscous; G″) moduli of in situ formed gels after reaching equilibrium with water at 37 ± 0,1 °C. They were calculated using and , respectively, where τ is the shear stress, γ is the deformation, and δ is the phase shift angle. (3) G ′ = ( τ / γ ) × cos δ (4) G ″ = ( τ / γ ) × sin δ In addition, complex viscosity (η*) was calculated according to , where τ is the shear stress, γ is the deformation, and ω is the angular frequency. (5) η ∗ = τ / ( γ × ω ) Rotational tests were performed using a cone and plate measuring system CP50-2 (cone diameter 49.961 mm, cone angle 2.001°, sample thickness 0.209 mm). The shear rate ranged from 1 s −1 to 100 s −1 . For the oscillatory tests, the stress sweep measurements were carried out at a constant frequency of 10.0 s −1 to determine the linear viscoelastic region. Afterward, the oscillatory shear measurements were performed as a function of frequency (0.1–100 s −1 ) at a small stress (0.1%) chosen within the linear region to provide the least disturbance of the microstructure.
A membraneless model, which enables direct contact between in situ formed gel and release medium, was applied for in vitro release testing. 1 g of Tα1-loaded precursor formulation containing 1.6 mg/g of peptide drug (i.e. dose of reference medicine) (Dominari et al., ) was injected with a 25-gauge needle directly into 15 mL of release medium preheated at 37 °C. Considering physiological conditions after SC administration and chemical stability of peptide drug Tα1, PBS (pH = 6.8) containing 5% (m/m) of ethanol was selected as the most appropriate release medium for the test completion in 2 weeks at 37 °C. At predetermined time points (1, 6, 12, 24, 36, 48 and 72 hours, and 7, 10 and 14 days) 1 mL aliquots of release medium were withdrawn and replaced by an equal volume of fresh preheated receptor medium to keep the volume constant. During the whole testing period samples were stored in an orbital shaker-incubator ES-20 (SIA Biosan, Riga, Latvia) set at 50 rpm and 37 °C. Experiments were performed in quadruplicate. The medium that was taken at each time point was analyzed quantitatively by ultra-high performance liquid chromatography (UHPLC) analysis described below. The cumulative amount of the released peptide drug Tα1 (Q t ) was plotted as a function of time and calculated according to where c t is the peptide drug Tα1 concentration of receptor medium at each sampling time, V rm is the volume of receptor medium, c i is the peptide drug Tα1 concentration at previous sampling times, and V i is the sampling volume. (6) Q t = c t × V rm + ∑ i = 0 t − 1 c i × V i
An Infinity 1290 ultra-high performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a diode array detector with a high-sensitivity Max-Light cartridge cell (60 mm) and an EZChrom acquisition system was used. Chromatographic separation was performed on a reversed-phase Synergi Hydro column 150 × 4.6 mm, 4 µm particle size (Phenomenex, Torrance, CA, USA) at 40 °C. The mobile phase consisted of solvent A: 0.1% H 3 PO 4 and solvent B: acetonitrile with a gradient elution of 12.0% to 16.5% solvent B in 12 min at a flow rate of 1 mL/min. The total run time was 14 min. The injection volume was 10 μL, and a detection wavelength of 214 nm was selected. The method was validated in terms of selectivity (no interference at the retention time of the Tα1), linearity ( R 2 = 1.000 in the concentration range between 1 and 100 mg/L), precision (RSD < 5%) and accuracy (10 0 ± 5%). Tα1 was stable in the samples for at least 4 days when 5% ethanol was added to the solution.
Circular dichroism (CD) measurements were performed using a Chirascan CD spectrometer equipped with a Peltier temperature controller (Applied Photophysics Ltd, London, United Kingdom). CD spectra were recorded in a 1-mm quartz cell (Hellma GmbH & Co, Müllheim, Germany) at 37 °C using 1-nm step, 1-nm bandwidth and 1-s sampling. The secondary structure of the peptide drug Tα1 was analyzed by scan measurement in the range from 200 nm to 260 nm. The results are the average of three scans.
The data and statistical analysis were performed with GraphPad Prism 10.2.0. All results, unless stated otherwise, were expressed as mean ± standard deviation (SD).
Pseudoternary phase diagram construction When developing a novel drug delivery system based on LCCs, phase behavior of systems with precisely defined components can be investigated using ternary or pseudoternary phase diagrams. In this study, four pseudoternary phase diagrams were constructed for the systems containing ethanol/glycerol monooleate/hydrophilic phase , ethanol/glycerol monolinoleate/hydrophilic phase , ethanol/lecithin/glycerol monooleate/hydrophilic phase , and ethanol/lecithin/glycerol monolinoleate/hydrophilic phase . Ethanol had a role of a hydrotropic substance for viscosity reduction (Ferreira et al., ) as well as peptide drug Tα1 stabilization. Lecithin was chosen as a biocompatible amphiphile capable of formation and stabilization of various LCCs mesophases depending on the remaining amphiphiles in the mix (Gosenca et al., ). Glycerol monooleate and glycerol monolinoleate were selected as hexagonal and/or cubic mesophases-forming amphiphilic lipids. They possess excellent biocompatible and biodegradable characteristics due to the presence of ester bonds in their structure, which undergo lipolytic degradation by endogenous lipases after SC injection (Zhang et al., ). Hydrophilic phase represented aqueous environment of the SC tissue. Macroscopic analysis of the systems formed across the constructed pseudoternary phase diagrams was initially performed. Different proportions and type of components resulted in formation of distinct systems. Transparent or semi-transparent and viscous gel-like systems were identified as LCCs. As our focus was on the formation of liquid crystalline mesophases, we did not explore in detail other regions of the diagrams where nonhomogeneous systems, coarse emulsions, or microemulsions were present. Pseudoternary phase diagram study showed that absence or presence of lecithin had a key influence on the formation of LCCs, while the type of lipid did not show any significant effect. Namely, in the diagrams from and employing only ethanol, lipid, and hydrophilic phase, a considerably smaller region of LCCs formation was observed compared to the diagrams from and utilizing ethanol/lecithin mixture, lipid, and hydrophilic phase. More specifically, in the case of the diagram from , adding hydrophilic phase at a level of 10–30% to precursor formulations containing 10–20% of ethanol resulted in the formation of LCCs. In parallel, a similarly small region of LCCs was distinguished in the diagram from , where addition of hydrophilic phase ranging from 50 to 70% to precursor formulations with 10–20% of ethanol led to LCCs formation. On the other hand, in the case of the diagrams from and , precursor formulations containing 50–80% of the ethanol/lecithin mixture formed a large region of LCCs after hydrophilic phase was added in the range of 10–90%. When comparing the systems across all diagrams from , it is important to note that the LCCs formed in the diagrams from and exhibited favorable macroscopic characteristics such as homogeneity and high viscosity. Phase transition analysis within pseudoternary phase diagram construction Systems identified as potential LCCs based on macroscopic analysis, utilizing pseudoternary phase diagrams, underwent subsequent microscopic analysis using polarized light microscope at room (25 °C) and body (37 °C) temperature. The acquired observations are schematically presented in , while the representative photomicrographs are depicted in . PLM is one of the most commonly used methods for investigating LCCs mesophases, offering valuable insight into their molecular organization and phase transitions. When subjected to polarized light, anisotropic systems such as lamellar and hexagonal LCCs exhibit characteristic birefringent pattern. Maltese crosses together with oily streaks denote the presence of lamellar mesophases, while fan-like textures indicate formation of hexagonal mesophases. A dark background, with no birefringence, suggests the presence of cubic mesophases, known for their isotropic liquid behavior (Manaia et al., ; Zhang et al., ). First, evaluation of the systems was performed at a temperature of 25 °C. In the case of the systems from the first and the second diagram, the black view under polarized microscope pointed to the presence of cubic LCCs being in good agreement with the investigation by Mei et al. . In contrast, the phase behavior of the systems depicted in the third and fourth diagram exhibited distinctive characteristics of LCCs, confirming that lecithin has a key function in formation of these LCCs. Namely, numerous and pronounced fan-like textures started to emerge in the dark background from precursor formulations containing 50%, 60%, and 70% of the ethanol/lecithin mixture, respectively, independent of the hydrophilic phase content. This observation is imperative as it draws us to two important findings. Firstly, it indicates the presence of hexagonal LCCs and in some areas coexistence with cubic LCCs; nevertheless, hexagonal mesophases were strongly prevailing. Secondly, it implies that absorption of only a small amount of hydrophilic phase was necessary for these precursor formulations to form hexagonal LCCs in situ . Further, mixed phases of hexagonal LCCs together with lamellar LCCs were formed from precursor formulations containing 80% of the ethanol/lecithin mixture, respectively, but only after a larger addition of hydrophilic phase, i.e. 40–90%. It should be noted that, for precursor formulations containing 80% of the ethanol/lecithin mixture, fan-like textures were less numerous and pronounced as well as that individual Maltese crosses were also observed. Further, comparable results were obtained at a temperature of 37 °C for all four diagrams. The only exception was that precursor formulations from the third and the fourth diagram containing 80% of the ethanol/lecithin mixture already showed fan-like textures along with Maltese crosses when the hydrophilic phase content reached 10%. This indicates that higher temperature induces, to some extent, the formation of hexagonal LCCs together with lamellar LCCs, which is desirable for SC administration. Based on all the findings obtained from the pseudoternary phase construction (i.e. macroscopic appearance) and the corresponding phase behavior analysis (i.e. microstructure), eight precursor formulations, capable of in situ phase transition to hexagonal LCCs upon addition of water, were selected for further characterization studies. The composition of the selected studied precursor formulations is reported in and highlighted in . Gelation time measurements Gelation time denotes the time required for a precursor formulation to transform into an in situ formed gel when exposed to excess aqueous medium (Mei et al., ). A rapid sol-gel transition is desired to minimize the possibility of initial burst release as in situ formation of gel retards the release of the incorporated drug. At room temperature, all precursor formulations were clear with good fluidity, but upon contact with aqueous medium heated to 37 °C, they quickly lost their flowability. Among all precursor formulations, a very short gelation time of a few seconds was measured for (E/L)Go50 (2.8 seconds), (E/L)Gl50 (2.5 seconds), (E/L)Go60 (4.1 seconds), and (E/L)Gl60 (3.6 seconds). Further, a slightly longer gelation time was measured for (E/L)Go70 (14.1 seconds) and (E/L)Gl70 (13.3 seconds). The longest sol-gel transition time was determined for (E/L)Go80 (70.0 seconds) and (E/L)Gl80 (45.4 seconds). These results indicate that all precursor formulations would spontaneously transform into an in situ gel at the site of administration upon exposure to the physiological fluid. However, it can be observed that there are certain differences among precursor formulations which are most likely related to their composition. Namely, increasing glycerol monooleate and glycerol monolinoleate content, respectively, leads to a decrease in gelation time, which is also consistent with the literature data (Mei et al., ). The phenomenon can be attributed to glycerol monooleate and glycerol monolinoleate being amphiphilic lipids capable of forming hexagonal LCCs. Consequently, they have the ability to rapidly induce the formation of these highly viscous mesophases. Gelation test The ability of a precursor formulation to form and maintain an in situ formed gel upon injection into excess aqueous medium heated to 37 °C was evaluated within gelation test. shows the visual appearance of in situ formed gels at selected predetermined time points, namely, immediately after injection, the initial and final time assessment points (1 hour and 14 days), the time points when equilibrium with water was established (24 and 72 hours), and the time point showing the most prominent morphological changes of in situ formed gels were observed (7 days). In addition, for better visualization, in situ formed gels in vials immediately after injection are also illustrated. Supplementary Figure S1 provides visual representation of in situ formed gels at other predetermined time points, namely 6, 12, 36, 48 hours, and 10 days. The intensity of color of all in situ formed gels was the highest at the first time point, i.e. 1 hour, due to the thorough uptake of the aqueous medium, which began immediately upon contact with it. The most compact forms with noticeably low quantity of uptook aqueous medium were formed by (E/L)Go50 and (E/L)Gl50, which, unlike the milky yellow (E/L)Go80 and (E/L)Gl80, were bright yellow. The least firm and visibly the biggest volumes of in situ formed gels were observed for (E/L)Go80 and (E/L)Gl80, as they uptook high quantity of aqueous medium. (E/L)Go60, (E/L)Gl60, (E/L)Go70, and (E/L)Gl70 typically represented an ‘intermediate stage”. They combined both clear and cloudy areas in color, and their consistency was softer than that of (E/L)Go50 and (E/L)Gl50 and firmer than that of (E/L)Go80 and (E/L)Gl80. With the exception of less intense color and slower process of swelling, after 6, 12, 24, 36, 48, and 72 hours, no significant changes in macroscopic appearance of all in situ formed gels were observed. However, after 7 days notable changes in color and consistency were detected for all in situ formed gels as they started to liquefy and decrease in their size. Over the following days, the process of liquification and erosion was slowly progressing. Interestingly, at the last time point, i.e. 14 days, remaining in situ formed gels of (E/L)Go80 and (E/L)Gl80 settled at the bottom of the vials, while remainings of the other systems were still floating. When comparing all precursor formulations, due to the yellow color of glycerol monooleate and glycerol monolinoleate, respectively, gels containing more lipid phase were more yellow. But what seems to be of major importance, we found that in situ formed gels with a higher lipid content were more compact and eroded more slowly, while in situ formed gels containing a higher amount of the ethanol/lecithin mixture were softer and degraded faster. Phase transition analysis within gelation test As the microstructure of in situ formed gel is a crucial factor influencing the drug release kinetics, the phase transition of precursor formulations upon contact with aqueous medium was monitored using PLM at 37 °C. The analysis was performed at the same time points as the gelation test was carried out. Photomicrographs obtained at the selected predetermine time points, i.e. 1, 24, 72 hours, and 7 and 14 days (see chapter Gelation test) are shown in . Supplementary Figure S2 provides photomicrographs taken at other predetermined time points, namely 6, 12, 36, 48 hours, and 10 days. The phase changes at post-hydration time of precursor formulations with excess aqueous medium revealed dynamic phase transitions, which were caused by rearrangement of molecules within precursor formulations. These microstructural changes can be understood in terms of aqueous self-assembly of amphiphile mixtures explained by their critical packing parameter (CPP). Namely, the self-assembly of single amphiphiles in aqueous medium is driven by a balance between the hydrophobic interactions of the tails and the geometrical packing constraints of the polar head groups. These factors are expressed as CPP = v/al , where v is the volume of the hydrophobic tail, a is the polar head group area, and l is the hydrophobic tail length of the amphiphilic molecule. As a guideline, amphiphiles with a CPP ∼1 usually self-assemble into lamellar LCCs (Engström & Engström, ), a CPP of ∼1.3 is characteristic for bicontinuous cubic mesophases, while amphiphiles with a CPP ∼1.7 form inverted hexagonal mesophases (Larsson, ). In regard to our results, clearly visible and numerous fan-like textures emerging from dark background, were observed for (E/L)Go50, (E/L)Gl50, (E/L)Go60, (E/L)Gl60, (E/L)Go70, and (E/L)Gl70 at the first time point of the assessment and they persisted until the conclusion of the analysis. It appears that hexagonal LCCs were quickly formed from these precursor formulations and that their microstructure was preserved until the final time point of the analysis. These findings can be attributed to the high content of glycerol monooleate and glycerol monolinoleate, respectively, in these precursor formulations. Namely, upon contact with aqueous medium, the polar head groups of the amphiphilic lipid from precursor formulations begin to move more freely. Consequently, these movements induce disorder in the hydrophobic chain of the amphiphilic lipid, leading to an increase of volume of the hydrophobic tail – v . However, the cross-sectional area of the polar head groups stays constant due to the strong hydrogen bonding. Therefore, CPP value increases as v increases and the polar head group area – a and the hydrophobic tail length of the amphiphilic molecule – l remain constant, thereby facilitating phase transition to hexagonal mesophases (Borgheti-Cardoso et al., ; Ferreira, ). When looking at (E/L)Go80 and (E/L)Gl80, hexagonal LCCs were also mainly present at all time points of the assessment. However, it should be noted that here fan-like structures were less pronounced. In addition, Maltese crosses indicative of lamellar LCCs were observed at the initial time point for (E/L)Go80 and (E/L)Gl80, with their presence slowly increasing throughout the analysis. Given that these precursor formulations contained a high content of lecithin/ethanol mixture, its effect was reflected in the resulting mesophases. Namely, a CPP value for lecithin, specifically for phosphatidylcholine as its main component, ranges from 0.5 to 1, meeting the requirement for bilayer formation of lamellar mesophases. Furthermore, ethanol molecules intercalated within phospholipid bilayers of lecithin additionally contributed to the lipid bilayer fluidity (Mkam Tsengam et al., ). As a result, in the case of the abovementioned precursor formulations, the self-assembly of lamellar mesophases was also observed along with formation of hexagonal mesophases. Water uptake evaluation Swelling behavior of in situ formed gels is another important characteristic influencing the drug release behavior. Therefore, their water uptake kinetics was evaluated at predetermined time points at temperature of 37 °C. The water uptake was monitored over time until equilibrium with water was reached. The determined value represented the maximum water absorption, referred to as water uptake capacity, shown in . The obtained results showed that the water uptake of all in situ formed gels increased rapidly in the first hour upon contact with excess aqueous medium and then gradually leveled off. The equilibrium water absorption for (E/L)Go50, (E/L)Gl50, (E/L)Go60, (E/L)Gl60, and (E/L)Gl70 was determined at 24 hours, while for (E/L)Go70, (E/L)Go80, and (E/L)Gl80 the equilibrium with water was reached after 72 hours. When considering the water capacities of in situ formed gels, the obtained data indicated that the chosen lipid played a pivotal role in their swelling behavior. The lowest water capacity was determined for (E/L)Go50 (5.4%) and (E/L)Gl50 (2.9%) consisting of the highest proportion of glycerol monooleate and glycerol monolinoleate, respectively. Slightly higher water capacities were observed for (E/L)Go60 (15.2%), (E/L)Gl60 (12.1%), and (E/L)Gl70 (13.4%). These results are in good agreement with phase transition analysis within gelation test, where it has been shown that fan-like textures, indicating hexagonal LCCs, are continually present in these in situ formed gels. According to the literature, water channels within hexagonal mesophases are closed to the external environment, hence water diffusion is retarded (Chavda et al., ). Further, moderately higher water capacity was determined for (E/L)Go70 (25.2%), while (E/L)Go80 (82.2%) and (E/L)Gl80 (74.3%) stood out with the highest water capacity. Again, these results correlate well with phase transition analysis within gelation test, revealing that in addition to hexagonal LCCs, lamellar mesophases are also present in (E/L)Go80 and (E/L)Gl80. It is known that lamellar LCCs usually absorb more water (Alfutimie et al., ). When looking at all the results together, another interesting finding can be observed. Namely, in situ formed gels containing glycerol monooleate appeared to absorb a higher amount of water compared to those containing glycerol monolinoleate. This overall trend is important to note, as it seems to be also reflected in the results of the in vitro release testing presented later in the study. Differential scanning calorimetry DSC analysis was performed to elucidate intermolecular interactions and water state within in situ formed gels after reaching equilibrium with water. Evaluation was performed based on the crystallization (T c ) and melting (T m ) temperatures visible in the crystallization and the melting curves as well as the enthalpies of crystallization (ΔH c ) and melting (ΔH m ) derived by integrating the areas under the corresponding peaks in the DSC thermograms. Initially, assessment was carried out for individual compounds, i.e. ethanol, lecithin, glycerol monooleate, and glycerol monolinoleate, and bidistilled water. On the crystallization curves of individual components , no thermal events were observed for ethanol or lecithin. However, for glycerol monooleate, a minor broad exothermic peak at T c1 = 15.7 °C (ΔH c1 = 0.35 J/g) plus a noticeable exothermic peak at T c2 = −0.8 °C (ΔH c2 = 133.0 J/g) were detected. For glycerol monolinoleate, a small exothermic triple peak appeared (T c1 = −16.3 °C, ΔH c1 = 1.2 J/g, T c2 = −22.9 °C, ΔH c2 = 6.7 J/g, T c3 = −29.2 °C, ΔH c3 = 5.6 J/g). The observed peaks of both lipids can be attributed to the rearrangement and/or crystallization of glycerol monooleate and glycerol monolinoleate molecules, respectively (Chauhan et al., ). Linolenic acid (C 18:2 ) has one more double bond than oleic acid (C 18:1 ), which contributes to its higher degree of unsaturation and greater mobility. This increased mobility is evidenced by more crystallization peaks, as observed in the DSC thermograms (Nyame Mendendy Boussambe et al., ). Regarding bidistilled water, at T c1 = −20.1 °C (ΔH c1 = 239.6 J/g) a sharp exothermic peak appeared, coinciding with the crystallization of supercooled water. Next, on the melting curves of individual components , again no thermal events were observed for lecithin. Nevertheless, in case of ethanol an endothermic peak was detected at T m1 = 76.2 °C (ΔH m1 = −775.5 J/g) corresponding to its evaporation. In the case of lipid components, their melting was characterized by small double endothermic peaks. More specifically, at T m1 = 3.3 °C (ΔH m1 = −15.4 J/g) and T m2 = 13.7 °C (ΔH m2 = −12.6 J/g) for glycerol monooleate, and at T m1 = −15.6 °C (ΔH m1 = −13.5 J/g) and T m2 = 4.9 °C (ΔH m2 = −6.9 J/g) for glycerol monolinoleate. The thermal events of bidistilled water were observed at T m1 = −0.3 °C (ΔH m1 = −278.4 J/g), attributed to ice melting, followed by a broad endothermic peak at T m2 = 97.9 °C (ΔH m2 = −1709.2 J/g), ascribed to its evaporation. In the next step, evaluation of in situ formed gels after reaching equilibrium with water was performed with special attention given to the water state within them. Water, located near the polar heads of amphiphilic molecules in LCCs, exhibits different thermal properties due to interactions that reduce its degrees of freedom when compared to water that is more distant from the polar heads. Consequently, water molecules forming stronger interactions with amphiphilic molecules solidify at lower temperatures compared to water with weaker interactions, resulting in a lower enthalpy of freezing, sometimes even below the detection limit. Based on this water is classified as non-freezable, freezable interlamellar bound water, and freezable bulk water (Ezrahi et al., ). displays the crystallization curves and shows the crystallization enthalpies of in situ formed gels after equilibrium with water was reached. As regards the crystallization curves of (E/L)Go50 and (E/L)Gl50, two wide exothermic peaks in the range of T c = −18.5 °C to T c = −43.1 °C with small areas under the curve appeared. It seems plausible that herein most of the water was located around the polar headgroups of ethanol and lecithin, with almost no free water in the in situ formed gel. Further, in the case of (E/L)Go60, (E/L)Gl60, (E/L)Go70, and (E/L)Gl70, we detected two exothermic peaks between in the range of T c = −16.2 °C to T c = −44.7 °C, representing the crystallization of free water and bound water from the second hydration layer. In these in situ formed gels, water was present around the polar headgroups of ethanol and lecithin in addition to free water within the water channels, indicating that the polar headgroups were already saturated with water molecules. Further, it should be emphasized that certain differences were observed among (E/L)Go70 and the other listed in situ formed gels. Namely, area under the first exothermic peak at approximately −20 °C, attributed to free water within the system, was 3- to 5-times larger in the case of (E/L)Go70, which corresponds well with the results of water uptake evaluation and is also shown in the in vitro release testing presented later in the study. In regard to the crystallization curves of (E/L)Go80 and (E/L)Gl80, only one exothermic peak appeared at −22.6 °C and 22.8 °C, respectively, which in terms of the size of the area under the curve and shape, most closely resembles the reference peak of bidistilled water. It can be postulated that the polar headgroups of ethanol and lecithin are already fully saturated with water molecules of the first and the second hydration layer and that a significant amount of absorbed water is in the form of free water within the water channels. It seems plausible that this free water mostly belongs to lamellar mesophases, which were detected in addition to hexagonal LCCs by PLM analysis of these in situ formed gels. To note, all of these findings are in good agreement with results of gelation test and water uptake evaluation, which confirm the lowest water absorption of (E/L)Go50 and (E/L)Gl50, contrary to (E/L)Go80 and (E/L)Gl80 with the highest water uptake. shows the melting curves, and presents the melting enthalpies of in situ formed gels after equilibrium with water was reached. Melting of ice formed within the cooling cycle of the analysis was noted at approximately 0 °C. In addition, evaporation of ethanol was detected at approximately 78 °C, while water evaporated at approximately 100 °C. The obtained melting curves confirm trends observed from the crystallization curves, where the positions and areas under the curves were directly proportional to the content of absorbed water within in situ formed gels. Rheological measurements Microstructure evaluation of precursor formulations as well as in situ formed gels after reaching equilibrium with water was further upgraded by rheological tests, which provided additional insights into their flow behavior after applied stress as well as viscoelastic characteristics. These measurements contributed to a comprehensive understanding of the structural changes going along with sol–gel transition in addition to structural analyses performed using PLM and DSC. Firstly, rotational measurements were performed to elucidate the flow behavior of a system subjected to applied stress, offering an insight into microstructural alterations upon SC administration. The viscosity curves of all precursor formulations obtained at 25 °C demonstrated a constant viscosity regardless of increasing shear rate. This finding confirm that all precursor formulations exhibited Newtonian fluid behavior, a desirable feature for injectables designed for SC administration. When comparing the viscosities of precursor formulations at the lowest shear rate, a positive correlation between lipid content and viscosity was revealed. However, it is important to note that viscosities of all precursor formulations ranged from 17.0 cP to 36.9 cP, being far below 50 cP, therefore confirming their suitability for SC injection (Miller et al., ). Precursor formulations with glycerol monolinoleate as a lipid phase generally exhibited lower viscosities. Further, rotational tests were also performed for in situ formed gels after reaching equilibrium with water at a temperature of 37 °C. Their viscosities decreased with increasing shear rate until they reached a constant value at high shear rates, hence all in situ formed gels can be classified as non-Newtonian pseudoplastic systems, which is also consistent with our expectations. Upon evaluating the viscosities of in situ formed gels, the viscosity values of in situ formed gels were prominently higher when compared to precursor formulations. In addition, if negligible variations in viscosity values at the lowest shear rate (1 s −1 ) for precursor formulations were detected, notable variations were observed for in situ formed gels which can be explained with their spontaneous formation. Therefore, viscosities for in situ formed gels are presented at 2 s −1 with similar trend to that observed for their respective precursor formulations. More specifically, viscosity values ranged from 698.0 Pa·s to 111.9 Pa·s. In situ formed gels containing glycerol monolinoleate as a lipid phase in general exhibited lower viscosities. All of these results correlate well with gelation test and the corresponding PLM analysis. Further, oscillatory shear frequency sweep measurements were performed for in situ formed gels after reaching equilibrium with water at 37 °C , as they provide important information regarding the viscoelastic properties of a system corresponding to its network structure. Therefore, rheological parameters, including storage (G′) and loss (G″) moduli, as well as complex viscosity (η*), across various angular frequencies were recorded. The G′ modulus reflects elastic properties of a system, with high values demonstrating a system with strong elasticity and structure, while high G’’ values suggest a predominantly viscous, liquid-like material. Depending on the dominant modulus, a system can be classified as either elastic or viscous. The G’ modulus was generally higher than the G″ modulus with increasing frequency, whereas complex viscosity was decreasing with increasing frequency in case of all in situ formed gels, indicating predominantly elastic behavior. This is characteristic of gel-like systems and can be attributed to the well-organized microstructure of the LCCs. More specifically, in the case of (E/L)Go50, (E/L)Gl50, (E/L)Go60, (E/L)Gl60, and (E/L)Gl70, the G′ and G″ moduli were strongly enhanced with increasing frequency. This observed rheological pattern is representative of hexagonal LCCs (Xingqi et al., ) and is good agreement with PLM photomicrographs. Furthermore, similar curves were also observed for (E/L)Go70, whereby the G′ and G″ moduli were less enhanced with increasing frequency, indicating a less dense network of hexagonal LCCs. To note, less strong interactions between surfactant molecules and water were also confirmed by DSC measurements for this in situ formed gel. Next, the rheological behavior of (E/L)Go80 and (E/L)Gl80 also correlated well with the results of other assessments. Namely, in the case of these two in situ formed gels, both the G′ and G″ moduli were nearly independent of the angular frequency over the entire range investigated with the large gap between the both curves, suggesting the coexistence of hexagonal mesophases along with lamellar LCCs (Mistry et al., ), as we had also anticipated based on PLM analysis. In vitro release testing Selection of the release medium The newly developed in situ forming liquid crystalline systems were designed for the sustained release of the peptide drug Tα1 with inherent poor stability. Therefore, to ensure an optimal in vitro release testing experiment for the period of 2 weeks, preliminary studies were conducted to assess the influence of the release medium on the Tα1’s stability. In addition, given that the experiment was carried out at 37 °C and that the samples were stored for subsequent UHPLC analysis after sampling, the effect of storage temperature on the Tα1’s stability was also evaluated . Within the assessment of release media, we examined the Tα1’s stability regarding the absence (ultrapure water) or the presence of ions in various buffers (PBS, simulated body fluid), the pH value (6.8 and 7.4) and the proportion of ethanol (5%, 100% (m/m)). Additionally, as part of the temperature stability testing, the Tα1’s stability was evaluated at the following temperatures for all release media: −20 °C (freezer temperature), 8 °C (refrigerator temperature), 25 °C (room temperature), and 37 °C (body temperature). At −20 °C and 8 °C, the Tα1’s stability was adequate within all tested combinations of release media. However, evident differences in stability were observed at elevated temperatures. To note, we found that by adding a small proportion of ethanol improved stability of the peptide drug Tα1 in the release medium was obtained, proving its key influence on Tα1’s stability. Considering the observed finding and the literature data reporting that a slightly acidic pH improves Tα1’s stability (Dai et al., ), PBS (pH = 6.8) containing 5% (m/m) of ethanol was selected as the most appropriate release medium at all tested temperatures over the entire testing period. The potential effect of ethanol on in situ depot formation was investigated by PLM microstructural examination of the in situ formed gels exposed to the release medium containing 5% (m/m) ethanol after equilibrium with water was reached (data not shown). It has been demonstrated that this proportion of ethanol had no effect on depot formation. In vitro release of the Tα1 from in situ formed gels Achieving the sustained release of the peptide drug Tα1 was one of the pivotal aspects we focused on in the development of the in situ forming liquid crystalline systems in this study. Thus , in vitro release testing was performed to evaluate their potential for minimization of the Tα1’s dosing frequency that could greatly improve patient compliance upon clinical translation of the systems. displays the cumulative release of the peptide drug Tα1 in vitro from the in situ formed gels over a period of 2 weeks. All the studied in situ formed gels demonstrated the sustained release profiles; however, noticeable differences were observed among them. (E/L)Go80 and (E/L)Gl80 exhibited the greatest total drug release after 2 weeks with 84.2% and 93.4%, respectively. Further, (E/L)Go70 demonstrated 19.1% of released Tα1 after 2 weeks. It is important to note that this represented 2- to 4-times greater total drug release when compared to other in situ formed gels. Namely, they exhibited comparable amount of released drug after 2 weeks, being 8.4% for (E/L)Gl70, 8.3% for (E/L)Go50, 7.8% for (E/L)Gl50, 5.8% for (E/L)Gl60 and 5.5% (E/L)Go60. The observed differences can be explained by bidirectional relationship among variables influencing the drug release mechanism from LCCs. Specifically, the hydrophilic characteristics of the peptide drug Tα1 (Goldstein et al., ), which determine the affinity for the water channels of LCCs, as well as the composition and the microstructure of the LCCs with the interrelated water uptake capacity. It is known from the literature that the release of hydrophilic drugs from lamellar LCCs, which are in general more highly hydrated mesophases, is more rapid than from hexagonal LCCs with relatively low water absorption. This phenomenon can be attributed to an increase in the water channels available for release of hydrophilic drugs with increasing water content within the system (Borgheti-Cardoso et al., ; Elnaggar et al., ). In the present study, the coexistence of hexagonal mesophases along with lamellar LCCs was confirmed by PLM analysis and oscillatory measurements for (E/L)Go80 and (E/L)Gl80. Consequently, the water uptake capacity of (E/L)Go80 and (E/L)Gl80 was exceptionally high and the release was greater than that of the other gels formed in situ . In keeping with this, their release profiles were also consistent with the explanation provided above. Namely, the other in situ formed gels formed only hexagonal mesophases, resulting in their noticeably sustained release profiles. Among these, (E/L)Go70 demonstrated a moderately greater total drug release, which corresponded with its higher water uptake capacity and the associated larger proportion of free water, as confirmed by DSC measurements as well. In other words, larger amount of free water within water channels of hexagonal mesophases present in (E/L)Go70 enabled moderately greater release of the hydrophilic peptide drug Tα1. However, it is still necessary to take into account that (E/L)Go70 formed only hexagonal mesophases and that water channels within them are closed to the external environment, hence water diffusion is retarded (Chavda et al., ). Other in situ formed gels exhibiting solely hexagonal mesophases showed similar water uptake capacities and similar intermolecular network, as identified by DSC analysis, their amount of the released peptide drug Tα1 was comparable. Further, the peptide drug Tα1’s secondary structure using CD spectroscopy was examined. Considering the literature indicating that Tα1 is an intrinsically disordered peptide at neutral pH and body temperature in water, with various solvents capable of inducing structural changes (Hoch & Volk, ), its structural stability was systematically evaluated in different samples throughout processing. Supplementary Figure S3A shows the dichroic profile of the peptide drug Tα1 in ethanol for incorporation into formulation, indicating β-sheet conformation (Greenfield, ). Further, the CD spectrum of the peptide drug Tα1 in the release medium post-drug release testing, shown in Supplementary Figure S3B , indicates that the peptide adopted a random coil conformation in the aqueous environment, which aligns well with findings from (Grottesi et al., ). In addition, it also correlates with the CD spectra obtained for the dissolved lyophilisate of the peptide drug Tα1 in the release medium and in PBS itself ( Supplementary Figure S3C and S3D ). Taken together, these results confirm that the peptide drug Tα1 adopts and maintains its native conformation, characteristic of aqueous environment, in the release medium after the completion of the in vitro release testing. Notably, the conformational changes in different environments may serve as structural prerequisites for Tα1’s interaction with lymphocyte membranes, potentially representing the initial event in lymphocyte activation during immune response modulation, thereby highlighting the functional relevance (Grottesi et al., ). To conclude, the results of the in vitro release testing demonstrated that adjusting the composition of precursor formulations facilitates the regulation of in situ formed gels’ microstructure, thereby controlling the release profiles of the incorporated peptide drug Tα1. Furthermore, the release profiles obtained over a period of 2 weeks imply the potential of the in situ formed gels innovated in this study to prolong the peptide drug Tα1’s release and notably minimize its dosing frequency. Nevertheless, it is important to note that the % of the released peptide drug Tα1 increases only slightly after initial release observed in the first days of the in vitro release testing. A similar release behavior has also been reported for the peptide drug leuprolide acetate from liquid crystalline hexagonal mesophases (Báez-Santos et al., ). Upon administration, the SC tissue pressure along with flow of the SC interstitial fluid perfusing the in situ formed depots is expected to assist the erosion of the in situ formed gel matrix and enhance the drug release, though (Torres-Terán et al., ).
When developing a novel drug delivery system based on LCCs, phase behavior of systems with precisely defined components can be investigated using ternary or pseudoternary phase diagrams. In this study, four pseudoternary phase diagrams were constructed for the systems containing ethanol/glycerol monooleate/hydrophilic phase , ethanol/glycerol monolinoleate/hydrophilic phase , ethanol/lecithin/glycerol monooleate/hydrophilic phase , and ethanol/lecithin/glycerol monolinoleate/hydrophilic phase . Ethanol had a role of a hydrotropic substance for viscosity reduction (Ferreira et al., ) as well as peptide drug Tα1 stabilization. Lecithin was chosen as a biocompatible amphiphile capable of formation and stabilization of various LCCs mesophases depending on the remaining amphiphiles in the mix (Gosenca et al., ). Glycerol monooleate and glycerol monolinoleate were selected as hexagonal and/or cubic mesophases-forming amphiphilic lipids. They possess excellent biocompatible and biodegradable characteristics due to the presence of ester bonds in their structure, which undergo lipolytic degradation by endogenous lipases after SC injection (Zhang et al., ). Hydrophilic phase represented aqueous environment of the SC tissue. Macroscopic analysis of the systems formed across the constructed pseudoternary phase diagrams was initially performed. Different proportions and type of components resulted in formation of distinct systems. Transparent or semi-transparent and viscous gel-like systems were identified as LCCs. As our focus was on the formation of liquid crystalline mesophases, we did not explore in detail other regions of the diagrams where nonhomogeneous systems, coarse emulsions, or microemulsions were present. Pseudoternary phase diagram study showed that absence or presence of lecithin had a key influence on the formation of LCCs, while the type of lipid did not show any significant effect. Namely, in the diagrams from and employing only ethanol, lipid, and hydrophilic phase, a considerably smaller region of LCCs formation was observed compared to the diagrams from and utilizing ethanol/lecithin mixture, lipid, and hydrophilic phase. More specifically, in the case of the diagram from , adding hydrophilic phase at a level of 10–30% to precursor formulations containing 10–20% of ethanol resulted in the formation of LCCs. In parallel, a similarly small region of LCCs was distinguished in the diagram from , where addition of hydrophilic phase ranging from 50 to 70% to precursor formulations with 10–20% of ethanol led to LCCs formation. On the other hand, in the case of the diagrams from and , precursor formulations containing 50–80% of the ethanol/lecithin mixture formed a large region of LCCs after hydrophilic phase was added in the range of 10–90%. When comparing the systems across all diagrams from , it is important to note that the LCCs formed in the diagrams from and exhibited favorable macroscopic characteristics such as homogeneity and high viscosity.
Systems identified as potential LCCs based on macroscopic analysis, utilizing pseudoternary phase diagrams, underwent subsequent microscopic analysis using polarized light microscope at room (25 °C) and body (37 °C) temperature. The acquired observations are schematically presented in , while the representative photomicrographs are depicted in . PLM is one of the most commonly used methods for investigating LCCs mesophases, offering valuable insight into their molecular organization and phase transitions. When subjected to polarized light, anisotropic systems such as lamellar and hexagonal LCCs exhibit characteristic birefringent pattern. Maltese crosses together with oily streaks denote the presence of lamellar mesophases, while fan-like textures indicate formation of hexagonal mesophases. A dark background, with no birefringence, suggests the presence of cubic mesophases, known for their isotropic liquid behavior (Manaia et al., ; Zhang et al., ). First, evaluation of the systems was performed at a temperature of 25 °C. In the case of the systems from the first and the second diagram, the black view under polarized microscope pointed to the presence of cubic LCCs being in good agreement with the investigation by Mei et al. . In contrast, the phase behavior of the systems depicted in the third and fourth diagram exhibited distinctive characteristics of LCCs, confirming that lecithin has a key function in formation of these LCCs. Namely, numerous and pronounced fan-like textures started to emerge in the dark background from precursor formulations containing 50%, 60%, and 70% of the ethanol/lecithin mixture, respectively, independent of the hydrophilic phase content. This observation is imperative as it draws us to two important findings. Firstly, it indicates the presence of hexagonal LCCs and in some areas coexistence with cubic LCCs; nevertheless, hexagonal mesophases were strongly prevailing. Secondly, it implies that absorption of only a small amount of hydrophilic phase was necessary for these precursor formulations to form hexagonal LCCs in situ . Further, mixed phases of hexagonal LCCs together with lamellar LCCs were formed from precursor formulations containing 80% of the ethanol/lecithin mixture, respectively, but only after a larger addition of hydrophilic phase, i.e. 40–90%. It should be noted that, for precursor formulations containing 80% of the ethanol/lecithin mixture, fan-like textures were less numerous and pronounced as well as that individual Maltese crosses were also observed. Further, comparable results were obtained at a temperature of 37 °C for all four diagrams. The only exception was that precursor formulations from the third and the fourth diagram containing 80% of the ethanol/lecithin mixture already showed fan-like textures along with Maltese crosses when the hydrophilic phase content reached 10%. This indicates that higher temperature induces, to some extent, the formation of hexagonal LCCs together with lamellar LCCs, which is desirable for SC administration. Based on all the findings obtained from the pseudoternary phase construction (i.e. macroscopic appearance) and the corresponding phase behavior analysis (i.e. microstructure), eight precursor formulations, capable of in situ phase transition to hexagonal LCCs upon addition of water, were selected for further characterization studies. The composition of the selected studied precursor formulations is reported in and highlighted in .
Gelation time denotes the time required for a precursor formulation to transform into an in situ formed gel when exposed to excess aqueous medium (Mei et al., ). A rapid sol-gel transition is desired to minimize the possibility of initial burst release as in situ formation of gel retards the release of the incorporated drug. At room temperature, all precursor formulations were clear with good fluidity, but upon contact with aqueous medium heated to 37 °C, they quickly lost their flowability. Among all precursor formulations, a very short gelation time of a few seconds was measured for (E/L)Go50 (2.8 seconds), (E/L)Gl50 (2.5 seconds), (E/L)Go60 (4.1 seconds), and (E/L)Gl60 (3.6 seconds). Further, a slightly longer gelation time was measured for (E/L)Go70 (14.1 seconds) and (E/L)Gl70 (13.3 seconds). The longest sol-gel transition time was determined for (E/L)Go80 (70.0 seconds) and (E/L)Gl80 (45.4 seconds). These results indicate that all precursor formulations would spontaneously transform into an in situ gel at the site of administration upon exposure to the physiological fluid. However, it can be observed that there are certain differences among precursor formulations which are most likely related to their composition. Namely, increasing glycerol monooleate and glycerol monolinoleate content, respectively, leads to a decrease in gelation time, which is also consistent with the literature data (Mei et al., ). The phenomenon can be attributed to glycerol monooleate and glycerol monolinoleate being amphiphilic lipids capable of forming hexagonal LCCs. Consequently, they have the ability to rapidly induce the formation of these highly viscous mesophases.
The ability of a precursor formulation to form and maintain an in situ formed gel upon injection into excess aqueous medium heated to 37 °C was evaluated within gelation test. shows the visual appearance of in situ formed gels at selected predetermined time points, namely, immediately after injection, the initial and final time assessment points (1 hour and 14 days), the time points when equilibrium with water was established (24 and 72 hours), and the time point showing the most prominent morphological changes of in situ formed gels were observed (7 days). In addition, for better visualization, in situ formed gels in vials immediately after injection are also illustrated. Supplementary Figure S1 provides visual representation of in situ formed gels at other predetermined time points, namely 6, 12, 36, 48 hours, and 10 days. The intensity of color of all in situ formed gels was the highest at the first time point, i.e. 1 hour, due to the thorough uptake of the aqueous medium, which began immediately upon contact with it. The most compact forms with noticeably low quantity of uptook aqueous medium were formed by (E/L)Go50 and (E/L)Gl50, which, unlike the milky yellow (E/L)Go80 and (E/L)Gl80, were bright yellow. The least firm and visibly the biggest volumes of in situ formed gels were observed for (E/L)Go80 and (E/L)Gl80, as they uptook high quantity of aqueous medium. (E/L)Go60, (E/L)Gl60, (E/L)Go70, and (E/L)Gl70 typically represented an ‘intermediate stage”. They combined both clear and cloudy areas in color, and their consistency was softer than that of (E/L)Go50 and (E/L)Gl50 and firmer than that of (E/L)Go80 and (E/L)Gl80. With the exception of less intense color and slower process of swelling, after 6, 12, 24, 36, 48, and 72 hours, no significant changes in macroscopic appearance of all in situ formed gels were observed. However, after 7 days notable changes in color and consistency were detected for all in situ formed gels as they started to liquefy and decrease in their size. Over the following days, the process of liquification and erosion was slowly progressing. Interestingly, at the last time point, i.e. 14 days, remaining in situ formed gels of (E/L)Go80 and (E/L)Gl80 settled at the bottom of the vials, while remainings of the other systems were still floating. When comparing all precursor formulations, due to the yellow color of glycerol monooleate and glycerol monolinoleate, respectively, gels containing more lipid phase were more yellow. But what seems to be of major importance, we found that in situ formed gels with a higher lipid content were more compact and eroded more slowly, while in situ formed gels containing a higher amount of the ethanol/lecithin mixture were softer and degraded faster.
As the microstructure of in situ formed gel is a crucial factor influencing the drug release kinetics, the phase transition of precursor formulations upon contact with aqueous medium was monitored using PLM at 37 °C. The analysis was performed at the same time points as the gelation test was carried out. Photomicrographs obtained at the selected predetermine time points, i.e. 1, 24, 72 hours, and 7 and 14 days (see chapter Gelation test) are shown in . Supplementary Figure S2 provides photomicrographs taken at other predetermined time points, namely 6, 12, 36, 48 hours, and 10 days. The phase changes at post-hydration time of precursor formulations with excess aqueous medium revealed dynamic phase transitions, which were caused by rearrangement of molecules within precursor formulations. These microstructural changes can be understood in terms of aqueous self-assembly of amphiphile mixtures explained by their critical packing parameter (CPP). Namely, the self-assembly of single amphiphiles in aqueous medium is driven by a balance between the hydrophobic interactions of the tails and the geometrical packing constraints of the polar head groups. These factors are expressed as CPP = v/al , where v is the volume of the hydrophobic tail, a is the polar head group area, and l is the hydrophobic tail length of the amphiphilic molecule. As a guideline, amphiphiles with a CPP ∼1 usually self-assemble into lamellar LCCs (Engström & Engström, ), a CPP of ∼1.3 is characteristic for bicontinuous cubic mesophases, while amphiphiles with a CPP ∼1.7 form inverted hexagonal mesophases (Larsson, ). In regard to our results, clearly visible and numerous fan-like textures emerging from dark background, were observed for (E/L)Go50, (E/L)Gl50, (E/L)Go60, (E/L)Gl60, (E/L)Go70, and (E/L)Gl70 at the first time point of the assessment and they persisted until the conclusion of the analysis. It appears that hexagonal LCCs were quickly formed from these precursor formulations and that their microstructure was preserved until the final time point of the analysis. These findings can be attributed to the high content of glycerol monooleate and glycerol monolinoleate, respectively, in these precursor formulations. Namely, upon contact with aqueous medium, the polar head groups of the amphiphilic lipid from precursor formulations begin to move more freely. Consequently, these movements induce disorder in the hydrophobic chain of the amphiphilic lipid, leading to an increase of volume of the hydrophobic tail – v . However, the cross-sectional area of the polar head groups stays constant due to the strong hydrogen bonding. Therefore, CPP value increases as v increases and the polar head group area – a and the hydrophobic tail length of the amphiphilic molecule – l remain constant, thereby facilitating phase transition to hexagonal mesophases (Borgheti-Cardoso et al., ; Ferreira, ). When looking at (E/L)Go80 and (E/L)Gl80, hexagonal LCCs were also mainly present at all time points of the assessment. However, it should be noted that here fan-like structures were less pronounced. In addition, Maltese crosses indicative of lamellar LCCs were observed at the initial time point for (E/L)Go80 and (E/L)Gl80, with their presence slowly increasing throughout the analysis. Given that these precursor formulations contained a high content of lecithin/ethanol mixture, its effect was reflected in the resulting mesophases. Namely, a CPP value for lecithin, specifically for phosphatidylcholine as its main component, ranges from 0.5 to 1, meeting the requirement for bilayer formation of lamellar mesophases. Furthermore, ethanol molecules intercalated within phospholipid bilayers of lecithin additionally contributed to the lipid bilayer fluidity (Mkam Tsengam et al., ). As a result, in the case of the abovementioned precursor formulations, the self-assembly of lamellar mesophases was also observed along with formation of hexagonal mesophases.
Swelling behavior of in situ formed gels is another important characteristic influencing the drug release behavior. Therefore, their water uptake kinetics was evaluated at predetermined time points at temperature of 37 °C. The water uptake was monitored over time until equilibrium with water was reached. The determined value represented the maximum water absorption, referred to as water uptake capacity, shown in . The obtained results showed that the water uptake of all in situ formed gels increased rapidly in the first hour upon contact with excess aqueous medium and then gradually leveled off. The equilibrium water absorption for (E/L)Go50, (E/L)Gl50, (E/L)Go60, (E/L)Gl60, and (E/L)Gl70 was determined at 24 hours, while for (E/L)Go70, (E/L)Go80, and (E/L)Gl80 the equilibrium with water was reached after 72 hours. When considering the water capacities of in situ formed gels, the obtained data indicated that the chosen lipid played a pivotal role in their swelling behavior. The lowest water capacity was determined for (E/L)Go50 (5.4%) and (E/L)Gl50 (2.9%) consisting of the highest proportion of glycerol monooleate and glycerol monolinoleate, respectively. Slightly higher water capacities were observed for (E/L)Go60 (15.2%), (E/L)Gl60 (12.1%), and (E/L)Gl70 (13.4%). These results are in good agreement with phase transition analysis within gelation test, where it has been shown that fan-like textures, indicating hexagonal LCCs, are continually present in these in situ formed gels. According to the literature, water channels within hexagonal mesophases are closed to the external environment, hence water diffusion is retarded (Chavda et al., ). Further, moderately higher water capacity was determined for (E/L)Go70 (25.2%), while (E/L)Go80 (82.2%) and (E/L)Gl80 (74.3%) stood out with the highest water capacity. Again, these results correlate well with phase transition analysis within gelation test, revealing that in addition to hexagonal LCCs, lamellar mesophases are also present in (E/L)Go80 and (E/L)Gl80. It is known that lamellar LCCs usually absorb more water (Alfutimie et al., ). When looking at all the results together, another interesting finding can be observed. Namely, in situ formed gels containing glycerol monooleate appeared to absorb a higher amount of water compared to those containing glycerol monolinoleate. This overall trend is important to note, as it seems to be also reflected in the results of the in vitro release testing presented later in the study.
DSC analysis was performed to elucidate intermolecular interactions and water state within in situ formed gels after reaching equilibrium with water. Evaluation was performed based on the crystallization (T c ) and melting (T m ) temperatures visible in the crystallization and the melting curves as well as the enthalpies of crystallization (ΔH c ) and melting (ΔH m ) derived by integrating the areas under the corresponding peaks in the DSC thermograms. Initially, assessment was carried out for individual compounds, i.e. ethanol, lecithin, glycerol monooleate, and glycerol monolinoleate, and bidistilled water. On the crystallization curves of individual components , no thermal events were observed for ethanol or lecithin. However, for glycerol monooleate, a minor broad exothermic peak at T c1 = 15.7 °C (ΔH c1 = 0.35 J/g) plus a noticeable exothermic peak at T c2 = −0.8 °C (ΔH c2 = 133.0 J/g) were detected. For glycerol monolinoleate, a small exothermic triple peak appeared (T c1 = −16.3 °C, ΔH c1 = 1.2 J/g, T c2 = −22.9 °C, ΔH c2 = 6.7 J/g, T c3 = −29.2 °C, ΔH c3 = 5.6 J/g). The observed peaks of both lipids can be attributed to the rearrangement and/or crystallization of glycerol monooleate and glycerol monolinoleate molecules, respectively (Chauhan et al., ). Linolenic acid (C 18:2 ) has one more double bond than oleic acid (C 18:1 ), which contributes to its higher degree of unsaturation and greater mobility. This increased mobility is evidenced by more crystallization peaks, as observed in the DSC thermograms (Nyame Mendendy Boussambe et al., ). Regarding bidistilled water, at T c1 = −20.1 °C (ΔH c1 = 239.6 J/g) a sharp exothermic peak appeared, coinciding with the crystallization of supercooled water. Next, on the melting curves of individual components , again no thermal events were observed for lecithin. Nevertheless, in case of ethanol an endothermic peak was detected at T m1 = 76.2 °C (ΔH m1 = −775.5 J/g) corresponding to its evaporation. In the case of lipid components, their melting was characterized by small double endothermic peaks. More specifically, at T m1 = 3.3 °C (ΔH m1 = −15.4 J/g) and T m2 = 13.7 °C (ΔH m2 = −12.6 J/g) for glycerol monooleate, and at T m1 = −15.6 °C (ΔH m1 = −13.5 J/g) and T m2 = 4.9 °C (ΔH m2 = −6.9 J/g) for glycerol monolinoleate. The thermal events of bidistilled water were observed at T m1 = −0.3 °C (ΔH m1 = −278.4 J/g), attributed to ice melting, followed by a broad endothermic peak at T m2 = 97.9 °C (ΔH m2 = −1709.2 J/g), ascribed to its evaporation. In the next step, evaluation of in situ formed gels after reaching equilibrium with water was performed with special attention given to the water state within them. Water, located near the polar heads of amphiphilic molecules in LCCs, exhibits different thermal properties due to interactions that reduce its degrees of freedom when compared to water that is more distant from the polar heads. Consequently, water molecules forming stronger interactions with amphiphilic molecules solidify at lower temperatures compared to water with weaker interactions, resulting in a lower enthalpy of freezing, sometimes even below the detection limit. Based on this water is classified as non-freezable, freezable interlamellar bound water, and freezable bulk water (Ezrahi et al., ). displays the crystallization curves and shows the crystallization enthalpies of in situ formed gels after equilibrium with water was reached. As regards the crystallization curves of (E/L)Go50 and (E/L)Gl50, two wide exothermic peaks in the range of T c = −18.5 °C to T c = −43.1 °C with small areas under the curve appeared. It seems plausible that herein most of the water was located around the polar headgroups of ethanol and lecithin, with almost no free water in the in situ formed gel. Further, in the case of (E/L)Go60, (E/L)Gl60, (E/L)Go70, and (E/L)Gl70, we detected two exothermic peaks between in the range of T c = −16.2 °C to T c = −44.7 °C, representing the crystallization of free water and bound water from the second hydration layer. In these in situ formed gels, water was present around the polar headgroups of ethanol and lecithin in addition to free water within the water channels, indicating that the polar headgroups were already saturated with water molecules. Further, it should be emphasized that certain differences were observed among (E/L)Go70 and the other listed in situ formed gels. Namely, area under the first exothermic peak at approximately −20 °C, attributed to free water within the system, was 3- to 5-times larger in the case of (E/L)Go70, which corresponds well with the results of water uptake evaluation and is also shown in the in vitro release testing presented later in the study. In regard to the crystallization curves of (E/L)Go80 and (E/L)Gl80, only one exothermic peak appeared at −22.6 °C and 22.8 °C, respectively, which in terms of the size of the area under the curve and shape, most closely resembles the reference peak of bidistilled water. It can be postulated that the polar headgroups of ethanol and lecithin are already fully saturated with water molecules of the first and the second hydration layer and that a significant amount of absorbed water is in the form of free water within the water channels. It seems plausible that this free water mostly belongs to lamellar mesophases, which were detected in addition to hexagonal LCCs by PLM analysis of these in situ formed gels. To note, all of these findings are in good agreement with results of gelation test and water uptake evaluation, which confirm the lowest water absorption of (E/L)Go50 and (E/L)Gl50, contrary to (E/L)Go80 and (E/L)Gl80 with the highest water uptake. shows the melting curves, and presents the melting enthalpies of in situ formed gels after equilibrium with water was reached. Melting of ice formed within the cooling cycle of the analysis was noted at approximately 0 °C. In addition, evaporation of ethanol was detected at approximately 78 °C, while water evaporated at approximately 100 °C. The obtained melting curves confirm trends observed from the crystallization curves, where the positions and areas under the curves were directly proportional to the content of absorbed water within in situ formed gels.
Microstructure evaluation of precursor formulations as well as in situ formed gels after reaching equilibrium with water was further upgraded by rheological tests, which provided additional insights into their flow behavior after applied stress as well as viscoelastic characteristics. These measurements contributed to a comprehensive understanding of the structural changes going along with sol–gel transition in addition to structural analyses performed using PLM and DSC. Firstly, rotational measurements were performed to elucidate the flow behavior of a system subjected to applied stress, offering an insight into microstructural alterations upon SC administration. The viscosity curves of all precursor formulations obtained at 25 °C demonstrated a constant viscosity regardless of increasing shear rate. This finding confirm that all precursor formulations exhibited Newtonian fluid behavior, a desirable feature for injectables designed for SC administration. When comparing the viscosities of precursor formulations at the lowest shear rate, a positive correlation between lipid content and viscosity was revealed. However, it is important to note that viscosities of all precursor formulations ranged from 17.0 cP to 36.9 cP, being far below 50 cP, therefore confirming their suitability for SC injection (Miller et al., ). Precursor formulations with glycerol monolinoleate as a lipid phase generally exhibited lower viscosities. Further, rotational tests were also performed for in situ formed gels after reaching equilibrium with water at a temperature of 37 °C. Their viscosities decreased with increasing shear rate until they reached a constant value at high shear rates, hence all in situ formed gels can be classified as non-Newtonian pseudoplastic systems, which is also consistent with our expectations. Upon evaluating the viscosities of in situ formed gels, the viscosity values of in situ formed gels were prominently higher when compared to precursor formulations. In addition, if negligible variations in viscosity values at the lowest shear rate (1 s −1 ) for precursor formulations were detected, notable variations were observed for in situ formed gels which can be explained with their spontaneous formation. Therefore, viscosities for in situ formed gels are presented at 2 s −1 with similar trend to that observed for their respective precursor formulations. More specifically, viscosity values ranged from 698.0 Pa·s to 111.9 Pa·s. In situ formed gels containing glycerol monolinoleate as a lipid phase in general exhibited lower viscosities. All of these results correlate well with gelation test and the corresponding PLM analysis. Further, oscillatory shear frequency sweep measurements were performed for in situ formed gels after reaching equilibrium with water at 37 °C , as they provide important information regarding the viscoelastic properties of a system corresponding to its network structure. Therefore, rheological parameters, including storage (G′) and loss (G″) moduli, as well as complex viscosity (η*), across various angular frequencies were recorded. The G′ modulus reflects elastic properties of a system, with high values demonstrating a system with strong elasticity and structure, while high G’’ values suggest a predominantly viscous, liquid-like material. Depending on the dominant modulus, a system can be classified as either elastic or viscous. The G’ modulus was generally higher than the G″ modulus with increasing frequency, whereas complex viscosity was decreasing with increasing frequency in case of all in situ formed gels, indicating predominantly elastic behavior. This is characteristic of gel-like systems and can be attributed to the well-organized microstructure of the LCCs. More specifically, in the case of (E/L)Go50, (E/L)Gl50, (E/L)Go60, (E/L)Gl60, and (E/L)Gl70, the G′ and G″ moduli were strongly enhanced with increasing frequency. This observed rheological pattern is representative of hexagonal LCCs (Xingqi et al., ) and is good agreement with PLM photomicrographs. Furthermore, similar curves were also observed for (E/L)Go70, whereby the G′ and G″ moduli were less enhanced with increasing frequency, indicating a less dense network of hexagonal LCCs. To note, less strong interactions between surfactant molecules and water were also confirmed by DSC measurements for this in situ formed gel. Next, the rheological behavior of (E/L)Go80 and (E/L)Gl80 also correlated well with the results of other assessments. Namely, in the case of these two in situ formed gels, both the G′ and G″ moduli were nearly independent of the angular frequency over the entire range investigated with the large gap between the both curves, suggesting the coexistence of hexagonal mesophases along with lamellar LCCs (Mistry et al., ), as we had also anticipated based on PLM analysis.
Selection of the release medium The newly developed in situ forming liquid crystalline systems were designed for the sustained release of the peptide drug Tα1 with inherent poor stability. Therefore, to ensure an optimal in vitro release testing experiment for the period of 2 weeks, preliminary studies were conducted to assess the influence of the release medium on the Tα1’s stability. In addition, given that the experiment was carried out at 37 °C and that the samples were stored for subsequent UHPLC analysis after sampling, the effect of storage temperature on the Tα1’s stability was also evaluated . Within the assessment of release media, we examined the Tα1’s stability regarding the absence (ultrapure water) or the presence of ions in various buffers (PBS, simulated body fluid), the pH value (6.8 and 7.4) and the proportion of ethanol (5%, 100% (m/m)). Additionally, as part of the temperature stability testing, the Tα1’s stability was evaluated at the following temperatures for all release media: −20 °C (freezer temperature), 8 °C (refrigerator temperature), 25 °C (room temperature), and 37 °C (body temperature). At −20 °C and 8 °C, the Tα1’s stability was adequate within all tested combinations of release media. However, evident differences in stability were observed at elevated temperatures. To note, we found that by adding a small proportion of ethanol improved stability of the peptide drug Tα1 in the release medium was obtained, proving its key influence on Tα1’s stability. Considering the observed finding and the literature data reporting that a slightly acidic pH improves Tα1’s stability (Dai et al., ), PBS (pH = 6.8) containing 5% (m/m) of ethanol was selected as the most appropriate release medium at all tested temperatures over the entire testing period. The potential effect of ethanol on in situ depot formation was investigated by PLM microstructural examination of the in situ formed gels exposed to the release medium containing 5% (m/m) ethanol after equilibrium with water was reached (data not shown). It has been demonstrated that this proportion of ethanol had no effect on depot formation. In vitro release of the Tα1 from in situ formed gels Achieving the sustained release of the peptide drug Tα1 was one of the pivotal aspects we focused on in the development of the in situ forming liquid crystalline systems in this study. Thus , in vitro release testing was performed to evaluate their potential for minimization of the Tα1’s dosing frequency that could greatly improve patient compliance upon clinical translation of the systems. displays the cumulative release of the peptide drug Tα1 in vitro from the in situ formed gels over a period of 2 weeks. All the studied in situ formed gels demonstrated the sustained release profiles; however, noticeable differences were observed among them. (E/L)Go80 and (E/L)Gl80 exhibited the greatest total drug release after 2 weeks with 84.2% and 93.4%, respectively. Further, (E/L)Go70 demonstrated 19.1% of released Tα1 after 2 weeks. It is important to note that this represented 2- to 4-times greater total drug release when compared to other in situ formed gels. Namely, they exhibited comparable amount of released drug after 2 weeks, being 8.4% for (E/L)Gl70, 8.3% for (E/L)Go50, 7.8% for (E/L)Gl50, 5.8% for (E/L)Gl60 and 5.5% (E/L)Go60. The observed differences can be explained by bidirectional relationship among variables influencing the drug release mechanism from LCCs. Specifically, the hydrophilic characteristics of the peptide drug Tα1 (Goldstein et al., ), which determine the affinity for the water channels of LCCs, as well as the composition and the microstructure of the LCCs with the interrelated water uptake capacity. It is known from the literature that the release of hydrophilic drugs from lamellar LCCs, which are in general more highly hydrated mesophases, is more rapid than from hexagonal LCCs with relatively low water absorption. This phenomenon can be attributed to an increase in the water channels available for release of hydrophilic drugs with increasing water content within the system (Borgheti-Cardoso et al., ; Elnaggar et al., ). In the present study, the coexistence of hexagonal mesophases along with lamellar LCCs was confirmed by PLM analysis and oscillatory measurements for (E/L)Go80 and (E/L)Gl80. Consequently, the water uptake capacity of (E/L)Go80 and (E/L)Gl80 was exceptionally high and the release was greater than that of the other gels formed in situ . In keeping with this, their release profiles were also consistent with the explanation provided above. Namely, the other in situ formed gels formed only hexagonal mesophases, resulting in their noticeably sustained release profiles. Among these, (E/L)Go70 demonstrated a moderately greater total drug release, which corresponded with its higher water uptake capacity and the associated larger proportion of free water, as confirmed by DSC measurements as well. In other words, larger amount of free water within water channels of hexagonal mesophases present in (E/L)Go70 enabled moderately greater release of the hydrophilic peptide drug Tα1. However, it is still necessary to take into account that (E/L)Go70 formed only hexagonal mesophases and that water channels within them are closed to the external environment, hence water diffusion is retarded (Chavda et al., ). Other in situ formed gels exhibiting solely hexagonal mesophases showed similar water uptake capacities and similar intermolecular network, as identified by DSC analysis, their amount of the released peptide drug Tα1 was comparable. Further, the peptide drug Tα1’s secondary structure using CD spectroscopy was examined. Considering the literature indicating that Tα1 is an intrinsically disordered peptide at neutral pH and body temperature in water, with various solvents capable of inducing structural changes (Hoch & Volk, ), its structural stability was systematically evaluated in different samples throughout processing. Supplementary Figure S3A shows the dichroic profile of the peptide drug Tα1 in ethanol for incorporation into formulation, indicating β-sheet conformation (Greenfield, ). Further, the CD spectrum of the peptide drug Tα1 in the release medium post-drug release testing, shown in Supplementary Figure S3B , indicates that the peptide adopted a random coil conformation in the aqueous environment, which aligns well with findings from (Grottesi et al., ). In addition, it also correlates with the CD spectra obtained for the dissolved lyophilisate of the peptide drug Tα1 in the release medium and in PBS itself ( Supplementary Figure S3C and S3D ). Taken together, these results confirm that the peptide drug Tα1 adopts and maintains its native conformation, characteristic of aqueous environment, in the release medium after the completion of the in vitro release testing. Notably, the conformational changes in different environments may serve as structural prerequisites for Tα1’s interaction with lymphocyte membranes, potentially representing the initial event in lymphocyte activation during immune response modulation, thereby highlighting the functional relevance (Grottesi et al., ). To conclude, the results of the in vitro release testing demonstrated that adjusting the composition of precursor formulations facilitates the regulation of in situ formed gels’ microstructure, thereby controlling the release profiles of the incorporated peptide drug Tα1. Furthermore, the release profiles obtained over a period of 2 weeks imply the potential of the in situ formed gels innovated in this study to prolong the peptide drug Tα1’s release and notably minimize its dosing frequency. Nevertheless, it is important to note that the % of the released peptide drug Tα1 increases only slightly after initial release observed in the first days of the in vitro release testing. A similar release behavior has also been reported for the peptide drug leuprolide acetate from liquid crystalline hexagonal mesophases (Báez-Santos et al., ). Upon administration, the SC tissue pressure along with flow of the SC interstitial fluid perfusing the in situ formed depots is expected to assist the erosion of the in situ formed gel matrix and enhance the drug release, though (Torres-Terán et al., ).
The newly developed in situ forming liquid crystalline systems were designed for the sustained release of the peptide drug Tα1 with inherent poor stability. Therefore, to ensure an optimal in vitro release testing experiment for the period of 2 weeks, preliminary studies were conducted to assess the influence of the release medium on the Tα1’s stability. In addition, given that the experiment was carried out at 37 °C and that the samples were stored for subsequent UHPLC analysis after sampling, the effect of storage temperature on the Tα1’s stability was also evaluated . Within the assessment of release media, we examined the Tα1’s stability regarding the absence (ultrapure water) or the presence of ions in various buffers (PBS, simulated body fluid), the pH value (6.8 and 7.4) and the proportion of ethanol (5%, 100% (m/m)). Additionally, as part of the temperature stability testing, the Tα1’s stability was evaluated at the following temperatures for all release media: −20 °C (freezer temperature), 8 °C (refrigerator temperature), 25 °C (room temperature), and 37 °C (body temperature). At −20 °C and 8 °C, the Tα1’s stability was adequate within all tested combinations of release media. However, evident differences in stability were observed at elevated temperatures. To note, we found that by adding a small proportion of ethanol improved stability of the peptide drug Tα1 in the release medium was obtained, proving its key influence on Tα1’s stability. Considering the observed finding and the literature data reporting that a slightly acidic pH improves Tα1’s stability (Dai et al., ), PBS (pH = 6.8) containing 5% (m/m) of ethanol was selected as the most appropriate release medium at all tested temperatures over the entire testing period. The potential effect of ethanol on in situ depot formation was investigated by PLM microstructural examination of the in situ formed gels exposed to the release medium containing 5% (m/m) ethanol after equilibrium with water was reached (data not shown). It has been demonstrated that this proportion of ethanol had no effect on depot formation.
Achieving the sustained release of the peptide drug Tα1 was one of the pivotal aspects we focused on in the development of the in situ forming liquid crystalline systems in this study. Thus , in vitro release testing was performed to evaluate their potential for minimization of the Tα1’s dosing frequency that could greatly improve patient compliance upon clinical translation of the systems. displays the cumulative release of the peptide drug Tα1 in vitro from the in situ formed gels over a period of 2 weeks. All the studied in situ formed gels demonstrated the sustained release profiles; however, noticeable differences were observed among them. (E/L)Go80 and (E/L)Gl80 exhibited the greatest total drug release after 2 weeks with 84.2% and 93.4%, respectively. Further, (E/L)Go70 demonstrated 19.1% of released Tα1 after 2 weeks. It is important to note that this represented 2- to 4-times greater total drug release when compared to other in situ formed gels. Namely, they exhibited comparable amount of released drug after 2 weeks, being 8.4% for (E/L)Gl70, 8.3% for (E/L)Go50, 7.8% for (E/L)Gl50, 5.8% for (E/L)Gl60 and 5.5% (E/L)Go60. The observed differences can be explained by bidirectional relationship among variables influencing the drug release mechanism from LCCs. Specifically, the hydrophilic characteristics of the peptide drug Tα1 (Goldstein et al., ), which determine the affinity for the water channels of LCCs, as well as the composition and the microstructure of the LCCs with the interrelated water uptake capacity. It is known from the literature that the release of hydrophilic drugs from lamellar LCCs, which are in general more highly hydrated mesophases, is more rapid than from hexagonal LCCs with relatively low water absorption. This phenomenon can be attributed to an increase in the water channels available for release of hydrophilic drugs with increasing water content within the system (Borgheti-Cardoso et al., ; Elnaggar et al., ). In the present study, the coexistence of hexagonal mesophases along with lamellar LCCs was confirmed by PLM analysis and oscillatory measurements for (E/L)Go80 and (E/L)Gl80. Consequently, the water uptake capacity of (E/L)Go80 and (E/L)Gl80 was exceptionally high and the release was greater than that of the other gels formed in situ . In keeping with this, their release profiles were also consistent with the explanation provided above. Namely, the other in situ formed gels formed only hexagonal mesophases, resulting in their noticeably sustained release profiles. Among these, (E/L)Go70 demonstrated a moderately greater total drug release, which corresponded with its higher water uptake capacity and the associated larger proportion of free water, as confirmed by DSC measurements as well. In other words, larger amount of free water within water channels of hexagonal mesophases present in (E/L)Go70 enabled moderately greater release of the hydrophilic peptide drug Tα1. However, it is still necessary to take into account that (E/L)Go70 formed only hexagonal mesophases and that water channels within them are closed to the external environment, hence water diffusion is retarded (Chavda et al., ). Other in situ formed gels exhibiting solely hexagonal mesophases showed similar water uptake capacities and similar intermolecular network, as identified by DSC analysis, their amount of the released peptide drug Tα1 was comparable. Further, the peptide drug Tα1’s secondary structure using CD spectroscopy was examined. Considering the literature indicating that Tα1 is an intrinsically disordered peptide at neutral pH and body temperature in water, with various solvents capable of inducing structural changes (Hoch & Volk, ), its structural stability was systematically evaluated in different samples throughout processing. Supplementary Figure S3A shows the dichroic profile of the peptide drug Tα1 in ethanol for incorporation into formulation, indicating β-sheet conformation (Greenfield, ). Further, the CD spectrum of the peptide drug Tα1 in the release medium post-drug release testing, shown in Supplementary Figure S3B , indicates that the peptide adopted a random coil conformation in the aqueous environment, which aligns well with findings from (Grottesi et al., ). In addition, it also correlates with the CD spectra obtained for the dissolved lyophilisate of the peptide drug Tα1 in the release medium and in PBS itself ( Supplementary Figure S3C and S3D ). Taken together, these results confirm that the peptide drug Tα1 adopts and maintains its native conformation, characteristic of aqueous environment, in the release medium after the completion of the in vitro release testing. Notably, the conformational changes in different environments may serve as structural prerequisites for Tα1’s interaction with lymphocyte membranes, potentially representing the initial event in lymphocyte activation during immune response modulation, thereby highlighting the functional relevance (Grottesi et al., ). To conclude, the results of the in vitro release testing demonstrated that adjusting the composition of precursor formulations facilitates the regulation of in situ formed gels’ microstructure, thereby controlling the release profiles of the incorporated peptide drug Tα1. Furthermore, the release profiles obtained over a period of 2 weeks imply the potential of the in situ formed gels innovated in this study to prolong the peptide drug Tα1’s release and notably minimize its dosing frequency. Nevertheless, it is important to note that the % of the released peptide drug Tα1 increases only slightly after initial release observed in the first days of the in vitro release testing. A similar release behavior has also been reported for the peptide drug leuprolide acetate from liquid crystalline hexagonal mesophases (Báez-Santos et al., ). Upon administration, the SC tissue pressure along with flow of the SC interstitial fluid perfusing the in situ formed depots is expected to assist the erosion of the in situ formed gel matrix and enhance the drug release, though (Torres-Terán et al., ).
This study has shown that lyotropic liquid crystals represent a flexible and versatile platform that enables the regulation of macro and microstructure, and thereby the release profile and overall performance, through minimal adjustments in the component ratios. We report here the development of an in situ forming system for SC administration for potential sustained release of the peptide drug Tα1. Through a systematic design, we accomplished all the main objectives that we set at the beginning of the study. Firstly, nonaqueous precursor formulations demonstrated optimal rheological properties for SC injection. Further, an easy and quick in situ phase transition of precursor formulations to hexagonal LCCs was obtained. The change was triggered by water absorption, which represents the least invasive stimulus for phase transition occurrence. Finally, the obtained release kinetics of the peptide drug Tα1 from in situ formed gels imply a prolonged release behavior that could notably minimize its dosing frequency. These results highlight the great potential of the newly developed in situ forming liquid crystalline systems as injectable long-acting depots for SC administration of the peptide drug Tα1, promoting patient adherence.
Supplementary_material.docx
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Health literacy and frailty: the mediating role of instrumental activities of daily living | e7b20a00-279b-4a32-8a91-923f88bd194c | 11810535 | Health Literacy[mh] | Populations across the world are ageing at an increasing rate, especially in Japan, which has the fastest ageing society in the world and high prevalence of frailty. , Thus, frailty is a significant social and medical issue, characterised by age‐related decline in physiological reserves, leading to adverse outcomes, including disability and death, often triggered by mild stress. , Early diagnosis and treatment may reverse frailty progression, highlighting the need for timely identification and intervention. As per the World Health Organization ‘Health literacy represents the cognitive and social skills which determine the motivation and ability of individuals to gain access to, understand and use information in ways which promote and maintain good health.’ Thus, health literacy has three levels: (1) functional—basic reading and writing skills; (2) communicative—applying new information to changing circumstances; and (3) critical—analyzing and using information to manage different circumstances. Low health literacy leads to increased healthcare utilisation and costs, while higher health literacy enables better information gathering and healthier behaviours. In older adults, higher health literacy is associated with fewer chronic conditions and better physical function; thus, health literacy may play a critical role in health management and quality of life maintenance. Despite these established associations, the direct relationship between health literacy and frailty remains understudied. A previous study demonstrated that higher health literacy was associated with a lower risk of frailty progression over 4 years. However, this study only used the Kihon Checklist as a frailty assessment, without incorporating physical frailty assessments such as the well‐validated Japanese version of the Cardiovascular Health Study (J‐CHS) criteria, which is considered the gold standard for frailty evaluation. Another study reported that limited health literacy increased the risk of pre‐frailty by 1.4 times over a period of 2 years; however, the study focused only on functional literacy. The role of health literacy in the management of older adults' health has been increasingly recognised. However, the mechanisms involved in the effect of health literacy on frailty progression are still not fully understood. Specifically, the role of instrumental activities of daily living (IADL) in the effect of health literacy on frailty, remains underexplored. IADL refers to a broader range of daily activities that reflect physical and social independence and has been reported to be associated with both health literacy and frailty. , , IADL decline increases frailty risk and impairs physical function. Higher health literacy may contribute to the maintenance and improvement of ADLs, thus, delaying frailty progression. However, the role of IADL as an associated factor or mediator in the relationship between health literacy and frailty, remains unclear. Understanding the role of IADL in this relationship is critical for the development of better frailty prevention and management strategies. Furthermore, focusing on communicative and critical health literacy (CCHL), rather than only functional literacy, may provide deeper insights into improving health management and preventing frailty. We hypothesised that: (1) older adults with lower CCHL literacy would show poorer frailty outcomes after 1 year, independent of potential confounders; and (2) IADL would serve as a mediating factor in the relationship between health literacy and frailty progression. Therefore, the aim of this study was to investigate the impact of CCHL on the progression of pre‐frailty and frailty in older adults over 1 year. We also characterised the role of IADL in this relationship. Study design and participants In this retrospective cohort study, data from surveys conducted for the Matsumoto City Frailty Prevention Project in 2021 and 2022 were used to assess the factors contributing to frailty progression. The program included a single frailty assessment and a one‐time frailty prevention lecture. The lecture provided information on key aspects of frailty prevention, including exercise, nutrition, medication management, and oral health. All participants attended both the assessment and the lecture, ensuring full participation in the program. The survey was focused on older adults residing in Matsumoto City, Nagano Prefecture, Japan. We included individuals residing in Matsumoto City aged ≥65 years who participated in frailty and health literacy assessments at community gathering places in Matsumoto City in 2021. The participants attended a 1‐day nutritional and exercise guidance session or a frailty‐related medication lecture. The inclusion criteria were: (1) aged ≥65 years; and (2) participation in both frailty and health literacy assessments at baseline. The exclusion criterion was non‐participation in the frailty assessment at the 1‐year follow‐up. The participant recruitment flowchart is presented in Fig. . The study was approved by the ethics committee of Matsumoto City Hospital (protocol number, 03–5; approval date, 22/06/2021), and conducted as per the guidelines of the Declaration of Helsinki. In this observational study, the data were anonymised and obtained from non‐invasive assessments conducted at community gathering places; therefore, the need for individual participant consent was waived. However, the participants were informed about the study and provided relevant information. Furthermore, the participants were informed that they could withdraw from the study at any time. Measurement Baseline assessment Health literacy was measured using the CCHL scale. This scale measures three aspects of health literacy, including ability to access, understand, and use health information. The participants were enquired about their ability to: (1) obtain health‐related information from various sources; (2) extract the required information; (3) understand and communicate the information obtained; (4) assess the reliability of the information; and (5) make decisions based on the information, specifically in the context of health‐related issues. Each item was rated on a five‐point Likert scale: 1 (strongly disagree); 2 (somewhat disagree); 3 (undecided); 4 (somewhat agree); and 5 (strongly agree). The scores of the five items were then averaged. Participants were classified into two groups based on their average score into high health literacy (high HL; with an average score of ≥4) and low health literacy (low HL; with an average score of <4) groups. This classification was based on a previous study that reported that the median health literacy score of the population was 4. Using this threshold, we aligned our classification with established research practices. Other measures assessed in the study were basic demographics (age, sex, and years of education) and the number of chronic diseases (heart disease, cerebrovascular disease, respiratory disease, diabetes, cancer, rheumatoid arthritis, and osteoarthritis). The years of education were categorised into four groups (Group 1–reference, approximately 9 years; Group 2, approximately 12 years; Group 3, approximately 15 years; and Group 4, others). Chronic diseases were defined as “yes” if diagnosed by a physician. Subjective cognitive function was assessed using three questions from the Kihon Checklist: “Do people around you say you keep repeating the same questions or make similar comments about your forgetfulness?”, “Can you look up phone numbers to make phone calls by yourself?”, and “Do you find yourself not knowing today's date?” Subjective cognitive decline was defined as at least one negative response to these questions. Social factors, including living arrangements (living alone or not) and employment status (currently employed or not), were also assessed. Additionally, at baseline, participants were assessed using the Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG‐IC) (five instrumental ADLs items) for IADL. If the answer was “yes,” one point was added; if the answer was “no,” zero points were added. The total score ranged 0–5, with higher scores indicating greater functional abilities. Normal gait speed and grip strength were assessed as indicators of physical function. Gait speed was calculated based on the time(s) required to walk a distance of 5 m. The participants were instructed to walk at a comfortable pace in an unobstructed hallway. Patients were permitted to use assistive devices including walkers and canes. Grip strength was measured on both sides of the body and calculated as the average of the left‐ and right‐hand grip strength measurements. Participants were also assessed for fatigue, physical activity, and weight loss of more than 2–3 kg within the last 6 months. The revised J‐CHS criteria were used to assess frailty. According to the revised J‐CHS criteria, participants were classified into three frailty status groups: frail (≥3 points), pre‐frail (1–2 points), and robust (0 points). The criteria included the following components: (i) shrinking, defined as unintentional weight loss of ≥2 kg in the past 6 months; (ii) weakness, defined as grip strength <28 kg for men and < 18 kg for women; (iii) exhaustion, defined as feeling tired without reason in the past 2 weeks; (iv) slowness, defined as gait speed <1.0 m/s; and (v) low activity, defined as not engaging in moderate or low levels of physical exercise to improve health. For this analysis, participants classified as pre‐frail or frail were combined into a single group, while, those classified as robust were analyzed separately, resulting in two comparison groups based on frailty status. Follow‐up assessment One year after the baseline assessment, the presence or absence of pre‐frailty and frailty was assessed again in community gathering places using the J‐CHS criteria. Medical and long‐term care costs from baseline to 1 year were identified using the health insurance claims data and long‐term care insurance claims data. The Kokuho Database (KDB) contains health insurance claims data (e.g., monthly claims for patients' diagnoses, procedures, and medications) and long‐term care insurance claims data. Statistical analyses We presented the sociodemographic characteristics and outcome measures of the high HL and low HL groups as descriptive statistics. For categorical variables, including sex, employment status, and living arrangements, we reported the number and percentage of participants. For continuous variables, including age and IADL, we provided means (standard deviation), and median (25th and 75th percentiles). We used regression analyses to assess the effects of health literacy on IADL and frailty. Linear regression was used to assess the relationship between health literacy and IADL, adjusting for confounders including age, comorbidities, education level, cognitive function, living arrangements, employment status, and baseline frailty. Results of these analyses are presented as estimates, standard errors, and P ‐values. We then applied logistic regression to examine the association between health literacy and frailty status after 1 year, using the same set of covariates. Results are reported as odds ratios (OR) with 95% confidence intervals (CI). Mediation analysis was conducted to assess how IADL affected frailty status changes. We decomposed the total effect of health literacy on frailty into the average direct effect (ADE) and the average causal mediation effect (ACME), with 95% CIs obtained through 1000 bootstrap replications. A mediation effect of IADL was deemed present if the bootstrap 95% CI did not include zero and the regression coefficients were statistically significant. The mediated proportion was calculated as the ratio of the ACME to the total effect. All statistical analyses were performed using R software (version 4.3.2; R Core Team, 2023). Missing values were imputed using the missForest algorithm, implemented via the “missForest” package. Mediation analyses were performed using the “mediation” package, and the threshold for statistical significance was set at P < 0.05, unless otherwise specified. In this retrospective cohort study, data from surveys conducted for the Matsumoto City Frailty Prevention Project in 2021 and 2022 were used to assess the factors contributing to frailty progression. The program included a single frailty assessment and a one‐time frailty prevention lecture. The lecture provided information on key aspects of frailty prevention, including exercise, nutrition, medication management, and oral health. All participants attended both the assessment and the lecture, ensuring full participation in the program. The survey was focused on older adults residing in Matsumoto City, Nagano Prefecture, Japan. We included individuals residing in Matsumoto City aged ≥65 years who participated in frailty and health literacy assessments at community gathering places in Matsumoto City in 2021. The participants attended a 1‐day nutritional and exercise guidance session or a frailty‐related medication lecture. The inclusion criteria were: (1) aged ≥65 years; and (2) participation in both frailty and health literacy assessments at baseline. The exclusion criterion was non‐participation in the frailty assessment at the 1‐year follow‐up. The participant recruitment flowchart is presented in Fig. . The study was approved by the ethics committee of Matsumoto City Hospital (protocol number, 03–5; approval date, 22/06/2021), and conducted as per the guidelines of the Declaration of Helsinki. In this observational study, the data were anonymised and obtained from non‐invasive assessments conducted at community gathering places; therefore, the need for individual participant consent was waived. However, the participants were informed about the study and provided relevant information. Furthermore, the participants were informed that they could withdraw from the study at any time. Baseline assessment Health literacy was measured using the CCHL scale. This scale measures three aspects of health literacy, including ability to access, understand, and use health information. The participants were enquired about their ability to: (1) obtain health‐related information from various sources; (2) extract the required information; (3) understand and communicate the information obtained; (4) assess the reliability of the information; and (5) make decisions based on the information, specifically in the context of health‐related issues. Each item was rated on a five‐point Likert scale: 1 (strongly disagree); 2 (somewhat disagree); 3 (undecided); 4 (somewhat agree); and 5 (strongly agree). The scores of the five items were then averaged. Participants were classified into two groups based on their average score into high health literacy (high HL; with an average score of ≥4) and low health literacy (low HL; with an average score of <4) groups. This classification was based on a previous study that reported that the median health literacy score of the population was 4. Using this threshold, we aligned our classification with established research practices. Other measures assessed in the study were basic demographics (age, sex, and years of education) and the number of chronic diseases (heart disease, cerebrovascular disease, respiratory disease, diabetes, cancer, rheumatoid arthritis, and osteoarthritis). The years of education were categorised into four groups (Group 1–reference, approximately 9 years; Group 2, approximately 12 years; Group 3, approximately 15 years; and Group 4, others). Chronic diseases were defined as “yes” if diagnosed by a physician. Subjective cognitive function was assessed using three questions from the Kihon Checklist: “Do people around you say you keep repeating the same questions or make similar comments about your forgetfulness?”, “Can you look up phone numbers to make phone calls by yourself?”, and “Do you find yourself not knowing today's date?” Subjective cognitive decline was defined as at least one negative response to these questions. Social factors, including living arrangements (living alone or not) and employment status (currently employed or not), were also assessed. Additionally, at baseline, participants were assessed using the Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG‐IC) (five instrumental ADLs items) for IADL. If the answer was “yes,” one point was added; if the answer was “no,” zero points were added. The total score ranged 0–5, with higher scores indicating greater functional abilities. Normal gait speed and grip strength were assessed as indicators of physical function. Gait speed was calculated based on the time(s) required to walk a distance of 5 m. The participants were instructed to walk at a comfortable pace in an unobstructed hallway. Patients were permitted to use assistive devices including walkers and canes. Grip strength was measured on both sides of the body and calculated as the average of the left‐ and right‐hand grip strength measurements. Participants were also assessed for fatigue, physical activity, and weight loss of more than 2–3 kg within the last 6 months. The revised J‐CHS criteria were used to assess frailty. According to the revised J‐CHS criteria, participants were classified into three frailty status groups: frail (≥3 points), pre‐frail (1–2 points), and robust (0 points). The criteria included the following components: (i) shrinking, defined as unintentional weight loss of ≥2 kg in the past 6 months; (ii) weakness, defined as grip strength <28 kg for men and < 18 kg for women; (iii) exhaustion, defined as feeling tired without reason in the past 2 weeks; (iv) slowness, defined as gait speed <1.0 m/s; and (v) low activity, defined as not engaging in moderate or low levels of physical exercise to improve health. For this analysis, participants classified as pre‐frail or frail were combined into a single group, while, those classified as robust were analyzed separately, resulting in two comparison groups based on frailty status. Follow‐up assessment One year after the baseline assessment, the presence or absence of pre‐frailty and frailty was assessed again in community gathering places using the J‐CHS criteria. Medical and long‐term care costs from baseline to 1 year were identified using the health insurance claims data and long‐term care insurance claims data. The Kokuho Database (KDB) contains health insurance claims data (e.g., monthly claims for patients' diagnoses, procedures, and medications) and long‐term care insurance claims data. Health literacy was measured using the CCHL scale. This scale measures three aspects of health literacy, including ability to access, understand, and use health information. The participants were enquired about their ability to: (1) obtain health‐related information from various sources; (2) extract the required information; (3) understand and communicate the information obtained; (4) assess the reliability of the information; and (5) make decisions based on the information, specifically in the context of health‐related issues. Each item was rated on a five‐point Likert scale: 1 (strongly disagree); 2 (somewhat disagree); 3 (undecided); 4 (somewhat agree); and 5 (strongly agree). The scores of the five items were then averaged. Participants were classified into two groups based on their average score into high health literacy (high HL; with an average score of ≥4) and low health literacy (low HL; with an average score of <4) groups. This classification was based on a previous study that reported that the median health literacy score of the population was 4. Using this threshold, we aligned our classification with established research practices. Other measures assessed in the study were basic demographics (age, sex, and years of education) and the number of chronic diseases (heart disease, cerebrovascular disease, respiratory disease, diabetes, cancer, rheumatoid arthritis, and osteoarthritis). The years of education were categorised into four groups (Group 1–reference, approximately 9 years; Group 2, approximately 12 years; Group 3, approximately 15 years; and Group 4, others). Chronic diseases were defined as “yes” if diagnosed by a physician. Subjective cognitive function was assessed using three questions from the Kihon Checklist: “Do people around you say you keep repeating the same questions or make similar comments about your forgetfulness?”, “Can you look up phone numbers to make phone calls by yourself?”, and “Do you find yourself not knowing today's date?” Subjective cognitive decline was defined as at least one negative response to these questions. Social factors, including living arrangements (living alone or not) and employment status (currently employed or not), were also assessed. Additionally, at baseline, participants were assessed using the Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG‐IC) (five instrumental ADLs items) for IADL. If the answer was “yes,” one point was added; if the answer was “no,” zero points were added. The total score ranged 0–5, with higher scores indicating greater functional abilities. Normal gait speed and grip strength were assessed as indicators of physical function. Gait speed was calculated based on the time(s) required to walk a distance of 5 m. The participants were instructed to walk at a comfortable pace in an unobstructed hallway. Patients were permitted to use assistive devices including walkers and canes. Grip strength was measured on both sides of the body and calculated as the average of the left‐ and right‐hand grip strength measurements. Participants were also assessed for fatigue, physical activity, and weight loss of more than 2–3 kg within the last 6 months. The revised J‐CHS criteria were used to assess frailty. According to the revised J‐CHS criteria, participants were classified into three frailty status groups: frail (≥3 points), pre‐frail (1–2 points), and robust (0 points). The criteria included the following components: (i) shrinking, defined as unintentional weight loss of ≥2 kg in the past 6 months; (ii) weakness, defined as grip strength <28 kg for men and < 18 kg for women; (iii) exhaustion, defined as feeling tired without reason in the past 2 weeks; (iv) slowness, defined as gait speed <1.0 m/s; and (v) low activity, defined as not engaging in moderate or low levels of physical exercise to improve health. For this analysis, participants classified as pre‐frail or frail were combined into a single group, while, those classified as robust were analyzed separately, resulting in two comparison groups based on frailty status. One year after the baseline assessment, the presence or absence of pre‐frailty and frailty was assessed again in community gathering places using the J‐CHS criteria. Medical and long‐term care costs from baseline to 1 year were identified using the health insurance claims data and long‐term care insurance claims data. The Kokuho Database (KDB) contains health insurance claims data (e.g., monthly claims for patients' diagnoses, procedures, and medications) and long‐term care insurance claims data. We presented the sociodemographic characteristics and outcome measures of the high HL and low HL groups as descriptive statistics. For categorical variables, including sex, employment status, and living arrangements, we reported the number and percentage of participants. For continuous variables, including age and IADL, we provided means (standard deviation), and median (25th and 75th percentiles). We used regression analyses to assess the effects of health literacy on IADL and frailty. Linear regression was used to assess the relationship between health literacy and IADL, adjusting for confounders including age, comorbidities, education level, cognitive function, living arrangements, employment status, and baseline frailty. Results of these analyses are presented as estimates, standard errors, and P ‐values. We then applied logistic regression to examine the association between health literacy and frailty status after 1 year, using the same set of covariates. Results are reported as odds ratios (OR) with 95% confidence intervals (CI). Mediation analysis was conducted to assess how IADL affected frailty status changes. We decomposed the total effect of health literacy on frailty into the average direct effect (ADE) and the average causal mediation effect (ACME), with 95% CIs obtained through 1000 bootstrap replications. A mediation effect of IADL was deemed present if the bootstrap 95% CI did not include zero and the regression coefficients were statistically significant. The mediated proportion was calculated as the ratio of the ACME to the total effect. All statistical analyses were performed using R software (version 4.3.2; R Core Team, 2023). Missing values were imputed using the missForest algorithm, implemented via the “missForest” package. Mediation analyses were performed using the “mediation” package, and the threshold for statistical significance was set at P < 0.05, unless otherwise specified. Overall, 373 older adults were included in the study and underwent frailty assessments over 2 years. Of these, 15.6% were male, 56.3% were classified as robust, and 43.7% were pre‐frail or frail at baseline (Table ). Participant characteristics were similar between the high and low HL groups, except for age and employment status. Those in the low HL group tended to be older (79.7 ± 5.8 vs. 76.9 ± 6.4 years) and have a lower employment rate (6.8% vs. 16.1%) compared with those in the high HL group (Table ). The baseline comparison between the two groups ( n = 372 and n = 373, respectively) revealed no statistically significant differences in participant characteristics between participants who could not be followed up for a frailty assessment after 1 year and those who could be followed up (Table ). Participants who could not be followed up were excluded from the final analysis. At the 1‐year follow‐up, 67.3% and 41.4% of participants in the high and low HL groups, respectively, were classified as robust ( P < 0.001) (Table ). In the logistic regression model predicting pre‐frailty or frailty, higher health literacy remained significantly associated with lower OR of being pre‐frail or frail after 1 year (OR = 0.546, P = 0.009), even after adjusting for the confounding factors (Table ). IADL was also a significant predictor of pre‐frailty or frailty (OR = 0.648, P = 0.043) (Table ). Regression analysis revealed that higher health literacy was positively associated with better IADL outcomes (coefficient = 0.127, P = 0.043), even after adjusting for potential confounders including background characteristics (age, comorbidity, cognitive function, and education) and social factors (family status and employment status) (Table ). Causal mediation analysis revealed that IADL mediated 10.7% of the effect of health literacy on frailty status (ACME = −0.010, P = 0.030), with both the direct effect (ADE = −0.085, P = 0.044) and total effect (estimate = −0.096, P = 0.014) remaining significant, even after adjusting for confounders (Table , Fig. ). Our study confirms that higher health literacy is associated with better IADL outcomes and a reduced risk of progression to pre‐frailty or frailty, as defined by the J‐CHS criteria, even after controlling for various confounding factors including age, comorbidities, cognitive function, and social factors, such as family status and employment. Health literacy and frailty Our results align with those of previous studies, demonstrating the importance of health literacy, including CCHL, in managing health and preventing frailty. , The HL scores observed in our study are comparable to those reported in a previous study by Yoshizawa (2021), which documented a mean HL score of approximately four. This similarity supports the generalisability of our findings to older populations in similar settings. Higher CCHL allows older adults to access, understand, and apply health‐related information more effectively, critical for maintaining physical independence. This is particularly relevant to the instrumental self‐maintenance aspect of IADL with tasks such as using public transportation, shopping for daily necessities, and managing finances. Individuals with higher health literacy may be better able to navigate these complex tasks, facilitating maintenance of autonomy and physical independence, and ultimately reducing frailty risk. Therefore, 67.3% of those in the high HL group remained robust after 1 year, as compared to only 41.4% in the low HL group. Further reinforcing the hypothesis that health literacy plays a key role in mitigating frailty risk, as individuals with better health literacy are more adept at engaging in preventive health behaviours and managing chronic conditions. IADL as a mediator A key finding of this study was the identification of IADL as a significant mediator in the relationship between health literacy and frailty. This IADL subset includes both cognitively and physically demanding essential tasks, such as handling transportation, preparing meals, and managing finances, all of which are critical for maintaining independence in older age. , Causal mediation analysis revealed that approximately 10.7% of the effect of health literacy on frailty status was mediated by IADL, indicating that maintaining or improving IADL may be a pathway involved in the effect of health literacy on frailty. The retrospective nature of this study limited its ability to fully account for other health behaviours that may act as mediators. Future prospective studies are needed to evaluate the impact of these additional factors and provide a more comprehensive understanding of the pathways linking health literacy and frailty. Despite this limitation, our findings suggest that interventions for improving health literacy may indirectly contribute to better IADL performance; thus, preventing or slowing frailty progression. Previous studies have reported an association between IADL decline and higher frailty, aligning with our finding that IADL was a significant predictor of frailty status after 1 year (OR = 0.648, P = 0.043). This highlights the importance of addressing IADL in frailty prevention strategies, particularly among individuals with low health literacy. Interventions that target both health literacy and IADL enhance individuals' abilities to manage their health while maintaining their physical independence. Implications for practice The findings of this study have implications in designing interventions aimed at preventing frailty. First, improving CCHL should be a key component of frailty prevention programs, especially in ageing populations. Health literacy interventions should focus on equipping older adults with the skills needed to access and apply health information to promote healthy behaviours and reduce frailty. Second, the mediating role of IADL highlights the role of interventions aimed at health literacy improvement and IADL maintenance and enhancement. Programs incorporating physical, cognitive, and social components, aimed at maintaining daily living skills, may be particularly effective in reducing frailty risk. Tailored interventions accounting for unique social and cognitive needs of older adults may be more successful in promoting independence and preventing frailty. Limitations This study has some limitations. First, the observational design limited our ability to establish causality between health literacy, IADL, and frailty. Although mediation analysis provides insights into the potential mechanisms, randomised controlled trials are needed to confirm causal relationships. Moreover, as this study was retrospective, the role of other health behaviours acting as mediators could not be extensively assessed. Future prospective studies are required to address these gaps and to fully evaluate the mediating effects of various health behaviours. Second, health literacy was assessed using the CCHL Scale, which may not identify all the dimensions of health literacy, especially functional literacy. Moreover, we accounted for various confounding factors including cognitive function and social factors; however, residual confounding may still be present. Third, the IADL was assessed using the five items from the TMIG‐IC, which may have a ceiling effect. This could potentially limit the ability to detect only small differences in IADL between groups with different health literacy levels. Future studies should consider using additional or alternative measures of IADL providing a wider range of scoring and sensitivity to detect variations in functional independence. Despite these limitations, this study provides valuable insights into the relationship between health literacy, IADL, and frailty. Future research addressing these limitations will further enhance our understanding of these complex interactions. Conclusion In conclusion, this study demonstrates that health literacy, particularly CCHL, plays a critical role in preventing frailty progression in older adults. Moreover, IADL acts as an important mediator in this relationship, suggesting that interventions aimed at improving health literacy and maintaining daily living skills may be key to reducing frailty risk. As IADL contributed to only a portion of this mediation effect, future research should explore other health behaviours that may serve as mediators. Furthermore, future research should explore how these findings can be translated into practical interventions and policies to promote healthy ageing and prevent frailty in older populations. Our results align with those of previous studies, demonstrating the importance of health literacy, including CCHL, in managing health and preventing frailty. , The HL scores observed in our study are comparable to those reported in a previous study by Yoshizawa (2021), which documented a mean HL score of approximately four. This similarity supports the generalisability of our findings to older populations in similar settings. Higher CCHL allows older adults to access, understand, and apply health‐related information more effectively, critical for maintaining physical independence. This is particularly relevant to the instrumental self‐maintenance aspect of IADL with tasks such as using public transportation, shopping for daily necessities, and managing finances. Individuals with higher health literacy may be better able to navigate these complex tasks, facilitating maintenance of autonomy and physical independence, and ultimately reducing frailty risk. Therefore, 67.3% of those in the high HL group remained robust after 1 year, as compared to only 41.4% in the low HL group. Further reinforcing the hypothesis that health literacy plays a key role in mitigating frailty risk, as individuals with better health literacy are more adept at engaging in preventive health behaviours and managing chronic conditions. as a mediator A key finding of this study was the identification of IADL as a significant mediator in the relationship between health literacy and frailty. This IADL subset includes both cognitively and physically demanding essential tasks, such as handling transportation, preparing meals, and managing finances, all of which are critical for maintaining independence in older age. , Causal mediation analysis revealed that approximately 10.7% of the effect of health literacy on frailty status was mediated by IADL, indicating that maintaining or improving IADL may be a pathway involved in the effect of health literacy on frailty. The retrospective nature of this study limited its ability to fully account for other health behaviours that may act as mediators. Future prospective studies are needed to evaluate the impact of these additional factors and provide a more comprehensive understanding of the pathways linking health literacy and frailty. Despite this limitation, our findings suggest that interventions for improving health literacy may indirectly contribute to better IADL performance; thus, preventing or slowing frailty progression. Previous studies have reported an association between IADL decline and higher frailty, aligning with our finding that IADL was a significant predictor of frailty status after 1 year (OR = 0.648, P = 0.043). This highlights the importance of addressing IADL in frailty prevention strategies, particularly among individuals with low health literacy. Interventions that target both health literacy and IADL enhance individuals' abilities to manage their health while maintaining their physical independence. The findings of this study have implications in designing interventions aimed at preventing frailty. First, improving CCHL should be a key component of frailty prevention programs, especially in ageing populations. Health literacy interventions should focus on equipping older adults with the skills needed to access and apply health information to promote healthy behaviours and reduce frailty. Second, the mediating role of IADL highlights the role of interventions aimed at health literacy improvement and IADL maintenance and enhancement. Programs incorporating physical, cognitive, and social components, aimed at maintaining daily living skills, may be particularly effective in reducing frailty risk. Tailored interventions accounting for unique social and cognitive needs of older adults may be more successful in promoting independence and preventing frailty. This study has some limitations. First, the observational design limited our ability to establish causality between health literacy, IADL, and frailty. Although mediation analysis provides insights into the potential mechanisms, randomised controlled trials are needed to confirm causal relationships. Moreover, as this study was retrospective, the role of other health behaviours acting as mediators could not be extensively assessed. Future prospective studies are required to address these gaps and to fully evaluate the mediating effects of various health behaviours. Second, health literacy was assessed using the CCHL Scale, which may not identify all the dimensions of health literacy, especially functional literacy. Moreover, we accounted for various confounding factors including cognitive function and social factors; however, residual confounding may still be present. Third, the IADL was assessed using the five items from the TMIG‐IC, which may have a ceiling effect. This could potentially limit the ability to detect only small differences in IADL between groups with different health literacy levels. Future studies should consider using additional or alternative measures of IADL providing a wider range of scoring and sensitivity to detect variations in functional independence. Despite these limitations, this study provides valuable insights into the relationship between health literacy, IADL, and frailty. Future research addressing these limitations will further enhance our understanding of these complex interactions. In conclusion, this study demonstrates that health literacy, particularly CCHL, plays a critical role in preventing frailty progression in older adults. Moreover, IADL acts as an important mediator in this relationship, suggesting that interventions aimed at improving health literacy and maintaining daily living skills may be key to reducing frailty risk. As IADL contributed to only a portion of this mediation effect, future research should explore other health behaviours that may serve as mediators. Furthermore, future research should explore how these findings can be translated into practical interventions and policies to promote healthy ageing and prevent frailty in older populations. Keisuke Nakamura: project administration, writing – original draft preparation, formal analysis. Tomohiro Sasaki: writing– review and editing. Yoshiharu Yokokawa: investigation. Shinobu Yokouchi: conceptualisation. This research received no specific grants from any funding agency in the public, commercial, or not‐for‐profit sectors. The study was approved by the ethics committee of Matsumoto City Hospital (protocol number, 03–5; approval date, 22/06/2021). The need for informed consent was waived due to data anonymisation and the retrospective nature of the study. This study is an observational study and was not registered as a clinical trial as it does not involve any intervention. Table S1. Patient characteristics at baseline. Table S2. Frailty status after 1 year. Table S3. Linear regression analysis of instrumental activities of daily living (IADL) stratified by health literacy at baseline. |
Study protocol for a multicenter randomized controlled trial on simulation-based communication training for pediatric cardiology trainees (SIMUL-CHD) | 3cdcec21-d2ba-40cb-bd87-eb546b005a68 | 11539632 | Internal Medicine[mh] | Congenital heart disease (CHD) affects 8:1000 live births with disease severity ranging from mild to severe and potentially life-threatening . The management of CHD has evolved significantly over the past century however despite significant advancements in medicine, many CHD diagnoses still lack a cure and substantial comorbidity persists (such as cardiovascular or neurodevelopmental sequelae). In recent times, there has been a shift in focus from survival to enhancing the quality of life for children with CHD and their families . Parents of children with CHD are at a higher risk of experiencing depression and anxiety . Moments of particular concern for these emotional challenges arise during the initial diagnosis and around the time of surgical interventions. “Bad news” in the context of diagnosis often refers to more than the diagnosis itself. For many parents, it includes the prospect of prolonged hospital stays, open-heart surgeries (often multiple in the child’s early years), potential heart transplantation, and ongoing medical complications, such as growth failure, feeding difficulties, and neurodevelopmental or behavioral issues. Parents of children with CHD frequently endure long periods away from home, which adds strain to family relationships, marriages, and finances . In addition, parents must cope with the emotional weight of mourning the loss of the “normal” child they had expected, often grappling with feelings of guilt, grief, and anger, which put them at an increased risk for mental health challenges such as depression, anxiety, and post-traumatic stress disorder . Heightened levels of stress have been observed following the postnatal diagnosis of CHD in newborns, during a period of postpartum vulnerability for couples . Parental stress and anxiety have an important physiological impact on children and have been associated with poorer neurological development in children with heart conditions . This risk is additionally exacerbated in cases where the mother experiences postpartum depression . Therefore, effective communication between the pediatric cardiologist and the caregivers of children with CHD plays a crucial role, not only in circumstances where a “cure” is not achievable. It is thus paramount that every physician with a holistic approach to healthcare should be able to develop “caring” skills. The way in which pediatric cardiologists communicate this diagnosis, manage expectations, and guide parents through treatment plays a pivotal role in helping families cope and prepare for the road ahead . However, such skills are significantly underrepresented in medical education and even more rarely addressed in research protocols. In France, approximately 65 to 70% of severe CHD is detected during prenatal screening. Consequently, a substantial portion of CHD diagnoses is anticipated to occur during the postnatal period . This pivotal first interaction often takes place at the newborn or child’s bedside, in the presence of exhausted parents, usually at the time of initial echocardiography and can be impacted by other commitments including on-call duties, and with minimal supervision. This practical training gap, focusing on medical skills rather than humanistic ones, is not aligned with the approach of new medical educations reforms in France. It also contradicts the commonly emphasized principle of “never the first time on a patient” ; which means that training is required before practicing . The importance of formal training in communication skills has also been acknowledged by the National Academy of Medicine . It is inevitable that pediatric cardiologists will face the challenging responsibility of delivering ‘bad news’ to patients and families, and it is suggested that these initial interactions between the physician and the family could potentially influence patient outcomes, including compliance during follow-up. Despite this, many pediatric cardiology fellowship programs fail to integrate formal communication skills in their curriculum, something that may be easily addressed by simulation-based training. Simulation-based education involves recreating realistic clinical scenarios in a safe, controlled environment. This allows healthcare professionals to practice practical skills, make critical decisions, and develop interpersonal abilities without risking patient safety. It is increasingly used in both adult and pediatric medicine for training in clinical skills, such as emergency management and resuscitation . While some studies have shown the benefits of simulation training for patient-physician communication, it is still underutilized in pediatrics . A few studies have demonstrated improved confidence and communication skills through simulation-based learning in oncology , as well as better communication after resuscitation . In pediatric cardiology, trainees can struggle with the emotional challenges of diagnosis counseling. Anxiety and lack of confidence can hinder their ability to communicate effectively with families. Poor communication during initial CHD diagnosis can lead to feelings of guilt and disappointment in the trainees, significantly affecting their psychological well-being . Research gap There have been no randomized controlled trials (RCT) published comparing a simulation-based communication training program in providing CHD counselling compared to traditional training methods. There have been no randomized controlled trials (RCT) published comparing a simulation-based communication training program in providing CHD counselling compared to traditional training methods. Study aims Our aim is to investigate the impact of a simulation-based education program on the effectiveness of initial counselling of postnatal diagnoses of CHD among pediatric cardiology trainees. The primary outcome of this study is the effect of our simulation program on communication skills. The secondary outcomes include the impact of the simulation program on performance anxiety prior to the diagnostic disclosure and the level of empathy experienced by participants before the announcement. Study design and settings Participants’ characteristics In the French medical system, there are two key stages. After completing medical school, doctors enter a stage called “ internat ,” which is equivalent to pediatric cardiology fellows in many countries. During this period, they are in specialized training within a specific medical field (e.g., pediatric cardiology). This training typically lasts 3 to 5 years, during which they rotate through various departments, gaining practical experience under the supervision of senior doctors. After completing their residency, they may take up the role of post-residency position that involves working as a fully licensed doctor but still within a structured training environment. During these two stages, trainees did not received specific communication training skills. All members of the French national congenital heart disease network (“M3C”) comprising 10 number of centers, with pediatric cardiology trainees within their institutions will be invited to take part in the study. The study period will be 6 months. All pediatric cardiology trainees (pediatric cardiology fellows, post-residency assistants) irrespective of the stage in training will be included within the study. Participants without a high proficiency in the French language will be excluded to control for any bias in communication attributed to language. Pediatric cardiology trainees who have recently undertaken training in diagnostic counselling in the year prior to assessment will also be excluded in order to maintain homogeneity of prior skills. Study design This is a multicenter, randomized, superiority trial conducted over 10 university hospitals and tertiary pediatric cardiac centers in France (SIMUL-CHD trial) with a study period of 6 months. Figure describes the study design. Participants are recruited into the study following initial eligibility assessment and consent procedures. The steering committee will proceed with randomization using a computer-generated allocation sequence. Randomization will be stratified by center and by the level achieved during the study, with two strata: pediatric cardiology fellows post-residency assistants. Simulated parents To enhance the authenticity of our simulation, we propose the inclusion of simulated parents, individuals who have personally faced the challenges associated with a diagnosis of CHD. Simulated parents in this study are standardized parents who have undergone training to be able to train medical students and young physicians : there are not actors but actual parents of the national association “Petit Coeur de Beurre”’. Family and caregiver involvement will be entirely voluntary, and they will not receive compensation for participating in the study. They will be selected by the association’s director based on the fact that their child’s CHD is in the past, allowing them the emotional distance needed to engage in our study. By incorporating the perspectives of simulated parents, we aim to provide a more authentic and emotionally resonant learning experience for pediatric cardiologists. This approach aligns with the ‘patient-centered’ medical philosophy endorsed by the World Health Organization, advocating for the active involvement of patients and the public in medical care, education, and research . Primary outcome definition The primary endpoint is the change from baseline between initial and final scores for dimension 2 “Breaking Bad News (BBN)” skills form (Fig. ) following intervention. The BBN skills will be completed by the evaluation committee using videos and blinded to the intervention. This mean variation will be compared between the two groups. The evaluation committee will be composed of a senior pediatric cardiologist, a clinical psychologist specializing in diagnostic counseling and a member of the national parents’ association (“Petit Coeur de Beurre”). The mean of the three scores will be calculated to evaluate the primary outcome. The BBN is a checklist of skills or actions to be performed during the breaking of bad news, which will be assessed with ‘yes’ or ‘no’ responses. Some items are reverse-scored. The score comprises 30 questions in total, but the maximum possible score is 24. This score is divided into five sections that correspond to the overall competencies expected during a diagnostic announcement. The first section addresses the preamble to delivering bad news and consists of eight items (two of which are reverse-scored). The second section pertains to the direct action of announcing the bad news and includes six items (two of which are also reverse-scored). The third section focuses on the attention given to the reactions of the patient or their relatives following the announcement, comprising five items, one of which is reverse-scored. The fourth section relates to the communication regarding the emotions of the patient (or their relatives) after the announcement and consists of five items (one of which is reverse-scored). Finally, the last section evaluates the patient’s or their relatives’ readiness to consider next steps and their preferences for communication following the announcement, comprising six items. The assessment form will be used within the study to evaluate the effectiveness of the diagnostic counselling session. The BBN Skills form was chosen due its comprehensive assessment of communication skills when delivering bad news . Initially used in adult oncology and in the English language, it will be translated into French and adapted for use in any diagnostic counselling scenario. Psychometric validation will be carried out in an ancillary study. Video recording evaluation The video recording will be standardized and the participants in the pre- and post-evaluation scenarios will be equipped with microphones to ensure good quality audio (Fig. ). The evaluation scenarios will include the diagnostic counselling of a simple form of CHD, for example : “postnatal detection of a ventricular septal defect requiring surgery in the first year of life”. The pediatric cardiology trainees will be expected to carry out this simulation with a “parent” acting within the scenario. The parent will have prior experience and training to perform and react appropriately within the scenario. The parents’ responses to the words and actions of the pediatric cardiology trainees will be standardized and reproducible. The scenario will begin with a visualization of several clips of transthoracic echocardiography. The diagnosis of the congenital heart disease will be revealed at the end of the video and the main principles of management will be outlined to the participant to standardize for any knowledge differences between the trainees. The aim is to assess communication skills and not CHD knowledge proficiency. Secondary outcome definition During the study duration, baseline and final evaluations will be performed and recording. Performance anxiety and empathy are key psychological factors that can influence performance and interactions in the medical field. These two secondary parameters will also be assessed by questionnaires and the change from baseline will be compared within the two groups: Empathy will be assessed using the Jefferson Scale of Physician’s Empathy (JSPE) questionnaire. Anxiety will be assessed using the self-assessment of performance anxiety with the State-Trait Anxiety Inventory (STAI) questionnaire. The JSPE is a self questionnaire consisting of 20 items, ten of which are inverted (i). Each item is rated from 1 to 7 on a Likert scale, corresponding respectively to “strongly disagree” and “strongly agree”. The items are divided as follows into the three sub-components of cognitive empathy: Perspecting taking (ability to understand the patient’s perspective): items 2, 4, 5, 9, 10, 13, 15, 16, 17 and 20 (sub-scores from 10 to 70). Compassionate care: items 1(i), 7(i), 8(i), 11(i), 12(i), 14(i), 18(i) and 19(i) (sub-score from 8 to 56). The ability to walk in the patient’s shoes: items 3(i) and 6(i) (sub-scores from 2 to 14). The overall score obtained from the questionnaire ranges from 20 to 140. The higher the score, the greater the empathy estimated by the participant. This scale is widely used throughout the world. Its reliability and validity have been verified by the authors at the time of its creation and in a follow-up cross-cultural study in 2014, confirming that the 3 components of this scale remain stable even in different cultures . Its internal consistency was determined by a Cronbach’s alpha coefficient of 0.80. The JSPE has been validated in French . In addition, the JSPE has already been used to assess the empathy of medical fellows in several other studies . In order to assess performance anxiety, we selected the STAI questionnaire .The STAI is a widely recognized psychometric tool for assessing anxiety in individuals in specific situations . The STAI consists of two distinct parts: the State Anxiety Scale (SAS) and the Trait Anxiety Scale (TAS). The SAS assesses the temporary or transient anxiety that individuals may experience in specific situations, while the TAS assesses general or lasting anxiety in individuals, regardless of the situation. The STAI questionnaire is composed of several items scored on a Likert scale, where participants indicate their degree of agreement with the proposed statements. It provides quantitative scores that can be used to measure the intensity of anxiety experienced by individuals. The STAI is widely used in psychological research and has demonstrated its reliability and validity. It has also been validated in French . We believe it can be used to assess performance anxiety specific to situations where patients are given bad news. Thus, using the STAI in this study will make it possible to collect objective data on the performance anxiety of the participants prior to delivering difficult news to the patient and their families. Ancillary studies Two ancillary study are planned Translation in French and psychometric validation of the BBN Skills communication performance score for future use. Qualitative evaluation of the simulation session in the SIMUL-CHD arm. The “SIMUL-CHD” program and the comparative group The “SIMUL-CHD” program The SIMUL-CHD training program is a comprehensive, one-day simulation-based workshop that aims to improve pediatric cardiology trainees’ communication skills. The training program integrates theoretical learning with hands-on simulation exercises, designed to foster empathy, improve clarity in communication, and provide participants with essential tools to handle emotionally charged medical situations. The design of the program reflects the feedback and input from senior cardiologists, psychologists, and actual parents of children with CHD. This collaboration ensures a holistic approach to communication training. The course is designed for small groups of five participants, allowing for intensive and personalized learning. These participants are pediatric cardiology trainees at various stages of their careers. This multidisciplinary setup ensures that the training is applicable to all levels of experience, while small group sizes enable each participant to engage deeply in both simulated scenarios and debriefing sessions. The SIMUL-CHD training is delivered by a multidisciplinary team, including: A senior pediatric cardiologist, who brings medical expertise and shares their experience in delivering difficult diagnoses. A psychologist, who focuses on the emotional and psychological impact of delivering and receiving bad news. Two simulated parents, real parents of children with CHD, who share their lived experiences and participate in the simulated scenarios. One parent engages directly in the role-play scenarios, while the other joins the debriefing to offer authentic insights into the parent’s perspective. Program Structure: The morning session focuses on theoretical knowledge, with contributions from each of the trainers: Senior Pediatric Cardiologist : The cardiologist explains the process of delivering bad news, focusing on organization, body language, verbal and non-verbal communication, and managing both the physician’s and the family’s expectations. Key points include how to manage complex medical information and the physician’s role in supporting the family emotionally. Psychologist : The psychologist provides insights into the psychological impact of these communications, emphasizing how stress and emotional states can affect both the physician’s delivery and the family’s reception of the news. They also discuss strategies for managing difficult emotional responses during these interactions. Simulated Parents : parents shares what they wish doctors would know when delivering a diagnosis. Their testimony is aimed at helping trainees understand the emotional weight and expectations parents have during these critical conversations. After a brief break, participants are encouraged to share their own experiences of delivering bad news, whether positive or negative. This open discussion allows for peer learning and reflection, with input from both the psychologist and the parent, providing an emotionally safe environment for trainees to discuss challenges they have faced. These experiences are then linked back to the theoretical concepts discussed earlier. The afternoon session is dedicated to hands-on simulated scenarios. Each participant will take on the role of the “main announcer,” or “co-announcer. Each simulation consists of three phases: the announcement, the debriefing, and peer feedback. Each scenario simulates a real-life situation where a trainee must communicate a difficult diagnosis. The trainees must explain the medical implications of the diagnosis, including possible treatments and outcomes, while managing the emotional needs of the parents. These scenarios are designed to cover a range of emotional and cognitive challenges, from parents in shock to those with numerous questions and fears about the future. After each simulation, participants receive structured feedback from the trainers and peers. The debriefing is structured using the “plus-delta” approach, focusing on what was done well and what could be improved. The presence of the partner parent in the debriefing adds an authentic perspective, highlighting the emotional nuances that may have been missed by the trainees. Formative assessment takes place throughout the day during the debriefing sessions. Trainees receive real-time feedback on their communication style, empathy, and ability to handle emotionally charged situations. These assessments are not graded but are meant to foster growth and reflection. All sites will use the same set of simulated scenarios, with scripts carefully designed to cover a variety of communication challenges. The trainers, including cardiologists, psychologists, and parents, will undergo a standardized preparation session to ensure consistency in feedback and debriefing methods. The same trainers will be used across all sites. To reduce bias, the parent/caregiver taking part in the training program will be different to those participating in the evaluation scenarios. Similarly, the scenarios used during the simulation program will be different from those used in the evaluation scenarios. The comparative group In order to avoid bias of over-evaluation of the effect of our simulation program by the “simple” fact of giving more time for training, we are going to implement an online training webinar sufficiently relevant for the control arm. The webinar will last approximately 3 h and will be organized into three to four presentations. These presentations will be delivered by pediatric cardiologists, psychologists, and parents, each offering unique perspectives on delivering difficult news. Also, employing a control group receiving standardized theoretical training can be regarded as ethically sound, ensuring access to a form of training for all study participants. Study design and statistical methods Sample size calculation The study by Gorniewicz et al. (2017) concluded that if dimension 2 (“Breaking Bad News”) is retained, a sample of 18 people in each group will have 90% power to detect a difference in mean of -1.3 (the difference between a control group mean, µ₁, of -0.25 and an intervention group mean, µ₂, of 1.05) assuming a common standard deviation of 1.14, using a Student’s t-test with a two-tailed significance level of 5% (NQuery software). With a non-exploitable data rate of around 10%, we concluded that it would be necessary to include 20 subjects per group. Study timeline and feasibility In France, resident and post-resident juniors rotate between departments every six months which provides a predictability with participant availability. The major pediatric cardiac referral centers enroll 5 to 10 pediatric cardiology trainees and the centers of pediatric cardiac expertise support 1 and 5 pediatric cardiology trainees. The feasibility of the study over a six-month period is therefore guaranteed. The study will start in in May 2025 and end in November 2025. During this six-month period, all participants will undergo an initial evaluation, followed by training based on randomization, and finally, a concluding evaluation. The participant timeline is presented in Table . Data analysis plan A descriptive analysis of the initial characteristics of each group will be conducted. Qualitative variables will include the sample size and the frequency of different modalities. Quantitative variables will include the sample size, mean, standard deviation, median, and extreme values (minimum and maximum). The comparability of the two groups before treatment will be assessed for all initial characteristics. In case of non-comparability on one or more parameters that may influence the efficacy criteria, an adjustment may be made on these parameters for the intergroup comparisons of the outcome measures as part of a sensitivity analysis. The analysis of efficacy outcome measures will be conducted on an intention-to-treat (ITT) basis. Each doctor will be analyzed to their randomized group regardless of protocol deviations. The difference between the initial and final scores for dimension 2 “Breaking Bad News” skills questionnaire will be compared between the two groups using a Student’s t-test if the distribution is parametric or using a Mann-Whitney test if the distribution is non-parametric. The effect size (Cohen’s d) and its 95% confidence interval will be assessed. The following secondary criteria will be analyzed using the same strategy as the primary criterion: difference between initial and final scores for the other 4 dimensions of “BBN Skills” evaluated by the evaluation committee self-assessment by the paediatric cardiology trainees of performance anxiety before and after the intervention self-assessment of trainee empathy and concordance between different assessments of the BBN Skills score. A test will be considered statistically significant if the p-value is less than the 5% significance threshold. The analyses will be conducted using the SAS or R software after the database is locked and the Statistical Analysis Plan is approved. Our aim is to investigate the impact of a simulation-based education program on the effectiveness of initial counselling of postnatal diagnoses of CHD among pediatric cardiology trainees. The primary outcome of this study is the effect of our simulation program on communication skills. The secondary outcomes include the impact of the simulation program on performance anxiety prior to the diagnostic disclosure and the level of empathy experienced by participants before the announcement. Participants’ characteristics In the French medical system, there are two key stages. After completing medical school, doctors enter a stage called “ internat ,” which is equivalent to pediatric cardiology fellows in many countries. During this period, they are in specialized training within a specific medical field (e.g., pediatric cardiology). This training typically lasts 3 to 5 years, during which they rotate through various departments, gaining practical experience under the supervision of senior doctors. After completing their residency, they may take up the role of post-residency position that involves working as a fully licensed doctor but still within a structured training environment. During these two stages, trainees did not received specific communication training skills. All members of the French national congenital heart disease network (“M3C”) comprising 10 number of centers, with pediatric cardiology trainees within their institutions will be invited to take part in the study. The study period will be 6 months. All pediatric cardiology trainees (pediatric cardiology fellows, post-residency assistants) irrespective of the stage in training will be included within the study. Participants without a high proficiency in the French language will be excluded to control for any bias in communication attributed to language. Pediatric cardiology trainees who have recently undertaken training in diagnostic counselling in the year prior to assessment will also be excluded in order to maintain homogeneity of prior skills. Study design This is a multicenter, randomized, superiority trial conducted over 10 university hospitals and tertiary pediatric cardiac centers in France (SIMUL-CHD trial) with a study period of 6 months. Figure describes the study design. Participants are recruited into the study following initial eligibility assessment and consent procedures. The steering committee will proceed with randomization using a computer-generated allocation sequence. Randomization will be stratified by center and by the level achieved during the study, with two strata: pediatric cardiology fellows post-residency assistants. Simulated parents To enhance the authenticity of our simulation, we propose the inclusion of simulated parents, individuals who have personally faced the challenges associated with a diagnosis of CHD. Simulated parents in this study are standardized parents who have undergone training to be able to train medical students and young physicians : there are not actors but actual parents of the national association “Petit Coeur de Beurre”’. Family and caregiver involvement will be entirely voluntary, and they will not receive compensation for participating in the study. They will be selected by the association’s director based on the fact that their child’s CHD is in the past, allowing them the emotional distance needed to engage in our study. By incorporating the perspectives of simulated parents, we aim to provide a more authentic and emotionally resonant learning experience for pediatric cardiologists. This approach aligns with the ‘patient-centered’ medical philosophy endorsed by the World Health Organization, advocating for the active involvement of patients and the public in medical care, education, and research . In the French medical system, there are two key stages. After completing medical school, doctors enter a stage called “ internat ,” which is equivalent to pediatric cardiology fellows in many countries. During this period, they are in specialized training within a specific medical field (e.g., pediatric cardiology). This training typically lasts 3 to 5 years, during which they rotate through various departments, gaining practical experience under the supervision of senior doctors. After completing their residency, they may take up the role of post-residency position that involves working as a fully licensed doctor but still within a structured training environment. During these two stages, trainees did not received specific communication training skills. All members of the French national congenital heart disease network (“M3C”) comprising 10 number of centers, with pediatric cardiology trainees within their institutions will be invited to take part in the study. The study period will be 6 months. All pediatric cardiology trainees (pediatric cardiology fellows, post-residency assistants) irrespective of the stage in training will be included within the study. Participants without a high proficiency in the French language will be excluded to control for any bias in communication attributed to language. Pediatric cardiology trainees who have recently undertaken training in diagnostic counselling in the year prior to assessment will also be excluded in order to maintain homogeneity of prior skills. This is a multicenter, randomized, superiority trial conducted over 10 university hospitals and tertiary pediatric cardiac centers in France (SIMUL-CHD trial) with a study period of 6 months. Figure describes the study design. Participants are recruited into the study following initial eligibility assessment and consent procedures. The steering committee will proceed with randomization using a computer-generated allocation sequence. Randomization will be stratified by center and by the level achieved during the study, with two strata: pediatric cardiology fellows post-residency assistants. To enhance the authenticity of our simulation, we propose the inclusion of simulated parents, individuals who have personally faced the challenges associated with a diagnosis of CHD. Simulated parents in this study are standardized parents who have undergone training to be able to train medical students and young physicians : there are not actors but actual parents of the national association “Petit Coeur de Beurre”’. Family and caregiver involvement will be entirely voluntary, and they will not receive compensation for participating in the study. They will be selected by the association’s director based on the fact that their child’s CHD is in the past, allowing them the emotional distance needed to engage in our study. By incorporating the perspectives of simulated parents, we aim to provide a more authentic and emotionally resonant learning experience for pediatric cardiologists. This approach aligns with the ‘patient-centered’ medical philosophy endorsed by the World Health Organization, advocating for the active involvement of patients and the public in medical care, education, and research . The primary endpoint is the change from baseline between initial and final scores for dimension 2 “Breaking Bad News (BBN)” skills form (Fig. ) following intervention. The BBN skills will be completed by the evaluation committee using videos and blinded to the intervention. This mean variation will be compared between the two groups. The evaluation committee will be composed of a senior pediatric cardiologist, a clinical psychologist specializing in diagnostic counseling and a member of the national parents’ association (“Petit Coeur de Beurre”). The mean of the three scores will be calculated to evaluate the primary outcome. The BBN is a checklist of skills or actions to be performed during the breaking of bad news, which will be assessed with ‘yes’ or ‘no’ responses. Some items are reverse-scored. The score comprises 30 questions in total, but the maximum possible score is 24. This score is divided into five sections that correspond to the overall competencies expected during a diagnostic announcement. The first section addresses the preamble to delivering bad news and consists of eight items (two of which are reverse-scored). The second section pertains to the direct action of announcing the bad news and includes six items (two of which are also reverse-scored). The third section focuses on the attention given to the reactions of the patient or their relatives following the announcement, comprising five items, one of which is reverse-scored. The fourth section relates to the communication regarding the emotions of the patient (or their relatives) after the announcement and consists of five items (one of which is reverse-scored). Finally, the last section evaluates the patient’s or their relatives’ readiness to consider next steps and their preferences for communication following the announcement, comprising six items. The assessment form will be used within the study to evaluate the effectiveness of the diagnostic counselling session. The BBN Skills form was chosen due its comprehensive assessment of communication skills when delivering bad news . Initially used in adult oncology and in the English language, it will be translated into French and adapted for use in any diagnostic counselling scenario. Psychometric validation will be carried out in an ancillary study. Video recording evaluation The video recording will be standardized and the participants in the pre- and post-evaluation scenarios will be equipped with microphones to ensure good quality audio (Fig. ). The evaluation scenarios will include the diagnostic counselling of a simple form of CHD, for example : “postnatal detection of a ventricular septal defect requiring surgery in the first year of life”. The pediatric cardiology trainees will be expected to carry out this simulation with a “parent” acting within the scenario. The parent will have prior experience and training to perform and react appropriately within the scenario. The parents’ responses to the words and actions of the pediatric cardiology trainees will be standardized and reproducible. The scenario will begin with a visualization of several clips of transthoracic echocardiography. The diagnosis of the congenital heart disease will be revealed at the end of the video and the main principles of management will be outlined to the participant to standardize for any knowledge differences between the trainees. The aim is to assess communication skills and not CHD knowledge proficiency. The video recording will be standardized and the participants in the pre- and post-evaluation scenarios will be equipped with microphones to ensure good quality audio (Fig. ). The evaluation scenarios will include the diagnostic counselling of a simple form of CHD, for example : “postnatal detection of a ventricular septal defect requiring surgery in the first year of life”. The pediatric cardiology trainees will be expected to carry out this simulation with a “parent” acting within the scenario. The parent will have prior experience and training to perform and react appropriately within the scenario. The parents’ responses to the words and actions of the pediatric cardiology trainees will be standardized and reproducible. The scenario will begin with a visualization of several clips of transthoracic echocardiography. The diagnosis of the congenital heart disease will be revealed at the end of the video and the main principles of management will be outlined to the participant to standardize for any knowledge differences between the trainees. The aim is to assess communication skills and not CHD knowledge proficiency. During the study duration, baseline and final evaluations will be performed and recording. Performance anxiety and empathy are key psychological factors that can influence performance and interactions in the medical field. These two secondary parameters will also be assessed by questionnaires and the change from baseline will be compared within the two groups: Empathy will be assessed using the Jefferson Scale of Physician’s Empathy (JSPE) questionnaire. Anxiety will be assessed using the self-assessment of performance anxiety with the State-Trait Anxiety Inventory (STAI) questionnaire. The JSPE is a self questionnaire consisting of 20 items, ten of which are inverted (i). Each item is rated from 1 to 7 on a Likert scale, corresponding respectively to “strongly disagree” and “strongly agree”. The items are divided as follows into the three sub-components of cognitive empathy: Perspecting taking (ability to understand the patient’s perspective): items 2, 4, 5, 9, 10, 13, 15, 16, 17 and 20 (sub-scores from 10 to 70). Compassionate care: items 1(i), 7(i), 8(i), 11(i), 12(i), 14(i), 18(i) and 19(i) (sub-score from 8 to 56). The ability to walk in the patient’s shoes: items 3(i) and 6(i) (sub-scores from 2 to 14). The overall score obtained from the questionnaire ranges from 20 to 140. The higher the score, the greater the empathy estimated by the participant. This scale is widely used throughout the world. Its reliability and validity have been verified by the authors at the time of its creation and in a follow-up cross-cultural study in 2014, confirming that the 3 components of this scale remain stable even in different cultures . Its internal consistency was determined by a Cronbach’s alpha coefficient of 0.80. The JSPE has been validated in French . In addition, the JSPE has already been used to assess the empathy of medical fellows in several other studies . In order to assess performance anxiety, we selected the STAI questionnaire .The STAI is a widely recognized psychometric tool for assessing anxiety in individuals in specific situations . The STAI consists of two distinct parts: the State Anxiety Scale (SAS) and the Trait Anxiety Scale (TAS). The SAS assesses the temporary or transient anxiety that individuals may experience in specific situations, while the TAS assesses general or lasting anxiety in individuals, regardless of the situation. The STAI questionnaire is composed of several items scored on a Likert scale, where participants indicate their degree of agreement with the proposed statements. It provides quantitative scores that can be used to measure the intensity of anxiety experienced by individuals. The STAI is widely used in psychological research and has demonstrated its reliability and validity. It has also been validated in French . We believe it can be used to assess performance anxiety specific to situations where patients are given bad news. Thus, using the STAI in this study will make it possible to collect objective data on the performance anxiety of the participants prior to delivering difficult news to the patient and their families. Two ancillary study are planned Translation in French and psychometric validation of the BBN Skills communication performance score for future use. Qualitative evaluation of the simulation session in the SIMUL-CHD arm. Translation in French and psychometric validation of the BBN Skills communication performance score for future use. Qualitative evaluation of the simulation session in the SIMUL-CHD arm. The “SIMUL-CHD” program The SIMUL-CHD training program is a comprehensive, one-day simulation-based workshop that aims to improve pediatric cardiology trainees’ communication skills. The training program integrates theoretical learning with hands-on simulation exercises, designed to foster empathy, improve clarity in communication, and provide participants with essential tools to handle emotionally charged medical situations. The design of the program reflects the feedback and input from senior cardiologists, psychologists, and actual parents of children with CHD. This collaboration ensures a holistic approach to communication training. The course is designed for small groups of five participants, allowing for intensive and personalized learning. These participants are pediatric cardiology trainees at various stages of their careers. This multidisciplinary setup ensures that the training is applicable to all levels of experience, while small group sizes enable each participant to engage deeply in both simulated scenarios and debriefing sessions. The SIMUL-CHD training is delivered by a multidisciplinary team, including: A senior pediatric cardiologist, who brings medical expertise and shares their experience in delivering difficult diagnoses. A psychologist, who focuses on the emotional and psychological impact of delivering and receiving bad news. Two simulated parents, real parents of children with CHD, who share their lived experiences and participate in the simulated scenarios. One parent engages directly in the role-play scenarios, while the other joins the debriefing to offer authentic insights into the parent’s perspective. Program Structure: The morning session focuses on theoretical knowledge, with contributions from each of the trainers: Senior Pediatric Cardiologist : The cardiologist explains the process of delivering bad news, focusing on organization, body language, verbal and non-verbal communication, and managing both the physician’s and the family’s expectations. Key points include how to manage complex medical information and the physician’s role in supporting the family emotionally. Psychologist : The psychologist provides insights into the psychological impact of these communications, emphasizing how stress and emotional states can affect both the physician’s delivery and the family’s reception of the news. They also discuss strategies for managing difficult emotional responses during these interactions. Simulated Parents : parents shares what they wish doctors would know when delivering a diagnosis. Their testimony is aimed at helping trainees understand the emotional weight and expectations parents have during these critical conversations. After a brief break, participants are encouraged to share their own experiences of delivering bad news, whether positive or negative. This open discussion allows for peer learning and reflection, with input from both the psychologist and the parent, providing an emotionally safe environment for trainees to discuss challenges they have faced. These experiences are then linked back to the theoretical concepts discussed earlier. The afternoon session is dedicated to hands-on simulated scenarios. Each participant will take on the role of the “main announcer,” or “co-announcer. Each simulation consists of three phases: the announcement, the debriefing, and peer feedback. Each scenario simulates a real-life situation where a trainee must communicate a difficult diagnosis. The trainees must explain the medical implications of the diagnosis, including possible treatments and outcomes, while managing the emotional needs of the parents. These scenarios are designed to cover a range of emotional and cognitive challenges, from parents in shock to those with numerous questions and fears about the future. After each simulation, participants receive structured feedback from the trainers and peers. The debriefing is structured using the “plus-delta” approach, focusing on what was done well and what could be improved. The presence of the partner parent in the debriefing adds an authentic perspective, highlighting the emotional nuances that may have been missed by the trainees. Formative assessment takes place throughout the day during the debriefing sessions. Trainees receive real-time feedback on their communication style, empathy, and ability to handle emotionally charged situations. These assessments are not graded but are meant to foster growth and reflection. All sites will use the same set of simulated scenarios, with scripts carefully designed to cover a variety of communication challenges. The trainers, including cardiologists, psychologists, and parents, will undergo a standardized preparation session to ensure consistency in feedback and debriefing methods. The same trainers will be used across all sites. To reduce bias, the parent/caregiver taking part in the training program will be different to those participating in the evaluation scenarios. Similarly, the scenarios used during the simulation program will be different from those used in the evaluation scenarios. The comparative group In order to avoid bias of over-evaluation of the effect of our simulation program by the “simple” fact of giving more time for training, we are going to implement an online training webinar sufficiently relevant for the control arm. The webinar will last approximately 3 h and will be organized into three to four presentations. These presentations will be delivered by pediatric cardiologists, psychologists, and parents, each offering unique perspectives on delivering difficult news. Also, employing a control group receiving standardized theoretical training can be regarded as ethically sound, ensuring access to a form of training for all study participants. The SIMUL-CHD training program is a comprehensive, one-day simulation-based workshop that aims to improve pediatric cardiology trainees’ communication skills. The training program integrates theoretical learning with hands-on simulation exercises, designed to foster empathy, improve clarity in communication, and provide participants with essential tools to handle emotionally charged medical situations. The design of the program reflects the feedback and input from senior cardiologists, psychologists, and actual parents of children with CHD. This collaboration ensures a holistic approach to communication training. The course is designed for small groups of five participants, allowing for intensive and personalized learning. These participants are pediatric cardiology trainees at various stages of their careers. This multidisciplinary setup ensures that the training is applicable to all levels of experience, while small group sizes enable each participant to engage deeply in both simulated scenarios and debriefing sessions. The SIMUL-CHD training is delivered by a multidisciplinary team, including: A senior pediatric cardiologist, who brings medical expertise and shares their experience in delivering difficult diagnoses. A psychologist, who focuses on the emotional and psychological impact of delivering and receiving bad news. Two simulated parents, real parents of children with CHD, who share their lived experiences and participate in the simulated scenarios. One parent engages directly in the role-play scenarios, while the other joins the debriefing to offer authentic insights into the parent’s perspective. Program Structure: The morning session focuses on theoretical knowledge, with contributions from each of the trainers: Senior Pediatric Cardiologist : The cardiologist explains the process of delivering bad news, focusing on organization, body language, verbal and non-verbal communication, and managing both the physician’s and the family’s expectations. Key points include how to manage complex medical information and the physician’s role in supporting the family emotionally. Psychologist : The psychologist provides insights into the psychological impact of these communications, emphasizing how stress and emotional states can affect both the physician’s delivery and the family’s reception of the news. They also discuss strategies for managing difficult emotional responses during these interactions. Simulated Parents : parents shares what they wish doctors would know when delivering a diagnosis. Their testimony is aimed at helping trainees understand the emotional weight and expectations parents have during these critical conversations. After a brief break, participants are encouraged to share their own experiences of delivering bad news, whether positive or negative. This open discussion allows for peer learning and reflection, with input from both the psychologist and the parent, providing an emotionally safe environment for trainees to discuss challenges they have faced. These experiences are then linked back to the theoretical concepts discussed earlier. The afternoon session is dedicated to hands-on simulated scenarios. Each participant will take on the role of the “main announcer,” or “co-announcer. Each simulation consists of three phases: the announcement, the debriefing, and peer feedback. Each scenario simulates a real-life situation where a trainee must communicate a difficult diagnosis. The trainees must explain the medical implications of the diagnosis, including possible treatments and outcomes, while managing the emotional needs of the parents. These scenarios are designed to cover a range of emotional and cognitive challenges, from parents in shock to those with numerous questions and fears about the future. After each simulation, participants receive structured feedback from the trainers and peers. The debriefing is structured using the “plus-delta” approach, focusing on what was done well and what could be improved. The presence of the partner parent in the debriefing adds an authentic perspective, highlighting the emotional nuances that may have been missed by the trainees. Formative assessment takes place throughout the day during the debriefing sessions. Trainees receive real-time feedback on their communication style, empathy, and ability to handle emotionally charged situations. These assessments are not graded but are meant to foster growth and reflection. All sites will use the same set of simulated scenarios, with scripts carefully designed to cover a variety of communication challenges. The trainers, including cardiologists, psychologists, and parents, will undergo a standardized preparation session to ensure consistency in feedback and debriefing methods. The same trainers will be used across all sites. To reduce bias, the parent/caregiver taking part in the training program will be different to those participating in the evaluation scenarios. Similarly, the scenarios used during the simulation program will be different from those used in the evaluation scenarios. In order to avoid bias of over-evaluation of the effect of our simulation program by the “simple” fact of giving more time for training, we are going to implement an online training webinar sufficiently relevant for the control arm. The webinar will last approximately 3 h and will be organized into three to four presentations. These presentations will be delivered by pediatric cardiologists, psychologists, and parents, each offering unique perspectives on delivering difficult news. Also, employing a control group receiving standardized theoretical training can be regarded as ethically sound, ensuring access to a form of training for all study participants. Sample size calculation The study by Gorniewicz et al. (2017) concluded that if dimension 2 (“Breaking Bad News”) is retained, a sample of 18 people in each group will have 90% power to detect a difference in mean of -1.3 (the difference between a control group mean, µ₁, of -0.25 and an intervention group mean, µ₂, of 1.05) assuming a common standard deviation of 1.14, using a Student’s t-test with a two-tailed significance level of 5% (NQuery software). With a non-exploitable data rate of around 10%, we concluded that it would be necessary to include 20 subjects per group. Study timeline and feasibility In France, resident and post-resident juniors rotate between departments every six months which provides a predictability with participant availability. The major pediatric cardiac referral centers enroll 5 to 10 pediatric cardiology trainees and the centers of pediatric cardiac expertise support 1 and 5 pediatric cardiology trainees. The feasibility of the study over a six-month period is therefore guaranteed. The study will start in in May 2025 and end in November 2025. During this six-month period, all participants will undergo an initial evaluation, followed by training based on randomization, and finally, a concluding evaluation. The participant timeline is presented in Table . Data analysis plan A descriptive analysis of the initial characteristics of each group will be conducted. Qualitative variables will include the sample size and the frequency of different modalities. Quantitative variables will include the sample size, mean, standard deviation, median, and extreme values (minimum and maximum). The comparability of the two groups before treatment will be assessed for all initial characteristics. In case of non-comparability on one or more parameters that may influence the efficacy criteria, an adjustment may be made on these parameters for the intergroup comparisons of the outcome measures as part of a sensitivity analysis. The analysis of efficacy outcome measures will be conducted on an intention-to-treat (ITT) basis. Each doctor will be analyzed to their randomized group regardless of protocol deviations. The difference between the initial and final scores for dimension 2 “Breaking Bad News” skills questionnaire will be compared between the two groups using a Student’s t-test if the distribution is parametric or using a Mann-Whitney test if the distribution is non-parametric. The effect size (Cohen’s d) and its 95% confidence interval will be assessed. The following secondary criteria will be analyzed using the same strategy as the primary criterion: difference between initial and final scores for the other 4 dimensions of “BBN Skills” evaluated by the evaluation committee self-assessment by the paediatric cardiology trainees of performance anxiety before and after the intervention self-assessment of trainee empathy and concordance between different assessments of the BBN Skills score. A test will be considered statistically significant if the p-value is less than the 5% significance threshold. The analyses will be conducted using the SAS or R software after the database is locked and the Statistical Analysis Plan is approved. The study by Gorniewicz et al. (2017) concluded that if dimension 2 (“Breaking Bad News”) is retained, a sample of 18 people in each group will have 90% power to detect a difference in mean of -1.3 (the difference between a control group mean, µ₁, of -0.25 and an intervention group mean, µ₂, of 1.05) assuming a common standard deviation of 1.14, using a Student’s t-test with a two-tailed significance level of 5% (NQuery software). With a non-exploitable data rate of around 10%, we concluded that it would be necessary to include 20 subjects per group. In France, resident and post-resident juniors rotate between departments every six months which provides a predictability with participant availability. The major pediatric cardiac referral centers enroll 5 to 10 pediatric cardiology trainees and the centers of pediatric cardiac expertise support 1 and 5 pediatric cardiology trainees. The feasibility of the study over a six-month period is therefore guaranteed. The study will start in in May 2025 and end in November 2025. During this six-month period, all participants will undergo an initial evaluation, followed by training based on randomization, and finally, a concluding evaluation. The participant timeline is presented in Table . A descriptive analysis of the initial characteristics of each group will be conducted. Qualitative variables will include the sample size and the frequency of different modalities. Quantitative variables will include the sample size, mean, standard deviation, median, and extreme values (minimum and maximum). The comparability of the two groups before treatment will be assessed for all initial characteristics. In case of non-comparability on one or more parameters that may influence the efficacy criteria, an adjustment may be made on these parameters for the intergroup comparisons of the outcome measures as part of a sensitivity analysis. The analysis of efficacy outcome measures will be conducted on an intention-to-treat (ITT) basis. Each doctor will be analyzed to their randomized group regardless of protocol deviations. The difference between the initial and final scores for dimension 2 “Breaking Bad News” skills questionnaire will be compared between the two groups using a Student’s t-test if the distribution is parametric or using a Mann-Whitney test if the distribution is non-parametric. The effect size (Cohen’s d) and its 95% confidence interval will be assessed. The following secondary criteria will be analyzed using the same strategy as the primary criterion: difference between initial and final scores for the other 4 dimensions of “BBN Skills” evaluated by the evaluation committee self-assessment by the paediatric cardiology trainees of performance anxiety before and after the intervention self-assessment of trainee empathy and concordance between different assessments of the BBN Skills score. A test will be considered statistically significant if the p-value is less than the 5% significance threshold. The analyses will be conducted using the SAS or R software after the database is locked and the Statistical Analysis Plan is approved. Our study protocol, SIMUL-CHD, addresses the unique challenges in successful communication during the period of initial diagnostic counselling of CHD by pediatric cardiologists to patients and their families. The intricacies of child/parent-physician relationships in the context of postnatal detection of CHD underscore the need for a nuanced and practical simulation-based training approach. Our protocol addresses this challenge by utilizing a simulation-based approach that combines both theoretical and practical elements, creating a comprehensive learning experience designed to improve physician-patient-family interactions. The inclusion of real parents as standardized patients in our simulations is a particularly innovative aspect of this study. While simulated patients have been employed in medical education since the 1960s, the specific use of parents in pediatric settings—particularly within cardiology—remains largely unexplored. This approach introduces a personal and experiential element that goes beyond the standard simulated patient model. The inclusion of parents who have personally experienced the challenges of raising a child with CHD adds an unparalleled layer of authenticity to the simulation. This experiential component allows trainees to engage with the emotional realities of families in a way that goes beyond traditional simulation models. To our knowledge, no prior studies have employed this approach specifically in pediatric cardiology. We anticipate that this model could be adapted for use in other pediatric subspecialties or chronic disease areas where effective communication is paramount. This study could shift the broader medical education paradigm toward integrating real patients and caregivers as standardized patients, fostering a deeper, more empathetic approach to clinical care. Despite the potential benefits, the SIMUL-CHD study has several limitations. First, the training of simulated parents may introduce variability, as it relies heavily on the personal experiences and emotional authenticity of the parents involved. Standardizing this component across multiple training sites is challenging and may affect the uniformity of the educational experience. Second, while the study's small group size allows for personalized feedback and interaction, it may limit the generalizability of our findings. Additionally, we must consider the possibility that the effects observed in a controlled, simulated environment may not fully translate to real-world clinical settings, where emotional and time pressures can vary significantly. Lastly, our study focuses primarily on the technical and emotional aspects of delivering a diagnosis. While this is crucial, further studies are needed to assess how these communication improvements impact long-term family outcomes, including parental coping, adherence to medical recommendations, and the child's health trajectory. The SIMUL-CHD protocol addresses the intricate challenges of communication skills training in pediatric cardiology, particularly when delivering a diagnosis of CHD. A key innovation of this study is the involvement of real parents of children with CHD as simulated parents, which we believe will provide trainees with a more realistic and impactful learning experience. This unique approach could pave the way for similar training models in pediatric specialties. |
An avenue to miscarriage: a case report | 82be95a7-ae46-4976-aed9-fc1044369c5c | 9587106 | Forensic Medicine[mh] | The victim A lived in a multi-occupancy flat in London with other Polish nationals, including one of the defendants B. A co-defendant, C, lived in an adjacent street and a further defendant, D, had been staying in the victim’s flat at the time of the incident which occurred in the early hours of the morning in August 2016. A was assaulted in the front room of his flat which was used as his bedroom. This room was on the ground floor of the property, above a basement flat, and was accessed via a set of steps leading up to the communal entrance of the property. A was attacked with a knife in the front room of his flat and sustained bleeding injuries. His cries for help were heard by other occupants of the house who went to his flat and knocked on the door which was opened by D. Following this initial assault, A escaped from the flat through the front bay window. On exiting the window, he then had to traverse the narrow window ledge and climb down onto the front steps leading onto the pavement. Defendants B and C were observed on CCTV leaving the flat and walking away in the opposite direction to A. The other occupant of the flat, D, followed A. Short time later, B returned to the property and into A’s room where he moved around and stood on a sofa which was positioned under the bay window. In doing so, he stepped in A’s blood, which was present on the floor, and transferred footwear marks. In the meantime, A had collapsed outside the front door of a residential property approximately 60 m away in another avenue. His blood-soaked T-shirt was found beside his body. He had been heard shouting for help in the street and is believed to have crouched behind a parked car. The occupant of the residential property, who was wakened by a disturbance outside, found A collapsed in a pool of blood on his doorstep.
The scene within the front room of the deceased’s flat was identified after the police followed a trail of blood spots from where the body of A was found. It was evident that the trail of blood back to the flat had a significant impact on how the crime scene examination strategy developed. The strategy employed by the police was based on the assumption that (i) A had been assaulted exclusively in the front room of the flat; (ii) all of his injuries had been sustained there; and (iii) the trail of blood demonstrated the route that he took after he left the property through the bay window, pausing behind the parked car before making his way into the avenue where he eventually collapsed outside a residential property. Part of his journey was corroborated by private CCTV footage. This showed that his hands were down by his side—an observation that had a significant impact on the expected blood loss from the fatal neck injury. These erroneous initial assumptions made by the police were then conveyed to others, including forensic science experts who attended the scene and documented bloodstaining, etc. within the flat and on the window and communal steps. Although the attending scientists were aware that a blood trail led from the flat to the area where the body was found, this area was not subject to specialist scientific examination and only general scene photographs were taken.
a – b) The inside of the flat was examined by a bloodstain scientist. The distribution of blood comprised a combination of transfer and spattered bloodstains. There was clear evidence of some form of assault having taken place on or near the bed with heavy saturated bloodstaining on the mattress. There was transfer bloodstaining, mixed with hair, on the wall behind the bed indicating that the deceased had sustained some injuries to his head—this was confirmed at autopsy by the finding of an extensive incised wound across the top and back of the scalp. The extent and severity of the scalp injuries were sufficient to account for the heavy bloodstaining within the flat.
a) The area immediately outside the flat was also examined by a bloodstain scientist. A limited number of drip stains were identified in the area of the bay window and transfer bloodstaining was present on the underside of the window frame. The findings corroborated the view that A , having already been injured, had left the property from the bay window. However, there was no apparent expirated bloodstaining or evidence of heavy bleeding from a severed major blood vessel.
b) All areas beyond the immediate vicinity of the flat were not subjected to specialist forensic examination. Scene photographs showed a small number of drip stains on the pavement along the initial part of the route taken by A after he left the flat. The relative paucity of dripped blood suggested that A was not bleeding heavily at this time. Bloodstains on the road at the rear of the parked car and on the rear bumper of the vehicle corroborated the view that A had stopped there and that he was bleeding more freely at that time.
a–c) The avenue, and an alleyway leading from it, was not subjected to a specialist forensic examination. Photographs demonstrated a substantial increase in bloodstaining in an alley off this avenue and large volume drips of blood could be seen on the ground and walls. The blood would appear to have dripped directly from A ’s injuries and/or from surfaces that had become saturated with blood. In places, the blood drops were present in clusters and were associated with pronounced secondary spatter, a feature that is typically associated with prolonged or large volume blood loss and can indicate ‘pause points’ and/or locations where a volume of blood has been suddenly released. In summary, the blood findings in the alley suggested that A had been there for a period of time and that blood loss had markedly increased there. Extremely heavy bloodstaining, including extensive pooling and drip patterns, was present outside the residential property where A was found slumped against a wall. Heavy bloodstaining was present on the door of the property including some expirated blood that showed pronounced bubbling. A number of large directional drops of blood were seen on the walls on each side of the door and appeared to comprise vomited/coughed blood (from A ’s mouth or neck injury), along with probable projected blood lost from his neck wound or cast from surfaces wet with blood. The features collectively indicated that A had sustained the fatal stab injury to his neck at this location resulting in significant amounts of blood entering his airways.
) The deceased was a healthy 25-year-old male (height 184 cm and weighing 72 kg). He had sustained a number of incised wounds to his head, neck, left shoulder, left wrist and right forearm. Sharp force trauma to the back of the head corresponded to bloodstains of Fig. a and b in the flat. The fatal wound was a 14-cm deep stab wound to the left side of his neck which passed across the neck through the muscles and trachea to the right side where it had divided the right carotid artery. Further details of this arterial stab wound were not provided in the autopsy report. This was deemed the fatal injury and would have caused rapid but not immediate death. The deceased had also sustained a number of blunt force injuries consistent with him having been assaulted. Blood had been aspirated into his air passages and lungs. Toxicology revealed some cannabis and amphetamine but the levels detected were not deemed significant.
Following the police investigation, persons B, C and D were charged with murder and sent for trial. Defendant B told the jury that he had seen D with a knife attacking the deceased in the front room and hence had left but had returned a short time later to look for the victim providing an explanation for his partial footwear marks in A’s blood. The prosecution however sought to attribute this as evidence that he had been present in the room at the time of the attack. Unfortunately, defendants B and C lied to the police and this tainted their later testimony and undermined their credibility as witnesses in respect of the actions of D. None of the medical or scientific experts involved in the initial investigation appeared to have questioned the assumption that an individual with a fatal stab wound to the neck would have been capable of climbing out through a window, negotiating a narrow ledge, jumping onto steps and then running over 60 m before collapsing and without leaving a significant trail of blood en route. It was ostensibly accepted that a young, healthy male could carry out strenuous physical activity after the fatal injury was sustained and that the lack of blood along the escape route could be accounted for by him using his T-shirt to stem the bleeding. After a trial lasting 6 weeks and based on the assumption that the fatal stab wound and all the other injuries were sustained whilst A was inside the flat when all 3 defendants were present, the jury convicted B and C of murder by a majority 10−2 verdict. The jury were unable to reach a verdict in respect of D who they had been told was of good character. He was subsequently re-tried but once again the jury failed to reach a verdict. The crown offered no further evidence against him and a ‘not guilty’ verdict was entered.
Following the verdict, work of newly instructed experts challenged the basis on which the crime scene examination strategy had been progressed. A review of the pathology findings, blood distribution and CCTV footage casts serious doubt on the assumption that the fatal neck wound had been sustained inside the flat. The opinion of the newly instructed pathologists was that A’s fatal neck injury would have bled massively immediately after infliction and would have rapidly limited his ability to carry out purposeful movements and activities. The right carotid artery had been divided and this would have resulted in rapid and massive blood loss leading to hypovolaemic shock and collapse, probably within a few minutes. Even if, as suggested, A was able to stem the flow of blood by applying pressure to his neck, blood would have entered the partially divided trachea causing difficulty breathing. This, and an incision through the larynx, would have rendered him incapable of screaming loudly despite a witness hearing loud screams after he had left the flat. Also, it would have been expected that expirated blood patterns would have been found inside the flat and in the area around the exit route through the window if the airways were filled with blood—these areas were subjected to a full examination by bloodstain pattern scientists and no expirated blood was found. It was the opinion of the new pathology experts that all the bloodstaining inside the flat could be accounted for by the scalp and back wounds which would have bled considerably. The forensic biologist who reviewed the case was of the opinion that the relative amount of bloodstaining outside the flat, as observed from the scene photographs, was initially limited but then increased substantially in the alley close to where A was found. Pronounced drip patterns were present there, and although in themselves could not be used to identify the actions or scenario which led to their development, they indicated that A was bleeding extensively at this location. Heavy areas of bloodstaining on and around the door of the property where A had collapsed and died, including pronounced exhaled blood from his mouth or neck (from the laryngeal and tracheal wounds), indicated that A had sustained the major stab wound to his neck and that significant amounts of blood were entering his airways at this time. Given A’s activities over the proposed timescale and the absence of significant bloodstaining, commensurate with the neck wound, around the bay window and behind the parked car, in combination with the paucity of dripped blood en route to the alley, all cast doubt on the initial assumption of where the fatal attack took place. The conclusion that the fatal stab wound was inflicted in the alley some distance from the flat effectively excluded defendants B and C as being responsible.
In January 2021, the convictions of B and C were quashed and a re-trial ordered.
B and C stood trial at the Central Criminal Court in August 2021. The new scientific and pathology evidence was presented to the court and, as a result, both men were acquitted of murder. They were released from custody and were free to return home to their families in Poland.
The incapacity to act differs individually and is dependent on age, pre-existing illnesses and injury pattern. A differentiation is made between immediate, faster, delayed, restricted and lack of incapacity to act, depending on the injury pattern . Immediate incapacity to act is given if the brain as the central control unit of the entire organism is affected. Rapid inability to act results from damage to vital structures such as the heart, pulmonary trunk or aorta, when severe blood loss causes rapid circulatory depression with subsequent hypoxia of the brain, whilst delayed inability to act is caused by damage to large vessels or organs (liver, lungs, kidneys). In forensic literature, however, exceptions are repeatedly reported when victims were able, for example, to shoot themselves in the head several times [ – ], to run long distances or to respond to fire despite gunshot-related heart rupture . In this case, the new pathology and scientific evidence was critical in reconstructing the sequence of events leading up to the death of A. The initial reconstruction of events as determined by the police investigation team, including the pathologist and scientific experts initially instructed was, as was later agreed, badly flawed. The underlying forensic strategy was based on the assumption that all of A’s wounds, including the fatal stab wound to the neck, had been inflicted within the flat. This error had a significant impact on how the investigation developed. Because the crime scene and forensic personnel did not question this assumption, on which their instructions were based, a detailed examination of the bloodstaining on the roadway, or in the alley, did not take place. That A would have been capable of climbing out of the window, negotiating the narrow window ledge and then jumping onto the steps before running a significant distance before collapsing was accepted without question and consequently it was felt that there was no reason to examine the bloodstains along that route in any detail. The absence of discernible bloodstains along the initial section of road leading from the flat and the relative paucity of blood around the bay window were explained by A being able to staunch the flow of blood from his injuries. The flawed strategy was inappropriately reinforced and inadequately challenged leading to a miscarriage of justice. There was no doubt that the attack on A started inside his flat as demonstrated by bloodstaining on the floor, walls and bed but it was also assumed, and not challenged, that the fatal neck wound was also inflicted here. This assumption allowed the crown to attribute responsibility for the killing to all 3 men who had been present in the flat. The review conducted by the newly instructed pathology and scientific experts challenged the basis on which the initial crime scene examination had progressed. The outcomes of their review were then subject to a joint conference between the pathologists and forensic scientists instructed on behalf of the prosecution and defense with the production of agreed joint statements. In this case, 6 independent medical and scientific experts, from 3 jurisdictions, concluded that A’s fatal injury had been sustained outside the flat and in the avenue or alley, 60 m away. This joint opinion was a crucial piece of evidence at the re-trial and, in no small measure, contributed to the acquittal of both B and C both of whom had already spent 4 years in prison. The development of the forensic strategy in this case started with an assumption that was not challenged or reviewed during the course of the investigation and nurtured an environment for confirmation bias. A more inclusive crime scene examination strategy would have mitigated the damage that those assumptions had on the evolving investigation. The importance of specialist scene examination by forensic experts in those cases where blood pattern analysis is required cannot be over emphasised . It is also essential that forensic scientists and pathologists work together sharing information to assess the significance of wounds and their relevance to scene interpretation. In this case, the focus of the investigation was misplaced and the scientific findings so fragmented that a miscarriage of justice was inevitable.
Crime scene examination prior to autopsy should, where possible, be carried in suspected cases of homicide. Scene examination and interpretation is crucial for forensic pathologists. Reliance solely on isolated autopsy findings may result in loss of key evidence. In suspected homicide cases, it is important that pathologists take into consideration scene findings and work in conjunction with forensic scientists when drawing up reports and conclusions. Whilst bloodstain pattern analysis is carried out by crime scene investigators and forensic scientists, it is recommended that forensic pathologists have specialised training in this field. Ability to act remarkably differs in individual subjects with regard to age, pre-existing illnesses and other confounding factors. Nonetheless, general statements can be made with respect to individual injury patterns and circumstances of the respective case.
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Comparing effects and application of telemedicine for different specialties in emergency medicine using the Emergency Talk Application (U-Sim ETA Trial) | f0240841-e059-489c-9a54-d70e7ad2028b | 10432512 | Internal Medicine[mh] | The field of Emergency Medicine is currently under pressure to develop new solutions for multiple challenges: Increasing numbers of emergencies to which ambulances respond, more patients that present themselves in emergency departments (ED) but also an aging population with increasingly complex and chronic medical histories. Furthermore, emerging diseases that can present themselves in extreme scenarios like the covid 19 pandemic are only a few but some of the reasons why the field of Emergency Medicine has to keep on evolving – . Economic pressure, a limited number of available staff and an increasing societal urge for a quicker visit and a possible diagnosis are other reasons why new paths to provide adequate and timely treatment are more than needed , , , . Also increasing accessibility to medical services by lowering boundaries especially for socially disadvantaged as well as patients from resource limited settings in an increasingly digitally connected world are challenges of our current time – . Therefore applying novel technologies within established clinical settings could be a path to approach these challenges. Telemedicine as a digital health technology can overcome geographical distances and can connect patients with healthcare providers , . Within the field of Emergency Medicine (EM) the use of telemedicine is as varied as the nature of the field of EM itself. There are a variety of applications, systems and combinations that either use a store and forward technology, provide a real-time video and audio transfer (videoconference-like solutions) or even a combination of both. Also vital signs can be transmitted the same way, allowing the remote monitoring of patients (telemonitoring), while communicating with paramedics in real time and at times even the patient. This variety allows a different delivery of telemedicine in different medical fields: The store and forward technology is often reported for the cardiovascular field, real-time video and audio transfer solutions for stroke treatment while for general and acute care a combination of both technologies is reported – . In Addition, the availability and application of the used technology can very: For communication and visualisation, cameras can be available within ambulances, in other settings paramedics are provided with bodycams or use smartphones and tablets for communication. But also vital signs can be transferred directly, or via monitoring devices or even a data transmission unit, as well as written orders and other relevant information can be sent directly to the scene or to a T-EP. Therefore the increasing amount of options that are available in the field of telemedicine offer healthcare providers many possibilities on how treatments as well as information can be transferred, received and applied – . Also the environment for telemedical system vary as they depends on regional emergency medical structures including geography but also on laws, data protection regulations but also on standardized medical treatment options which paramedics are allowed to provide – , , – . Therefore the concepts of telemedical system varies and so the position from which the TEP participates (that is consulted by paramedics which provide treatment) is different: The TEP can be located at an emergency dispatch center, in an other prehospital setting (like an emergency physician vehicle, like in Germany), within an ED or can be available only as a specialist for certain medical areas and questions regarding pediatrics, cardiology, palliative care, chronic or acute care but also as a general practitioner , – . To improve the application of telemedicine, lower the boundaries as well as improve access for low- and middle income healthcare systems we planned to provide an open-access solution so that different structures, regions and already applied concepts, could be included: By putting the patient in the center and allowing quick but safe access to a telemedical emergency network, patients can be provided with relevant medical expertise while being treated at their site of emergency. By developing an Emergency Talk Network (ETN) with a focus on the individual patient and adding technology to the already available regional structure of EM, patients might be treated quicker and more effectively. While offering non-urgent emergencies a redirection to a different healthcare provider instead of an ED and f.ex a treatment by GP or an other regionally available facility (i.e. Fig. ). This solution strategy builds on available regional structures and includes their strengths while adding technology to improve the patient’s pathway through the healthcare system. This could mean that after a live telemedical consultation, patients could be referred for later treatment or if needed transferred to a hospital or specialist, if treatment via the network was not enough , – . Many telemedical system are used and evaluated for only one disease (like stroke or myocardial infarction) but a broader view must be taken as effectivities of emergency medical systems could be improved. Also unnecessary transports to hospitals could be reduced while improving cost-effectiveness and even reducing costs , , , , . To support this concept we developed a telemedical system called the Emergency Talk Application (ETA). This bidirection live video and audio communication solution is based on an open-source and standardized web based communication technology as defined by the W3 Consortium, while being over-compliant and encrypted with current data protection and safety standards including EU-GDPR and HIPAA . This involves constantly updated security technologies that can be used with WebRTC like SSL/TLS certificates (Secure Sockets Layer/Transport Layer Security) but can also provide Transport Layer Security (TLS) like the Secure Real-time Transport Protocol (SRTP) as security threats need to be constantly monitored and managed in telemedical technologies . But is also conform to different international telemedical guidelines from the field of emergency medicine like the German guideline on tele-emergency medicine or the American Telemedicine Association’s Telestroke Guidelines as well as ethical considerations in telemedicine , , . Combining this technical and medical approach puts the patient at the center while being accessible and usable worldwide. To test this application and concept, we focused not like other studies in the field of pre-hospital emergency telemedicine only on one tracer diagnosis like stroke, trauma or myocardial infarction, but also on common diseases like hypertensive emergencies, exacerbations of a chronic lung disease (like COPD), hypoglycemia or generalized seizures as a general application of a telemedical solution in the field of EM should be possible .
Trial design and setting In this first application of ETA we performed a randomized usability and simulation trial (U-Sim ETA Trial) at the Prehospital Emergency Medical Education Center Wetzlar. As no potential harm was to be expected, the local ethics committee (University Hospital Giessen, Germany) solely required informed consent from the participants (AZ 90/21). After written consent including a data-privacy agreement was signed, the participants received an initial lecture and introduction to ETA. Overall there were 16 scenarios (i.e. Table ) from the medical areas of internal medicine, trauma and neurology. These were all standardized including sequences of events, vital parameters, predefined patient answers to questions as well as complications if mistakes or a certain therapy was not performed. These scenarios are also used for the paramedic board examination in the state of Hesse. High-fidelity simulators and actors were used as standardized patients (SP), as well as standard equipment. After the introduction participants were allowed to practice with the equipment until they felt comfortable. During the scenarios an instructor was present to enable help in case of technical problems but offered no medical support. All instructors were state certified educators for paramedic education. The teams were instructed to examine the SP like in clinical routine. If vital signs or an examination could not be performed these were announced by an instructor, but only if they were measured or examined. Observers were also present, but they were only allowed to document the scenario. Therefore comparable and uniform scenarios were ensured. The scenario ended if the paramedics were finished with their treatment and would begin the transport of the patient. This was chosen as some participants could chose to treat the patient during a transport or within the ambulance, which could result in a lower scenario specific scoring, although an adequate therapy might have been performed “on the go”. Also a maximum time of 45 min per scenario was defined. Participant recruitment and randomization Emergency medical staff and Emergency Physicians (EP) received invitations via local email lists and announcements for this simulation study and could participate voluntarily if they currently worked in the German state of Hessen. Depending on their qualification eligibility was evaluated. Certified EMTs and Paramedics with a valid board certificate could participate. Physicians could participate if they held a valid board qualification as EPs. After completing the registration process the participants were randomized into teams by sealed envelopes. Each Emergency Medical Service (EMS) team consisted of 2 paramedics, acting as an ambulance team and one EP, acting as the Tele Emergency Physician (TEP). The TEP was not present at the Emergency Medical Education Center Wetzlar (trial site) but was digitally available via ETA at their clinical worksite which could be an ED, intensive care unit or a normal ward. This allowed the TEP to support the paramedics, while being able to do their regular clinical work. Participants therefore did not know the TEP before the scenario. A week prior to the study participating TEP received a standardized introduction and practical training with the telemedicine system. Randomization of the scenario was performed on entering the simulation room. The ambulance team pointed to one of 16 envelopes which were lying on a table. The instructor took the pointed-out envelope and took out the scenario description. The description was only visual to the instructor. Each team could then complete one of 16 randomised scenarios and not perform the same scenario more than once. Telemedical system The paramedics were equipped with an Ipad Mini Series 5 (Apple Inc., California USA ) that had a standard browser (Safari Version 15 ). The browsers starting page was the ETA website. Every Team received a username and a Password, so that an individual login was performed. The TEP could use a Tablet, PC or Smartphone of their choice that was available during the time of the trial. The device had to be tested beforehand during the standardized introduction. Once the Paramedics needed the support of a TEP, they logged into the website and chose an TEP that was shown as “available”. The TEP then received an alarm on their device. The Alarm included one of three buttons: The “accept” button allowed to accept the call right away and opened a tab in which a continuous two-way audio and video communication was directly possible. A second Tab “accept, but later” accepted the call, but opening a tab would be postponed and only a button with “available alarm” was shown. Choosing this would open a tab in which the audio and video communication was directly possible. The third button “deny” would stop and deny the alarm and no communication would be possible. Once the Alarm was accepted a standard screen was available for all participants (e.g. Fig. ). Audio and Video communication is shown in the middle. The left side allows a live messenger communication. Every sent message can be confirmed by the receiver via a “confirm” button. Every Message and confirmation has a time and date marker. The right side shows a document viewer. The document viewer can show uploaded PDF documents like standard operating procedures or checklists. During this trial only the algorithms that were available and developed for the use within the state of Hesse were available and could be viewed by the participants . If the TEP help wasn’t needed by the paramedics anymore, he could leave by exiting the tab or window. Data sources and outcomes To compare the performance of the teams a set of scenario-specific scoring items for each scenario were developed (i.e. Table ). These items represented guideline based diagnostic and therapeutic skills which are based on medical algorithms for treatment by paramedics in the state of Hesse , . Depending on the scenario these were 8–10 skills (i.e. Supplement Table ). These where then calculated to receive a performance percentage value (example: 7 out of 9 performed = 0.78 or 78%). This would allow a relative scenario specific comparison. Relevant time marks like the beginning, the end of a scenario, the time of diagnosis, arrival and departure of the TEP were documented. Therefore the length of time for each scenario, time a TEP was bound, time of TEP arrival after being called by paramedics and the time until a diagnosis was made could be analyzed. Also if a TEP was called and which participant (Paramedic or TEP) made the diagnosis was documented. Each scenario was analyzed by two faculty observers using the mentioned scoring items and chronologically documented the scenario by using a prepared form. Both observers had to reach a common decision before all data was transferred to a database (Microsoft Excel, Version 22.10, Vermont, USA ). Figure shows the trial design. Statistical methods Due to the fact that this was a pilot study and the first use of ETA, no alpha-adjustment was performed and p-values < 0.05 were considered to be significant. Categorical data were presented by frequencies and percentage. The analysis was then performed per scenario (n = 16) and medical specialty (internal medicine, trauma or neurology; n = 3). For the durations and scenario-specific scoring an Analysis of Variance (ANOVA) or Welch’s-ANOVA (W-test) was planned with Post-Hoc Games–Howell or Tukey analysis depending on the Homogeneity of variance (Levene’s test, p > 0.05.) Normal distribution was assessed by the Shapiro–Wilk test (α = 0.05) or if a sample size of n > 30 was available . If a non-parametrical test was to be performed because of a small or non-normally distributed sample the Kruskal–Wallis-Test H-Test was chosen as an alternative with Post-hoc analysis by Dunn–Bonferroni-Tests. Omega squared (ω 2 ) was used as a measure of effect size, as eta squared contains positive bias , . Correlation analysis between scenario length and the time a TEP was bound, scenario length and scenario-specific scoring, duration of TEP bound time and scenario-specific scoring was performed by Pearson product-moment correlation coefficient (PPMCC). If correlation analysis showed a medium to strong correlation, a regression analysis would be performed. Regression analysis was conducted to analyse if the performance depended on the length of the scenario or the length of time the TEP was bound with a (simple) linear regression. If more than one medium or strong correlation could be analyzed a multiple linear regression was planned. (Omega squared (ω 2 ) was used as a measure of effect size). A two-sided binominal test was performed to analyse if the diagnosis was made by the TEP or paramedics. The investigators suspected that 75% were to be made by paramedics and 25% by the TEP. Epidemiologic data was also evaluated: Participants age, gender, experience and qualification was evaluated in an anonymized questionnaire. The Data Analysis was then performed with IBM SPSS Statistics (Version 28.0.1.1 (14), IBM Corp., Armonk, New York, USA). Visualisation with R (Version 4.2.2 (2022-10-31), The R Foundation for Statistical Computing). All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the ethics committee of the Justus Liebig University Giessen (AZ 90/21). Ethics approval and consent to participate As no potential harm was to be expected, the local ethics committee (University Hospital Giessen, Germany) solely required informed consent from the participants. Participation was only possible after written consent including a data-privacy agreement was signed. Informed consent Written informed consent for the publication of images and details was obtained from all participants.
In this first application of ETA we performed a randomized usability and simulation trial (U-Sim ETA Trial) at the Prehospital Emergency Medical Education Center Wetzlar. As no potential harm was to be expected, the local ethics committee (University Hospital Giessen, Germany) solely required informed consent from the participants (AZ 90/21). After written consent including a data-privacy agreement was signed, the participants received an initial lecture and introduction to ETA. Overall there were 16 scenarios (i.e. Table ) from the medical areas of internal medicine, trauma and neurology. These were all standardized including sequences of events, vital parameters, predefined patient answers to questions as well as complications if mistakes or a certain therapy was not performed. These scenarios are also used for the paramedic board examination in the state of Hesse. High-fidelity simulators and actors were used as standardized patients (SP), as well as standard equipment. After the introduction participants were allowed to practice with the equipment until they felt comfortable. During the scenarios an instructor was present to enable help in case of technical problems but offered no medical support. All instructors were state certified educators for paramedic education. The teams were instructed to examine the SP like in clinical routine. If vital signs or an examination could not be performed these were announced by an instructor, but only if they were measured or examined. Observers were also present, but they were only allowed to document the scenario. Therefore comparable and uniform scenarios were ensured. The scenario ended if the paramedics were finished with their treatment and would begin the transport of the patient. This was chosen as some participants could chose to treat the patient during a transport or within the ambulance, which could result in a lower scenario specific scoring, although an adequate therapy might have been performed “on the go”. Also a maximum time of 45 min per scenario was defined.
Emergency medical staff and Emergency Physicians (EP) received invitations via local email lists and announcements for this simulation study and could participate voluntarily if they currently worked in the German state of Hessen. Depending on their qualification eligibility was evaluated. Certified EMTs and Paramedics with a valid board certificate could participate. Physicians could participate if they held a valid board qualification as EPs. After completing the registration process the participants were randomized into teams by sealed envelopes. Each Emergency Medical Service (EMS) team consisted of 2 paramedics, acting as an ambulance team and one EP, acting as the Tele Emergency Physician (TEP). The TEP was not present at the Emergency Medical Education Center Wetzlar (trial site) but was digitally available via ETA at their clinical worksite which could be an ED, intensive care unit or a normal ward. This allowed the TEP to support the paramedics, while being able to do their regular clinical work. Participants therefore did not know the TEP before the scenario. A week prior to the study participating TEP received a standardized introduction and practical training with the telemedicine system. Randomization of the scenario was performed on entering the simulation room. The ambulance team pointed to one of 16 envelopes which were lying on a table. The instructor took the pointed-out envelope and took out the scenario description. The description was only visual to the instructor. Each team could then complete one of 16 randomised scenarios and not perform the same scenario more than once.
The paramedics were equipped with an Ipad Mini Series 5 (Apple Inc., California USA ) that had a standard browser (Safari Version 15 ). The browsers starting page was the ETA website. Every Team received a username and a Password, so that an individual login was performed. The TEP could use a Tablet, PC or Smartphone of their choice that was available during the time of the trial. The device had to be tested beforehand during the standardized introduction. Once the Paramedics needed the support of a TEP, they logged into the website and chose an TEP that was shown as “available”. The TEP then received an alarm on their device. The Alarm included one of three buttons: The “accept” button allowed to accept the call right away and opened a tab in which a continuous two-way audio and video communication was directly possible. A second Tab “accept, but later” accepted the call, but opening a tab would be postponed and only a button with “available alarm” was shown. Choosing this would open a tab in which the audio and video communication was directly possible. The third button “deny” would stop and deny the alarm and no communication would be possible. Once the Alarm was accepted a standard screen was available for all participants (e.g. Fig. ). Audio and Video communication is shown in the middle. The left side allows a live messenger communication. Every sent message can be confirmed by the receiver via a “confirm” button. Every Message and confirmation has a time and date marker. The right side shows a document viewer. The document viewer can show uploaded PDF documents like standard operating procedures or checklists. During this trial only the algorithms that were available and developed for the use within the state of Hesse were available and could be viewed by the participants . If the TEP help wasn’t needed by the paramedics anymore, he could leave by exiting the tab or window.
To compare the performance of the teams a set of scenario-specific scoring items for each scenario were developed (i.e. Table ). These items represented guideline based diagnostic and therapeutic skills which are based on medical algorithms for treatment by paramedics in the state of Hesse , . Depending on the scenario these were 8–10 skills (i.e. Supplement Table ). These where then calculated to receive a performance percentage value (example: 7 out of 9 performed = 0.78 or 78%). This would allow a relative scenario specific comparison. Relevant time marks like the beginning, the end of a scenario, the time of diagnosis, arrival and departure of the TEP were documented. Therefore the length of time for each scenario, time a TEP was bound, time of TEP arrival after being called by paramedics and the time until a diagnosis was made could be analyzed. Also if a TEP was called and which participant (Paramedic or TEP) made the diagnosis was documented. Each scenario was analyzed by two faculty observers using the mentioned scoring items and chronologically documented the scenario by using a prepared form. Both observers had to reach a common decision before all data was transferred to a database (Microsoft Excel, Version 22.10, Vermont, USA ). Figure shows the trial design.
Due to the fact that this was a pilot study and the first use of ETA, no alpha-adjustment was performed and p-values < 0.05 were considered to be significant. Categorical data were presented by frequencies and percentage. The analysis was then performed per scenario (n = 16) and medical specialty (internal medicine, trauma or neurology; n = 3). For the durations and scenario-specific scoring an Analysis of Variance (ANOVA) or Welch’s-ANOVA (W-test) was planned with Post-Hoc Games–Howell or Tukey analysis depending on the Homogeneity of variance (Levene’s test, p > 0.05.) Normal distribution was assessed by the Shapiro–Wilk test (α = 0.05) or if a sample size of n > 30 was available . If a non-parametrical test was to be performed because of a small or non-normally distributed sample the Kruskal–Wallis-Test H-Test was chosen as an alternative with Post-hoc analysis by Dunn–Bonferroni-Tests. Omega squared (ω 2 ) was used as a measure of effect size, as eta squared contains positive bias , . Correlation analysis between scenario length and the time a TEP was bound, scenario length and scenario-specific scoring, duration of TEP bound time and scenario-specific scoring was performed by Pearson product-moment correlation coefficient (PPMCC). If correlation analysis showed a medium to strong correlation, a regression analysis would be performed. Regression analysis was conducted to analyse if the performance depended on the length of the scenario or the length of time the TEP was bound with a (simple) linear regression. If more than one medium or strong correlation could be analyzed a multiple linear regression was planned. (Omega squared (ω 2 ) was used as a measure of effect size). A two-sided binominal test was performed to analyse if the diagnosis was made by the TEP or paramedics. The investigators suspected that 75% were to be made by paramedics and 25% by the TEP. Epidemiologic data was also evaluated: Participants age, gender, experience and qualification was evaluated in an anonymized questionnaire. The Data Analysis was then performed with IBM SPSS Statistics (Version 28.0.1.1 (14), IBM Corp., Armonk, New York, USA). Visualisation with R (Version 4.2.2 (2022-10-31), The R Foundation for Statistical Computing). All methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the ethics committee of the Justus Liebig University Giessen (AZ 90/21).
As no potential harm was to be expected, the local ethics committee (University Hospital Giessen, Germany) solely required informed consent from the participants. Participation was only possible after written consent including a data-privacy agreement was signed.
Written informed consent for the publication of images and details was obtained from all participants.
Study population The demographic data of the 96 Participants showed some differences: 64 participated as paramedics and 32 as TEP. Paramedics: age 22.88 (range 18–33) years, 55.26% female, 44.74% male, clinical experience 37.55 (range 12–133) months, 61.28% Paramedics, 38.72% EMTs; TEP: age 37.06 (range 29–59) years, 33.09% female, 59.56% male, clinical experience 138.22 (range 12–360) months, 75.74% emergency physicians, 24.26% emergency physicians with a medical officers qualification (German equivalent: “Leitender Notarzt”). Specializations were described as anesthesiology (n = 8), internal medicine (n = 8), general practitioner (n = 6), trauma surgery and orthopedics (n = 5) and neurology (n = 5). Oberserved outcomes 141 scenarios were performed with 129 (91.49%) having used ETA while 12 (8.51%) didn’t need the support of a TEP. Of the 32 teams, 19 teams performed 4 scenarios (n = 76), while 13 teams performed 5 scenarios overall (n = 65). One case in the telemedical group and one in the non-telemedical had to be excluded for not being clearly assigned to one of the 16 scenarios. Therefore 128 telemedical and 11 non-telemedical scenarios could be analyzed. Because the amount of cases per scenario with telemedical support ranged from 7 to 12, these were grouped in to the 3 medical fields of internal medicine (n = 83; 64.32%), trauma (n = 33; 25.58%) and neurology (n = 12; 9.3%) for further analysis. With a median of 8 for internal medicine, trauma 8 and neurology 12. Overall the amount of scenarios ranged from 7 to 10 for the field of internal medicine, trauma 8 to 10 and neurology at 13 (e.g. Supplement Table ). Differences When comparing the medical specialties there were statistically significant differences found for the length of a scenario ( p = 0.033), the Durations of time the TEP was on scene ( p = 0.005), TEP arrival after scenario start ( p = 0.028), duration until TEP was called ( p = 0.039) and the duration until a diagnosis was made ( p = 0.001) (e.g. Fig. ). When comparing task and procedures, there were also significant differences ( p = 0.001) between the specialties (i.e. Table ). In the Post-Hoc analysis significant between group differences could be shown for the duration of time the TEP was on scene, from scenario start to calling of TEP, scenario start to TEP arrival and the time until a diagnosis was made. This could also be shown for Tasks and procedures (i.e. Table ). The previously significant result for length of a scenario ( p = 0.033), couldn’t be shown in the following Games–Howel Post-Hoc Analysis. Correlation and regressions There was a strong positive and significant correlation between duration of the scenario and the time a Tele-Emergency Physician (TEP) was bound, r = 0.570, p < 0.001 (i.e. Table ). Between scenario length and the tasks and procedures ( r = 0.008, p < 0.929) there was no correlation. Between the length of time a TEP was on scene and performed tasks and procedures was a weak but non significantly correlation ( r = 0.166, p = 0.062). Simple linear regression was used to test if the scenario length predicted the length a TEP would be bound. The regression model was: TEP on scene = 0.502 × scenario length − 88.208. The overall regression was statistically significant ( R 2 = 0.32, F (1,126) = 60.541, p < 0.001) and had a strong effect ( f = 0.68442). It was found that scenario length significantly predicted the length a TEP would be bound (β = 0 . 570, p < 0.001) (e.g. Fig. ). The exact binominal test indicated that the proportion of diagnosis made by paramedics (92.8%) was higher than the expected (75%), p < 0.001, n = 128, 2-sided.
The demographic data of the 96 Participants showed some differences: 64 participated as paramedics and 32 as TEP. Paramedics: age 22.88 (range 18–33) years, 55.26% female, 44.74% male, clinical experience 37.55 (range 12–133) months, 61.28% Paramedics, 38.72% EMTs; TEP: age 37.06 (range 29–59) years, 33.09% female, 59.56% male, clinical experience 138.22 (range 12–360) months, 75.74% emergency physicians, 24.26% emergency physicians with a medical officers qualification (German equivalent: “Leitender Notarzt”). Specializations were described as anesthesiology (n = 8), internal medicine (n = 8), general practitioner (n = 6), trauma surgery and orthopedics (n = 5) and neurology (n = 5).
141 scenarios were performed with 129 (91.49%) having used ETA while 12 (8.51%) didn’t need the support of a TEP. Of the 32 teams, 19 teams performed 4 scenarios (n = 76), while 13 teams performed 5 scenarios overall (n = 65). One case in the telemedical group and one in the non-telemedical had to be excluded for not being clearly assigned to one of the 16 scenarios. Therefore 128 telemedical and 11 non-telemedical scenarios could be analyzed. Because the amount of cases per scenario with telemedical support ranged from 7 to 12, these were grouped in to the 3 medical fields of internal medicine (n = 83; 64.32%), trauma (n = 33; 25.58%) and neurology (n = 12; 9.3%) for further analysis. With a median of 8 for internal medicine, trauma 8 and neurology 12. Overall the amount of scenarios ranged from 7 to 10 for the field of internal medicine, trauma 8 to 10 and neurology at 13 (e.g. Supplement Table ).
When comparing the medical specialties there were statistically significant differences found for the length of a scenario ( p = 0.033), the Durations of time the TEP was on scene ( p = 0.005), TEP arrival after scenario start ( p = 0.028), duration until TEP was called ( p = 0.039) and the duration until a diagnosis was made ( p = 0.001) (e.g. Fig. ). When comparing task and procedures, there were also significant differences ( p = 0.001) between the specialties (i.e. Table ). In the Post-Hoc analysis significant between group differences could be shown for the duration of time the TEP was on scene, from scenario start to calling of TEP, scenario start to TEP arrival and the time until a diagnosis was made. This could also be shown for Tasks and procedures (i.e. Table ). The previously significant result for length of a scenario ( p = 0.033), couldn’t be shown in the following Games–Howel Post-Hoc Analysis.
There was a strong positive and significant correlation between duration of the scenario and the time a Tele-Emergency Physician (TEP) was bound, r = 0.570, p < 0.001 (i.e. Table ). Between scenario length and the tasks and procedures ( r = 0.008, p < 0.929) there was no correlation. Between the length of time a TEP was on scene and performed tasks and procedures was a weak but non significantly correlation ( r = 0.166, p = 0.062). Simple linear regression was used to test if the scenario length predicted the length a TEP would be bound. The regression model was: TEP on scene = 0.502 × scenario length − 88.208. The overall regression was statistically significant ( R 2 = 0.32, F (1,126) = 60.541, p < 0.001) and had a strong effect ( f = 0.68442). It was found that scenario length significantly predicted the length a TEP would be bound (β = 0 . 570, p < 0.001) (e.g. Fig. ). The exact binominal test indicated that the proportion of diagnosis made by paramedics (92.8%) was higher than the expected (75%), p < 0.001, n = 128, 2-sided.
The application and use of telemedicine varies in different specialties, which makes the implementation of a new solution into clinical routine and processes even more demanding in an interdisciplinary field like emergency medicine. Although there were significant differences between the different medical fields, the mean scenario length was between 20–25 min. We could show that within a few minutes paramedics could be supported by a TEP and live bidirectional communication with a patient was possible. These durations are comparable with results from other studies , , , although this has been a simulation setting. The support of a TEP was not needed for the whole length of a scenario but only for a short duration, while paramedics already started treatment and monitoring of the patient. This approach allows EP to be more flexible and can therefore support more paramedics and patients in the field, by using such a sophisticated and adaptable telemedicine system like ETA. This user-based approach could therefore generally allow a more dynamic but also a specific integration of telemedicine in the field of EM allowing an improved usability of such systems. As the landscape in pre-hospital emergency medicine differs regionally the use of telemedicine can optimize the treatment of patients and reduce the interval until a physician can perform or support the currently needed therapy to improve patient outcomes and increase clinical processes . In physician based system like Germany, in which Emergency Physician ideally arrive at the scene of emergency in an own vehicle within 15 min , while in paramedic based systems, emergency physicians mainly work in Emergency Departments, the time until a patient is seen by a physician could therefore effectively be shortened to a few minutes with this technology in both systems allowing a global application of a system like ETA. In this trial paramedics made the correct diagnosis within few minutes and initiated the needed therapy. When comparing the different medical fields like internal medicine to trauma there were more correct task and procedures performed, but this did not differ significantly between these two groups. Nor could a correlation be described in regard of the length of scenario time or longer TEP availability at the scene of emergency. This could indicate that the quality of treatment is not automatically increased by a longer availability of a TEP or increased scenario length, but by other individual and interpersonal factors like education and experience which is a relevant issue in the use of telemedicine for clinical examination , . When comparing the length of time until a diagnosis was made it was shown that this took longer during the internal medicine scenarios, compared to trauma, but once the TEP was on scene, this changed. The longer stay of the TEP could have had an impact on the correct tasks and procedures during these scenarios. Usually TEP are called for further advice or to supervise more invasive procedures that paramedics aren’t allowed to perform by themselves (e. g. because of state regulations) although these are essential parts of a three-year curriculum and are lifesaving procedures , . Telemedicine could be a solution to close this gap. Supervising paramedics via telemedicine could therefore be a technology that supports these in the field, while allowing patients to safely receive lifesaving procedures as well as medication and reducing time delays in treatment . Especially when rapid interventions are need like in the management of seizures improving response times and increasing diagnostic accuracy could prevent and reduce the risk of Sudden Unexplained Death in Epilepsy Patients (SUDEP) – . In such a case, difficult airway management is clearly a field that requires specialist expertise and skills, but introduction of a supraglottic airway or bag mask ventilation could prevent hypoxia and is a skill paramedics are trained in. Regarding the application of medication by paramedics, these are only allowed to provide midazolam as a medical option for seizures in Hesse , , but an advanced medical treatment could be needed in such cases and include medication that could be provided by paramedics (e.g. levetiracetam, propofol etc.) . Telemedical support and a supervision of a T-EP could increase the response times, increase the safety of correct dosages and the choice of medication, especially in special populations like children. This shows that telemedicine is not only a solution to reduce a physician free interval, but also to supervise and safely perform therapies that manually can be performed by qualified paramedics, which national or local regulations often do not allow to be performed. Therefore further research is needed to answer question about the safety and management of relevant clinical complications in these procedures and their management in regard of the availability of telemedical communication solutions and a possibility of supervision by T-EP . Furthermore effectively managing the limited available staff could be a reason for law-makers and healthcare policy regulators to introduce or even improve regional telemedical technologies. With solutions like ETA independent, modern, safe and GDPR conform as well as a low-cost and open-source based web technology is now available for healthcare providers and patients. Limitations As this was a simulation trial, results cannot be directly transferred to clinical application. Many external, but also regional and individual factors influence the therapy of patients. But when comparing the length of time consultations were comparable with other results. Therefore an implementation of such a system in an ED must be evaluated, as this might increase the workload, availability of physicians and processes would have to be adapted . Also the qualification of paramedics varies and depends mainly on the emergency medical system, which can lead to differences in therapy options. This was the first time ETA was used as a telemedical solution. As this solution was not compared with other systems the results cannot directly be replicated to other systems. Comparing telemedical system could allow a comparison of response times, but also in the length of stay as the medical capabilities within different systems vary. Also for this first application of ETA we wanted to test it in the most standardized, structured and comparable field of emergency medicine, which are urgent emergencies. As the applied scenarios are standardized and approved by local healthcare authorities for the use in board examinations of paramedics in Hesse, we saw this as a way of improving standardization of medical simulations as much as possible. This involves not only the medical histories, expected and allowed therapies by paramedics, but also all the surroundings and the environment of each scenario. But of course non-urgent emergencies are a relevant factor in EM and could be a field where telemedicine could be applied as accessible, digital and convenient solution. Such a technology could allow patient flows to be directed away from the ED or ambulance services directly to specialists, general practitioners or other healthcare providers, as overcrowding and an increasing number of visits for non-emergencies are an increasing global trend , , , , , . Therefore further testing in the field of Telemedicine has to involve non-urgent emergencies and the effects such technologies could have on regional structures and systems. A bias towards using ETA could be seen, as many participants decided to use the telemedical system. Although telemedicine as a technology has been around for a while, a broad implementation in emergency medical systems has happened in many but not in all regions in Germany and might have lead participants to the decision to try this novel technology. Although the sample sizes for internal medicine and trauma are not small, the design of a following study would have to allow a comparison with non-telemedically supported scenarios as well as balanced group sizes for each medical field. Especially the results for the field of neurology can only be limited to the scenario “status epilepticus”. We chose not to perform stroke scenarios as paramedics in Hesse do not require an EP for the treatment and transport of these cases. But as Quadflieg et al. describes, seizures are often mistakenly diagnosed as strokes and a telemedical consultation could result in a better diagnostic quality by supporting neurological examination, using questionnaires and video transmissions , . Similarly paramedics in Hessen can be perform analgesia for the treatment of fractures without an EP and theoretically have the choice between six different analgetics , . Different studies have shown that analgesia can be safely performed using telemedicine but the option of having a TEP as a back-up solution can increase patient safety – . Technical performance indicators have been described in the area of telemedicine but medically this field is also understudied and needs further research .
As this was a simulation trial, results cannot be directly transferred to clinical application. Many external, but also regional and individual factors influence the therapy of patients. But when comparing the length of time consultations were comparable with other results. Therefore an implementation of such a system in an ED must be evaluated, as this might increase the workload, availability of physicians and processes would have to be adapted . Also the qualification of paramedics varies and depends mainly on the emergency medical system, which can lead to differences in therapy options. This was the first time ETA was used as a telemedical solution. As this solution was not compared with other systems the results cannot directly be replicated to other systems. Comparing telemedical system could allow a comparison of response times, but also in the length of stay as the medical capabilities within different systems vary. Also for this first application of ETA we wanted to test it in the most standardized, structured and comparable field of emergency medicine, which are urgent emergencies. As the applied scenarios are standardized and approved by local healthcare authorities for the use in board examinations of paramedics in Hesse, we saw this as a way of improving standardization of medical simulations as much as possible. This involves not only the medical histories, expected and allowed therapies by paramedics, but also all the surroundings and the environment of each scenario. But of course non-urgent emergencies are a relevant factor in EM and could be a field where telemedicine could be applied as accessible, digital and convenient solution. Such a technology could allow patient flows to be directed away from the ED or ambulance services directly to specialists, general practitioners or other healthcare providers, as overcrowding and an increasing number of visits for non-emergencies are an increasing global trend , , , , , . Therefore further testing in the field of Telemedicine has to involve non-urgent emergencies and the effects such technologies could have on regional structures and systems. A bias towards using ETA could be seen, as many participants decided to use the telemedical system. Although telemedicine as a technology has been around for a while, a broad implementation in emergency medical systems has happened in many but not in all regions in Germany and might have lead participants to the decision to try this novel technology. Although the sample sizes for internal medicine and trauma are not small, the design of a following study would have to allow a comparison with non-telemedically supported scenarios as well as balanced group sizes for each medical field. Especially the results for the field of neurology can only be limited to the scenario “status epilepticus”. We chose not to perform stroke scenarios as paramedics in Hesse do not require an EP for the treatment and transport of these cases. But as Quadflieg et al. describes, seizures are often mistakenly diagnosed as strokes and a telemedical consultation could result in a better diagnostic quality by supporting neurological examination, using questionnaires and video transmissions , . Similarly paramedics in Hessen can be perform analgesia for the treatment of fractures without an EP and theoretically have the choice between six different analgetics , . Different studies have shown that analgesia can be safely performed using telemedicine but the option of having a TEP as a back-up solution can increase patient safety – . Technical performance indicators have been described in the area of telemedicine but medically this field is also understudied and needs further research .
Developing a globally applicable and usable telemedical solution for emergency medicine requires high standards not only performance wise but especially on the technical side regarding data protection and usability. Testing our developed solution, the Emergency Talk Application (ETA), in multiple different scenarios to provide information about medical performance can provide relevant information about using one solution in the interdisciplinary field of EM. As emergency medical system are changing globally such results can provide further detailed evidence on how regional structures can benefit from telemedicine and how a possible application of the Emergency Talk Application could improve response times, quality of treatment and of patient outcomes.
Supplementary Tables.
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Effect of driving pressure-guided individualized positive end-expiratory pressure (PEEP) ventilation strategy on postoperative atelectasis in patients undergoing laparoscopic surgery as assessed by ultrasonography: study protocol for a prospective randomized controlled trial | 1056f5f7-f3f3-498c-baec-cbe876245804 | 11948704 | Surgery[mh] | Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http://www.equator-network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-clinical-trials/ ).
Background and rationale {6a} Ventilator-induced lung injury (VILI) caused by mechanical ventilation under general anesthesia as well as CO 2 pneumoperitoneum and special positions for laparoscopy may increase the risk of postoperative atelectasis , because the abdominal cavity is separated from the thoracic cavity only by the diaphragm, surgical procedures and pressure changes in the abdominal cavity, as well as anesthesia-related factors, can directly affect the function of the respiratory system. Brandão et al. showed that the combined use of pneumoperitoneum and Trendelenburg position significantly increased driving pressure and transpulmonary pressure (TPP), which can aggravate the degree of atelectasis in laparoscopic surgery. Even with the use of lung protective ventilation strategy (LPVS) in gynecologic laparoscopic surgery, the incidence of postoperative atelectasis in postanesthesia care unit (PACU) remained 40 ~ 55% . Impaired gas exchange, hypoxemia, and reduced functional residual capacity (FRC) caused by atelectasis may increase the risk of postoperative pulmonary complications (PPCs) , which leads to higher morbidity and mortality, as well as prolonged hospitalization . Previous study found that the use of LPVS during general anesthesia including small tidal volume (VT), appropriate positive end-expiratory pressure (PEEP), and recruitment maneuvers (RM) showed advantages in patients with healthy lungs and had a better prognosis for patients with lung injury. Appropriate PEEP has the advantages of increasing static lung compliance, improving oxygenation, and reducing VILI and PPCs . However, a fixed PEEP is not suitable for all surgical patients, especially to specific populations such as those undergoing laparoscopic surgery or obese patients, that lower PEEP does not play a role in lung protection and higher PEEP may cause hemodynamic instability [ – ]. Compared with setting a fixed PEEP, individualized PEEP that conforms to the individual physiology of patients can better improve intraoperative oxygenation, produce greater lung protective effects, and have a positive impact on postoperative prognosis. Individualized PEEP is the most recommended LPVS in the 2019 International Expert Group Consensus . Nevertheless, there is still a lack of consistent criteria for the optimal level of PEEP and the titration method of individualized PEEP. Driving pressure reflects the overall respiratory strain, which can be calculated as the difference between plateau pressure and positive end-expiratory pressure (Pplat − PEEP) , so driving pressure can be adjusted by regulating PEEP. Lower driving pressure indicates higher respiratory compliance , which effectively avoids atelectasis and lung hyperinflation and improves perioperative oxygenation and respiratory mechanics. According to the result of a meta-analysis , intraoperative high driving pressure in patients without pulmonary disease is associated with a higher incidence of PPCs. High driving pressure is one of the independent factors associated with the occurrence of PPCs, and only LPVS that reduces driving pressure has a protective effect . Previous prospective randomized controlled trials showed that individualized PEEP based on minimal driving pressure reduced the incidence of PPCs in patients undergoing thoracic surgery and open abdominal surgery . Therefore, we hypothesize that the use of individualized PEEP guided by driving pressure for laparoscopic surgery is a beneficial new option which can reduce incidence of PPCs. Objectives {7} The objective of this study is to explore whether driving pressure-guided individualized PEEP ventilation can reduce the incidence of postoperative atelectasis based on ultrasound assessment consequently the incidence of PPCs by improving intraoperative respiratory mechanics in laparoscopic surgery compared with the conventional fixed PEEP ventilation strategy. Trial design {8} The study is an investigator-initiated, prospective, single-center, randomized controlled clinical study. The framework for the trial is difference trial. Patients will be randomly assigned in a 1:1 ratio to receive an individualized PEEP guided by minimum drive pressure (group D) or a fixed PEEP of 5 cmH 2 O (group C).
Ventilator-induced lung injury (VILI) caused by mechanical ventilation under general anesthesia as well as CO 2 pneumoperitoneum and special positions for laparoscopy may increase the risk of postoperative atelectasis , because the abdominal cavity is separated from the thoracic cavity only by the diaphragm, surgical procedures and pressure changes in the abdominal cavity, as well as anesthesia-related factors, can directly affect the function of the respiratory system. Brandão et al. showed that the combined use of pneumoperitoneum and Trendelenburg position significantly increased driving pressure and transpulmonary pressure (TPP), which can aggravate the degree of atelectasis in laparoscopic surgery. Even with the use of lung protective ventilation strategy (LPVS) in gynecologic laparoscopic surgery, the incidence of postoperative atelectasis in postanesthesia care unit (PACU) remained 40 ~ 55% . Impaired gas exchange, hypoxemia, and reduced functional residual capacity (FRC) caused by atelectasis may increase the risk of postoperative pulmonary complications (PPCs) , which leads to higher morbidity and mortality, as well as prolonged hospitalization . Previous study found that the use of LPVS during general anesthesia including small tidal volume (VT), appropriate positive end-expiratory pressure (PEEP), and recruitment maneuvers (RM) showed advantages in patients with healthy lungs and had a better prognosis for patients with lung injury. Appropriate PEEP has the advantages of increasing static lung compliance, improving oxygenation, and reducing VILI and PPCs . However, a fixed PEEP is not suitable for all surgical patients, especially to specific populations such as those undergoing laparoscopic surgery or obese patients, that lower PEEP does not play a role in lung protection and higher PEEP may cause hemodynamic instability [ – ]. Compared with setting a fixed PEEP, individualized PEEP that conforms to the individual physiology of patients can better improve intraoperative oxygenation, produce greater lung protective effects, and have a positive impact on postoperative prognosis. Individualized PEEP is the most recommended LPVS in the 2019 International Expert Group Consensus . Nevertheless, there is still a lack of consistent criteria for the optimal level of PEEP and the titration method of individualized PEEP. Driving pressure reflects the overall respiratory strain, which can be calculated as the difference between plateau pressure and positive end-expiratory pressure (Pplat − PEEP) , so driving pressure can be adjusted by regulating PEEP. Lower driving pressure indicates higher respiratory compliance , which effectively avoids atelectasis and lung hyperinflation and improves perioperative oxygenation and respiratory mechanics. According to the result of a meta-analysis , intraoperative high driving pressure in patients without pulmonary disease is associated with a higher incidence of PPCs. High driving pressure is one of the independent factors associated with the occurrence of PPCs, and only LPVS that reduces driving pressure has a protective effect . Previous prospective randomized controlled trials showed that individualized PEEP based on minimal driving pressure reduced the incidence of PPCs in patients undergoing thoracic surgery and open abdominal surgery . Therefore, we hypothesize that the use of individualized PEEP guided by driving pressure for laparoscopic surgery is a beneficial new option which can reduce incidence of PPCs.
The objective of this study is to explore whether driving pressure-guided individualized PEEP ventilation can reduce the incidence of postoperative atelectasis based on ultrasound assessment consequently the incidence of PPCs by improving intraoperative respiratory mechanics in laparoscopic surgery compared with the conventional fixed PEEP ventilation strategy.
The study is an investigator-initiated, prospective, single-center, randomized controlled clinical study. The framework for the trial is difference trial. Patients will be randomly assigned in a 1:1 ratio to receive an individualized PEEP guided by minimum drive pressure (group D) or a fixed PEEP of 5 cmH 2 O (group C).
Study setting {9} The study will be performed in Beijing Tongren Hospital, Capital Medical University, Beijing, China. Patients are recruited in the surgical ward, including those scheduled for elective laparoscopic surgery. Patients are considered for inclusion if they meet the criteria as defined below. The specific flowchart is shown in Fig. . Eligibility criteria {10} Inclusion criteria Participants who meet the following criteria will be eligible for the study: Patients scheduled for elective laparoscopic surgery with an expected operation duration longer than 2 h. Age > 18 years old. The body mass index (BMI) < 35 kg·m −2 . American Society of Anesthesiologists (ASA) class I–III. Identified as medium to high risk of PPCs according to the ARISCAT risk score (ARISCAT score ≥ 26). Exclusion criteria If the participants meet any of the following criteria, they will not be eligible for the study: Patients with severe acute and chronic lung diseases, including respiratory failure, pneumothorax, pulmonary bullae, acute exacerbation of chronic obstructive pulmonary disease, and poorly controlled asthma. Patients with severe cardiovascular complications defined as NYHA class III–IV. Patients who have undergone thoracic surgery or have a history of mechanical ventilation within 4 weeks. Patients with other contraindications for PEEP, including high intracranial pressure, bronchopleural fistula, and hypovolemic shock. Who will take informed consent? {26a} Patients scheduled for elective laparoscopic surgery will be screened for eligibility to participate in this study based on the abovementioned criteria. After screening of patients who meet the criteria, a specialized investigator will explain all the details of the clinical trial, explain any remaining questions patients have, and obtain those informed consent on the day prior to surgery. Additional consent provisions for collection and use of participant data and biological specimens {26b} N/A. All participant data and biological specimens collected in the study will not be used in ancillary studies.
The study will be performed in Beijing Tongren Hospital, Capital Medical University, Beijing, China. Patients are recruited in the surgical ward, including those scheduled for elective laparoscopic surgery. Patients are considered for inclusion if they meet the criteria as defined below. The specific flowchart is shown in Fig. .
Inclusion criteria Participants who meet the following criteria will be eligible for the study: Patients scheduled for elective laparoscopic surgery with an expected operation duration longer than 2 h. Age > 18 years old. The body mass index (BMI) < 35 kg·m −2 . American Society of Anesthesiologists (ASA) class I–III. Identified as medium to high risk of PPCs according to the ARISCAT risk score (ARISCAT score ≥ 26).
Participants who meet the following criteria will be eligible for the study: Patients scheduled for elective laparoscopic surgery with an expected operation duration longer than 2 h. Age > 18 years old. The body mass index (BMI) < 35 kg·m −2 . American Society of Anesthesiologists (ASA) class I–III. Identified as medium to high risk of PPCs according to the ARISCAT risk score (ARISCAT score ≥ 26).
If the participants meet any of the following criteria, they will not be eligible for the study: Patients with severe acute and chronic lung diseases, including respiratory failure, pneumothorax, pulmonary bullae, acute exacerbation of chronic obstructive pulmonary disease, and poorly controlled asthma. Patients with severe cardiovascular complications defined as NYHA class III–IV. Patients who have undergone thoracic surgery or have a history of mechanical ventilation within 4 weeks. Patients with other contraindications for PEEP, including high intracranial pressure, bronchopleural fistula, and hypovolemic shock.
Patients scheduled for elective laparoscopic surgery will be screened for eligibility to participate in this study based on the abovementioned criteria. After screening of patients who meet the criteria, a specialized investigator will explain all the details of the clinical trial, explain any remaining questions patients have, and obtain those informed consent on the day prior to surgery.
N/A. All participant data and biological specimens collected in the study will not be used in ancillary studies.
Explanation for the choice of comparators {6b} To reduce the risk of PPCs in surgical patients, the guidelines recommend lung protective ventilation including small VT, appropriate PEEP, and RM during general anesthesia. However, lower PEEP does not play a role in lung protection and higher PEEP may cause hemodynamic instability in patients. There is still a lot of debate about the setting and application of PEEP. In this study, the PEEP of group C will be set at a fixed 5 cmH 2 O, because this is currently considered to be a relatively safe and effective PEEP value, which will not have a negative impact on the majority of surgical patients and be widely used in clinical anesthesia. Intervention description {11a} Anesthesia and analgesia All participants will be routinely fasted from food and water for 8 h preoperatively. Upon entering the operating room, patients will open peripheral venous access, monitor non-invasive blood pressure, heart rate (HR), pulse oxygen saturation (SpO 2 ), bispectral index (BIS), radial artery puncture catheterization under local anesthesia, and monitor invasive arterial blood pressure (ABP). All patients will be monitored neuromuscular blockade and preoxygenated 3 min using 100% fraction of inspired oxygen (FiO 2 ) before induction. Anesthesia will be induced sequentially with midazolam 0.02 mg·kg −1 , sufentanil 0.3 ~ 0.5 μg·kg −1 , propofol 1.5 ~ 2.5 mg·kg −1 , and rocuronium 0.6 ~ 1.0 mg·kg −1 . When T1 = 0 and TOF count = 0, the anesthesiologist will choose the appropriate type of reinforced endotracheal tube and complete endotracheal intubation under the guidance of visual laryngoscope. Intraoperative anesthesia will be maintained with combined intravenous and inhalation anesthesia including inhalation of sevoflurane 0.7 ~ 1.0 MAC and continuous infusion of propofol 4 ~ 6 mg·kg −1 ·h −1 to maintain the BIS of patients at 40 ~ 60. Intraoperative analgesia will be achieved by the addition of sufentanil and the continuous infusion of remifentanil 0.1 ~ 0.2 μg·kg −1 ·min −1 according to the patient’s needs. Additional rocuronium 0.1 mg·kg −1 will be administered according to neuromuscular blockade monitoring to maintain deep neuromuscular blockade. During the operation, patients will be placed in Trendelenburg position or anti-Trendelenburg position according to the different surgical procedures, and the pneumoperitoneum pressure will be set at 12 mmHg; the CO 2 flow rate during pneumoperitoneum is 1 L·min −1 . When the cavity of the abdomen closes, sufentanil 5 μg will be given for analgesia. At the end of the operation, neuromuscular blockade antagonists (neostigmine 0.04 mg·kg −1 + atropine 0.02 mg·kg −1 ) will be routinely administered when T2 appear, the endotracheal tube will be removed when TOFr > 90% and VT > 6 ml·kg −1 , and the patients will be sent to the PACU. The anesthesiologist will determine the fluid therapy regimen based on intraoperative conditions to maintain the HR range of 50 ~ 80 bpm and the ABP fluctuations no more than 20% of baseline. Ephedrine, phenylephrine, and norepinephrine may be used as appropriate to maintain circulatory stability and be recorded. Fluid therapy details such as crystalloid solution, colloidal solution, and blood products were will be also recorded. The patient controlled intravenous analgesia (PCIA) will be used in the postoperative period, with a formulation of sufentanil 1.5 μg·kg −1 + ondansetron 24 mg + 0.9% normal saline to 100 ml. The background dose will be 2 ml·h −1 , and a single dose of 2 ml will be given with a locking time of 15 min. Lung protective ventilation strategy (LPVS) All patients will use LPVS during general anesthesia mechanical ventilation. After endotracheal intubation, volume control ventilation (VCV) will be used, with VT of 7 ml·kg −1 of predicted body weight (PBW), PEEP 5 cmH 2 O, inspiratory to expiratory ratio (I:E) of 1:2, end-inspiratory pause (EIP) of 10%, fresh oxygen flow of 2 L·min −1 , FiO 2 ≥ 40% to maintain SpO 2 ≥ 95%, regulate respiratory rate (RR) to maintain partial pressure of end-expiratory carbon dioxide (P ET CO 2 ) at 35 ~ 45 mmHg. RM will be performed in both groups after establishing the pneumoperitoneum-Trendelenburg position, after stopping the pneumoperitoneum and resuming the supine position, and one more time in group D after completing the PEEP titration. RM will be performed using the incremental PEEP method: The driving pressure will be set at 20 cmH 2 O in the PCV mode, PEEP will be increased 5 cmH 2 O for every 5 respiratory cycles to reach 40 cmH 2 O Ppeak and maintain for 10 respiratory cycles, and then PEEP will be decreased 5 cmH 2 O for every 5 respiratory cycles to the pre-RM level before changing back to the VCV mode and parameters. Hemodynamic stability of patients should be ensured before RM, which should maintain MAP ≥ 80 mmHg, HR ≥ 60 bpm. If hemodynamic instability occurs during RM, the procedure will be suspended and vasoactive drugs will be administered. Intervention In the conventional LPVS group (group C), PEEP will be maintained at 5 cmH 2 O throughout mechanical ventilation. In the driving pressure-guided individualized PEEP group (group D), an incremental PEEP titration protocol will be performed to identify the optimal individualized PEEP that resulted in minimum driving pressure (calculated as Pplat − PEEP) after the pneumoperitoneum-Trendelenburg position is established and RM is performed, which is shown in Fig. . PEEP will be gradually increased by 1 cmH 2 O starting from the lowest PEEP allowed by the anesthesia machine (3 cmH 2 O) to 12 cmH 2 O, and each PEEP level will be maintained for 10 respiratory cycles and the driving pressure values will be recorded. When driving pressure increased with increasing PEEP, downward PEEP titration will be performed until the minimum driving pressure appears. After another RM, this optimal individualized PEEP will be maintained throughout the procedure. Lung ultrasound All lung ultrasound (LUS) assessment will be performed by a blinded well-trained anesthesiologist, using a Wisonic ultrasound machine (Wisonic Medical Co., Ltd., Shenzhen, China) with a 5 to 12 MHz linear transducer. All the intercostal space of 12 regions will be scanned sequentially from right to left, cranial to caudal, and anterior to posterior of patients’ chest in the supine position, as described previously . Each hemithorax is divided into six regions by anterior axillary line, posterior axillary line, and pectoral nipple line, with a total of 12 regions bilaterally. Ultrasound images of each region will be saved for analysis. An independent investigator who is blinded to group allocations and time points will analyze the ultrasound images. The lung aeration in each region will be assigned a score of 0 to 3 that is previously described based on the following scoring criteria: ① normal aeration (0): lung sliding sign and A-lines or isolated B-lines (< 3); ② mild reduction in lung ventilation (1): multiple, vertical, laser-like B-lines (≥ 3) or one or more small subpleural consolidations; ③ severe reduction in lung ventilation (2): multiple coalescent B-lines spaced ≤ 3 mm and continuous fusion or multiple small subpleural consolidations; ④ complete aeration loss (3): localized consolidation as subpleural tissue-like signs, fragment signs, and bronchial inflation signs. The LUS score (0 ~ 36) is calculated by adding up the scores of the 12 regions with higher scores indicating more severe aeration loss. Criteria for discontinuing or modifying allocated interventions {11b} Treatment of elevated peak airway pressure (Ppeak ≥ 30 cmH 2 O) A. First, eliminate diseases leading to increased airway resistance, endotracheal tube displacement, ventilator failure, neuromuscular blockade recovery, and then take intervention measures according to the situation. B. If group D appears elevated peak airway pressure during titration, stop increasing PEEP and select PEEP that has currently produced the minimum driving pressure. Treatment of severe hemodynamic instability (≥ 20% of baseline) A. If it occurs during RM, stop performing RM. B. If it occurs during titration in group D, stop increasing PEEP and select PEEP that has currently produced the minimum driving pressure. C. If recovery is not possible after exclusion of the above conditions, use vasoactive drugs (ephedrine, phenylephrine, and norepinephrine) as appropriate. Treatment of hypoxemia (SpO 2 ≤ 92%, lasting > 1 min) A. First, eliminate diseases leading to increased airway resistance, pneumothorax, hemodynamic instability, ventilator failure, and then take intervention measures according to the groups. B. Group C: First, gradually increase FiO 2 , when FiO 2 increases to 100% and SpO 2 still does not reach 92%, gradually increase PEEP levels. If there is still no improvement, then increase RMs. If the blood oxygen still does not the standard, adjust position to the supine position. C. Group D: First, perform individualized PEEP titration again after RM. If SpO 2 still does not reach 92%, gradually increase FiO 2 . If the blood oxygen still does not the standard, adjust position to the supine position. Strategies to improve adherence to interventions {11c} During the recruitment and preoperative visits, specialized investigators will fully explain the purpose of this study, the intervention, and the postoperative follow-up to the patients so that they are informed and their consent is obtained. The intervention of this study will be during mechanical ventilation under general anesthesia, and the information to be collected in this study is based on routine medical procedures, which will not add additional injury or cooperative work to the patients and will not incur additional costs. During the patient’s hospitalization and the postoperative follow-up period, the investigator will follow up with the participants and provide diagnostic and treatment recommendations to improve patient compliance. Relevant concomitant care permitted or prohibited during the trial {11d} Patients will be encouraged to perform activities and deep breathing exercises early after surgery. Patients who require additional respiratory support during postoperative hospitalization should be clearly diagnosed and recorded. Provisions for post-trial care {30} Participants will have access to study clinics for post-trial care through the routine health system. Outcomes {12} Primary outcomes The primary outcome is the LUS score at 24 h after surgery (T6). Secondary outcomes A. The LUS scores at other time points: enter the operation room (T0), 30 min after extubation (T5), 48 h after surgery (T7), 72 h after surgery (T8). B. Intraoperative respiratory mechanics: Ppeak, Pplat, the driving pressure, the dynamic lung compliance (Cdyn) at 15 min after intubation (T1), 10 min after pneumoperitoneum establishment or after individualized PEEP titration (T2), 2 h after pneumoperitoneum establishment or before the end of pneumoperitoneum (T3), the end of surgery (T4). VT, PEEP, SpO 2 , FiO 2 , RR, and P ET CO 2 mean values at the above intraoperative time points. C. Oxygenation index (PaO 2 /FiO 2 ) at T1, T3, and T4. D. The incidence and specific types of PPCs within 7 days after surgery or before discharge. The clinical outcome definitions of PPCs will adopt European joint taskforce guidelines published in 2015 which include respiratory infection, respiratory failure, atelectasis, pleural effusion, pneumothorax, bronchospasm, aspiration pneumonia, pulmonary edema, ARDS, tracheobronchitis, pulmonary edema, exacerbation of pre-existing lung disease, and pulmonary embolism. Other outcomes A. Baseline characteristics: age, sex, height, weight, BMI, ASA, preoperative complications, ARISCAT score, smoking history, preoperative pulmonary complications, SpO 2 , etc. B. Intraoperative indicators: type of operation, position of operation, duration of operation, duration of anesthesia, duration of mechanical ventilation, duration of pneumoperitoneum, dosage of anesthetic drugs, intraoperative hemodynamics (HR, MAP, SpO 2 and BIS value), quantity of liquid intake, urine and blood loss, intraoperative adverse events, use of vasoactive drugs, etc. C. Postoperative recovery indicators: extubation time, PACU stay time, incidence of hypoxemia in PACU, ICU admission and duration, postoperative analgesia assessment, extrapulmonary complications (extrapulmonary infection, arrhythmia, cardiovascular complications, cerebrovascular complications, cerebrovascular complications, postoperative cognitive dysfunction, liver and kidney function injury, etc.), in-hospital mortality, early activity, length of stay, postoperative hospitalization duration, hospitalization expense, etc. D. Long-term prognostic indicators: the mortality rate, the incidence of PPCs and extrapulmonary complications, the recurrence rate of tumor at 1, 3, and 6 months after operation. Participant timeline {13} Table shows the participant timeline. Sample size {14} PASS 15.0 was used for sample size calculation. The pretest of 10 patients in each group was completed according to randomization. Based on the results of the LUS score at T6 (6.1 ± 3.1 in group C and 4.3 ± 1.8 in group D), a two independent samples t -test with unequal variance was selected for sample size calculation with 90% power and an alpha of 0.05 (two-sided). The estimated sample size was 48 cases in each group. Accounting for the 10% probability of loss to follow-up, the sample size was increased to 53 cases in each group for a total of 106 cases. Recruitment {15} Patients will be recruited in the surgical ward of Beijing Tongren Hospital, Capital Medical University (CMU). The hospital has mature surgical technology and large laparoscopic operation volume, which can meet the patient recruitment for this study.
To reduce the risk of PPCs in surgical patients, the guidelines recommend lung protective ventilation including small VT, appropriate PEEP, and RM during general anesthesia. However, lower PEEP does not play a role in lung protection and higher PEEP may cause hemodynamic instability in patients. There is still a lot of debate about the setting and application of PEEP. In this study, the PEEP of group C will be set at a fixed 5 cmH 2 O, because this is currently considered to be a relatively safe and effective PEEP value, which will not have a negative impact on the majority of surgical patients and be widely used in clinical anesthesia.
Anesthesia and analgesia All participants will be routinely fasted from food and water for 8 h preoperatively. Upon entering the operating room, patients will open peripheral venous access, monitor non-invasive blood pressure, heart rate (HR), pulse oxygen saturation (SpO 2 ), bispectral index (BIS), radial artery puncture catheterization under local anesthesia, and monitor invasive arterial blood pressure (ABP). All patients will be monitored neuromuscular blockade and preoxygenated 3 min using 100% fraction of inspired oxygen (FiO 2 ) before induction. Anesthesia will be induced sequentially with midazolam 0.02 mg·kg −1 , sufentanil 0.3 ~ 0.5 μg·kg −1 , propofol 1.5 ~ 2.5 mg·kg −1 , and rocuronium 0.6 ~ 1.0 mg·kg −1 . When T1 = 0 and TOF count = 0, the anesthesiologist will choose the appropriate type of reinforced endotracheal tube and complete endotracheal intubation under the guidance of visual laryngoscope. Intraoperative anesthesia will be maintained with combined intravenous and inhalation anesthesia including inhalation of sevoflurane 0.7 ~ 1.0 MAC and continuous infusion of propofol 4 ~ 6 mg·kg −1 ·h −1 to maintain the BIS of patients at 40 ~ 60. Intraoperative analgesia will be achieved by the addition of sufentanil and the continuous infusion of remifentanil 0.1 ~ 0.2 μg·kg −1 ·min −1 according to the patient’s needs. Additional rocuronium 0.1 mg·kg −1 will be administered according to neuromuscular blockade monitoring to maintain deep neuromuscular blockade. During the operation, patients will be placed in Trendelenburg position or anti-Trendelenburg position according to the different surgical procedures, and the pneumoperitoneum pressure will be set at 12 mmHg; the CO 2 flow rate during pneumoperitoneum is 1 L·min −1 . When the cavity of the abdomen closes, sufentanil 5 μg will be given for analgesia. At the end of the operation, neuromuscular blockade antagonists (neostigmine 0.04 mg·kg −1 + atropine 0.02 mg·kg −1 ) will be routinely administered when T2 appear, the endotracheal tube will be removed when TOFr > 90% and VT > 6 ml·kg −1 , and the patients will be sent to the PACU. The anesthesiologist will determine the fluid therapy regimen based on intraoperative conditions to maintain the HR range of 50 ~ 80 bpm and the ABP fluctuations no more than 20% of baseline. Ephedrine, phenylephrine, and norepinephrine may be used as appropriate to maintain circulatory stability and be recorded. Fluid therapy details such as crystalloid solution, colloidal solution, and blood products were will be also recorded. The patient controlled intravenous analgesia (PCIA) will be used in the postoperative period, with a formulation of sufentanil 1.5 μg·kg −1 + ondansetron 24 mg + 0.9% normal saline to 100 ml. The background dose will be 2 ml·h −1 , and a single dose of 2 ml will be given with a locking time of 15 min. Lung protective ventilation strategy (LPVS) All patients will use LPVS during general anesthesia mechanical ventilation. After endotracheal intubation, volume control ventilation (VCV) will be used, with VT of 7 ml·kg −1 of predicted body weight (PBW), PEEP 5 cmH 2 O, inspiratory to expiratory ratio (I:E) of 1:2, end-inspiratory pause (EIP) of 10%, fresh oxygen flow of 2 L·min −1 , FiO 2 ≥ 40% to maintain SpO 2 ≥ 95%, regulate respiratory rate (RR) to maintain partial pressure of end-expiratory carbon dioxide (P ET CO 2 ) at 35 ~ 45 mmHg. RM will be performed in both groups after establishing the pneumoperitoneum-Trendelenburg position, after stopping the pneumoperitoneum and resuming the supine position, and one more time in group D after completing the PEEP titration. RM will be performed using the incremental PEEP method: The driving pressure will be set at 20 cmH 2 O in the PCV mode, PEEP will be increased 5 cmH 2 O for every 5 respiratory cycles to reach 40 cmH 2 O Ppeak and maintain for 10 respiratory cycles, and then PEEP will be decreased 5 cmH 2 O for every 5 respiratory cycles to the pre-RM level before changing back to the VCV mode and parameters. Hemodynamic stability of patients should be ensured before RM, which should maintain MAP ≥ 80 mmHg, HR ≥ 60 bpm. If hemodynamic instability occurs during RM, the procedure will be suspended and vasoactive drugs will be administered. Intervention In the conventional LPVS group (group C), PEEP will be maintained at 5 cmH 2 O throughout mechanical ventilation. In the driving pressure-guided individualized PEEP group (group D), an incremental PEEP titration protocol will be performed to identify the optimal individualized PEEP that resulted in minimum driving pressure (calculated as Pplat − PEEP) after the pneumoperitoneum-Trendelenburg position is established and RM is performed, which is shown in Fig. . PEEP will be gradually increased by 1 cmH 2 O starting from the lowest PEEP allowed by the anesthesia machine (3 cmH 2 O) to 12 cmH 2 O, and each PEEP level will be maintained for 10 respiratory cycles and the driving pressure values will be recorded. When driving pressure increased with increasing PEEP, downward PEEP titration will be performed until the minimum driving pressure appears. After another RM, this optimal individualized PEEP will be maintained throughout the procedure. Lung ultrasound All lung ultrasound (LUS) assessment will be performed by a blinded well-trained anesthesiologist, using a Wisonic ultrasound machine (Wisonic Medical Co., Ltd., Shenzhen, China) with a 5 to 12 MHz linear transducer. All the intercostal space of 12 regions will be scanned sequentially from right to left, cranial to caudal, and anterior to posterior of patients’ chest in the supine position, as described previously . Each hemithorax is divided into six regions by anterior axillary line, posterior axillary line, and pectoral nipple line, with a total of 12 regions bilaterally. Ultrasound images of each region will be saved for analysis. An independent investigator who is blinded to group allocations and time points will analyze the ultrasound images. The lung aeration in each region will be assigned a score of 0 to 3 that is previously described based on the following scoring criteria: ① normal aeration (0): lung sliding sign and A-lines or isolated B-lines (< 3); ② mild reduction in lung ventilation (1): multiple, vertical, laser-like B-lines (≥ 3) or one or more small subpleural consolidations; ③ severe reduction in lung ventilation (2): multiple coalescent B-lines spaced ≤ 3 mm and continuous fusion or multiple small subpleural consolidations; ④ complete aeration loss (3): localized consolidation as subpleural tissue-like signs, fragment signs, and bronchial inflation signs. The LUS score (0 ~ 36) is calculated by adding up the scores of the 12 regions with higher scores indicating more severe aeration loss.
All participants will be routinely fasted from food and water for 8 h preoperatively. Upon entering the operating room, patients will open peripheral venous access, monitor non-invasive blood pressure, heart rate (HR), pulse oxygen saturation (SpO 2 ), bispectral index (BIS), radial artery puncture catheterization under local anesthesia, and monitor invasive arterial blood pressure (ABP). All patients will be monitored neuromuscular blockade and preoxygenated 3 min using 100% fraction of inspired oxygen (FiO 2 ) before induction. Anesthesia will be induced sequentially with midazolam 0.02 mg·kg −1 , sufentanil 0.3 ~ 0.5 μg·kg −1 , propofol 1.5 ~ 2.5 mg·kg −1 , and rocuronium 0.6 ~ 1.0 mg·kg −1 . When T1 = 0 and TOF count = 0, the anesthesiologist will choose the appropriate type of reinforced endotracheal tube and complete endotracheal intubation under the guidance of visual laryngoscope. Intraoperative anesthesia will be maintained with combined intravenous and inhalation anesthesia including inhalation of sevoflurane 0.7 ~ 1.0 MAC and continuous infusion of propofol 4 ~ 6 mg·kg −1 ·h −1 to maintain the BIS of patients at 40 ~ 60. Intraoperative analgesia will be achieved by the addition of sufentanil and the continuous infusion of remifentanil 0.1 ~ 0.2 μg·kg −1 ·min −1 according to the patient’s needs. Additional rocuronium 0.1 mg·kg −1 will be administered according to neuromuscular blockade monitoring to maintain deep neuromuscular blockade. During the operation, patients will be placed in Trendelenburg position or anti-Trendelenburg position according to the different surgical procedures, and the pneumoperitoneum pressure will be set at 12 mmHg; the CO 2 flow rate during pneumoperitoneum is 1 L·min −1 . When the cavity of the abdomen closes, sufentanil 5 μg will be given for analgesia. At the end of the operation, neuromuscular blockade antagonists (neostigmine 0.04 mg·kg −1 + atropine 0.02 mg·kg −1 ) will be routinely administered when T2 appear, the endotracheal tube will be removed when TOFr > 90% and VT > 6 ml·kg −1 , and the patients will be sent to the PACU. The anesthesiologist will determine the fluid therapy regimen based on intraoperative conditions to maintain the HR range of 50 ~ 80 bpm and the ABP fluctuations no more than 20% of baseline. Ephedrine, phenylephrine, and norepinephrine may be used as appropriate to maintain circulatory stability and be recorded. Fluid therapy details such as crystalloid solution, colloidal solution, and blood products were will be also recorded. The patient controlled intravenous analgesia (PCIA) will be used in the postoperative period, with a formulation of sufentanil 1.5 μg·kg −1 + ondansetron 24 mg + 0.9% normal saline to 100 ml. The background dose will be 2 ml·h −1 , and a single dose of 2 ml will be given with a locking time of 15 min.
All patients will use LPVS during general anesthesia mechanical ventilation. After endotracheal intubation, volume control ventilation (VCV) will be used, with VT of 7 ml·kg −1 of predicted body weight (PBW), PEEP 5 cmH 2 O, inspiratory to expiratory ratio (I:E) of 1:2, end-inspiratory pause (EIP) of 10%, fresh oxygen flow of 2 L·min −1 , FiO 2 ≥ 40% to maintain SpO 2 ≥ 95%, regulate respiratory rate (RR) to maintain partial pressure of end-expiratory carbon dioxide (P ET CO 2 ) at 35 ~ 45 mmHg. RM will be performed in both groups after establishing the pneumoperitoneum-Trendelenburg position, after stopping the pneumoperitoneum and resuming the supine position, and one more time in group D after completing the PEEP titration. RM will be performed using the incremental PEEP method: The driving pressure will be set at 20 cmH 2 O in the PCV mode, PEEP will be increased 5 cmH 2 O for every 5 respiratory cycles to reach 40 cmH 2 O Ppeak and maintain for 10 respiratory cycles, and then PEEP will be decreased 5 cmH 2 O for every 5 respiratory cycles to the pre-RM level before changing back to the VCV mode and parameters. Hemodynamic stability of patients should be ensured before RM, which should maintain MAP ≥ 80 mmHg, HR ≥ 60 bpm. If hemodynamic instability occurs during RM, the procedure will be suspended and vasoactive drugs will be administered.
In the conventional LPVS group (group C), PEEP will be maintained at 5 cmH 2 O throughout mechanical ventilation. In the driving pressure-guided individualized PEEP group (group D), an incremental PEEP titration protocol will be performed to identify the optimal individualized PEEP that resulted in minimum driving pressure (calculated as Pplat − PEEP) after the pneumoperitoneum-Trendelenburg position is established and RM is performed, which is shown in Fig. . PEEP will be gradually increased by 1 cmH 2 O starting from the lowest PEEP allowed by the anesthesia machine (3 cmH 2 O) to 12 cmH 2 O, and each PEEP level will be maintained for 10 respiratory cycles and the driving pressure values will be recorded. When driving pressure increased with increasing PEEP, downward PEEP titration will be performed until the minimum driving pressure appears. After another RM, this optimal individualized PEEP will be maintained throughout the procedure.
All lung ultrasound (LUS) assessment will be performed by a blinded well-trained anesthesiologist, using a Wisonic ultrasound machine (Wisonic Medical Co., Ltd., Shenzhen, China) with a 5 to 12 MHz linear transducer. All the intercostal space of 12 regions will be scanned sequentially from right to left, cranial to caudal, and anterior to posterior of patients’ chest in the supine position, as described previously . Each hemithorax is divided into six regions by anterior axillary line, posterior axillary line, and pectoral nipple line, with a total of 12 regions bilaterally. Ultrasound images of each region will be saved for analysis. An independent investigator who is blinded to group allocations and time points will analyze the ultrasound images. The lung aeration in each region will be assigned a score of 0 to 3 that is previously described based on the following scoring criteria: ① normal aeration (0): lung sliding sign and A-lines or isolated B-lines (< 3); ② mild reduction in lung ventilation (1): multiple, vertical, laser-like B-lines (≥ 3) or one or more small subpleural consolidations; ③ severe reduction in lung ventilation (2): multiple coalescent B-lines spaced ≤ 3 mm and continuous fusion or multiple small subpleural consolidations; ④ complete aeration loss (3): localized consolidation as subpleural tissue-like signs, fragment signs, and bronchial inflation signs. The LUS score (0 ~ 36) is calculated by adding up the scores of the 12 regions with higher scores indicating more severe aeration loss.
Treatment of elevated peak airway pressure (Ppeak ≥ 30 cmH 2 O) A. First, eliminate diseases leading to increased airway resistance, endotracheal tube displacement, ventilator failure, neuromuscular blockade recovery, and then take intervention measures according to the situation. B. If group D appears elevated peak airway pressure during titration, stop increasing PEEP and select PEEP that has currently produced the minimum driving pressure. Treatment of severe hemodynamic instability (≥ 20% of baseline) A. If it occurs during RM, stop performing RM. B. If it occurs during titration in group D, stop increasing PEEP and select PEEP that has currently produced the minimum driving pressure. C. If recovery is not possible after exclusion of the above conditions, use vasoactive drugs (ephedrine, phenylephrine, and norepinephrine) as appropriate. Treatment of hypoxemia (SpO 2 ≤ 92%, lasting > 1 min) A. First, eliminate diseases leading to increased airway resistance, pneumothorax, hemodynamic instability, ventilator failure, and then take intervention measures according to the groups. B. Group C: First, gradually increase FiO 2 , when FiO 2 increases to 100% and SpO 2 still does not reach 92%, gradually increase PEEP levels. If there is still no improvement, then increase RMs. If the blood oxygen still does not the standard, adjust position to the supine position. C. Group D: First, perform individualized PEEP titration again after RM. If SpO 2 still does not reach 92%, gradually increase FiO 2 . If the blood oxygen still does not the standard, adjust position to the supine position.
2 O) A. First, eliminate diseases leading to increased airway resistance, endotracheal tube displacement, ventilator failure, neuromuscular blockade recovery, and then take intervention measures according to the situation. B. If group D appears elevated peak airway pressure during titration, stop increasing PEEP and select PEEP that has currently produced the minimum driving pressure.
A. If it occurs during RM, stop performing RM. B. If it occurs during titration in group D, stop increasing PEEP and select PEEP that has currently produced the minimum driving pressure. C. If recovery is not possible after exclusion of the above conditions, use vasoactive drugs (ephedrine, phenylephrine, and norepinephrine) as appropriate.
2 ≤ 92%, lasting > 1 min) A. First, eliminate diseases leading to increased airway resistance, pneumothorax, hemodynamic instability, ventilator failure, and then take intervention measures according to the groups. B. Group C: First, gradually increase FiO 2 , when FiO 2 increases to 100% and SpO 2 still does not reach 92%, gradually increase PEEP levels. If there is still no improvement, then increase RMs. If the blood oxygen still does not the standard, adjust position to the supine position. C. Group D: First, perform individualized PEEP titration again after RM. If SpO 2 still does not reach 92%, gradually increase FiO 2 . If the blood oxygen still does not the standard, adjust position to the supine position.
During the recruitment and preoperative visits, specialized investigators will fully explain the purpose of this study, the intervention, and the postoperative follow-up to the patients so that they are informed and their consent is obtained. The intervention of this study will be during mechanical ventilation under general anesthesia, and the information to be collected in this study is based on routine medical procedures, which will not add additional injury or cooperative work to the patients and will not incur additional costs. During the patient’s hospitalization and the postoperative follow-up period, the investigator will follow up with the participants and provide diagnostic and treatment recommendations to improve patient compliance.
Patients will be encouraged to perform activities and deep breathing exercises early after surgery. Patients who require additional respiratory support during postoperative hospitalization should be clearly diagnosed and recorded.
Participants will have access to study clinics for post-trial care through the routine health system.
Primary outcomes The primary outcome is the LUS score at 24 h after surgery (T6). Secondary outcomes A. The LUS scores at other time points: enter the operation room (T0), 30 min after extubation (T5), 48 h after surgery (T7), 72 h after surgery (T8). B. Intraoperative respiratory mechanics: Ppeak, Pplat, the driving pressure, the dynamic lung compliance (Cdyn) at 15 min after intubation (T1), 10 min after pneumoperitoneum establishment or after individualized PEEP titration (T2), 2 h after pneumoperitoneum establishment or before the end of pneumoperitoneum (T3), the end of surgery (T4). VT, PEEP, SpO 2 , FiO 2 , RR, and P ET CO 2 mean values at the above intraoperative time points. C. Oxygenation index (PaO 2 /FiO 2 ) at T1, T3, and T4. D. The incidence and specific types of PPCs within 7 days after surgery or before discharge. The clinical outcome definitions of PPCs will adopt European joint taskforce guidelines published in 2015 which include respiratory infection, respiratory failure, atelectasis, pleural effusion, pneumothorax, bronchospasm, aspiration pneumonia, pulmonary edema, ARDS, tracheobronchitis, pulmonary edema, exacerbation of pre-existing lung disease, and pulmonary embolism. Other outcomes A. Baseline characteristics: age, sex, height, weight, BMI, ASA, preoperative complications, ARISCAT score, smoking history, preoperative pulmonary complications, SpO 2 , etc. B. Intraoperative indicators: type of operation, position of operation, duration of operation, duration of anesthesia, duration of mechanical ventilation, duration of pneumoperitoneum, dosage of anesthetic drugs, intraoperative hemodynamics (HR, MAP, SpO 2 and BIS value), quantity of liquid intake, urine and blood loss, intraoperative adverse events, use of vasoactive drugs, etc. C. Postoperative recovery indicators: extubation time, PACU stay time, incidence of hypoxemia in PACU, ICU admission and duration, postoperative analgesia assessment, extrapulmonary complications (extrapulmonary infection, arrhythmia, cardiovascular complications, cerebrovascular complications, cerebrovascular complications, postoperative cognitive dysfunction, liver and kidney function injury, etc.), in-hospital mortality, early activity, length of stay, postoperative hospitalization duration, hospitalization expense, etc. D. Long-term prognostic indicators: the mortality rate, the incidence of PPCs and extrapulmonary complications, the recurrence rate of tumor at 1, 3, and 6 months after operation.
The primary outcome is the LUS score at 24 h after surgery (T6).
A. The LUS scores at other time points: enter the operation room (T0), 30 min after extubation (T5), 48 h after surgery (T7), 72 h after surgery (T8). B. Intraoperative respiratory mechanics: Ppeak, Pplat, the driving pressure, the dynamic lung compliance (Cdyn) at 15 min after intubation (T1), 10 min after pneumoperitoneum establishment or after individualized PEEP titration (T2), 2 h after pneumoperitoneum establishment or before the end of pneumoperitoneum (T3), the end of surgery (T4). VT, PEEP, SpO 2 , FiO 2 , RR, and P ET CO 2 mean values at the above intraoperative time points. C. Oxygenation index (PaO 2 /FiO 2 ) at T1, T3, and T4. D. The incidence and specific types of PPCs within 7 days after surgery or before discharge. The clinical outcome definitions of PPCs will adopt European joint taskforce guidelines published in 2015 which include respiratory infection, respiratory failure, atelectasis, pleural effusion, pneumothorax, bronchospasm, aspiration pneumonia, pulmonary edema, ARDS, tracheobronchitis, pulmonary edema, exacerbation of pre-existing lung disease, and pulmonary embolism.
A. Baseline characteristics: age, sex, height, weight, BMI, ASA, preoperative complications, ARISCAT score, smoking history, preoperative pulmonary complications, SpO 2 , etc. B. Intraoperative indicators: type of operation, position of operation, duration of operation, duration of anesthesia, duration of mechanical ventilation, duration of pneumoperitoneum, dosage of anesthetic drugs, intraoperative hemodynamics (HR, MAP, SpO 2 and BIS value), quantity of liquid intake, urine and blood loss, intraoperative adverse events, use of vasoactive drugs, etc. C. Postoperative recovery indicators: extubation time, PACU stay time, incidence of hypoxemia in PACU, ICU admission and duration, postoperative analgesia assessment, extrapulmonary complications (extrapulmonary infection, arrhythmia, cardiovascular complications, cerebrovascular complications, cerebrovascular complications, postoperative cognitive dysfunction, liver and kidney function injury, etc.), in-hospital mortality, early activity, length of stay, postoperative hospitalization duration, hospitalization expense, etc. D. Long-term prognostic indicators: the mortality rate, the incidence of PPCs and extrapulmonary complications, the recurrence rate of tumor at 1, 3, and 6 months after operation.
Table shows the participant timeline.
PASS 15.0 was used for sample size calculation. The pretest of 10 patients in each group was completed according to randomization. Based on the results of the LUS score at T6 (6.1 ± 3.1 in group C and 4.3 ± 1.8 in group D), a two independent samples t -test with unequal variance was selected for sample size calculation with 90% power and an alpha of 0.05 (two-sided). The estimated sample size was 48 cases in each group. Accounting for the 10% probability of loss to follow-up, the sample size was increased to 53 cases in each group for a total of 106 cases.
Patients will be recruited in the surgical ward of Beijing Tongren Hospital, Capital Medical University (CMU). The hospital has mature surgical technology and large laparoscopic operation volume, which can meet the patient recruitment for this study.
Sequence generation {16a} The study will adopt a simple randomized design, and the randomization will be conducted by a computer-generated random number list (IBM SPSS Statistics 26.0). Patients will be randomly assigned in a 1:1 ratio to receive an individualized PEEP guided by minimum drive pressure (group D) or a fixed PEEP of 5 cmH 2 O (group C). Concealment mechanism {16b} Group information will be concealed in sequentially numbered, opaque, sealed envelopes to achieve allocation concealment. Implementation {16c} Randomization and allocation concealment will be done by independent investigators. When the patients enter the operating room on the day of surgery, the sealed envelope containing random numbers will be given to the anesthesiologist, who will open it to obtain the group information, and others will not know the allocation result.
The study will adopt a simple randomized design, and the randomization will be conducted by a computer-generated random number list (IBM SPSS Statistics 26.0). Patients will be randomly assigned in a 1:1 ratio to receive an individualized PEEP guided by minimum drive pressure (group D) or a fixed PEEP of 5 cmH 2 O (group C).
Group information will be concealed in sequentially numbered, opaque, sealed envelopes to achieve allocation concealment.
Randomization and allocation concealment will be done by independent investigators. When the patients enter the operating room on the day of surgery, the sealed envelope containing random numbers will be given to the anesthesiologist, who will open it to obtain the group information, and others will not know the allocation result.
Who will be blinded {17a} Due to the different mechanical ventilation parameter settings in this study, the chief anesthesiologist will not be blinded to ensure medical safety, but they will not be involved in subsequent procedures. Lung ultrasound assessment will be done by a well-trained anesthesiologist and the ultrasound images will be analyzed by an independent investigator, both of whom are blinded to the interventions and not involved in the anesthesia procedure, the follow-up, and statistical analysis. In addition, participants, surgeons, follow-up investigators, data collectors, and statisticians will be blinded. Procedure for unblinding if needed {17b} No special criteria for revealing blinding. If a major adverse event that poses a serious threat to the safety of the participant occurs during the perioperative period, an authorized blind participant may open an emergency blind envelope, record the time and cause of the event, and name of the blind participant.
Due to the different mechanical ventilation parameter settings in this study, the chief anesthesiologist will not be blinded to ensure medical safety, but they will not be involved in subsequent procedures. Lung ultrasound assessment will be done by a well-trained anesthesiologist and the ultrasound images will be analyzed by an independent investigator, both of whom are blinded to the interventions and not involved in the anesthesia procedure, the follow-up, and statistical analysis. In addition, participants, surgeons, follow-up investigators, data collectors, and statisticians will be blinded.
No special criteria for revealing blinding. If a major adverse event that poses a serious threat to the safety of the participant occurs during the perioperative period, an authorized blind participant may open an emergency blind envelope, record the time and cause of the event, and name of the blind participant.
Plans for assessment and collection of outcomes {18a} Data will be derived from electronic patient records and collected with Case Report Form (CRF). Baseline data for participants will be collected preoperatively, intraoperative data will be collected by investigators who will not know the group of participants, laboratory tests are performed by the central diagnostic laboratory, and radiological examination will be made at the Department of Radiology of Beijing Tongren Hospital. All data acquired during the study will be anonymized and saved in a study folder on our protected research server. Only the study team has access to this specific study folder. Plans to promote participant retention and complete follow-up {18b} The patients will receive extensive information about the study set-up and requirements during the recruitment. The importance of completion of the follow-up will be stressed. Throughout the follow-up period, the researchers will check responses and if necessary contact patients for completion of their follow-up. Patients are allowed to stop at any time during the study and are not obliged to give a reason to discontinue. Data management {19} Data will be derived from electronic patient records and collected with CRF. Informed consent of signed paper forms will be stored within the anesthesia department of our hospital in a locked room. Output will be stored in SPSS. To avoid human errors in entry, two data entry personnel will be used to enter data independently and synchronously, and then the machine will compare the data to extract duplicates. All raw data and all steps taken in the analysis will be documented in SPSS. Confidentiality {27} Research data will be stored using a study identification code for each participant. All data acquired during the study will be anonymized and saved in a study folder on our protected research server. This specific study folder will only be available to the research team during the study and will be documented and safeguarded by the principal investigator according to research guidelines after completion of the study. No patient identification details will be reported in publications. Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} N/A. All biological specimens collected in the study will not be used for genetic or molecular analysis in ancillary studies.
Data will be derived from electronic patient records and collected with Case Report Form (CRF). Baseline data for participants will be collected preoperatively, intraoperative data will be collected by investigators who will not know the group of participants, laboratory tests are performed by the central diagnostic laboratory, and radiological examination will be made at the Department of Radiology of Beijing Tongren Hospital. All data acquired during the study will be anonymized and saved in a study folder on our protected research server. Only the study team has access to this specific study folder.
The patients will receive extensive information about the study set-up and requirements during the recruitment. The importance of completion of the follow-up will be stressed. Throughout the follow-up period, the researchers will check responses and if necessary contact patients for completion of their follow-up. Patients are allowed to stop at any time during the study and are not obliged to give a reason to discontinue.
Data will be derived from electronic patient records and collected with CRF. Informed consent of signed paper forms will be stored within the anesthesia department of our hospital in a locked room. Output will be stored in SPSS. To avoid human errors in entry, two data entry personnel will be used to enter data independently and synchronously, and then the machine will compare the data to extract duplicates. All raw data and all steps taken in the analysis will be documented in SPSS.
Research data will be stored using a study identification code for each participant. All data acquired during the study will be anonymized and saved in a study folder on our protected research server. This specific study folder will only be available to the research team during the study and will be documented and safeguarded by the principal investigator according to research guidelines after completion of the study. No patient identification details will be reported in publications.
N/A. All biological specimens collected in the study will not be used for genetic or molecular analysis in ancillary studies.
Statistical methods for primary and secondary outcomes {20a} IBM SPSS 26.0 will be used to analyze the data. Shapiro–Wilk test will be used to test normality. Continuous variables with a normal distribution will be expressed as mean ± standard deviation (x ± s), and variance analysis or independent samples t -test will be used for appropriate comparisons between and within groups. LUS scores as repeated continuous variables with a non-normal distribution will be expressed as median and interquartile range (IQR) and will be compared within groups using the Friedman test and between groups using the Mann–Whitney U test. Categorical variables will be expressed as frequencies or percentages (%) and will be compared with the χ 2 test or Fisher exact test. The rank sum test will be used for grade data. A two-sided P value < 0.05 will be considered statistically significant. Interim analyses {21b} N/A. There are no interim analyses planned. Methods for additional analyses (e.g., subgroup analyses) {20b} N/A. There are no subgroup analyses planned. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} The primary outcome will be assessed using an intention-to-treat analysis. Missing data will be reduced to a minimum by using the appropriate measures described above. If any statistical method is needed to account for missing data in the secondary outcomes, multiple imputation will be used. Plans to give access to the full protocol, participant-level data, and statistical code {31c} The datasets used and analyzed during the current study can be made available by the corresponding author upon reasonable request.
IBM SPSS 26.0 will be used to analyze the data. Shapiro–Wilk test will be used to test normality. Continuous variables with a normal distribution will be expressed as mean ± standard deviation (x ± s), and variance analysis or independent samples t -test will be used for appropriate comparisons between and within groups. LUS scores as repeated continuous variables with a non-normal distribution will be expressed as median and interquartile range (IQR) and will be compared within groups using the Friedman test and between groups using the Mann–Whitney U test. Categorical variables will be expressed as frequencies or percentages (%) and will be compared with the χ 2 test or Fisher exact test. The rank sum test will be used for grade data. A two-sided P value < 0.05 will be considered statistically significant.
N/A. There are no interim analyses planned.
N/A. There are no subgroup analyses planned.
The primary outcome will be assessed using an intention-to-treat analysis. Missing data will be reduced to a minimum by using the appropriate measures described above. If any statistical method is needed to account for missing data in the secondary outcomes, multiple imputation will be used.
The datasets used and analyzed during the current study can be made available by the corresponding author upon reasonable request.
Composition of the coordinating center and trial steering committee {5d} This is a single-center study designed, performed and coordinated in the Beijing Tongren Hospital, CMU. Day to day support for the trial is provided by: Principle investigator: takes supervision of the trial and medical responsibility of the patients. Data manager: organizes data capture, safeguards quality and data. Study coordinator: trial registration, coordinates study visits, safety reports. Study physician: identifies potential recruits, takes informed consent, ensures procedures according to protocol. Composition of the data monitoring committee, its role and reporting structure {21a} The data monitoring committee (DMC) consists of an ethics expert, an anesthesiologist, a radiologist, a statistician, and a clinical trial management expert. All team members are independent of the sponsor, and there are no conflicts of interest. The study team meets monthly to review research progress, check data integrity, and monitor the occurrence of serious adverse events (SAEs). Adverse event reporting and harms {22} All adverse events reported by the participants or observed by the investigators will be recorded. When a serious adverse event occurs, the investigator will immediately report it to the DMC and ethics committee. The investigators will record the symptoms, severity, duration, relevant medical measures taken, outcome, and correlation with intervention measures in the CRF form and submit a complete SAE report form within 24 h. Investigators will follow up on SAEs until the adverse event disappears or returns to baseline level. Frequency and plans for auditing trial conduct {23} The study will be completed under the supervision of the ethics committee throughout. The DMC will conduct monthly reviews at various stages of the initiation phase, patient recruitment, and statistical analysis. Because our study is a single-center clinical trial with mature and low-risk intervention measures, no additional auditing procedures are set except for the DMC monthly meetings. Plans for communicating important protocol amendments to relevant parties (e.g., trial participants, ethical committees) {25} Any revisions to the plan or the informed consent form will be sent for approval by the ethics committee. The sponsor and funders will be notified first, then the PI will notify the centers. The copy of the revised protocol will be sent to the PI to add to the Investigator Site File. In addition, any deviations from the agreement will be fully documented using the Violation Report Form. If necessary, additional consent from the participants will be obtained. Dissemination plans {31a} Results of this research will be disclosed completely in international peer-reviewed journals. Both positive and negative results will be reported.
This is a single-center study designed, performed and coordinated in the Beijing Tongren Hospital, CMU. Day to day support for the trial is provided by: Principle investigator: takes supervision of the trial and medical responsibility of the patients. Data manager: organizes data capture, safeguards quality and data. Study coordinator: trial registration, coordinates study visits, safety reports. Study physician: identifies potential recruits, takes informed consent, ensures procedures according to protocol.
The data monitoring committee (DMC) consists of an ethics expert, an anesthesiologist, a radiologist, a statistician, and a clinical trial management expert. All team members are independent of the sponsor, and there are no conflicts of interest. The study team meets monthly to review research progress, check data integrity, and monitor the occurrence of serious adverse events (SAEs).
All adverse events reported by the participants or observed by the investigators will be recorded. When a serious adverse event occurs, the investigator will immediately report it to the DMC and ethics committee. The investigators will record the symptoms, severity, duration, relevant medical measures taken, outcome, and correlation with intervention measures in the CRF form and submit a complete SAE report form within 24 h. Investigators will follow up on SAEs until the adverse event disappears or returns to baseline level.
The study will be completed under the supervision of the ethics committee throughout. The DMC will conduct monthly reviews at various stages of the initiation phase, patient recruitment, and statistical analysis. Because our study is a single-center clinical trial with mature and low-risk intervention measures, no additional auditing procedures are set except for the DMC monthly meetings.
Any revisions to the plan or the informed consent form will be sent for approval by the ethics committee. The sponsor and funders will be notified first, then the PI will notify the centers. The copy of the revised protocol will be sent to the PI to add to the Investigator Site File. In addition, any deviations from the agreement will be fully documented using the Violation Report Form. If necessary, additional consent from the participants will be obtained.
Results of this research will be disclosed completely in international peer-reviewed journals. Both positive and negative results will be reported.
This study protocol is a prospective, single-center, randomized, controlled trial in patients undergoing laparoscopic surgery to determine whether the driving pressure-guided individualized PEEP ventilation strategy reduces the postoperative LUS scores and the incidence of postoperative atelectasis, and to assess clinical parameters related to lung injury, postoperative recovery indicators, and long-term prognostic indicators. In laparoscopic surgery, many factors directly affect the respiratory system. First, anesthetics used during general anesthesia can loss the muscle tone and decrease FRC, leading to the formation of atelectasis . Once patients under general anesthesia receive mechanical ventilation, VILI may occur due to inappropriate VT, asynchronous respiratory movements and increased TPP caused by mechanical ventilation, and inflammatory response caused by repeated stimulation. During the surgical procedure, the abdominal pressure caused by CO 2 pneumoperitoneum is significantly increased so that the diaphragm moves upwards, the thoracic expansion and the respiratory movement is significantly limited . At the same time, the lung tissue adjacent to the diaphragm is deformed or even collapsed by compression, resulting in reduced lung volume and functional residual capacity. In addition, different positions are used for different sites of the operation. The Trendelenburg position, which is often used for laparoscopic surgery in the lower abdomen and pelvis, causes the intra-abdominal organs to move to the cephalic side under the effect of gravity, increasing the compression on the diaphragm and further restricting the diaphragmatic activity, which can further reduce lung volume, decrease lung compliance, and increase airway pressure and driving pressure . All these factors increase the risk of perioperative atelectasis and PPCs, which will affect the postoperative recovery of patients . Therefore, how to reduce the incidence of PPCs has always been a problem to be solved in clinical practice. The advantages of the LPVS based on appropriate PEEP for thoracoscopic and laparoscopic surgeries have been demonstrated [ – ]. A study of laparoscopic surgery in obese patients showed that appropriate individualized PEEP can re-expand collapsed alveoli, increase FRC and lung compliance, maintain a certain pressure at end of expiration to prevent alveoli from collapsing completely, and improve ventilation and oxygenation . However, there are still great controversies about the titration method of individualized PEEP. Electrical impedance tomography (EIT)-guided titration of individualized PEEP can monitor local lung ventilation and perfusion in real time, but it is expensive, complicated to operate, and difficult to provide intraoperative bedside guidance, which is not conducive to the popularization of individualized PEEP . TPP is used to optimize PEEP, but commonly used esophageal manometry requires additional equipment and professional training for its placement, hindering its clinical application ; and the esophageal pressure is influenced by many factors. In patients with normal chest wall compliance, the driving pressure is equal to the pressure that promotes alveolar opening during mechanical ventilation and represents the pressure at which the pulmonary parenchyma is subjected to cyclic strain during each ventilatory cycle , and changes in driving pressure can appropriately replace changes in TPP and pulmonary strain. Therefore, the individualized PEEP titration based on minimum driving pressure can improve the compliance and oxygenation of the respiratory system to achieve lung protection. Furthermore, compared with EIT and TPP, driving pressure-guided PEEP titration is a relatively simple and straightforward strategy that does not require special equipment but is an equally effective method. The occurrence of PPCs is closely associated with postoperative hospitalization, short and long-term mortality, so early and accurate assessment is essential for the prevention and treatment of perioperative atelectasis and PPCs. Previous studies on individualized PEEP focused on the incidence of PPCs in surgical patients diagnosed by CT [ , , ]. Although the gold standard for the diagnosis of lung diseases is still lung CT, it is difficult to ensure that each patient will have a CT examination after surgery because of radiation exposure, inconvenient transport, economic costs, and other factors in clinical practice, resulting in that many PPCs with insignificant symptoms may be misdiagnosed and ignored. However, as a safe and feasible bedside imaging method, LUS has been widely used in the diagnosis of lung diseases because of its non-invasive, radiation-free, immediate and dynamic observation of disease changes . LUS can be scored by identifying ultrasound signs in the lungs and used as a tool for semi-quantitative assessment of perioperative lung atelectasis . Studies indicated that using CT as a reference, LUS has a sensitivity of 87.7%, specificity of 92.1%, and accuracy of 90.8% in detecting atelectasis, which was in good agreement with atelectasis diagnosed by CT . Recent research showed that, as a novel auxiliary diagnostic tool, LUS can be used for the diagnosis of perioperative pulmonary ventilation loss and atelectasis . Currently, there are fewer studies related to the use of ultrasound to assess driving pressure-guided individualized PEEP ventilation strategy for postoperative lung ventilation loss in patients undergoing laparoscopic surgery. In summary, this study will better evaluate the effect of individualized PEEP application guided by driving pressure based on ultrasound assessment on the incidence of postoperative atelectasis consequently the incidence of PPCs in patients undergoing prolonged laparoscopic surgery. The results may provide a clinical evidence for optimizing perioperative lung protection strategies. Trial status The current protocol is version 2.0 of November 16, 2023. Recruiting started in January 2024. Patient recruitment is estimated to be completed around August 2024.
The current protocol is version 2.0 of November 16, 2023. Recruiting started in January 2024. Patient recruitment is estimated to be completed around August 2024.
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Non-targeted metabolomics-based molecular networking enables the chemical characterization of | 0f1e0c5a-fb5f-47c2-a95f-dd856903b7bd | 11761831 | Biochemistry[mh] | By 2050, the world population is predicted to have grown to 10 billion people, and, as a consequence, the food demand will dramatically increase. Over the past few decades, agriculture has primarily focused on enhancing crop productivity to meet the growing demand for food. However, the innovative farming techniques may not be sufficient to guarantee market requirements in the future. Moreover, today more than ever, great attention is paid not only to agricultural yield but also to food quality. We know more than 50,000 plants that are considered edible, but only few of them have been cultivated as food crops (The Plants That Feed the World, ). In this scenario, the utilization of wild edible plants (WEPs) can have an interesting role in making the food system healthier, sustainable and resilient to the current climate changes. WEPs gathering has been a habitual practice since ancient times all over the world, however they continue to attract the scientific interest for different reasons even now. Indeed, WEPs are a good source of specialized/secondary metabolites, which can have health-promoting effects (Ceccanti et al., ) and are generally more tolerant to adverse climate conditions in comparison to crops. The identification of effective sources of stress tolerance is an important aspect for an effective plant protection strategy. In this sense, wild plants constitute a rich natural reservoir of genes involved in plant stress tolerance. Despite this, WEPs remain understudied, leading to a scarcity of knowledge regarding their bioactive constituents and nutritional properties. In particular, the genus of Rumex , belonging to the polygonaceae family, includes approximately 200 species and it is notable among the herbaceous plants of temperate climatic zones. In fact, Rumex species have several advantages, such as a short vegetative period, rapid biomass accumulation, and adaptable growth requirements (Van Assche et al., ). Plants belonging to Rumex genus are also great producer of phenolic compounds thus making these plants interesting source of bioactive molecules (Mishra et al., ). Moreover, several plants belonging to Rumex genus are also included in the pharmacopoeias of different countries (Ernst, ; Shaikh et al., ; Shikov et al., ). In Europe, Rumex acetosa and Rumex acetosella (Korpelainen & Pietiläinen, ) are routinely incorporated into dietary practices whereas R. sanguineus, commonly known as bloody dock or red-veined sorrel, still remains relatively underexplored in terms of nutritional values and health benefits (Feduraev et al., ). Leaves were traditionally consumed fresh or cooked. While the tender, young leaves are enjoyed in their raw state as a fresh, leafy vegetable, the mature leaves require blanching, due to the presence of oxalic acid in adult leaves, which may cause adverse effects. While there is scientific evidence to support the traditional use (both medicinal and culinary) of many Rumex species (Jeon et al., ) less is known about its metabolic profile. Scientific evidences are available regarding its content of anthocyanins vitamin C, iron, and other minerals (Feduraev et al., ; Vasas et al., ) However, a comprehensive characterization of the secondary metabolites (Panche et al., ) of the different organs of this plant, roots, stems and leaves, is yet to be uncovered (Korpelainen & Pietiläinen, ). Up to now, targeted methods, developed for quantification of a given class of metabolites, have been exclusively applied to investigate R. sanguineus chemical composition (Feduraev et al., ), mainly focusing on a restricted metabolite’s class. Nevertheless, nowadays, advanced analytical tools permit the simultaneous analysis of hundreds of metabolites, allowing a better characterization of small molecules (up to 1200 Da), therefore, the composition of complex plant matrices can be investigated in depth. In fact, in the last decade, the applicability of metabolomics to food science and nutrition research has strongly emerged (Charria-Girón et al., ; Zdouc et al., ). However, one of the major bottlenecks of the non-targeted metabolomics workflow is the annotation step. Indeed, on average, only 5% of the aligned features can be annotated, while the 95% remains unknowns, limiting our view of the metabolome. Recently, several approaches to improve feature annotation have been proposed (Aron et al., ; Zdouc et al., ). Among these tools, Feature-Based Molecular Networking (FBMN) (Watrous et al., ) has emerged as computational approach to improve feature annotation and visualization of the relationships between different compounds based on their structural similarities. The basic idea behind classical molecular networking (MN) is to group together features with MS/MS spectra that have similar mass fragmentation patterns, and with each feature represented as a node in a network, construct clusters or families of chemically-related features (Nothias et al., ). FBMN improves upon classical MN by leveraging MS1 information such as isotope patterns and retention time. This enables the differentiation of isomers with similar MS2 spectra and facilitates the inclusion of relative quantitative data for robust downstream metabolomics statistical analysis. Additionally, FBMN aids in data redundancy reduction that simplifies the discovery of structurally related compounds. The added value of this approach has been already demonstrated in different metabolomics application fields (e.g. natural products, food safety, (Li et al., )) leading to an increase in annotated compounds up to 10% (de Jonge et al., ; Wang et al., ). In this study, we aimed to take advantage of the potential of such a powerful approach, schematically represented in Fig. , to comprehensively characterize the chemical profile of R. sanguineus . Chemicals and reagents LC–MS grade methanol and acetonitrile were purchased from Biosolve Chimie (Netherlands). Bi-distilled water was obtained using Milli-Q System (Millipore, Bedford, MA, USA), formic acid (CAS 64-18-6), from Fisher Chemical (Thermo Fisher Scientific Inc., San Jose, CA, USA) was also used. The following standards were purchased from Sigma-Aldrich Chemie GmbH (Germany): emodin (CAS 518-82-1) emodin-8-glucoside (CAS 23313-21-5), aloin A (CAS 1415-73-2), aloin B (CAS 28371-16-6) and L-triptofan-d5 (CAS 62595-11-3). Fluka Chemie GmbH (Buchs, Switzerland) provided rutin (CAS 153-18-4). Kaempferol (CAS 520-18-3) and quercetin (CAS 849061-97-8) were obtained from PhytoLab GmbH & Co. KG (Germany). Plant material For this study , Rumex sanguineus seeds were stratified at 4 °C in dark for 3 days before sowing them on soil. Seeds (2–4 per pot) were sowed in pots (8 × 8 cm in size) in a 6:1 mix of soil and perlite, germinated and grown at + 25 °C in the C.N.R. (National Research Council) greenhouse in Montelibretti (Monterotondo, Italy), following the protocol previously reported (Van Assche et al., ). Subsequently, seedlings were transferred to larger pots (30 × 40 × 20 cm), where 28 plants grew for 1 year until the end of the experiment. The pots were kept at 40% humidity, 16/8 h photoperiod at + 25 °C in the greenhouse for thirteen months. The watering of the plants was repeated every 3 days. During the vegetative stage, one year after germination, the initial sampling was conducted. At this stage, adult leaves were collected separately from young leaves, with adult leaves measuring between 7.34 and 15.27 cm, and young leaves measuring between 2.47 and 7.34 cm (Figure ). In this first stage, 3 biological replicates each for old and young leaves were collected. For the subsequent sampling, adult leaves, stems and roots were collected to obtain 3 biological replicates for each organ. After each sampling, the plant material was frozen using liquid nitrogen, grounded into a homogeneous and fine powder and freeze-dried for 72 h. Non-targeted metabolomics: sample extraction and LC-HRMS analysis Sample extraction of roots, stems and leaves was performed as previously reported (Salem et al., ). Briefly, 25 mg of homogenized tissues were extracted with 1650 µl of extraction solvent mix (water, methanol, methyl tert-butyl ether). After centrifugation, 600 µl of supernatant were dried down under nitrogen flow and then reconstructed in 300 µL UPLC-grade methanol/water (1:1, vol/vol). 100 µL of the extract was transferred into a vial and added with the internal standard at 1 mg/L concentration (L-tryptophan d-5). To measure performance and system stability and assess the reproducibility of the sample treatment procedure, Quality Control samples (QC) were injected during the analyses. QCs were obtained by mixing equal volumes (10 µL) of all 24 sample extracts and following the same procedure as for the other samples. QCs were injected at the beginning of the run and after every 8 real samples. The data acquisition was performed using Agilent 1290 UHPLC system coupled to a Thermo Fisher Q-Exactive Focus Quadrupole-Orbitrap Mass Spectrometer equipped with a heated electrospray ionization (HESI) interface. 24 samples were analyzed, comprising 4 biological replicates extracted in duplicate (8 roots, 8 stems and 8 leaves). Sample extracts were injected (5 µL) and chromatographically separated using a reversed-phase C18 HSS T3 Acquity column 2.1 × 100 mm, 1.8 µm particle size (Waters, Milford, MA, USA). The mobile phases used for chromatographic separation were water containing 0.1% formic acid (Buffer A) and acetonitrile containing 0.1% formic acid (Buffer B). A gradient profile was applied using water (eluent A) and acetonitrile (eluent B), both acidified with 0.1% formic acid as mobile phases. A multi-step elution dual-mode gradient was developed as follow: at 0.0 min (5% B; 0.40 mL/min) a gradient begun as follows: 11 min 35% B, 12.5 min 70% B, 13.5 min 99% B, held for 1.5 min until 15.0 min, and then in 1 min the initial conditions were restored to 5% B; the total run time was 21 min including 5 min of re-equilibration. The column was maintained at 40 ℃ and a flow rate of 0.4 mL/min used. The settings of the interface, HESI source, were configured to operate effectively in both negative and positive ion modes, and the parameters were set as follows: sheath gas flow rate 30 mL/min, aux gas flow rate at 15 mL/min and sweep gas flow rate at 33 mL/min, spray voltage 3.2 kV, capillary temperature 320 ℃, S-lens RF level 47.0, aux gas heater temp 0 ℃. The mass spectra were acquired in DDA (Stincone et al., ) (Data Dependent Analysis) by full scan MS1 and MS2 in positive and negative ionization mode on a Q-Exactive high resolution Orbitrap-type MS (ThermoFisher, Bremen, Germany) in a scan range from 100 to 1000 m /z ; the full MS resolution was 70,000 FWHM, AGC target 3e6, maximum IT 200 ms and with a profile spectrum data type. The dd/MS 2 analysis was performed with a resolution of 17,500 FWHM with three different collision energy: 15,30,45 eV, AGC target 2e5, maximum IT 50 ms, loop count of 3, minimum AGC target of 8.00e3, intensity threshold 1.6e5, apex trigger 2 to 15 s, dynamic exclusion of 4.0 s, charge exclusion 2–8, > 8 and a profile spectrum data file. Non-targeted metabolomics: multivariate data analysis and metabolite annotation Multivariate data analysis was conducted using Metaboanalyst 5 (Pang et al., ), with the aim of confirming the absence of any anomalies in the instrumental analysis process, thus enhancing the overall accuracy and reliability of the data collected. The data set was normalized on the internal standard (L-tryptophan d-5), and then principal component analysis (PCA) score scatter plots were obtained, as depicted in Fig. . Data acquired in the RAW Thermo format, were converted to.mzXML using Proteowizard’s MSConvertGUI (64-bit) tool (Chambers et al., ). Converted.mzXML data were then imported to MZmine 3.9.0 (Katajamaa et al., ), to perform data pre-processing using ADAP (Automated Data Analysis Pipeline) including EIC (Extracted Ion Chromatogram) construction, chromatographic peak detection, spectral deconvolution and alignment with the following parameters: (1) Mass detection = retention time, auto, MS1 noise level 5.0E3, MS2 noise level 1.0E3. (2) ADAP chromatogram builder = retention time 2.00–15.00 min, MS1 level minimum consecutive scans 8, minimum intensity for consecutive scans 2.0E4, minimum absolute height 3.0E4, m/z tolerance scan-to-scan 5.0 ppm; (3) Smoothing = Savitzky Golay; (4) Local minimum feature resolver = minimum relative feature height 25%, chromatographic threshold 75%, Minimum search range RT/Mobility 0.050, minimum absolute height 5E4, peak duration 0.10–3.00 min; (5) Isotopic filtering = retention time tolerance 0.020 s, m/z tolerance 0.0010 or 5.0 ppm, monotonic shape no, maximum charge 2, representative isotope, most intense; (6) Isotopic peaks finder = m/z tolerance 0.0010 or 5.0 ppm, maximum charge of isotope m/z 1; Join aligner = 0.0015 or 5.0 ppm, weight for m/z 3, retention time tolerance 0,200 min, weight for RT 1, (7) Feature list rows filter = Minimum aligned features 1, retention time filter 2.00–15.00 min, Chromatographic FWHM (Full Width at Half Maximum) 0.00–6.00, never remove feature with MS2, on; (8) Peak finder = intensity tolerance 25%, m/z tolerance 0.002 or 5.0 ppm, retention time tolerance 0.200 min, minimum scans 4. Features with retention time below 2.0 min and above 15.00 min were excluded. The resultant feature list comprised 2060 features for ESI positive ion mode and 1118 for ESI negative ion mode, were exported to a format compatible with GNPS and SIRIUS (.csv and.mgf with a metadata file) utilizing the dedicated "Export for GNPS" or “Export for SIRIUS” feature provided within the software. A Feature-Based Molecular Networking (Nothias et al., ) (FBMN) version, (Release_27), was generated by exporting the output data from MZmine 3.9.0. and uploading.mgf,.csv, and metadata files onto the GNPS platform. The parameters employed for generating the Feature-Based Molecular Network are as follows: precursor ion mass tolerance of 0.01 Da, fragment ion mass tolerance 0.01 Da, minimum cosine score that must occur between a pair of MS/MS spectra in order to form an edge in the molecular network of 0.70, minimum number of fragment ions that are shared between pairs of related MS/MS spectra, in order to be connected by an edge in the molecular network of 7, maximum shift between precursors 500 Da, maximum number of neighbour nodes for one single node of 20, maximum size of nodes allowed in a single connected network 100; search analogs, on, minimum number of fragments that MS/MS spectra should contain in order to be considered for annotation of 6, score threshold of 0.7, maximum analog search mass difference 100 Da, filtering options, off. The created FBMN was then downloaded as a Cytoscape (Shannon et al., ) file and then visualized using the same tool (Cytoscape 3.10.1). The FBMN job of the positive ionization mode dataset is publicly available at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f3363189286c428bae3470d9fbd371c6 , while the FBMN job of the negative ionization mode dataset is available at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=4e3d798eb99e42a58f0401ca78399186 . The MS data were deposited on public repository (Dataset: MSV000092024), https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?accession=MSV000092024 . This approach enabled us to visually organize spectral data and gain insights into the distribution of the same compound among the three distinct organs. All the detected features were computed using SIRIUS 4 both in positive and negative ionization modes with the following parameters: mass accuracy 5 ppm (Orbitrap), all possible adducts; ZODIAC (Identification of molecular formulas) use 2 step approach, edge thresholds 0.95, min local connections 10; CSI:FingerID (predicts molecular fingerprint from MS/MS and fragmentation tree for each compound) all fallbacks adducts, search molecular structure in all databases, tag lipids, off; CANOPUS (predicts compounds categories for each compound individually predicted based on its predicted molecular fingerprint) on. The identification level of the annotated metabolites is reported following the Chemical Analysis Working Group (CAWG)(Sumner et al., ) criteria, including level I (comparison against an authentic standard) and level II (putatively identified molecule) annotations (for a full list, see Zenodo upload https://doi.org/ 10.5281/zenodo.14236385). For the annotation, both spectral matching (comparison with identified spectra, in Biocyc, Pubchem, HMDB, COCONUT, CHEBI, KEGG, Natural Products, SuperNatural) and literature search were used. Targeted analysis of anthraquinones With the aim to quantify the amount of emodin and emodin 8-glucoside, samples were extracted following the protocol previously described by Shi et al., (Shi et al., ). Briefly, 10 mg of roots, stems and leaves were extracted with 1 ml of LC-grade methanol/water (1:1, vol/vol) followed by ultrasonication for 30 min. Samples were then centrifuged (14,000 rpm, 5 min, 4 °C) and the supernatants were collected and diluted (100-fold dilution) prior to UHPLC-HRMS analysis. Calibration curves were set up using external standards (range 0.05 mg/L to 1 mg/L for emodin and 0.01 mg/L to 0.5 mg/L for emodin-8-glucoside) for target analyte quantification. Satisfactory linearity was obtained for each compound (emodin R 2 = 0.9996, y = 6E + 06x + 222,814; emodin-8-glucoside R 2 = 0.9993, y = 1E + 07x − 17,982). Data acquisition was performed by Thermo Xcalibur 2.2 software (Thermo Fisher Scientific, Waltham, MA, USA). A one-way ANOVA with a p-value cutoff of 0.05 followed by a Tukey’s HSD post hoc was performed using XLStat to determine statistically significant accumulation of emodin and its glucoside across root, stem and leaf samples. LC–MS grade methanol and acetonitrile were purchased from Biosolve Chimie (Netherlands). Bi-distilled water was obtained using Milli-Q System (Millipore, Bedford, MA, USA), formic acid (CAS 64-18-6), from Fisher Chemical (Thermo Fisher Scientific Inc., San Jose, CA, USA) was also used. The following standards were purchased from Sigma-Aldrich Chemie GmbH (Germany): emodin (CAS 518-82-1) emodin-8-glucoside (CAS 23313-21-5), aloin A (CAS 1415-73-2), aloin B (CAS 28371-16-6) and L-triptofan-d5 (CAS 62595-11-3). Fluka Chemie GmbH (Buchs, Switzerland) provided rutin (CAS 153-18-4). Kaempferol (CAS 520-18-3) and quercetin (CAS 849061-97-8) were obtained from PhytoLab GmbH & Co. KG (Germany). For this study , Rumex sanguineus seeds were stratified at 4 °C in dark for 3 days before sowing them on soil. Seeds (2–4 per pot) were sowed in pots (8 × 8 cm in size) in a 6:1 mix of soil and perlite, germinated and grown at + 25 °C in the C.N.R. (National Research Council) greenhouse in Montelibretti (Monterotondo, Italy), following the protocol previously reported (Van Assche et al., ). Subsequently, seedlings were transferred to larger pots (30 × 40 × 20 cm), where 28 plants grew for 1 year until the end of the experiment. The pots were kept at 40% humidity, 16/8 h photoperiod at + 25 °C in the greenhouse for thirteen months. The watering of the plants was repeated every 3 days. During the vegetative stage, one year after germination, the initial sampling was conducted. At this stage, adult leaves were collected separately from young leaves, with adult leaves measuring between 7.34 and 15.27 cm, and young leaves measuring between 2.47 and 7.34 cm (Figure ). In this first stage, 3 biological replicates each for old and young leaves were collected. For the subsequent sampling, adult leaves, stems and roots were collected to obtain 3 biological replicates for each organ. After each sampling, the plant material was frozen using liquid nitrogen, grounded into a homogeneous and fine powder and freeze-dried for 72 h. Sample extraction of roots, stems and leaves was performed as previously reported (Salem et al., ). Briefly, 25 mg of homogenized tissues were extracted with 1650 µl of extraction solvent mix (water, methanol, methyl tert-butyl ether). After centrifugation, 600 µl of supernatant were dried down under nitrogen flow and then reconstructed in 300 µL UPLC-grade methanol/water (1:1, vol/vol). 100 µL of the extract was transferred into a vial and added with the internal standard at 1 mg/L concentration (L-tryptophan d-5). To measure performance and system stability and assess the reproducibility of the sample treatment procedure, Quality Control samples (QC) were injected during the analyses. QCs were obtained by mixing equal volumes (10 µL) of all 24 sample extracts and following the same procedure as for the other samples. QCs were injected at the beginning of the run and after every 8 real samples. The data acquisition was performed using Agilent 1290 UHPLC system coupled to a Thermo Fisher Q-Exactive Focus Quadrupole-Orbitrap Mass Spectrometer equipped with a heated electrospray ionization (HESI) interface. 24 samples were analyzed, comprising 4 biological replicates extracted in duplicate (8 roots, 8 stems and 8 leaves). Sample extracts were injected (5 µL) and chromatographically separated using a reversed-phase C18 HSS T3 Acquity column 2.1 × 100 mm, 1.8 µm particle size (Waters, Milford, MA, USA). The mobile phases used for chromatographic separation were water containing 0.1% formic acid (Buffer A) and acetonitrile containing 0.1% formic acid (Buffer B). A gradient profile was applied using water (eluent A) and acetonitrile (eluent B), both acidified with 0.1% formic acid as mobile phases. A multi-step elution dual-mode gradient was developed as follow: at 0.0 min (5% B; 0.40 mL/min) a gradient begun as follows: 11 min 35% B, 12.5 min 70% B, 13.5 min 99% B, held for 1.5 min until 15.0 min, and then in 1 min the initial conditions were restored to 5% B; the total run time was 21 min including 5 min of re-equilibration. The column was maintained at 40 ℃ and a flow rate of 0.4 mL/min used. The settings of the interface, HESI source, were configured to operate effectively in both negative and positive ion modes, and the parameters were set as follows: sheath gas flow rate 30 mL/min, aux gas flow rate at 15 mL/min and sweep gas flow rate at 33 mL/min, spray voltage 3.2 kV, capillary temperature 320 ℃, S-lens RF level 47.0, aux gas heater temp 0 ℃. The mass spectra were acquired in DDA (Stincone et al., ) (Data Dependent Analysis) by full scan MS1 and MS2 in positive and negative ionization mode on a Q-Exactive high resolution Orbitrap-type MS (ThermoFisher, Bremen, Germany) in a scan range from 100 to 1000 m /z ; the full MS resolution was 70,000 FWHM, AGC target 3e6, maximum IT 200 ms and with a profile spectrum data type. The dd/MS 2 analysis was performed with a resolution of 17,500 FWHM with three different collision energy: 15,30,45 eV, AGC target 2e5, maximum IT 50 ms, loop count of 3, minimum AGC target of 8.00e3, intensity threshold 1.6e5, apex trigger 2 to 15 s, dynamic exclusion of 4.0 s, charge exclusion 2–8, > 8 and a profile spectrum data file. Multivariate data analysis was conducted using Metaboanalyst 5 (Pang et al., ), with the aim of confirming the absence of any anomalies in the instrumental analysis process, thus enhancing the overall accuracy and reliability of the data collected. The data set was normalized on the internal standard (L-tryptophan d-5), and then principal component analysis (PCA) score scatter plots were obtained, as depicted in Fig. . Data acquired in the RAW Thermo format, were converted to.mzXML using Proteowizard’s MSConvertGUI (64-bit) tool (Chambers et al., ). Converted.mzXML data were then imported to MZmine 3.9.0 (Katajamaa et al., ), to perform data pre-processing using ADAP (Automated Data Analysis Pipeline) including EIC (Extracted Ion Chromatogram) construction, chromatographic peak detection, spectral deconvolution and alignment with the following parameters: (1) Mass detection = retention time, auto, MS1 noise level 5.0E3, MS2 noise level 1.0E3. (2) ADAP chromatogram builder = retention time 2.00–15.00 min, MS1 level minimum consecutive scans 8, minimum intensity for consecutive scans 2.0E4, minimum absolute height 3.0E4, m/z tolerance scan-to-scan 5.0 ppm; (3) Smoothing = Savitzky Golay; (4) Local minimum feature resolver = minimum relative feature height 25%, chromatographic threshold 75%, Minimum search range RT/Mobility 0.050, minimum absolute height 5E4, peak duration 0.10–3.00 min; (5) Isotopic filtering = retention time tolerance 0.020 s, m/z tolerance 0.0010 or 5.0 ppm, monotonic shape no, maximum charge 2, representative isotope, most intense; (6) Isotopic peaks finder = m/z tolerance 0.0010 or 5.0 ppm, maximum charge of isotope m/z 1; Join aligner = 0.0015 or 5.0 ppm, weight for m/z 3, retention time tolerance 0,200 min, weight for RT 1, (7) Feature list rows filter = Minimum aligned features 1, retention time filter 2.00–15.00 min, Chromatographic FWHM (Full Width at Half Maximum) 0.00–6.00, never remove feature with MS2, on; (8) Peak finder = intensity tolerance 25%, m/z tolerance 0.002 or 5.0 ppm, retention time tolerance 0.200 min, minimum scans 4. Features with retention time below 2.0 min and above 15.00 min were excluded. The resultant feature list comprised 2060 features for ESI positive ion mode and 1118 for ESI negative ion mode, were exported to a format compatible with GNPS and SIRIUS (.csv and.mgf with a metadata file) utilizing the dedicated "Export for GNPS" or “Export for SIRIUS” feature provided within the software. A Feature-Based Molecular Networking (Nothias et al., ) (FBMN) version, (Release_27), was generated by exporting the output data from MZmine 3.9.0. and uploading.mgf,.csv, and metadata files onto the GNPS platform. The parameters employed for generating the Feature-Based Molecular Network are as follows: precursor ion mass tolerance of 0.01 Da, fragment ion mass tolerance 0.01 Da, minimum cosine score that must occur between a pair of MS/MS spectra in order to form an edge in the molecular network of 0.70, minimum number of fragment ions that are shared between pairs of related MS/MS spectra, in order to be connected by an edge in the molecular network of 7, maximum shift between precursors 500 Da, maximum number of neighbour nodes for one single node of 20, maximum size of nodes allowed in a single connected network 100; search analogs, on, minimum number of fragments that MS/MS spectra should contain in order to be considered for annotation of 6, score threshold of 0.7, maximum analog search mass difference 100 Da, filtering options, off. The created FBMN was then downloaded as a Cytoscape (Shannon et al., ) file and then visualized using the same tool (Cytoscape 3.10.1). The FBMN job of the positive ionization mode dataset is publicly available at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=f3363189286c428bae3470d9fbd371c6 , while the FBMN job of the negative ionization mode dataset is available at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=4e3d798eb99e42a58f0401ca78399186 . The MS data were deposited on public repository (Dataset: MSV000092024), https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?accession=MSV000092024 . This approach enabled us to visually organize spectral data and gain insights into the distribution of the same compound among the three distinct organs. All the detected features were computed using SIRIUS 4 both in positive and negative ionization modes with the following parameters: mass accuracy 5 ppm (Orbitrap), all possible adducts; ZODIAC (Identification of molecular formulas) use 2 step approach, edge thresholds 0.95, min local connections 10; CSI:FingerID (predicts molecular fingerprint from MS/MS and fragmentation tree for each compound) all fallbacks adducts, search molecular structure in all databases, tag lipids, off; CANOPUS (predicts compounds categories for each compound individually predicted based on its predicted molecular fingerprint) on. The identification level of the annotated metabolites is reported following the Chemical Analysis Working Group (CAWG)(Sumner et al., ) criteria, including level I (comparison against an authentic standard) and level II (putatively identified molecule) annotations (for a full list, see Zenodo upload https://doi.org/ 10.5281/zenodo.14236385). For the annotation, both spectral matching (comparison with identified spectra, in Biocyc, Pubchem, HMDB, COCONUT, CHEBI, KEGG, Natural Products, SuperNatural) and literature search were used. With the aim to quantify the amount of emodin and emodin 8-glucoside, samples were extracted following the protocol previously described by Shi et al., (Shi et al., ). Briefly, 10 mg of roots, stems and leaves were extracted with 1 ml of LC-grade methanol/water (1:1, vol/vol) followed by ultrasonication for 30 min. Samples were then centrifuged (14,000 rpm, 5 min, 4 °C) and the supernatants were collected and diluted (100-fold dilution) prior to UHPLC-HRMS analysis. Calibration curves were set up using external standards (range 0.05 mg/L to 1 mg/L for emodin and 0.01 mg/L to 0.5 mg/L for emodin-8-glucoside) for target analyte quantification. Satisfactory linearity was obtained for each compound (emodin R 2 = 0.9996, y = 6E + 06x + 222,814; emodin-8-glucoside R 2 = 0.9993, y = 1E + 07x − 17,982). Data acquisition was performed by Thermo Xcalibur 2.2 software (Thermo Fisher Scientific, Waltham, MA, USA). A one-way ANOVA with a p-value cutoff of 0.05 followed by a Tukey’s HSD post hoc was performed using XLStat to determine statistically significant accumulation of emodin and its glucoside across root, stem and leaf samples. Extracts from the roots, stems and leaves, of R. sanguineus were subjected to non-targeted metabolomics workflow to comprehensively annotate its metabolites composition. The water/methanol phase allowed for the extraction of the main plant biochemical classes of both primary (i.e. carbohydrates) and specialized metabolites (i.e. flavonoids, anthraquinones). The resulting UHPLC-HRMS raw datasets, acquired both in positive and in the negative ionization modes, underwent several data processing steps, as schematically represented in Fig. . Overall, 8896 and 10,589 features were aligned in positive and negative mode respectively, using MZmine 3.9.0. At first, unsupervised models were employed, specifically PCA, to reduce the dimensionality of our dataset while maximizing the retention of its inherent variability. The PCA score plots (depicted in Figure of supplementary information) reveals different metabolomic composition for Rumex organs, due to a distinctive clustering pattern among the three different organs. Afterwards, the aligned datasets (ESI positive and negative) were independently analyzed using the feature-based molecular networking workflow of GNPS and visualized using Cytoscape software. The resulting molecular networks are reported in Figures , for ESI positive and negative ion modes, respectively. Overview of metabolite annotations: identification levels, biochemical groups, and tissue distributions Overall, the annotation process in both positive and negative ionization mode yielded 449 annotated features, including 102 overlapping annotations, resulting in 347 unique metabolites. Of these, 200 metabolites were annotated in positive ESI mode, and 143 metabolites were annotated in negative ESI mode. Within all the annotated features, 2% (7) were identified at level I (Schymanski et al., ) indicating the highest confidence level of identification, while 98% (442) were categorized as level II, denoting a reliable matching with existing libraries. The lists of annotated features are uploaded to Zenodo: https://doi.org/ 10.5281/zenodo.14236385. The 347 primary and specialized metabolites were grouped into 8 biochemical classes, as depicted in Fig. . This categorization offers a comprehensive snapshot of the chemical diversity within the analysed compounds under different ionization modes. As an example, some chemical classes such as alkaloids and disaccharides ionize exclusively in positive mode while polyphenols ionized mainly in negative mode. Among the annotated compounds, well-established constituents found in other Rumex species, such as polyphenols and anthraquinones, were found. In addition, our analysis unveiled a significant presence of coumarins, surpassing what is typically reported in literature for other Rumex spp. (Vasas et al., ). Notably, coumarin presence has been rarely reported in Rumex species, with only R. roseus (Saoudi et al., ) showing some derivatives. Several cinnamic acids were also annotated. These antioxidant compounds were previously identified and found responsible for the biological activity of the R. dentatus extract (Khaliq et al., ). However, most annotated metabolites (60%) still belong to the polyphenols and anthraquinones classes. Cyanidins and proanthocyanidins were previously found only in R. acetosa (Bicker et al., ). Flavonoids, which are prevalent in the leaves of the Rumex genus, are primarily renowned for their beneficial attributes (Rana et al., ). A wide range of glycosylated quercetin derivatives like rutin were annotated in this study. Furthermore, while flavonol derivatives like kaempferol are present in subgenus species such as R. confertus and R. sanguineus , they are notably absent in R. acetosa and R. acetosella . Kaempferols and quercetins play a crucial role as health promoters due to their potent antioxidant properties and their potential to mitigate oxidative damage in biological systems (Zhang & Tsao, ). Therefore, the accumulation of these bioactive phytochemicals in the edible part of R. sanguineus underscores its significance as a food source of these beneficial compounds. Although the nature of our analysis does not permit conclusions about the absolute concentrations of compounds, it does reveal distribution trends. Notably, most of the annotated compounds (90%) are present in all three plant organs, as shown in the Venn diagram. (Fig. ). However, what sets them apart is the variation in their proportions. In other words, a single compound found in all three organs can either be detected in all organs of Rumex but accumulate at different ratios (Fig. ), or in some rare cases can be accumulated into a specific one (organ-specific). In our study, we observed that anthraquinones are generally accumulated in leaves, while aloin A, aloin B and some emodin derivatives, were predominantly found in the roots (Fig. ). It is worth mentioning that many plant-specialized metabolites (anthraquinones and phenolic compounds) accumulate as glycosides as storage of bioactive molecules, hence, it is not surprising that aloin A and B were predominantly found in roots. These compounds are synthesized in the roots through the action of enzymes called glycosyltransferases (Yamada et al., ). However, further research is necessary to determine whether hydroxyanthracene glucoside derivatives are directly produced in the roots or if they are synthesized elsewhere in the plant and then stored in the roots. In the leaves, emodin appears to function as a deterrent to herbivores, especially vertebrates, at certain concentrations. Studies, (Trial & Dimond, ) suggest that emodin can be toxic to animals, which likely contributes to its role in protecting plants from herbivory. The glucosylation of emodin in the roots could be linked to the plant’s allelopathic strategy. By producing emodin glucosides, the plant may inhibit the growth of neighbouring plant species (Izhaki, ). This allelopathic effect could give the plant a competitive advantage in crowded environments. While both biotic (e.g., interactions with other organisms) and abiotic (e.g., environmental factors) forces may influence the evolution of emodin, the distribution and concentration of emodin in various parts of a plant likely reflect a complex interplay of these factors. These interactions shape how emodin is produced, stored, and utilized across different species. Consequently, the presence of emodin in varying concentrations in different plant organs reflects not only the ecological conditions in which the plant thrives but also its diverse adaptive functions. Besides hydroxyanthracenes derivatives, it’s also important to consider the distribution of other bioactive compounds in plants, such as flavonols. While emodin glucosides are primarily concentrated in the roots, flavonols, including quercetin and kaempferol, tend to accumulate preferentially in the leaves. This is in line with the results reported for another Rumex spp. , namely R. acetosella (Feduraev et al., ) . Despite leveraging on advanced annotation tools, a significant proportion of features within R. sanguineus extracts, still remain unidentified, accounting for approximately 90% of the total. The molecular networking approach to decode the Rumex chemical composition FBMN allowed the visualization of clusters of annotated compounds. The FBMN built with ESI positive data (Fig. ) is a graphical representation of the relationships between chemical compounds, based on their spectral similarities. Each compound is represented by a node and the edges between the nodes indicate the similarity of their mass spectra on the basis of a modified cosine score. A total of 1118 features (nodes), interconnected by 2060 edges, were organized into 88 distinct clusters, while 507 remained without any connection indicating a lack of chemical class connection between them based on their shared fragmentation pathway with the used topology filter parameter settings to construct the molecular network. Table provides a summary of all the annotated compounds clustered in the five sub-networks as thoroughly explained in the corresponding subsections (referred to as "sub-networks") highlighted in our analysis. The batch annotation spreadsheet can be found in the supplementary information as Table . This spreadsheet has been made available on both Zenodo and MASSIVE platforms. It serves as a GNPS metadata-derived table, providing insights into molecule identities annotated through SIRIUS and manual compound annotation. By adopting a common machine-readable format, this resource offers valuable spectral annotation data to the scientific community. Additionally, all analysed fragmentation spectra can be accessed on MASSIVE in the form of mzXML files. Each annotated compound is associated with a distinct scan number, facilitating its retrieval within the molecular network uploaded on Zenodo. Sub-network exploration Among all clusters depicted in Fig. , we focused on the two largest classes, namely anthraquinones and polyphenols, because of their significance in plant metabolism and their potential implications in various biological activities. The anthraquinones sub-network: focus on emodin and its derivatives (1) The anthraquinones family is largely present in roots, stems and leaves of Rumex ; several anthraquinones derivatives were annotated thanks to their common fragmentation pattern. Starting with sub-network 1 (Figure for ESI positive and Figure for ESI negative), an emodin derivative was putatively annotated with feature number 6662 ( m/z 271.0601, [M + H] + ion adduct), based on the match of its fragmentation spectra with that of the emodin reference standard (Table , Supplementary Information). The substantial difference in their retention times (11.5 min for feature number 6662 vs 13.8 min of the reference standard, Table ), indicates a different interaction of the compound with the stationary phase and a consequently difference in the distribution of emodin functional groups around the anthracene skeleton. At retention time of 12.7 min, we annotated emodin anthrone (feature number 7293) with [M + H] + ion adduct of m/z 257.0811, and the data presented in Table , provides evidence of its characteristic fragmentation pattern as reported in library databases and literature (Bo et al., ). Notably, in this sub-network we annotated emodin anthrone hydrogen sulfate (feature number 5623, m/z 337.0375 [M + H] + ion adduct, Table ), based on the match between the fragmentation pattern of emodin anthrone, and to the characteristic hydrogen sulfite loss of 79.9578. Emodin hydrogen sulfate [M + H] + ion adduct ( m/z 351.0172), was putatively annotated with feature number 5708, at retention time of 9.8 min in the same sub-network (Table ). Due to their functional groups, anthraquinones easily produce [M–H] − ion adducts in the negative ESI source. Therefore, the annotation of anthraquinones derivatives was conducted by investigating the ESI negative mode data (Zhao et al., ) (Fig. , ). Among these, emodin stands out as a toxic compound (Younes et al., ). Given the coexistence of other emodin isomers with different retention times in our samples, particularly in the edible leaves, we aimed to confirm its presence with a reference standard. Emodin was unambiguously identified at retention time of 13.8 min with feature number of 10,182 (Table ) and [M–H] − ion adduct of m/z 269.0455. Moreover, compound with [M–H] − ion adduct and m/z 285.0408 (Table ), was annotated with feature number 10585 as a 3-methyl-1,4,5,7-tetrahydroxy-anthraquinone with a retention time of 14.96 min, indicating that it is an emodin derivative bearing a different rearrangement of the hydroxyl and methyl groups on the hydroxyanthracene scaffold. Dihydroxy-dimethylxanthone (Table ) was tentatively identified with feature number 8935 at retention time of 12.9 min, and [M–H] − ion adduct of m/z 255.0666; notably, its fragmentation spectra align with the characteristic fragmentation pattern observed for anthraquinones. Moreover, two carboxylic acid derivatives, namely dihydroxy- methylanthraquinone-carboxylic acid (feature number of 8702, rt 12.8 min, m/z 297.0409) and endocrocin (feature number of 7036, rt 11.5 min and m/z 313.0360) were annotated (Table ). Endocrocin is an emodin derivative that forms a conjugate with a carboxylic acid, as evidenced by the fragmentation spectra uploaded on massIVE. The fragmentation pattern matches with that of emodin after the cleavage of the carboxylic acid, confirming the structural relationship between the two compounds. It is worth emphasizing that both compounds are predominantly concentrated in the roots (see yellow nodes in Fig. ), indicating their specific accumulation in this plant organ. Here, we introduce the negative molecular network of glycosylated anthraquinones (Fig. ), which is closely linked to the previous network. Similar to free anthraquinones, anthraquinone conjugates easily produced [M–H] − ion adduct in the negative ESI source and gave rise to [aglycone-H] − ion as the MS/MS base peak by elimination of the glucosyl residue. At a retention time of 9.8 min, emodin-8-glucopyranosyl sulfate (Table ) was detected with [M–H] − ion adduct of m/z 511.0554 and feature number 5264. This compound can be considered the glycosylated corresponding ion of emodin hydrogen sulfate (Table ). This may be attributed to the cleavage of the glucoside unit within the compound during the ESI ionization process (Es-Safi et al., ). Another noteworthy compound, emodin-8-glucoside, was identified (level I) with reference standard confirming its retention time at 10.7 min, feature number of 6115 and [M–H] − ion adduct of m/z 431.0985. The structure elucidation in supplementary Table highlights the loss of the glucoside unit and the subsequent fragmentation pattern typical of anthraquinones, aligning well with the reference standard. Another putative candidate was tentatively identified as dihydroxy-methyl-dioxoanthracen-yl)-oxy-trihydroxyoxan-yl-methyl acetate which is an emodin derivative. It is characterized by feature number 6998, a retention time of 11.6 min, and a [M–H] − ion adduct with m/z 473.1092, as depicted in the fragmentation spectra uploaded on massIVE. Furthermore, the glucosyl derivative, kwanzoquinone D, (Table ), was tentatively annotated with feature number 7183 at a retention time of 11.6 min and a [M–H] − ion adduct of m/z 517.0990. It shares an identical fragmentation pattern with the previous candidate, but the key distinction lies in the presence of a carboxylic acid, which is bonded to the methyl acetate group in kwanzoquinone D (Cichewicz et al., ). The anthraquinones sub-network: focus on aloin and its derivatives (2) Sub-network number 2 (Fig. ) was a focal point of our investigation due to its distinct composition of anthraquinone derivatives, notably the presence of aloin derivatives. Aloin A and aloin B were confirmed (level I) with reference standards with feature number of 5401 and 5188 respectively (Table ), with a retention time of 9.2 min for aloin A and of 8.8 min for aloin B. Both of these compounds share the same [M + H] + ion adduct of m/z 419.1336 and an identical fragmentation spectra since they are isomers. The distinguishing factor between the two spectra lies in the relative abundance of fragment ions m/z 239.0967 and m/z 211.0749. In aloin B, the former fragment is more prevalent than the latter, whereas in aloin A, these two fragments exhibit equivalent relative abundances. A compound directly associated with aloin B has an m/z of 463.1238 ([M + H] + ion adduct). This compound has been tentatively annotated as nodososide, identified by feature number 4431 and a retention time of 7.6 min, as outlined in Table . The most abundant fragment is m/z 283.0600 which corresponds to the cleavage of the glucopyranoside unit (neutral loss of 180.0641) as indicated by the fragmentation spectra. The [M + H] + ion adduct with a m/z of 447.1293, was putatively annotated as glucoobtusifolin (Table ), feature number 5247 and retention time of 8.9 min. The fragmentation of glucoobtusifolin involves the same cleavage of a glucopyranoside unit, resulting in a neutral loss of 180.0641, as elucidated by the fragmentation spectra uploaded on MassIVE. This process leads to the generation of its most predominant fragment with an m/z ratio of 267.0650. Overall, 37 anthraquinones were annotated (level II) in ESI positive (N = 19) and ESI negative (N = 18) modes, respectively, while 4 were identified (level I), namely emodin, emodin-8-glucoside, aloin A and aloin B. Emodin and emodin-8-glucoside exhibited highest accumulation in leaves, while other derivatives, such as carboxylic acid and sulfate derivatives, were predominantly accumulated in roots. The polyphenols sub-network: focus on kaempferol and quercetin (3) Phenolic compounds are ubiquitously distributed among plants, including various Rumex species, displaying substantial diversity in both type and concentration. Their presence serves multifaceted ecological and physiological functions across the plant genus (Feduraev et al., ). Firstly, kaempferol was annotated based on a [M + H] + ion adduct ( m/z 287.0553), with feature number 6917, and then confirmed with a reference standard by matching fragmentation spectra and retention times at 12.1 min (Table ). The compounds closely connected with identified kaempferol ( m/z 287.0553), have been annotated as kaempferol derivatives (class II) with feature numbers 4680, 4526 and 7816. The structure elucidation is based to the comparison of the feature fragmentation spectra to that of the reference standard. Their presence can be explained as in-source fragmentation of glycosylated forms of kaempferol, particularly feature 4680, whose aglycone retention time aligns with that of kaempferol-3-glucuronide. Additionally, feature 4526 could potentially be an in-source fragment derivative of kaempferol-3-glucoside, as evidenced by its matching retention time (7.6 min in kaempferol-3glucoside and 7.6 min in feature 4526) and the alignment of seven key fragments provides strong evidence of clear structural similarity (Barnes & Schug, ). Fisetin was putatively annotated (Table ) within this sub network, with a feature number of 3601 ( m/z 287.0552, [M + H] + ion adduct) and, based on the similarity of its structure to that of kaempferol. The fragmentation spectra is similar to that of kaempferol, but the ions ratio is different (Chambers et al., ). Quercetin was also annotated (feature number 7757, m/z 303.0498 , [M + H] + ) and confirmed with reference standard (Table ). Compounds with feature number and 4263 (rt 7.16 min) and feature 4057 (rt 6.88 min), were putatively annotated as quercetin isomers based on the similarity of their fragmentation spectra and the difference in their retention times as showed in on massIVE upload. Both features may be interpreted as an in-source fragments of a glycosylated forms of quercetin, as all annotated forms of quercetin exhibit the same retention times and given that elute earlier than the reference standard of quercetin (Barnes & Schug, ; Li et al., ). The polyphenols sub-network: focus on kaempferol and quercetin glycosides (4) Three quercetin derivatives, namely quercetin 3-[6″-(3-hydroxy-3-methylglutaryl) glucoside feature number 4505, m/z 609.1449), quercetin-3-glucuronide (feature number 4184, m/z 479.0819) and quercetin-3-glucoside (feature 4143, m/z 465.1028) were annotated in positive ESI mode ([M + H] + ion adduct) as their MS/MS were predominated by the elimination of the sugar unit giving rise to the [aglicone + H] + ion. In particular, the neutral losses of 306.1096 (162.0521 glucoside + 144.0437 methylglutaryl portion), 176.3270 (glucuronide) and 162.0521 (glucoside), respectively, enabled the annotation of the quercetin derivatives (Delcambre et al., ; Nagy et al., ). Feature number 4194 with an m/z of 957.1586 [M + H] + ion adduct, was reported as a quercetin 3-glucuronide dimer (yet not annotated) formed in ESI source (Pan, ). Evidences are that quercetin 3-glucuronide and its dimer have the same retention time at 7.1 min; if the dimer is formed in-solution, the m/z of the monomeric species would be eluted at a different retention time with respect to the one of the dimeric species, due to the quite different molecular properties of monomer and dimer and the different interactions of the two species with the stationary phase of the column (Alcalde-Eon et al., ). Similarly, as for quercetin, four kaempferol derivatives were annotated based on the neutral losses. Feature number 5002 has close correlation to quercetin3-[6″-(3-hydroxy-3-methylglutaryl) glucoside (Table ), thus was annotated as kaempferol 3-[6″-(3-hydroxy-3-methylglutaryl) glucoside (rt 8.54 min, m/z 593.1497, [M + H] + ion adduct). Kaempferol is characterized by a precursor ion of m/z 287.0548 and as for the quercetin derivatives, the MS/MS shows the same neutral loss of 306.0952 (feature 4505) consistent with the cleavage of the sugar unit bonded to 3-hydroxy-3-methylglutaryl sub-unit. Along with its congeners, the ion m/z of 449.1087 (feature 4533) was annotated as kaempferol-3-glucoside; within the fragmentation spectra, the glucoside unit produces a neutral loss of 162.0521 while the aglycone unit confirms kaempferol structure (precursor ion of m/z 287.0548). Furthermore, kaempferol-3-rutinoside (feature number 4513, m/z 595.1653, [M + H] + ion adduct) was annotated as a disaccharide form of kaempferol. As for the other glycosylated forms, we can observe a neutral loss (in this case of 308.1114) corresponding to the O-glycoside portion bonded to kaempferol. Feature number 4660 was tentatively annotated as kaempferol-3-glucuronide with a rt of 7.98 min and m/z of 463.0874 [M + H] + . Feature number 4039 ( m/z 611.1607, [M + H] + ion adduct) was unambiguously identified as rutin with the reference standard and confirmed with the rt (6.7 min, Table ). Cyanidin-3-glucoside (feature 2095, m/z 449.1085, [M] + ) was tentatively identified monitoring the flavylium glycosylated cation (Barnes & Schug, ) and the unique fragment ion of m/z 149.0232, which enable to distinguish cyanidin derivatives from quercetin and kaempferol. Based on a similar assumption, feature 4302 ( m/z 577.1337, [M] + ion adduct, rt 7.2 min) was putatively described as a cyanidin-catechin dimer This identification is supported by the observed neutral loss of 290.0799 corresponding to the loss of catechin, and by the fact that cyanidin mass spectra are dominated by the molecular ion [M] + rather than [M + H] + due to anthocyanins existing as flavylium cations under acidic conditions (Li et al., ). This conclusion is further corroborated by the fragmentation spectra (uploaded to MassIVE) as described in the literature (Delcambre et al., ; Nagy et al., ). The anthocyanidin sub-network: focus on proanthocyanidins (5) Previous literature reported high content of proanthocyanidins (Spencer et al., ) in R. obtusifolius (Sganzerla et al., ), hence we aimed to verify their presence also in R. sanguineus . An unequivocal network emerged, including proanthocyanidin B2, proanthocyanidin B2-gallate, and a trimer of proanthocyanidin B2 (Fig. ). Proanthocyanidin B2, was putatively annotated (feature number 1695, rt 3.9 min, m/z 579.1510, [M + H] + ) based on the fragmentation pattern, which indicates a potential cleavage corresponding to an epicatechin (a single monomer forming proanthocyanidin B2). This results in the formation of the fragment ion m/z 287.0480. Similarly, proanthocyanidin B2-gallate was annotated (feature number of 3328, rt 5.5 min, m/z 731.1621, [M + H] + ) matching his fragments with two neutral losses for the cleavage of propyl gallate (neutral loss of 152.0528 and of 292.0953 leading to the formation of the m/z 287.0480 as reported in literature (Rue et al., )). Furthermore, we proposed the annotation of a proanthocyanidin B2 trimer, characterized by the presence of three individual epicatechin monomers connected via C–C bonds. This trimeric compound (feature number 2108, rt 4.3 min, m/z 867.2146, [M + H] + ) is subject to similar fragmentation pattern, with two epicatechin cleavages and consequently neutral losses of 292.0953 as reported in massIVE upload. As indicated in the literature, the occurrence of trimers is more widespread than commonly thought. A-type trimers (Li et al., ) are commonly found in cranberry (Foo et al., ), while trimeric and tetrameric proanthocyanidins have been reported in the aerial parts of Rumex acetosa (Derksen et al., ). Quantitative analysis of emodin and emodin-8-glucoside in roots, stems and leaves Our metabolomic analysis showed that several anthraquinones (19 annotated in ESI positive ionization mode and 18 annotated in ESI negative), including emodin and emodin-8-glucoside, are differently distributed across the three different plant organs of Rumex . Despite the lack of precise concentration data, we observed that these compounds are present in varying amounts in each organ, highlighting specific patterns of accumulation. Because of their know toxicity (Paneitz et al., ) and the recent EFSA opinion on the presence of emodin and aloe-emodin in edible plants (Younes et al., ) a quantitative analysis was performed. As shown in Fig. A the content of emodin was significantly (p < 0.0001) higher in leaves (8 mg/g of dry weight), compared to stems and roots (leaves > stems > roots) considering an adult growth stage and a winter harvesting period. A different accumulation trend was observed for emodin-8-glucoside (Fig. A), whose content was significantly higher (p < 0.0005) in stems (2 mg/g of dry weight) followed by leaves and roots (stems > leaves > roots). This difference across organs distribution may be elucidated considering that emodin-8-glucoside serves as a storage form of emodin. Activation of this storage form involves the action of glycosyltransferases, which facilitate the removal of a sugar unit from the anthraquinone ring. The observed variation in distribution across plant organs, with potentially higher expression in stems, suggests the involvement of glycosyltransferase activity. However, further studies are necessary to conclusively affirm this hypothesis (Izhaki, ; Paneitz et al., ). Considering that leaves constitute the edible part of the plant and that young leaves are the most suitable for eating consumption, we quantified the emodin content in leaves at distinct growth stages (young and adult leaves). When comparing the levels of emodin between young and adult leaves, a notable difference was observed: the concentration of emodin was found to be significantly higher in young leaves (Fig. ) showing an increase of approximately 30% compared to adult leaves. This result could be explained considering that synthesis and accumulation of secondary metabolites in plants is affected the growing stage and by abiotic environmental factors, such as light intensity, soil minerals, osmotic stresses (drought and salinity) and seasonality (Izhaki, ). Studies supporting this hypothesis indicate that emodin levels in plants depend on season and light intensity. The leaves of Rheum undulatum in Europe exhibited the highest concentration of total anthraquinones, with approximately 50% being emodin, during the spring season (April). Subsequently, there was a consistent decline in anthraquinone levels throughout the summer, reaching their lowest amounts in late summer (September). This observed seasonal variation could be indicative of a trade-off between the plant’s developmental processes and its defence mechanisms (Paneitz et al., ). The accumulation of emodin, emodin-8-glucoside and other derivatives such as aloin A and B, has been reported also for other Rumex species, such as R. confertus, R. crispus, R. obtusifolius and R. aquaticus (Eom et al., ; Pelzer et al., ; Poluyanov et al., ). Our findings correspond well within the wide range documented in existing literature (2 to 160 mg/g) (Wegiera et al., ), despite the considerable variability in emodin content observed across seasons, organs, and Rumex species. The variability of emodin concentration in the roots shows significant differences among various Rumex species. In certain species, such as R. acetosella, R. confertus, and R. crispus , the emodin content is higher in the roots than in the leaves. Conversely, in other species, the emodin levels are higher in the leaves. Notably, in R. obtusifolius , the proportional distribution aligns with our findings, with emodin levels being higher in the leaves compared to the roots. Given the absence of R. sanguineus from EFSA’s plant list containing approved hydroxyanthracene derivatives in food supplements, our research gains heightened significance. The EFSA opinion classifies hydroxyanthracene derivatives (including emodin, aloin and derivatives) as potentially genotoxic and carcinogenic. Notably, EFSA faces challenges in providing precise recommendations for the daily intake of hydroxyanthracene derivatives that would not raise concerns regarding potential adverse health effects. This underscores the need for continued investigation and assessment of the potential implications associated with the presence of hydroxyanthracene derivatives in R. sanguineus . As R. sanguineus demonstrates the ability to biosynthesize anthraquinone, particularly emodin and its derivatives, the intricate and at times contradictory pharmacological activities associated with emodin necessitate thorough investigation. In 2021 the European Commission considered the significant adverse health effects linked to the consumption of aloe-emodin, emodin, danthron, and aloe extracts containing hydroxyanthracene derivatives in food, and given the absence of a defined daily intake level that does not raise concerns for human health, prohibited these substances (Annex III, Part A of Regulation (EC) No 1925/20069) (L96, ). The implementation of non-targeted metabolomics in our investigation has enhanced our understanding of the chemical profile of R. sanguineus highlighting its potential for applications in contemporary nutrition and agriculture. From a food nutritional and safety perspective, Rumex leaves were found to be rich in bioactive compounds, including phenolic, coumarins and cinnamic acids. When it comes to safety aspects, cooking Rumex leaves prior to consumption is expected to mitigate concerns regarding emodin content, as thermal processing induces the degradation of emodin (Narayanan et al., ). However, rigorous analytical investigations are essential to elucidate the kinetics of emodin degradation under various cooking conditions, thereby facilitating informed decision making to determine the safety and nutritional value of cooked Rumex leaves. Overall, the annotation process in both positive and negative ionization mode yielded 449 annotated features, including 102 overlapping annotations, resulting in 347 unique metabolites. Of these, 200 metabolites were annotated in positive ESI mode, and 143 metabolites were annotated in negative ESI mode. Within all the annotated features, 2% (7) were identified at level I (Schymanski et al., ) indicating the highest confidence level of identification, while 98% (442) were categorized as level II, denoting a reliable matching with existing libraries. The lists of annotated features are uploaded to Zenodo: https://doi.org/ 10.5281/zenodo.14236385. The 347 primary and specialized metabolites were grouped into 8 biochemical classes, as depicted in Fig. . This categorization offers a comprehensive snapshot of the chemical diversity within the analysed compounds under different ionization modes. As an example, some chemical classes such as alkaloids and disaccharides ionize exclusively in positive mode while polyphenols ionized mainly in negative mode. Among the annotated compounds, well-established constituents found in other Rumex species, such as polyphenols and anthraquinones, were found. In addition, our analysis unveiled a significant presence of coumarins, surpassing what is typically reported in literature for other Rumex spp. (Vasas et al., ). Notably, coumarin presence has been rarely reported in Rumex species, with only R. roseus (Saoudi et al., ) showing some derivatives. Several cinnamic acids were also annotated. These antioxidant compounds were previously identified and found responsible for the biological activity of the R. dentatus extract (Khaliq et al., ). However, most annotated metabolites (60%) still belong to the polyphenols and anthraquinones classes. Cyanidins and proanthocyanidins were previously found only in R. acetosa (Bicker et al., ). Flavonoids, which are prevalent in the leaves of the Rumex genus, are primarily renowned for their beneficial attributes (Rana et al., ). A wide range of glycosylated quercetin derivatives like rutin were annotated in this study. Furthermore, while flavonol derivatives like kaempferol are present in subgenus species such as R. confertus and R. sanguineus , they are notably absent in R. acetosa and R. acetosella . Kaempferols and quercetins play a crucial role as health promoters due to their potent antioxidant properties and their potential to mitigate oxidative damage in biological systems (Zhang & Tsao, ). Therefore, the accumulation of these bioactive phytochemicals in the edible part of R. sanguineus underscores its significance as a food source of these beneficial compounds. Although the nature of our analysis does not permit conclusions about the absolute concentrations of compounds, it does reveal distribution trends. Notably, most of the annotated compounds (90%) are present in all three plant organs, as shown in the Venn diagram. (Fig. ). However, what sets them apart is the variation in their proportions. In other words, a single compound found in all three organs can either be detected in all organs of Rumex but accumulate at different ratios (Fig. ), or in some rare cases can be accumulated into a specific one (organ-specific). In our study, we observed that anthraquinones are generally accumulated in leaves, while aloin A, aloin B and some emodin derivatives, were predominantly found in the roots (Fig. ). It is worth mentioning that many plant-specialized metabolites (anthraquinones and phenolic compounds) accumulate as glycosides as storage of bioactive molecules, hence, it is not surprising that aloin A and B were predominantly found in roots. These compounds are synthesized in the roots through the action of enzymes called glycosyltransferases (Yamada et al., ). However, further research is necessary to determine whether hydroxyanthracene glucoside derivatives are directly produced in the roots or if they are synthesized elsewhere in the plant and then stored in the roots. In the leaves, emodin appears to function as a deterrent to herbivores, especially vertebrates, at certain concentrations. Studies, (Trial & Dimond, ) suggest that emodin can be toxic to animals, which likely contributes to its role in protecting plants from herbivory. The glucosylation of emodin in the roots could be linked to the plant’s allelopathic strategy. By producing emodin glucosides, the plant may inhibit the growth of neighbouring plant species (Izhaki, ). This allelopathic effect could give the plant a competitive advantage in crowded environments. While both biotic (e.g., interactions with other organisms) and abiotic (e.g., environmental factors) forces may influence the evolution of emodin, the distribution and concentration of emodin in various parts of a plant likely reflect a complex interplay of these factors. These interactions shape how emodin is produced, stored, and utilized across different species. Consequently, the presence of emodin in varying concentrations in different plant organs reflects not only the ecological conditions in which the plant thrives but also its diverse adaptive functions. Besides hydroxyanthracenes derivatives, it’s also important to consider the distribution of other bioactive compounds in plants, such as flavonols. While emodin glucosides are primarily concentrated in the roots, flavonols, including quercetin and kaempferol, tend to accumulate preferentially in the leaves. This is in line with the results reported for another Rumex spp. , namely R. acetosella (Feduraev et al., ) . Despite leveraging on advanced annotation tools, a significant proportion of features within R. sanguineus extracts, still remain unidentified, accounting for approximately 90% of the total. FBMN allowed the visualization of clusters of annotated compounds. The FBMN built with ESI positive data (Fig. ) is a graphical representation of the relationships between chemical compounds, based on their spectral similarities. Each compound is represented by a node and the edges between the nodes indicate the similarity of their mass spectra on the basis of a modified cosine score. A total of 1118 features (nodes), interconnected by 2060 edges, were organized into 88 distinct clusters, while 507 remained without any connection indicating a lack of chemical class connection between them based on their shared fragmentation pathway with the used topology filter parameter settings to construct the molecular network. Table provides a summary of all the annotated compounds clustered in the five sub-networks as thoroughly explained in the corresponding subsections (referred to as "sub-networks") highlighted in our analysis. The batch annotation spreadsheet can be found in the supplementary information as Table . This spreadsheet has been made available on both Zenodo and MASSIVE platforms. It serves as a GNPS metadata-derived table, providing insights into molecule identities annotated through SIRIUS and manual compound annotation. By adopting a common machine-readable format, this resource offers valuable spectral annotation data to the scientific community. Additionally, all analysed fragmentation spectra can be accessed on MASSIVE in the form of mzXML files. Each annotated compound is associated with a distinct scan number, facilitating its retrieval within the molecular network uploaded on Zenodo. Among all clusters depicted in Fig. , we focused on the two largest classes, namely anthraquinones and polyphenols, because of their significance in plant metabolism and their potential implications in various biological activities. The anthraquinones sub-network: focus on emodin and its derivatives (1) The anthraquinones family is largely present in roots, stems and leaves of Rumex ; several anthraquinones derivatives were annotated thanks to their common fragmentation pattern. Starting with sub-network 1 (Figure for ESI positive and Figure for ESI negative), an emodin derivative was putatively annotated with feature number 6662 ( m/z 271.0601, [M + H] + ion adduct), based on the match of its fragmentation spectra with that of the emodin reference standard (Table , Supplementary Information). The substantial difference in their retention times (11.5 min for feature number 6662 vs 13.8 min of the reference standard, Table ), indicates a different interaction of the compound with the stationary phase and a consequently difference in the distribution of emodin functional groups around the anthracene skeleton. At retention time of 12.7 min, we annotated emodin anthrone (feature number 7293) with [M + H] + ion adduct of m/z 257.0811, and the data presented in Table , provides evidence of its characteristic fragmentation pattern as reported in library databases and literature (Bo et al., ). Notably, in this sub-network we annotated emodin anthrone hydrogen sulfate (feature number 5623, m/z 337.0375 [M + H] + ion adduct, Table ), based on the match between the fragmentation pattern of emodin anthrone, and to the characteristic hydrogen sulfite loss of 79.9578. Emodin hydrogen sulfate [M + H] + ion adduct ( m/z 351.0172), was putatively annotated with feature number 5708, at retention time of 9.8 min in the same sub-network (Table ). Due to their functional groups, anthraquinones easily produce [M–H] − ion adducts in the negative ESI source. Therefore, the annotation of anthraquinones derivatives was conducted by investigating the ESI negative mode data (Zhao et al., ) (Fig. , ). Among these, emodin stands out as a toxic compound (Younes et al., ). Given the coexistence of other emodin isomers with different retention times in our samples, particularly in the edible leaves, we aimed to confirm its presence with a reference standard. Emodin was unambiguously identified at retention time of 13.8 min with feature number of 10,182 (Table ) and [M–H] − ion adduct of m/z 269.0455. Moreover, compound with [M–H] − ion adduct and m/z 285.0408 (Table ), was annotated with feature number 10585 as a 3-methyl-1,4,5,7-tetrahydroxy-anthraquinone with a retention time of 14.96 min, indicating that it is an emodin derivative bearing a different rearrangement of the hydroxyl and methyl groups on the hydroxyanthracene scaffold. Dihydroxy-dimethylxanthone (Table ) was tentatively identified with feature number 8935 at retention time of 12.9 min, and [M–H] − ion adduct of m/z 255.0666; notably, its fragmentation spectra align with the characteristic fragmentation pattern observed for anthraquinones. Moreover, two carboxylic acid derivatives, namely dihydroxy- methylanthraquinone-carboxylic acid (feature number of 8702, rt 12.8 min, m/z 297.0409) and endocrocin (feature number of 7036, rt 11.5 min and m/z 313.0360) were annotated (Table ). Endocrocin is an emodin derivative that forms a conjugate with a carboxylic acid, as evidenced by the fragmentation spectra uploaded on massIVE. The fragmentation pattern matches with that of emodin after the cleavage of the carboxylic acid, confirming the structural relationship between the two compounds. It is worth emphasizing that both compounds are predominantly concentrated in the roots (see yellow nodes in Fig. ), indicating their specific accumulation in this plant organ. Here, we introduce the negative molecular network of glycosylated anthraquinones (Fig. ), which is closely linked to the previous network. Similar to free anthraquinones, anthraquinone conjugates easily produced [M–H] − ion adduct in the negative ESI source and gave rise to [aglycone-H] − ion as the MS/MS base peak by elimination of the glucosyl residue. At a retention time of 9.8 min, emodin-8-glucopyranosyl sulfate (Table ) was detected with [M–H] − ion adduct of m/z 511.0554 and feature number 5264. This compound can be considered the glycosylated corresponding ion of emodin hydrogen sulfate (Table ). This may be attributed to the cleavage of the glucoside unit within the compound during the ESI ionization process (Es-Safi et al., ). Another noteworthy compound, emodin-8-glucoside, was identified (level I) with reference standard confirming its retention time at 10.7 min, feature number of 6115 and [M–H] − ion adduct of m/z 431.0985. The structure elucidation in supplementary Table highlights the loss of the glucoside unit and the subsequent fragmentation pattern typical of anthraquinones, aligning well with the reference standard. Another putative candidate was tentatively identified as dihydroxy-methyl-dioxoanthracen-yl)-oxy-trihydroxyoxan-yl-methyl acetate which is an emodin derivative. It is characterized by feature number 6998, a retention time of 11.6 min, and a [M–H] − ion adduct with m/z 473.1092, as depicted in the fragmentation spectra uploaded on massIVE. Furthermore, the glucosyl derivative, kwanzoquinone D, (Table ), was tentatively annotated with feature number 7183 at a retention time of 11.6 min and a [M–H] − ion adduct of m/z 517.0990. It shares an identical fragmentation pattern with the previous candidate, but the key distinction lies in the presence of a carboxylic acid, which is bonded to the methyl acetate group in kwanzoquinone D (Cichewicz et al., ). The anthraquinones sub-network: focus on aloin and its derivatives (2) Sub-network number 2 (Fig. ) was a focal point of our investigation due to its distinct composition of anthraquinone derivatives, notably the presence of aloin derivatives. Aloin A and aloin B were confirmed (level I) with reference standards with feature number of 5401 and 5188 respectively (Table ), with a retention time of 9.2 min for aloin A and of 8.8 min for aloin B. Both of these compounds share the same [M + H] + ion adduct of m/z 419.1336 and an identical fragmentation spectra since they are isomers. The distinguishing factor between the two spectra lies in the relative abundance of fragment ions m/z 239.0967 and m/z 211.0749. In aloin B, the former fragment is more prevalent than the latter, whereas in aloin A, these two fragments exhibit equivalent relative abundances. A compound directly associated with aloin B has an m/z of 463.1238 ([M + H] + ion adduct). This compound has been tentatively annotated as nodososide, identified by feature number 4431 and a retention time of 7.6 min, as outlined in Table . The most abundant fragment is m/z 283.0600 which corresponds to the cleavage of the glucopyranoside unit (neutral loss of 180.0641) as indicated by the fragmentation spectra. The [M + H] + ion adduct with a m/z of 447.1293, was putatively annotated as glucoobtusifolin (Table ), feature number 5247 and retention time of 8.9 min. The fragmentation of glucoobtusifolin involves the same cleavage of a glucopyranoside unit, resulting in a neutral loss of 180.0641, as elucidated by the fragmentation spectra uploaded on MassIVE. This process leads to the generation of its most predominant fragment with an m/z ratio of 267.0650. Overall, 37 anthraquinones were annotated (level II) in ESI positive (N = 19) and ESI negative (N = 18) modes, respectively, while 4 were identified (level I), namely emodin, emodin-8-glucoside, aloin A and aloin B. Emodin and emodin-8-glucoside exhibited highest accumulation in leaves, while other derivatives, such as carboxylic acid and sulfate derivatives, were predominantly accumulated in roots. The polyphenols sub-network: focus on kaempferol and quercetin (3) Phenolic compounds are ubiquitously distributed among plants, including various Rumex species, displaying substantial diversity in both type and concentration. Their presence serves multifaceted ecological and physiological functions across the plant genus (Feduraev et al., ). Firstly, kaempferol was annotated based on a [M + H] + ion adduct ( m/z 287.0553), with feature number 6917, and then confirmed with a reference standard by matching fragmentation spectra and retention times at 12.1 min (Table ). The compounds closely connected with identified kaempferol ( m/z 287.0553), have been annotated as kaempferol derivatives (class II) with feature numbers 4680, 4526 and 7816. The structure elucidation is based to the comparison of the feature fragmentation spectra to that of the reference standard. Their presence can be explained as in-source fragmentation of glycosylated forms of kaempferol, particularly feature 4680, whose aglycone retention time aligns with that of kaempferol-3-glucuronide. Additionally, feature 4526 could potentially be an in-source fragment derivative of kaempferol-3-glucoside, as evidenced by its matching retention time (7.6 min in kaempferol-3glucoside and 7.6 min in feature 4526) and the alignment of seven key fragments provides strong evidence of clear structural similarity (Barnes & Schug, ). Fisetin was putatively annotated (Table ) within this sub network, with a feature number of 3601 ( m/z 287.0552, [M + H] + ion adduct) and, based on the similarity of its structure to that of kaempferol. The fragmentation spectra is similar to that of kaempferol, but the ions ratio is different (Chambers et al., ). Quercetin was also annotated (feature number 7757, m/z 303.0498 , [M + H] + ) and confirmed with reference standard (Table ). Compounds with feature number and 4263 (rt 7.16 min) and feature 4057 (rt 6.88 min), were putatively annotated as quercetin isomers based on the similarity of their fragmentation spectra and the difference in their retention times as showed in on massIVE upload. Both features may be interpreted as an in-source fragments of a glycosylated forms of quercetin, as all annotated forms of quercetin exhibit the same retention times and given that elute earlier than the reference standard of quercetin (Barnes & Schug, ; Li et al., ). The polyphenols sub-network: focus on kaempferol and quercetin glycosides (4) Three quercetin derivatives, namely quercetin 3-[6″-(3-hydroxy-3-methylglutaryl) glucoside feature number 4505, m/z 609.1449), quercetin-3-glucuronide (feature number 4184, m/z 479.0819) and quercetin-3-glucoside (feature 4143, m/z 465.1028) were annotated in positive ESI mode ([M + H] + ion adduct) as their MS/MS were predominated by the elimination of the sugar unit giving rise to the [aglicone + H] + ion. In particular, the neutral losses of 306.1096 (162.0521 glucoside + 144.0437 methylglutaryl portion), 176.3270 (glucuronide) and 162.0521 (glucoside), respectively, enabled the annotation of the quercetin derivatives (Delcambre et al., ; Nagy et al., ). Feature number 4194 with an m/z of 957.1586 [M + H] + ion adduct, was reported as a quercetin 3-glucuronide dimer (yet not annotated) formed in ESI source (Pan, ). Evidences are that quercetin 3-glucuronide and its dimer have the same retention time at 7.1 min; if the dimer is formed in-solution, the m/z of the monomeric species would be eluted at a different retention time with respect to the one of the dimeric species, due to the quite different molecular properties of monomer and dimer and the different interactions of the two species with the stationary phase of the column (Alcalde-Eon et al., ). Similarly, as for quercetin, four kaempferol derivatives were annotated based on the neutral losses. Feature number 5002 has close correlation to quercetin3-[6″-(3-hydroxy-3-methylglutaryl) glucoside (Table ), thus was annotated as kaempferol 3-[6″-(3-hydroxy-3-methylglutaryl) glucoside (rt 8.54 min, m/z 593.1497, [M + H] + ion adduct). Kaempferol is characterized by a precursor ion of m/z 287.0548 and as for the quercetin derivatives, the MS/MS shows the same neutral loss of 306.0952 (feature 4505) consistent with the cleavage of the sugar unit bonded to 3-hydroxy-3-methylglutaryl sub-unit. Along with its congeners, the ion m/z of 449.1087 (feature 4533) was annotated as kaempferol-3-glucoside; within the fragmentation spectra, the glucoside unit produces a neutral loss of 162.0521 while the aglycone unit confirms kaempferol structure (precursor ion of m/z 287.0548). Furthermore, kaempferol-3-rutinoside (feature number 4513, m/z 595.1653, [M + H] + ion adduct) was annotated as a disaccharide form of kaempferol. As for the other glycosylated forms, we can observe a neutral loss (in this case of 308.1114) corresponding to the O-glycoside portion bonded to kaempferol. Feature number 4660 was tentatively annotated as kaempferol-3-glucuronide with a rt of 7.98 min and m/z of 463.0874 [M + H] + . Feature number 4039 ( m/z 611.1607, [M + H] + ion adduct) was unambiguously identified as rutin with the reference standard and confirmed with the rt (6.7 min, Table ). Cyanidin-3-glucoside (feature 2095, m/z 449.1085, [M] + ) was tentatively identified monitoring the flavylium glycosylated cation (Barnes & Schug, ) and the unique fragment ion of m/z 149.0232, which enable to distinguish cyanidin derivatives from quercetin and kaempferol. Based on a similar assumption, feature 4302 ( m/z 577.1337, [M] + ion adduct, rt 7.2 min) was putatively described as a cyanidin-catechin dimer This identification is supported by the observed neutral loss of 290.0799 corresponding to the loss of catechin, and by the fact that cyanidin mass spectra are dominated by the molecular ion [M] + rather than [M + H] + due to anthocyanins existing as flavylium cations under acidic conditions (Li et al., ). This conclusion is further corroborated by the fragmentation spectra (uploaded to MassIVE) as described in the literature (Delcambre et al., ; Nagy et al., ). The anthocyanidin sub-network: focus on proanthocyanidins (5) Previous literature reported high content of proanthocyanidins (Spencer et al., ) in R. obtusifolius (Sganzerla et al., ), hence we aimed to verify their presence also in R. sanguineus . An unequivocal network emerged, including proanthocyanidin B2, proanthocyanidin B2-gallate, and a trimer of proanthocyanidin B2 (Fig. ). Proanthocyanidin B2, was putatively annotated (feature number 1695, rt 3.9 min, m/z 579.1510, [M + H] + ) based on the fragmentation pattern, which indicates a potential cleavage corresponding to an epicatechin (a single monomer forming proanthocyanidin B2). This results in the formation of the fragment ion m/z 287.0480. Similarly, proanthocyanidin B2-gallate was annotated (feature number of 3328, rt 5.5 min, m/z 731.1621, [M + H] + ) matching his fragments with two neutral losses for the cleavage of propyl gallate (neutral loss of 152.0528 and of 292.0953 leading to the formation of the m/z 287.0480 as reported in literature (Rue et al., )). Furthermore, we proposed the annotation of a proanthocyanidin B2 trimer, characterized by the presence of three individual epicatechin monomers connected via C–C bonds. This trimeric compound (feature number 2108, rt 4.3 min, m/z 867.2146, [M + H] + ) is subject to similar fragmentation pattern, with two epicatechin cleavages and consequently neutral losses of 292.0953 as reported in massIVE upload. As indicated in the literature, the occurrence of trimers is more widespread than commonly thought. A-type trimers (Li et al., ) are commonly found in cranberry (Foo et al., ), while trimeric and tetrameric proanthocyanidins have been reported in the aerial parts of Rumex acetosa (Derksen et al., ). The anthraquinones family is largely present in roots, stems and leaves of Rumex ; several anthraquinones derivatives were annotated thanks to their common fragmentation pattern. Starting with sub-network 1 (Figure for ESI positive and Figure for ESI negative), an emodin derivative was putatively annotated with feature number 6662 ( m/z 271.0601, [M + H] + ion adduct), based on the match of its fragmentation spectra with that of the emodin reference standard (Table , Supplementary Information). The substantial difference in their retention times (11.5 min for feature number 6662 vs 13.8 min of the reference standard, Table ), indicates a different interaction of the compound with the stationary phase and a consequently difference in the distribution of emodin functional groups around the anthracene skeleton. At retention time of 12.7 min, we annotated emodin anthrone (feature number 7293) with [M + H] + ion adduct of m/z 257.0811, and the data presented in Table , provides evidence of its characteristic fragmentation pattern as reported in library databases and literature (Bo et al., ). Notably, in this sub-network we annotated emodin anthrone hydrogen sulfate (feature number 5623, m/z 337.0375 [M + H] + ion adduct, Table ), based on the match between the fragmentation pattern of emodin anthrone, and to the characteristic hydrogen sulfite loss of 79.9578. Emodin hydrogen sulfate [M + H] + ion adduct ( m/z 351.0172), was putatively annotated with feature number 5708, at retention time of 9.8 min in the same sub-network (Table ). Due to their functional groups, anthraquinones easily produce [M–H] − ion adducts in the negative ESI source. Therefore, the annotation of anthraquinones derivatives was conducted by investigating the ESI negative mode data (Zhao et al., ) (Fig. , ). Among these, emodin stands out as a toxic compound (Younes et al., ). Given the coexistence of other emodin isomers with different retention times in our samples, particularly in the edible leaves, we aimed to confirm its presence with a reference standard. Emodin was unambiguously identified at retention time of 13.8 min with feature number of 10,182 (Table ) and [M–H] − ion adduct of m/z 269.0455. Moreover, compound with [M–H] − ion adduct and m/z 285.0408 (Table ), was annotated with feature number 10585 as a 3-methyl-1,4,5,7-tetrahydroxy-anthraquinone with a retention time of 14.96 min, indicating that it is an emodin derivative bearing a different rearrangement of the hydroxyl and methyl groups on the hydroxyanthracene scaffold. Dihydroxy-dimethylxanthone (Table ) was tentatively identified with feature number 8935 at retention time of 12.9 min, and [M–H] − ion adduct of m/z 255.0666; notably, its fragmentation spectra align with the characteristic fragmentation pattern observed for anthraquinones. Moreover, two carboxylic acid derivatives, namely dihydroxy- methylanthraquinone-carboxylic acid (feature number of 8702, rt 12.8 min, m/z 297.0409) and endocrocin (feature number of 7036, rt 11.5 min and m/z 313.0360) were annotated (Table ). Endocrocin is an emodin derivative that forms a conjugate with a carboxylic acid, as evidenced by the fragmentation spectra uploaded on massIVE. The fragmentation pattern matches with that of emodin after the cleavage of the carboxylic acid, confirming the structural relationship between the two compounds. It is worth emphasizing that both compounds are predominantly concentrated in the roots (see yellow nodes in Fig. ), indicating their specific accumulation in this plant organ. Here, we introduce the negative molecular network of glycosylated anthraquinones (Fig. ), which is closely linked to the previous network. Similar to free anthraquinones, anthraquinone conjugates easily produced [M–H] − ion adduct in the negative ESI source and gave rise to [aglycone-H] − ion as the MS/MS base peak by elimination of the glucosyl residue. At a retention time of 9.8 min, emodin-8-glucopyranosyl sulfate (Table ) was detected with [M–H] − ion adduct of m/z 511.0554 and feature number 5264. This compound can be considered the glycosylated corresponding ion of emodin hydrogen sulfate (Table ). This may be attributed to the cleavage of the glucoside unit within the compound during the ESI ionization process (Es-Safi et al., ). Another noteworthy compound, emodin-8-glucoside, was identified (level I) with reference standard confirming its retention time at 10.7 min, feature number of 6115 and [M–H] − ion adduct of m/z 431.0985. The structure elucidation in supplementary Table highlights the loss of the glucoside unit and the subsequent fragmentation pattern typical of anthraquinones, aligning well with the reference standard. Another putative candidate was tentatively identified as dihydroxy-methyl-dioxoanthracen-yl)-oxy-trihydroxyoxan-yl-methyl acetate which is an emodin derivative. It is characterized by feature number 6998, a retention time of 11.6 min, and a [M–H] − ion adduct with m/z 473.1092, as depicted in the fragmentation spectra uploaded on massIVE. Furthermore, the glucosyl derivative, kwanzoquinone D, (Table ), was tentatively annotated with feature number 7183 at a retention time of 11.6 min and a [M–H] − ion adduct of m/z 517.0990. It shares an identical fragmentation pattern with the previous candidate, but the key distinction lies in the presence of a carboxylic acid, which is bonded to the methyl acetate group in kwanzoquinone D (Cichewicz et al., ). Sub-network number 2 (Fig. ) was a focal point of our investigation due to its distinct composition of anthraquinone derivatives, notably the presence of aloin derivatives. Aloin A and aloin B were confirmed (level I) with reference standards with feature number of 5401 and 5188 respectively (Table ), with a retention time of 9.2 min for aloin A and of 8.8 min for aloin B. Both of these compounds share the same [M + H] + ion adduct of m/z 419.1336 and an identical fragmentation spectra since they are isomers. The distinguishing factor between the two spectra lies in the relative abundance of fragment ions m/z 239.0967 and m/z 211.0749. In aloin B, the former fragment is more prevalent than the latter, whereas in aloin A, these two fragments exhibit equivalent relative abundances. A compound directly associated with aloin B has an m/z of 463.1238 ([M + H] + ion adduct). This compound has been tentatively annotated as nodososide, identified by feature number 4431 and a retention time of 7.6 min, as outlined in Table . The most abundant fragment is m/z 283.0600 which corresponds to the cleavage of the glucopyranoside unit (neutral loss of 180.0641) as indicated by the fragmentation spectra. The [M + H] + ion adduct with a m/z of 447.1293, was putatively annotated as glucoobtusifolin (Table ), feature number 5247 and retention time of 8.9 min. The fragmentation of glucoobtusifolin involves the same cleavage of a glucopyranoside unit, resulting in a neutral loss of 180.0641, as elucidated by the fragmentation spectra uploaded on MassIVE. This process leads to the generation of its most predominant fragment with an m/z ratio of 267.0650. Overall, 37 anthraquinones were annotated (level II) in ESI positive (N = 19) and ESI negative (N = 18) modes, respectively, while 4 were identified (level I), namely emodin, emodin-8-glucoside, aloin A and aloin B. Emodin and emodin-8-glucoside exhibited highest accumulation in leaves, while other derivatives, such as carboxylic acid and sulfate derivatives, were predominantly accumulated in roots. Phenolic compounds are ubiquitously distributed among plants, including various Rumex species, displaying substantial diversity in both type and concentration. Their presence serves multifaceted ecological and physiological functions across the plant genus (Feduraev et al., ). Firstly, kaempferol was annotated based on a [M + H] + ion adduct ( m/z 287.0553), with feature number 6917, and then confirmed with a reference standard by matching fragmentation spectra and retention times at 12.1 min (Table ). The compounds closely connected with identified kaempferol ( m/z 287.0553), have been annotated as kaempferol derivatives (class II) with feature numbers 4680, 4526 and 7816. The structure elucidation is based to the comparison of the feature fragmentation spectra to that of the reference standard. Their presence can be explained as in-source fragmentation of glycosylated forms of kaempferol, particularly feature 4680, whose aglycone retention time aligns with that of kaempferol-3-glucuronide. Additionally, feature 4526 could potentially be an in-source fragment derivative of kaempferol-3-glucoside, as evidenced by its matching retention time (7.6 min in kaempferol-3glucoside and 7.6 min in feature 4526) and the alignment of seven key fragments provides strong evidence of clear structural similarity (Barnes & Schug, ). Fisetin was putatively annotated (Table ) within this sub network, with a feature number of 3601 ( m/z 287.0552, [M + H] + ion adduct) and, based on the similarity of its structure to that of kaempferol. The fragmentation spectra is similar to that of kaempferol, but the ions ratio is different (Chambers et al., ). Quercetin was also annotated (feature number 7757, m/z 303.0498 , [M + H] + ) and confirmed with reference standard (Table ). Compounds with feature number and 4263 (rt 7.16 min) and feature 4057 (rt 6.88 min), were putatively annotated as quercetin isomers based on the similarity of their fragmentation spectra and the difference in their retention times as showed in on massIVE upload. Both features may be interpreted as an in-source fragments of a glycosylated forms of quercetin, as all annotated forms of quercetin exhibit the same retention times and given that elute earlier than the reference standard of quercetin (Barnes & Schug, ; Li et al., ). Three quercetin derivatives, namely quercetin 3-[6″-(3-hydroxy-3-methylglutaryl) glucoside feature number 4505, m/z 609.1449), quercetin-3-glucuronide (feature number 4184, m/z 479.0819) and quercetin-3-glucoside (feature 4143, m/z 465.1028) were annotated in positive ESI mode ([M + H] + ion adduct) as their MS/MS were predominated by the elimination of the sugar unit giving rise to the [aglicone + H] + ion. In particular, the neutral losses of 306.1096 (162.0521 glucoside + 144.0437 methylglutaryl portion), 176.3270 (glucuronide) and 162.0521 (glucoside), respectively, enabled the annotation of the quercetin derivatives (Delcambre et al., ; Nagy et al., ). Feature number 4194 with an m/z of 957.1586 [M + H] + ion adduct, was reported as a quercetin 3-glucuronide dimer (yet not annotated) formed in ESI source (Pan, ). Evidences are that quercetin 3-glucuronide and its dimer have the same retention time at 7.1 min; if the dimer is formed in-solution, the m/z of the monomeric species would be eluted at a different retention time with respect to the one of the dimeric species, due to the quite different molecular properties of monomer and dimer and the different interactions of the two species with the stationary phase of the column (Alcalde-Eon et al., ). Similarly, as for quercetin, four kaempferol derivatives were annotated based on the neutral losses. Feature number 5002 has close correlation to quercetin3-[6″-(3-hydroxy-3-methylglutaryl) glucoside (Table ), thus was annotated as kaempferol 3-[6″-(3-hydroxy-3-methylglutaryl) glucoside (rt 8.54 min, m/z 593.1497, [M + H] + ion adduct). Kaempferol is characterized by a precursor ion of m/z 287.0548 and as for the quercetin derivatives, the MS/MS shows the same neutral loss of 306.0952 (feature 4505) consistent with the cleavage of the sugar unit bonded to 3-hydroxy-3-methylglutaryl sub-unit. Along with its congeners, the ion m/z of 449.1087 (feature 4533) was annotated as kaempferol-3-glucoside; within the fragmentation spectra, the glucoside unit produces a neutral loss of 162.0521 while the aglycone unit confirms kaempferol structure (precursor ion of m/z 287.0548). Furthermore, kaempferol-3-rutinoside (feature number 4513, m/z 595.1653, [M + H] + ion adduct) was annotated as a disaccharide form of kaempferol. As for the other glycosylated forms, we can observe a neutral loss (in this case of 308.1114) corresponding to the O-glycoside portion bonded to kaempferol. Feature number 4660 was tentatively annotated as kaempferol-3-glucuronide with a rt of 7.98 min and m/z of 463.0874 [M + H] + . Feature number 4039 ( m/z 611.1607, [M + H] + ion adduct) was unambiguously identified as rutin with the reference standard and confirmed with the rt (6.7 min, Table ). Cyanidin-3-glucoside (feature 2095, m/z 449.1085, [M] + ) was tentatively identified monitoring the flavylium glycosylated cation (Barnes & Schug, ) and the unique fragment ion of m/z 149.0232, which enable to distinguish cyanidin derivatives from quercetin and kaempferol. Based on a similar assumption, feature 4302 ( m/z 577.1337, [M] + ion adduct, rt 7.2 min) was putatively described as a cyanidin-catechin dimer This identification is supported by the observed neutral loss of 290.0799 corresponding to the loss of catechin, and by the fact that cyanidin mass spectra are dominated by the molecular ion [M] + rather than [M + H] + due to anthocyanins existing as flavylium cations under acidic conditions (Li et al., ). This conclusion is further corroborated by the fragmentation spectra (uploaded to MassIVE) as described in the literature (Delcambre et al., ; Nagy et al., ). Previous literature reported high content of proanthocyanidins (Spencer et al., ) in R. obtusifolius (Sganzerla et al., ), hence we aimed to verify their presence also in R. sanguineus . An unequivocal network emerged, including proanthocyanidin B2, proanthocyanidin B2-gallate, and a trimer of proanthocyanidin B2 (Fig. ). Proanthocyanidin B2, was putatively annotated (feature number 1695, rt 3.9 min, m/z 579.1510, [M + H] + ) based on the fragmentation pattern, which indicates a potential cleavage corresponding to an epicatechin (a single monomer forming proanthocyanidin B2). This results in the formation of the fragment ion m/z 287.0480. Similarly, proanthocyanidin B2-gallate was annotated (feature number of 3328, rt 5.5 min, m/z 731.1621, [M + H] + ) matching his fragments with two neutral losses for the cleavage of propyl gallate (neutral loss of 152.0528 and of 292.0953 leading to the formation of the m/z 287.0480 as reported in literature (Rue et al., )). Furthermore, we proposed the annotation of a proanthocyanidin B2 trimer, characterized by the presence of three individual epicatechin monomers connected via C–C bonds. This trimeric compound (feature number 2108, rt 4.3 min, m/z 867.2146, [M + H] + ) is subject to similar fragmentation pattern, with two epicatechin cleavages and consequently neutral losses of 292.0953 as reported in massIVE upload. As indicated in the literature, the occurrence of trimers is more widespread than commonly thought. A-type trimers (Li et al., ) are commonly found in cranberry (Foo et al., ), while trimeric and tetrameric proanthocyanidins have been reported in the aerial parts of Rumex acetosa (Derksen et al., ). Our metabolomic analysis showed that several anthraquinones (19 annotated in ESI positive ionization mode and 18 annotated in ESI negative), including emodin and emodin-8-glucoside, are differently distributed across the three different plant organs of Rumex . Despite the lack of precise concentration data, we observed that these compounds are present in varying amounts in each organ, highlighting specific patterns of accumulation. Because of their know toxicity (Paneitz et al., ) and the recent EFSA opinion on the presence of emodin and aloe-emodin in edible plants (Younes et al., ) a quantitative analysis was performed. As shown in Fig. A the content of emodin was significantly (p < 0.0001) higher in leaves (8 mg/g of dry weight), compared to stems and roots (leaves > stems > roots) considering an adult growth stage and a winter harvesting period. A different accumulation trend was observed for emodin-8-glucoside (Fig. A), whose content was significantly higher (p < 0.0005) in stems (2 mg/g of dry weight) followed by leaves and roots (stems > leaves > roots). This difference across organs distribution may be elucidated considering that emodin-8-glucoside serves as a storage form of emodin. Activation of this storage form involves the action of glycosyltransferases, which facilitate the removal of a sugar unit from the anthraquinone ring. The observed variation in distribution across plant organs, with potentially higher expression in stems, suggests the involvement of glycosyltransferase activity. However, further studies are necessary to conclusively affirm this hypothesis (Izhaki, ; Paneitz et al., ). Considering that leaves constitute the edible part of the plant and that young leaves are the most suitable for eating consumption, we quantified the emodin content in leaves at distinct growth stages (young and adult leaves). When comparing the levels of emodin between young and adult leaves, a notable difference was observed: the concentration of emodin was found to be significantly higher in young leaves (Fig. ) showing an increase of approximately 30% compared to adult leaves. This result could be explained considering that synthesis and accumulation of secondary metabolites in plants is affected the growing stage and by abiotic environmental factors, such as light intensity, soil minerals, osmotic stresses (drought and salinity) and seasonality (Izhaki, ). Studies supporting this hypothesis indicate that emodin levels in plants depend on season and light intensity. The leaves of Rheum undulatum in Europe exhibited the highest concentration of total anthraquinones, with approximately 50% being emodin, during the spring season (April). Subsequently, there was a consistent decline in anthraquinone levels throughout the summer, reaching their lowest amounts in late summer (September). This observed seasonal variation could be indicative of a trade-off between the plant’s developmental processes and its defence mechanisms (Paneitz et al., ). The accumulation of emodin, emodin-8-glucoside and other derivatives such as aloin A and B, has been reported also for other Rumex species, such as R. confertus, R. crispus, R. obtusifolius and R. aquaticus (Eom et al., ; Pelzer et al., ; Poluyanov et al., ). Our findings correspond well within the wide range documented in existing literature (2 to 160 mg/g) (Wegiera et al., ), despite the considerable variability in emodin content observed across seasons, organs, and Rumex species. The variability of emodin concentration in the roots shows significant differences among various Rumex species. In certain species, such as R. acetosella, R. confertus, and R. crispus , the emodin content is higher in the roots than in the leaves. Conversely, in other species, the emodin levels are higher in the leaves. Notably, in R. obtusifolius , the proportional distribution aligns with our findings, with emodin levels being higher in the leaves compared to the roots. Given the absence of R. sanguineus from EFSA’s plant list containing approved hydroxyanthracene derivatives in food supplements, our research gains heightened significance. The EFSA opinion classifies hydroxyanthracene derivatives (including emodin, aloin and derivatives) as potentially genotoxic and carcinogenic. Notably, EFSA faces challenges in providing precise recommendations for the daily intake of hydroxyanthracene derivatives that would not raise concerns regarding potential adverse health effects. This underscores the need for continued investigation and assessment of the potential implications associated with the presence of hydroxyanthracene derivatives in R. sanguineus . As R. sanguineus demonstrates the ability to biosynthesize anthraquinone, particularly emodin and its derivatives, the intricate and at times contradictory pharmacological activities associated with emodin necessitate thorough investigation. In 2021 the European Commission considered the significant adverse health effects linked to the consumption of aloe-emodin, emodin, danthron, and aloe extracts containing hydroxyanthracene derivatives in food, and given the absence of a defined daily intake level that does not raise concerns for human health, prohibited these substances (Annex III, Part A of Regulation (EC) No 1925/20069) (L96, ). The implementation of non-targeted metabolomics in our investigation has enhanced our understanding of the chemical profile of R. sanguineus highlighting its potential for applications in contemporary nutrition and agriculture. From a food nutritional and safety perspective, Rumex leaves were found to be rich in bioactive compounds, including phenolic, coumarins and cinnamic acids. When it comes to safety aspects, cooking Rumex leaves prior to consumption is expected to mitigate concerns regarding emodin content, as thermal processing induces the degradation of emodin (Narayanan et al., ). However, rigorous analytical investigations are essential to elucidate the kinetics of emodin degradation under various cooking conditions, thereby facilitating informed decision making to determine the safety and nutritional value of cooked Rumex leaves. Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 4208 KB) |
Factors influencing the utilization of dental services in East Java, Indonesia | 37d31b64-5bfc-45d3-8708-e6a0b0ded691 | 8082568 | Dental[mh] | Health is a fundamental right of every human being without discrimination related to race, religion, and socioeconomic status ( ). Oral health is integral to the overall health of human beings ( ; ). However, in most countries, access to and utilization of oral health services are limited ( ; ; ). Lack of access to such services can have a detrimental impact on people’s general health and quality of life ( ). Tooth loss is mainly the result of accumulated dental diseases as a product of low utilization of dental services ( ). One of the commonly used indices to assess the utilization of dental services is the percentage of the population attending a dental visit in the previous year ( ). The utilization of dental services is varied across countries. In developing countries, the majority of people only visits the dentist for pain relief rather than preventive care ( ), while in developed countries about 40–80% of the adults visit a dentist in a given year ( ). Previous research has indicated certain factors that influence dental service utilization ( ; ; ; ). For example, socio-demographic factors related to dental service use include age, sex, education, and residential location ( ; ; ). Moreover, poor health behaviors are usually clustered in the same person wherein a person with a bad tooth brushing habit also rarely accesses dental treatments ( ). Furthermore, dental clinical condition such as dental status (dentate vs edentulous) could influence the utilization of dental services as it differentiates the extent of dental treatment need. So far, utilization of dental services and factors related to it have been mainly reported in developed countries, while such reporting in developing countries has been limited. Oral health is a much neglected field of research in developing countries, including in Indonesia. Indonesia is the fourth most populated country in the world after China, India, and the United States of America ( ). Cases of dental caries in this country is high; for example, more than 88% of the population has been estimated to have experienced caries, with 45% having untreated caries ( ). Currently, Indonesia is in the process of establishing universal health coverage through Jaminan Kesehatan Nasional (JKN), wherein basic dental health is included in the insurance coverage. This insurance covers seven services: 1. dental examination, medication and consultation, 2. premedication, 3. dental emergency, 4. extraction of deciduous or permanent tooth without difficulties, 5. medication after extraction, 6. glass ionomer and composite dental fillings resulting from disease (not cosmetic reasons), and 7. scaling (once a year) ( ). This universal health coverage program was implemented as a capitation system whereby the dentists are paid a fixed amount for the number of people who were under their care ( ). The participants in the insurance scheme include Contribution Assistance Recipients (“ Penerima Bantuan Iuran ,” PBI) and non-PBI members ( ). PBI include poorer citizen whose insurance is funded by the government through taxes. Non-PBI members include other citizens not categorized as poor, who need to subscribe to the insurance scheme by paying for it monthly (e.g., through deduction directly from their income) ( ). A comprehensive assessment of the JKN program conducted by the Government of Indonesia in 2017 found that JKN has managed to bring 76% of Indonesia’s population under the program; this is considered an impressive coverage rate ( ). Despite the high level of dental problems and the high insurance coverage, the utilization of dental services among the Indonesian population is very low ( ). Indonesian Basic Health Research (Riset Kesehatan Dasar/RISKESDAS) 2018 showed that only 8.1% of Indonesians used dental services ( ). In Indonesia, East Java is the second most populated province with a slightly below average national dentist-population ratio ( ). The insurance coverage in this province is 80%. However, the utilization of dental services in this province is similar to the national estimate which is 8.6% ( ). Understanding the factors influencing dental service utilization among East Java residents is needed as a fundamental step to develop policy to increase the utilization of the services. This study aimed to explore the associations of socio-demographic characteristics, behavioral factors, and clinical condition with the utilization of dental services among East Java residents.
Study population and research design This secondary data analysis used data from the 2013 Indonesian Basic Health Survey (Riskesdas 2013). Riskesdas 2013 was a cross sectional national survey. It was part of a serial Indonesian national basic health survey conducted every six years. As the latest Riskesdas data is not currently open to the public, the Riskesdas 2013 data was used in this analysis. Riskesdas 2013 used a three-stage, stratified cluster sampling design to select a representative sample of Indonesian residents. The sampling frame was households recorded in the 2010 bloc census database, revalidated by the 2013 enumerator team. Indonesia was stratified into metropolitan and non-metropolitan areas by provincial status, with clusters based on district or municipality, which were selected with probability proportional to size. All persons in the household were included in the census. Final respondents were 294,959 households with the mean number of residents equal to 3.8. Response rate for the Indonesian residents was 93%. Details of the 2013 Indonesian national basic health research report has been published elsewhere ( ). For the purpose of this analysis, a subset of East Java participants 5 to 100 years old who participated in the survey was analyzed. Data collection and management Data was collected through a questionnaire in the Indonesian language. The outcome of interest was utilization of dental services. It was self-reported by respondents by answering a single question “Have you received dental treatment(s) during the last twelve months?” The response options were yes or no. Potential indicators of the dental service utilization in this study were socio-demographic characteristics, behavioral factors, and clinical condition, as informed by the existing literature ( ; ; ). Socio-demographic characteristics included age, sex, education and residential location. For inclusion in the analysis, the respondent had to be between 5 and 100 years old and must have completed the Riskesdas 2013 oral examination. Age was then categorized into <25, 25–<50, and ≥50 years old. The choice of the cut-off points for the age categorization was based in the distribution of the age. Sex was recorded as male or female. Education was measured by the highest level of school, post-school, or tertiary educational attainment and dichotomized into junior high school or less vs senior high school or higher. East Java province consists of 29 districts and nine municipalities, which differ due to the size of the area, capital, and development such as in the economy and education. Municipalities are usually ahead of the districts in terms of socio-capital development. Thus, residential location was dichotomized into districts and municipalities. The behavioral factor was tooth brushing habit (self-reported by the respondents as good vs bad tooth brushing habit). Respondents were categorized as having a good tooth brushing habit if they answered yes to the question “do you brush your teeth daily?”. Clinical condition was measured through dental status (self-reported by respondents as dentate [having one or more teeth] vs edentulous). Statistical analysis Statistical analysis was performed using SAS version 9.4-callable SUDAAN version 11.0.3 (Research Triangle Institute, North Carolina, USA). Characteristics of the study participants were presented using descriptive statistics. Bivariate analyses of the association between utilization of dental treatment and each of the potential indicators were performed using chi square tests. The potential indicators include socio-demographic characteristics (age, sex, education and residential location), dental behavior (tooth brushing habit), and clinical (dental) condition. Multivariable logistic regression analysis was conducted to model together these factors influencing dental treatment utilization among a sample with information on all study variables (N=79,322); no imputation was done for missing data. The statistical significance of the associations was evaluated at P < 0.05. Ethical review Ethical approval of Riskesdas 2013 was granted by the Ministry of Health Republic of Indonesia’s Human Research Ethics Committee. However, this particular study involved secondary analysis of anonymized data, and no new ethical clearance was required.
This secondary data analysis used data from the 2013 Indonesian Basic Health Survey (Riskesdas 2013). Riskesdas 2013 was a cross sectional national survey. It was part of a serial Indonesian national basic health survey conducted every six years. As the latest Riskesdas data is not currently open to the public, the Riskesdas 2013 data was used in this analysis. Riskesdas 2013 used a three-stage, stratified cluster sampling design to select a representative sample of Indonesian residents. The sampling frame was households recorded in the 2010 bloc census database, revalidated by the 2013 enumerator team. Indonesia was stratified into metropolitan and non-metropolitan areas by provincial status, with clusters based on district or municipality, which were selected with probability proportional to size. All persons in the household were included in the census. Final respondents were 294,959 households with the mean number of residents equal to 3.8. Response rate for the Indonesian residents was 93%. Details of the 2013 Indonesian national basic health research report has been published elsewhere ( ). For the purpose of this analysis, a subset of East Java participants 5 to 100 years old who participated in the survey was analyzed.
Data was collected through a questionnaire in the Indonesian language. The outcome of interest was utilization of dental services. It was self-reported by respondents by answering a single question “Have you received dental treatment(s) during the last twelve months?” The response options were yes or no. Potential indicators of the dental service utilization in this study were socio-demographic characteristics, behavioral factors, and clinical condition, as informed by the existing literature ( ; ; ). Socio-demographic characteristics included age, sex, education and residential location. For inclusion in the analysis, the respondent had to be between 5 and 100 years old and must have completed the Riskesdas 2013 oral examination. Age was then categorized into <25, 25–<50, and ≥50 years old. The choice of the cut-off points for the age categorization was based in the distribution of the age. Sex was recorded as male or female. Education was measured by the highest level of school, post-school, or tertiary educational attainment and dichotomized into junior high school or less vs senior high school or higher. East Java province consists of 29 districts and nine municipalities, which differ due to the size of the area, capital, and development such as in the economy and education. Municipalities are usually ahead of the districts in terms of socio-capital development. Thus, residential location was dichotomized into districts and municipalities. The behavioral factor was tooth brushing habit (self-reported by the respondents as good vs bad tooth brushing habit). Respondents were categorized as having a good tooth brushing habit if they answered yes to the question “do you brush your teeth daily?”. Clinical condition was measured through dental status (self-reported by respondents as dentate [having one or more teeth] vs edentulous).
Statistical analysis was performed using SAS version 9.4-callable SUDAAN version 11.0.3 (Research Triangle Institute, North Carolina, USA). Characteristics of the study participants were presented using descriptive statistics. Bivariate analyses of the association between utilization of dental treatment and each of the potential indicators were performed using chi square tests. The potential indicators include socio-demographic characteristics (age, sex, education and residential location), dental behavior (tooth brushing habit), and clinical (dental) condition. Multivariable logistic regression analysis was conducted to model together these factors influencing dental treatment utilization among a sample with information on all study variables (N=79,322); no imputation was done for missing data. The statistical significance of the associations was evaluated at P < 0.05.
Ethical approval of Riskesdas 2013 was granted by the Ministry of Health Republic of Indonesia’s Human Research Ethics Committee. However, this particular study involved secondary analysis of anonymized data, and no new ethical clearance was required.
shows the characteristics of the total study participants and the final participants included in the multivariable analysis. From a total of 90,551 respondents, 79,322 respondents with information on all variables were included in the final analysis. The included respondents were similar in all characteristics to the total number of respondents except in the age and educational level. Those included in the final analysis had a higher education level when compared with the total respondent population. The results from the bivariate analyses are presented in . All the indicators showed a significant relationship with the utilization of dental services. In terms of age, people over 50 years old showed the lowest utilization of dental treatment, followed by people less than 25 years old. Respondents that received the highest number of dental treatments were people between 25 and 50 years old. Furthermore, males, people with lower educational background, residents of districts, people reporting bad tooth brushing habit, and those with edentulism showed lower utilization of dental treatments compared to their counterparts. The results of the multivariable model are presented in . The model showed that dental services utilization differed by age. Respondents less than 25 years of age received lower dental treatment than respondents ≥50 years old (PR [95% CI] = 0.74 [0.71–0.78]). However, respondents 25–<50 years old showed more utilization of dental treatments than respondents ≥50 years old (PR [95% CI] = 1.23 [1.18–1.28]). Being male and having lower education were indicators for having a lower utilization of dental treatment (PR [95% CI] = 0.81 [0.79–0.84] and PR [95% CI] = 0.89 [0.86–0.93], respectively). Similarly, people living in districts showed lower utilization of dental treatment than those living in municipalities. Respondents with bad tooth brushing habit showed lower utilization of dental treatment. Having teeth was associated higher utilization of dental treatment (PR [95% CI] = 1.39 [1.16–1.66]).
This study showed that the utilization of dental services by East Java residents in Indonesia was very low. Only 9% of the East Java population received dental treatments during the last 12 months, which is slightly higher than the national average (8.1%) ( ). Dental service utilization was low among respondents who were less than 25 years old, male, district residents, edentulous, and had lower education and poor toothbrushing habit. Among Indonesians, the fact that 95.5% of the population never utilize dental services was revealed in the 2018 national health survey ( ). The percentage of the population that never accesses dental services in Indonesia was far higher than that has been reported in rural India, another developing country ( ). Research in a rural population of western Rajasthan, revealed that around 55% of the population never visited a dentist while only 1.5% visited a dentist in the last 6 months ( ). However, these estimates contradict some research findings from economically developed countries where approximately 40%–80% of the population have accessed dental services within the last 12 months ( ; ). Lack of awareness about oral health could be a reason behind the lower utilization of dental services in East Java province, Indonesia. In Indonesia, the secondary data analysis from the National Socioeconomic Survey in 2013 also showed that a high proportion of the Indonesian people had no perceived need for dental services (98.36%) and had never utilized dental services, resulting in considerable unmet need for dental treatments (97.7%). Of those who had unmet need for dental treatments, 94.8% had no perceived need for dental services ( ). Perceived need for dental care is one of the best predictors of dental service utilization ( ). Individuals’ perceived dental health may influence their perceived need for dental care ( ). In general, people will not attend health services unless they have health problems, and they demand health care as they believe that health service can solve their health problems ( ). In East Java, utilization of dental services varied according to socio-demographic, behavioural, and clinical factors. People aged 25–<50 years had the highest utilization of dental services in this study, followed by those ≥50 year old. Those less than 25 years old had the lowest utilization of dental treatment. Among Indonesians, people in 25–<50 years of age are considered the productive workforce, at which point they have usually progressed in their careers to a point and are earning enough to allow them to have insurance or utilize dental services privately. This could be the reason for greater utilization of dental treatment in this age group. The lowest utilization of dental services among people less than 25 years old could also be influenced by dental anxiety. Previous study in the Indonesian population reveals that people less than 25 years old have higher dental anxiety than their counterparts ( ). People aged younger than 25 years old still had limited self-control and were less focussed on long-term consequences relative to people aged 25 years and older. These characteristics lead to an increased risk of unhealthy behaviours among people in this age group ( ), including the lack of dental service utilization to maintain their oral health. However, high levels of self-concept among people aged 25 and older may help them maintain their oral health and motivate them to improve dental service utilization. Similarly, for baby boomers (people aged 50 years and older), physical attractiveness is important, as evidenced by the growing number of people in this age group seeking cosmetic dental care ( ). This study finds that females have greater utilization of dental services, supporting previous studies ( ; ; ; ; ), while also contradicting another study ( ). Women’s greater use of oral health service providers is likely because they pay more attention to esthetics and oral hygiene. Research shows that women pay more attention to their appearance and health ( ). On the other hand, men tend not to seek dental service due to the lack of perception of their need ( ). However, each gender’s perspectives and beliefs may differ culturally, explaining the contradictory results. Higher dental service use was also observed among working women than working men in a study in Japan ( ). One possible explanation is that working women were more likely than working men to allocate time to use dental service. Compared to men, women had a greater interest in health and literacy and put more attention to oral health ( ). Education is also a significant contributing factor to dental service utilization. People with lower educational background have lower access to dental treatment, supporting previous research. Considering that the analysis sample in this study were better educated than the total respondent population only underestimates this problem. A study showed that higher educated individuals visited the dentist 10 times more than those with low education ( ). A previous study has demonstrated a higher level of oral health knowledge among people with a high educational background than those with low educational background. Having adequate oral health knowledge is attributed to positive attitudes towards dental service utilization, resulting in more educated people using dental services than less educated people ( ). Our results indicate that prevention programs would benefit by specifically targeting less educated people. East Java province is one of the provinces located in the main island, Java. Among other islands, the Java island is categorized as the most developed. Thus, socioeconomic inequality among each area in East Java could be considered lower than other less developed islands. However, this research still finds that dental service utilization is lower among the district residents than the municipality residents in East Java. This finding supports previous research ( ). It is known that public transport networks are less developed in rural areas than in urban areas. Lack of transport access to dental facilities can be an obstacle to routine dental visit ( ; ). Moreover, the use of a service also depends on the perception of user’s needs, influenced by their values, beliefs and cultures ( ). A study in Istanbul ( ) found that more than half of the population surveyed did not feel the need or have the desire to visit a dentist, although their dental conditions were not ideal. This study finds that people who have bad tooth brushing habit utilize dental services less, supporting previous research ( ). Jessor’s problem behaviour theory ( ) proposes that various risk behaviours are inter-related. Research ( ) has affirmed this theory in the dental behaviors field, finding that there are clustering patterns in dental health behaviors whereby less frequent tooth brushing is clustered with high sugar intake, current smoking, and lack of dental visits. Moreover, dentate people in East Java utilize more dental services than edentulous people, similar with other research finding ( ). In many countries, the reason for dental service use is to mainly undergo dental treatment. This is especially true in developing countries where most of the people visit dental care services only when they are in pain ( ). Edentulousness in some parts of the world has been thought of as a healthy condition without pain even though this condition could reduce the ability to chew certain types of food, reducing the quality of life. Elders were also found to be more resilient to poor clinical status compared to younger people ( ). The importance of dentate status in predicting dental service utilization is evident. It has also been argued that age will not be a significant predictor of dental service utilization if individuals were grouped by dentate status ( ). The strength of this study lies in the nature of data collected in a national survey, allowing a representative data of the East Java population. The cross-sectional study design is a limitation as it precludes causal explanations. Self-reported utilization of dental services and lack of data on insurance coverage and income could be other limitations of this study as associations between lower socioeconomic status and decreased access to dental services have been found in several countries ( ; ; ; ; ; ). Some potential bias could arise due to self-reported data and residual confounder. However, the study findings are in agreement with prior research that assessed dental care utilization, and it provides avenues for future research. This study has the potential to inform guidelines, or specific changes to existing policy or practice in Indonesia. Recognizing significant indicators that influence dental care utilization may aid in the planning and provision of programs for dental services and resource allocation. Strategies for the improvements in dental care utilization may require a multidimensional approach. The results may also inform oral health practitioners and policymakers about specific target groups requiring oral health intervention programs.
This first detailed population-based study in the East Java province of Indonesia has demonstrated that the use of dental services is influenced by socio-demographic factors. People less than 25 years old, male, those with lower educational background, those living in a district, those with poor tooth brushing habit and being edentulous are associated with lower dental service utilization.
Data used for this analysis are available by a written request to the Ministry of Health Republic of Indonesia. Source data The available variables of Riskesdas 2013 dataset could be learnt from the national report of Riskesdas 2013 produced by Indonesian Ministry of Health, available online in http://labdata.litbang.kemkes.go.id/images/download/laporan/RKD/2013/Laporan_riskesdas_2013_final.pdf . A written request of a sub data set should be sent to the Ministry of Health Republic of Indonesia (sent to the head of research and development division of the Ministry of Health Republic of Indonesia at Jl. Percetakan Negara no 29 Jakarta Pusat, Indonesia) along with a proposal detailing the proposed analysis. After approval, the proposal will be analyzed by the data management laboratory. Successful applicants will get the data by email after signing a letter of agreement about the data management, including an agreement to neither send the data to other party nor using it for other reason than that has been agreed by the Ministry. The instruction of how to apply for the data are available from the Indonesian Ministry of Health’s website: http://labmandat.litbang.kemkes.go.id/images/download/peraturan/alur.pdf
The available variables of Riskesdas 2013 dataset could be learnt from the national report of Riskesdas 2013 produced by Indonesian Ministry of Health, available online in http://labdata.litbang.kemkes.go.id/images/download/laporan/RKD/2013/Laporan_riskesdas_2013_final.pdf . A written request of a sub data set should be sent to the Ministry of Health Republic of Indonesia (sent to the head of research and development division of the Ministry of Health Republic of Indonesia at Jl. Percetakan Negara no 29 Jakarta Pusat, Indonesia) along with a proposal detailing the proposed analysis. After approval, the proposal will be analyzed by the data management laboratory. Successful applicants will get the data by email after signing a letter of agreement about the data management, including an agreement to neither send the data to other party nor using it for other reason than that has been agreed by the Ministry. The instruction of how to apply for the data are available from the Indonesian Ministry of Health’s website: http://labmandat.litbang.kemkes.go.id/images/download/peraturan/alur.pdf
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Every bite counts to achieve oral health: a scoping review on diet and oral health preventive practices | dec6e883-bb62-463c-a676-374acf989308 | 11613938 | Dentistry[mh] | Oral health is a frequently overlooked yet essential aspect of overall well-being. Described as a “silent epidemic,” poor oral health impacts nearly 4 billion people worldwide, resulting in substantial economic losses . In the United States (U.S.), oral diseases, including both inflammatory and degenerative conditions, lead to an annual financial impact of approximately $136 billion, emphasizing the urgency of integrating oral health into broader health policy frameworks and enhancing accessible, cost-effective preventive measures, particularly among children, older adults, and underserved populations . Oral diseases not only compromise masticatory function, which is essential for proper nutrition and quality of life (QoL), but are associated with increased risk for systemic conditions and noncommunicable chronic diseases (NCDs), such as diabetes and cardiovascular disease . Such systemic manifestations, often heightened by dietary deficiencies, further complicate the burden of oral diseases and potentially lead to severe and life-threatening health conditions . A key objective for both public and private health entities in the U.S. is the reduction of health disparities, as underscored the 2000 Surgeon General's report, which highlighted oral health as essential to overall well-being and pointed out disparities in dental care access. Later, the 2011 Institute of Medicine (IOM) report acknowledged that efforts to date have been insufficient in eliminating these disparities. Recently, the 2021 NIDCR's “Oral Health in America: Advances and Challenges” report noted the minimal progress made, particularly for the most disadvantaged groups, reaffirming the urgent need for focused action . Whilst the World Health Organization (WHO) champions the concept of health as a holistic state encompassing physical, mental, and social well-being, wherein oral health is recognized as a crucial component . Building on this foundation, the WHO’s “Global Oral Health Action Plan 2023 – 2030” underscores the integration of oral health into overall health strategies . Additionally, in 2022, the WHO, along with the Food and Agriculture Organization of the United Nations, the World Organization for Animal Health, and the United Nations Environment Programme, forged a collaborative agreement to propel the One Health initiative to the forefront of global health priorities, emphasizing the interconnectedness of human, animal, and environmental health . Concurrently, the United Nations Agenda 2030 introduced the 17 Sustainable Development Goals (SDG) aimed at securing peace, prosperity, and well-being for all by 2030. Notably, SDG 3, which seeks to ensure good health and promote well-being for all ages, implicitly encompasses oral health, despite not targeting it explicitly . Oral health extends beyond the mere condition of one’s teeth, influencing and being influenced by broader social, environmental, and economic factors. These strategic partnerships and action plans highlight the upstream and downstream social determinants of health that impact oral health outcomes, addressing factors from global policy initiatives to individual behaviors, also mirrored in the US where significant socioeconomic impacts and health disparities persist . Good oral health ensures the dignity of performing daily activities without pain or discomfort, which is paramount for maintaining QoL . Tooth loss and other oral health problems can directly compromise QoL, dietary intake and nutritional quality, leading to broader health consequences. Most oral diseases are preventable, and factors involved in caries are highly modifiable with the right support . Sustained neglect of oral health not only diminishes QoL, but also poses risks to micronutrient deficiencies and overall systemic health, highlighting the critical need for integrated approaches such as the One Health initiative . The prevailing dental restorative approach proves unfeasible in many regions, especially in low-income countries where over 90% of caries remains untreated, highlighting the urgent need for accessible, affordable, and sustainable oral health solutions . Established links between chronic oral conditions and systemic diseases, including heart disease, diabetes, and neurodegenerative diseases such as Alzheimer’s and related dementias (AD/ADRDs), are likely due to inflammation caused by oral bacteria and occlusal dysfunction . Rethinking oral and planetary health can redefine global oral health strategies by fostering sustainable oral health outcomes, ensuring today's needs for the most vulnerable and underserved are met without compromising future capabilities . Socioeconomic factors and access to care are significant determinants of oral health, with vulnerable groups facing the greatest challenges . The interplay of oral health with nutrition is pivotal; diets high in sugars and fermentable carbohydrates exacerbate the risk for dental caries and disease. However, dental caries a preventable condition characterized by long duration and slow progression, directly influences food choices and an individual’s ability to eat “chew” a balanced and healthy diet. Individuals often perceive foods rather than individual nutrients, highlighting the need for dietary considerations and modifiable risk factors in oral health promotion . Approximately one in six children aged 6–11 have experienced caries, and a similar proportion of adolescents have untreated cavities, while a significant number of older adults have lost all their teeth due to caries . The impact of caries, tooth loss, and oral function on the ability to follow the Dietary Guidelines for Americans (DGAs) is profound, with poor oral health limiting the intake of more healthful food choices, thereby influencing long-term health outcomes . This scoping review (SR) aims to bridge the gap between oral health and dietary guidelines by examining the landscape of preventive strategies and interventions to achieve oral health equity, with particular attention given to dental caries prevention, diet, and socioeconomic determinants across various demographic and geographic regions.
We conducted an SR according to the framework described by Peters et al. to identify relevant studies that center efforts on behavioral and educational programs/interventions to support effective preventive oral health practices in reducing caries and cariogenic bacteria . This review has been reported according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses – Scoping Review extension (PRISMA-ScR).
Inclusion criteria Population: Studies including pregnant women, young children, and older adults, with subgroup analysis where applicable; Study Design: Cross-sectional, case‒control, cohort, and interventional studies, including randomized controlled trials and systematic reviews; Impact on Health: Studies reporting a clear, positive impact on individual and/or population-level oral health outcomes, behaviors, or health promotion with a focus on health equity. Relevance to DGAs: Evidence that can inform DGAs with actionable recommendations to address preventable oral diseases. Equity Focus: Research focused on health disparities and social determinants of health. Language and Publication Date: English-language articles published between 2011 and 2022. Exclusion criteria Language Barriers: Non-English articles. Publication Type: Case reports/series, opinions, commentaries, editorials, abstracts, and other non-peer-reviewed materials. Irrelevant Results: Studies reporting nonsignificant findings that are not relevant to the research question or those with inadequate or small sample sizes. Methodological issues: Articles with reported methodological flaws. Generalizability: Studies with results that cannot be generalized to broader populations. Accessibility and Sustainability: Studies involving interventions or technologies (excluding mobile phones) are not feasible for implementation among marginalized or low socioeconomic communities (e.g., expensive dental equipment). Search strategy and data abstraction An electronic database search of the literature was conducted on October 7, 2022, by a librarian (CW). The search was developed in PubMed (National Library of Medicine) using controlled vocabulary and keywords for four concepts (Table ). The searches were translated into four other databases using database-specific controlled vocabulary and keyword adjustments—Embase (Elsevier), Dentistry and Oral Sciences Source (EBSCO), ERIC (EBSCO), and the Cochrane Central Register of Trials (Wiley). (Appendix. 1) Results were deduplicated in EndNote before being screened for eligibility in Covidence. The SR was conducted by a team comprising six independent reviewers: Mona Alhassan (MA), Alhassan Hameedaldeen (AH), Adam Yang (AY), Shivangi Kaplish (SK), Steffany Chamut (SC), and Tejasvita Chandel (TJ). The preliminary screening of titles and abstracts to determine the articles’ eligibility for comprehensive review was performed by MA and AH. Once an agreement was reached on which abstracts and studies were suitable for full-text review, MA, AH, AY, and SK proceeded to evaluate the full texts according to the predefined eligibility criteria. In instances of discord, SC reviewed the abstracts and full text in each stage to reach a consensus . An adapted Population, Intervention, Comparator/s, and Outcomes (PICO) framework was used to carry out the data extraction strategy and charting for this SR. MA, AH, AY, and SK generated the data from the chosen articles. TJ conducted a random verification of 10% of the collated data to confirm its precision. SC and TJ, who organized the information by author, publication year, country, type of publication, outcomes of the program, and key results, evaluated the full-text data according to the eligibility criteria and primary concepts (Tables , and ).
Population: Studies including pregnant women, young children, and older adults, with subgroup analysis where applicable; Study Design: Cross-sectional, case‒control, cohort, and interventional studies, including randomized controlled trials and systematic reviews; Impact on Health: Studies reporting a clear, positive impact on individual and/or population-level oral health outcomes, behaviors, or health promotion with a focus on health equity. Relevance to DGAs: Evidence that can inform DGAs with actionable recommendations to address preventable oral diseases. Equity Focus: Research focused on health disparities and social determinants of health. Language and Publication Date: English-language articles published between 2011 and 2022.
Language Barriers: Non-English articles. Publication Type: Case reports/series, opinions, commentaries, editorials, abstracts, and other non-peer-reviewed materials. Irrelevant Results: Studies reporting nonsignificant findings that are not relevant to the research question or those with inadequate or small sample sizes. Methodological issues: Articles with reported methodological flaws. Generalizability: Studies with results that cannot be generalized to broader populations. Accessibility and Sustainability: Studies involving interventions or technologies (excluding mobile phones) are not feasible for implementation among marginalized or low socioeconomic communities (e.g., expensive dental equipment).
An electronic database search of the literature was conducted on October 7, 2022, by a librarian (CW). The search was developed in PubMed (National Library of Medicine) using controlled vocabulary and keywords for four concepts (Table ). The searches were translated into four other databases using database-specific controlled vocabulary and keyword adjustments—Embase (Elsevier), Dentistry and Oral Sciences Source (EBSCO), ERIC (EBSCO), and the Cochrane Central Register of Trials (Wiley). (Appendix. 1) Results were deduplicated in EndNote before being screened for eligibility in Covidence. The SR was conducted by a team comprising six independent reviewers: Mona Alhassan (MA), Alhassan Hameedaldeen (AH), Adam Yang (AY), Shivangi Kaplish (SK), Steffany Chamut (SC), and Tejasvita Chandel (TJ). The preliminary screening of titles and abstracts to determine the articles’ eligibility for comprehensive review was performed by MA and AH. Once an agreement was reached on which abstracts and studies were suitable for full-text review, MA, AH, AY, and SK proceeded to evaluate the full texts according to the predefined eligibility criteria. In instances of discord, SC reviewed the abstracts and full text in each stage to reach a consensus . An adapted Population, Intervention, Comparator/s, and Outcomes (PICO) framework was used to carry out the data extraction strategy and charting for this SR. MA, AH, AY, and SK generated the data from the chosen articles. TJ conducted a random verification of 10% of the collated data to confirm its precision. SC and TJ, who organized the information by author, publication year, country, type of publication, outcomes of the program, and key results, evaluated the full-text data according to the eligibility criteria and primary concepts (Tables , and ).
A. Overview of included studies The PRISMA flowchart illustrates the process of this SR, which initially identified a total of 9,114 articles through electronic databases, and after removing 1,129 duplicates, 1,940 articles remained for screening for full-text review (Fig. , Tables , and ). A total of 1,833 articles did not meet the inclusion criteria and were excluded. A total of 107 articles published between 2011 and 2022 were selected and included in the SR. The studies originated from Asia ( N = 54), Europe ( N = 24), North America ( N = 10), South America ( N = 7), Africa ( N = 5), Oceania ( N = 4), and others with multiple locations ( N = 3). Most of the articles ( N = 36) were cross-sectional studies, with a sample size range of 116–99,07. The remaining studies were case‒control studies ( N = 10); cohort studies ( N = 3); clinical trials ( N = 5); cluster randomized studies ( N = 1); comparative studies ( N = 1); cost-effectiveness analyses ( N = 1); descriptive epidemiological studies ( N = 1); interventional studies ( N = 4); literature reviews ( N = 1); mixed methods investigations ( N = 1); narratives ( N = 1); pilot programs ( N = 1); prospective cohort studies ( N = 1); prospective observational studies ( N = 1); quasirandomized field studies ( N = 1); quasi-experimental studies ( N = 2); reviews ( N = 7); randomized controlled trials ( N = 17); randomized controlled trials ( N = 1); randomized cluster trials ( N = 1); and systematic reviews ( N = 10). B. Types/themes of effective interventions Tables , and describes the included articles that are categorized under the themes of effective interventions with the primary objective of addressing various facets of oral health, with a particular emphasis on reducing the prevalence of caries, enhancing oral hygiene practices, and enhancing overall oral health knowledge among the target populations. 1) Behavioral practices ( N = 33; Table ); 2) educational intervention ( N = 39; Table ); 3) dietary best practices ( N = 28; Table ), including sugar-free gums (SFGs) ( N = 7; Table ). Behavioral interventions were investigated in 33 studies (Table ) . Overall, these studies revealed various strategies implemented across diverse demographics and geographies. These interventions were primarily focused on reducing the prevalence of caries, improving oral hygiene practices, and enhancing overall dental health knowledge . Studies in Tonga and Thailand revealed that school-based fluoride mouth-rinsing programs significantly improved children’s oral health, with long-term participants showing fewer caries lesions . A family-centered oral health intervention targeting new parents significantly enhanced feeding and toothbrushing practices, reducing toddlers’ risk of caries . Additionally, motivational interviewing within healthcare environments has proven to be a successful strategy for preventing caries in children from lower-income families . Oral health education in urban schools plays a pivotal role in recognizing key factors such as brushing duration and the oral hygiene index as critical predictors of caries . Gao and Jain highlighted the importance of identifying key risk factors for early childhood caries (ECC) to prevent the progression of this condition . Educational interventions. Education interventions were a prominent focus in our SR, with 39 studies exploring their effects by highlighting a variety of effective strategies that have improved oral health outcomes . These strategies target different groups, from caregivers and parents to schoolchildren and teenagers. We observed that interventions involving oral hygiene training and engaging in healthy oral hygiene practices resulted in a notably lower incidence of caries . Similarly, a positive correlation was found between the mother’s level of education and the frequency of her children’s dental visits and tooth brushing . The outcomes include improved oral hygiene practices, reduced caries progression, and better oral health-related QoL. Moreover, articles mentioned that the effectiveness of oral health education programs is influenced by factors such as the source of education, frequency of dental flossing, parental encouragement, and maternal education level. Targeted interventions, especially those involving caretakers and parents, have shown long-term benefits in reducing caries and improving oral health . Dietary interventions described in 35 reported that unhealthy dietary behaviors increase the prevalence of caries, revealing significant insights into the relationship between diet and oral health . Out of the 35 studies, 7 reported that oral health-related knowledge and awareness of sugar-free gum (SFG) are low, but its consumption is associated with a lower incidence of caries . It was also found that feeding methods contribute substantially to caries risk during early deciduous dentition stages, while sugary habits become more influential in later stages . Additionally, frequent consumption of sweet snacks was associated with a greater incidence of caries, even among individuals with otherwise healthy oral habits . Notably, fluoridated water was shown to provide superior protection against caries compared to fluoridated household salt in schoolchildren from developing countries . Jaghasi reported that comprehensive educational programs emphasizing dietary patterns and oral health relationships are crucial for children and their guardians , while Davis reported that a balanced diet, including the consumption of vegetables, can mitigate the adverse effects of sugary food intake . High caries prevalence and related risk factors, such as low maternal education and inappropriate oral health behavior, were observed among kindergarten children . Maternal xylitol consumption demonstrated promising preventive effects on salivary mutans streptococci and caries levels in children, while xylitol gum and erythritol showed potential for reducing caries-related factors . In low- and middle-income countries, socioeconomic status, education, high sugar consumption, and low maternal education were identified as risk factors for caries, emphasizing the importance of oral health education and limiting sugar intake . Unhealthy dietary behaviors, such as frequent intake of carbonated drinks and confectionery, were linked to an increased incidence of caries .
The PRISMA flowchart illustrates the process of this SR, which initially identified a total of 9,114 articles through electronic databases, and after removing 1,129 duplicates, 1,940 articles remained for screening for full-text review (Fig. , Tables , and ). A total of 1,833 articles did not meet the inclusion criteria and were excluded. A total of 107 articles published between 2011 and 2022 were selected and included in the SR. The studies originated from Asia ( N = 54), Europe ( N = 24), North America ( N = 10), South America ( N = 7), Africa ( N = 5), Oceania ( N = 4), and others with multiple locations ( N = 3). Most of the articles ( N = 36) were cross-sectional studies, with a sample size range of 116–99,07. The remaining studies were case‒control studies ( N = 10); cohort studies ( N = 3); clinical trials ( N = 5); cluster randomized studies ( N = 1); comparative studies ( N = 1); cost-effectiveness analyses ( N = 1); descriptive epidemiological studies ( N = 1); interventional studies ( N = 4); literature reviews ( N = 1); mixed methods investigations ( N = 1); narratives ( N = 1); pilot programs ( N = 1); prospective cohort studies ( N = 1); prospective observational studies ( N = 1); quasirandomized field studies ( N = 1); quasi-experimental studies ( N = 2); reviews ( N = 7); randomized controlled trials ( N = 17); randomized controlled trials ( N = 1); randomized cluster trials ( N = 1); and systematic reviews ( N = 10).
Tables , and describes the included articles that are categorized under the themes of effective interventions with the primary objective of addressing various facets of oral health, with a particular emphasis on reducing the prevalence of caries, enhancing oral hygiene practices, and enhancing overall oral health knowledge among the target populations. 1) Behavioral practices ( N = 33; Table ); 2) educational intervention ( N = 39; Table ); 3) dietary best practices ( N = 28; Table ), including sugar-free gums (SFGs) ( N = 7; Table ). Behavioral interventions were investigated in 33 studies (Table ) . Overall, these studies revealed various strategies implemented across diverse demographics and geographies. These interventions were primarily focused on reducing the prevalence of caries, improving oral hygiene practices, and enhancing overall dental health knowledge . Studies in Tonga and Thailand revealed that school-based fluoride mouth-rinsing programs significantly improved children’s oral health, with long-term participants showing fewer caries lesions . A family-centered oral health intervention targeting new parents significantly enhanced feeding and toothbrushing practices, reducing toddlers’ risk of caries . Additionally, motivational interviewing within healthcare environments has proven to be a successful strategy for preventing caries in children from lower-income families . Oral health education in urban schools plays a pivotal role in recognizing key factors such as brushing duration and the oral hygiene index as critical predictors of caries . Gao and Jain highlighted the importance of identifying key risk factors for early childhood caries (ECC) to prevent the progression of this condition . Educational interventions. Education interventions were a prominent focus in our SR, with 39 studies exploring their effects by highlighting a variety of effective strategies that have improved oral health outcomes . These strategies target different groups, from caregivers and parents to schoolchildren and teenagers. We observed that interventions involving oral hygiene training and engaging in healthy oral hygiene practices resulted in a notably lower incidence of caries . Similarly, a positive correlation was found between the mother’s level of education and the frequency of her children’s dental visits and tooth brushing . The outcomes include improved oral hygiene practices, reduced caries progression, and better oral health-related QoL. Moreover, articles mentioned that the effectiveness of oral health education programs is influenced by factors such as the source of education, frequency of dental flossing, parental encouragement, and maternal education level. Targeted interventions, especially those involving caretakers and parents, have shown long-term benefits in reducing caries and improving oral health . Dietary interventions described in 35 reported that unhealthy dietary behaviors increase the prevalence of caries, revealing significant insights into the relationship between diet and oral health . Out of the 35 studies, 7 reported that oral health-related knowledge and awareness of sugar-free gum (SFG) are low, but its consumption is associated with a lower incidence of caries . It was also found that feeding methods contribute substantially to caries risk during early deciduous dentition stages, while sugary habits become more influential in later stages . Additionally, frequent consumption of sweet snacks was associated with a greater incidence of caries, even among individuals with otherwise healthy oral habits . Notably, fluoridated water was shown to provide superior protection against caries compared to fluoridated household salt in schoolchildren from developing countries . Jaghasi reported that comprehensive educational programs emphasizing dietary patterns and oral health relationships are crucial for children and their guardians , while Davis reported that a balanced diet, including the consumption of vegetables, can mitigate the adverse effects of sugary food intake . High caries prevalence and related risk factors, such as low maternal education and inappropriate oral health behavior, were observed among kindergarten children . Maternal xylitol consumption demonstrated promising preventive effects on salivary mutans streptococci and caries levels in children, while xylitol gum and erythritol showed potential for reducing caries-related factors . In low- and middle-income countries, socioeconomic status, education, high sugar consumption, and low maternal education were identified as risk factors for caries, emphasizing the importance of oral health education and limiting sugar intake . Unhealthy dietary behaviors, such as frequent intake of carbonated drinks and confectionery, were linked to an increased incidence of caries .
Metabolic and systemic diseases, such as diabetes and cardiovascular disorders, are major contributors to mortality and disability in the U.S. and are significantly influenced by dietary habits . The critical role of the mouth in food intake, digestion, and neurostimulation underscores the importance of oral health. Compromised oral health, as evidenced by caries and tooth loss, can disrupt vital functions of daily living, leading to a preference for softer, carbohydrate-rich, and nutritionally inadequate foods. These dietary shifts can heighten the risk of broader health complications and potentially exacerbate metabolic and systemic disorders . This SR emerges from the urgent need to address the silent epidemic of oral diseases and provides an expansive view of how preventive oral health practices can be integrated into DGAs, with a particular focus on health equity and noninvasive evidence-based interventions. This SR explores the complex effects of behavioral, educational, and dietary interventions and synthesizes evidence from numerous studies informing policies that can pivot the nation toward better oral and systemic health. Most people cannot afford to see a dentist due to cost, and current dental financing mechanisms do not meet the needs of the U.S. population equitably . However, there is an opportunity for collaboration among other healthcare professionals to promote oral health. Registered dietitians can play a pivotal role in promoting oral health by utilizing DGAs as guidance. Their expertise in nutrition can complement dental care efforts, fostering interprofessional teamwork and expanding access to preventive care, thus promoting oral health equity. Our findings highlight the critical link between diet and oral health, reinforcing the American Dental Association’s recommendations on regular dental care, increasing fluoride use, and, when snacking, giving preference to selecting nutritious snacks and reduced sugar, as advised by the DGAs . This SR underscores the effectiveness of a comprehensive approach to improving oral health and preventing diseases such as caries, which may also reduce the risk of NCDs and enhance broader health promotion initiatives. Substantiated by data from the Centers for Disease Control and Prevention (CDC), the National Health and Nutrition Examination Survey (NHANES), and Healthy People 2030, our findings emphasize the direct effects of dietary choices, particularly high and frequent sugar intake, on caries and its broader implications for social determinants of health (SDOH) . In line with national health initiatives such as the National Plan to Address Alzheimer's Disease (NAPA) by the US Health and Human Services (HHS), our findings highlight the critical role of comprehensive oral health approaches in addressing not only caries but also the prevention and management of NCDs . Such alignment has the potential to not only improve oral health but also contribute to “accelerate action to promote healthy aging and reduce risk factors for AD/ADRD” , a goal that the NAPA and similar initiatives aim to achieve through their multifaceted public health strategies . Behavioral interventions for preventing caries and improving oral health Behavioral interventions and modification strategies have emerged as essential strategies for mitigating the public health challenge of reducing the prevalence of caries and enhancing overall oral health (Fig. ) . Impact of regular oral hygiene on oral health The foundation of behavioral interventions lies in regular dental hygiene practices , such as toothbrushing and flossing, and regular access to fluoride sources . Numerous studies have consistently shown their positive impact on reducing the prevalence of caries and improving oral health. Research has consistently highlighted the positive impact of these practices on oral health. Notably, interdental cleaning devices are associated with decreased oral disease incidence and fewer missing teeth. Effective brushing habits correlate with reduced cariogenic bacteria and lower caries incidence, emphasizing the need to modify daily oral hygiene behaviors to achieve substantial benefits in oral health . Programs focusing on early interventions , such as supervised toothbrushing initiatives, are critical and have proven to be valuable preventive techniques, especially for young children. For example, the “National Supervised Toothbrushing Program” in Scotland illustrates the effectiveness and substantial benefits of such early intervention, emphasizing the value of advocacy in instilling good oral hygiene habits from a young age . Integrating oral health education across settings A parent/child approach that integrates nutrition and oral health education underscores the need for comprehensive, family-oriented initiatives to ensure the continuity and effectiveness of oral health practices . School-based interventions have also shown a positive impact, especially among schoolchildren, in fostering proper oral hygiene practices, indicating their potential to significantly enhance oral health outcomes when implemented within the educational system . Research consistently indicates that motivational interviewing is particularly impactful for adolescents and members of lower-income families, showcasing its potential as a powerful catalyst for change (Fig. ). Reinforcing oral health through positive parenting and fluoridation In line with the above interventions, positive parenting practices , such as engagement, encouragement, and problem solving, are fundamental for reducing the prevalence of childhood caries . For instance, learning the distinct patterns of risk factors associated with ECC, as identified in our study, enables parents and caregivers to tailor their approaches to better suit their children’s specific needs, thus enhancing the effectiveness of preventive measures and ultimately leading to a significant decrease in the incidence of ECC . Such practices not only benefit the immediate oral health of children but also contribute to establishing lifelong healthful habits. Our review is consistent with the thrust of behavioral modification for sustained oral health and supports the daily use of high-fluoride products in conjunction with standard toothpaste, particularly for high-risk older children and adults, which is not advised in young children due to fluorosis concerns. This recommendation aligns with the broader theme of comprehensive behavior modification and is supported by studies such as Sonbul et al. (2011), which validate the effectiveness of high-fluoride products in enhancing and sustaining oral health . Furthermore, while advocating for the integration of fluoride-based products as a cornerstone of preventive oral health measures, it is essential to acknowledge existing controversies surrounding fluoridation. Addressing concerns and promoting informed discussions regarding the safety and efficacy of fluoride interventions are paramount. Additionally, ongoing research into alternative clinical solutions that complement or supplement traditional fluoride approaches can offer valuable insights into enhancing oral health outcomes for diverse populations. Educational interventions for enhanced oral health (Fig. ) In addition to established behavioral interventions, educational programs play a pivotal role in oral health promotion. The evidence suggests that interactive and contextually tailored education leads to significant improvements in oral hygiene behaviors, increased utilization of preventive services, and a reduction in the incidence of caries (Fig. ). Enhancement of oral hygiene behaviors Educational interventions have demonstrated their ability to significantly improve oral hygiene behaviors. Studies have consistently shown that individuals who receive oral health education demonstrate better oral hygiene practices than those who do not receive such education. The effectiveness of hands-on demonstrations and integration into school curricula is particularly noteworthy, resulting in improved oral hygiene and a decrease in caries among students. This finding reinforces the value of incorporating oral health education into early learning environments . Innovation in teaching methods The use of gamified mobile applications and other educational tools for oral health and nutrition has made learning more engaging and effective. Practical demonstrations, animated screenings, and experiential learning have helped individuals better understand the practical aspects of maintaining good oral health through proper nutrition and hygiene practices . School-based and community-level programs Educational programs that incorporate hands-on demonstrations and are integrated into school curricula are instrumental in fostering better oral hygiene and reducing the incidence of caries. The strategic integration of oral health education within the general health curriculum has emerged as a pivotal approach to enhancing oral health literacy and outcomes (Fig. ). Additionally, school-based and community-level programs are vital forums for the widespread dissemination of oral health and nutrition education. Often, these initiatives are geared toward promoting healthy eating habits and understanding the nutritional value of foods. By embedding effective oral hygiene education and practices within a comprehensive health framework, these programs highlight the interdependence of good oral health and overall health and nutrition. Acknowledging that optimal nutrition can be compromised by poor oral health further accentuates the significance of maintaining oral hygiene as a cornerstone of nutritional and overall well-being. Involving schoolteachers as health educators who regularly promote healthy dietary and oral health habits and practices within standard school curricula is key to providing extensive health education, offering substantial benefits to both students and the wider community . However, it is essential to recognize the potential challenges faced by schoolteachers when assuming additional responsibilities in health education. Limited time within the curriculum, competing educational priorities, and varying levels of teacher training in oral health and nutrition may pose obstacles to effective implementation. Strategies such as providing specialized training for teachers, integrating oral health and nutrition education into existing subject areas, and collaborating with external healthcare professionals can help mitigate these barriers, ensuring that comprehensive health education remains a priority within school settings. To further bolster these efforts, policy support and incentives are essential. Implementing policies that support the integration of oral health and nutrition education into school curricula, coupled with incentives such as professional development opportunities and recognition for teachers who excel in health education, can effectively motivate and support educators in delivering high-quality health education to students. Community-level considerations Effective oral health promotion extends beyond individual practices, connecting broader oral health and nutrition challenges to the overall wellbeing of communities. Health program planners can foster a culture of health by leveraging local resources to advocate for healthy lifestyle choices and spread positive messages about nutrition and oral health, thereby mitigating negative influences and promoting healthy habits within the community (Fig. ). Recognizing that people spend a significant amount of time in settings such as preschools, long-term care facilities, churches, and programs such as the Head Start and Special Supplemental Nutrition Program for Women, Infants, & Children (WIC), integrating oral health and nutrition education into these community-based settings can have a profound and lasting impact on improving oral and overall health outcomes and well-being for children and adults. Family-centered education Targeting the family unit, particularly caregivers and parents, is essential for the prevention of ECC . Education targeting families acknowledges the critical role that parents play in shaping their children's oral health routines, emphasizing how educational attainment correlates with health outcomes. Research has highlighted the nexus between a mother’s education level, the frequency of her child’s dental visits, and her tooth brushing habits, underscoring the influence of socioeconomic status on oral health practices (Fig. ). The role of diet in caries prevention and oral health enhancement (Fig. ) In addition to behavioral and educational measures, dietary strategies are crucial for preventing caries and enhancing overall oral health. By improving nutritional intake and reducing sugar consumption, these interventions not only lower the incidence of caries but also promote broader oral wellness . Beyond dietary interventions Effective oral health promotion involves more than just reducing sugar and fermentable carbohydrate intake. The incorporation of nutritious alternatives such as high-quality proteins and vitamins (i.e. A, B, and C) and other micronutrients, which significantly improve oral health outcomes, is needed to reduce chronic inflammation and the burden of oral diseases . Public health policies must emphasize comprehensive dietary improvements, not just reducing sugar consumption. Recent studies, demonstrate the significant impact of taste preferences, age, oral microbiota, and use of dental prostheses on dietary choices, which in turn influence oral health behaviors and outcomes, underscoring the need for integrated strategies that consider both taste perception and nutritional education in oral health promotion and identify populations at risk . By increasing public awareness of the harmful effects of frequent sugar intake and promoting a balanced diet, these policies can broaden the scope of oral disease prevention strategies and enhance overall oral health . Sugar-free gum (SFG) The role of SFG, particularly that containing xylitol, in caries prevention is well documented. The regular use of SFG contributes significantly to reducing caries risk by stimulating saliva flow, which neutralizes acids produced by bacteria in biofilms (plaques), helps to remineralize tooth enamel, and aids in managing and improving oral health outcomes . Studies suggest that maternal use of SFGs can effectively reduce caries risk in mothers and their children older than 4 years . This preventive dietary ally, though not a source of nutrition, can serve as an adjunct to good dietary practices. Its cost-effectiveness suggests the potential for broader public health applications with potential national savings on dental care costs. Thus, consideration should be given to how various public health and nutrition assistance programs can promote SFG use, emphasizing its role as a supplement to a balanced healthy diet and oral care practices . One potential avenue could be to make SFG available at schools or in vending machines alongside soda and chips or to provide it to students for free, similar to feminine hygiene or birth control products; however, further studies are necessary to implement such a recommendation effectively. Breastfeeding and early dietary effects While breastfeeding offers numerous health benefits, its relationship with caries, especially prolonged breastfeeding, is complex. It is essential to maintain good oral hygiene practices for children breastfed beyond one year to mitigate caries risk and ECC. This underscores the need for comprehensive recommendations from healthcare providers for reducing nocturnal breastfeeding, and cleaning “wipes” the child’s mouth after feeding is vital for balancing the benefits of breastfeeding with oral health . Diet quality and oral health A high-quality diet rich in dairy, proteins, fruits, and vegetables can protect against caries. The Healthy Eating Index (HEI) shows an inverse relationship with ECC risk, suggesting that dietary patterns are more indicative of oral health outcomes than individual nutrients . Post-sugary meal practices, such as consuming dairy or xylitol gum, can further reduce caries risk. On the other hand, combining sugar with starch or having a dietary pattern high in desserts and crackers would intensify the cariogenic effect . Social determinants of health and dietary choices Access to a healthy diet and fluoridated water, which are crucial for caries prevention, is significantly influenced by social determinants of health (SDOHs), which also shape health behaviors . Our review strongly supports the effectiveness of community water fluoridation, as seen in Australia's National Oral Health Plan, and advocates for its broader implementation to help prevent caries . Economic barriers, however, often make sugary foods and starches more accessible, challenging healthy dietary choices . Added sugar consumption is notably affected by factors such as socioeconomic status, household dietary habits, the locality of sugar sources, and peer influences . Policies aimed at limiting sugary food consumption and enhancing fluoridation access are vital for reducing oral health disparities . Additionally, ensuring access to affordable, nutritious food is crucial, particularly in underserved communities. Educational strategies should provide practical guidance on budget-friendly, healthy food choices and must consider the cultural relevance of dietary recommendations to effectively promote long-term dietary changes and enhance oral health . Implications for practice and policy Proper advocacy and implementation of these solutions and strategies within the DGAs framework can address some of the underlying upstream factors contributing to social gradients in oral diseases, thus, significantly contributing to equitable health outcomes and reducing the strain on the national healthcare system . We recommend that policymakers integrate these evidence-based strategies into public health policies to address oral health disparities and lessen the overall healthcare burden (Figs. , ). Effective public health strategies should not only aim to improve food security and promote healthy eating, but also ensure access to oral health education, literacy, and services for all groups, particularly high-risk groups (Fig. ). Collaborative efforts among dietitians, allied health professionals, dental-behavioral-mental health experts, and policymakers, especially those representing underrepresented minorities, are essential to developing and implementing comprehensive oral-nutrition guidelines, thereby preventing metabolic and systemic disorders and easing healthcare burdens . Targeting oral-nutrition public health initiatives is crucial for preventing not only caries, but also enhancing quality-adjusted life years (QALYs) and reducing disability-adjusted life years (DALYs) . Unfortunately, oral health continues to be overlooked within the current healthcare and policy frameworks . In light of recognizing quality healthcare as a fundamental human right , and in alignment with the WHO Global Oral Health Action Plan, it is imperative to fully integrate oral health within the healthcare system to achieve comprehensive systemic, planetary, and One Health outcomes . Oral health is as critical as any other aspect of healthcare; and systemic health cannot be fully realized without incorporating oral health into the equations for healthy eating habits and healthcare management . Considering socioeconomic and cultural contexts, local authorities can raise awareness of how eating habits influence oral health and the broader spectrum of NCDs. By integrating public health campaigns and regulatory measures tailored to the unique demographic and geographic variations in oral health practices and resources, authorities can ensure effective and inclusive health promotion strategies . Strengths and limitations While this scoping review aimed for comprehensive coverage, the literature search may not have been exhaustive, possibly omitting relevant studies. The included literature encompassed articles focusing on equity principles, health disparities, and SDOH because we aimed to identify and highlight cost-effective, accessible strategies that benefit the broader population, especially those with constrained resources. The focus on equity and inclusivity may have introduced biases or overlooked certain articles. These limitations underscore areas for future research efforts. The review process itself is subject to potential selection biases and the heterogeneity of study designs, cautioning against overinterpretation of findings and suggesting methodological improvements for future studies. Inconsistencies in research data comparability further challenge conclusive interpretations, calling for methodological advancements in data collection and analysis. Despite these limitations, this review lays a foundation for future studies by highlighting the critical role of dietary choices in achieving equitable oral and overall health. Further research is warranted to explore the long-term effects of interventions across diverse populations, including those with varying socioeconomic backgrounds and access to dental care. Our findings can inform targeted interventions and policy changes to improve oral health outcomes and guide future research directions. The SR’s commitment to transparency through comprehensive documentation enhances the integrity and utility of its conclusions. Moving forward, long-term studies and randomized controlled trials are needed to elucidate the mechanisms and effectiveness of interventions, such as sugar-free gum, and to develop tailored oral health promotion programs adaptable to individual needs and contexts.
Behavioral interventions and modification strategies have emerged as essential strategies for mitigating the public health challenge of reducing the prevalence of caries and enhancing overall oral health (Fig. ) .
The foundation of behavioral interventions lies in regular dental hygiene practices , such as toothbrushing and flossing, and regular access to fluoride sources . Numerous studies have consistently shown their positive impact on reducing the prevalence of caries and improving oral health. Research has consistently highlighted the positive impact of these practices on oral health. Notably, interdental cleaning devices are associated with decreased oral disease incidence and fewer missing teeth. Effective brushing habits correlate with reduced cariogenic bacteria and lower caries incidence, emphasizing the need to modify daily oral hygiene behaviors to achieve substantial benefits in oral health . Programs focusing on early interventions , such as supervised toothbrushing initiatives, are critical and have proven to be valuable preventive techniques, especially for young children. For example, the “National Supervised Toothbrushing Program” in Scotland illustrates the effectiveness and substantial benefits of such early intervention, emphasizing the value of advocacy in instilling good oral hygiene habits from a young age .
A parent/child approach that integrates nutrition and oral health education underscores the need for comprehensive, family-oriented initiatives to ensure the continuity and effectiveness of oral health practices . School-based interventions have also shown a positive impact, especially among schoolchildren, in fostering proper oral hygiene practices, indicating their potential to significantly enhance oral health outcomes when implemented within the educational system . Research consistently indicates that motivational interviewing is particularly impactful for adolescents and members of lower-income families, showcasing its potential as a powerful catalyst for change (Fig. ).
In line with the above interventions, positive parenting practices , such as engagement, encouragement, and problem solving, are fundamental for reducing the prevalence of childhood caries . For instance, learning the distinct patterns of risk factors associated with ECC, as identified in our study, enables parents and caregivers to tailor their approaches to better suit their children’s specific needs, thus enhancing the effectiveness of preventive measures and ultimately leading to a significant decrease in the incidence of ECC . Such practices not only benefit the immediate oral health of children but also contribute to establishing lifelong healthful habits. Our review is consistent with the thrust of behavioral modification for sustained oral health and supports the daily use of high-fluoride products in conjunction with standard toothpaste, particularly for high-risk older children and adults, which is not advised in young children due to fluorosis concerns. This recommendation aligns with the broader theme of comprehensive behavior modification and is supported by studies such as Sonbul et al. (2011), which validate the effectiveness of high-fluoride products in enhancing and sustaining oral health . Furthermore, while advocating for the integration of fluoride-based products as a cornerstone of preventive oral health measures, it is essential to acknowledge existing controversies surrounding fluoridation. Addressing concerns and promoting informed discussions regarding the safety and efficacy of fluoride interventions are paramount. Additionally, ongoing research into alternative clinical solutions that complement or supplement traditional fluoride approaches can offer valuable insights into enhancing oral health outcomes for diverse populations.
) In addition to established behavioral interventions, educational programs play a pivotal role in oral health promotion. The evidence suggests that interactive and contextually tailored education leads to significant improvements in oral hygiene behaviors, increased utilization of preventive services, and a reduction in the incidence of caries (Fig. ).
Educational interventions have demonstrated their ability to significantly improve oral hygiene behaviors. Studies have consistently shown that individuals who receive oral health education demonstrate better oral hygiene practices than those who do not receive such education. The effectiveness of hands-on demonstrations and integration into school curricula is particularly noteworthy, resulting in improved oral hygiene and a decrease in caries among students. This finding reinforces the value of incorporating oral health education into early learning environments .
The use of gamified mobile applications and other educational tools for oral health and nutrition has made learning more engaging and effective. Practical demonstrations, animated screenings, and experiential learning have helped individuals better understand the practical aspects of maintaining good oral health through proper nutrition and hygiene practices .
Educational programs that incorporate hands-on demonstrations and are integrated into school curricula are instrumental in fostering better oral hygiene and reducing the incidence of caries. The strategic integration of oral health education within the general health curriculum has emerged as a pivotal approach to enhancing oral health literacy and outcomes (Fig. ). Additionally, school-based and community-level programs are vital forums for the widespread dissemination of oral health and nutrition education. Often, these initiatives are geared toward promoting healthy eating habits and understanding the nutritional value of foods. By embedding effective oral hygiene education and practices within a comprehensive health framework, these programs highlight the interdependence of good oral health and overall health and nutrition. Acknowledging that optimal nutrition can be compromised by poor oral health further accentuates the significance of maintaining oral hygiene as a cornerstone of nutritional and overall well-being. Involving schoolteachers as health educators who regularly promote healthy dietary and oral health habits and practices within standard school curricula is key to providing extensive health education, offering substantial benefits to both students and the wider community . However, it is essential to recognize the potential challenges faced by schoolteachers when assuming additional responsibilities in health education. Limited time within the curriculum, competing educational priorities, and varying levels of teacher training in oral health and nutrition may pose obstacles to effective implementation. Strategies such as providing specialized training for teachers, integrating oral health and nutrition education into existing subject areas, and collaborating with external healthcare professionals can help mitigate these barriers, ensuring that comprehensive health education remains a priority within school settings. To further bolster these efforts, policy support and incentives are essential. Implementing policies that support the integration of oral health and nutrition education into school curricula, coupled with incentives such as professional development opportunities and recognition for teachers who excel in health education, can effectively motivate and support educators in delivering high-quality health education to students.
Effective oral health promotion extends beyond individual practices, connecting broader oral health and nutrition challenges to the overall wellbeing of communities. Health program planners can foster a culture of health by leveraging local resources to advocate for healthy lifestyle choices and spread positive messages about nutrition and oral health, thereby mitigating negative influences and promoting healthy habits within the community (Fig. ). Recognizing that people spend a significant amount of time in settings such as preschools, long-term care facilities, churches, and programs such as the Head Start and Special Supplemental Nutrition Program for Women, Infants, & Children (WIC), integrating oral health and nutrition education into these community-based settings can have a profound and lasting impact on improving oral and overall health outcomes and well-being for children and adults.
Targeting the family unit, particularly caregivers and parents, is essential for the prevention of ECC . Education targeting families acknowledges the critical role that parents play in shaping their children's oral health routines, emphasizing how educational attainment correlates with health outcomes. Research has highlighted the nexus between a mother’s education level, the frequency of her child’s dental visits, and her tooth brushing habits, underscoring the influence of socioeconomic status on oral health practices (Fig. ).
) In addition to behavioral and educational measures, dietary strategies are crucial for preventing caries and enhancing overall oral health. By improving nutritional intake and reducing sugar consumption, these interventions not only lower the incidence of caries but also promote broader oral wellness .
Effective oral health promotion involves more than just reducing sugar and fermentable carbohydrate intake. The incorporation of nutritious alternatives such as high-quality proteins and vitamins (i.e. A, B, and C) and other micronutrients, which significantly improve oral health outcomes, is needed to reduce chronic inflammation and the burden of oral diseases . Public health policies must emphasize comprehensive dietary improvements, not just reducing sugar consumption. Recent studies, demonstrate the significant impact of taste preferences, age, oral microbiota, and use of dental prostheses on dietary choices, which in turn influence oral health behaviors and outcomes, underscoring the need for integrated strategies that consider both taste perception and nutritional education in oral health promotion and identify populations at risk . By increasing public awareness of the harmful effects of frequent sugar intake and promoting a balanced diet, these policies can broaden the scope of oral disease prevention strategies and enhance overall oral health .
The role of SFG, particularly that containing xylitol, in caries prevention is well documented. The regular use of SFG contributes significantly to reducing caries risk by stimulating saliva flow, which neutralizes acids produced by bacteria in biofilms (plaques), helps to remineralize tooth enamel, and aids in managing and improving oral health outcomes . Studies suggest that maternal use of SFGs can effectively reduce caries risk in mothers and their children older than 4 years . This preventive dietary ally, though not a source of nutrition, can serve as an adjunct to good dietary practices. Its cost-effectiveness suggests the potential for broader public health applications with potential national savings on dental care costs. Thus, consideration should be given to how various public health and nutrition assistance programs can promote SFG use, emphasizing its role as a supplement to a balanced healthy diet and oral care practices . One potential avenue could be to make SFG available at schools or in vending machines alongside soda and chips or to provide it to students for free, similar to feminine hygiene or birth control products; however, further studies are necessary to implement such a recommendation effectively.
While breastfeeding offers numerous health benefits, its relationship with caries, especially prolonged breastfeeding, is complex. It is essential to maintain good oral hygiene practices for children breastfed beyond one year to mitigate caries risk and ECC. This underscores the need for comprehensive recommendations from healthcare providers for reducing nocturnal breastfeeding, and cleaning “wipes” the child’s mouth after feeding is vital for balancing the benefits of breastfeeding with oral health .
A high-quality diet rich in dairy, proteins, fruits, and vegetables can protect against caries. The Healthy Eating Index (HEI) shows an inverse relationship with ECC risk, suggesting that dietary patterns are more indicative of oral health outcomes than individual nutrients . Post-sugary meal practices, such as consuming dairy or xylitol gum, can further reduce caries risk. On the other hand, combining sugar with starch or having a dietary pattern high in desserts and crackers would intensify the cariogenic effect .
Access to a healthy diet and fluoridated water, which are crucial for caries prevention, is significantly influenced by social determinants of health (SDOHs), which also shape health behaviors . Our review strongly supports the effectiveness of community water fluoridation, as seen in Australia's National Oral Health Plan, and advocates for its broader implementation to help prevent caries . Economic barriers, however, often make sugary foods and starches more accessible, challenging healthy dietary choices . Added sugar consumption is notably affected by factors such as socioeconomic status, household dietary habits, the locality of sugar sources, and peer influences . Policies aimed at limiting sugary food consumption and enhancing fluoridation access are vital for reducing oral health disparities . Additionally, ensuring access to affordable, nutritious food is crucial, particularly in underserved communities. Educational strategies should provide practical guidance on budget-friendly, healthy food choices and must consider the cultural relevance of dietary recommendations to effectively promote long-term dietary changes and enhance oral health .
Proper advocacy and implementation of these solutions and strategies within the DGAs framework can address some of the underlying upstream factors contributing to social gradients in oral diseases, thus, significantly contributing to equitable health outcomes and reducing the strain on the national healthcare system . We recommend that policymakers integrate these evidence-based strategies into public health policies to address oral health disparities and lessen the overall healthcare burden (Figs. , ). Effective public health strategies should not only aim to improve food security and promote healthy eating, but also ensure access to oral health education, literacy, and services for all groups, particularly high-risk groups (Fig. ). Collaborative efforts among dietitians, allied health professionals, dental-behavioral-mental health experts, and policymakers, especially those representing underrepresented minorities, are essential to developing and implementing comprehensive oral-nutrition guidelines, thereby preventing metabolic and systemic disorders and easing healthcare burdens . Targeting oral-nutrition public health initiatives is crucial for preventing not only caries, but also enhancing quality-adjusted life years (QALYs) and reducing disability-adjusted life years (DALYs) . Unfortunately, oral health continues to be overlooked within the current healthcare and policy frameworks . In light of recognizing quality healthcare as a fundamental human right , and in alignment with the WHO Global Oral Health Action Plan, it is imperative to fully integrate oral health within the healthcare system to achieve comprehensive systemic, planetary, and One Health outcomes . Oral health is as critical as any other aspect of healthcare; and systemic health cannot be fully realized without incorporating oral health into the equations for healthy eating habits and healthcare management . Considering socioeconomic and cultural contexts, local authorities can raise awareness of how eating habits influence oral health and the broader spectrum of NCDs. By integrating public health campaigns and regulatory measures tailored to the unique demographic and geographic variations in oral health practices and resources, authorities can ensure effective and inclusive health promotion strategies .
While this scoping review aimed for comprehensive coverage, the literature search may not have been exhaustive, possibly omitting relevant studies. The included literature encompassed articles focusing on equity principles, health disparities, and SDOH because we aimed to identify and highlight cost-effective, accessible strategies that benefit the broader population, especially those with constrained resources. The focus on equity and inclusivity may have introduced biases or overlooked certain articles. These limitations underscore areas for future research efforts. The review process itself is subject to potential selection biases and the heterogeneity of study designs, cautioning against overinterpretation of findings and suggesting methodological improvements for future studies. Inconsistencies in research data comparability further challenge conclusive interpretations, calling for methodological advancements in data collection and analysis. Despite these limitations, this review lays a foundation for future studies by highlighting the critical role of dietary choices in achieving equitable oral and overall health. Further research is warranted to explore the long-term effects of interventions across diverse populations, including those with varying socioeconomic backgrounds and access to dental care. Our findings can inform targeted interventions and policy changes to improve oral health outcomes and guide future research directions. The SR’s commitment to transparency through comprehensive documentation enhances the integrity and utility of its conclusions. Moving forward, long-term studies and randomized controlled trials are needed to elucidate the mechanisms and effectiveness of interventions, such as sugar-free gum, and to develop tailored oral health promotion programs adaptable to individual needs and contexts.
Effective management of oral health is essential for promoting long-term health equity. This scoping review emphasizes the importance of an integrated approach that includes behavior change, education, and dietary modifications for optimal oral and overall health outcomes. The strategic use of sugar-free chewing gum, such as those containing xylitol, offers a practical and cost-effective alternative to traditional oral hygiene and dietary practices, providing protective benefits against caries and potential healthcare savings. A robust strategy that combines behavior modification, oral-nutrition health literacy enhancement, and targeted dietary practices, especially in reducing sugar consumption, is imperative. This tripartite approach, which emphasizes personal habits, informed choices, and consumption, serves as the cornerstone for preventing caries and promoting general-systemic health while reducing healthcare expenditures. Integrating educational enhancement, behavioral change, nutritional management, and dietary regulation, particularly in managing sugar intake, forms the foundation for improving oral health. Incorporating oral health recommendations within the DGAs highlights the critical relationship between societal factors and individual behaviors, holding the potential to enhance QoL and foster sustainable practices that lead to improved health outcomes and positively impact the nation’s economic landscape. This global-to-local perspective emphasizes the urgent need for integrated, multidisciplinary approaches that consider oral health not just as an isolated issue, but as an integral part of overall health and well-being. These efforts are crucial for both improving global health outcomes and addressing specific challenges within the U.S., ensuring that all individuals have access to the necessary resources and care to maintain optimal oral health. Choosing the right foods not only supports overall health, but also safeguards the wellness of the mouth. Committing to this comprehensive approach not only ensures that optimal oral health becomes an achievable goal for everyone, but also fosters transdisciplinary, multi-level, and cross-sector collaboration essential for promoting equitable health and well-being. This approach contributes to a future where oral health achievements are possible, even in context where dental care is limited, absent, or significant oral health inequalities exist.
Supplementary Material 1.
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Elevated Plasma Complement Factors in | e2bbeff5-2ebd-4074-bf90-2820507a254c | 11855139 | Biochemistry[mh] | Patients This study was performed in compliance with the guidelines of the Declaration of Helsinki and has the approval of the local Institutional Review Board (University Medical Center Utrecht [UMCU]). In total, 30 patients were referred to the UMCU from the Amsterdam University Medical Centers, Leiden University Medical Center, Bartiméus Diagnostic Center for complex visual disorders, The Rotterdam Eye Hospital and Rotterdam Ophthalmic Institute, and Groningen University Medical Center for recruitment at the outpatient clinic of the Department of Ophthalmology of the UMCU (MEC-14-065). Each patient provided written informed consent before participation and blood collection. The CRB1 -IRD diagnosis was established by ophthalmic examination, imaging, full-field electroretinography, and next-generation sequencing or whole-exome sequencing. Patients were considered to have a molecularly confirmed CRB1 -IRD when they harbored two or more rare functional variants (i.e., disease-causing CRB1 variants, such as missense, deletions, loss of function, or splice altering variants) affecting both gene copies of CRB1 . We excluded patients with systemic inflammatory conditions at the time of sampling and/or patients who had received systemic immunomodulatory treatment. On the day of inclusion, patients were examined by visual acuity measurement, and an experienced uveitis specialist performed slit-lamp examination to assess vitreous cells and vitreous haze. Next, we obtained blood from 29 anonymous age-matched healthy blood bank donors with no history of ocular inflammatory disease who gave broad written informed consent at the research blood bank of the University Medical Center Utrecht (The “Mini Donor Dienst”). Targeted Blood Proteomics Venous blood samples were collected in EDTA Lavender Top Tubes (#362084; BD Biosciences, Franklin Lakes, USA) and then centrifuged for 10 minutes (400 × g ) within an hour of blood collection . Plasma was transferred to a Falcon 15 mL Conical Centrifuge Tube (Corning Inc., New York, USA) and centrifuged for 10 minutes (1500 × g ), after which the cleared plasma was transferred and stored in Micronic 1.4-mL round-bottom tubes (#MP32022; Micronic, Lelystad, the Netherlands) at –80°C. For proteomic analysis of plasma, patient and control samples were randomized over three 96-well plates (#72.1980; Sarstedt, Nümbrecht, Germany) and sealed (#232698; Thermo Fisher, Waltham, MA, USA) before shipment on dry ice to the Olink Proteomic facility at the Erasmus Medical Center, Rotterdam, the Netherlands. We used the Olink Explore 384 Inflammation II panel for targeted proteomics of blood plasma. This Olink technology is based on proximity extension assays coupled with next-generation sequencing (NGS) and is capable of the simultaneous relative quantification of 370 protein analytes. TaqMan Single-Nucleotide Polymorphism Genotyping Genomic DNA of 59 peripheral mononuclear blood samples in an RLT Plus lysis buffer (#1053393; Qiagen, Hilden, Germany) was isolated with the AllPrep DNA/RNA/miRNA Universal Kit (#80224; Qiagen). Next, we determined the rs7535263 variant with the TaqMan single-nucleotide polymorphism (SNP) genotyping technology (#4351379; Thermo Fisher). Genotypes were called using QuantStudio 12K Flex software (Thermo Fisher). Linkage disequilibrium ( r 2 or D′) data for rs7535263 in the CEU population of the 1000 genomes were obtained with the LDproxy tool with genome build GRCh37 in LDlink . The estimated recombination rate for Utah residents with Northern and Western European ancestry CEU (Utah residents (CEPH) with Northern and Western European ancestry, build GRCh37 ) generated by Adam Auton was obtained via the 1000 Genomes ftp site ( ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/working/20130507_omni_recombination_rates/ ). Using phased whole-genome sequencing data of ∼150,000 individuals of the UK Biobank (UKB), we calculated the linkage disequilibrium (LD) between rs7535263 in CFH and CRB1 missense variants p.(Cys948Tyr) and p.(Arg764Cys), using the –ld function in plink 2.0. , These are two relatively more commonly found CRB1 missense variants with allele frequencies in the UKB that permit meaningful LD calculations (i.e., others are too rare). , The UKB has ethical approval from North West–Haydock Research Ethics Committee (REC reference: 16/NW/0274). Details of the UK Biobank study have been described in detail previously. This research has been conducted using the UK Biobank Resource under Application Number 24711. TaqMan genotyping of rs7535263 was also performed in an independent validation cohort (cohort II) of 123 CRB1 -IRD patients from the United Kingdom, Australia, Palestine, Italy, France, Belgium, the Netherlands, Canada, Poland, Switzerland, South Africa, Lithuania, Germany, and Ireland (MEC-2010-359) and 1292 Dutch controls from the amyotrophic lateral sclerosis study. All patients provided written informed consent for genetic testing at the center of recruitment. CRB1 -associated genetic diagnoses were made by targeted gene Sanger sequencing or established recently through a sequencing panel for retinitis pigmentosa and Leber congenital amaurosis using a single-molecule molecular inversion probes (smMIP) method. TaqMan SNP genotyping (#4351379; Thermo Fisher) was performed on genomic DNA following the same procedure as described above by the laboratory of the Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands. Statistical Analyses Proteome analyses were performed on Normalized Protein eXpression (NPX) units from the output of the Olink analysis in R and RStudio (version 4.2.2) using the Olink Analyze R package. One protein, TNFSF9, had 13 missing data points, and 10 proteins did not pass quality control (Olink assay report). These proteins were removed before group analyses. The prcomp function of R base was used to perform principal component analysis (PCA). Differential expression analysis was performed using a likelihood ratio test (LRT) with age and sex as covariates in the models, and we used the qvalue package from Bioconductor to correct P values for multiple testing. Overrepresentation analysis (or enrichment) was performed on proteins with nominal significant differential expression levels ( P < 0.05) with the ClusterProfiler R package and using the WikiPathways database as a reference. The results were plotted using the dotplot function of the enrichplot R package, and the P values from enrichment analysis were adjusted with the Benjamini–Hochberg (BH) procedure, using the p.adjust function in R base, and P values ( P adj) < 0.05 were considered statistically significant. Disease association for rs7535263 was assessed with Plink using Fisher's exact test and expressed as the odds ratio (OR) with a 95% confidence interval (CI). We performed an LRT to assess the relation between the genotype and the protein expression levels and visualized the results using the corrplot R package. Other figures were generated with the ggplot2 package. The explained variance ( R 2 ) by the genotype of rs7535263 of plasma proteins was calculated using the squared Pearson correlation coefficient ( r 2 ) using the cor function in R base. This study was performed in compliance with the guidelines of the Declaration of Helsinki and has the approval of the local Institutional Review Board (University Medical Center Utrecht [UMCU]). In total, 30 patients were referred to the UMCU from the Amsterdam University Medical Centers, Leiden University Medical Center, Bartiméus Diagnostic Center for complex visual disorders, The Rotterdam Eye Hospital and Rotterdam Ophthalmic Institute, and Groningen University Medical Center for recruitment at the outpatient clinic of the Department of Ophthalmology of the UMCU (MEC-14-065). Each patient provided written informed consent before participation and blood collection. The CRB1 -IRD diagnosis was established by ophthalmic examination, imaging, full-field electroretinography, and next-generation sequencing or whole-exome sequencing. Patients were considered to have a molecularly confirmed CRB1 -IRD when they harbored two or more rare functional variants (i.e., disease-causing CRB1 variants, such as missense, deletions, loss of function, or splice altering variants) affecting both gene copies of CRB1 . We excluded patients with systemic inflammatory conditions at the time of sampling and/or patients who had received systemic immunomodulatory treatment. On the day of inclusion, patients were examined by visual acuity measurement, and an experienced uveitis specialist performed slit-lamp examination to assess vitreous cells and vitreous haze. Next, we obtained blood from 29 anonymous age-matched healthy blood bank donors with no history of ocular inflammatory disease who gave broad written informed consent at the research blood bank of the University Medical Center Utrecht (The “Mini Donor Dienst”). Venous blood samples were collected in EDTA Lavender Top Tubes (#362084; BD Biosciences, Franklin Lakes, USA) and then centrifuged for 10 minutes (400 × g ) within an hour of blood collection . Plasma was transferred to a Falcon 15 mL Conical Centrifuge Tube (Corning Inc., New York, USA) and centrifuged for 10 minutes (1500 × g ), after which the cleared plasma was transferred and stored in Micronic 1.4-mL round-bottom tubes (#MP32022; Micronic, Lelystad, the Netherlands) at –80°C. For proteomic analysis of plasma, patient and control samples were randomized over three 96-well plates (#72.1980; Sarstedt, Nümbrecht, Germany) and sealed (#232698; Thermo Fisher, Waltham, MA, USA) before shipment on dry ice to the Olink Proteomic facility at the Erasmus Medical Center, Rotterdam, the Netherlands. We used the Olink Explore 384 Inflammation II panel for targeted proteomics of blood plasma. This Olink technology is based on proximity extension assays coupled with next-generation sequencing (NGS) and is capable of the simultaneous relative quantification of 370 protein analytes. Genomic DNA of 59 peripheral mononuclear blood samples in an RLT Plus lysis buffer (#1053393; Qiagen, Hilden, Germany) was isolated with the AllPrep DNA/RNA/miRNA Universal Kit (#80224; Qiagen). Next, we determined the rs7535263 variant with the TaqMan single-nucleotide polymorphism (SNP) genotyping technology (#4351379; Thermo Fisher). Genotypes were called using QuantStudio 12K Flex software (Thermo Fisher). Linkage disequilibrium ( r 2 or D′) data for rs7535263 in the CEU population of the 1000 genomes were obtained with the LDproxy tool with genome build GRCh37 in LDlink . The estimated recombination rate for Utah residents with Northern and Western European ancestry CEU (Utah residents (CEPH) with Northern and Western European ancestry, build GRCh37 ) generated by Adam Auton was obtained via the 1000 Genomes ftp site ( ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/working/20130507_omni_recombination_rates/ ). Using phased whole-genome sequencing data of ∼150,000 individuals of the UK Biobank (UKB), we calculated the linkage disequilibrium (LD) between rs7535263 in CFH and CRB1 missense variants p.(Cys948Tyr) and p.(Arg764Cys), using the –ld function in plink 2.0. , These are two relatively more commonly found CRB1 missense variants with allele frequencies in the UKB that permit meaningful LD calculations (i.e., others are too rare). , The UKB has ethical approval from North West–Haydock Research Ethics Committee (REC reference: 16/NW/0274). Details of the UK Biobank study have been described in detail previously. This research has been conducted using the UK Biobank Resource under Application Number 24711. TaqMan genotyping of rs7535263 was also performed in an independent validation cohort (cohort II) of 123 CRB1 -IRD patients from the United Kingdom, Australia, Palestine, Italy, France, Belgium, the Netherlands, Canada, Poland, Switzerland, South Africa, Lithuania, Germany, and Ireland (MEC-2010-359) and 1292 Dutch controls from the amyotrophic lateral sclerosis study. All patients provided written informed consent for genetic testing at the center of recruitment. CRB1 -associated genetic diagnoses were made by targeted gene Sanger sequencing or established recently through a sequencing panel for retinitis pigmentosa and Leber congenital amaurosis using a single-molecule molecular inversion probes (smMIP) method. TaqMan SNP genotyping (#4351379; Thermo Fisher) was performed on genomic DNA following the same procedure as described above by the laboratory of the Department of Human Genetics, Radboud University Medical Center, Nijmegen, the Netherlands. Proteome analyses were performed on Normalized Protein eXpression (NPX) units from the output of the Olink analysis in R and RStudio (version 4.2.2) using the Olink Analyze R package. One protein, TNFSF9, had 13 missing data points, and 10 proteins did not pass quality control (Olink assay report). These proteins were removed before group analyses. The prcomp function of R base was used to perform principal component analysis (PCA). Differential expression analysis was performed using a likelihood ratio test (LRT) with age and sex as covariates in the models, and we used the qvalue package from Bioconductor to correct P values for multiple testing. Overrepresentation analysis (or enrichment) was performed on proteins with nominal significant differential expression levels ( P < 0.05) with the ClusterProfiler R package and using the WikiPathways database as a reference. The results were plotted using the dotplot function of the enrichplot R package, and the P values from enrichment analysis were adjusted with the Benjamini–Hochberg (BH) procedure, using the p.adjust function in R base, and P values ( P adj) < 0.05 were considered statistically significant. Disease association for rs7535263 was assessed with Plink using Fisher's exact test and expressed as the odds ratio (OR) with a 95% confidence interval (CI). We performed an LRT to assess the relation between the genotype and the protein expression levels and visualized the results using the corrplot R package. Other figures were generated with the ggplot2 package. The explained variance ( R 2 ) by the genotype of rs7535263 of plasma proteins was calculated using the squared Pearson correlation coefficient ( r 2 ) using the cor function in R base. Generation of a High-Quality Plasma Proteome of CRB1 -IRD Patients To explore the composition of inflammatory mediators in the plasma proteome of CRB1 -IRD patients, we conducted 370-plex targeted proteomics using proximity extension assays combined with NGS in a cohort of 30 patients and 29 healthy controls. To ensure high-quality data for quantitative proteomic analysis, we first applied a stringent quality control process. We first filtered out low-quality analytes by removing proximity extension assays that did not pass quality control in all plasma samples, resulting in 358 (97%) proteins that passed this filtering step. We detected no outlier plasma samples as shown by the relatively comparable sample median and sample interquartile range as well as NPX (the protein expression unit, which is in log 2 scale) distribution between plasma samples ( A, B). However, nine plasma samples exhibited internal controls deviating >0.3 NPX from the plate median, which were also removed from the data set ( B). PCA on the 50 remaining plasma samples revealed a moderate plate effect that we mitigated by using the Combat algorithm to account for batch effects ( C, D). As a result, a high-quality proteomic data set was generated for downstream analysis. Elevated Concentrations of Plasma Complement Factors in CRB1 -IRD Patients We then compared the plasma proteome of patients to controls using linear models that include age and sex (likelihood ratio test). At a false discovery rate of 5% ( q < 0.05), we detected 10 proteins with significantly different abundance between patients and controls ( A and ). Among these, elevated levels were observed for complement factor I (CFI), complement factor H (CFH), protein S (PROS1), serpin family D member 1 (SERPIND1), and a disintegrin-like and metalloproteinase with thrombospondin type 1 (ADAMTS1) in patients compared to healthy controls ( B). We also detected significantly decreased levels for serine protease 22 (PRSS22) and microfibrillar-associated protein 4 (MFAP4) in CRB1 -IRD patients. The 62 proteins most associated with CRB1 -IRD patients (nominal P < 0.05) showed overall considerable positive correlation in expression levels, with close clustering of CFI, CFH, C3, and SERPIND1 ( C). These 62 proteins were strongly enriched for pathways involving platelet biology, the complement system, and coagulation cascades ( D and ). In detail, the most enriched pathway was the “Complement cascade” (e.g., Reactome pathway R-HSA-166658, P adj = P = 3.03 × 10 −15 , D). In total, 12/62 CRB1 -IRD associated proteins were linked to this pathway, including the significantly elevated proteins CFH, CFI, and PROS1. In addition, PROS1 and SERPIND1 were also linked to coagulation pathways (i.e., “Formation of Fibrin Clot (Clotting Cascade),” P adj = P = 4.69 × 10 −7 ). Another enriched pathway included the “Extracellular matrix organization” (R-HSA-1474244, P adj = P = 5.84 × 10 −3 ) based on the significantly altered plasma proteins ADAMTS1 and MFAP4 . Collectively, these results show that elevated levels of complement factors and inflammation-related proteins characterized the plasma proteome of patients with CRB1 -IRDs. The CFH Genotype Is Altered in Dutch CRB1 -IRD Patients On chromosome 1, the CRB1 gene is located adjacent to the CFH-CFHR locus, which is implicated in other retinal conditions by influencing plasma complement protein levels and other proteins detected in our study. Therefore, we hypothesized that linkage between CRB1 and the CFH-CFHR locus could influence the plasma proteome of CRB1 -IRD patients. To investigate this, we genotyped the common intronic SNP rs7535263 (LD r 2 = 1.0 with AMD risk variant rs1410996 in the CEU population of the 1000 Genomes). The genotype of rs7535263 was shown to have the strongest correlation with the gene expression levels of CFH-related 1 to 4 (CFHR1–4) in the Genotype-Tissue Expression project and is associated with multiple retinal diseases (e.g., AMD, multifocal choroiditis, and serious chorioretinopathy), as well as with other immune mediators on our array, such as CFP. – The LD signatures of rs7535263 ( r 2 and D′ with linked SNPs) and genetic recombination rates support that CRB1 variants and CFH-CFHR variants may reside in the same haplotype block ( A, B). As expected, the allele frequency of rs7535263 of Dutch controls (A allele of rs7535263 = 0.34) was similar to that of the comparable North European populations of the 1000 genomes (0.36 in GBR [British in England and Scotland] population). In contrast, the A allele of CFH -rs7535263 was significantly higher in frequency in patients compared to controls (A allele of rs7535263; OR = 2.85; 95% CI, 1.35–6.02; P = 6.19 × 10 −3 ) ( C, D). The most common CRB1 variant in our cohort was the homozygous p.(Met1041Thr) missense variants ( CRB1 c.3122T > C; p.(Met1041Thr)) found in 11 of 30 patients (37% of patients), which has been previously linked to a large consanguineous pedigree originating from a genetically isolated population in the Netherlands ( E, F). While this variant is rare in the general European population (allele frequency = 0.00001 for rs62635656 in the gnomAD database), the variant is found to be relatively more common (>1%) in this specific geographical Dutch area . We suspected that this deviation in allele frequency may therefore be the result of biological relatedness among cases (i.e., founder effect). All patients in our cohort homozygous for p.(Met1041Thr) were also homozygous for the A allele of rs7535263, which results in an excess of homozygotes for the A allele of the CFH variant rs7535263 and provides circumstantial evidence that biological relatedness between cases may likely influence our analysis of plasma complement factors. In a validation cohort of 123 CRB1 -IRD cases from multiple countries and 1292 Dutch control participants, the A allele of rs7535263 was not significantly more frequent in patients compared to controls (A allele of rs7535263; OR = 1.24; 95% CI, 0.95–1.61; P = 0.12). However, similar to the first cohort, a skewed allele frequency was observed for rs7535263 specifically in relation to the most common CRB1 variant, p.(Cys948Tyr). This variant is also a common CRB1 variant in previous reports, indicating possible linkage disequilibrium between specific variants of CRB1 and CFH . Utilizing data from large population databases such as the UKB may allow more accurate LD calculation between rare missense variants in CRB1 and rs7535263 in the CFH gene. To this end, we used phased whole-genome sequencing data from the UKB from ∼150,000 individuals and calculated LD for CRB1 missense variants. Two CRB1 missense variants commonly found in CRB1 -IRD cases with UKB allele frequencies that allow meaningful LD calculations (allele frequency >0.0001) display strong LD with rs7535263 in CFH ; the p.(Cys948Tyr) variant is in LD with the G allele of rs7535263 (D′ = 0.97, UKB), and the p.(Arg764Cys) variant is in full LD (D′ = 1.0 in UKB) with the A allele of rs7535263. Collectively, these results confirm our initial observation that patients with CRB1 missense variants may coinherit functional variants in the adjacent CFH locus, which should be considered in quantitative analysis of plasma complement factors in CRB1 -IRD patients. CRB1 -IRD Patients Show Elevated Plasma CFHR2 After Adjusting for the CFH Genotype We investigated the effect of rs7535263 on the plasma concentrations of immune mediators in healthy controls. In line with previous studies, the genotype of rs7535263 explained a significant proportion of the plasma levels of several proteins encoded by genes in the extended CFH-CFHR locus, including CFH-related protein 2 (CFHR2) ( R 2 = 0.36) and CFHR4 ( R 2 = 0.24), but not CFH ( R 2 = 0.003) ( with R 2 values), indicating that the genotype of this SNP may interfere with our analysis of some proteins in CRB1 patients. We therefore repeated our proteomic analysis for cohort 1 while correcting for the genotype of rs7535263 (along with sex and age). This analysis revealed that the association with the 10 significantly altered plasma proteins ( q < 0.05) in our initial group comparison was not significantly influenced by the genotype, such as CFI ( q rs7535263 = 0.003) and CFH ( q rs7535263 = 0.06) ( A, B and ), and all 10 remained within a false discovery rate of 7% ( q < 0.07). However, adjusting for rs7535263 revealed additional proteins associated with CRB1 -IRDs, most likely by improving statistical power by reducing residual variance. Among these plasma proteins was CFHR2 ( q rs7535263 = 0.04), further supporting dysregulation of complement pathways in CRB1 -IRDs ( A, B). Correcting for rs7535263 also revealed a significant association ( q rs7535263 < 0.05) with the DNA binding protein ZBP1, and RNA helicase DDX39A, which was previously also associated with the genotype of rs7535263 in multifocal choroiditis patients. The identified complement factors CFI, CFH, and CFHR2 all target C3, , an important downstream effector of the complement system, which was also increased in CRB1 -IRD patients, albeit at nominal significance ( q = 0.064) ( D). Based on the profile of complement factors in our array ( n = 18), we performed a correlation analysis to identify the factors associated with plasma C3 levels in our cohort. This analysis revealed that CFH and CFI, but not CFHR2, strongly correlated with the levels of plasma C3 ( C, D). CRB1 -IRD Patients To explore the composition of inflammatory mediators in the plasma proteome of CRB1 -IRD patients, we conducted 370-plex targeted proteomics using proximity extension assays combined with NGS in a cohort of 30 patients and 29 healthy controls. To ensure high-quality data for quantitative proteomic analysis, we first applied a stringent quality control process. We first filtered out low-quality analytes by removing proximity extension assays that did not pass quality control in all plasma samples, resulting in 358 (97%) proteins that passed this filtering step. We detected no outlier plasma samples as shown by the relatively comparable sample median and sample interquartile range as well as NPX (the protein expression unit, which is in log 2 scale) distribution between plasma samples ( A, B). However, nine plasma samples exhibited internal controls deviating >0.3 NPX from the plate median, which were also removed from the data set ( B). PCA on the 50 remaining plasma samples revealed a moderate plate effect that we mitigated by using the Combat algorithm to account for batch effects ( C, D). As a result, a high-quality proteomic data set was generated for downstream analysis. CRB1 -IRD Patients We then compared the plasma proteome of patients to controls using linear models that include age and sex (likelihood ratio test). At a false discovery rate of 5% ( q < 0.05), we detected 10 proteins with significantly different abundance between patients and controls ( A and ). Among these, elevated levels were observed for complement factor I (CFI), complement factor H (CFH), protein S (PROS1), serpin family D member 1 (SERPIND1), and a disintegrin-like and metalloproteinase with thrombospondin type 1 (ADAMTS1) in patients compared to healthy controls ( B). We also detected significantly decreased levels for serine protease 22 (PRSS22) and microfibrillar-associated protein 4 (MFAP4) in CRB1 -IRD patients. The 62 proteins most associated with CRB1 -IRD patients (nominal P < 0.05) showed overall considerable positive correlation in expression levels, with close clustering of CFI, CFH, C3, and SERPIND1 ( C). These 62 proteins were strongly enriched for pathways involving platelet biology, the complement system, and coagulation cascades ( D and ). In detail, the most enriched pathway was the “Complement cascade” (e.g., Reactome pathway R-HSA-166658, P adj = P = 3.03 × 10 −15 , D). In total, 12/62 CRB1 -IRD associated proteins were linked to this pathway, including the significantly elevated proteins CFH, CFI, and PROS1. In addition, PROS1 and SERPIND1 were also linked to coagulation pathways (i.e., “Formation of Fibrin Clot (Clotting Cascade),” P adj = P = 4.69 × 10 −7 ). Another enriched pathway included the “Extracellular matrix organization” (R-HSA-1474244, P adj = P = 5.84 × 10 −3 ) based on the significantly altered plasma proteins ADAMTS1 and MFAP4 . Collectively, these results show that elevated levels of complement factors and inflammation-related proteins characterized the plasma proteome of patients with CRB1 -IRDs. CFH Genotype Is Altered in Dutch CRB1 -IRD Patients On chromosome 1, the CRB1 gene is located adjacent to the CFH-CFHR locus, which is implicated in other retinal conditions by influencing plasma complement protein levels and other proteins detected in our study. Therefore, we hypothesized that linkage between CRB1 and the CFH-CFHR locus could influence the plasma proteome of CRB1 -IRD patients. To investigate this, we genotyped the common intronic SNP rs7535263 (LD r 2 = 1.0 with AMD risk variant rs1410996 in the CEU population of the 1000 Genomes). The genotype of rs7535263 was shown to have the strongest correlation with the gene expression levels of CFH-related 1 to 4 (CFHR1–4) in the Genotype-Tissue Expression project and is associated with multiple retinal diseases (e.g., AMD, multifocal choroiditis, and serious chorioretinopathy), as well as with other immune mediators on our array, such as CFP. – The LD signatures of rs7535263 ( r 2 and D′ with linked SNPs) and genetic recombination rates support that CRB1 variants and CFH-CFHR variants may reside in the same haplotype block ( A, B). As expected, the allele frequency of rs7535263 of Dutch controls (A allele of rs7535263 = 0.34) was similar to that of the comparable North European populations of the 1000 genomes (0.36 in GBR [British in England and Scotland] population). In contrast, the A allele of CFH -rs7535263 was significantly higher in frequency in patients compared to controls (A allele of rs7535263; OR = 2.85; 95% CI, 1.35–6.02; P = 6.19 × 10 −3 ) ( C, D). The most common CRB1 variant in our cohort was the homozygous p.(Met1041Thr) missense variants ( CRB1 c.3122T > C; p.(Met1041Thr)) found in 11 of 30 patients (37% of patients), which has been previously linked to a large consanguineous pedigree originating from a genetically isolated population in the Netherlands ( E, F). While this variant is rare in the general European population (allele frequency = 0.00001 for rs62635656 in the gnomAD database), the variant is found to be relatively more common (>1%) in this specific geographical Dutch area . We suspected that this deviation in allele frequency may therefore be the result of biological relatedness among cases (i.e., founder effect). All patients in our cohort homozygous for p.(Met1041Thr) were also homozygous for the A allele of rs7535263, which results in an excess of homozygotes for the A allele of the CFH variant rs7535263 and provides circumstantial evidence that biological relatedness between cases may likely influence our analysis of plasma complement factors. In a validation cohort of 123 CRB1 -IRD cases from multiple countries and 1292 Dutch control participants, the A allele of rs7535263 was not significantly more frequent in patients compared to controls (A allele of rs7535263; OR = 1.24; 95% CI, 0.95–1.61; P = 0.12). However, similar to the first cohort, a skewed allele frequency was observed for rs7535263 specifically in relation to the most common CRB1 variant, p.(Cys948Tyr). This variant is also a common CRB1 variant in previous reports, indicating possible linkage disequilibrium between specific variants of CRB1 and CFH . Utilizing data from large population databases such as the UKB may allow more accurate LD calculation between rare missense variants in CRB1 and rs7535263 in the CFH gene. To this end, we used phased whole-genome sequencing data from the UKB from ∼150,000 individuals and calculated LD for CRB1 missense variants. Two CRB1 missense variants commonly found in CRB1 -IRD cases with UKB allele frequencies that allow meaningful LD calculations (allele frequency >0.0001) display strong LD with rs7535263 in CFH ; the p.(Cys948Tyr) variant is in LD with the G allele of rs7535263 (D′ = 0.97, UKB), and the p.(Arg764Cys) variant is in full LD (D′ = 1.0 in UKB) with the A allele of rs7535263. Collectively, these results confirm our initial observation that patients with CRB1 missense variants may coinherit functional variants in the adjacent CFH locus, which should be considered in quantitative analysis of plasma complement factors in CRB1 -IRD patients. -IRD Patients Show Elevated Plasma CFHR2 After Adjusting for the CFH Genotype We investigated the effect of rs7535263 on the plasma concentrations of immune mediators in healthy controls. In line with previous studies, the genotype of rs7535263 explained a significant proportion of the plasma levels of several proteins encoded by genes in the extended CFH-CFHR locus, including CFH-related protein 2 (CFHR2) ( R 2 = 0.36) and CFHR4 ( R 2 = 0.24), but not CFH ( R 2 = 0.003) ( with R 2 values), indicating that the genotype of this SNP may interfere with our analysis of some proteins in CRB1 patients. We therefore repeated our proteomic analysis for cohort 1 while correcting for the genotype of rs7535263 (along with sex and age). This analysis revealed that the association with the 10 significantly altered plasma proteins ( q < 0.05) in our initial group comparison was not significantly influenced by the genotype, such as CFI ( q rs7535263 = 0.003) and CFH ( q rs7535263 = 0.06) ( A, B and ), and all 10 remained within a false discovery rate of 7% ( q < 0.07). However, adjusting for rs7535263 revealed additional proteins associated with CRB1 -IRDs, most likely by improving statistical power by reducing residual variance. Among these plasma proteins was CFHR2 ( q rs7535263 = 0.04), further supporting dysregulation of complement pathways in CRB1 -IRDs ( A, B). Correcting for rs7535263 also revealed a significant association ( q rs7535263 < 0.05) with the DNA binding protein ZBP1, and RNA helicase DDX39A, which was previously also associated with the genotype of rs7535263 in multifocal choroiditis patients. The identified complement factors CFI, CFH, and CFHR2 all target C3, , an important downstream effector of the complement system, which was also increased in CRB1 -IRD patients, albeit at nominal significance ( q = 0.064) ( D). Based on the profile of complement factors in our array ( n = 18), we performed a correlation analysis to identify the factors associated with plasma C3 levels in our cohort. This analysis revealed that CFH and CFI, but not CFHR2, strongly correlated with the levels of plasma C3 ( C, D). In this study, we examined the plasma proteome of patients with CRB1 -IRDs, whose levels of inflammation-related proteins and complement factors were notably elevated compared to healthy controls. A remarkable observation was the detection of elevated CFH and CFHR2 proteins in CRB1 -IRDs, which is interesting because elevated plasma levels of CFHR2 have been linked with other eye conditions, including AMD and multifocal choroiditis patients. , , , , CFH/CFHR proteins are all involved in the regulation of the complement component C3, , which was supported by our observation of a strong correlation between CFI and CFH with C3 in plasma. Elevated CFH/CFHR proteins may be a response to increased levels of C3, a compensatory mechanism in order to reduce complement activation. Our results are also in line with animal models of IRDs (rd10 mice), which show an increase in C3 protein levels and C3 activation, as well as an increase of mRNA of Cfh and Cfi in the retina. While C3 is important for complement activation and can cause microglial activation and photoreceptor cell injury, it has also been shown to mediate retina-protective signaling in other murine models of retinal degeneration but will require human trials with (intravitreal) anti-C3 therapy to elucidate its potential as a therapeutic target for CRB1 -IRDs. , , However, our study did not measure complement activation markers directly but rather essential complement components. Increased levels of essential complement components do not necessarily indicate increased activation, as depletion could also reflect activation through increased cleavage. Additionally, targeted proteomics, while powerful, has inherent (selection) bias and limited coverage. The panel we have used, however, provides an extensive panel of proteins related to inflammation. Regardless, the increased levels of complement factors and inflammation-related proteins that we found in patients support immune-mediated pathways involved in CRB1 -IRDs. Recent research with models of CRB1 -associated inherited retinal dystrophy shows that a “leaky gut,” or increased gut permeability, may play a role in CRB1 -IRDs. Bacterial translocation from the gut, which occurs when the gut barrier is compromised, leads to inflammation in the retina. Systemic treatment with antibiotics also prevented the onset and progression of ocular disease in CRB1 -IRD models. A compromised gut barrier in CRB1 -IRD patients might, through systemic inflammation, lead to complement activation, further implicating the role of the complement cascade in CRB1 -IRDs. Among the plasma proteins that were decreased in the plasma of CRB1 -IRD patients was the extracellular protein MFAP4, which is ubiquitously expressed and plays a role in intracellular interactions and cell adhesion. Macrophages drive local immune responses in retinal degeneration, , and MFAP4 loss affects macrophage differentiation in animal models. The elevated plasma levels of ADAMTS1 detected in CRB1 -IRD patients may be related to angiogenic responses due to the vascular impairment secondary to degenerative changes in the retina. – The ADAMTS1 enzyme is an angiogenic matrix-modifying enzyme that is upregulated by proinflammatory conditions in the retinal pigment epithelium (RPE). We also detected elevated levels of PROS1, a protein involved in the homeostasis of RPE. Whole-exome sequencing has previously identified homozygous pathogenic variants in the PROS1 gene as causing retinal dystrophy in two unrelated families, potentially disrupting the vitamin K–dependent activities of PROS1, leading to RPE degradation. Further investigation is needed to determine whether the elevated levels of PROS1 are caused by retinal damage or by other biological events involving PROS1. While the plasma proteome may reflect inflammation at the retinal level, it may also be influenced by functional variants in complement factor coding genes. The CRB1 gene is located on chromosome 1 immediately downstream of the CFH locus. SNPs in the CFH locus have been associated with several retinal conditions in which CFH/CFHR proteins were also altered, including AMD, central serous chorioretinopathy, and multifocal choroiditis, – indicating that this locus could be an important disease-modifying locus for eye conditions. Using TaqMan SNP genotyping technology, we found evidence for a skewed distribution of functional variants in the CFH gene, which may predispose to altered complement factor levels in CRB1 -IRDs or obscure relevant disease associations with plasma proteins, as demonstrated by our work. The significant skewing of the common CFH variants across CRB1 pathogenic variants in our Dutch cohort is likely the result of frequent consanguineous marriages in geographically more isolated populations, such as the strict geographical location of the p.(Met1041Thr) in the Netherlands, and predisposes to homozygosity of harmful recessive alleles. Genotype frequencies, Hardy–Weinberg equilibrium assumptions, and allele frequencies can be affected by relatedness, and our study demonstrates that addressing relatedness is crucial to ensure the accuracy of genotype–disease relationships and assessment of quantification of the complement factors in CRB1 -IRDs and other family-based monogenic conditions. We believe this genetic “confounding” of common variants is currently strongly underappreciated in molecular profiling studies of CRB1 -IRDs. While we show that in a second global cohort, there is no association with the CFH/CFHR locus in CRB1 cases in general, data from the well-powered UK Biobank demonstrated a linkage between variants in CFH and CRB1 . Several key missense variants (e.g., p.(Cys948Tyr)) are nearly exclusively inherited with one of the two alleles of rs7535263, which tag functionally distinct CFH/CFHR haplotypes and predispose to altered composition of plasma complement factors. , This linkage is expected from their proximity on the genome and LD signatures in the general population and has important implications for future research and treatment modalities: (1) Our study demonstrates that complement factor components are involved in CRB1 -IRDs but that relative biological relatedness (i.e., founder effects) between individuals or linkage with functional variants in the CFH locus should be considered in follow-up studies of complement dysregulation in human studies of CRB1 -IRDs. (2) While we demonstrate that correcting for the genotype of rs7535263 revealed an otherwise obscured increase in CFHR2 levels in patients, it is not unlikely that additional CFH(R) gene variants that genetically predispose to altered complement factor levels contribute to the pathophysiology of CRB1 -IRDs. The limited sample size of our study and the lack of probing for other CFH-related factors encoded by genes upstream of the CRB1 locus (e.g., CFHR1 and CFHR3 levels correlate with the genotype of rs7535263 according to gtexportal ) may have resulted in an underestimation of the differences in the levels of complement factors in CRB1 -IRD patients compared to controls. (3) Many more common variants in immune genes are likely involved in disease-modifying pathways that may influence the severity and disease course of CRB1 -IRDs. The use of genome-wide association studies using well-defined clinical endpoints, preferably based on microperimetry, may aid in the discovery of these common variants and could improve genotype–phenotype correlations to better predict disease course and perhaps lead to therapeutic modalities that can alter the disease course. The latter may also be of interest for emerging CRB1 gene augmentation therapy or mutational correction by allelic substitution via CRISPR/Cas9-mediated homology-directed repair or base editing , because these therapeutic approaches are accompanied by immune activation when administered intravitreally or subretinally. It would be important to determine to what extent the inflammatory markers identified in this study may help understand the side effects of gene therapy, such as increased chorioretinal atrophy, which has recently been described after gene therapy using voretigene neparvovec for RPE65 -associated retinal dystrophies. , A major source of plasma complement components is the liver, but complement proteins are also produced and expressed in the retina, as well as by immune cells and various tissues. Complement activation may occur in the retina due to loss of tissue integrity caused by CRB1 dysfunction, and modulating the complement system without addressing this primary cause may not have a clinically significant impact on the progression of the disease. Regardless, a complement-targeted therapy may be useful as a combination therapy to minimize adverse reactions from CRB1 gene-correcting strategies in retinal degeneration and potentially even in other gene therapy strategies for IRDs. In future studies, it might be informative to determine the immune profile of patients before commencing gene therapy interventions to determine to what extent complement activation and plasma concentrations influence outcome in patients. In conclusion, our results implicate the involvement of the complement cascade in CRB1 -IRDs. For future research, gene association studies of all SNPs in the genome with a categorization of patients based on disease severity may provide more insight into the pathophysiology of CRB1 -IRDs and IRDs in general. Supplement 1 |
The abdominal compliance index and postoperative pain after laparoscopic gynecologic surgery: a preliminary observational cohort study | 808029c0-2ecc-4941-8d4a-0faecfb91717 | 11931810 | Laparoscopy[mh] | Elevated intraabdominal pressure has gained increased attention in surgical and medical settings over recent decades, including in intensive care . A pneumoperitoneum at 12 mmHg is an iatrogenically created situation that leads to short-term abdominal hypertension. Full expansion for every patient has certainly not been certainly achieved at one pressure setting. Abdominal compliance is the measure of abdominal expansion and defined as the change in intraabdominal volume per change in intraabdominal pressure (mL/mmHg), which is unique to each patient . The elasticity of the abdomen and diaphragm are the major determinants . An increase in intraabdominal pressure exceeding the limits of abdominal compliance reduces blood flow and impairs perfusion, resulting in ischemia, hypoxia, and increased oxidative stress . Reduced abdominal compliance at higher insufflation pressures indicates that the surrounding tissues and organs are exposed to disproportional stress, without substantial improvement of surgical workspace . Normal abdominal compliance is in the range 250–450 mL/mmHg. The accommodation of the abdominal cavity follows phases of reshaping, stretching, and pressurization . The stretching capacity of abdominal expansion is influenced by numerous factors such as age, obesity, gender, fat distribution (visceral vs. subcutaneous), history of abdominal surgery, and chronic medical problems like ascites, peritoneal dialysis, and severe chronic obstructive pulmonary disease. In female patients, parity also plays a role [ – ]. Chronic increased intraabdominal pressure may damage the viscoelastic components of the abdominal wall, thereby increasing abdominal compliance. The pneumoperitoneum of laparoscopic surgery is determined by biomechanical rules of compliance and pressure as well as physical principles like Pascal’s and Laplace’s laws . The gas pressure settings for a laparoscopic pneumoperitoneum only give rough estimations for abdominal compliance . Currently, a simple and practical method for measuring and continuously monitoring abdominal compliance during laparoscopic surgery is not available . Endoscopic oscillometry permits concurrent monitoring of the changes in abdominal compliance throughout laparoscopic surgery in an animal model. It has been validated in opposition to compliance arising from computed tomographic (CT) imaging. The abdominal compliance was calculated from the subsequent pressure and flow in the insufflation circuit by using small oscillations . Selecting the lowest possible intraabdominal pressure is recommended to avoid the probable adverse effects of insufflation pressure on circulation . Nevertheless, in clinical practice, a practical methodology to achieve this has not yet been defined. Diaz-Cambronero et al. suggested a personalized pressure approach where the abdomen is prestretched in 15 mmHg and then the pressure is progressively decreased to the minimum intraabdominal pressure that still preserves optimal surgical workspace . Postoperative pain following laparoscopic surgery comprises incisional trocar pain and pneumoperitoneum pain . Abdominal wall expansion due to the insufflation and diaphragmatic irritation from the acidity of the carbon gas are the contributing factors to pneumoperitoneum pain . Postoperative shoulder pain is one of the most frequent and troublesome problems, with an incidence rate of 34%–82% after laparoscopic gynecologic surgery . The cause of this shoulder pain has not been fully elucidated and is thought to be multifactorial, likely referred pain. The central and diaphragmatic pleura are innervated by the phrenic nerve (C3–C5), while the acromion process is innervated by the supraclavicular nerve (C3–C4). When the phrenic nerve is irritated, pain appears in the neck or scapula . One theory suggests this pain in caused by irritation of the peritoneal and diaphragmatic nerves by carbonic acid, which reduces peritoneal pH . Another theory posits that retained gas and delayed absorption of CO 2 leads to strain on the triangular and coronary ligaments of the liver, causing subdiaphragmatic pain when patients sit or mobilize, usually more than four hours after the operation . The neuropraxia theory suggests that stretching and/or peritoneal and diaphragmatic injury leads to tearing of blood vessels, strain on the phrenic nerve, and the formation of inflammatory mediators, which evoke referred pain in the shoulder . More recently, a surrogate clinical index for abdominal compliance has been proposed as a feasible predictor of postoperative pain in male patients undergoing laparoscopic inguinal hernia repair . It was verified that the abdominal compliance index (ACI) could be obtained from precisely calculated abdominal compliance with high predictive correctness. We speculate that in patients with higher ACI values, greater expansion of the abdomen leads to extreme stretching of the abdominal wall and causes the patient to experience more severe pain. The ACI has probable clinical suggestions for adjusting intraabdominal pressure. It could be a useful surrogate clinical index to predict optimal pressure settings while reducing surgical stress in association with pneumoperitoneum. We aim to investigate a newly defined surrogate index of abdominal compliance in relation to postoperative abdominal and shoulder pain in patients undergoing elective laparoscopic gynecologic surgery.
This study was approved by the Ethics Committee of Bilkent City Hospital, Ankara, Türkiye with decision number (TABED1-24-321) dated 06/06/2024. Informed consent was acquired from all participating patients to use their clinical data and personal information upon admission. 2.1. Study design The study was a prospective observational cohort study including patients over 18 years of age with an American Society of Anesthesiologists (ASA) score of 1–3 undergoing elective laparoscopic gynecologic surgery. Exclusion criteria included conversion to open surgery; having a body mass index (BMI) >35 kg/m 2 ; loss of data during follow-up, chronic pain syndromes like fibromyalgia, neck, or shoulder pain; a history of opioid use; allergies to the study drugs; cognitive impairment; unstable cardiovascular disease; severe chronic obstructive pulmonary disease; liver or kidney dysfunction; or inability to communicate. 2.2. Data collection Postoperative pain was evaluated using a VAS of 0–10 recorded at 30 min, 6 h, 24 h, and 36 h postoperatively. Postoperative pain was classified as abdominal or shoulder pain. Abdominal pain was defined as away from the trocar site and was regarded as visceral (nonincisional) pain. Postoperative analgesic consumption was also documented at the same time intervals. 2.3. Anesthesia protocol and surgical procedure Similar general anesthesia and surgical protocols were applied to all patients. None of the patients were premedicated. Heart rate, noninvasive blood pressure, and oxygen saturation were monitored upon entrance to the operation room. After preoxygenation, general anesthesia was induced with propofol (2.5 mg/kg) and fentanyl (2 μg/kg). Tracheal intubation and muscle relaxation was facilitated by rocuronium (0.9 mg/kg) and repeated as necessary at periodic intervals. Anesthesia was maintained with 1 MAC desflurane with oxygen in the air (1:2 ratio) and remifentanil infusion (0–1–0.5 μg/kg/min). All patients were ventilated to maintain end tidal CO 2 at 35–40 mmHg. Respiratory rate and tidal volume were adjusted accordingly. Bispectral index levels were kept at 40–60. Ondansetron (8 mg IV) was administered for nausea and vomiting prophylaxis when the surgeons started to close the umbilical trocar sites. Neuromuscular blockade was reversed with sugammadex (2 mg/kg). Patients were set in the steep Trendelenburg and lithotomy positions during surgery. All surgeries were performed by experienced laparoscopic surgeons with abdominal insufflation at 12 mmHg using a standard automatic insufflator unit with a flow rate of 3 L/min (Karl Storz, Germany). The insufflated intraabdominal volume (IAV) was measured from the cumulative CO 2 volume gauge on the insufflation module of the laparoscopy system. A standard technique was used with one 10-mm trocar and two 5-mm trocars. The insufflated gas was not heated or humidified with additional devices. The initial insufflation volume was used to measure abdominal compliance after equilibrium and divided by body surface area (BSA) using the Du Bois formula to adjust for body constitution. The resulting surrogate index represented the abdominal expansion capacity per unit area under 12 mmHg insufflation pressure . The ACI was calculated as follows: ACI ( L / m 2 ) = insufflated IAV ( L ) / ( 0.007184 × height 0.725 ( cm ) × weight 0.425 ( kg ) ) For all patients, the remainder of the surgery was performed with intraabdominal gas pressure held at 12 mmHg, and residual carbon dioxide was completely evacuated at the end of the procedure by manual squeezing of the abdomen with open trocars. Patients were monitored in the postoperative care unit (PACU) until their conditions stabilized. Patient demographics, operation duration, and pneumoperitoneum time were recorded. 2.4. Postoperative analgesia The use of a patient-controlled analgesia (PCA) device and the VAS for postoperative pain assessment were explained to all patients during the preoperative visit. Abdominal pain and shoulder pain were questioned separately at 30 min after surgery and then at predefined time intervals (6, 12, 24, and 36 h postoperatively) using a 10-cm VAS scaled from 0 = no pain to 10 = unbearable pain. The standard analgesic protocol of tramadol (2 mg/kg) and paracetamol (1 g IV) was applied approximately 20 min before the end of the surgery. Tilcotil (20 mg IV) was administered upon request. For all patients, a PCA device was started in the PACU to deliver a bolus dose of 10 mg tramadol with a 15-min lock interval without a basal infusion. Ondansetron (IV) was given every 8 h for 24 h postoperatively. For VAS scores >3, patients were treated with rescue analgesics: paracetamol (1g IV) or tilcotil (20 mg IV). Tramadol requests, cumulative amounts, and total tramadol requirements were recorded from PCA memory and additional analgesic uses were recorded at the specified time intervals. 2.5. Statistical analysis A power analysis was conducted using G*Power v.3.1.9.7 (Franz Faul, Universitat Kiel, Germany), resulting in n1 = 15 (3.2 ± 0.4), n2 = 16 (4.3 ± 0.4), standard deviation = 0.7, α = 0.05, and effect size (d) = 1.57. The power was found to be 98%. Preliminary analyses were completed for frequencies, means, standard errors (SE), and percentages as applicable. A histogram and the Shapiro–Wilk test were used to analyze the variable distributions. Categorical variables were analyzed using the chi-square test, and continuous variables were analyzed using the Mann–Whitney U test. Statistical significance was set at p < 0.05 for all analyses. The statistical analyses were done using SPSS v.17.0 (SPSS Inc.; Chicago, IL, USA).
The study was a prospective observational cohort study including patients over 18 years of age with an American Society of Anesthesiologists (ASA) score of 1–3 undergoing elective laparoscopic gynecologic surgery. Exclusion criteria included conversion to open surgery; having a body mass index (BMI) >35 kg/m 2 ; loss of data during follow-up, chronic pain syndromes like fibromyalgia, neck, or shoulder pain; a history of opioid use; allergies to the study drugs; cognitive impairment; unstable cardiovascular disease; severe chronic obstructive pulmonary disease; liver or kidney dysfunction; or inability to communicate.
Postoperative pain was evaluated using a VAS of 0–10 recorded at 30 min, 6 h, 24 h, and 36 h postoperatively. Postoperative pain was classified as abdominal or shoulder pain. Abdominal pain was defined as away from the trocar site and was regarded as visceral (nonincisional) pain. Postoperative analgesic consumption was also documented at the same time intervals.
Similar general anesthesia and surgical protocols were applied to all patients. None of the patients were premedicated. Heart rate, noninvasive blood pressure, and oxygen saturation were monitored upon entrance to the operation room. After preoxygenation, general anesthesia was induced with propofol (2.5 mg/kg) and fentanyl (2 μg/kg). Tracheal intubation and muscle relaxation was facilitated by rocuronium (0.9 mg/kg) and repeated as necessary at periodic intervals. Anesthesia was maintained with 1 MAC desflurane with oxygen in the air (1:2 ratio) and remifentanil infusion (0–1–0.5 μg/kg/min). All patients were ventilated to maintain end tidal CO 2 at 35–40 mmHg. Respiratory rate and tidal volume were adjusted accordingly. Bispectral index levels were kept at 40–60. Ondansetron (8 mg IV) was administered for nausea and vomiting prophylaxis when the surgeons started to close the umbilical trocar sites. Neuromuscular blockade was reversed with sugammadex (2 mg/kg). Patients were set in the steep Trendelenburg and lithotomy positions during surgery. All surgeries were performed by experienced laparoscopic surgeons with abdominal insufflation at 12 mmHg using a standard automatic insufflator unit with a flow rate of 3 L/min (Karl Storz, Germany). The insufflated intraabdominal volume (IAV) was measured from the cumulative CO 2 volume gauge on the insufflation module of the laparoscopy system. A standard technique was used with one 10-mm trocar and two 5-mm trocars. The insufflated gas was not heated or humidified with additional devices. The initial insufflation volume was used to measure abdominal compliance after equilibrium and divided by body surface area (BSA) using the Du Bois formula to adjust for body constitution. The resulting surrogate index represented the abdominal expansion capacity per unit area under 12 mmHg insufflation pressure . The ACI was calculated as follows: ACI ( L / m 2 ) = insufflated IAV ( L ) / ( 0.007184 × height 0.725 ( cm ) × weight 0.425 ( kg ) ) For all patients, the remainder of the surgery was performed with intraabdominal gas pressure held at 12 mmHg, and residual carbon dioxide was completely evacuated at the end of the procedure by manual squeezing of the abdomen with open trocars. Patients were monitored in the postoperative care unit (PACU) until their conditions stabilized. Patient demographics, operation duration, and pneumoperitoneum time were recorded.
The use of a patient-controlled analgesia (PCA) device and the VAS for postoperative pain assessment were explained to all patients during the preoperative visit. Abdominal pain and shoulder pain were questioned separately at 30 min after surgery and then at predefined time intervals (6, 12, 24, and 36 h postoperatively) using a 10-cm VAS scaled from 0 = no pain to 10 = unbearable pain. The standard analgesic protocol of tramadol (2 mg/kg) and paracetamol (1 g IV) was applied approximately 20 min before the end of the surgery. Tilcotil (20 mg IV) was administered upon request. For all patients, a PCA device was started in the PACU to deliver a bolus dose of 10 mg tramadol with a 15-min lock interval without a basal infusion. Ondansetron (IV) was given every 8 h for 24 h postoperatively. For VAS scores >3, patients were treated with rescue analgesics: paracetamol (1g IV) or tilcotil (20 mg IV). Tramadol requests, cumulative amounts, and total tramadol requirements were recorded from PCA memory and additional analgesic uses were recorded at the specified time intervals.
A power analysis was conducted using G*Power v.3.1.9.7 (Franz Faul, Universitat Kiel, Germany), resulting in n1 = 15 (3.2 ± 0.4), n2 = 16 (4.3 ± 0.4), standard deviation = 0.7, α = 0.05, and effect size (d) = 1.57. The power was found to be 98%. Preliminary analyses were completed for frequencies, means, standard errors (SE), and percentages as applicable. A histogram and the Shapiro–Wilk test were used to analyze the variable distributions. Categorical variables were analyzed using the chi-square test, and continuous variables were analyzed using the Mann–Whitney U test. Statistical significance was set at p < 0.05 for all analyses. The statistical analyses were done using SPSS v.17.0 (SPSS Inc.; Chicago, IL, USA).
A total of 31 patients participated in the study. Three patients were excluded, two due to conversion to open surgery and one due to surgery cancellation. The patient and procedural demographics are shown in . The age range of the patients was 27–78, with a mean age of 53.03 ± 11.9 years. The patients were grouped by median ACI level, which ranged from 1.37 to 2.73 L/m 2 . Group 1 included those with an ACI ≤ 2.16 L/m 2 , and group 2 included those with an ACI > 2.16 L/m 2 . There were no significant differences in age, height, weight, BMI, BSA, surgery history, comorbidities, delivery history, mode of delivery, abdominal drainage placement, ASA score, or pneumoperitoneum time between the two groups (p > 0.05). The total amount of tramadol infusion and the insufflated volume (TV) were significantly lower in group 1 than in group 2 (p = 0.049 and p < 0.001, respectively) ( ). Additional analgesic use was higher in group 2 than group 1 (p = 0.001). Abdominal pain VAS scores at 30 m postoperative were significantly higher in group 2 than in group 1 (p < 0.001). Shoulder pain VAS scores at 24 h and 36 h postoperative were significantly higher in group 2 than in group 1 (p = 0.021 and p = 0.002, respectively). The number of patients experiencing abdominal and shoulder pain with VAS scores over 3 at different time points is shown in . A comparison of abdominal VAS scores at different time points is presented in , and a similar comparison for shoulder pain VAS scores is presented in .
The impact of pneumoperitoneum pressure on postoperative pain is broadly emphasized, but the relationship between pain after pneumoperitoneum and abdominal compliance due to expansion has thus far not been evaluated in laparoscopic gynecologic surgery. Abdominal compliance is a largely overlooked factor in intensive care, robotic surgery, and laparoscopic surgery . The relationship between intraabdominal pressure with IAV is not linear as the pressure-volume curve is nonlinear. Its slope shows the facility of abdominal expansion defined as abdominal compliance . Until complete expansion is reached, more gas means more space. After maximal compliance is attained, more gas increases pressure without any improvement in operating workspace and influences end organ perfusion for a limited time, creating short term abdominal hypertension . One pressure setting does not fit all patients. In other words, the amount of gas needed to establish the optimum surgical workspace is specific for any given patient. As a first in the literature, the findings of this study demonstrate that high ACI is significantly related to postoperative abdominal pain at 30 min and to postoperative shoulder pain at 24 h and 36 h after laparoscopic gynecologic surgery. Postoperative analgesic consumption and additional analgesic requirements were also increased in the high ACI group, meaning patients with greater abdominal expansion at 12 mmHg intraabdominal pressure. The orientation of connective tissue fibers, the biomechanics of the abdominal wall, and physical principles combine to form abdominal compliance. It is an anisotropic, nonlinear, and dynamic entity . Insufflation of the abdominal wall during pneumoperitoneum switches the potential intraabdominal space from a flattened sphere to an elliptical one. It has overlapping phases of reshaping, stretching, and pressurization. The abdominal cavity, which is a dome-shaped container, acts as a hydraulic system . Normal abdominal pressure at rest in a supine position is 5–7 mmHg. Stretching of the anterolateral abdominal muscles increases the anteroposterior diameter of the abdomen and decreases the transverse diameter. After the maximal stretch and elastic expansion of the abdominal wall is reached, small increments in volume leads to dramatic increments in intraabdominal pressure in the pressurization phase [ , , ]. The disadvantages of CO 2 insufflation might be attenuated by avoiding the use of extreme pressures through monitoring abdominal compliance. Lower pneumoperitoneum pressure is related to lower postoperative pain [ – ]. The lowest intraabdominal pressure under maximal abdominal wall compliance creates ideal circumstances for laparoscopic surgery by minimizing the pathophysiological effects of excessive pressurization while providing sufficient surgical exposure . Accurate calculation of abdominal compliance requires multiple measurement sites with special instruments and equipment such as flowmeters, tensiometers, and computer recording systems, making it unfeasible in a clinical setting . There is no clinically available practical method to measure and guide the tension/stress balance exerted on the intraabdominal organs and tissues . Calculating the insufflated CO 2 gas volume has disadvantages, such as gas leakage through trocars, removal through suction, and the absorption of gas by the peritoneum over time. Magnetic resonance imaging and CT have been used experimentally to obtain precise and repeatable calculations, but their added benefit is disclaimed by their drawbacks. Interference with the surgery, additional time required for measurements, and the presence of ionizing radiation or powerful magnetic fields prevent their routine use in clinical settings. Endoscopic oscillometry, a new strategy offering quantitative analysis of abdominal compliance alterations through stepwise insufflation, may also not be clinically practical since it requires preparations for endoscopic measurements . Females perceive postoperative pain more severely than males. Parity and history of past laparoscopic and abdominal surgery affects the intensity of postoperative pain [ , , , ]. Kinoshita et al. recently noted that novel ACI might not correctly predict postoperative pain in patients with a history of laparoscopic surgery or pregnancy since intraabdominal capacity increases and abdominal elasticity decreases . Actual ACI is decreased, while ACI would presumably increase . No differences were found between the groups in this study in terms of parity and history of abdominal and laparoscopic surgery. Jiang et al. found a negative correlation between pain scores and BMI, with BMI values <24 being an independent risk factor for shoulder pain after laparoscopic surgery . One possible explanation for this finding is that the stretching of the CO 2 gas irritating the diaphragm fills a larger space in the upper abdomen of thin patients than those of obese patients, whose upper abdominal area is covered by omentum . Patients with BMI values >35 kg/m 2 were not included in this study. BMI neither affected the results nor differed between the groups. Kim et al. emphasized that the risk of postoperative shoulder pain increases with decreased height, weight, and BMI and with greater abdominal circumference difference in a univariate analysis of patients after laparoscopic appendectomy . High ACI patients, who have larger IAV per unit body area, may have increased residual gas pockets in the abdominal cavity. More abdominal expansion might result in more tensile stress, causing higher postoperative pain in these patients. There is a positive correlation between the intensity of shoulder pain and volume of residual gas . No direct measurement of the residual gas was done in the current study. The abdominal pain VAS score at 30 min postoperative is significantly higher in group 2. This might be due to excessive stretching of the pneumoperitoneum and more retained gas in the abdomen. Several studies have emphasized that time characteristics of postoperative abdominal and shoulder pain differ after laparoscopic surgery. Abdominal pain peaks in the first 24 h postoperative and then decreases, while shoulder pain that is minimal on the day of surgery increases the following day . This study followed that same pattern, with postoperative shoulder pain worsening over time. Li et al. found that shoulder pain is the most resistant type of pain to nonsteroidal anti-inflammatory drugs and opioid analgesics , which is consistent with our findings. Despite the use of additional analgesics, shoulder pain increased over time in our patients. Analgesic administration protocols should be organized according to the characteristics of shoulder pain occurrence . The precise mechanism of shoulder pain after laparoscopic surgery has not been elucidated. A main explanation for referred pain to the shoulder mediated by the phrenic nerve is the extreme stretching of the diaphragm caused by the pressure of the insufflated gas to establish pneumoperitoneum. Subcostal residual carbonic gas is also thought to be responsible, irritating phrenic nerves with its acidic nature . A current review emphasized several methods for reducing or preventing the severity of shoulder pain. These strategies embrace the use of alternative insufflating gases to establish pneumoperitoneum, the use of warmed and humidified gas, the use of drugs preventatively or therapeutically, intraperitoneal application of local anesthetic agents, the use of intraperitoneal drains, and the instillation of intraperitoneal fluid. For the ejection of gas from the abdominal cavity, techniques such as the pulmonary recruitment maneuver, a postoperative Trendelenburg position, or active suction and manual evacuation of gas have been used [ , – ]. Identifying patients most likely develop shoulder pain after laparoscopy may allow for sustainable and individualized strategies to minimize pain. Unfortunately, the literature has shown diverse and occasionally contradictory results concerning the efficacy of these interventions. One limitation of this study is that we used the Du Bois BSA formula, which is calculated using only height and weight. Thus, adipose tissue and muscle mass could not be differentiated. Other limitations were that the study population was composed of patients with both benign and malign gynecological diseases, and there was a small sample size. This study is the first preliminary prospective cohort study to show a high association of ACI with postoperative abdominal pain, shoulder pain, and postoperative analgesic consumption after laparoscopic gynecological surgery. ACI, a surrogate index of abdominal expansion capacity, may be used to guide individualization of insufflation pressures by identifying female patients at risk of higher postoperative pain.
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Infrared monitoring-based optimization of interventional lumbar sympathectomy outcomes evaluation in peripheral vascular disease patients: Experimental trial thermovision-guided lumbar sympathectomy | d607cfcd-6314-49d5-84ee-94987172dde6 | 11835127 | Surgical Procedures, Operative[mh] | Chronic limb-threatening ischemia (CLTI) represents the end stage of peripheral arterial disease (PAD). The accurate determination of CLTI epidemiology is not possible, particularly due to developing countries and secondarily due to a lack of high-quality data, especially from developing countries. In an analysis of the US MarketScan database, the prevalence and annual incidence of CLTI were estimated at 1.33% and 0.35%, respectively. Another recently published study reported an estimated annual incidence and an estimated 2-year prevalence of 0.33% and 0.74%, respectively. The prognosis of CLTI patients is alarming, resulting in significant mortality, limb loss, pain, and a diminished health-related quality of life. Outcomes are even more devastating when revascularization is impossible. There is a high risk of ischemic events in the CLTI population; thus, treatment must include optimal medical therapy consisting of lifestyle modifications, that is, smoking cessation, healthy diet, and weight loss, and pharmacological treatment, that is, antithrombotic therapy, antihypertensive therapy, lipid-lowering therapy, and diabetes management. In addition, rest pain, optimal wound care, and infection control should be managed. Exercise training is not recommended in patients with CLTI and wounds. The endovascular or surgical revascularization is recommended for the limb salvage when it is possible. Intermittent pneumatic compression or spinal cord stimulation can be considered in carefully selected patients (e.g., rest pain and minor tissue loss) in whom revascularization is not possible. Prostanoids can be considered selectively for patients with rest pain or minor tissue loss and in whom revascularization is not possible. The administration of vasoactive drugs (naftidrofuryl, pentoxifylline, and cilostazol) for these patients is not recommended. Although endovascular treatment has made remarkable progress in CLTI patient management, the risk of amputation persists even after successful revascularization. In the study by Baubeta et al , the amputation rate reached 12% during the first 6 months despite successful revascularization. According to another review of recent literature, amputation rates exceeded 15–20% within the first year. Persistent microvascular dysfunction may continue to exist even after successful endovascular treatment, and it may be a factor influencing wound healing. Microvascular dysfunction is associated with impaired autoregulation of blood flow and vascular tone, leading to reduced oxygen delivery to the tissue, increased oxidative stress, and capillary rarefaction. The lumbar sympathectomy (LS) still represents one of the supportive treatment possibilities for CLTI patients. The interventional variant of LS is a minimally invasive procedure that irreversibly damages a part of the sympathetic trunk and adjacent ganglia between the L1 and L5 vertebrae, resulting in a reduction of peripheral resistance controlled by sympathetic activity. This potentially increases blood flow at the level of microcirculation. Decrease of pain intensity is another beneficial effect of LS. Despite the positive facts mentioned above, the use of LS has gradually declined. One of the possible explanations for this can be contradictory results of studies dealing with evidence of tissue perfusion improvement after LS. However, one has to highlight that the relevance of commonly selected tests for perfusion assessment at the level of microcirculation after LS is debatable in studies. In recent years, thermal imaging has been used to monitor and diagnose patients with PAD, primarily to assess blood circulation in the limbs. Several studies show that infrared thermography (IRT) can detect temperature changes in areas of impaired blood flow and can serve as a noninvasive indicator of improvement after treatment such as rehabilitation exercise or revascularization. One of the relevant studies followed patients with PAD who underwent a home exercise program. IRT was used to measure the temperature of the foot before and after the exercise program. The results showed that exercise increased the average temperature of the foot, indicating an improvement in blood flow and thus peripheral perfusion, with no differences between genders. Another study monitored the temperature of the feet of patients with critical limb ischemia before and after endovascular revascularization. The average difference in temperatures between less and more affected limbs was approximately 1.7°C before revascularization. It was found that after successful revascularization, there was a significant increase in temperature only in the more affected foot on average 2°C. A systematic review of studies on the use of IRT in the diagnosis and monitoring of PAD confirmed that IRT can be an effective tool for the assessment of blood flow. However, it was pointed out the need for further development and standardization so that this technology can be effectively integrated into clinical practice. IRT offers a practical possibility of evaluating the success of endovascular revascularization in patients with critical limb ischemia, in monitoring the temperature of the foot in patients with diabetic foot and PADs. The aim of our study is to determine the efficacy of lumbar sympathetic block on tissue perfusion evaluated by precise thermal measurements of skin zones (dermatomes) related to peripheral nerves and their associated angiosomes of the foot. In addition, another aim of this study is to find out whether there is any association between the patency of below-the-knee (BTK) arteries and temperatures of dermatomes before and after the LS procedure. In the presented study, we measured changes of foot temperature in dedicated skin zones (dermatomes) using IRT before the LS procedure, and subsequently 10 and 20 minutes after LS in 2 CLTI patients (A, B) with chronic lower limb wound and ischemia at least grade 1 according to Wound, Ischemia, and foot Infection classification. We did not include patients with diabetes mellitus due to the potential sympathectomy-related risks associated with diabetic neuropathy. The ultrasonographic examination of lower limb arteries was performed before admission to the hospital. The angiography of both lower limbs using a retrograde femoral approach was performed in all patients before LS. The side for the femoral approach was chosen based on prior ultrasonographic findings. The presented study is experimental in nature; therefore, revascularization of the BTK arteries was not performed during angiography to avoid the influence of revascularization on the results of thermographic measurements before and after the LS procedure. Revascularization of the BTK arteries was performed using an antegrade femoral approach a few days later. The LS procedure was performed at least 1 day after angiography and endovascular treatment of above-the-knee arteries. The measurement of foot temperature changes using IRT was performed a few minutes before the LS procedure, that is, at least 1 day after angiography and subsequently 10 and 20 minutes after the LS procedure. Ethical approval to report this case series was obtained from the Ethics Committee of the East Slovak Institute of Cardiovascular Diseases, Košice (approval number 4/2023/VUSCH/EK) and was registered on clinicaltrials.gov with registration ID NCT06111599. Written informed consent was obtained from the patients for their anonymized information to be published in this article. 2.1. Technical aspects of a lumbar sympathetic blockade The procedure was conducted under X-ray guidance. The position of the needle was controlled gradually in the anterior-posterior projection and was verified by a small amount of contrast. After visualizing the lumbar sympathetic structures with contrast, the medication (local anesthetic) was administered (Fig. ). 2.2. Infrared thermography measurements—technical aspects We utilized a FLIR SC660 thermal imaging camera (FLIR, Sweden, 640 × 480 pixels) to monitor temperature changes. Thermal imaging with the camera was conducted before LS, 10 minutes after LS, and 20 minutes after LS. We observed temperature changes in selected areas of the foot, that is, dermatomes as depicted in Figure B. The patient was in a supine position during the scan and the measured area was exposed. The ambient temperature remained between 22°C and 23°C during the measurement. The distance between the thermal imaging camera and the measured area was 0.7 to 0.8 meters. Subsequently, we evaluated the thermal imaging images using the FLIR Reporter program (FLIR, Sweden). During the evaluation, the area of interest was outlined by a polygon, and the minimum, maximum, and average temperatures were assessed. Clinically relevant change of temperature was arbitrarily considered an increase of more than 1°C after the LS procedure. The procedure was conducted under X-ray guidance. The position of the needle was controlled gradually in the anterior-posterior projection and was verified by a small amount of contrast. After visualizing the lumbar sympathetic structures with contrast, the medication (local anesthetic) was administered (Fig. ). We utilized a FLIR SC660 thermal imaging camera (FLIR, Sweden, 640 × 480 pixels) to monitor temperature changes. Thermal imaging with the camera was conducted before LS, 10 minutes after LS, and 20 minutes after LS. We observed temperature changes in selected areas of the foot, that is, dermatomes as depicted in Figure B. The patient was in a supine position during the scan and the measured area was exposed. The ambient temperature remained between 22°C and 23°C during the measurement. The distance between the thermal imaging camera and the measured area was 0.7 to 0.8 meters. Subsequently, we evaluated the thermal imaging images using the FLIR Reporter program (FLIR, Sweden). During the evaluation, the area of interest was outlined by a polygon, and the minimum, maximum, and average temperatures were assessed. Clinically relevant change of temperature was arbitrarily considered an increase of more than 1°C after the LS procedure. We measured the intensity of pain before the operation and 20 minutes, 12 hours, 24 hours, and 48 hours after the lumbar sympathetic blockade procedure using a numerical rating scale (0–10). All data generated or analyzed during this study are included in this published article [and its supplementary information files]. The clinical characteristics of the treated patients are presented in Table . The assessment of pain in all patients showed the most significant improvement 12 hours after the procedure, as measured by the numerical rating scale, and this improvement persisted with only slight changes within 48 hours (Table ). The angiography showed stenosis of the superficial femoral artery, occlusion of anterior tibial artery and peroneal artery (PA) on the right limb, and occlusion of posterior tibial artery (PTA) on the left limb in Patient A. PTA artery was occluded on both limbs in patient B and in addition, PA was occluded on the right limb. The popliteal artery was stenosed on the left limb in patient B. The distribution of lesions affecting the lower limb arteries in patients A and B is shown in Table for better orientation. Figures and show the angiographic findings and distribution of lesions affecting BTK arteries in patients A and B. The scoring balloon angioplasty of superficial femoral artery stenosis in patient A and popliteal artery in patient B was performed. BTK arteries were left without treatment. Comparing the left and right foot, the relevant increase of temperature was observed in patient A on the left foot in the dermatome of lateral and medial plantar nerve ranging from 1.4°C to 2.4°C (Figures – and Table ), and on the right foot in the dermatome of the superficial peroneal nerve and sural nerve ranging from 1.7°C to 2.4°C (Figures – and Table ). In patient B, there was an apparent clinically relevant increase of temperature from 1.6°C up to 2.3°C in the dermatome of the lateral plantar, medial plantar, and tibial nerve on the left foot (Figures – and Table ). On the right foot, the clinically relevant increase of temperature ranging from 1.8°C up to 2.4 °C was found in the dermatome of the lateral plantar, medial plantar, tibial, and sural nerve (Figures – and Table ). One month after the LS procedure all wounds on lower limbs were healed up. Denervation of the lumbar sympathetic ganglia increases blood flow to the lower limb by inducing vasodilation through the reduction of sympathetic tone. This, in turn, improves tissue oxygenation, reduces tissue damage, and alleviates pain. Despite the positives of LS mentioned above, several randomized trials have failed to identify objective parameters for evaluating the effect of sympathectomy. However, it is worth noting that the effectiveness of sympathectomy was assessed based on changes in pain intensity, wound healing (or amputations), and ankle-brachial index (ABI). Pain intensity is a highly subjective parameter. Although ABI is a fast, simple, and easily reproducible test, it is primarily intended for vascular screening. In addition, ABI may be less useful in the settings of noncompressible vessels due to Mönckeberg medial calcific sclerosis. However, among patients with diabetic foot ulceration, the measurement of skin perfusion pressures, toe pressures, and transcutaneous oxygen pressure appears to be more useful in predicting ulcer healing than ankle pressures or the ABI. IRT is an instrumental tool that is contact-free, noninvasive, low-cost, simple to use, and not influenced by the operator’s experience. It offers a fast, painless, and radiation-free method of photographically imaging temperature differences of the skin surface, allowing arbitrary repetitions of recording. Thermal imaging of the body surface can detect minute changes in the underlying tissues, which indicates neurological and vascular as well as metabolic pathologies. For this purpose, modern infrared thermal imaging cameras are used, which have a high-temperature sensitivity and produce high-resolution thermal images. Thermographic devices detect the infrared radiation emitted by the body surface, which is invisible to humans, convert it into electrical signals, and a thermogram can be created, which shows temperatures pictorially in different colors or gray tones. Temperature emitted from the skin directly depends on the quality of tissue blood perfusion. A reduction in tissue blood perfusion due to arterial vessel obstruction compromises cellular metabolism, resulting in decreased heat production. IRT has proven to be a reliable tool for assessing the severity of atherosclerotic disease in a patient’s limbs, showing lower temperature values in the less perfused limb. It is a safe, reliable, and easy-to-use tool, making it a valuable instrument for assessing the clinical condition and severity of foot blood perfusion in symptomatic patients with lower extremity arterial disease. It has been demonstrated that thermographic measurements are associated with wound healing and can serve as an independent predictor for achieving 6-month freedom from major amputation in clinical practice. The angiosome concept, initially introduced in plastic surgery by Taylor and Palmer in 1987, was also adopted by vascular medicine for the purpose of targeted revascularization and this way improving wound healing. It is defined as a three-dimensional network of vessels not only in the skin but also in all tissue layers between the skin and the bone. In the zones between adjacent angiosomes, 2 types of anastomotic arteries were identified: reduced-caliber (“choke”) and similar-caliber (“true”) anastomotic arteries. These “choke vessels” typically remain in a state of reduced caliber during routine circulation, essentially lying dormant. However, they have the capacity to dilate and become active during times of increased demand to facilitate greater blood flow. Choke vessels can serve as crucial safety conduits, enabling one angiosome to receive blood from a neighboring angiosome in the event of an occlusion of the original source artery. These vessels are controlled by hormonal, nervous, or biochemical factors. In the most common angiosome model, the foot consists of 6 angiosomes originating from the 3 main BTK arteries, that is, anterior tibial artery, PTA, and PA (Fig. A). In our pilot clinical trial, we decided to evaluate and compare temperature changes using IRT in dedicated skin areas—dermatomes on the foot in CLTI patients after the LS procedure adjusted to the patency of the source BTK artery. Dermatomes represent the surface area of angiosomes; therefore, it makes sense to evaluate perfusion in angiosomes by temperature changes in dermatomes. The anatomical relationship between dermatomes and angiosomes of the foot is shown in Figure A (Angiosomes) and Figure B (Dermatomes). We observed a clinically relevant increase of temperature in patient A on the left foot in dermatome of lateral and medial plantar nerve that corresponds to the angiosome of PTA and in the dermatome of the superficial peroneal nerve and sural nerve on the right foot corresponding to the anterior tibial and PA. In patient B, a clinically relevant increase of temperature was documented in the dermatomes corresponding to PTA on both feet and PA on the right one. Our results suggest that a clinically relevant increase of temperature was observed only in dermatomes corresponding to angiosomes with occluded source artery. One potential explanation of this could be our hypothesis that alteration in sympathetic tone by the LS procedure could cause dilation and reorientation of flow in choke vessels, and this way the blood flow from the adjacent angiosome with patent source artery to angiosome with occluded source artery could increase. This increase of blood flow could be objectified using IRT as an increase of temperature in dedicated dermatome corresponding to underlying angiosome. Our study did not demonstrate the clinically relevant increase of temperature in all dermatomes of the foot after the LS procedure equally, but the area-specific increase of temperature was found, that is, an increase of temperature in dermatomes corresponding to angiosomes without direct flow due to source artery occlusion. This could be considered as further proof supporting the use of LS for CLTI patients without diabetes, especially when revascularization is not possible. In addition, LS provides a pain-relieving effect. Our results also suggest that IRT could be an effective and more appropriate method for evaluating LS procedure outcomes compared with commonly used hemodynamic tests. On the other hand, the decision to use the increase of temperature in the dermatome of more than 1°C for the criterion of LS efficacy may be considered as the most disputable part of the presented study. Two main arguments can be used for the explanation of this: technical parameters of thermal imaging cameras and results of other studies dealing with the use of IRT in medicine. First, the thermal sensitivity and accuracy of the FLIR SC660 thermal imaging camera used in the presented study is 0.045°C at 30°C and ± 1°C, respectively. Second, there is no wide consensus in the literature on what exact value of temperature difference should be considered clinically relevant. The vast majority of studies just compared temperatures of healthy and affected limb/feet or more and less affected limb by the atherosclerosis at the rest, or mean increase of limb/feet temperatures before and after the revascularization procedure. The reported mean difference in temperature of feet in the studies varies approximately at the level of 0.7–1.3°C between the affected and nonaffected limbs by atherosclerosis at baseline, and 0.7–1.7°C between more and less impaired limbs in patients with PAD. The mean increase in temperature of feet before and after revascularization varies between 0.4 and 2.1°C in patients with PAD. In conclusion, our findings suggest that lumbar sympathetic blockade may be beneficial for CLTI patients, especially for those without diabetes. However, it must be emphasized that the mentioned results are too preliminary, and further randomized trials are necessary to assess the effect of lumbar sympathetic blockade in CLTI patients for whom endovascular treatment or surgery is not feasible due to technical or medical reasons. The use of new and progressive techniques of LS, like cryoablation or radiofrequency ablation, may potentially extend the duration of sympathetic denervation, and this way the short-term effect, that is, the most criticized shortcoming of LS, could be overcome. The decision to consider the 1°C increase in temperature over time as a relevant factor for the evaluation of the effectiveness of LS represents a significant methodological limitation of this study that could be a potential source of bias. This could be considered as the main limitation of this study. We also identified 2 further limitations of our study: nonrandomized design and small sample size; thus the documented beneficial effect of LS is based mainly on our experimental hypothesis. Methodology: Ladislav Kočan, Viktória Rajťúková. Writing—original draft: Ladislav Kočan, Marek Hudák. Conceptualization: Marek Hudák. Data curation: Viktória Rajťúková. Investigation: Viktória Rajťúková, Vasil Šatnik. Supervision: Mária Rašiová, Igor Martuliak. Writing—review & editing: Mária Rašiová, Janka Vašková, Vasil Šatnik. Formal analysis: Hana Kočanová, Janka Vašková. Validation: Hana Kočanová, Igor Martuliak. Funding acquisition: Radovan Hudák. Project administration: Radovan Hudák. |
Chirurgisches Nahtmaterial – Grundlagen | b3f46aa7-77eb-49a3-b465-b43450136e9c | 10520208 | Suturing[mh] | Nach der Lektüre dieses Beitrags ... kennen Sie die verschiedenen Arten und Formen von Nahtmaterialien, können Sie resorbierbare von nichtresorbierbaren Materialien unterscheiden, sind Ihnen die Eigenschaften, Vor- und Nachteile von monofilem und polyfilem Nahtmaterial bekannt, sind Sie in der Lage, die Techniken der chirurgischen Knoten zu beschreiben und nach Übung zu praktizieren, ist Ihnen die Technik des Fadenzugs bekannt, und Sie können diese durchführen.
Chirurgische Nähte werden zur Adaptation von Wundrändern und Gewebe verwendet, ohne dabei übermäßige, schädigende Spannung im Sinne von Ischämie zu verursachen . Das Nahtmaterial Nahtmaterial muss dabei in der Lage sein, das Gewebe bis zur vollständigen Heilung zusammenhalten zu können. Bei resorbierbaren Nähten resorbierbaren Nähten darf die Resorption erst nach der Heilung einsetzen. Im Weiteren soll das Nahtmaterial für den Chirurgen sicher und einfach anwendbar und an die zu nähenden Strukturen angepasst sein. Das Nahtmaterial selbst sollte dabei möglichst geringe Interaktion mit dem Gewebe hervorrufen. Oft setzt der Patient die sichtbare Wunde (Hautnaht) respektive die später sichtbare Narbe mit der Qualität oder Erfolg der Operation gleich. Wir wissen jedoch, und dies sollte dem Patienten vor jeder Operation schon gesagt werden, dass nicht jeder Mensch eine identische Narbenbildung Narbenbildung hat. Kinder und Jugendliche neigen viel stärker zur hypertrophen Narbenbildung (bei uns fälschlicherweise als Keloid bezeichnet), wogegen ältere Menschen aufgrund der Atrophie des Gewebes kosmetisch sehr ansprechende Narben aufweisen können. Das innere funktionelle Ergebnis wie auch die äußere sichtbare Narbe können durch nichtadäquates Nahtmaterial und Nahttechnik, aber auch durch eine inadäquate Inzision (Hautlinien) sehr stark leiden. Im Weiteren hängt das Resultat der Narbe nicht zuletzt auch von der Wundspannung Wundspannung bei Schwellung oder durch übermäßige Hautexzision (z. B. Narbenkorrektur) ab . Prinzipiell können folgende Faktoren die Qualität (Wahrnehmung) einer Naht beeinflussen: Entstehungsart der Wunde (elektiv oder traumatisch), Lokalisation der Wunde, Hautdicke/Hautbeschaffenheit, Spannung der Weichteile, kosmetisches Ziel/Erwartungshaltung (dies ist eine Wahrnehmung), Infekte, Anordnung der Einstichstellen: Distanz zum Wundrand, konstanter Abstand der einzelnen Einstichstellen, Alter des Patienten. Nahtmaterial Die Wahl des Nahtmaterials hängt einerseits von der Körperregion, der zu erwartenden Spannung des Gewebes bzw. der Wunde ab, andererseits aber auch vom Zustand des Gewebes selbst. Die verschiedenen Nahtmaterialien lassen sich nach Zugfestigkeit, Knotenfestigkeit, Handhabung und Gewebereaktion sowie nach Resorbierbarkeit einteilen. Welchen Ansprüchen das ideale Nahtmaterial ideale Nahtmaterial in Bezug auf den zu erfüllenden Zweck genügen muss, hat der Chirurg zu definieren und danach zu wählen. Wunsch des Operateurs ist die Kombination von hoher Zugfestigkeit, einfacher Handhabung, geringer bis keiner Gewebereaktivität und minimalem Infektionsrisiko. Das Material soll gut im Gewebe sichtbar sein, und es soll aus nichtkapillarem, nichtallergenem und nichtkarzinogenem Material bestehen . Es werden hauptsächlich 2 Gruppen unterschieden: resorbierbares und nichtresorbierbares Nahtmaterial. Resorbierbare Fäden Resorbierbare Fäden verlieren ihre Zugfestigkeit meist in weniger als 60 Tagen. Diese werden in der Regel nicht entfernt. Nichtresorbierbare Fäden Nichtresorbierbare Fäden behalten den Hauptteil der Zugfestigkeit über 2 Monate hinaus. Diese auch für Hautnähte gebräuchlichen Fäden werden entfernt. Auf Wundverschluss mit Klammersystemen wird in dieser Arbeit nicht eingegangen. Wundheilung Es wird die primäre von der sekundären Wundheilung unterschieden. In diesem Beitrag soll es weitgehend um chirurgische Wunden nach operativem Management also primäre Wundheilung primäre Wundheilung gehen. Die ersten 3 Tage nach Haut- und Weichteilverschluss werden als exsudative oder inflammatorische Phase inflammatorische Phase charakterisiert. Es folgt die proliferative Phase proliferative Phase an Tag 4 bis 7 gefolgt von der reparativen Phase reparativen Phase ab Tag 8 bis zu Monaten. Auf die Darstellung der Details der pathophysiologischen Abläufe wird hier verzichtet. Das Nahtmaterial ist in der ersten Phase für den Zusammenhalt der Wunde verantwortlich. Nahtmaterialien können im Gewebe Fremdkörperreaktionen Fremdkörperreaktionen auslösen. Daher sollten bei der Aufklärung zur Operation die Patienten nach bisherigen Reaktionen auf Nahtmaterial gefragt werden. Merke Es empfiehlt sich, möglichst den dünnsten erforderlichen Faden zu verwenden, um Fremdkörperreaktionen so gering wie möglich zu halten.
Die Wahl des Nahtmaterials hängt einerseits von der Körperregion, der zu erwartenden Spannung des Gewebes bzw. der Wunde ab, andererseits aber auch vom Zustand des Gewebes selbst. Die verschiedenen Nahtmaterialien lassen sich nach Zugfestigkeit, Knotenfestigkeit, Handhabung und Gewebereaktion sowie nach Resorbierbarkeit einteilen. Welchen Ansprüchen das ideale Nahtmaterial ideale Nahtmaterial in Bezug auf den zu erfüllenden Zweck genügen muss, hat der Chirurg zu definieren und danach zu wählen. Wunsch des Operateurs ist die Kombination von hoher Zugfestigkeit, einfacher Handhabung, geringer bis keiner Gewebereaktivität und minimalem Infektionsrisiko. Das Material soll gut im Gewebe sichtbar sein, und es soll aus nichtkapillarem, nichtallergenem und nichtkarzinogenem Material bestehen . Es werden hauptsächlich 2 Gruppen unterschieden: resorbierbares und nichtresorbierbares Nahtmaterial. Resorbierbare Fäden Resorbierbare Fäden verlieren ihre Zugfestigkeit meist in weniger als 60 Tagen. Diese werden in der Regel nicht entfernt. Nichtresorbierbare Fäden Nichtresorbierbare Fäden behalten den Hauptteil der Zugfestigkeit über 2 Monate hinaus. Diese auch für Hautnähte gebräuchlichen Fäden werden entfernt. Auf Wundverschluss mit Klammersystemen wird in dieser Arbeit nicht eingegangen.
Es wird die primäre von der sekundären Wundheilung unterschieden. In diesem Beitrag soll es weitgehend um chirurgische Wunden nach operativem Management also primäre Wundheilung primäre Wundheilung gehen. Die ersten 3 Tage nach Haut- und Weichteilverschluss werden als exsudative oder inflammatorische Phase inflammatorische Phase charakterisiert. Es folgt die proliferative Phase proliferative Phase an Tag 4 bis 7 gefolgt von der reparativen Phase reparativen Phase ab Tag 8 bis zu Monaten. Auf die Darstellung der Details der pathophysiologischen Abläufe wird hier verzichtet. Das Nahtmaterial ist in der ersten Phase für den Zusammenhalt der Wunde verantwortlich. Nahtmaterialien können im Gewebe Fremdkörperreaktionen Fremdkörperreaktionen auslösen. Daher sollten bei der Aufklärung zur Operation die Patienten nach bisherigen Reaktionen auf Nahtmaterial gefragt werden. Merke Es empfiehlt sich, möglichst den dünnsten erforderlichen Faden zu verwenden, um Fremdkörperreaktionen so gering wie möglich zu halten.
Es empfiehlt sich, möglichst den dünnsten erforderlichen Faden zu verwenden, um Fremdkörperreaktionen so gering wie möglich zu halten.
Bei der Beurteilung der Eigenschaften von Nahtmaterial sind verschiedene Parameter zu beachten: physikalische Parameter, Flüssigkeitsaufnahme und Kapillarität, Kaliber oder Durchmesser, Zugfestigkeit, Torsion, Absorptionsfähigkeit, Elastizität, Plastizität, Gedächtnis, Reibungskoeffizient und Knotensicherheit. Die optimalen Bereiche für jede dieser Eigenschaften sind für die meisten Nahtmaterialien und Indikationen noch nicht definiert . Das Nahtmaterial muss auch eine hohe Biegsamkeit hohe Biegsamkeit (Geschmeidigkeit) und Flexibilität aufweisen, damit es sich beim Nähen besser handhaben lässt. Darüber hinaus sind eine einfache Knotenplatzierung, eine hohe Knotensicherheit, aber auch Reizfreiheit und Schutz vor Infektionen weitere wichtige und zu fordernde Eigenschaften. . Die verwendeten Materialien verwendeten Materialien unterscheiden sich hinsichtlich: Reißfestigkeit/Zugfestigkeit (Zugspannung im Augenblick des Reißens des Nahtmaterials), Elastizität (Fähigkeit, nach Dehnung wieder in die Ursprungslänge zurück zu gelangen), Plastizität (Eigenschaft, nach Dehnung die neue Länge beizubehalten ), Memory-Effekt (nach Formveränderung wieder in den ursprünglichen Zustand zurückzukehren ), bestimmter Durchmesser (Kaliber), Festigkeit der Knoten-Oberfläche, Aufnahme von Flüssigkeit ins Material (Fähigkeit des Nahtmaterials, Flüssigkeit aufzusaugen, zu absorbieren ), Kapillarität (Fähigkeit des Nahtmaterials, Flüssigkeit aufzunehmen und im Material weiterzuleiten ), Eigenschaften in der Handhabung, Biegsamkeit (lässt sich leicht biegen) (Geschmeidigkeit), Reibungskoeffizient (leichtes Gleiten) – zur Verhinderung von Gewebewiderstand, Verrutschen von Knoten und zur Erleichterung des Knüpfens von Knoten, Eigenschaft der Gewebereaktion, nicht allergen, nicht krebserregend, (minimale) Gewebereaktionen, physikalische Eigenschaften, Reibungskoeffizient, Monofilament oder Multifilament, Nahtmaterial mit Widerhaken, Kapillarität, Absorptionsfähigkeit. Reißfestigkeit. Reißfestigkeit (Zugfestigkeit) ist der Kraftaufwand, um einen linear gestreckten Faden zu zerreißen . Knoten. Mit Knotensitz Knotensitz wird das sichere Halten des Knotens auf dem Faden nach Einbringung ins Gewebe bezeichnet. Der Knoten soll im Endzustand nicht auf dem Faden rutschen. Knotenreißkraft Knotenreißkraft ist der Kraftaufwand, der benötigt wird, einen geknoteten Faden zu zerreißen. Es ergeben sich definierte Prüfwerte. Die Knotenbruchfestigkeit Knotenbruchfestigkeit sagt aus, bei welcher Kraft der Faden im Knoten reißt. Im Knoten hat der Faden den schwächsten Punkt der Naht. Torsion. Sie wird durch die Anzahl der Verdrehungen im Faden dargestellt. Diese steht in umgekehrtem Verhältnis zur Zugfestigkeit der Naht. Eine Vergrößerung des Durchmessers einer Naht führt zu einer Erhöhung der Längskraft Längskraft , die erforderlich ist, um die Naht zu zerreißen; eine Verdoppelung des Durchmessers der Naht erfordert eine Vervierfachung des zum Zerreißen der Naht erforderlichen Gewichts . Dochtwirkung. Dochtwirkung fußt auf der Kapillarität des Fadens. Nur die monofile Fadenkonstruktion monofile Fadenkonstruktion gibt zuverlässigen Schutz vor Bakterientransport oder -migration . Die multifilen und pseudomonofilen Fadenkonstruktionen fördern ausnahmslos die Übertragung von Bakterien, wenn auch in unterschiedlichem Maße . Das Eindringen von Flüssigkeiten und Bakterien ist abhängig von den Absorptionseigenschaften, der Beschichtung und dem Vorhandensein eines offenen Fadenendes . Auch die Bindung von Bakterien an das Nahtmaterial ist abhängig von dem Fadentyp, von Material und Oberfläche. Geflochtenes, also polyfiles resorbierbares Nahtmaterial sollte nicht bei infektiösem Gewebe verwendet werden . Quellung. Quellung ist das Aufsaugen von Flüssigkeit ( Wundsekret Wundsekret ) vom Nahtmaterial. Gewebedurchzug. Hiermit ist das Gleiten des Nahtmaterials durch das Gewebe charakterisiert. Sterilität. Sterilität wird durch Sterilisationsverfahren Sterilisationsverfahren erzielt, Chromierung z. B. bei Katgut. Elongation. Elongation ist die Ausdehnung des Nahtmaterials. Diese kann temporär oder auch dauerhaft auftreten. Resorption. Die Resorptionszeit Resorptionszeit gibt den Zeitpunkt an, bis zu dem das gesamte Material abgebaut wird. Die Halbwertszeit gibt den Zeitpunkt an, bis zu dem noch 50 % der Ausgangsreißkraft vorliegt (s. oben).
Reißfestigkeit (Zugfestigkeit) ist der Kraftaufwand, um einen linear gestreckten Faden zu zerreißen .
Mit Knotensitz Knotensitz wird das sichere Halten des Knotens auf dem Faden nach Einbringung ins Gewebe bezeichnet. Der Knoten soll im Endzustand nicht auf dem Faden rutschen. Knotenreißkraft Knotenreißkraft ist der Kraftaufwand, der benötigt wird, einen geknoteten Faden zu zerreißen. Es ergeben sich definierte Prüfwerte. Die Knotenbruchfestigkeit Knotenbruchfestigkeit sagt aus, bei welcher Kraft der Faden im Knoten reißt. Im Knoten hat der Faden den schwächsten Punkt der Naht.
Sie wird durch die Anzahl der Verdrehungen im Faden dargestellt. Diese steht in umgekehrtem Verhältnis zur Zugfestigkeit der Naht. Eine Vergrößerung des Durchmessers einer Naht führt zu einer Erhöhung der Längskraft Längskraft , die erforderlich ist, um die Naht zu zerreißen; eine Verdoppelung des Durchmessers der Naht erfordert eine Vervierfachung des zum Zerreißen der Naht erforderlichen Gewichts .
Dochtwirkung fußt auf der Kapillarität des Fadens. Nur die monofile Fadenkonstruktion monofile Fadenkonstruktion gibt zuverlässigen Schutz vor Bakterientransport oder -migration . Die multifilen und pseudomonofilen Fadenkonstruktionen fördern ausnahmslos die Übertragung von Bakterien, wenn auch in unterschiedlichem Maße . Das Eindringen von Flüssigkeiten und Bakterien ist abhängig von den Absorptionseigenschaften, der Beschichtung und dem Vorhandensein eines offenen Fadenendes . Auch die Bindung von Bakterien an das Nahtmaterial ist abhängig von dem Fadentyp, von Material und Oberfläche. Geflochtenes, also polyfiles resorbierbares Nahtmaterial sollte nicht bei infektiösem Gewebe verwendet werden .
Quellung ist das Aufsaugen von Flüssigkeit ( Wundsekret Wundsekret ) vom Nahtmaterial.
Hiermit ist das Gleiten des Nahtmaterials durch das Gewebe charakterisiert.
Sterilität wird durch Sterilisationsverfahren Sterilisationsverfahren erzielt, Chromierung z. B. bei Katgut.
Elongation ist die Ausdehnung des Nahtmaterials. Diese kann temporär oder auch dauerhaft auftreten.
Die Resorptionszeit Resorptionszeit gibt den Zeitpunkt an, bis zu dem das gesamte Material abgebaut wird. Die Halbwertszeit gibt den Zeitpunkt an, bis zu dem noch 50 % der Ausgangsreißkraft vorliegt (s. oben).
Die Unterscheidung zwischen traumatischem und atraumatischem Nahtmaterial bezieht sich auf die Nadel-Faden-Verbindung Nadel-Faden-Verbindung . Traumatische Fäden Traumatische Fäden werden in das Öhr der Nadel eingefädelt, während atraumatisches Nahtmaterial atraumatisches Nahtmaterial in der Nadel mittels Quetschung, Verklebung oder Laserung quasi ohne Durchmesseränderung fixiert ist . Bei der Auswahl des Nahtmaterials gilt der Grundsatz, mit einem Minimum an unerwünschten Gewebereaktionen und infektiösem Potenzial eine ausreichende Festigkeit der Wunde Festigkeit der Wunde für die notwendige Dauer der Wundheilung zu erreichen. Stärkeres oder zugfesteres Nahtmaterial ist jedoch nicht immer besser, da das Fadenkaliber erhöht werden muss und es zu einer unbeabsichtigten Strangulierung des Gewebes kommen kann, was einerseits die Durchblutung gefährdet, möglicherweise auch die Entzündungsreaktivität erhöht . Die einzelnen Gewebe, Schichten und Indikationen erfordern Konditionen von der Sehnenreparatur bis zur feinen kosmetischen Naht im Gesicht (Abb. ). Aufbau des Nahtmaterials Nahtmaterial wird in verschiedenen Konfigurationen angeboten. Beim Aufbau von Nahtmaterial wird unterschieden zwischen monofilem, poly- oder multifilem Aufbau. Dabei können auch zusätzlich Beschichtungen oder Ummantelungen aufgebracht sein (s. Tabelle A2; Abb. ). Monofil Monofil bezeichnet man ein einzelsträngiges Filamentnahtmaterial einzelsträngiges Filamentnahtmaterial (z. B. Polyamid 6 oder Nylon). Diese Fäden werden mit dem Verfahren der Extrudierung hergestellt. Kapillarität innerhalb des Nahtmaterials tritt nicht auf, die Oberfläche ist glatt, wodurch das Infektionsrisiko geringer ist. Die Gewebepenetration bzw. der Gewebedurchzug wird durch die glatte Oberfläche erleichtert. Aufgrund verminderter Reibung aufgrund der glatten Oberfläche ist dagegen die Haltekraft der Knoten vermindert, sodass normalerweise mehr Knoten als bei multifilem Material gelegt werden müssen. Das Fadenmaterial kann bei der Handhabung etwas rigide erscheinen. Polyfil/multifil Bei den multiplen Fäden werden mehrere Einzelfäden (Filamente) verdreht, geflochten oder verzwirnt. Auf dem Boden der vermehrten Rauigkeit vermehrten Rauigkeit und damit höheren Reibung halten Knoten dieses Materials besser. Merke Bei geflochtenen Fäden halten die Knoten aufgrund der Oberflächenrauigkeit und damit höheren Reibehaftung besser. Die rauere Oberflächenbeschaffenheit führt beim Durchzug des Fadens durch das Gewebe zu einer Sägewirkung des Fadens. Werden diese Fäden in steilem Winkel durch das Gewebe gezogen, ist die Sägewirkung des Nahtmaterials deutlich geringer als im flachen Winkel. Merke Bei geflochtenem Nahtmaterial wird mit einem steilen Eintrittswinkel zur Oberfläche die Sägewirkung des Fadens im Gewebe verringert. Bei diesen Fäden kann auch auf dem Boden der Kapillarität eine Dochtwirkung Dochtwirkung entstehen, die den Transport von Keimen in die Wunde entlang des Fadens begünstigen kann. Je glatter die Oberflache ist – z. B. Vergleich monofiler Polyesterfaden und polyfiler Polyesterfaden –, desto geringer ist die Anzahl an Bindegewebe- und Fremdkörperriesenzellen, die sich in und um den Faden nach Implantation ansammeln . Hierbei ist die Dochtwirkung bei geflochtenen Fäden geringer als bei gezwirnten, da die Einzelfilamente beim Flechten fast quer zur Längsachse liegen. Insgesamt ist bei geflochtenem Nahtmaterial die Oberfläche größer, und Todräume innerhalb des Fadens sind vermehrt, sodass dadurch die potenzielle Bakterienansiedlung gefördert wird . Das Material wirkt beim Knoten geschmeidig, der Knotensitz aufgrund der Reibung ist gut, die Reißkraft ist hoch. Durch Beschichtung multipler Fäden wird die Reibekraft der Oberfläche reduziert und ein besserer Durchzug erzielt. Selbstsichernde Nahtmaterialien Wird die Oberfläche der Nahtmaterialien mit Widerhaken Widerhaken versehen, die so angeordnet sind, dass sie beim Einstechen an der Oberfläche anliegen, bei entgegengesetzter Bewegung heraustreten, kann auf Knotung verzichtet werden [ , , ]. Andere Autoren weisen auf die Zeitökonomie beim Einsatz dieser Materialien hin [ , , ] und auf ein geringeres Gesamtkomplikationsrisiko bei (arthroskopischen) Knieoperationen . Nahtmaterialfarben Gefärbtes Material kann dazu beitragen, während der Operation besser sichtbar zu sein, was bei Sehnennähten von Vorteil sein kann. Bei Intrakutannähten ist farbiges Nahtmaterial unvorteilhaft, da es durchscheinen kann und wie eine Hauttätowierung imponieren kann. Bei Hautnähten, die ja meist gezogen werden, erleichtert das Anfärben des Nahtmaterials durch Kontrastierung gegenüber der Haut das Fadenziehen. Merke Bei Intrakutannähten wird farbloses Nahtmaterial verwendet. Farbmarkierung. Die Farbkodierungen des Nahtmaterials auf den Verpackungen der verschiedenen Hersteller sind nicht kompatibel. Fadenstärke, -durchmesser, Kaliber In Europa wird verbindlich nach der Europäischen Pharmakopöe Europäischen Pharmakopöe (Ph.Eur.) metrisch die Nahtmaterialstärke festgelegt. Auf den Nahtmaterialverpackungen finden sich zusätzlich Bezeichnungen der United States Pharmakopöe (USP) , die kein direktes Verhältnis zum Fadendurchmesser erkennen lassen (Tab. ). Für USP gilt, je größer die Zahl des Nahtmaterials ist, desto kleiner ist der Durchmesser, z. B. ist ein 7‑0-Nahtmaterial kleiner als ein 4‑0-Nahtmaterial. Im Markt haben sich die historisch gewachsenen Stärkeangaben der USP weltweit durchgesetzt. Unabhängig davon, ob es sich um einen ein- oder mehrschichtigen Wundverschluss handelt, sollte die kleinste Fadenstärke bzw. der kleinste Durchmesser des Nahtmaterials gewählt werden, die bzw. der den jeweiligen Zweck erfüllt, um sowohl das Gewebetrauma Gewebetrauma bei jedem Nadeldurchgang als auch die Menge des zurückbleibenden Fremdmaterials zu minimieren. Materialien aus natürlichen und synthetischen Stoffen, aber auch aus Metall kommen zum Einsatz . Merke Benutze die kleinste Fadenstärke, die die Voraussetzungen erfüllt. Resorption Die Begriffe Resorption und Absorption werden synonym verwendet. Die wichtigsten Merkmale für den biologischen Abbau und die Absorption von resorbierbarem Nahtmaterial sind das Festigkeits- und Massenverlustprofil sowie die Biokompatibilität der Abbaumaterialien. Bei Nahtmaterial auf Proteinbasis Nahtmaterial auf Proteinbasis , das durch proteolytische Enzyme und Phagozyten abgebaut wird, haften die Bakterien besser am Nahtmaterial . Eine verstärkte Kapillarität erhöht ebenfalls das Infektionsrisiko, da sich die Mikroorganismen leichter bewegen und ausbreiten können . Als Resorptionszeit Resorptionszeit wird die Zeitspanne benannt, in der das Material 50 % der Reißkraft des Knotens verliert. Von Massenresorption Massenresorption spricht man, wenn das gesamte Nahtmaterial vom Gewebe abgebaut ist. Abbau durch Hydrolyse. Synthetisches resorbierbares Nahtmaterial kann durch einen hydrolytischen Mechanismus über die Spaltung von Esterbindungen im Polymergerüst abgebaut werden. Sie werden durch natürliche Stoffwechselprozesse natürliche Stoffwechselprozesse fast rückstandsfrei abgebaut . Wenn die Moleküle hydrophob sind, wird ihre Hydrolyse verzögert und ihre Absorptionszeit verlängert . Eindringendes Wasser in das Nahtmaterial zerstört die Polymerstruktur des Fadens. Die Aufrechterhaltung des Gleichgewichts zwischen schneller Absorption und der Verlängerung der Zugfestigkeit wurde durch Behandlungen und chemische Strukturierung unterstützt, die die Absorptionszeit verlängern . Die Art des Abbaus, den das Material erfährt, seine Kapillarität und seine physikalische Konfiguration beeinflussen das Infektionsrisiko . Bei der Hydrolyse kommt es zu einer geringeren Gewebereaktion als beim enzymatischen Abbauprozess . Enzymatischer Abbau. Beim Wundverschluss mit resorbierbarem Nahtmaterial nimmt die Zugfestigkeit in den ersten Wochen allmählich und meist linear ab. Es findet physiologisch eine Gewebe- und Zellreaktion statt, um Zelltrümmer und abbaubare Anteile des Nahtmaterials zu entfernen. Diese Phasen können durch Infektionen und Eiweißmangel beeinträchtigt werden, wobei die Zugfestigkeit schneller verloren geht und eine Wunddehiszenz klinisch sichtbar werden kann. Nach dem primären Zusammenhalt des Gewebes werden diese Nahtmaterialien durch enzymatische oder hydrolytische Prozesse im Gewebe abgebaut. Die Abbaurate Abbaurate variiert in Abhängigkeit vom Material, der Lokalisation und patientenabhängigen Faktoren (Tab. ). Durch diese Prozesse werden Zugfestigkeit und Reißkraft konstant meist nach einer vom Material abhängigen Latenzzeit vermindert.
Nahtmaterial wird in verschiedenen Konfigurationen angeboten. Beim Aufbau von Nahtmaterial wird unterschieden zwischen monofilem, poly- oder multifilem Aufbau. Dabei können auch zusätzlich Beschichtungen oder Ummantelungen aufgebracht sein (s. Tabelle A2; Abb. ). Monofil Monofil bezeichnet man ein einzelsträngiges Filamentnahtmaterial einzelsträngiges Filamentnahtmaterial (z. B. Polyamid 6 oder Nylon). Diese Fäden werden mit dem Verfahren der Extrudierung hergestellt. Kapillarität innerhalb des Nahtmaterials tritt nicht auf, die Oberfläche ist glatt, wodurch das Infektionsrisiko geringer ist. Die Gewebepenetration bzw. der Gewebedurchzug wird durch die glatte Oberfläche erleichtert. Aufgrund verminderter Reibung aufgrund der glatten Oberfläche ist dagegen die Haltekraft der Knoten vermindert, sodass normalerweise mehr Knoten als bei multifilem Material gelegt werden müssen. Das Fadenmaterial kann bei der Handhabung etwas rigide erscheinen. Polyfil/multifil Bei den multiplen Fäden werden mehrere Einzelfäden (Filamente) verdreht, geflochten oder verzwirnt. Auf dem Boden der vermehrten Rauigkeit vermehrten Rauigkeit und damit höheren Reibung halten Knoten dieses Materials besser. Merke Bei geflochtenen Fäden halten die Knoten aufgrund der Oberflächenrauigkeit und damit höheren Reibehaftung besser. Die rauere Oberflächenbeschaffenheit führt beim Durchzug des Fadens durch das Gewebe zu einer Sägewirkung des Fadens. Werden diese Fäden in steilem Winkel durch das Gewebe gezogen, ist die Sägewirkung des Nahtmaterials deutlich geringer als im flachen Winkel. Merke Bei geflochtenem Nahtmaterial wird mit einem steilen Eintrittswinkel zur Oberfläche die Sägewirkung des Fadens im Gewebe verringert. Bei diesen Fäden kann auch auf dem Boden der Kapillarität eine Dochtwirkung Dochtwirkung entstehen, die den Transport von Keimen in die Wunde entlang des Fadens begünstigen kann. Je glatter die Oberflache ist – z. B. Vergleich monofiler Polyesterfaden und polyfiler Polyesterfaden –, desto geringer ist die Anzahl an Bindegewebe- und Fremdkörperriesenzellen, die sich in und um den Faden nach Implantation ansammeln . Hierbei ist die Dochtwirkung bei geflochtenen Fäden geringer als bei gezwirnten, da die Einzelfilamente beim Flechten fast quer zur Längsachse liegen. Insgesamt ist bei geflochtenem Nahtmaterial die Oberfläche größer, und Todräume innerhalb des Fadens sind vermehrt, sodass dadurch die potenzielle Bakterienansiedlung gefördert wird . Das Material wirkt beim Knoten geschmeidig, der Knotensitz aufgrund der Reibung ist gut, die Reißkraft ist hoch. Durch Beschichtung multipler Fäden wird die Reibekraft der Oberfläche reduziert und ein besserer Durchzug erzielt. Selbstsichernde Nahtmaterialien Wird die Oberfläche der Nahtmaterialien mit Widerhaken Widerhaken versehen, die so angeordnet sind, dass sie beim Einstechen an der Oberfläche anliegen, bei entgegengesetzter Bewegung heraustreten, kann auf Knotung verzichtet werden [ , , ]. Andere Autoren weisen auf die Zeitökonomie beim Einsatz dieser Materialien hin [ , , ] und auf ein geringeres Gesamtkomplikationsrisiko bei (arthroskopischen) Knieoperationen .
Monofil bezeichnet man ein einzelsträngiges Filamentnahtmaterial einzelsträngiges Filamentnahtmaterial (z. B. Polyamid 6 oder Nylon). Diese Fäden werden mit dem Verfahren der Extrudierung hergestellt. Kapillarität innerhalb des Nahtmaterials tritt nicht auf, die Oberfläche ist glatt, wodurch das Infektionsrisiko geringer ist. Die Gewebepenetration bzw. der Gewebedurchzug wird durch die glatte Oberfläche erleichtert. Aufgrund verminderter Reibung aufgrund der glatten Oberfläche ist dagegen die Haltekraft der Knoten vermindert, sodass normalerweise mehr Knoten als bei multifilem Material gelegt werden müssen. Das Fadenmaterial kann bei der Handhabung etwas rigide erscheinen.
Bei den multiplen Fäden werden mehrere Einzelfäden (Filamente) verdreht, geflochten oder verzwirnt. Auf dem Boden der vermehrten Rauigkeit vermehrten Rauigkeit und damit höheren Reibung halten Knoten dieses Materials besser. Merke Bei geflochtenen Fäden halten die Knoten aufgrund der Oberflächenrauigkeit und damit höheren Reibehaftung besser. Die rauere Oberflächenbeschaffenheit führt beim Durchzug des Fadens durch das Gewebe zu einer Sägewirkung des Fadens. Werden diese Fäden in steilem Winkel durch das Gewebe gezogen, ist die Sägewirkung des Nahtmaterials deutlich geringer als im flachen Winkel. Merke Bei geflochtenem Nahtmaterial wird mit einem steilen Eintrittswinkel zur Oberfläche die Sägewirkung des Fadens im Gewebe verringert. Bei diesen Fäden kann auch auf dem Boden der Kapillarität eine Dochtwirkung Dochtwirkung entstehen, die den Transport von Keimen in die Wunde entlang des Fadens begünstigen kann. Je glatter die Oberflache ist – z. B. Vergleich monofiler Polyesterfaden und polyfiler Polyesterfaden –, desto geringer ist die Anzahl an Bindegewebe- und Fremdkörperriesenzellen, die sich in und um den Faden nach Implantation ansammeln . Hierbei ist die Dochtwirkung bei geflochtenen Fäden geringer als bei gezwirnten, da die Einzelfilamente beim Flechten fast quer zur Längsachse liegen. Insgesamt ist bei geflochtenem Nahtmaterial die Oberfläche größer, und Todräume innerhalb des Fadens sind vermehrt, sodass dadurch die potenzielle Bakterienansiedlung gefördert wird . Das Material wirkt beim Knoten geschmeidig, der Knotensitz aufgrund der Reibung ist gut, die Reißkraft ist hoch. Durch Beschichtung multipler Fäden wird die Reibekraft der Oberfläche reduziert und ein besserer Durchzug erzielt.
Bei geflochtenen Fäden halten die Knoten aufgrund der Oberflächenrauigkeit und damit höheren Reibehaftung besser. Die rauere Oberflächenbeschaffenheit führt beim Durchzug des Fadens durch das Gewebe zu einer Sägewirkung des Fadens. Werden diese Fäden in steilem Winkel durch das Gewebe gezogen, ist die Sägewirkung des Nahtmaterials deutlich geringer als im flachen Winkel.
Bei geflochtenem Nahtmaterial wird mit einem steilen Eintrittswinkel zur Oberfläche die Sägewirkung des Fadens im Gewebe verringert. Bei diesen Fäden kann auch auf dem Boden der Kapillarität eine Dochtwirkung Dochtwirkung entstehen, die den Transport von Keimen in die Wunde entlang des Fadens begünstigen kann. Je glatter die Oberflache ist – z. B. Vergleich monofiler Polyesterfaden und polyfiler Polyesterfaden –, desto geringer ist die Anzahl an Bindegewebe- und Fremdkörperriesenzellen, die sich in und um den Faden nach Implantation ansammeln . Hierbei ist die Dochtwirkung bei geflochtenen Fäden geringer als bei gezwirnten, da die Einzelfilamente beim Flechten fast quer zur Längsachse liegen. Insgesamt ist bei geflochtenem Nahtmaterial die Oberfläche größer, und Todräume innerhalb des Fadens sind vermehrt, sodass dadurch die potenzielle Bakterienansiedlung gefördert wird . Das Material wirkt beim Knoten geschmeidig, der Knotensitz aufgrund der Reibung ist gut, die Reißkraft ist hoch. Durch Beschichtung multipler Fäden wird die Reibekraft der Oberfläche reduziert und ein besserer Durchzug erzielt.
Wird die Oberfläche der Nahtmaterialien mit Widerhaken Widerhaken versehen, die so angeordnet sind, dass sie beim Einstechen an der Oberfläche anliegen, bei entgegengesetzter Bewegung heraustreten, kann auf Knotung verzichtet werden [ , , ]. Andere Autoren weisen auf die Zeitökonomie beim Einsatz dieser Materialien hin [ , , ] und auf ein geringeres Gesamtkomplikationsrisiko bei (arthroskopischen) Knieoperationen .
Gefärbtes Material kann dazu beitragen, während der Operation besser sichtbar zu sein, was bei Sehnennähten von Vorteil sein kann. Bei Intrakutannähten ist farbiges Nahtmaterial unvorteilhaft, da es durchscheinen kann und wie eine Hauttätowierung imponieren kann. Bei Hautnähten, die ja meist gezogen werden, erleichtert das Anfärben des Nahtmaterials durch Kontrastierung gegenüber der Haut das Fadenziehen. Merke Bei Intrakutannähten wird farbloses Nahtmaterial verwendet. Farbmarkierung. Die Farbkodierungen des Nahtmaterials auf den Verpackungen der verschiedenen Hersteller sind nicht kompatibel.
Bei Intrakutannähten wird farbloses Nahtmaterial verwendet.
Die Farbkodierungen des Nahtmaterials auf den Verpackungen der verschiedenen Hersteller sind nicht kompatibel.
In Europa wird verbindlich nach der Europäischen Pharmakopöe Europäischen Pharmakopöe (Ph.Eur.) metrisch die Nahtmaterialstärke festgelegt. Auf den Nahtmaterialverpackungen finden sich zusätzlich Bezeichnungen der United States Pharmakopöe (USP) , die kein direktes Verhältnis zum Fadendurchmesser erkennen lassen (Tab. ). Für USP gilt, je größer die Zahl des Nahtmaterials ist, desto kleiner ist der Durchmesser, z. B. ist ein 7‑0-Nahtmaterial kleiner als ein 4‑0-Nahtmaterial. Im Markt haben sich die historisch gewachsenen Stärkeangaben der USP weltweit durchgesetzt. Unabhängig davon, ob es sich um einen ein- oder mehrschichtigen Wundverschluss handelt, sollte die kleinste Fadenstärke bzw. der kleinste Durchmesser des Nahtmaterials gewählt werden, die bzw. der den jeweiligen Zweck erfüllt, um sowohl das Gewebetrauma Gewebetrauma bei jedem Nadeldurchgang als auch die Menge des zurückbleibenden Fremdmaterials zu minimieren. Materialien aus natürlichen und synthetischen Stoffen, aber auch aus Metall kommen zum Einsatz . Merke Benutze die kleinste Fadenstärke, die die Voraussetzungen erfüllt.
Benutze die kleinste Fadenstärke, die die Voraussetzungen erfüllt.
Die Begriffe Resorption und Absorption werden synonym verwendet. Die wichtigsten Merkmale für den biologischen Abbau und die Absorption von resorbierbarem Nahtmaterial sind das Festigkeits- und Massenverlustprofil sowie die Biokompatibilität der Abbaumaterialien. Bei Nahtmaterial auf Proteinbasis Nahtmaterial auf Proteinbasis , das durch proteolytische Enzyme und Phagozyten abgebaut wird, haften die Bakterien besser am Nahtmaterial . Eine verstärkte Kapillarität erhöht ebenfalls das Infektionsrisiko, da sich die Mikroorganismen leichter bewegen und ausbreiten können . Als Resorptionszeit Resorptionszeit wird die Zeitspanne benannt, in der das Material 50 % der Reißkraft des Knotens verliert. Von Massenresorption Massenresorption spricht man, wenn das gesamte Nahtmaterial vom Gewebe abgebaut ist. Abbau durch Hydrolyse. Synthetisches resorbierbares Nahtmaterial kann durch einen hydrolytischen Mechanismus über die Spaltung von Esterbindungen im Polymergerüst abgebaut werden. Sie werden durch natürliche Stoffwechselprozesse natürliche Stoffwechselprozesse fast rückstandsfrei abgebaut . Wenn die Moleküle hydrophob sind, wird ihre Hydrolyse verzögert und ihre Absorptionszeit verlängert . Eindringendes Wasser in das Nahtmaterial zerstört die Polymerstruktur des Fadens. Die Aufrechterhaltung des Gleichgewichts zwischen schneller Absorption und der Verlängerung der Zugfestigkeit wurde durch Behandlungen und chemische Strukturierung unterstützt, die die Absorptionszeit verlängern . Die Art des Abbaus, den das Material erfährt, seine Kapillarität und seine physikalische Konfiguration beeinflussen das Infektionsrisiko . Bei der Hydrolyse kommt es zu einer geringeren Gewebereaktion als beim enzymatischen Abbauprozess . Enzymatischer Abbau. Beim Wundverschluss mit resorbierbarem Nahtmaterial nimmt die Zugfestigkeit in den ersten Wochen allmählich und meist linear ab. Es findet physiologisch eine Gewebe- und Zellreaktion statt, um Zelltrümmer und abbaubare Anteile des Nahtmaterials zu entfernen. Diese Phasen können durch Infektionen und Eiweißmangel beeinträchtigt werden, wobei die Zugfestigkeit schneller verloren geht und eine Wunddehiszenz klinisch sichtbar werden kann. Nach dem primären Zusammenhalt des Gewebes werden diese Nahtmaterialien durch enzymatische oder hydrolytische Prozesse im Gewebe abgebaut. Die Abbaurate Abbaurate variiert in Abhängigkeit vom Material, der Lokalisation und patientenabhängigen Faktoren (Tab. ). Durch diese Prozesse werden Zugfestigkeit und Reißkraft konstant meist nach einer vom Material abhängigen Latenzzeit vermindert.
Synthetisches resorbierbares Nahtmaterial kann durch einen hydrolytischen Mechanismus über die Spaltung von Esterbindungen im Polymergerüst abgebaut werden. Sie werden durch natürliche Stoffwechselprozesse natürliche Stoffwechselprozesse fast rückstandsfrei abgebaut . Wenn die Moleküle hydrophob sind, wird ihre Hydrolyse verzögert und ihre Absorptionszeit verlängert . Eindringendes Wasser in das Nahtmaterial zerstört die Polymerstruktur des Fadens. Die Aufrechterhaltung des Gleichgewichts zwischen schneller Absorption und der Verlängerung der Zugfestigkeit wurde durch Behandlungen und chemische Strukturierung unterstützt, die die Absorptionszeit verlängern . Die Art des Abbaus, den das Material erfährt, seine Kapillarität und seine physikalische Konfiguration beeinflussen das Infektionsrisiko . Bei der Hydrolyse kommt es zu einer geringeren Gewebereaktion als beim enzymatischen Abbauprozess .
Beim Wundverschluss mit resorbierbarem Nahtmaterial nimmt die Zugfestigkeit in den ersten Wochen allmählich und meist linear ab. Es findet physiologisch eine Gewebe- und Zellreaktion statt, um Zelltrümmer und abbaubare Anteile des Nahtmaterials zu entfernen. Diese Phasen können durch Infektionen und Eiweißmangel beeinträchtigt werden, wobei die Zugfestigkeit schneller verloren geht und eine Wunddehiszenz klinisch sichtbar werden kann. Nach dem primären Zusammenhalt des Gewebes werden diese Nahtmaterialien durch enzymatische oder hydrolytische Prozesse im Gewebe abgebaut. Die Abbaurate Abbaurate variiert in Abhängigkeit vom Material, der Lokalisation und patientenabhängigen Faktoren (Tab. ). Durch diese Prozesse werden Zugfestigkeit und Reißkraft konstant meist nach einer vom Material abhängigen Latenzzeit vermindert.
Bei den Nahtmaterialien wird unterschieden zwischen natürlichen und synthetischen resorbierbaren Materialien. Die European Association of the Surgical Suture Industry ( EASSI EASSI ) hat für die verschiedenen Materialien und Spezifikationen Vorgaben erlassen, die in DIN EN ISO 15223‑1 dokumentiert sind (, Tab. A1). Resorbierbare Materialien Nahtmaterial aus natürlichen organischen Ausgangsmaterialien Natürliche Nahtmaterialien können in organische resorbierbare Materialien (aus Kollagen gewonnen, Katgut Katgut ) und nicht resorbierbare (Seide, Zwirn) unterteilt werden. Sie werden seltener verwendet, da sie tendenziell eine stärkere Gewebereaktion stärkere Gewebereaktion hervorrufen. Nähseide Nähseide wird jedoch immer noch regelmäßig bei der Sicherung von chirurgischen Drainagen verwendet. Kollagen. In der Vergangenheit wurden organische Nahtstoffe gewählt, zu dieser ältesten, ursprünglichen Gruppe gehörten Katgut, mit Chromsalz gegerbtes Chromkatgut und Kollagenbänder Kollagenbänder . Diese wurden zuerst aus der Submukosa des Schafdarms und später der Serosa des Rinderdarms gewonnen . Dieses Material wurde gezwirnt, war also multifilamentär, oder wurde beschichtet, z. B. mit Silikon . Katgut aus Rindern wurde Anfang der 2000er-Jahre vom Markt genommen. Rinderkollagen von BSE(bovine spongiforme Enzephalopathie)-freien Rindern wird inzwischen wieder angeboten. Seide. Die Grundsubstanz der chirurgischen Seidenfäden wird aus dem Kokon der Seidenraupenlarve gewonnen und besteht aus Fibroin der Rohseidenfaser, dem die Kittsubstanz Sericin entzogen wurde . Seidenfäden sind geflochten und können durch Beschichtungen, z. B. Bienenwachs oder Silikon, zum pseudomonofilen Nahtmaterial pseudomonofilen Nahtmaterial werden . Zwirne. Zwirnfäden – in Europa aus Flachs, in den USA aus der Zellulose von Baumwolle hergestellt – werden immer in multipler Konfiguration angeboten . Diese Materialien werden in Orthopädie und Unfallchirurgie bei uns kaum eingesetzt. Synthetische resorbierbare Materialien Sie bestehen aus künstlich hergestellten Materialien. Die Eigenschaften sind meist konstanter als bei den natürlichen Nahtmaterialien, insbesondere was den Verlust der Zugfestigkeit und die Absorption betrifft. Sie bestehen meist aus chemischen Polymeren chemischen Polymeren , die durch Hydrolyse resorbiert werden. Diese Materialien haben durchweg eine geringere Gewebereaktion als natürliche Stoffe. Bei den resorbierbaren synthetisch hergestellten Nahtmaterialien finden sich folgende Monomaterialien oder auch Copolymere (s. auch Tab. A1 und A2 online). Lactomer-Copolymer/Polyglactin 910. Dieses Nahtmaterial ist ein geflochtenes Multifilamentnahtmaterial geflochtenes Multifilamentnahtmaterial , das mit einem Copolymer aus Lactid (10 %) und Glycolid (90 %) besteht und teilweise beschichtet angeboten wird mit Polyglactin 370 plus Calciumstearat. Die wasserabweisende Eigenschaft von Lactid Lactid verlangsamt den Verlust der Zugfestigkeit, und die Bauschigkeit von Lactid führt zu einer schnellen Resorption des Nahtmaterials, sobald die Zugfestigkeit verloren geht. Hiermit können unterschiedliche Eigenschaften in einem Nahtmaterial verbunden werden. Glycolid Glycolid sorgt für eine hohe anfängliche Zugfestigkeit, wird aber im Gewebe schnell hydrolysiert . Lactid hat eine langsamere und kontrolliertere Hydrolyserate und somit auch einen langsameren Zugfestigkeitsverlust . Eine Beschichtung aus einer resorbierbaren Mischung auf dem Nahtmaterial aus Caprolacton-Glycolid-Copolymer und Calciumstearyl-Lactylat erzeugt eine Reduzierung der Oberflächenreibung, eine präzise Knotenplatzierung und ein reibungsloses Ligieren. Die Zugfestigkeit des Polyglactin 910-Nahtmaterials liegt am Tag 14 nach der Implantation bei ca. 65 %. Durch Hydrolyse ist nach 56 bis 120 Tagen vollständig erfolgt (s. Tab. A2 online). Diese Fäden verursachen nur eine minimale Gewebereaktion. Sie werden bei der allgemeinen Weichteilapproximation und Gefäßligatur eingesetzt. Polyglycolsäure. Polyglycolsäure (PGS) wird mit Polycaprolat oder Polyol beschichtet. Diese wird ungefärbt und farbig in violett angeboten. Das Material wird meist geflochten angeboten. Zugfestigkeit und Resorptionsschnelligkeit verhalten sich ähnlich wie bei Polyglactin 910 nach 90 bis 120 Tagen. Poliglecaprone 25. Poliglecaprone 25 ist ein monofiles Nahtmaterial monofiles Nahtmaterial . Es ist ein Copolymer aus Glycolid und ε‑Caprolacton. Die Biegsamkeit ist sehr hoch, was eine einfache Handhabung ermöglicht. Die Zugfestigkeit ist anfänglich hoch, 50–60 % am Tag 7 nach der Implantation und nimmt am Tag 21 ab. Die Resorption ist nach 91 bis 119 Tagen abgeschlossen. Poliglecaprone 25-Nähte werden für subkutane Nähte und Weichteilapproximationen und Ligaturen verwendet. Poliglecapron 25-Nähte verursachen signifikant weniger Fadenextrusion als Polyglactin 910 . Die Knotenfestigkeit wird als gut eingestuft. Es wird für schnell heilende Gewebe genutzt. Die Gewebereaktion ist gering. Polydioxanon. Dieses monofile Nahtmaterial ist aus Polyester hergestellt, bietet eine längere Wundunterstützung längere Wundunterstützung und ruft nur eine geringe Gewebereaktion hervor. Die Zugfestigkeit beträgt 70 % an Tag 14 und 25 % an Tag 42. Die Wundunterstützung bleibt bis zu 6 Wochen erhalten. Die Resorption durch Hydrolyse ist in den ersten 90 Tagen minimal und innerhalb von 6 Monaten im Wesentlichen abgeschlossen. Wie andere monofile Nahtmaterialien hat Polydioxanon eine geringe Affinität zu Mikroorganismen. Es wird für die Approximation von Weichteilen verwendet. Poly(glycolid)trimethylencarbonat. Polytrimethylencarbonat hat ähnliche Zugfestigkeit und Absorption wie Polydioxanon. Glycomer 631. Polymer aus Glysolid, Dioxanon, Trimethylen-Karbonat. Polygytone 6211. Polymer aus Glysolid, Caprolacton, Trimethylen-Karbonat und Lactid. Nichtresorbierbare Materialien Diese Materialien verfügen über eine gute Festigkeit gute Festigkeit , können aber im Gewebe Fremdkörperreaktionen hervorrufen. Gewebereaktionen Gewebereaktionen führen dabei im Verlauf zu Ummantelung des Nahtmaterials. Diese Abkapselung erfolgt durch Fibroblasten. Auch Makrophagen und Riesenzellen finden sich in der Umgebung . Nichtresorbierbare Materialien können durch Beschichtung eine Friktionsverbesserung und/oder auch eine geringere Kapillarität erhalten. Diese nichtresorbierbaren Fäden werden einerseits in langsamer heilenden Geweben eingesetzt, aber auch dort, wo starker Halt gefordert wird, z. B. Sehnen. Unter Verfallszeit Verfallszeit versteht man bei nichtresorbierbarem Nahtmaterial die Zeit, in der durch Degradation das Material in Abschnitte zerfällt. Natürliche nichtresorbierbare Materialien Zu den natürlichen, nichtresorbierbaren Nahtmaterialien gehören chirurgische Seide, Zwirn und Baumwolle. Sie werden seltener verwendet, da sie tendenziell eine stärkere Gewebereaktion hervorrufen. Nähseide wird in einigen Regionen immer noch regelmäßig bei der Sicherung von chirurgischen Drainagen verwendet. Chirurgische Seide. Aus Rohseide, also den Kokons der Seidenraupen, wird dieses Nahtmaterial gesponnen. Eine Beschichtung kann mit Wachs, z. B. Bienenwachs oder Silikon, erfolgen. Seide lässt sich gut knoten, und vielen gilt die Handhabung mit Seide als der Standard. Bei längerer Proteolyse kann Seide vom Gewebe innerhalb von 2 Jahren resorbiert werden. Die Zugfestigkeit nimmt mit der Feuchtigkeitsaufnahme ab und geht nach 1 Jahr verloren. Das Hauptproblem bei Seidenfäden ist eine akute Entzündungsreaktion akute Entzündungsreaktion . Das umliegende Gewebe verkapselt das Nahtmaterial durch faseriges Bindegewebe. Seidenfäden haben sehr gute Knüpfeigenschaften, sind geschmeidig und haben damit eine gute Handhabung. Seide hat eine hohe Kapillarität und ermöglicht einen hohen interfilamentären Bakterientransport . Chirurgische Baumwolle. Chirurgische Baumwolle wird in den deutschsprachigen Ländern so gut wie nicht eingesetzt. Sie wird hier nur der Vollständigkeit halber genannt. Dieses Nahtmaterial wird aus gedrehten, langstapeligen Baumwollfasern hergestellt. Die Zugfestigkeit beträgt 50 % innerhalb von 6 Monaten und 30–40 % nach 2 Jahren. Die nichtresorbierbare Baumwolle wird im Körpergewebe eingekapselt. Als nachteilig angesehen werden Kapillarität, Gewebereaktion und Bakteriophilie. Natürliche anorganische Ausgangsmaterialien Meist werden Edelstahle Edelstahle verwendet. Tantal ist im Gebrauch. Korrosionsbeständige Metalle sind notwendig, da durch die Körperflüssigkeiten die Metalloberflächen angegriffen werden, was sogar bei hochwertigen Titanimplantaten als Metallose Metallose imponieren kann. Nahtmaterial aus Stahl wird monofil oder polyfil angeboten. Synthetische nichtresorbierbare Materialien Polyester und Polyolefine sind 2 Vertreter dieser Gruppe. Polyester. Das unauflösbare Polyestermaterial unauflösbare Polyestermaterial besteht aus den Rohstoffen Polyethylenterephatalat oder Polybutylenterephtalat. Es werden monofile, multifile, geflochtene und pseudomonofile Fäden angeboten. Sie werden mit Polytetrafluoräthylen, Polytetramethylenadipat, Silikonkautschuk, Äthylcellulose, Wachs oder Polyolefine beschichtet, um bei den geflochtenen Konfigurationen eine pseudomonofile Eigenschaft zu erreichen . Monofile Fäden weisen keinen interfilamentären Bakterientransport auf . Polyester hat eine hohe Knotenzugfestigkeit, gute Flexibilität und geringe chemische Degradation. Polyolefine. Sie bestehen aus Polypropylen oder Polyäthylen und werden durchweg als monofiles Material angeboten . Die Oberfläche ist so glatt, dass eine Beschichtung nicht erforderlich ist. Polypropylen hat einen ausgezeichneten Gewebewiderstand und Stabilität Stabilität . Polyamide. Nahtmaterialien aus der Gruppe der Polyamide werden nicht resorbiert oder absorbiert, sie zerfallen nach längerer Liegezeit. Sie werden hergestellt aus Polyamid 6.6 (Hexamethylendiamin und Adipinsäure) [Nylon], oder aus Polyamid 6 Aminocapronsäure. Sie werden in folgenden Konfigurationen angeboten: monofil, geflochten, multifil, pseudomonofil. Als Beschichtung dient Polyamid 6 oder eine Imprägnierung mit Bienenwachs . Die Hauptnachteile von Polyamid-Nahtmaterial sind seine schlechten Handhabungseigenschaften schlechten Handhabungseigenschaften und seine Knotensicherheit . Auswahl von Material und Fadenstärke Beim Verschluss von Wunden oder chirurgischen Zugängen werden üblicherweise in der Subkutis und Subdermis resorbierbare Nähte und für den Hautverschluss folgende monofile nichtresorbierbare Nähte verwendet: oberflächliche Läsionen im Gesicht: 6‑0, andere oberflächliche Hautläsionen: in Bereichen mit niedriger Hautspannung: 5‑0, in Bereichen mit höherer Hautspannung: 4‑0. Differenziert man nach Fadenstärke, schlagen die Autoren in folgende Auswahl vor (s. Tab. ). Die vorgeschlagenen Fadenstärken Fadenstärken müssen dem Alter, der Körperkonstitution und dem Gewebezustand angepasst werden. Beim Wundverschluss wird das Material abhängig von der Schicht verwendet: in der Tiefe eher resorbierbar und polyfil, in der Haut selbst monofil nicht resorbierbar (Tab. und ). Beschichtung von Nahtmaterial mit Trägerstoffen Antibiotikabeschichtung. Metaanalysen konnten zeigen, dass die Anwendung von mit Triclosan Triclosan antimikrobiell beschichtetem Nahtmaterial die Inzidenz von „surgical site infections“ nach sauberen, sauber-kontaminierten und kontaminierten Operationen reduzierte . In Orthopädie und Unfallchirurgie werden diese Fäden selten genutzt, da keine Evidenz vorliegt . Neuere Beschichtungen auf Chlorhexidin-Basis oder Octenidindihydrochlorid sind in der Entwicklung. m(„messenger“)RNA(Ribonukleinsäure)-Beschichtung. Experimentelle Untersuchungen lassen für die Zukunft die Entwicklung von mRNA-beschichtetem Nahtmaterial beim chirurgischen Wundverschluss in den Fokus rücken. Zellen im Wundbereich könnten direkt übertragen werden, wodurch die Wundheilung beschleunigt und verbessert werden könnte . Handhabung von Verpackung und Nahtmaterial Obwohl es für Verpackungen und Beschriftung ISO-Normen gibt (ISO 20417) ISO Nr. 15223 , finden sich auf dem Markt keine standardisierten Verpackungen keine standardisierten Verpackungen . Dies bedeutet für den Anwender, dass er je nach Hersteller sich über die Handhabung der Verpackungen informieren muss. Die primäre Innenverpackung liegt in einer äußeren, meist durchsichtigen Peel-Back-Verpackung Peel-Back-Verpackung . Die Sterilität bleibt erhalten, bis sie geöffnet wird oder das Verfallsdatum erreicht ist. Die sterilen Inhalte sterilen Inhalte der Verpackungen werden von der OP-Pflegekraft oder dem Arzt durch Aufreißen der Umverpackung angereicht. Die sterile Verpackung lässt dann sich über Laschen öffnen. Der Inhalt wird nach Umklappen einer Lasche geöffnet. Die Nadel liegt frei und kann mit dem Nadelhalter in der Verpackung gefasst werden (s. hierzu ). Nach Aufklappen des Fadenträgers (Wickelträgers) wird die freigelegte Nadel mit anhängendem Faden entnommen. Diese kann vorsichtig gestreckt werden, um den Fadendrill zu vermindern. Hierbei sollte der Faden nicht zwischen Nadel und Fadenende, sondern nur innerhalb des Fadens gestreckt werden (Abb. ). Bei Ligaturen oder nadelfreien Fäden liegt das Material dementsprechend in der Verpackung.
Nahtmaterial aus natürlichen organischen Ausgangsmaterialien Natürliche Nahtmaterialien können in organische resorbierbare Materialien (aus Kollagen gewonnen, Katgut Katgut ) und nicht resorbierbare (Seide, Zwirn) unterteilt werden. Sie werden seltener verwendet, da sie tendenziell eine stärkere Gewebereaktion stärkere Gewebereaktion hervorrufen. Nähseide Nähseide wird jedoch immer noch regelmäßig bei der Sicherung von chirurgischen Drainagen verwendet. Kollagen. In der Vergangenheit wurden organische Nahtstoffe gewählt, zu dieser ältesten, ursprünglichen Gruppe gehörten Katgut, mit Chromsalz gegerbtes Chromkatgut und Kollagenbänder Kollagenbänder . Diese wurden zuerst aus der Submukosa des Schafdarms und später der Serosa des Rinderdarms gewonnen . Dieses Material wurde gezwirnt, war also multifilamentär, oder wurde beschichtet, z. B. mit Silikon . Katgut aus Rindern wurde Anfang der 2000er-Jahre vom Markt genommen. Rinderkollagen von BSE(bovine spongiforme Enzephalopathie)-freien Rindern wird inzwischen wieder angeboten. Seide. Die Grundsubstanz der chirurgischen Seidenfäden wird aus dem Kokon der Seidenraupenlarve gewonnen und besteht aus Fibroin der Rohseidenfaser, dem die Kittsubstanz Sericin entzogen wurde . Seidenfäden sind geflochten und können durch Beschichtungen, z. B. Bienenwachs oder Silikon, zum pseudomonofilen Nahtmaterial pseudomonofilen Nahtmaterial werden . Zwirne. Zwirnfäden – in Europa aus Flachs, in den USA aus der Zellulose von Baumwolle hergestellt – werden immer in multipler Konfiguration angeboten . Diese Materialien werden in Orthopädie und Unfallchirurgie bei uns kaum eingesetzt.
Natürliche Nahtmaterialien können in organische resorbierbare Materialien (aus Kollagen gewonnen, Katgut Katgut ) und nicht resorbierbare (Seide, Zwirn) unterteilt werden. Sie werden seltener verwendet, da sie tendenziell eine stärkere Gewebereaktion stärkere Gewebereaktion hervorrufen. Nähseide Nähseide wird jedoch immer noch regelmäßig bei der Sicherung von chirurgischen Drainagen verwendet. Kollagen. In der Vergangenheit wurden organische Nahtstoffe gewählt, zu dieser ältesten, ursprünglichen Gruppe gehörten Katgut, mit Chromsalz gegerbtes Chromkatgut und Kollagenbänder Kollagenbänder . Diese wurden zuerst aus der Submukosa des Schafdarms und später der Serosa des Rinderdarms gewonnen . Dieses Material wurde gezwirnt, war also multifilamentär, oder wurde beschichtet, z. B. mit Silikon . Katgut aus Rindern wurde Anfang der 2000er-Jahre vom Markt genommen. Rinderkollagen von BSE(bovine spongiforme Enzephalopathie)-freien Rindern wird inzwischen wieder angeboten. Seide. Die Grundsubstanz der chirurgischen Seidenfäden wird aus dem Kokon der Seidenraupenlarve gewonnen und besteht aus Fibroin der Rohseidenfaser, dem die Kittsubstanz Sericin entzogen wurde . Seidenfäden sind geflochten und können durch Beschichtungen, z. B. Bienenwachs oder Silikon, zum pseudomonofilen Nahtmaterial pseudomonofilen Nahtmaterial werden . Zwirne. Zwirnfäden – in Europa aus Flachs, in den USA aus der Zellulose von Baumwolle hergestellt – werden immer in multipler Konfiguration angeboten . Diese Materialien werden in Orthopädie und Unfallchirurgie bei uns kaum eingesetzt.
In der Vergangenheit wurden organische Nahtstoffe gewählt, zu dieser ältesten, ursprünglichen Gruppe gehörten Katgut, mit Chromsalz gegerbtes Chromkatgut und Kollagenbänder Kollagenbänder . Diese wurden zuerst aus der Submukosa des Schafdarms und später der Serosa des Rinderdarms gewonnen . Dieses Material wurde gezwirnt, war also multifilamentär, oder wurde beschichtet, z. B. mit Silikon . Katgut aus Rindern wurde Anfang der 2000er-Jahre vom Markt genommen. Rinderkollagen von BSE(bovine spongiforme Enzephalopathie)-freien Rindern wird inzwischen wieder angeboten.
Die Grundsubstanz der chirurgischen Seidenfäden wird aus dem Kokon der Seidenraupenlarve gewonnen und besteht aus Fibroin der Rohseidenfaser, dem die Kittsubstanz Sericin entzogen wurde . Seidenfäden sind geflochten und können durch Beschichtungen, z. B. Bienenwachs oder Silikon, zum pseudomonofilen Nahtmaterial pseudomonofilen Nahtmaterial werden .
Zwirnfäden – in Europa aus Flachs, in den USA aus der Zellulose von Baumwolle hergestellt – werden immer in multipler Konfiguration angeboten . Diese Materialien werden in Orthopädie und Unfallchirurgie bei uns kaum eingesetzt.
Sie bestehen aus künstlich hergestellten Materialien. Die Eigenschaften sind meist konstanter als bei den natürlichen Nahtmaterialien, insbesondere was den Verlust der Zugfestigkeit und die Absorption betrifft. Sie bestehen meist aus chemischen Polymeren chemischen Polymeren , die durch Hydrolyse resorbiert werden. Diese Materialien haben durchweg eine geringere Gewebereaktion als natürliche Stoffe. Bei den resorbierbaren synthetisch hergestellten Nahtmaterialien finden sich folgende Monomaterialien oder auch Copolymere (s. auch Tab. A1 und A2 online). Lactomer-Copolymer/Polyglactin 910. Dieses Nahtmaterial ist ein geflochtenes Multifilamentnahtmaterial geflochtenes Multifilamentnahtmaterial , das mit einem Copolymer aus Lactid (10 %) und Glycolid (90 %) besteht und teilweise beschichtet angeboten wird mit Polyglactin 370 plus Calciumstearat. Die wasserabweisende Eigenschaft von Lactid Lactid verlangsamt den Verlust der Zugfestigkeit, und die Bauschigkeit von Lactid führt zu einer schnellen Resorption des Nahtmaterials, sobald die Zugfestigkeit verloren geht. Hiermit können unterschiedliche Eigenschaften in einem Nahtmaterial verbunden werden. Glycolid Glycolid sorgt für eine hohe anfängliche Zugfestigkeit, wird aber im Gewebe schnell hydrolysiert . Lactid hat eine langsamere und kontrolliertere Hydrolyserate und somit auch einen langsameren Zugfestigkeitsverlust . Eine Beschichtung aus einer resorbierbaren Mischung auf dem Nahtmaterial aus Caprolacton-Glycolid-Copolymer und Calciumstearyl-Lactylat erzeugt eine Reduzierung der Oberflächenreibung, eine präzise Knotenplatzierung und ein reibungsloses Ligieren. Die Zugfestigkeit des Polyglactin 910-Nahtmaterials liegt am Tag 14 nach der Implantation bei ca. 65 %. Durch Hydrolyse ist nach 56 bis 120 Tagen vollständig erfolgt (s. Tab. A2 online). Diese Fäden verursachen nur eine minimale Gewebereaktion. Sie werden bei der allgemeinen Weichteilapproximation und Gefäßligatur eingesetzt. Polyglycolsäure. Polyglycolsäure (PGS) wird mit Polycaprolat oder Polyol beschichtet. Diese wird ungefärbt und farbig in violett angeboten. Das Material wird meist geflochten angeboten. Zugfestigkeit und Resorptionsschnelligkeit verhalten sich ähnlich wie bei Polyglactin 910 nach 90 bis 120 Tagen. Poliglecaprone 25. Poliglecaprone 25 ist ein monofiles Nahtmaterial monofiles Nahtmaterial . Es ist ein Copolymer aus Glycolid und ε‑Caprolacton. Die Biegsamkeit ist sehr hoch, was eine einfache Handhabung ermöglicht. Die Zugfestigkeit ist anfänglich hoch, 50–60 % am Tag 7 nach der Implantation und nimmt am Tag 21 ab. Die Resorption ist nach 91 bis 119 Tagen abgeschlossen. Poliglecaprone 25-Nähte werden für subkutane Nähte und Weichteilapproximationen und Ligaturen verwendet. Poliglecapron 25-Nähte verursachen signifikant weniger Fadenextrusion als Polyglactin 910 . Die Knotenfestigkeit wird als gut eingestuft. Es wird für schnell heilende Gewebe genutzt. Die Gewebereaktion ist gering. Polydioxanon. Dieses monofile Nahtmaterial ist aus Polyester hergestellt, bietet eine längere Wundunterstützung längere Wundunterstützung und ruft nur eine geringe Gewebereaktion hervor. Die Zugfestigkeit beträgt 70 % an Tag 14 und 25 % an Tag 42. Die Wundunterstützung bleibt bis zu 6 Wochen erhalten. Die Resorption durch Hydrolyse ist in den ersten 90 Tagen minimal und innerhalb von 6 Monaten im Wesentlichen abgeschlossen. Wie andere monofile Nahtmaterialien hat Polydioxanon eine geringe Affinität zu Mikroorganismen. Es wird für die Approximation von Weichteilen verwendet. Poly(glycolid)trimethylencarbonat. Polytrimethylencarbonat hat ähnliche Zugfestigkeit und Absorption wie Polydioxanon. Glycomer 631. Polymer aus Glysolid, Dioxanon, Trimethylen-Karbonat. Polygytone 6211. Polymer aus Glysolid, Caprolacton, Trimethylen-Karbonat und Lactid.
Dieses Nahtmaterial ist ein geflochtenes Multifilamentnahtmaterial geflochtenes Multifilamentnahtmaterial , das mit einem Copolymer aus Lactid (10 %) und Glycolid (90 %) besteht und teilweise beschichtet angeboten wird mit Polyglactin 370 plus Calciumstearat. Die wasserabweisende Eigenschaft von Lactid Lactid verlangsamt den Verlust der Zugfestigkeit, und die Bauschigkeit von Lactid führt zu einer schnellen Resorption des Nahtmaterials, sobald die Zugfestigkeit verloren geht. Hiermit können unterschiedliche Eigenschaften in einem Nahtmaterial verbunden werden. Glycolid Glycolid sorgt für eine hohe anfängliche Zugfestigkeit, wird aber im Gewebe schnell hydrolysiert . Lactid hat eine langsamere und kontrolliertere Hydrolyserate und somit auch einen langsameren Zugfestigkeitsverlust . Eine Beschichtung aus einer resorbierbaren Mischung auf dem Nahtmaterial aus Caprolacton-Glycolid-Copolymer und Calciumstearyl-Lactylat erzeugt eine Reduzierung der Oberflächenreibung, eine präzise Knotenplatzierung und ein reibungsloses Ligieren. Die Zugfestigkeit des Polyglactin 910-Nahtmaterials liegt am Tag 14 nach der Implantation bei ca. 65 %. Durch Hydrolyse ist nach 56 bis 120 Tagen vollständig erfolgt (s. Tab. A2 online). Diese Fäden verursachen nur eine minimale Gewebereaktion. Sie werden bei der allgemeinen Weichteilapproximation und Gefäßligatur eingesetzt.
Polyglycolsäure (PGS) wird mit Polycaprolat oder Polyol beschichtet. Diese wird ungefärbt und farbig in violett angeboten. Das Material wird meist geflochten angeboten. Zugfestigkeit und Resorptionsschnelligkeit verhalten sich ähnlich wie bei Polyglactin 910 nach 90 bis 120 Tagen.
Poliglecaprone 25 ist ein monofiles Nahtmaterial monofiles Nahtmaterial . Es ist ein Copolymer aus Glycolid und ε‑Caprolacton. Die Biegsamkeit ist sehr hoch, was eine einfache Handhabung ermöglicht. Die Zugfestigkeit ist anfänglich hoch, 50–60 % am Tag 7 nach der Implantation und nimmt am Tag 21 ab. Die Resorption ist nach 91 bis 119 Tagen abgeschlossen. Poliglecaprone 25-Nähte werden für subkutane Nähte und Weichteilapproximationen und Ligaturen verwendet. Poliglecapron 25-Nähte verursachen signifikant weniger Fadenextrusion als Polyglactin 910 . Die Knotenfestigkeit wird als gut eingestuft. Es wird für schnell heilende Gewebe genutzt. Die Gewebereaktion ist gering.
Dieses monofile Nahtmaterial ist aus Polyester hergestellt, bietet eine längere Wundunterstützung längere Wundunterstützung und ruft nur eine geringe Gewebereaktion hervor. Die Zugfestigkeit beträgt 70 % an Tag 14 und 25 % an Tag 42. Die Wundunterstützung bleibt bis zu 6 Wochen erhalten. Die Resorption durch Hydrolyse ist in den ersten 90 Tagen minimal und innerhalb von 6 Monaten im Wesentlichen abgeschlossen. Wie andere monofile Nahtmaterialien hat Polydioxanon eine geringe Affinität zu Mikroorganismen. Es wird für die Approximation von Weichteilen verwendet.
Polytrimethylencarbonat hat ähnliche Zugfestigkeit und Absorption wie Polydioxanon.
Polymer aus Glysolid, Dioxanon, Trimethylen-Karbonat.
Polymer aus Glysolid, Caprolacton, Trimethylen-Karbonat und Lactid.
Diese Materialien verfügen über eine gute Festigkeit gute Festigkeit , können aber im Gewebe Fremdkörperreaktionen hervorrufen. Gewebereaktionen Gewebereaktionen führen dabei im Verlauf zu Ummantelung des Nahtmaterials. Diese Abkapselung erfolgt durch Fibroblasten. Auch Makrophagen und Riesenzellen finden sich in der Umgebung . Nichtresorbierbare Materialien können durch Beschichtung eine Friktionsverbesserung und/oder auch eine geringere Kapillarität erhalten. Diese nichtresorbierbaren Fäden werden einerseits in langsamer heilenden Geweben eingesetzt, aber auch dort, wo starker Halt gefordert wird, z. B. Sehnen. Unter Verfallszeit Verfallszeit versteht man bei nichtresorbierbarem Nahtmaterial die Zeit, in der durch Degradation das Material in Abschnitte zerfällt. Natürliche nichtresorbierbare Materialien Zu den natürlichen, nichtresorbierbaren Nahtmaterialien gehören chirurgische Seide, Zwirn und Baumwolle. Sie werden seltener verwendet, da sie tendenziell eine stärkere Gewebereaktion hervorrufen. Nähseide wird in einigen Regionen immer noch regelmäßig bei der Sicherung von chirurgischen Drainagen verwendet. Chirurgische Seide. Aus Rohseide, also den Kokons der Seidenraupen, wird dieses Nahtmaterial gesponnen. Eine Beschichtung kann mit Wachs, z. B. Bienenwachs oder Silikon, erfolgen. Seide lässt sich gut knoten, und vielen gilt die Handhabung mit Seide als der Standard. Bei längerer Proteolyse kann Seide vom Gewebe innerhalb von 2 Jahren resorbiert werden. Die Zugfestigkeit nimmt mit der Feuchtigkeitsaufnahme ab und geht nach 1 Jahr verloren. Das Hauptproblem bei Seidenfäden ist eine akute Entzündungsreaktion akute Entzündungsreaktion . Das umliegende Gewebe verkapselt das Nahtmaterial durch faseriges Bindegewebe. Seidenfäden haben sehr gute Knüpfeigenschaften, sind geschmeidig und haben damit eine gute Handhabung. Seide hat eine hohe Kapillarität und ermöglicht einen hohen interfilamentären Bakterientransport . Chirurgische Baumwolle. Chirurgische Baumwolle wird in den deutschsprachigen Ländern so gut wie nicht eingesetzt. Sie wird hier nur der Vollständigkeit halber genannt. Dieses Nahtmaterial wird aus gedrehten, langstapeligen Baumwollfasern hergestellt. Die Zugfestigkeit beträgt 50 % innerhalb von 6 Monaten und 30–40 % nach 2 Jahren. Die nichtresorbierbare Baumwolle wird im Körpergewebe eingekapselt. Als nachteilig angesehen werden Kapillarität, Gewebereaktion und Bakteriophilie. Natürliche anorganische Ausgangsmaterialien Meist werden Edelstahle Edelstahle verwendet. Tantal ist im Gebrauch. Korrosionsbeständige Metalle sind notwendig, da durch die Körperflüssigkeiten die Metalloberflächen angegriffen werden, was sogar bei hochwertigen Titanimplantaten als Metallose Metallose imponieren kann. Nahtmaterial aus Stahl wird monofil oder polyfil angeboten. Synthetische nichtresorbierbare Materialien Polyester und Polyolefine sind 2 Vertreter dieser Gruppe. Polyester. Das unauflösbare Polyestermaterial unauflösbare Polyestermaterial besteht aus den Rohstoffen Polyethylenterephatalat oder Polybutylenterephtalat. Es werden monofile, multifile, geflochtene und pseudomonofile Fäden angeboten. Sie werden mit Polytetrafluoräthylen, Polytetramethylenadipat, Silikonkautschuk, Äthylcellulose, Wachs oder Polyolefine beschichtet, um bei den geflochtenen Konfigurationen eine pseudomonofile Eigenschaft zu erreichen . Monofile Fäden weisen keinen interfilamentären Bakterientransport auf . Polyester hat eine hohe Knotenzugfestigkeit, gute Flexibilität und geringe chemische Degradation. Polyolefine. Sie bestehen aus Polypropylen oder Polyäthylen und werden durchweg als monofiles Material angeboten . Die Oberfläche ist so glatt, dass eine Beschichtung nicht erforderlich ist. Polypropylen hat einen ausgezeichneten Gewebewiderstand und Stabilität Stabilität . Polyamide. Nahtmaterialien aus der Gruppe der Polyamide werden nicht resorbiert oder absorbiert, sie zerfallen nach längerer Liegezeit. Sie werden hergestellt aus Polyamid 6.6 (Hexamethylendiamin und Adipinsäure) [Nylon], oder aus Polyamid 6 Aminocapronsäure. Sie werden in folgenden Konfigurationen angeboten: monofil, geflochten, multifil, pseudomonofil. Als Beschichtung dient Polyamid 6 oder eine Imprägnierung mit Bienenwachs . Die Hauptnachteile von Polyamid-Nahtmaterial sind seine schlechten Handhabungseigenschaften schlechten Handhabungseigenschaften und seine Knotensicherheit .
Zu den natürlichen, nichtresorbierbaren Nahtmaterialien gehören chirurgische Seide, Zwirn und Baumwolle. Sie werden seltener verwendet, da sie tendenziell eine stärkere Gewebereaktion hervorrufen. Nähseide wird in einigen Regionen immer noch regelmäßig bei der Sicherung von chirurgischen Drainagen verwendet. Chirurgische Seide. Aus Rohseide, also den Kokons der Seidenraupen, wird dieses Nahtmaterial gesponnen. Eine Beschichtung kann mit Wachs, z. B. Bienenwachs oder Silikon, erfolgen. Seide lässt sich gut knoten, und vielen gilt die Handhabung mit Seide als der Standard. Bei längerer Proteolyse kann Seide vom Gewebe innerhalb von 2 Jahren resorbiert werden. Die Zugfestigkeit nimmt mit der Feuchtigkeitsaufnahme ab und geht nach 1 Jahr verloren. Das Hauptproblem bei Seidenfäden ist eine akute Entzündungsreaktion akute Entzündungsreaktion . Das umliegende Gewebe verkapselt das Nahtmaterial durch faseriges Bindegewebe. Seidenfäden haben sehr gute Knüpfeigenschaften, sind geschmeidig und haben damit eine gute Handhabung. Seide hat eine hohe Kapillarität und ermöglicht einen hohen interfilamentären Bakterientransport . Chirurgische Baumwolle. Chirurgische Baumwolle wird in den deutschsprachigen Ländern so gut wie nicht eingesetzt. Sie wird hier nur der Vollständigkeit halber genannt. Dieses Nahtmaterial wird aus gedrehten, langstapeligen Baumwollfasern hergestellt. Die Zugfestigkeit beträgt 50 % innerhalb von 6 Monaten und 30–40 % nach 2 Jahren. Die nichtresorbierbare Baumwolle wird im Körpergewebe eingekapselt. Als nachteilig angesehen werden Kapillarität, Gewebereaktion und Bakteriophilie.
Aus Rohseide, also den Kokons der Seidenraupen, wird dieses Nahtmaterial gesponnen. Eine Beschichtung kann mit Wachs, z. B. Bienenwachs oder Silikon, erfolgen. Seide lässt sich gut knoten, und vielen gilt die Handhabung mit Seide als der Standard. Bei längerer Proteolyse kann Seide vom Gewebe innerhalb von 2 Jahren resorbiert werden. Die Zugfestigkeit nimmt mit der Feuchtigkeitsaufnahme ab und geht nach 1 Jahr verloren. Das Hauptproblem bei Seidenfäden ist eine akute Entzündungsreaktion akute Entzündungsreaktion . Das umliegende Gewebe verkapselt das Nahtmaterial durch faseriges Bindegewebe. Seidenfäden haben sehr gute Knüpfeigenschaften, sind geschmeidig und haben damit eine gute Handhabung. Seide hat eine hohe Kapillarität und ermöglicht einen hohen interfilamentären Bakterientransport .
Chirurgische Baumwolle wird in den deutschsprachigen Ländern so gut wie nicht eingesetzt. Sie wird hier nur der Vollständigkeit halber genannt. Dieses Nahtmaterial wird aus gedrehten, langstapeligen Baumwollfasern hergestellt. Die Zugfestigkeit beträgt 50 % innerhalb von 6 Monaten und 30–40 % nach 2 Jahren. Die nichtresorbierbare Baumwolle wird im Körpergewebe eingekapselt. Als nachteilig angesehen werden Kapillarität, Gewebereaktion und Bakteriophilie.
Meist werden Edelstahle Edelstahle verwendet. Tantal ist im Gebrauch. Korrosionsbeständige Metalle sind notwendig, da durch die Körperflüssigkeiten die Metalloberflächen angegriffen werden, was sogar bei hochwertigen Titanimplantaten als Metallose Metallose imponieren kann. Nahtmaterial aus Stahl wird monofil oder polyfil angeboten.
Polyester und Polyolefine sind 2 Vertreter dieser Gruppe. Polyester. Das unauflösbare Polyestermaterial unauflösbare Polyestermaterial besteht aus den Rohstoffen Polyethylenterephatalat oder Polybutylenterephtalat. Es werden monofile, multifile, geflochtene und pseudomonofile Fäden angeboten. Sie werden mit Polytetrafluoräthylen, Polytetramethylenadipat, Silikonkautschuk, Äthylcellulose, Wachs oder Polyolefine beschichtet, um bei den geflochtenen Konfigurationen eine pseudomonofile Eigenschaft zu erreichen . Monofile Fäden weisen keinen interfilamentären Bakterientransport auf . Polyester hat eine hohe Knotenzugfestigkeit, gute Flexibilität und geringe chemische Degradation. Polyolefine. Sie bestehen aus Polypropylen oder Polyäthylen und werden durchweg als monofiles Material angeboten . Die Oberfläche ist so glatt, dass eine Beschichtung nicht erforderlich ist. Polypropylen hat einen ausgezeichneten Gewebewiderstand und Stabilität Stabilität . Polyamide. Nahtmaterialien aus der Gruppe der Polyamide werden nicht resorbiert oder absorbiert, sie zerfallen nach längerer Liegezeit. Sie werden hergestellt aus Polyamid 6.6 (Hexamethylendiamin und Adipinsäure) [Nylon], oder aus Polyamid 6 Aminocapronsäure. Sie werden in folgenden Konfigurationen angeboten: monofil, geflochten, multifil, pseudomonofil. Als Beschichtung dient Polyamid 6 oder eine Imprägnierung mit Bienenwachs . Die Hauptnachteile von Polyamid-Nahtmaterial sind seine schlechten Handhabungseigenschaften schlechten Handhabungseigenschaften und seine Knotensicherheit .
Das unauflösbare Polyestermaterial unauflösbare Polyestermaterial besteht aus den Rohstoffen Polyethylenterephatalat oder Polybutylenterephtalat. Es werden monofile, multifile, geflochtene und pseudomonofile Fäden angeboten. Sie werden mit Polytetrafluoräthylen, Polytetramethylenadipat, Silikonkautschuk, Äthylcellulose, Wachs oder Polyolefine beschichtet, um bei den geflochtenen Konfigurationen eine pseudomonofile Eigenschaft zu erreichen . Monofile Fäden weisen keinen interfilamentären Bakterientransport auf . Polyester hat eine hohe Knotenzugfestigkeit, gute Flexibilität und geringe chemische Degradation.
Sie bestehen aus Polypropylen oder Polyäthylen und werden durchweg als monofiles Material angeboten . Die Oberfläche ist so glatt, dass eine Beschichtung nicht erforderlich ist. Polypropylen hat einen ausgezeichneten Gewebewiderstand und Stabilität Stabilität .
Nahtmaterialien aus der Gruppe der Polyamide werden nicht resorbiert oder absorbiert, sie zerfallen nach längerer Liegezeit. Sie werden hergestellt aus Polyamid 6.6 (Hexamethylendiamin und Adipinsäure) [Nylon], oder aus Polyamid 6 Aminocapronsäure. Sie werden in folgenden Konfigurationen angeboten: monofil, geflochten, multifil, pseudomonofil. Als Beschichtung dient Polyamid 6 oder eine Imprägnierung mit Bienenwachs . Die Hauptnachteile von Polyamid-Nahtmaterial sind seine schlechten Handhabungseigenschaften schlechten Handhabungseigenschaften und seine Knotensicherheit .
Beim Verschluss von Wunden oder chirurgischen Zugängen werden üblicherweise in der Subkutis und Subdermis resorbierbare Nähte und für den Hautverschluss folgende monofile nichtresorbierbare Nähte verwendet: oberflächliche Läsionen im Gesicht: 6‑0, andere oberflächliche Hautläsionen: in Bereichen mit niedriger Hautspannung: 5‑0, in Bereichen mit höherer Hautspannung: 4‑0. Differenziert man nach Fadenstärke, schlagen die Autoren in folgende Auswahl vor (s. Tab. ). Die vorgeschlagenen Fadenstärken Fadenstärken müssen dem Alter, der Körperkonstitution und dem Gewebezustand angepasst werden. Beim Wundverschluss wird das Material abhängig von der Schicht verwendet: in der Tiefe eher resorbierbar und polyfil, in der Haut selbst monofil nicht resorbierbar (Tab. und ).
Antibiotikabeschichtung. Metaanalysen konnten zeigen, dass die Anwendung von mit Triclosan Triclosan antimikrobiell beschichtetem Nahtmaterial die Inzidenz von „surgical site infections“ nach sauberen, sauber-kontaminierten und kontaminierten Operationen reduzierte . In Orthopädie und Unfallchirurgie werden diese Fäden selten genutzt, da keine Evidenz vorliegt . Neuere Beschichtungen auf Chlorhexidin-Basis oder Octenidindihydrochlorid sind in der Entwicklung. m(„messenger“)RNA(Ribonukleinsäure)-Beschichtung. Experimentelle Untersuchungen lassen für die Zukunft die Entwicklung von mRNA-beschichtetem Nahtmaterial beim chirurgischen Wundverschluss in den Fokus rücken. Zellen im Wundbereich könnten direkt übertragen werden, wodurch die Wundheilung beschleunigt und verbessert werden könnte .
Metaanalysen konnten zeigen, dass die Anwendung von mit Triclosan Triclosan antimikrobiell beschichtetem Nahtmaterial die Inzidenz von „surgical site infections“ nach sauberen, sauber-kontaminierten und kontaminierten Operationen reduzierte . In Orthopädie und Unfallchirurgie werden diese Fäden selten genutzt, da keine Evidenz vorliegt . Neuere Beschichtungen auf Chlorhexidin-Basis oder Octenidindihydrochlorid sind in der Entwicklung.
Experimentelle Untersuchungen lassen für die Zukunft die Entwicklung von mRNA-beschichtetem Nahtmaterial beim chirurgischen Wundverschluss in den Fokus rücken. Zellen im Wundbereich könnten direkt übertragen werden, wodurch die Wundheilung beschleunigt und verbessert werden könnte .
Obwohl es für Verpackungen und Beschriftung ISO-Normen gibt (ISO 20417) ISO Nr. 15223 , finden sich auf dem Markt keine standardisierten Verpackungen keine standardisierten Verpackungen . Dies bedeutet für den Anwender, dass er je nach Hersteller sich über die Handhabung der Verpackungen informieren muss. Die primäre Innenverpackung liegt in einer äußeren, meist durchsichtigen Peel-Back-Verpackung Peel-Back-Verpackung . Die Sterilität bleibt erhalten, bis sie geöffnet wird oder das Verfallsdatum erreicht ist. Die sterilen Inhalte sterilen Inhalte der Verpackungen werden von der OP-Pflegekraft oder dem Arzt durch Aufreißen der Umverpackung angereicht. Die sterile Verpackung lässt dann sich über Laschen öffnen. Der Inhalt wird nach Umklappen einer Lasche geöffnet. Die Nadel liegt frei und kann mit dem Nadelhalter in der Verpackung gefasst werden (s. hierzu ). Nach Aufklappen des Fadenträgers (Wickelträgers) wird die freigelegte Nadel mit anhängendem Faden entnommen. Diese kann vorsichtig gestreckt werden, um den Fadendrill zu vermindern. Hierbei sollte der Faden nicht zwischen Nadel und Fadenende, sondern nur innerhalb des Fadens gestreckt werden (Abb. ). Bei Ligaturen oder nadelfreien Fäden liegt das Material dementsprechend in der Verpackung.
Bei geflochtenem Nahtmaterial ist es aufgrund der größeren Reibehaftung normalerweise ausreichend, 3 Knoten 3 Knoten zu legen und anzuziehen, wobei einer gegenläufig sein sollte. Bei monofilem Nahtmaterial ist die Knotenzahl zu erhöhen, da die Reibehaftung aufgrund der glatten Oberfläche geringer ist. Hier gilt im Allgemeinen die Regel Fadenstärke in USP plus mindestens 1 zusätzlicher Knoten.
Eine sichere chirurgische Knotentechnik ist eine Grundlage für den Erfolg einer chirurgischen Maßnahme. Der fertige Knoten sollte fest, straff und nicht rutschend sein. Bei der Hautnaht Hautnaht liegt der Knoten nicht in der Inzisionslinie, um das Infektionsrisiko zu verringern. Merke Bei der Hautnaht liegt der Knoten immer seitlich der Inzisionslinie. Der chirurgische Knoten ist ein modifizierter Riffknoten modifizierter Riffknoten mit einer zusätzlichen Drehung im ersten Wurf, die die Zugfestigkeit des Knotens erhöht. Es ist darauf zu achten, dass der Knoten flach aufliegt; dies kommt beim sog. Samariter-Knoten oder auch Kreuzknoten genannt zustande (Abb. ). Die Knoten sollten klein sein. Am Schluss werden die Enden kurz geschnitten (2–4 mm). Überkreuzter Knoten in Einhandtechnik Um Gegenläufigkeit zu erreichen, wird normalerweise mit beiden Händen geknotet (Abb. a–m), einhändige Technik kann in manchen Situationen aber auch erforderlich werden (Abb. a–d). Die Legenden geben die Details wieder. Überkreuzter Knoten in Zweihandtechnik Dieser Knoten ist einfach zu knoten und hat einen guten Knotensitz (s. auch Abb. e–m) Die Legenden geben die Details wieder.
Bei der Hautnaht liegt der Knoten immer seitlich der Inzisionslinie. Der chirurgische Knoten ist ein modifizierter Riffknoten modifizierter Riffknoten mit einer zusätzlichen Drehung im ersten Wurf, die die Zugfestigkeit des Knotens erhöht. Es ist darauf zu achten, dass der Knoten flach aufliegt; dies kommt beim sog. Samariter-Knoten oder auch Kreuzknoten genannt zustande (Abb. ). Die Knoten sollten klein sein. Am Schluss werden die Enden kurz geschnitten (2–4 mm).
Um Gegenläufigkeit zu erreichen, wird normalerweise mit beiden Händen geknotet (Abb. a–m), einhändige Technik kann in manchen Situationen aber auch erforderlich werden (Abb. a–d). Die Legenden geben die Details wieder.
Dieser Knoten ist einfach zu knoten und hat einen guten Knotensitz (s. auch Abb. e–m) Die Legenden geben die Details wieder.
Zeitpunkt des Fadenzugs Hautfäden werden am zügigsten im Kopf-Hals-Bereich und bei Kindern gezogen. Bei Immunsuppression, z. B. Cortison-Einnahme, Diabetes mellitus sollte der Fadenzug prolongiert wegen der verzögerten Wundheilung erfolgen (s. auch Tab. ). Fadenzug Zum Fadenzug eignen sich verschiedene Techniken verschiedene Techniken . Ein Ende der Naht wird mit einer anatomischen Pinzette gefasst und vorsichtig angehoben oder zu einer Seite der Wunde gezogen. Bei Verwendung normaler Skalpellklingen wird beachtet, dass die Schneide hautabgewandt ist. Identisches Vorgehen kann auch mit einer spitzen Schere oder einem speziellen Fadenmesser erfolgen (Abb. , , , und ).
Hautfäden werden am zügigsten im Kopf-Hals-Bereich und bei Kindern gezogen. Bei Immunsuppression, z. B. Cortison-Einnahme, Diabetes mellitus sollte der Fadenzug prolongiert wegen der verzögerten Wundheilung erfolgen (s. auch Tab. ).
Zum Fadenzug eignen sich verschiedene Techniken verschiedene Techniken . Ein Ende der Naht wird mit einer anatomischen Pinzette gefasst und vorsichtig angehoben oder zu einer Seite der Wunde gezogen. Bei Verwendung normaler Skalpellklingen wird beachtet, dass die Schneide hautabgewandt ist. Identisches Vorgehen kann auch mit einer spitzen Schere oder einem speziellen Fadenmesser erfolgen (Abb. , , , und ).
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Arbuscular mycorrhizal fungi and | bbb78e64-8eb6-422d-9497-16c72ed96253 | 11080229 | Microbiology[mh] | Bacteria and fungi coexist in a wide variety of environments, and their interactions (so-called bacterial-fungal interactions (BFIs)) are crucial in the functioning of many agroecosystems, driving biogeochemical cycles and contributing to plant nutrition and health . Recently, co-occurrence analysis between bacteria and fungi revealed complex ecological processes such as cross-feeding, competition, and predation . Interactions involving keystone species — defined as “highly interconnected species that drive community responses through microbe-microbe interactions” — have been reported in the phyllosphere microbiome as well as in leaf litter, soils, and plants . The hyphosphere — “the microhabitat surrounding hyphal cells” — also provides niches that are colonized by bacterial communities that may play key roles in hyphal growth, production of secondary metabolites, and reproduction and are therefore important in processes such as the fermentation of foods (e.g., cheese), beverages (e.g., beer), and cultivation of edible mushrooms . In the last decade, increasing attention has been devoted to the hyphosphere of arbuscular mycorrhizal (AM) fungi, which are obligatory symbionts of plants. Given that AM fungal mycelia vastly extend in soil (from 82 to 111 m cm −3 in prairie and 52 to 81 m cm −3 in ungrazed pasture ) and mobilize between 4 and 20% of the total carbon (C) synthesized by plants , they can be considered as key players in ecosystems, enabling bacterial activity (e.g., solubilization of organic phosphorus (P)) by excretion of compounds used as nutrients or acting as signals at their surface . This hyphosphere interface thus represents a huge biological market for C and nutrients (especially P) with trade-offs between AM fungi and bacteria . This is important for the function of AM fungi, as they have little capacity to utilize organic nutrients and thus must cooperate with bacteria in the hyphosphere . For instance, recent studies have reported that some bacteria (e.g., Rahnella aquatilis — a phosphate solubilizing bacterium (PSB)) are stimulated at the hyphal surface of AM fungi, further allowing AM fungi to obtain P from organic P substrates . Interestingly, AM fungi can specifically enrich/select some bacterial groups and reject others at their hyphal surface . These selected bacteria might constitute the keystone candidate taxa of the microbial communities evolving in the hyphosphere. However, it is still unknown whether such bacterial taxa may shape hyphosphere communities, impacting fitness of AM fungi. Current knowledge on the AM fungal hyphosphere is still limited, since the study of BFI in this niche requires specific methodologies . Among them, the in vitro cultivation of AM fungi on root organs or on whole plants has allowed the production of a large amount of AM hyphae in a root-free and contaminant-free compartment. Here, we used the in vitro cultivation system on carrot root organs to investigate BFI in the AM fungal hyphosphere, with phytate as the unique P source. The results were then confirmed with synthetic communities of bacteria (SynComs) under pot cultivation systems in the greenhouse. Three hypotheses were specifically addressed: (1) AM fungi recruited specific bacteria from a community of in vitro culturable bacteria as keystone taxa; (2) the selected keystone taxa had strong abilities to mobilize organic P, exchanging benefit with AM fungi, e.g., acquiring C source; and (3) the keystone taxa could inhibit other bacteria from competition with the AM fungus for P source, therefore influencing the microbial community evolving at the hyphal surface.
The genus Streptomyces was a keystone taxon in the in vitro culturable bacterial community of the hyphosphere of AM fungi Bacterial suspensions extracted from the soils (bulk soil) of five long-term field experiments (soil SH, BJ, QY, WL, and TA) across China were spread on the surface of the hyphal compartment (HC) covered by extraradical hyphae (ERH) of Rhizophagus irregularis MUCL 43194 (i.e., ERH treatments) or free of hyphae (i.e., control treatments). Community composition was assessed by 16S rRNA gene profiling, 6 days after inoculation (DAI). Irrespective of soil origin, bacterial communities in the ERH treatments were dominated by the genus Streptomyces (Fig. A). Averaged over the five soils, this genus accounted for 77.5% ± 6.0% of the bacterial community. In the control treatments, a more even community (with Shannon evenness of 0.49 significantly higher than the Shannon evenness of 0.21 associated with ERH treatments) composition was noticed with the genus Streptomyces accounting for only 24.3% ± 4.2% of the bacterial community (Fig. A). The same approach was conducted comparing the bacterial community composition of ERH of four AM fungi (i.e., R. irregularis MUCL 43194, R . irregularis MUCL 41833, Rhizophagus clarus MUCL 46238, and R . intraradices MUCL 49410) and of the control treatments at 3 and 6 DAI. The soil BJ was considered. Regardless of DAI and AM fungus, the ERH treatments were dominated by the genus Streptomyces , while control treatments were dominated by the genus Pseudomonas (Fig. A). Averaged over the four AM fungal treatments, the genus Streptomyces accounted for 89.9% ± 1.1% and 67.0% ± 2.7% of the bacterial community at 3 and 6 DAI, respectively, while in the control treatments, it accounted for 4.7% ± 1.5% and 4.1% ± 0.7% of the bacterial community at 3 and 6 DAI, respectively. We then collected data from six publications to explore whether the preference for the genus Streptomyces is a widespread phenomenon. We compared the relative abundance of this genus in bulk and hyphosphere soil. The results showed that the relative abundance of the genus Streptomyces increased in the presence of AM fungi (Fig. B) suggesting a preponderance of AM fungi to recruit bacteria belonging to this genus. Two bacterial community co-occurrence network analyses were further constructed in the presence versus the absence of ERH. In the presence of ERH, 17 keystone amplicon sequence variants (ASVs) were identified among which 11 belonged to the genus Streptomyces (Fig. C). The keystone ASVs of this genus showed more than 48 degrees of co-occurrence network, suggesting that they might influence the presence and distribution of other ASVs in the network. In the absence of ERH, 31 keystone ASVs were identified that mainly belonged to the genera Variovorax , Allo-Neo-Para-Rhizobium , Pseudomonas , and Nocardioides . Ten other genera, also including Streptomyces , were represented by only one keystone ASV (Fig. C). To study the mechanisms involved in AM fungi and bacteria interaction, 62 bacteria were isolated from the ERH surface of R . irregularis MUCL 43194 (from the soil BJ), and 20 were selected by clustering at 97% similarity in 16S rRNA gene sequences to eliminate redundancy. The relative abundance of the 20 isolates on the ERH surface ranged from < 0.01 to 10.01% (Fig. D and Table S ). Interestingly, the mean relative abundance of ASVs that matched Streptomyces sp. D1 increased (from 5.62 to 10.01%) between the control treatments and the ERH treatments (Fig. D). Finally, two SynComs were constructed, composed of 20 bacterial isolates (see for composition Fig. E), thus including (SynCom20) or not (SynCom19) Streptomyces sp. D1. The two SynComs were spread on the surface of the hyphal compartment (HC) covered by ERH of R. irregularis MUCL 43194 or free of hyphae. In the absence of Streptomyces sp. D1 (i.e., SynCom19), 12 isolates were enriched at the surface of the ERH, while in the presence of this bacterium (i.e., SynCom20), only 6 isolates were enriched (Fig. E). Due to the presence of Streptomyces sp. D1, 7 out of the 12 isolates that were enriched by ERH in SynCom19 were subsequently disfavored by ERH treatment, as indicated by a lack of bacterial growth (Fig. E, labelled with star). The SynCom20 was inoculated in the HC of the pot microcosm. In the presence of ERH, the relative abundance of Streptomyces sp. D1 was above 50%, while in the absence of ERH it was less than 25% (Fig. F). The Streptomyces sp. D1 was the most abundant in the ERH of the soil environment (Fig. F). Collectively, the above results clearly showed that the genus Streptomyces was a keystone taxon in the early stage of interaction in the hyphosphere bacterial community of AM fungi under in vitro culture conditions. Streptomyces sp. D1 showed preference for trehalose as C source The carbon (C) assimilation profile of the 20 bacterial isolates was determined using six kinds of C sources (i.e., fructose, glucose, trehalose, inositol, citric acid, and succinic acid), which are among the major compounds found in the exudates of AM fungal hyphae . Most bacteria were able to grow in the presence of at least one kind of C source and show preference for glucose, succinic acid, or citric acid (Figure S ). Streptomyces sp. D1 could use all six kinds of C sources, with a preference for trehalose (Fig. A). The whole-genome sequencing of Streptomyces sp. D1 showed the presence of genes encoding the proteins involved in trehalose transport ( ThuE , ThuF , ThuG , and MalK ) and metabolism ( OtsB , TREH , TreZ , TreS , and glvA ) (Fig. B and Table S ). The transcriptional sequence results showed that in the presence of ERH of R. irregularis MUCL 43194, the expression of genes from trehalose to glucose-6-P of Streptomyces sp. D1, including α-trehalase gene TREH (converting trehalose to glucose) and polyphosphate glucokinase gene ppgK (converting glucose to glucose-6P), was significantly increased (Fig. B). Streptomyces sp. D1 had a strong ability to mineralize organic P in the hyphosphere The alkaline phosphatase (AP) activity of the 20 bacterial isolates was measured under C (10-mM glucose) and P (KH 2 PO 4 , 50 mM) limited condition via the release of para -nitrophenol ( p NP) from para -nitrophenol phosphate ( p NPP). The bacteria were arbitrarily separated in three groups (i.e., high > 0.8, 0.4 < medium < 0.8, and low < 0.4-mM p NP hr −1 OD 600 −1 ) according to their efficacy to produce AP (Fig. C), with bacteria having a high AP being significantly different from those with low AP (Tukey’s HSD, P < 0.05). Streptomyces sp. D1 ranked in the highest AP production efficiency, while Pseudomonas sp. H2 had nearly the lowest AP production efficiency (Fig. C). From the genome analysis, Streptomyces sp. D1 had abundant P metabolism genes, including 10 genes related to organic P mineralization, 9 genes related to inorganic P solubilization, 14 genes related to P transporters, and 2 genes related to P metabolism regulation (Fig. D). Streptomyces sp. D1 also had genes (e.g., GH6, PL1, PL3, and PL9) involved in the degradation of plant cell wall polysaccharides (Fig. D), which are absent in the genome of R . irregularis (Additional file ). Overall, and with the exception of the regulatory genes, the number of genes involved in P metabolism (inorganic P solubilization, organic P mineralization, and P transporters), cellulose or hemicellulose degradation (glycoside hydrolases, GHs; glycosyl transferases, GTs; polysaccharide lyases, PLs; carbohydrate esterases, CEs; auxiliary activities, AAs; carbohydrate-binding modules, CBMs) was higher in the genome of Streptomyces sp. D1 compared to the genome of Pseudomonas sp. H2 (Fig. D, Table S , Table S , and Additional file ). Streptomyces sp. D1 and Pseudomonas sp. H2 were the dominant organisms in the bacterial community in the presence and absence of AM fungal hyphae, respectively, in soil BJ. Therefore, to determine the influence of AM fungi on the organic P mobilization ability of Streptomyces sp. D1 and Pseudomonas sp. H2, a comparative mRNA transcriptomic analysis was performed on both bacteria in the presence or absence of ERH of R . irregularis MUCL 43194. Overall, 768 and 1159 genes were significantly differentially expressed ( P < 0.05) between these two conditions in Streptomyces sp. D1 and Pseudomonas sp. H2, respectively. Numerous genes associated with energy production were up-regulated in the glycolysis and citrate cycles in both microorganisms, indicating that a huge flow of C was transferred from the AM fungus to the bacteria (Figure S A, B). The up-regulation of fructose phosphotransferase gene fruA and alpha-trehalase gene TREH (Fig. A) suggested that the hyphae released fructose and trehalose were taken up by Streptomyces sp. D1 resulting in genes up-regulated in the glycolysis and citrate cycle pathways. Furthermore, the inorganic P transporters, including pstS , pstC , and pstB , were down-regulated in Streptomyces sp. D1 (Fig. A). Most of the genes involved in glycolysis, citrate cycle, and pentose phosphate pathway were significantly increased in Pseudomonas sp. H2 (Fig. B and Figure S B), as well as its inorganic P transporter genes ( pstS , pstC , pstA , and pstB ) (Fig. B). The inorganic P solubilization gene gcd / gdh and organic P mineralization gene phoD were not significantly changed in both strains grown in contact with the ERH (Fig. A, B; Figure S A, B). Much more genes of C (glycolysis, citrate cycle, and pentose phosphate) and P (purine metabolism and pyrimidine metabolism) consumption pathways were up-regulated in Pseudomonas sp. H2 than in Streptomyces sp. D1 (Fig. C). The Sec secretion pathway (including genes: secA , the ATPase motor; ftsY , membrane receptor; secYEG , transmembrane SecYEG channel; secDF , yajC , and yidC , auxiliary component enhance translocation efficiency) is ubiquitous in all domains of bacteria that can secrete enzymes (e.g., AP). The genes of secY and yidC were significantly up-regulated in both bacteria (Fig. A, B). This was consistent with the stronger activity of AP observed in the two bacteria in contact with ERH compared to the control. Streptomyces sp. D1 showed much stronger AP (more than 37 times) than Pseudomonas sp. H2 in contact with ERH (Fig. D). Furthermore, 17 out of 89 cell motility genes were up-regulated in Pseudomonas sp. H2 indicating that motility was activated in the presence of ERH exudates (Table S and Additional file ). Streptomyces sp. D1 stimulated organic P utilization by AM fungi and gene expression of C-P exchange in plants The phytate-P consumption of Streptomyces sp. D1 and Pseudomonas sp. H2 was significantly enhanced when the bacteria were grown in contact with the ERH of R. irregularis MUCL 43194 versus their growth in the absence of ERH (Fig. A). Moreover, in the presence of the ERH, the phytate consumption of Streptomyces sp. D1 was nearly doubled as compared to Pseudomonas sp. H2, and in the absence of ERH, it was six-folds greater (Fig. A). Gene expression of Pi-transporter ( Pho84 ), vacuolar phosphate transporter ( Pho91 ), and vacuolar transporter chaperone ( VTC2 and VTC4 ) was significantly greater in the ERH inoculated with Streptomyces sp. D1 as compared to the control (i.e., the ERH without the bacteria — Fig. B). Reversely, in the presence of Pseudomonas sp. H2, a significant down-regulation was noticed for VTC2 and VTC4 compared to the control. No differences in gene expression in the presence/absence of Pseudomonas sp. H2 were noticed for Pho84 and Pho91 compared to the control (Fig. B). A comparative mRNA transcriptome was conducted on triplicate ERH samples (each replicate was a mix of 5 plates, 15 plates for each treatment) of R. irregularis MUCL 43194 associated or not to Streptomyces sp. D1. More than 1.6% of the AM fungal gene repertoire (434 out of 26143 genes) was affected by the bacterium. Twenty genes, known to be necessary for phosphate and polyphosphate transport and metabolism in R. irregularis , were scrutinized. A general increase of expression of genes involved in phosphate limitation regulated gene ( pho80 ) phosphate transport ( pho84 ) and polyphosphate synthesis/decomposition ( VTC2 , VTC4 , and PPN 1) was noticed in the presence of Streptomyces sp. D1 (Fig. C). In addition, the expression of polyphosphate-related ion transporter genes including ZRT1 (zinc-regulated transporter 1), MatA (magnesium transporting ATPase), and VIT1 (vacuolar iron transporter 1) was up-regulated in the presence of the bacterium (Fig. C). A general increase in NAD(P)H synthesis and oxidoreduction was also observed (Fig. C). The differentially expressed genes were divided into Gene Ontology (GO) functional categories. This included 112 genes associated to cellular components, 49 genes to biological processes, and 136 genes to molecular functions (Fig. D). The genes most up- or down-regulated belong to the integral component of membrane, the ATP binding, and protein kinase activity. This included 46 genes up-regulated and 48 genes down-regulated (integral component of membrane), 24 genes up-regulated and 21 genes down-regulated (ATP binding), and 17 genes up-regulated and 20 genes down-regulated (protein kinase activity) in the ERH of the AM fungus inoculated with Streptomyces sp. D1 as compared to the fungus grown in the absence of the bacterium, respectively (Fig. D). The effect of bacteria on AM fungi could be extended to the plant. After inoculation of Streptomyces sp. D1 in the hyphosphere, a significant increase in the expression of the ATPase gene MtHA1 that is essential for P transport from the symbiotic interface to the plant was observed. Similarly, the expression of genes related to fatty acid synthesis ( WRI5a and MtFatM ) and transport ( STR2 ) from plants to symbiotic interface was significantly increased (Fig. E). The inoculation of Pseudomonas sp. H2 in the hyphosphere also significantly increased the expression of WRI5a and STR2 in the plant, while no differences were noticed for the expression of genes MtFatM and MtHA1 (Fig. E). Streptomyces sp. D1 impacted the bacterial community in the hyphosphere Exudates of Streptomyces sp. D1 were tested for growth inhibition on 19 bacteria from the surface of ERH grown with soil BJ bacterial suspension. Fourteen out of 19 isolates, including Pseudomonas sp. H2, were inhibited by the exudates (Fig. A). Notably, six isolates with higher AP production efficiency (> 0.4 mM p NP h −1 OD 600 −1 ) were not impacted or minimally inhibited (Fig. B). A negative correlation was found between Streptomyces sp. D1 and Pseudomonas sp. H2 in soil BJ. qPCR showed a significant decrease in the absolute abundance ratio of Streptomyces sp. D1 and Pseudomonas sp. H2 in the absence of ERH. Streptomyces sp. D1 had greater colony development in ERH treatment in single or dual culture than in the control treatment (Fig. C, D). Both the culture cells and exudates of Streptomyces sp. D1 were able to inhibit the normal growth of Pseudomonas sp. H2 (Fig. E). The Streptomyces sp. D1 harbored the entire albaflavenone (bactericidal antibiotic) synthesis pathway genes (Fig. F, G and Table S ). Consistently, a general increase of genes involved in terpenoid backbone biosynthesis in Streptomyces sp. D1 was observed, which were necessary for synthesis of albaflavenone precursor substances (Fig. G and Figure S ). Additionally, albaflavenone (C 15 H 22 O) at the concentration of 0.93 µg L −1 ( R 2 = 0.998) was identified by UPLC-MSMS against the standard (CAS: 157078–47-2) on the 1/2 TSB medium cultured Streptomyces sp. D1 (Fig. H).
Streptomyces was a keystone taxon in the in vitro culturable bacterial community of the hyphosphere of AM fungi Bacterial suspensions extracted from the soils (bulk soil) of five long-term field experiments (soil SH, BJ, QY, WL, and TA) across China were spread on the surface of the hyphal compartment (HC) covered by extraradical hyphae (ERH) of Rhizophagus irregularis MUCL 43194 (i.e., ERH treatments) or free of hyphae (i.e., control treatments). Community composition was assessed by 16S rRNA gene profiling, 6 days after inoculation (DAI). Irrespective of soil origin, bacterial communities in the ERH treatments were dominated by the genus Streptomyces (Fig. A). Averaged over the five soils, this genus accounted for 77.5% ± 6.0% of the bacterial community. In the control treatments, a more even community (with Shannon evenness of 0.49 significantly higher than the Shannon evenness of 0.21 associated with ERH treatments) composition was noticed with the genus Streptomyces accounting for only 24.3% ± 4.2% of the bacterial community (Fig. A). The same approach was conducted comparing the bacterial community composition of ERH of four AM fungi (i.e., R. irregularis MUCL 43194, R . irregularis MUCL 41833, Rhizophagus clarus MUCL 46238, and R . intraradices MUCL 49410) and of the control treatments at 3 and 6 DAI. The soil BJ was considered. Regardless of DAI and AM fungus, the ERH treatments were dominated by the genus Streptomyces , while control treatments were dominated by the genus Pseudomonas (Fig. A). Averaged over the four AM fungal treatments, the genus Streptomyces accounted for 89.9% ± 1.1% and 67.0% ± 2.7% of the bacterial community at 3 and 6 DAI, respectively, while in the control treatments, it accounted for 4.7% ± 1.5% and 4.1% ± 0.7% of the bacterial community at 3 and 6 DAI, respectively. We then collected data from six publications to explore whether the preference for the genus Streptomyces is a widespread phenomenon. We compared the relative abundance of this genus in bulk and hyphosphere soil. The results showed that the relative abundance of the genus Streptomyces increased in the presence of AM fungi (Fig. B) suggesting a preponderance of AM fungi to recruit bacteria belonging to this genus. Two bacterial community co-occurrence network analyses were further constructed in the presence versus the absence of ERH. In the presence of ERH, 17 keystone amplicon sequence variants (ASVs) were identified among which 11 belonged to the genus Streptomyces (Fig. C). The keystone ASVs of this genus showed more than 48 degrees of co-occurrence network, suggesting that they might influence the presence and distribution of other ASVs in the network. In the absence of ERH, 31 keystone ASVs were identified that mainly belonged to the genera Variovorax , Allo-Neo-Para-Rhizobium , Pseudomonas , and Nocardioides . Ten other genera, also including Streptomyces , were represented by only one keystone ASV (Fig. C). To study the mechanisms involved in AM fungi and bacteria interaction, 62 bacteria were isolated from the ERH surface of R . irregularis MUCL 43194 (from the soil BJ), and 20 were selected by clustering at 97% similarity in 16S rRNA gene sequences to eliminate redundancy. The relative abundance of the 20 isolates on the ERH surface ranged from < 0.01 to 10.01% (Fig. D and Table S ). Interestingly, the mean relative abundance of ASVs that matched Streptomyces sp. D1 increased (from 5.62 to 10.01%) between the control treatments and the ERH treatments (Fig. D). Finally, two SynComs were constructed, composed of 20 bacterial isolates (see for composition Fig. E), thus including (SynCom20) or not (SynCom19) Streptomyces sp. D1. The two SynComs were spread on the surface of the hyphal compartment (HC) covered by ERH of R. irregularis MUCL 43194 or free of hyphae. In the absence of Streptomyces sp. D1 (i.e., SynCom19), 12 isolates were enriched at the surface of the ERH, while in the presence of this bacterium (i.e., SynCom20), only 6 isolates were enriched (Fig. E). Due to the presence of Streptomyces sp. D1, 7 out of the 12 isolates that were enriched by ERH in SynCom19 were subsequently disfavored by ERH treatment, as indicated by a lack of bacterial growth (Fig. E, labelled with star). The SynCom20 was inoculated in the HC of the pot microcosm. In the presence of ERH, the relative abundance of Streptomyces sp. D1 was above 50%, while in the absence of ERH it was less than 25% (Fig. F). The Streptomyces sp. D1 was the most abundant in the ERH of the soil environment (Fig. F). Collectively, the above results clearly showed that the genus Streptomyces was a keystone taxon in the early stage of interaction in the hyphosphere bacterial community of AM fungi under in vitro culture conditions.
sp. D1 showed preference for trehalose as C source The carbon (C) assimilation profile of the 20 bacterial isolates was determined using six kinds of C sources (i.e., fructose, glucose, trehalose, inositol, citric acid, and succinic acid), which are among the major compounds found in the exudates of AM fungal hyphae . Most bacteria were able to grow in the presence of at least one kind of C source and show preference for glucose, succinic acid, or citric acid (Figure S ). Streptomyces sp. D1 could use all six kinds of C sources, with a preference for trehalose (Fig. A). The whole-genome sequencing of Streptomyces sp. D1 showed the presence of genes encoding the proteins involved in trehalose transport ( ThuE , ThuF , ThuG , and MalK ) and metabolism ( OtsB , TREH , TreZ , TreS , and glvA ) (Fig. B and Table S ). The transcriptional sequence results showed that in the presence of ERH of R. irregularis MUCL 43194, the expression of genes from trehalose to glucose-6-P of Streptomyces sp. D1, including α-trehalase gene TREH (converting trehalose to glucose) and polyphosphate glucokinase gene ppgK (converting glucose to glucose-6P), was significantly increased (Fig. B).
sp. D1 had a strong ability to mineralize organic P in the hyphosphere The alkaline phosphatase (AP) activity of the 20 bacterial isolates was measured under C (10-mM glucose) and P (KH 2 PO 4 , 50 mM) limited condition via the release of para -nitrophenol ( p NP) from para -nitrophenol phosphate ( p NPP). The bacteria were arbitrarily separated in three groups (i.e., high > 0.8, 0.4 < medium < 0.8, and low < 0.4-mM p NP hr −1 OD 600 −1 ) according to their efficacy to produce AP (Fig. C), with bacteria having a high AP being significantly different from those with low AP (Tukey’s HSD, P < 0.05). Streptomyces sp. D1 ranked in the highest AP production efficiency, while Pseudomonas sp. H2 had nearly the lowest AP production efficiency (Fig. C). From the genome analysis, Streptomyces sp. D1 had abundant P metabolism genes, including 10 genes related to organic P mineralization, 9 genes related to inorganic P solubilization, 14 genes related to P transporters, and 2 genes related to P metabolism regulation (Fig. D). Streptomyces sp. D1 also had genes (e.g., GH6, PL1, PL3, and PL9) involved in the degradation of plant cell wall polysaccharides (Fig. D), which are absent in the genome of R . irregularis (Additional file ). Overall, and with the exception of the regulatory genes, the number of genes involved in P metabolism (inorganic P solubilization, organic P mineralization, and P transporters), cellulose or hemicellulose degradation (glycoside hydrolases, GHs; glycosyl transferases, GTs; polysaccharide lyases, PLs; carbohydrate esterases, CEs; auxiliary activities, AAs; carbohydrate-binding modules, CBMs) was higher in the genome of Streptomyces sp. D1 compared to the genome of Pseudomonas sp. H2 (Fig. D, Table S , Table S , and Additional file ). Streptomyces sp. D1 and Pseudomonas sp. H2 were the dominant organisms in the bacterial community in the presence and absence of AM fungal hyphae, respectively, in soil BJ. Therefore, to determine the influence of AM fungi on the organic P mobilization ability of Streptomyces sp. D1 and Pseudomonas sp. H2, a comparative mRNA transcriptomic analysis was performed on both bacteria in the presence or absence of ERH of R . irregularis MUCL 43194. Overall, 768 and 1159 genes were significantly differentially expressed ( P < 0.05) between these two conditions in Streptomyces sp. D1 and Pseudomonas sp. H2, respectively. Numerous genes associated with energy production were up-regulated in the glycolysis and citrate cycles in both microorganisms, indicating that a huge flow of C was transferred from the AM fungus to the bacteria (Figure S A, B). The up-regulation of fructose phosphotransferase gene fruA and alpha-trehalase gene TREH (Fig. A) suggested that the hyphae released fructose and trehalose were taken up by Streptomyces sp. D1 resulting in genes up-regulated in the glycolysis and citrate cycle pathways. Furthermore, the inorganic P transporters, including pstS , pstC , and pstB , were down-regulated in Streptomyces sp. D1 (Fig. A). Most of the genes involved in glycolysis, citrate cycle, and pentose phosphate pathway were significantly increased in Pseudomonas sp. H2 (Fig. B and Figure S B), as well as its inorganic P transporter genes ( pstS , pstC , pstA , and pstB ) (Fig. B). The inorganic P solubilization gene gcd / gdh and organic P mineralization gene phoD were not significantly changed in both strains grown in contact with the ERH (Fig. A, B; Figure S A, B). Much more genes of C (glycolysis, citrate cycle, and pentose phosphate) and P (purine metabolism and pyrimidine metabolism) consumption pathways were up-regulated in Pseudomonas sp. H2 than in Streptomyces sp. D1 (Fig. C). The Sec secretion pathway (including genes: secA , the ATPase motor; ftsY , membrane receptor; secYEG , transmembrane SecYEG channel; secDF , yajC , and yidC , auxiliary component enhance translocation efficiency) is ubiquitous in all domains of bacteria that can secrete enzymes (e.g., AP). The genes of secY and yidC were significantly up-regulated in both bacteria (Fig. A, B). This was consistent with the stronger activity of AP observed in the two bacteria in contact with ERH compared to the control. Streptomyces sp. D1 showed much stronger AP (more than 37 times) than Pseudomonas sp. H2 in contact with ERH (Fig. D). Furthermore, 17 out of 89 cell motility genes were up-regulated in Pseudomonas sp. H2 indicating that motility was activated in the presence of ERH exudates (Table S and Additional file ).
sp. D1 stimulated organic P utilization by AM fungi and gene expression of C-P exchange in plants The phytate-P consumption of Streptomyces sp. D1 and Pseudomonas sp. H2 was significantly enhanced when the bacteria were grown in contact with the ERH of R. irregularis MUCL 43194 versus their growth in the absence of ERH (Fig. A). Moreover, in the presence of the ERH, the phytate consumption of Streptomyces sp. D1 was nearly doubled as compared to Pseudomonas sp. H2, and in the absence of ERH, it was six-folds greater (Fig. A). Gene expression of Pi-transporter ( Pho84 ), vacuolar phosphate transporter ( Pho91 ), and vacuolar transporter chaperone ( VTC2 and VTC4 ) was significantly greater in the ERH inoculated with Streptomyces sp. D1 as compared to the control (i.e., the ERH without the bacteria — Fig. B). Reversely, in the presence of Pseudomonas sp. H2, a significant down-regulation was noticed for VTC2 and VTC4 compared to the control. No differences in gene expression in the presence/absence of Pseudomonas sp. H2 were noticed for Pho84 and Pho91 compared to the control (Fig. B). A comparative mRNA transcriptome was conducted on triplicate ERH samples (each replicate was a mix of 5 plates, 15 plates for each treatment) of R. irregularis MUCL 43194 associated or not to Streptomyces sp. D1. More than 1.6% of the AM fungal gene repertoire (434 out of 26143 genes) was affected by the bacterium. Twenty genes, known to be necessary for phosphate and polyphosphate transport and metabolism in R. irregularis , were scrutinized. A general increase of expression of genes involved in phosphate limitation regulated gene ( pho80 ) phosphate transport ( pho84 ) and polyphosphate synthesis/decomposition ( VTC2 , VTC4 , and PPN 1) was noticed in the presence of Streptomyces sp. D1 (Fig. C). In addition, the expression of polyphosphate-related ion transporter genes including ZRT1 (zinc-regulated transporter 1), MatA (magnesium transporting ATPase), and VIT1 (vacuolar iron transporter 1) was up-regulated in the presence of the bacterium (Fig. C). A general increase in NAD(P)H synthesis and oxidoreduction was also observed (Fig. C). The differentially expressed genes were divided into Gene Ontology (GO) functional categories. This included 112 genes associated to cellular components, 49 genes to biological processes, and 136 genes to molecular functions (Fig. D). The genes most up- or down-regulated belong to the integral component of membrane, the ATP binding, and protein kinase activity. This included 46 genes up-regulated and 48 genes down-regulated (integral component of membrane), 24 genes up-regulated and 21 genes down-regulated (ATP binding), and 17 genes up-regulated and 20 genes down-regulated (protein kinase activity) in the ERH of the AM fungus inoculated with Streptomyces sp. D1 as compared to the fungus grown in the absence of the bacterium, respectively (Fig. D). The effect of bacteria on AM fungi could be extended to the plant. After inoculation of Streptomyces sp. D1 in the hyphosphere, a significant increase in the expression of the ATPase gene MtHA1 that is essential for P transport from the symbiotic interface to the plant was observed. Similarly, the expression of genes related to fatty acid synthesis ( WRI5a and MtFatM ) and transport ( STR2 ) from plants to symbiotic interface was significantly increased (Fig. E). The inoculation of Pseudomonas sp. H2 in the hyphosphere also significantly increased the expression of WRI5a and STR2 in the plant, while no differences were noticed for the expression of genes MtFatM and MtHA1 (Fig. E).
sp. D1 impacted the bacterial community in the hyphosphere Exudates of Streptomyces sp. D1 were tested for growth inhibition on 19 bacteria from the surface of ERH grown with soil BJ bacterial suspension. Fourteen out of 19 isolates, including Pseudomonas sp. H2, were inhibited by the exudates (Fig. A). Notably, six isolates with higher AP production efficiency (> 0.4 mM p NP h −1 OD 600 −1 ) were not impacted or minimally inhibited (Fig. B). A negative correlation was found between Streptomyces sp. D1 and Pseudomonas sp. H2 in soil BJ. qPCR showed a significant decrease in the absolute abundance ratio of Streptomyces sp. D1 and Pseudomonas sp. H2 in the absence of ERH. Streptomyces sp. D1 had greater colony development in ERH treatment in single or dual culture than in the control treatment (Fig. C, D). Both the culture cells and exudates of Streptomyces sp. D1 were able to inhibit the normal growth of Pseudomonas sp. H2 (Fig. E). The Streptomyces sp. D1 harbored the entire albaflavenone (bactericidal antibiotic) synthesis pathway genes (Fig. F, G and Table S ). Consistently, a general increase of genes involved in terpenoid backbone biosynthesis in Streptomyces sp. D1 was observed, which were necessary for synthesis of albaflavenone precursor substances (Fig. G and Figure S ). Additionally, albaflavenone (C 15 H 22 O) at the concentration of 0.93 µg L −1 ( R 2 = 0.998) was identified by UPLC-MSMS against the standard (CAS: 157078–47-2) on the 1/2 TSB medium cultured Streptomyces sp. D1 (Fig. H).
The roots of most soil-grown plants are colonized by AM fungi with their ERH inhabited by structured communities of bacteria that provide direct benefits to their fungal host (e.g., solubilization of organic P) in exchange for C resources . As a result, the interaction between AM fungi and bacteria along the soil-hyphae-root continuum and microbe-microbe interactions on the hyphal surface has gradually received increased attention in recent years. Besides the endobacterium living inside an AM fungus which has been demonstrated to have a strong impact on both the fungal and plant associates , the hyphosphere bacteria have been shown to have a significant impact on the nutrient acquisition and fitness of AM fungi . The study of the hyphosphere microbiome (hyphobiome) of AM fungi requires to consider not only the interactions between AM fungi and their associated bacteria but also those between different species or functional groups of bacteria living at the hyphal surface . Here, we showed that 4 AM fungal strains are capable to preferentially recruit a specific bacterial taxon (i.e., Streptomyces sp. D1), from a community of in vitro culturable bacteria, in their hyphosphere, improving their nutrition and rewarding it with hyphal exudates. This selection further benefits the association between AM fungi and the plant by increasing the expression of genes involved in C-P exchange at the symbiotic interface. Moreover, we demonstrated that Streptomyces sp. D1 is a keystone taxon that plays an important role in shaping the bacterial community structure in the early stage of interaction at the surface of AM fungal hyphae, partly inhibiting the growth of bacteria with weak P-mineralizing ability, and noted that its removal can lead to significant changes in microbiome composition and functioning. AM fungi preferentially recruits Streptomyces sp. from a community of in vitro culturable bacteria Our study demonstrated that the ERH of AM fungi harbors a highly diverse assemblage of bacteria, further supporting evidence that hyphae release exudates that provide an energy-rich microhabitat shaping bacterial community composition in the same way as root exudates . Thus, AM fungal hyphae can be an important source of nutrients for a wide range of bacteria. The leachates of five soils inoculated in vitro on the ERH of R. irregularis MUCL 43194 or a single leachate of soil inoculated on the ERH of four different AM fungi showed a predominance of the genus Streptomyces over the other bacterial taxa. This was supported by data collected in other studies conducted in vitro and in soil showing a clear enrichment of the genus Streptomyces by the presence of AM fungi, which can be considered as “fungiphile”, as suggested by Warmink and van Elsas for other bacteria closely associated with soil fungi. In the present study, we analyzed the bacterial community at the early stage of interaction with the hyphae of AM fungi and found that Streptomyces sp. was a bacterium that grew rapidly in response to the presence of AM fungi. These early bacterial communities play a decisive role during the “establishment phase”, setting the stage for later community assemblages. Understanding this nascent community is crucial to understand the selection pressures exerted by AM fungi. The dynamics of early establishment on the hyphal surface reflects AM fungal selection, before other environmental factors affect these interactions. Long-term interaction studies are needed to determine whether these bacteria will remain dominant in the hyphosphere bacterial community. It should be noted, however, that under soil conditions, studies have shown that Proteobacteria, Actinobacteria, and Myxococcota phyla are enriched by AM fungi. In addition, a considerable proportion of Actinobacteria have been attributed C by AM fungi or have been defined as core species . The differences noticed between our in vitro culture system and the studies conducted under soil conditions may be attributed to the following reasons: First, the bacterial community in soil is much larger than in in vitro that only comprise culturable bacteria. Second, the environmental conditions markedly differ between soil and in vitro systems. Indeed, the soil is a complex micro-niche composed of solid, liquid, and gas phases and may also comprise a number of uncontrolled or less controlled factors (e.g., unwanted microbial contaminants). In its defense, the in vitro culture system is the only that makes it possible to study the bacteria-bacteria and bacteria-AM fungi interactions in a very rigorous way (i.e., under strictly controlled conditions). Thirdly, the bacterial community investigated in the present study is at the very early stage of interaction on the hyphal surface. It is therefore suggested that early growers may be more likely to dominate the bacterial community. The preponderance of Streptomyces sp. D1 in our study could be related partly to its ability to assimilate fructose, glucose, trehalose, inositol, citric acid, and succinic acid, which are among the major compounds found in the exudates of AM fungal hyphae . Most of the other bacteria tested (e.g., Roseateles , Pseudomonas , Allo-Neo-Para-Rhizobium , see Figure S ) were able to grow in the presence of at least one C source and show preference for glucose, succinic acid, or citric acid (Figure S ). Streptomyces sp. D1 showed preference for trehalose, suggesting that this compound was favorably used by this bacterium in the hyphosphere. This was supported by the trehalose transporter clusters found in the genome of Streptomyces sp. D1. The genes for synthesis of trehalose, trehalose 6-phosphate synthase ( otsA and TPS ), are present in the genome of R . irregularis MUCL 43194. However, no significant change was observed at the transcriptomic level. Previous studies have detected glucose, fructose, and trehalose in the secretion of hyphae. In particular, trehalose is highly abundant within hyphae and possesses the potential to be released extracellularly. The preponderance of Streptomyces sp. D1 on the hyphal surface may also be related to the nutritive advantage that the AM fungi may derive from this association. Zhang et al. have already demonstrated that the PSB Rahnella aquatilis was able to mineralize organic P (i.e., phytate) into inorganic P, stimulating the processes involved in P uptake by R . irregularis . In exchange, the AM fungus rewarded the bacterium with fructose, which could be considered as a C source but also as a signal molecule triggering bacteria-mediated organic P mineralization processes. On the basis of the growth curve and the AP production efficiency of the bacteria, it was observed that at a glucose concentration of 10 mM, the bacterial culture remained stable for 48 h, indicating glucose depletion. In addition, Streptomyces sp. D1 showed the highest efficiency of AP production compared to the other bacteria. This implied that if the AM fungus was able to invest the same amount of C in Streptomyces sp. D1, it would obtain the greatest reward in terms of P mobilization from this bacterium. In addition, as a consequence of the organic P mobilization by Streptomyces , the AM fungal genes involved in P uptake and polyP synthesis were up-regulated. Collectively, these results suggested that the AM fungi receive direct benefits from Streptomyces sp. D1 by increasing P availability. Streptomyces sp. D1 is a keystone taxon shaping the bacterial community structure at the surface of AM fungal hyphae We constructed microbial co-occurrence networks based on soil samples to investigate the interactions among bacteria. Streptomyces species were found to be keystones taxa in the network and played a major role in constructing the microbial network. Synthetic microbial communities were also designed to verify the results based on co-occurrence network. Streptomyces sp. D1 strongly altered the bacterial community and inhibited the growth of certain bacterial isolates, especially those with low AP production efficiency. For instance, the relative abundance of Paenarthrobacter sp. 31 decreased from 19.4% in the ERH-SynCom without Streptomyces sp. D1 to 1.7% in the presence of Streptomyces sp. D1. Besides the C for P exchange demonstrated between the AM fungi and Streptomyces sp. D1 (see above), the AM fungi could indirectly reward Streptomyces sp. D1 with C for its ability to compete with other bacteria using inorganic P sources for their own needs when they utilize the hyphal exudates , thereby competing with AM fungi for P availability . Although no significant negative correlation was noticed between the AP production efficiency of the bacterial isolates and the growth inhibition by the exudates of Streptomyces sp. D1 ( R 2 = 0.033, P = 0.22, Fig. B), all the isolates having an AP production efficiency above 0.4 mM p NP h −1 OD 600 −1 had their growth either stimulated, not impacted, or only slightly inhibited by the exudates of Streptomyces sp. D1. This suggested that Streptomyces sp. D1 may spare or at least not impact too heavily the bacteria with the highest organic P mobilization capability and thus those bacteria that were not truly competing with AM fungi for P. Interestingly, in the interaction between Streptomyces sp. D1 and Pseudomonas sp. H2, the ratio of absolute abundance of both bacteria at the hyphal surface was in favor of Streptomyces sp. D1, while in the absence of hyphae, the contrary was noticed. Pseudomonas sp. H2 has a very low AP production efficiency, therefore relying on the P mineralized by other bacteria with strong AP production efficiency. Furthermore, this bacterium inhibited polyP synthesis and transport processes in the ERH of the AM fungus. The exudates of Streptomyces sp. D1 inhibited the growth of Pseudomonas sp. H2 by nearly 40%, therefore decreasing the competitive pressure of this bacterium with the AM fungus for P. Using the whole genome sequence of Streptomyces sp. D1, multitudinous secondary metabolism genes were identified, related to antibiotics, including types I, II, and III polyketide synthases, non-ribosomal peptide synthases, and terpene (Additional file Table S6). Albaflavenone (C 15 H 22 O), a tricyclic sesquiterpene, was detected in the exudates of Streptomyces sp. D1 (Fig. F and Additional file Table S6) and reported to have strong antibacterial efficiently. Streptomyces sp. D1 could thus assist the AM fungus which lack the ability to inhibit the weak P-mineralizing bacteria . AM fungi and Streptomyces sp. D1: a global perspective Together, our results demonstrate the complex interactions between AM fungi and bacteria and bacterial-bacterial interactions on the surface of AM fungal hyphae. We showed that under the organic P condition, AM fungi are able to preferentially recruit from a community of in vitro culturable bacteria, species that improve their P nutrition and compete with weak P-mineralizing bacteria in the early stage (Fig. ). Thus, a privileged interaction was established with Streptomyces sp. D1; the ERH of AM fungi release C compounds, which are acquired by Streptomyces sp. D1 in exchange for mineralized organic P. This keystone bacterium also impacted the bacterial community residing on the hyphal surface, primarily by inhibiting bacteria with low AP production efficiency, thus competing with AM fungi for P. Furthermore, we shed light on the role of hyphae in connecting plants to bacteria. Indeed, the benefits of bacteria to AM fungi can be transmitted to plants via the hyphae, which greatly enhances our understanding of a novel paradigm: AM fungi serving as a bridge to connect plants and bacterial communities.
Streptomyces sp. from a community of in vitro culturable bacteria Our study demonstrated that the ERH of AM fungi harbors a highly diverse assemblage of bacteria, further supporting evidence that hyphae release exudates that provide an energy-rich microhabitat shaping bacterial community composition in the same way as root exudates . Thus, AM fungal hyphae can be an important source of nutrients for a wide range of bacteria. The leachates of five soils inoculated in vitro on the ERH of R. irregularis MUCL 43194 or a single leachate of soil inoculated on the ERH of four different AM fungi showed a predominance of the genus Streptomyces over the other bacterial taxa. This was supported by data collected in other studies conducted in vitro and in soil showing a clear enrichment of the genus Streptomyces by the presence of AM fungi, which can be considered as “fungiphile”, as suggested by Warmink and van Elsas for other bacteria closely associated with soil fungi. In the present study, we analyzed the bacterial community at the early stage of interaction with the hyphae of AM fungi and found that Streptomyces sp. was a bacterium that grew rapidly in response to the presence of AM fungi. These early bacterial communities play a decisive role during the “establishment phase”, setting the stage for later community assemblages. Understanding this nascent community is crucial to understand the selection pressures exerted by AM fungi. The dynamics of early establishment on the hyphal surface reflects AM fungal selection, before other environmental factors affect these interactions. Long-term interaction studies are needed to determine whether these bacteria will remain dominant in the hyphosphere bacterial community. It should be noted, however, that under soil conditions, studies have shown that Proteobacteria, Actinobacteria, and Myxococcota phyla are enriched by AM fungi. In addition, a considerable proportion of Actinobacteria have been attributed C by AM fungi or have been defined as core species . The differences noticed between our in vitro culture system and the studies conducted under soil conditions may be attributed to the following reasons: First, the bacterial community in soil is much larger than in in vitro that only comprise culturable bacteria. Second, the environmental conditions markedly differ between soil and in vitro systems. Indeed, the soil is a complex micro-niche composed of solid, liquid, and gas phases and may also comprise a number of uncontrolled or less controlled factors (e.g., unwanted microbial contaminants). In its defense, the in vitro culture system is the only that makes it possible to study the bacteria-bacteria and bacteria-AM fungi interactions in a very rigorous way (i.e., under strictly controlled conditions). Thirdly, the bacterial community investigated in the present study is at the very early stage of interaction on the hyphal surface. It is therefore suggested that early growers may be more likely to dominate the bacterial community. The preponderance of Streptomyces sp. D1 in our study could be related partly to its ability to assimilate fructose, glucose, trehalose, inositol, citric acid, and succinic acid, which are among the major compounds found in the exudates of AM fungal hyphae . Most of the other bacteria tested (e.g., Roseateles , Pseudomonas , Allo-Neo-Para-Rhizobium , see Figure S ) were able to grow in the presence of at least one C source and show preference for glucose, succinic acid, or citric acid (Figure S ). Streptomyces sp. D1 showed preference for trehalose, suggesting that this compound was favorably used by this bacterium in the hyphosphere. This was supported by the trehalose transporter clusters found in the genome of Streptomyces sp. D1. The genes for synthesis of trehalose, trehalose 6-phosphate synthase ( otsA and TPS ), are present in the genome of R . irregularis MUCL 43194. However, no significant change was observed at the transcriptomic level. Previous studies have detected glucose, fructose, and trehalose in the secretion of hyphae. In particular, trehalose is highly abundant within hyphae and possesses the potential to be released extracellularly. The preponderance of Streptomyces sp. D1 on the hyphal surface may also be related to the nutritive advantage that the AM fungi may derive from this association. Zhang et al. have already demonstrated that the PSB Rahnella aquatilis was able to mineralize organic P (i.e., phytate) into inorganic P, stimulating the processes involved in P uptake by R . irregularis . In exchange, the AM fungus rewarded the bacterium with fructose, which could be considered as a C source but also as a signal molecule triggering bacteria-mediated organic P mineralization processes. On the basis of the growth curve and the AP production efficiency of the bacteria, it was observed that at a glucose concentration of 10 mM, the bacterial culture remained stable for 48 h, indicating glucose depletion. In addition, Streptomyces sp. D1 showed the highest efficiency of AP production compared to the other bacteria. This implied that if the AM fungus was able to invest the same amount of C in Streptomyces sp. D1, it would obtain the greatest reward in terms of P mobilization from this bacterium. In addition, as a consequence of the organic P mobilization by Streptomyces , the AM fungal genes involved in P uptake and polyP synthesis were up-regulated. Collectively, these results suggested that the AM fungi receive direct benefits from Streptomyces sp. D1 by increasing P availability.
sp. D1 is a keystone taxon shaping the bacterial community structure at the surface of AM fungal hyphae We constructed microbial co-occurrence networks based on soil samples to investigate the interactions among bacteria. Streptomyces species were found to be keystones taxa in the network and played a major role in constructing the microbial network. Synthetic microbial communities were also designed to verify the results based on co-occurrence network. Streptomyces sp. D1 strongly altered the bacterial community and inhibited the growth of certain bacterial isolates, especially those with low AP production efficiency. For instance, the relative abundance of Paenarthrobacter sp. 31 decreased from 19.4% in the ERH-SynCom without Streptomyces sp. D1 to 1.7% in the presence of Streptomyces sp. D1. Besides the C for P exchange demonstrated between the AM fungi and Streptomyces sp. D1 (see above), the AM fungi could indirectly reward Streptomyces sp. D1 with C for its ability to compete with other bacteria using inorganic P sources for their own needs when they utilize the hyphal exudates , thereby competing with AM fungi for P availability . Although no significant negative correlation was noticed between the AP production efficiency of the bacterial isolates and the growth inhibition by the exudates of Streptomyces sp. D1 ( R 2 = 0.033, P = 0.22, Fig. B), all the isolates having an AP production efficiency above 0.4 mM p NP h −1 OD 600 −1 had their growth either stimulated, not impacted, or only slightly inhibited by the exudates of Streptomyces sp. D1. This suggested that Streptomyces sp. D1 may spare or at least not impact too heavily the bacteria with the highest organic P mobilization capability and thus those bacteria that were not truly competing with AM fungi for P. Interestingly, in the interaction between Streptomyces sp. D1 and Pseudomonas sp. H2, the ratio of absolute abundance of both bacteria at the hyphal surface was in favor of Streptomyces sp. D1, while in the absence of hyphae, the contrary was noticed. Pseudomonas sp. H2 has a very low AP production efficiency, therefore relying on the P mineralized by other bacteria with strong AP production efficiency. Furthermore, this bacterium inhibited polyP synthesis and transport processes in the ERH of the AM fungus. The exudates of Streptomyces sp. D1 inhibited the growth of Pseudomonas sp. H2 by nearly 40%, therefore decreasing the competitive pressure of this bacterium with the AM fungus for P. Using the whole genome sequence of Streptomyces sp. D1, multitudinous secondary metabolism genes were identified, related to antibiotics, including types I, II, and III polyketide synthases, non-ribosomal peptide synthases, and terpene (Additional file Table S6). Albaflavenone (C 15 H 22 O), a tricyclic sesquiterpene, was detected in the exudates of Streptomyces sp. D1 (Fig. F and Additional file Table S6) and reported to have strong antibacterial efficiently. Streptomyces sp. D1 could thus assist the AM fungus which lack the ability to inhibit the weak P-mineralizing bacteria .
Streptomyces sp. D1: a global perspective Together, our results demonstrate the complex interactions between AM fungi and bacteria and bacterial-bacterial interactions on the surface of AM fungal hyphae. We showed that under the organic P condition, AM fungi are able to preferentially recruit from a community of in vitro culturable bacteria, species that improve their P nutrition and compete with weak P-mineralizing bacteria in the early stage (Fig. ). Thus, a privileged interaction was established with Streptomyces sp. D1; the ERH of AM fungi release C compounds, which are acquired by Streptomyces sp. D1 in exchange for mineralized organic P. This keystone bacterium also impacted the bacterial community residing on the hyphal surface, primarily by inhibiting bacteria with low AP production efficiency, thus competing with AM fungi for P. Furthermore, we shed light on the role of hyphae in connecting plants to bacteria. Indeed, the benefits of bacteria to AM fungi can be transmitted to plants via the hyphae, which greatly enhances our understanding of a novel paradigm: AM fungi serving as a bridge to connect plants and bacterial communities.
Soil material and extraction of bacteria Soils were sampled in five long-term field experiments (soil SH: Shihezi 44°19′N, 86°00′E; soil BJ: Beijing 40°08′N, 116°10′E; soil QY: Qiyang 26°45′N, 111°52′E; soil WL: Wulumuqi 43°57′N, 87°46′E; soil TA: Taian 36°10′N, 117°09′E), which are located west (SH and WL), east (BJ and TA), and south (QY) of China. In each location, five subsamples of circa 300 g of soil were taken with an auger at a depth of 0 to 20 cm, mixed together, then sieved to 2 mm, and finally homogenized to constitute the soils SH, WL, BJ, TA, and QY. Soils were transported to the laboratory in sterile plastic bags on ice and temporarily stored at 4°C until extraction of bacterial suspensions. The texture, N, P, K, and organic C contents of the soils are detailed in Table S . Bacterial suspensions were extracted from the soils using Tween 80/tetrasodium yrophosphate (TTSP) and Nycodenz (for details, see Methods in the Additional file ). Arbuscular mycorrhizal fungi Four AM fungal stains, i.e., Rhizophagus irregularis MUCL 43194, Rhizophagus irregularis MUCL 41833, Rhizophagus clarus MUCL 46238, and Rhizophagus intraradices MUCL 49410, were provided by the Glomeromycota in vitro collection (GINCO, Belgium). The fungi were maintained in vitro on Ri T-DNA transformed roots of carrot ( Daucus carota L.) clone DC2 on the modified Strullu-Romand (MSR) medium as described in Cranenbrouck et al. (2005) . In vitro culture system design Bi-compartmented Petri plates (90 × 15 mm) were used to grow the carrot roots and AM fungi as detailed in St Arnaud et al. (1996) (Figure S A, B). Briefly, in one compartment (i.e., the root compartment — RC), the root was associated to the AM fungus, while in the other compartment (i.e., the hyphal compartment — HC), only the extraradical hyphae (ERH) of the AM fungus were allowed to growth. Twenty-five milliliters of sterilized (121°C for 15 min) MSR medium solidified with 3 g L −1 Phytagel (Gelzan™ CM, G3251, CP Kelco, USA) was added in the RC. In the HC, 20 mL of the modified MSR medium, termed M-MSR (without sucrose, inorganic P, EDTA, calcium, and vitamin sources), was added. The medium was supplemented with 280-μM organic P in the form of Na-phytate (phytic acid sodium salt hydrate, 68388, Sigma-Aldrich, MO, USA). A control treatment was included that consisted only of carrot roots growing in the RC. Impact of AM fungal strains and soil types on the bacterial community associated with extraradical hyphae Bi-compartmented Petri plates were prepared as described above with the AM fungus R. irregularis MUCL 43194. Seven weeks after association to the carrot root, the ERH in the RC crossed the partition wall separating the RC from HC and developed profusely in the HC. Two-hundred microliters of bacterial suspension from the five soils described above was inoculated on the surface of the HC. Thus, five treatments and their respective controls (i.e., the HC inoculated with the bacterial suspensions of the five soils without ERH, see Figure S ) were set up with three replicates each. After another 6 days, the M-MSR medium in the HC was dissolved with 35-mL aseptic sodium citrate buffer (10 mM, pH 6) , and the hyphae with their surface-associated bacteria were collected. Briefly, the M-MSR medium of the HC was cut into four pieces and distributed equally in two sterile centrifuge tubes of 50 mL, containing 35 mL of sodium citrate buffer, and vortexed for 4 min for dissolution of the phytagel. The hyphae from the two tubes were assembled, forming a pellet, and rinsed gently twice in a Petri plate containing fresh sodium citrate buffer to eliminate the remaining medium. Sterile blotting papers were used to soak up the excess buffer in the hyphal pellet. The controls that contained only the bacterial suspension in the HC were scraped from the surface of the M-MSR medium. The ERH and control samples were collected in 2-mL centrifuge tubes stored at − 80°C for DNA extraction. The total genomic DNA extraction of ERH and M-MSR medium was done using the FastDNA SPIN Kit for Soil (MP Biochemicals, Solon, OH, USA), following the manufacturer’s instructions. PCR amplification of the bacterial 16S rRNA gene V3–V4 region was performed. Pair-end 2 × 250 bp sequencing was performed using the Illumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd (see Methods in the Additional file ). The same approach as above was conducted with the four AM fungal strains (i.e., R. irregularis MUCL 43194, R . irregularis MUCL 41833, R . clarus MUCL 46238, and R . intraradices MUCL 49410) plus one control that consist of carrot roots not associated with an AM fungus, inoculated in the HC with 200 µL bacterial suspension from soil BJ. The BJ soil was selected because it had been collected as part of a long-term experiment with appropriate P application for over 20 years, representing the typical cultivation system of the North China Plain. Three replicates were considered per treatment. After 3 and 6 days, the M-MSR medium in the HC was dissolved with 35-mL sterilized sodium citrate buffer and then processed for DNA extraction as above. Isolation and identification of bacteria from extraradical hyphae of R . irregularis MUCL 43194 Bi-compartmented Petri plates were prepared with carrot roots clone DC2 associated with R. irregularis MUCL 43194 as described above. Bacterial suspension of BJ was inoculated in the HC for 6 days. The tiny layers of bacteria that developed around the hyphae were detached carefully with pipette tips under a stereomicroscope at 56 × magnification (Olympus SZX7, Japan). The bacteria were then streaked with the pipette tips on 10-mL solid tryptone soy broth (TSB, CM0129, Thermo Scientific, USA) in 60 × 15 mm plates (for details, see Methods in the Additional file ). To identify the bacteria, the V3–V4 regions of the 16S rRNA gene were applied on the DNAs extracted using degenerate primers (338F and 806R, Table S ). The PCR products obtained were subsequently sequenced by Sanger sequencing. To recover cultivated bacteria for further studies, the Sanger sequencing results were blasted (BLAST + 2.10.1) to the 16S rRNA gene amplicon sequence variants (ASVs) in the high-throughput sequencing data, allowing to relate the isolated bacteria to relevant ASVs. SynCom construction Bi-compartmented Petri plates were prepared with carrot roots clone DC2 associated with R. irregularis MUCL 43194 as described above. Two SynComs were constructed which consist of 20 bacterial isolates (including Streptomyces sp. D1) or 19 bacterial isolates (without Streptomyces sp. D1). The bacterial isolates were from the ERH of R. irregularis MUCL 43194 after inoculation with BJ bacteria suspension (Table S ). Each bacterium was cultured separately in 50-mL tubes, washed, and OD 600 adjusted to 0.6. The cultures were then mixed in 1:1 ratio and spread onto the HC containing the ERH. Non-mycorrhizal carrot roots plated in the RC and each of the two SynComs inoculated in the HC were considered as controls. After 3 days, the bacterial community on the ERH and on the medium (i.e., the controls) were collected in 2-mL centrifuge tubes stored at − 80°C for DNA extraction. The total genomic DNA was extracted and 16S rRNA gene amplified and analyzed. In parallel, a similar experiment was conducted in pots under 12-h light–dark cycle at 22°C night and 24°C day (for detailed description of the pot system, see Figure S ). The treatment consisted of the 20 bacterial isolates (thus with Streptomyces sp. D1) inoculated in the HC containing the ERH or devoid of ERH (SynCom + AM fungi and SynCom–AM fungi treatments, respectively). Maize ( Zea mays L., cv. Zhengdan 958) was used as the host plant and R. irregularis MUCL 43194 as the AM fungal strain. The soil from Changping, BJ was air-dried, sieved (2 mm), and sterilized by 25 kGy 60 Co gamma irradiation. The plant compartment was filled with a sterilized mixture of soil and river sand (4:1 v/v). The HC was filled with a sterilized mixture of soil and river sand (1:1, v:v). Ten weeks after planting, the ERH developed into the HC. At that time, the SynCom mixed with Na‐phytate was injected though the tube protruding outside the HC. The solution arrived at the top nylon mesh and then permeated to the soil homogeneously. After another 2 weeks, the soil, river sand, and associated fungal material in the HCs were transferred to a sieve with a 30-μm mesh. The soil was carefully washed through the mesh with filter-sterilized deionized water, leaving the mycelia and river sand on the sieve. To separate the ERH from the river sand and to clean it, the mixture was transferred into a 1-L beaker, and filter-sterilized deionized water was added. The mixture was then stirred gently and poured back into the sieve, leaving the river sand in the beaker. This procedure was repeated five times. The ERH was rinsed with filter-sterilized deionized water before it was collected from the sieve using forceps and placed into 2-mL centrifuge tubes. The control soil samples were also collected in 2-mL centrifuge tubes. The total genomic DNA extraction of ERH and bulk soil was applied for 16S rRNA gene amplification and analysis. Quantification of alkaline phosphatase activity of the bacterial isolates The bacterial isolates were cultured in 1/2 liquid TSB medium at 28°C in the dark. A modified minimal A medium was used to measure AP activity (see Methods in the Additional file ). Quantification of alkaline phosphatase activity of Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of R . irregularis MUCL 43194 Zero-point 5-mL cell cultures ( n = 3) of the two bacteria (for detail, see below — transcriptome landscape of Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of R . irregularis MUCL 43194) were inoculated in the HC in contact with the ERH or in the absence of ERH. After 72 h, the bacteria were collected and resuspended in a Tris–HCl buffer adjusted to pH 9.4 incubated at 30 °C in the dark with 4-mM (final concentration) para -nitrophenyl phosphate ( p NPP). The reaction was stopped using 2-mM (final concentration) NaOH once visible production of the colorimetric product para -nitrophenol ( p NP) was observed. Cell debris and precipitants were removed via centrifugation (3 min, 10,000 g) prior to iMark™ Microplate Reader (optical density 405 nm, Bio-Rad, Hercules, CA, USA). A standard curve for p NP was generated using a range of concentrations (0, 2, 4, 12.5, 25, 37.5, 50 µg mL −1 ). Transcriptome landscape of Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of R . irregularis MUCL 43194 Transformed carrot roots clone DC2 associated with R . irregularis MUCL 43194 were grown in bi-compartmented Petri plates as described above with minor modification in the HC. At week 7, the ERH of the AM fungus developed extensively on the slope in the HC. Ten milliliters of liquid M-MSR, containing 280-μM Na‐phytate, was added in the HC allowing the ERH to grow from the slope into the whole HC. After another 4 weeks, the HC was covered by actively growing hyphae. The HC was inoculated with 500-µL Streptomyces sp. D1 or Pseudomonas sp. H2 at OD 600 = 0.6. A control that consists of a carrot root without AM fungus grown on the MSR medium was included in the design. Before inoculation, each bacterium was pre-cultured for 16 h in liquid M-MSR medium containing 400-µM inorganic P and then washed three times with M-MSR medium to prevent any excess storage of phosphate that could hinder results. At 3 DAI, bacteria cell cultures in the HC were transferred to a 50-mL tube. After vortexing 4 min, 5 mL of bacteria cell cultures was stored at − 4°C for quantification of AP activity (see above). The rest of bacteria cell cultures was centrifuged (3 min, 10,000 g) to remove medium, and the bacteria cells were stored at − 80°C for RNA extraction (see Methods in the Additional file ). The transcriptome profile of glucose, fructose, and trehalose metabolism genes in Streptomyces sp. D1 was established. The glycolysis, pentose phosphate pathway, citrate cycle, fructose and mannose metabolism, starch and sucrose metabolism, fatty acid biosynthesis, two-component system, pyruvate metabolism, phosphotransferase system, purine metabolism, pyrimidine metabolism terpenoid backbone biosynthesis, sesquiterpenoid triterpenoid biosynthesis, and bacteria secretion system pathways were described in Streptomyces sp. D1 or Pseudomonas sp. H2, based on KEGG gene annotations. Transcriptome landscape of R . irregularis MUCL 43194 in the presence of Streptomyces sp. D1 A similar experimental design as above was followed. The ERH of R. irregularis MUCL 43194 was inoculated with Streptomyces sp. D1. A control that consists of an AM fungus-colonized carrot root without bacteria was included in the design. At 3 DAI on the ERH, the hyphae were collected, vortexed for 4 min, and stored at − 80°C for RNA extraction (see Methods in the Additional file ). All the known phosphate-associated genes (including pho84 , pho89 , VTC1/2/4 , pho80 , pho85 , pho81 , pho4 , pho91 , ppn1 ; Table S ), some metal ion transporter genes (including VIT1 , ZRT1 and MatA ; Table S ) and energy production genes (including Idh2 , NDUFA4 , ssuE , and wrbA ; Table S ) that were significantly differentially expressed based on KEGG gene annotations were described (see Table S ). All the genes to Terms in the Gene Ontology database were mapped and the numbers of differentially enriched genes in each Term calculated. TopGO was used to perform GO enrichment analysis on the genes. P -value was calculated by hypergeometric distribution method (the standard of significant enrichment is P -value < 0.05). Finally, the GO term with significantly differentially enriched genes was determined to assess the main biological functions performed by the genes. Phytate consumption of Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of ERH Transformed carrot roots clone DC2 associated with R . irregularis MUCL 43194 were grown in bi-compartmented Petri plates (90 × 15 mm) as described above with minor modifications in the HC. At week 7, the ERH of the AM fungus developed extensively on the slope in the HC. Ten milliliters of liquid M-MSR medium, containing 280-μM Na‐phytate, was added in the HC allowing the ERH to grow from the slope into the whole HC. After another 4 weeks, the HC was covered by actively growing hyphae. The HC was inoculated with 500-µL Streptomyces sp. D1 or Pseudomonas sp. H2 at OD 600 = 0.6. A control that consists of carrot roots in the RC without AM fungus and thus ERH development in the HC was considered in the design. The two bacterial strains were pre-cultured for 16 h on M-MSR medium containing 400-µM inorganic P and then washed three times with M-MSR medium to prevent any excess storage of phosphate that could hinder results. At 3 DAI, the ERH were collected in sterile 1.5-mL centrifuge tubes and stored at − 80°C for RNA extraction (see Additional file ). Relative quantitative RT-PCR was performed to quantify the genes expression in hyphae. The ΔCt was calculated by subtracting the Ct value of a reference gene (5.8S rRNA gene) from the Ct value of each target gene. Relative fold-change of each target gene was normalized by the 2 −ΔΔCt method, with reference to the ΔCt value in the control. The medium in the HC was passed through an Acrodisc® Syringe Filter (0.2-μm Supor® Membrane, Pall Corporation, New York, USA) to remove the bacterial cells and stored at − 20°C for analysis of inorganic and total P. Inorganic P concentration was determined by the molybdate-blue method . Total P concentration was evaluated by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Phytate-P concentration was calculated by subtracting the inorganic P from the total P concentration. Phytate-P consumption was then calculated with the Phytate-P of each control treatment minus each bacterium inoculation treatment. Impact of Streptomyces sp. D1 on organic P utilization by R . irregularis MUCL 43194 and stimulation of gene expression in plants Three-compartmented pots were constructed, consisting of a central root compartment (RC), separated from an external hyphal compartment (HC) by a buffer compartment (BC) (for detail, see Figure S ). The sides of the two tubes were covered with a nylon mesh of 30-μm pore size (Anping Wire Mesh Industrial Ltd., Hebei, China) preventing the outgrowth of roots from the RC to the BC and HC but allowing the spreading of hyphae in both compartments. The soil from Changping, BJ, was used, air-dried, sieved (2 mm), and sterilized at 25-kGy 60 Co gamma irradiation. The substrate in the RC and BC was a mixture of river sand and soil, thoroughly rinsed with deionized water, in the ratio of 4:1 (v/v). The HC was filled with a sterilized mixture of river sand and soil (1:1, v:v). A total of 250, 200, and 900 g of substrate was added into the RC, BC, and HC, respectively. Three surface-sterilized seeds of Medicago truncatula were placed in the RC. The pots were maintained in a growth chamber under 12-h light–dark cycle at 22°C night and 24°C day. Seven weeks after planting, the hyphae developed into the HC. At that time, the Streptomyces sp. D1 or Pseudomonas sp. H2 suspension mixed with Na‐phytate was inoculated in the HC. After 7 days, the M. truncatula plants were harvested, and roots washed tree times with sterile 1 × PBS buffer and one time with sterile deionized water. The roots were then frozen with liquid nitrogen and preserved at − 80°C for evaluation of the relative expression of genes involved in P transport as well as genes involved in fatty acid biosynthesis, transport, and regulation in roots using relative quantitative RT-PCR. Impact of Streptomyces sp. D1 exudates on bacteria isolated from ERH of R . irregularis MUCL 43194 Two-hundred microliters of Streptomyces sp. D1 cells suspension (OD 600 = 0.6) was washed three times in 0.9% NaCl (w:v) solution and incubated on cellophane membranes (35-mm diam., 0.22 µm) in Petri plates (90-mm diam.) containing nutrient broth (0.5% peptone, 0.3% yeast extract, 0.5% NaCl, 0.8% agar). After 5 days, the bacteria covered the cellophane membranes which were gently removed with forceps and placed in the center of larger cellophane membranes (85-mm diam., 0.22 µm) in the middle of Petri plates (90-mm diam.) containing the same nutrient broth medium as above. These membranes were removed after 24 h. The bacterial isolates isolated from the surface of the AM fungal hyphae in contact with the bacterial suspension of soil BJ were incubated at the places where the membranes were removed, using 4 µL of cells suspension (OD 600 = 0.6). A treatment that consists of cellophane membranes covered with 200 µL of 0.9% NaCl (w:v) solution was included as control. Bacteria were similarly incubated on the medium after the membranes were removed. The diameter of bacterial colonies was measured for both treatments and controls to calculate growth inhibition. The medium containing nutrient broth was used in pairwise interactions of strains. Streptomyces sp. D1 or its zymotic was spotted in the center of Petri plates (90-mm diam.). Pseudomonas sp. H2 was inoculated at both sides, 1 cm from the center. Four microliters of cell suspension at an OD 600 = 0.6 of each stain was used. Four microliters of 0.9% NaCl (w:v) solution or 4-µL TSB liquid medium was inoculated in the center of the Petri plates as the control treatments. Identification of antibacterial activity of Streptomyces sp. D1 AntiSMASH v.6.0 was used to analyze the genome of Streptomyces sp. D1. To detect well-defined clusters containing all secondary metabolism genes, the “strict” detection strictness was chosen in antiSMASH. Streptomyces sp. D1 culture broth was extracted with 85% ethanol, evaporated with an N 2 stream, and dissolved in methanol to analyze metabolites. UPLC-MSMS analysis was performed with an ACQUITY UPLC H-Class system (Waters Alliance, Milford, MA, USA) equipped with a mass spectrometer (API 4000, Applied Biosystems). The UPLC conditions were as follows: flow rate, 0.3 mL min −1 ; solvent A, ammonium acetate water; and solvent B, acetonitrile.
Soils were sampled in five long-term field experiments (soil SH: Shihezi 44°19′N, 86°00′E; soil BJ: Beijing 40°08′N, 116°10′E; soil QY: Qiyang 26°45′N, 111°52′E; soil WL: Wulumuqi 43°57′N, 87°46′E; soil TA: Taian 36°10′N, 117°09′E), which are located west (SH and WL), east (BJ and TA), and south (QY) of China. In each location, five subsamples of circa 300 g of soil were taken with an auger at a depth of 0 to 20 cm, mixed together, then sieved to 2 mm, and finally homogenized to constitute the soils SH, WL, BJ, TA, and QY. Soils were transported to the laboratory in sterile plastic bags on ice and temporarily stored at 4°C until extraction of bacterial suspensions. The texture, N, P, K, and organic C contents of the soils are detailed in Table S . Bacterial suspensions were extracted from the soils using Tween 80/tetrasodium yrophosphate (TTSP) and Nycodenz (for details, see Methods in the Additional file ).
Four AM fungal stains, i.e., Rhizophagus irregularis MUCL 43194, Rhizophagus irregularis MUCL 41833, Rhizophagus clarus MUCL 46238, and Rhizophagus intraradices MUCL 49410, were provided by the Glomeromycota in vitro collection (GINCO, Belgium). The fungi were maintained in vitro on Ri T-DNA transformed roots of carrot ( Daucus carota L.) clone DC2 on the modified Strullu-Romand (MSR) medium as described in Cranenbrouck et al. (2005) .
Bi-compartmented Petri plates (90 × 15 mm) were used to grow the carrot roots and AM fungi as detailed in St Arnaud et al. (1996) (Figure S A, B). Briefly, in one compartment (i.e., the root compartment — RC), the root was associated to the AM fungus, while in the other compartment (i.e., the hyphal compartment — HC), only the extraradical hyphae (ERH) of the AM fungus were allowed to growth. Twenty-five milliliters of sterilized (121°C for 15 min) MSR medium solidified with 3 g L −1 Phytagel (Gelzan™ CM, G3251, CP Kelco, USA) was added in the RC. In the HC, 20 mL of the modified MSR medium, termed M-MSR (without sucrose, inorganic P, EDTA, calcium, and vitamin sources), was added. The medium was supplemented with 280-μM organic P in the form of Na-phytate (phytic acid sodium salt hydrate, 68388, Sigma-Aldrich, MO, USA). A control treatment was included that consisted only of carrot roots growing in the RC.
Bi-compartmented Petri plates were prepared as described above with the AM fungus R. irregularis MUCL 43194. Seven weeks after association to the carrot root, the ERH in the RC crossed the partition wall separating the RC from HC and developed profusely in the HC. Two-hundred microliters of bacterial suspension from the five soils described above was inoculated on the surface of the HC. Thus, five treatments and their respective controls (i.e., the HC inoculated with the bacterial suspensions of the five soils without ERH, see Figure S ) were set up with three replicates each. After another 6 days, the M-MSR medium in the HC was dissolved with 35-mL aseptic sodium citrate buffer (10 mM, pH 6) , and the hyphae with their surface-associated bacteria were collected. Briefly, the M-MSR medium of the HC was cut into four pieces and distributed equally in two sterile centrifuge tubes of 50 mL, containing 35 mL of sodium citrate buffer, and vortexed for 4 min for dissolution of the phytagel. The hyphae from the two tubes were assembled, forming a pellet, and rinsed gently twice in a Petri plate containing fresh sodium citrate buffer to eliminate the remaining medium. Sterile blotting papers were used to soak up the excess buffer in the hyphal pellet. The controls that contained only the bacterial suspension in the HC were scraped from the surface of the M-MSR medium. The ERH and control samples were collected in 2-mL centrifuge tubes stored at − 80°C for DNA extraction. The total genomic DNA extraction of ERH and M-MSR medium was done using the FastDNA SPIN Kit for Soil (MP Biochemicals, Solon, OH, USA), following the manufacturer’s instructions. PCR amplification of the bacterial 16S rRNA gene V3–V4 region was performed. Pair-end 2 × 250 bp sequencing was performed using the Illumina NovaSeq platform with NovaSeq 6000 SP Reagent Kit (500 cycles) at Shanghai Personal Biotechnology Co., Ltd (see Methods in the Additional file ). The same approach as above was conducted with the four AM fungal strains (i.e., R. irregularis MUCL 43194, R . irregularis MUCL 41833, R . clarus MUCL 46238, and R . intraradices MUCL 49410) plus one control that consist of carrot roots not associated with an AM fungus, inoculated in the HC with 200 µL bacterial suspension from soil BJ. The BJ soil was selected because it had been collected as part of a long-term experiment with appropriate P application for over 20 years, representing the typical cultivation system of the North China Plain. Three replicates were considered per treatment. After 3 and 6 days, the M-MSR medium in the HC was dissolved with 35-mL sterilized sodium citrate buffer and then processed for DNA extraction as above.
R . irregularis MUCL 43194 Bi-compartmented Petri plates were prepared with carrot roots clone DC2 associated with R. irregularis MUCL 43194 as described above. Bacterial suspension of BJ was inoculated in the HC for 6 days. The tiny layers of bacteria that developed around the hyphae were detached carefully with pipette tips under a stereomicroscope at 56 × magnification (Olympus SZX7, Japan). The bacteria were then streaked with the pipette tips on 10-mL solid tryptone soy broth (TSB, CM0129, Thermo Scientific, USA) in 60 × 15 mm plates (for details, see Methods in the Additional file ). To identify the bacteria, the V3–V4 regions of the 16S rRNA gene were applied on the DNAs extracted using degenerate primers (338F and 806R, Table S ). The PCR products obtained were subsequently sequenced by Sanger sequencing. To recover cultivated bacteria for further studies, the Sanger sequencing results were blasted (BLAST + 2.10.1) to the 16S rRNA gene amplicon sequence variants (ASVs) in the high-throughput sequencing data, allowing to relate the isolated bacteria to relevant ASVs.
Bi-compartmented Petri plates were prepared with carrot roots clone DC2 associated with R. irregularis MUCL 43194 as described above. Two SynComs were constructed which consist of 20 bacterial isolates (including Streptomyces sp. D1) or 19 bacterial isolates (without Streptomyces sp. D1). The bacterial isolates were from the ERH of R. irregularis MUCL 43194 after inoculation with BJ bacteria suspension (Table S ). Each bacterium was cultured separately in 50-mL tubes, washed, and OD 600 adjusted to 0.6. The cultures were then mixed in 1:1 ratio and spread onto the HC containing the ERH. Non-mycorrhizal carrot roots plated in the RC and each of the two SynComs inoculated in the HC were considered as controls. After 3 days, the bacterial community on the ERH and on the medium (i.e., the controls) were collected in 2-mL centrifuge tubes stored at − 80°C for DNA extraction. The total genomic DNA was extracted and 16S rRNA gene amplified and analyzed. In parallel, a similar experiment was conducted in pots under 12-h light–dark cycle at 22°C night and 24°C day (for detailed description of the pot system, see Figure S ). The treatment consisted of the 20 bacterial isolates (thus with Streptomyces sp. D1) inoculated in the HC containing the ERH or devoid of ERH (SynCom + AM fungi and SynCom–AM fungi treatments, respectively). Maize ( Zea mays L., cv. Zhengdan 958) was used as the host plant and R. irregularis MUCL 43194 as the AM fungal strain. The soil from Changping, BJ was air-dried, sieved (2 mm), and sterilized by 25 kGy 60 Co gamma irradiation. The plant compartment was filled with a sterilized mixture of soil and river sand (4:1 v/v). The HC was filled with a sterilized mixture of soil and river sand (1:1, v:v). Ten weeks after planting, the ERH developed into the HC. At that time, the SynCom mixed with Na‐phytate was injected though the tube protruding outside the HC. The solution arrived at the top nylon mesh and then permeated to the soil homogeneously. After another 2 weeks, the soil, river sand, and associated fungal material in the HCs were transferred to a sieve with a 30-μm mesh. The soil was carefully washed through the mesh with filter-sterilized deionized water, leaving the mycelia and river sand on the sieve. To separate the ERH from the river sand and to clean it, the mixture was transferred into a 1-L beaker, and filter-sterilized deionized water was added. The mixture was then stirred gently and poured back into the sieve, leaving the river sand in the beaker. This procedure was repeated five times. The ERH was rinsed with filter-sterilized deionized water before it was collected from the sieve using forceps and placed into 2-mL centrifuge tubes. The control soil samples were also collected in 2-mL centrifuge tubes. The total genomic DNA extraction of ERH and bulk soil was applied for 16S rRNA gene amplification and analysis.
The bacterial isolates were cultured in 1/2 liquid TSB medium at 28°C in the dark. A modified minimal A medium was used to measure AP activity (see Methods in the Additional file ).
Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of R . irregularis MUCL 43194 Zero-point 5-mL cell cultures ( n = 3) of the two bacteria (for detail, see below — transcriptome landscape of Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of R . irregularis MUCL 43194) were inoculated in the HC in contact with the ERH or in the absence of ERH. After 72 h, the bacteria were collected and resuspended in a Tris–HCl buffer adjusted to pH 9.4 incubated at 30 °C in the dark with 4-mM (final concentration) para -nitrophenyl phosphate ( p NPP). The reaction was stopped using 2-mM (final concentration) NaOH once visible production of the colorimetric product para -nitrophenol ( p NP) was observed. Cell debris and precipitants were removed via centrifugation (3 min, 10,000 g) prior to iMark™ Microplate Reader (optical density 405 nm, Bio-Rad, Hercules, CA, USA). A standard curve for p NP was generated using a range of concentrations (0, 2, 4, 12.5, 25, 37.5, 50 µg mL −1 ).
Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of R . irregularis MUCL 43194 Transformed carrot roots clone DC2 associated with R . irregularis MUCL 43194 were grown in bi-compartmented Petri plates as described above with minor modification in the HC. At week 7, the ERH of the AM fungus developed extensively on the slope in the HC. Ten milliliters of liquid M-MSR, containing 280-μM Na‐phytate, was added in the HC allowing the ERH to grow from the slope into the whole HC. After another 4 weeks, the HC was covered by actively growing hyphae. The HC was inoculated with 500-µL Streptomyces sp. D1 or Pseudomonas sp. H2 at OD 600 = 0.6. A control that consists of a carrot root without AM fungus grown on the MSR medium was included in the design. Before inoculation, each bacterium was pre-cultured for 16 h in liquid M-MSR medium containing 400-µM inorganic P and then washed three times with M-MSR medium to prevent any excess storage of phosphate that could hinder results. At 3 DAI, bacteria cell cultures in the HC were transferred to a 50-mL tube. After vortexing 4 min, 5 mL of bacteria cell cultures was stored at − 4°C for quantification of AP activity (see above). The rest of bacteria cell cultures was centrifuged (3 min, 10,000 g) to remove medium, and the bacteria cells were stored at − 80°C for RNA extraction (see Methods in the Additional file ). The transcriptome profile of glucose, fructose, and trehalose metabolism genes in Streptomyces sp. D1 was established. The glycolysis, pentose phosphate pathway, citrate cycle, fructose and mannose metabolism, starch and sucrose metabolism, fatty acid biosynthesis, two-component system, pyruvate metabolism, phosphotransferase system, purine metabolism, pyrimidine metabolism terpenoid backbone biosynthesis, sesquiterpenoid triterpenoid biosynthesis, and bacteria secretion system pathways were described in Streptomyces sp. D1 or Pseudomonas sp. H2, based on KEGG gene annotations.
R . irregularis MUCL 43194 in the presence of Streptomyces sp. D1 A similar experimental design as above was followed. The ERH of R. irregularis MUCL 43194 was inoculated with Streptomyces sp. D1. A control that consists of an AM fungus-colonized carrot root without bacteria was included in the design. At 3 DAI on the ERH, the hyphae were collected, vortexed for 4 min, and stored at − 80°C for RNA extraction (see Methods in the Additional file ). All the known phosphate-associated genes (including pho84 , pho89 , VTC1/2/4 , pho80 , pho85 , pho81 , pho4 , pho91 , ppn1 ; Table S ), some metal ion transporter genes (including VIT1 , ZRT1 and MatA ; Table S ) and energy production genes (including Idh2 , NDUFA4 , ssuE , and wrbA ; Table S ) that were significantly differentially expressed based on KEGG gene annotations were described (see Table S ). All the genes to Terms in the Gene Ontology database were mapped and the numbers of differentially enriched genes in each Term calculated. TopGO was used to perform GO enrichment analysis on the genes. P -value was calculated by hypergeometric distribution method (the standard of significant enrichment is P -value < 0.05). Finally, the GO term with significantly differentially enriched genes was determined to assess the main biological functions performed by the genes.
Streptomyces sp. D1 and Pseudomonas sp. H2 in the presence of ERH Transformed carrot roots clone DC2 associated with R . irregularis MUCL 43194 were grown in bi-compartmented Petri plates (90 × 15 mm) as described above with minor modifications in the HC. At week 7, the ERH of the AM fungus developed extensively on the slope in the HC. Ten milliliters of liquid M-MSR medium, containing 280-μM Na‐phytate, was added in the HC allowing the ERH to grow from the slope into the whole HC. After another 4 weeks, the HC was covered by actively growing hyphae. The HC was inoculated with 500-µL Streptomyces sp. D1 or Pseudomonas sp. H2 at OD 600 = 0.6. A control that consists of carrot roots in the RC without AM fungus and thus ERH development in the HC was considered in the design. The two bacterial strains were pre-cultured for 16 h on M-MSR medium containing 400-µM inorganic P and then washed three times with M-MSR medium to prevent any excess storage of phosphate that could hinder results. At 3 DAI, the ERH were collected in sterile 1.5-mL centrifuge tubes and stored at − 80°C for RNA extraction (see Additional file ). Relative quantitative RT-PCR was performed to quantify the genes expression in hyphae. The ΔCt was calculated by subtracting the Ct value of a reference gene (5.8S rRNA gene) from the Ct value of each target gene. Relative fold-change of each target gene was normalized by the 2 −ΔΔCt method, with reference to the ΔCt value in the control. The medium in the HC was passed through an Acrodisc® Syringe Filter (0.2-μm Supor® Membrane, Pall Corporation, New York, USA) to remove the bacterial cells and stored at − 20°C for analysis of inorganic and total P. Inorganic P concentration was determined by the molybdate-blue method . Total P concentration was evaluated by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Phytate-P concentration was calculated by subtracting the inorganic P from the total P concentration. Phytate-P consumption was then calculated with the Phytate-P of each control treatment minus each bacterium inoculation treatment.
Streptomyces sp. D1 on organic P utilization by R . irregularis MUCL 43194 and stimulation of gene expression in plants Three-compartmented pots were constructed, consisting of a central root compartment (RC), separated from an external hyphal compartment (HC) by a buffer compartment (BC) (for detail, see Figure S ). The sides of the two tubes were covered with a nylon mesh of 30-μm pore size (Anping Wire Mesh Industrial Ltd., Hebei, China) preventing the outgrowth of roots from the RC to the BC and HC but allowing the spreading of hyphae in both compartments. The soil from Changping, BJ, was used, air-dried, sieved (2 mm), and sterilized at 25-kGy 60 Co gamma irradiation. The substrate in the RC and BC was a mixture of river sand and soil, thoroughly rinsed with deionized water, in the ratio of 4:1 (v/v). The HC was filled with a sterilized mixture of river sand and soil (1:1, v:v). A total of 250, 200, and 900 g of substrate was added into the RC, BC, and HC, respectively. Three surface-sterilized seeds of Medicago truncatula were placed in the RC. The pots were maintained in a growth chamber under 12-h light–dark cycle at 22°C night and 24°C day. Seven weeks after planting, the hyphae developed into the HC. At that time, the Streptomyces sp. D1 or Pseudomonas sp. H2 suspension mixed with Na‐phytate was inoculated in the HC. After 7 days, the M. truncatula plants were harvested, and roots washed tree times with sterile 1 × PBS buffer and one time with sterile deionized water. The roots were then frozen with liquid nitrogen and preserved at − 80°C for evaluation of the relative expression of genes involved in P transport as well as genes involved in fatty acid biosynthesis, transport, and regulation in roots using relative quantitative RT-PCR.
Streptomyces sp. D1 exudates on bacteria isolated from ERH of R . irregularis MUCL 43194 Two-hundred microliters of Streptomyces sp. D1 cells suspension (OD 600 = 0.6) was washed three times in 0.9% NaCl (w:v) solution and incubated on cellophane membranes (35-mm diam., 0.22 µm) in Petri plates (90-mm diam.) containing nutrient broth (0.5% peptone, 0.3% yeast extract, 0.5% NaCl, 0.8% agar). After 5 days, the bacteria covered the cellophane membranes which were gently removed with forceps and placed in the center of larger cellophane membranes (85-mm diam., 0.22 µm) in the middle of Petri plates (90-mm diam.) containing the same nutrient broth medium as above. These membranes were removed after 24 h. The bacterial isolates isolated from the surface of the AM fungal hyphae in contact with the bacterial suspension of soil BJ were incubated at the places where the membranes were removed, using 4 µL of cells suspension (OD 600 = 0.6). A treatment that consists of cellophane membranes covered with 200 µL of 0.9% NaCl (w:v) solution was included as control. Bacteria were similarly incubated on the medium after the membranes were removed. The diameter of bacterial colonies was measured for both treatments and controls to calculate growth inhibition. The medium containing nutrient broth was used in pairwise interactions of strains. Streptomyces sp. D1 or its zymotic was spotted in the center of Petri plates (90-mm diam.). Pseudomonas sp. H2 was inoculated at both sides, 1 cm from the center. Four microliters of cell suspension at an OD 600 = 0.6 of each stain was used. Four microliters of 0.9% NaCl (w:v) solution or 4-µL TSB liquid medium was inoculated in the center of the Petri plates as the control treatments.
Streptomyces sp. D1 AntiSMASH v.6.0 was used to analyze the genome of Streptomyces sp. D1. To detect well-defined clusters containing all secondary metabolism genes, the “strict” detection strictness was chosen in antiSMASH. Streptomyces sp. D1 culture broth was extracted with 85% ethanol, evaporated with an N 2 stream, and dissolved in methanol to analyze metabolites. UPLC-MSMS analysis was performed with an ACQUITY UPLC H-Class system (Waters Alliance, Milford, MA, USA) equipped with a mass spectrometer (API 4000, Applied Biosystems). The UPLC conditions were as follows: flow rate, 0.3 mL min −1 ; solvent A, ammonium acetate water; and solvent B, acetonitrile.
All statistical analyses were conducted in R v.4.0.3 and IBM SPSS Statistics v.21. Co-occurrence networks were constructed based on the relative abundance of ASVs > 0.01%. Robust correlations with Spearman’s correlation coefficients ( ρ ) > 0.65 and false discovery rate-corrected P -values < 0.01 were used to construct networks. The networks were visualized with the Fruchterman-Reingold layout with 10 4 permutations in igraph. Keystone ASVs were identified separately for the − AM fungi and + AM fungi treatments and defined as those nodes within the top 5% of node degree values of each network. The growth curves were fitted by the ggplot2 R package, method = “gam”. For the transcriptome, the filtered reads were mapped to the reference genome using HISAT2 v2.0.5 (Fungi) or Bowtie2 v2.2.6 (bacteria). The HTSeq v0.9.1 statistics were used to compare the read count values on each gene as the original expression of the gene, and FPKM was used to standardize the expression. Then, difference expression of genes was analyzed by DESeq v1.30.0 with screened conditions as follows: expression difference multiple |log2FoldChange|> 1 and significant P -value < 0.05. TopGO was used to conduct an analysis of differential genes. The P -value was calculated using the hypergeometric distribution method, with a significance threshold set at P < 0.05 to determine significant enrichment.
Additional file 1. Methods: Biological material - Extraction of bacterial suspensions from soil. Isolation and identification of bacteria from the extraradical hyphae network of R. irregularis MUCL 43194. Whole genome sequencing of Streptomyces sp. D1 and Pseudomonas sp. H2. Competition of Streptomyces sp. D1 and Pseudomonas sp. H2 in the hyphosphere. Carbon assimilation profiles of bacterial isolates. Pot system set up to study the impact of Streptomyces sp. D1 on organic P utilization by R. irregularis MUCL 43194 and stimulation of gene expression in plants. Liquid M-MSR medium in the hyphal compartment of the in vitro culture system. Quantification of alkaline phosphatase activity of the bacterial isolates. Competition of Streptomyces sp. and Pseudomonas sp. on hyphae. Identification of antibacterial activity of Streptomyces sp. D1. RNA extraction for RT-PCR and RT-PCR protocol. RNA extraction for transcriptome and transcriptome protocol. DNA extraction. 16S rRNA gene sequence analysis. Figure S1. Growth curves of 20 bacterial isolates grown in M9 minimal salts medium with 6 carbon sources (i.e., fructose, glucose, inositol, citric acid, trehalose, or succinic acid) as sole carbohydrate source. Error bars represent the standard deviation of four independent replicates. Figure S2. Reconstruction of (A) the Streptomyces sp. D1 and (B) Pseudomonas sp. H2 major carbohydrate metabolic pathway and phosphate metabolic pathway mapped with transcriptomic data. The reconstructed metabolic pathways of the two bacteria were based on KEGG genes annotations and their relative differential expression profiles of triplicates (each replicate was a mix of 5 culture plates). The genes with a significant ( P < 0.05) differential expression of >1 |log2FC| were indicated with an arrow (pathway) and a star (heatmap) in green (up-regulated in absence of the extraradical hyphae), red (up-regulated in the presence of the extraradical hyphae). The number in the pathways correspond to the number in the heatmaps. Gray arrow represents the absence of genes identified in the pathway. Figure S3. Reconstruction of the Streptomyces sp. D1 albaflavenone synthesis pathway mapped with transcriptomic data. The reconstructed metabolic pathway was based on KEGG genes annotations and their relative differential expression profiles of triplicates (each replicate was a mix of 5 culture plates, 15 plates for every treatment). The pathways with genes significant ( P < 0.05) differential expression of |log2FC| > 1 are indicated with pathway in green (down-regulated in the presence of the extraradical hyphae), red (up-regulated in absence of the extraradical hyphae). Figure S4. Bi-compartmented in vitro cultivation system. (A) Ri T-DNA transformed root of carrot developing single in the root compartment (RC) and bacterial community plated in the hyphal compartment (HC), representing the control treatment (RC/HC –AMF ). (B) Ri T-DNA transformed root of carrot associated with an AM fungus in the RC, with extraradical hyphae (ERH) and spores (in blue) extending in the HC in contact with the bacterial community, representing the ERH treatment (RC/HC +AMF ). (C) Detailed picture of the bi-compartmented in vitro cultivation system with a carrot root and AM fungus in the RC and ERH crossing the plastic barrier separating the RC from the HC and developing profusely in the HC in contact with a suspension of bacteria. Figure S5. Schematic representation of the hyphal compartment (HC). The HC was cuboid (6.8 cm in length, 6.0 cm in width, 5.8 cm in height) and made of PVC plates. In the plate on the top of the HC, a hole (6 mm in diameter) was made and a plastic tube (5 mm in diameter, 50 cm in length) was inserted and fixed with PVC glue. One centimeter below the top plate, another PVC plate with holes (1 mm in diameter) was fixed and covered with a 30 μm nylon mesh. On one side of the HC, a PVC plate with holes (4 mm in diameter) and covered with 30 μm or 0.45 μm nylon mesh was fixed to allow or not the AM fungal hyphae to grow into the HC. Two HCs were buried in the soil at both sides of the pots containing three maize plants associated to an AM fungus. The soil composition in the pot and HCs is detailed (see Methods in Additional file 1). The bacterial suspension was then injected with a syringe through the plastic tube, allowing the bacteria to diffuse homogeneously in the soil of the HC in contact or not with the ERH. Figure S6. (A) Schematic representation of the three-compartmented pot experimental system. The inner compartment contains the roots and fungal hyphae (RC), the middle compartment is a buffer compartment (BC) and the outer compartment is the hyphal compartment (HC) allowing only the extraradical hyphae (ERH) to extend. The BC, with a width of 1 cm, was placed between the two compartments to prevent the movement of soluble organic P from HC to RC. A nylon mesh of 30 μm separating the RC from the BC and the BC from the HC was used to avoid roots to extend from RC to HC, while allowing the hyphae to extend from RC to HC. The bacterium was inoculated in the HC in contact with the ERH of Rhizophagus irregularis MUCL 43194. Each treatment consisted of four replicates with three Medicago truncatula plants per RC. Red lines, extraradical hyphae of R. irregularis MUCL 43194; red dots, spores of R. irregularis MUCL 43194. (B) Detailed picture of the three-compartmented pot system. Table S1. Mean relative abundance of 20 bacterial isolates of all hyphal samples. Table S2. Trehalose metabolism and transporter genes in the genome of Streptomyces sp. D1 identified with KEGG. Table S3. Phosphate metabolism genes in the genome of Streptomyces sp. D1 based on KEGG gene annotations. Table S4. Phosphate metabolism genes in the genome of Pseudomonas sp. H2 based on KEGG gene annotations. Table S5. Number of genes significantly differentially expressed for each KEGG metabolic pathways in Pseudomonas sp. H2. Table S6. Secondary metabolites gene clusters in the genome of Streptomyces sp. D1 detected by antiSMASH6 pipeline. Table S7. Physico-chemical properties of the soils used to extract bacterial suspensions. Table S8. Summary of primers used in the study. Table S9. Phosphate, polyphosphate and associated genes expression significantly increased in Rhizophagus irregularis MUCL 43194 inoculated with Streptomyces sp. D1 as compared to the fungus grown in absence of the bacteria. Additional file 2. CAZy genes in D1 and CAZy genes in H2. Additional file 3. Flagellar assembly genes in D1 and Flagellar assembly genes in H2.
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How to follow the guidelines, when the appropriate fluid is missing? | 8a82d4eb-fb4d-4757-bf21-e4b004adc3b9 | 11098858 | Pediatrics[mh] | Intravenous maintenance fluid therapy (IV-MFT) is probably the most prescribed drug in paediatric hospital care . It has been used for more than a century and yet, it is only recently that paediatric societies have produced evidence-based practice guidelines to guide the use of IV fluids in clinical practice . These guidelines recommend the use of balanced isotonic fluid when prescribing IV-MFT in both acute and critical paediatric care. Those recommendations were based on the fact that balanced isotonic fluids were less likely to cause hyponatremia and metabolic hyperchloremic acidosis, which have been associated with several severe, potentially deadly, complications in the pediatric intensive care unit (PICU), such as neurological impairment, kidney injury or organ dysfunction . Besides, balanced solutions have also been shown to reduce the length of both PICU and hospital stay . It is also recommended to provide the appropriate amounts of potassium and glucose to prevent children from presenting hypokalaemia and hypoglycaemia . However, even though two international paediatric societies (the American Academic of Pediatrics (AAP) and the European Society of Paediatric and Neonatal Intensive Care (ESPNIC) ) are to be commended for these long-awaited guidelines, the applicability and implementation of these guidelines are threatened by a lack of these fluids available. During the last decade, the growing interest in the use of balanced crystalloids to prevent patients from developing clinical complications and mortality related to hyperchloremia and metabolic acidosis has been associated with a growing availability of balanced IV fluids . Unfortunately, amongst this variety of available balanced IV fluids, very few, or even none in certain countries, contain glucose. However, glucose content is fundamental for paediatric IV-MFT. In 2022, Morice et al. showed that the absence of glucose in the solution was the main reason for not prescribing a balanced fluid by 29.4% of the respondents . The main objective of this study was to describe the availability of glucose-containing balanced isotonic fluids in European and Middle Eastern paediatric acute and critical care settings, performing a complementary analysis of the Morice et al. survey. The secondary objective was to evaluate the impact of the absence of paediatric appropriate ready-to-use fluids on IV-MFT declarative practice. This work was an ancillary study of the survey dedicated to IV-MFT practice in the paediatric acute and critical care settings in 35 countries in Europe and Middle East . The study design, the included population and the survey instrument development, content and data collection have previously been published . This survey was designed to collect a single response per centre. Data analysis Data were analysed according to the country of the responders and according to the availability of balanced isotonic fluids, with or without glucose 5%. The data analysis was focused on the questions related to the use of balanced fluids (Q13, 14, 15, 16) and fluid choices (Q17, 18, 19, 20). We used a summative score to summarize the results from Likert scale questions for each participant. Variables distributions were assessed by the Shapiro–Wilk comparison test and continuous variables were presented as median (min-max). Categorical variables were presented as number (percentage). Comparisons between both groups were made by a Mann–Whitney U test or a Kruskal-Wallis’s test for continuous variables as appropriate and by a chi-square test with Monte Carlo simulation with 2000 replicates for categorical variables. The level of statistical significance was set at p < 0.05. Statistical analyses were performed using open-access R software (Version 4.2.1; R Foundation for Statistical Computing, Vienna, Austria). Ethical approval was obtained from the Caen-France institutional review board (reference number 2474). Data were analysed according to the country of the responders and according to the availability of balanced isotonic fluids, with or without glucose 5%. The data analysis was focused on the questions related to the use of balanced fluids (Q13, 14, 15, 16) and fluid choices (Q17, 18, 19, 20). We used a summative score to summarize the results from Likert scale questions for each participant. Variables distributions were assessed by the Shapiro–Wilk comparison test and continuous variables were presented as median (min-max). Categorical variables were presented as number (percentage). Comparisons between both groups were made by a Mann–Whitney U test or a Kruskal-Wallis’s test for continuous variables as appropriate and by a chi-square test with Monte Carlo simulation with 2000 replicates for categorical variables. The level of statistical significance was set at p < 0.05. Statistical analyses were performed using open-access R software (Version 4.2.1; R Foundation for Statistical Computing, Vienna, Austria). Ethical approval was obtained from the Caen-France institutional review board (reference number 2474). Participants’ characteristics Participants’ characteristics were presented in . The response rate related to contacted centres was 63%, with 153 centres represented, over 240 contacted. The responses represented 35 (82%) of the 43 countries surveyed. One participant was excluded for practicing in Australia. Fluid availability according to country Fluid availability according to the country of the responders is presented in Table . Balanced isotonic fluid with glucose 5% was declared available for only 32 (21%) responders. Balanced isotonic fluid with glucose 5% was consistently available only in the UK (90%) and totally absent from France, Greece, The Netherlands and Turkey. The most widely available fluids were balanced (93.5%) and unbalanced (87.6%) isotonic fluids without glucose. Impact of country on prescriptions' practices Responders’ consideration for the importance of balanced fluids varies considerably between countries in both conventional and critical care unit (SDC ). Prescription practices varied considerably between countries (SDC ). Balanced isotonic fluid was considered in 45.0% of the clinical situations (from 6.5% in Greece to 83.3% in Poland) and unbalanced isotonic fluid in 42.8% (from 11.1% in Poland to 78.5% in Turkey). Hypotonic unbalanced fluid was considered in 10.5% of the clinical situations (from 0% in the UK to 30% in Greece). It was consistently the less prescribed fluid, except in France and in Greece, where it was prescribed more than balanced isotonic fluid. Impact of fluid availability on prescriptions’ practices Among the 32 responders who declared having access to a balanced isotonic fluid with glucose 5%, 23 (71.9%) reported that balanced isotonic fluid should be always considered vs 42/121 (34.7%) ( p < 0.001) in the case of unavailability of a balanced isotonic fluid with glucose 5% (SDC ). The availability of a balanced isotonic fluid with glucose 5% was systematically and significantly associated with a preference for prescribing this fluid over unbalanced isotonic or hypotonic crystalloids, notwithstanding the clinical situation studied (SDC ). Participants’ characteristics were presented in . The response rate related to contacted centres was 63%, with 153 centres represented, over 240 contacted. The responses represented 35 (82%) of the 43 countries surveyed. One participant was excluded for practicing in Australia. Fluid availability according to the country of the responders is presented in Table . Balanced isotonic fluid with glucose 5% was declared available for only 32 (21%) responders. Balanced isotonic fluid with glucose 5% was consistently available only in the UK (90%) and totally absent from France, Greece, The Netherlands and Turkey. The most widely available fluids were balanced (93.5%) and unbalanced (87.6%) isotonic fluids without glucose. Responders’ consideration for the importance of balanced fluids varies considerably between countries in both conventional and critical care unit (SDC ). Prescription practices varied considerably between countries (SDC ). Balanced isotonic fluid was considered in 45.0% of the clinical situations (from 6.5% in Greece to 83.3% in Poland) and unbalanced isotonic fluid in 42.8% (from 11.1% in Poland to 78.5% in Turkey). Hypotonic unbalanced fluid was considered in 10.5% of the clinical situations (from 0% in the UK to 30% in Greece). It was consistently the less prescribed fluid, except in France and in Greece, where it was prescribed more than balanced isotonic fluid. Among the 32 responders who declared having access to a balanced isotonic fluid with glucose 5%, 23 (71.9%) reported that balanced isotonic fluid should be always considered vs 42/121 (34.7%) ( p < 0.001) in the case of unavailability of a balanced isotonic fluid with glucose 5% (SDC ). The availability of a balanced isotonic fluid with glucose 5% was systematically and significantly associated with a preference for prescribing this fluid over unbalanced isotonic or hypotonic crystalloids, notwithstanding the clinical situation studied (SDC ). Performing a complementary analysis on the declarative data of Morice et al. survey , focusing on the declared type of available IV fluids, we have realised that only 21% of responders have access to a commercialized balanced isotonic fluid containing 5% glucose, which is considered as the current recommended IV fluid for paediatric IV-MFT. We have shown that the availability of such a solution varies from one country to another but can also be inconsistent within the same country. In addition, we have observed that the availability of a balanced isotonic fluid with 5% glucose was associated with a higher declarative use of balanced isotonic fluid in almost all the assessed clinical situations. This inconsistency regarding the availability of these ready-made balanced solutions is a significant barrier to the implementation of the recent ESPNIC IV-MFT guidelines into clinical practices and could explain the obsolete but still current use of hypotonic IV fluids . This should be reassessed once a specific model for disseminating these guidelines in clinical practice has been implemented. In the absence of a ready-to-use appropriate IV fluid for children, local compounding to make solutions that comply with the recommendations is often required (Fig. ). Such manipulations give rise to significant patient risks regarding the uncertainty in physico-chemical stability, microbial contamination, prescription and preparation errors while manipulating electrolytes, as well as alterations of the tonicity and/or the balanced nature of the original fluid . Some clinicians have considered using paediatric IV fluids marketed for the peri-operative period as alternatives that are balanced isotonic fluids with 1% glucose. ISOPEDIA © (FRESENIUS KABI FRANCE) and BENELYTE © (FRESENIUS KABI POLSKA) are the only balanced isotonic glucose-containing crystalloids available in many European countries. Their marketing authorisation was obtained in 2017, based on perioperative IV-MFT guidelines in children, which recommended a 1 to 2.5% glucose concentration . However, this glucose-containing fluid is probably not appropriate for use outside of the perioperative setting, as they provide insufficient amount of glucose. No clear consensus exists on the optimal glucose concentration for paediatric IV-MFT. In the general paediatric setting, 5% glucose concentration solutions are common and recommended by some medical societies probably based on Holliday and Segar guidance . Likewise, adult guidelines suggest considering a daily glucose intake of 1 to 1.5 g/kg/day to prevent fasting ketonemia . We consider that isotonic balanced solutions which would provide different ranges of glucose (from 1 to 10%) should be favoured and made readily available on the market to ensure safe IV fluid therapy for children. In addition, as the insufficient amount of potassium in some balanced fluids has been called into question and may contribute to impairing the applicability of the guidelines, those fluids should be available with a sufficient amount of potassium for use in standard paediatric IV maintenance therapy . Specific considerations should be made regarding potassium content when bolus fluids are used or in case of renal failure. Finally, consideration should be given to cost and packaging. If these recommendations are to be applicable worldwide, including in low- and middle-income countries, the recommended fluids must be available at a reasonable price . In addition, to overcoming the wide variability in patient characteristics encountered in paediatric practice, the recommended fluids should be available in a range of packaging formats, in order to reduce waste as well as the environmental footprint of plastic packaging . The limitations inherent to the original survey were presented in . This study’s specific limitations mainly lie within the fact that the survey was not originally dedicated to determining the different fluid availability. It is therefore difficult to confirm that unavailability of the appropriate fluid in responding centres of one country reflects the absence of marketing of the fluid within the country or the simple lack of product referencing in the responding centre (due to cost issues or poor regard to the necessity of the product). This study was not designed to identify potential stakeholders in the availability of balanced fluids. Ready-to-use isotonic balanced IV solutions containing glucose in sufficient amounts exist but are inconsistently available throughout Europe. National and European Medication Safety Incentives should guarantee the availability of the most appropriate and safest IV-MFT solution for all children. Our expert group is calling for the rapid commercialization of appropriate solutions worldwide. Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 46 KB) Supplementary file2 (DOCX 43 KB) |
Soil Microbial Recovery to the Rubber Tree Replanting Process in Ivory Coast | b5a85559-725d-4bf1-8b89-54f146209ef9 | 11906521 | Microbiology[mh] | The soil microbiome represents a key component of belowground functioning and plant productivity . Soil microbial communities play major roles, for instance, in organic matter decomposition , soil structure formation and carbon and nitrogen cycles . Soil community assembly and microbial interactions are crucial for understanding the mechanisms that regulate ecosystem services . Microbial communities respond quickly to environmental modification (i.e. changes in land use, temperature or water stress, [ – ], and the extent and rate of microbiome responses to perturbations provide important information about soil functional stability and sustainability) . The consequences of shifts in community structures following soil perturbations (i.e. tillage , tree logging , compaction ) on ecosystem functioning are well documented in agroecosystems, but less is known about the drivers of resistance and resilience (i.e. ability to tolerate and recover from disturbances, respectively ) of microbial communities . Soil microbial functioning is often considered as the sum of individual functions, while soil microorganisms form complex networks and act in concert . Thus, insights into community reassembly after soil perturbation over time are becoming essential for understand agroecosystem sustainability and designing relevant agroecological practices . The respective roles and interdependencies of soil biotic and abiotic components in maintaining soil biological activities are increasingly studied . Soil functioning in agroecosystems depends on microbial processes such as the mineralization of organic nitrogen (N), carbon (C) and phosphorus (P), the transformation of organic matter (OM) in the soil and N 2 fixation by soil microbes . Previous studies have shown significant dependencies of the biogeographical distribution of soil microorganisms and their associated functions on major environmental filters, such as soil pH and redox status, OM quality and quantity, temperature and moisture , as well as soil texture and structure. In equivalent agricultural systems subjected to similar climates, soil texture is thus likely to impact soil functioning balance depending on the applied agricultural practices . Soil texture characterizes the distribution mineral particles, which is a key driver for soil OM protection, spatial distribution and associated biological activities . Bacteria and fungi act in concert for soil functioning, and their respective responses (resistance and resilience patterns) to soil perturbation may thus be either exacerbated or alleviated with respect depending on edaphic properties. Tropical tree plantations are a relevant model for studying the impact of a given perturbation on soil communities because of the disturbances occurring during the replanting process, such as tree clear-cutting, uprooting and soil compaction. In our study, we focus on rubber plantations ( Hevea brasiliensis ). The current and rapid expansion of rubber plantations to meet existing global demand for rubber puts soil biodiversity at risk . Studies have shown that forest replacement by rubber plantation results in an overall loss and extensive replacement of both aboveground (i.e. birds and bats ) and belowground (i.e. spiders, arthropods or nematodes [ – ]) biodiversity. However, rubber plantations are no longer a main cause of deforestation. The current trend is to replace annual crops (such as cassava and sugarcane) with rubber plantations and to repeat rubber plantations on the same soils after clear-cutting . After a rotation period ranging from 25 to 40 years , rubber plantations require tree replacement, and the replanting process constitutes a significant soil disturbance. This period is characterised by (1) the opening of the canopy following tree clear-cutting, (2) the removal of logging residues with machines and (3) subsoiling (i.e. breaking up of compacted layers) by heavy machinery. Thus, successive rubber plantation cycles impact soil diversity due to long-term modifications of soil physico-chemical properties. For example, Panklang et al. evidenced strong losses of N, K, Ca and Mg in the soil, and a 50% decrease in SOC content between forest and third rotation of rubber trees. Clear-cutting and soil compaction also severely impact nutrient content , soil functions (such as structure maintenance, carbon transformation and nutrient cycling ) and significantly reduce the biomass while altering the composition of soil microbial communities . Cultural practices such as organic matter inputs and cover crops play a fundamental role in soil functioning. Legumes are commonly grown in industrial rubber plantations after tree replacement to fix nitrogen in the soil, control weeds and limit soil erosion, particularly during the immature phase (0–5 years before latex extraction) [ – ]. The restitution of tree-logging residues (i.e. trunk, branches, leaves and roots of the logged plantation) also represents a relevant and responsible method for releasing organic matter and potentially improving soil biological quality and associated functioning . The restitution of logging residues has multiple benefits : it mitigates long-term changes in soil communities in forest soil , significantly increases both soil carbon stock and nutrient availability and stabilizes soil pH, moisture and temperature . The positive impact of retaining logging residues has also been demonstrated on soil fauna resilience , soil microbial community abundance and composition , topsoil nematode and soil organic carbon and nutrients levels . Recently, Perron et al. showed that the input of logging residues in the inter-rows sustains major soil functions such as soil structure maintenance, carbon transformation and nutrient cycling, after clear-cutting in immature rubber plantations in Ivory Coast. Here, we aim to address the effect of agroecological practices on both the resistance and resilience of soil microbial communities throughout a replantation cycle. In particular, our objectives are (1) to assess the impact of rubber tree clear-cutting on soil microbial resistance and (2) to determine how agroecological practices (such as logging residues and legume input) modulate microbial resilience over time, and (3) whether and how microbial responses vary across different edaphic contexts. We hypothesize that organic matter inputs improve soil microbial resistance and accelerate resilience, and that the recovery rate may vary according to both organic matter quality and pedological context. Thus, we conducted a diachronic monitoring in two large-scale field experiments in Ivory Coast and described soil microbial communities (including taxonomic and functional diversity, composition and co-occurrence networks) based on 16S and 18S rRNA gene investigations, before rubber tree clear-cutting and then every 6 months over a 24-month period following the perturbation.
Study Sites This study was conducted in two industrial rubber plantations in Ivory Coast (Fig. A). The Bongo plantation, belonging to the Société Africaine des Plantations d’Hévéas , is located in the Grand Bassam region and covers an area of 5700 ha in the South-East of the country (latitude 5°30′32.364′′ N, longitude 3°32′51.755′′ W). The Société des Caoutchoucs de Grand-Béréby is located in the South-West (latitude 4°43′9.696′′ N, longitude 7°6′41.795′′ W) and owns 16,300 ha of rubber plantations. Both sites are subjected to a sub-equatorial hot and humid climate, which is well-adapted to rubber tree plantations. The ranges provided (annual rainfall of 1700–1900 mm and annual average temperatures of 24–27 °C) are general references for the region and apply similarly to both sites. There are no significant differences in climate between the two sites. Soil physico-chemical properties have been described at both sites in a previous study (see Perron et al. ): the soil at the Bongo plantation is classified as a yellow ferralic Arenosol (FAO soil classification ) with a sandy loam texture (10% clay in the topsoil) and is characterized by an acidic pH (4.3). At the Grand-Béréby plantation, the soil is classified as a red Ferralsol with a clayey loam texture (23% clay in the topsoil) and is characterized by a slightly less acidic pH (around 4.7). Hereafter, the Bongo plantation will thus be referred as the sandy site, and the Grand-Béréby plantation as the clayey site . Total carbon and nitrogen content, and CEC are higher in the clayey site, and available phosphorus concentration in the topsoil layer is higher in the sandy site. The sandy site is characterized by slight slopes (< 5%), whereas the clayey site features hilly areas (slopes of 10–25%). Experimental Design The experiment began before the logging of the old rubber trees (38–40 years old) by bulldozers, in November 2017. At both sites, the logged stands were the first cycle of rubber plantation replacing natural rainforest. Similar designs were applied at both sites, testing four agroecological practices combining tree logging residues and legume growth (hereafter referred to as treatments): no residues and the legume Pueraria phaseoloides (R0L1); fine residues (i.e. leaves, fine branches with a diameter < 20 cm and stumps) with the legume Pueraria phaseoloides (R1L1); all residues (i.e. including branches > 20 cm diameter and trunks) with the legume Pueraria phaseoloides (R2L1); and a control treatment without legume nor residues (R0L0). Each treatment was replicated into 4 randomized blocks, resulting in a total of 16 plots (40 × 40 m) per site (Fig. B). Logging residues were placed between each new rubber row. P . phaseoloides, was sown by broadcasting (10 kg ha −1 of humidified seeds) in February 2018. Fine logging residues composing the R1L1 treatment amounted to 36 and 51 t ha −1 of carbon per hectare in the sandy and the clayey sites, respectively. Input composed of all residues (i.e. both fine and coarse, R2L1 treatment) amounted to 97 and 245 t ha −1 of carbon per hectare in the sandy and the clayey sites, respectively. More details about the experimental design is available in Perron et al. and Kouakou et al. . Soil Sampling Ten soil samples were collected between rows at 0–10 cm depth in each plot and then pooled to form a composite soil sample (Fig. C). A first campaign was conducted in October 2017, before the old plantation logging, to assess initial soil properties before clear-cutting: one composite soil sample per block, i.e. 4 samples per site was collected. Sampling was then carried out every 6 months to monitor the temporal changes in soil microbial communities according to the application of logging residues and legume. To limit the influence of climate, especially rainfall, sampling was carried out in October and in April. These periods respectively correspond to the start of the dry and beginning of the wet seasons in Ivory Coast. A total of five sampling campaigns were carried out from October 2017 to October 2019. Soil Functions We analysed 8 properties related to soil functioning from the Biofunctool® set of indicators [ – ]. Biofunctool® consists of a core set of expert-selected indicators, assessing carbon transformation, nutrient cycling and soil structure maintenance. Briefly, three indicators describe the soil nutrient status: available ammonium (NH 4 + ) and nitrate (NO 3 − ) from soil extraction with 1 M KCl, as well as NO 3 − dynamics with ion exchange membranes (AEMNO + ) . Permanganate oxidizable carbon (POXC) was assessed to estimate soil available carbon content. Four indicators evaluated the soil structure: aggregate stability at 0–2 cm depth (AggSurf) and at 2–10 cm depth (AggSoil) , water infiltration speed with the Beerkan method and the VESS method (Visual Evaluation of Soil Structure) . Soil moisture (H) and bulk density (BD) were also assessed to describe soil properties. For these analyses, soil samples were collected on the 0–10 cm soil layer, except for VESS that required a sampling at 0–25 cm depth. DNA Extraction, Amplification and Sequencing Soil used for the DNA extraction was sampled simultaneously and nearby soil collected for Biofunctool® analysis. DNA extraction was performed using the “FastDNA™ SPIN kit for soil” extraction kit from MP Biomedicals™ following the manufacturer’s instructions. Extracted DNA extracted were then sent to the ADNid company (Qualtech groupe, Montpellier, France) for quantification, library preparation and sequencing. The purity and integrity of the DNA recovered was also verified after migration on a 1% agarose gel. Amplicons were generated by PCR amplification using the primer pair 338F/806R specific to the V3-V4 region of the 16S gene and the primer pair FF390/FR1specific to the V7-V8 region of the 18S RNA gene . PCR was conducted on 10 ng of template DNA into 15 μL total mix, submitted to an initial denaturation of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 1 min at 55 °C and 30 s at 72 °C. A final extension of 30 min at 60 °C was also included to complete the reaction. Amplicons were verified on 1.5% agarose gel, purified using Agencourt® AMPure® XP kit (Beckman Coulter, Italy, Milano), and quantified by fluorimetry (Quant-iT™ Pico Green DNA Assay Kit). PCR products concentration was adjusted to 10 ng/μL. A second PCR step was performed using specific Illumina adapter, the index and the Illumina overhang adapter primers into 2 × KAPA Hifi Hotstart Ready Mix (Roche Molecular Systems, Inc). A volume of 5 μL of PCR products previously prepared was used into a 50 μL final volume of PCR mix. A 3 mn at 95 °C denaturation step was performed followed by 12 cycles of 30 s at 95 °C, 30 s at 55 °C, 30 s at 72 °C before a 5 mn at 72 °C final extension step. PCR2 products were purified and pooled together at 15 μg/μL in a final library. The library quality was controlled using a fragment analyzer. Sequencing runs, generating 2 × 250 bp, reads were performed on an Illumina MiSeq using V2 chemistry. Sequence Data Processing The raw sequencing data were processed using a custom script from the DADA2 pipeline , which is designed to resolve amplicon sequence variants (ASVs) . Raw sequences were first demultiplexed by comparing index reads with a key, and paired sequences were trimmed to uniform lengths. Sequences were dereplicated, and the unique sequence pairs were denoised using the dada function. Primers and adapters were screened and removed using the cutadapt script . Paired-end sequences were merged, and chimeras removed. Taxonomy assignments were determined with the IDtaxa function from the Decipher R package against the SILVA taxonomic database for both 16S and 18S sequences (release 132) . Sequence data were then manipulated using the phyloseq R package . ASVs affiliated to chloroplasts, mitochondria (16S rRNA gene dataset) were removed in order to keep only microbial sequences. The isContaminant function (default parameters) from the decontam package was used to identify contaminant sequences from reagents or introduced during the manipulation of the samples . A total of 9 prokaryote ASVs and 12 microeukaryote ASVs were identified as contaminants and removed from the datasets. Samples were rarefied to 5130 and 13,500 reads for 16S and 18S amplicon datasets, respectively, to account for differences in sequencing depth (rarefaction curves are presented in Fig. ). Two out of the 290 sample did not quite reach the asymptote but were kept because, as rare taxa were further filtered, the contribution did not introduce biais. ASVs present in only one sample and with less than 3 observations in the entire data set were excluded. The taxonomic affiliation of prevalent ASVs (i.e. observed in more than 5% of samples) was refined using nucleotide basic local alignment search tool (BLASTn) analyses on NCBI nr database based on the best hit or the last common ancestor between best hits. The FAPROTAX database was then used to assign prokaryote function to the filtered 16S data, and 51% of sequence variants (representing 36% of the total prokaryote abundance) were assigned to at least one group. We focused on key functions involved in nutrient cycling (i.e. nitrate denitrification, nitrogen fixation, nitrification, ureolysis), plant organic matter decomposition (i.e. aromatic compound degradation, chitinolysis, cellulolysis, xylanolysis, fermentation) and other key functions related with plant health (i.e. plant pathogens) and affected by soil compaction (i.e. dark sulphur oxidation). Co-occurrence Network Computation To further describe structures of microbial communities, cross-domain co-occurrence networks were computed per site for each treatment first and then for each time point using the SPIEC-EASI (SParse InversE Covariance Estimation for Ecological Association Inference) pipeline . Before computation, 16S and 18S ASV datasets were filtered to keep taxa seen in at least 20% of the samples, as network inference requires sufficient data to estimate correlations. We identified hubs (i.e. the most connected ASVs that are potential keystone taxa) based on their node parameters (method developed by Berry and Widder ): a low betweenness centrality (lower quantile, < 0.9), and a high closeness centrality (higher quantile, > 0.8), transitivity (higher quantile, > 0.25) and degree (higher quantile, > 0.8). Microbial networks were then described based on key topological parameters: the node mean degree (i.e. connectivity), betweenness and closeness centralities, transitivity and the respective number of prokaryotic and microeukaryotic hubs and total nodes. We used the igraph R package to evaluate the topological properties of the co-occurrence network. Statistical Analysis All bioinformatic and statistical analyses were performed using R version 4.2.0 . All graphical presentations have been created with the ggplot function of the ggplot2 package . Changes in β-diversity induced by the sites, time, treatments, and their interactions were assessed using a Permutational Multivariate ANalysis Of Variance (hereafter PERMANOVA), implemented in the adonis function from the vegan package on a Bray–Curtis dissimilarity matrix. To account for non-independence of samples collected from the same blocks, we included blocks as a random effect in the model using the strata option. Pairwise group comparisons were then tested using the pairwise.adonis2 function from the pairwiseAdonis package . Multivariate homogeneity of group dispersion was first tested with the betadisper function ( vegan package). Community structures were then presented with principal coordinates analysis (PCoA), and the envfit function from the vegan package was used to fit significant correlation with edaphic properties onto the ordination. The same procedure was used to describe prokaryote functional profiles. Processes at play in community assembly were described by partitioning the turnover and nestedness components of β-diversity from communities before perturbation (T0), measured respectively as Simpson pairwise dissimilarity and nestedness-fraction of Sorensen pairwise dissimilarity . Mixed linear models (using the lmer function from lme4 package ) were then used to assess the effect of time, sites and treatment on the Sorensen dissimilarity as well as on the true turnover (i.e. change in community composition) and the nestedness (i.e. differences in species assemblages due to one community being a subset of the more diverse community). When required, data were transformed using the bestNormalize R package to meet model assumption needs (homogeneity of variance and residuals normality). To account for non-independence of samples collected from the same blocks, we included blocks as a random effect in the model. Differences between groups were further determined by Tukey Post hoc tests (using the glht function of the multcomp package . Changes in α-diversity were addressed by calculating Hill numbers , which describe the total number of species ( q = 0), the Shannon–Wiener ( q = 1) and the Simpson ( q = 2) diversity indices . High values of q give much more weight to the most abundant species, whereas low values consider the diversity of all species. Hill numbers were computed using the entropart R package . Arithmetic mean was used to create diversity profiles for each combination of site, time, and treatment. Mixed linear models ( lmer function from lme4 R package ) accounting for block as a random effect were used to assess the effects of time, treatment and their interaction on hill numbers at q = 0, q = 1and q = 2. To describe the nature of diversity changes, differential abundances of microbial phyla were calculated (package DESeq2 ) across time, and across treatment at 24 months. Results with an adjusted p value < 0.05 and an absolute log2fold > 0.5 were considered as significant. In this study, resistance (disturbance rate) was approximated by comparisons between the pre- (i.e. at T0) and post-disturbance (i.e. after 6 months) soil microbial properties, and resilience (recovery rate) refers to the closeness between the final states (i.e. after 24 months) and the initial state (i.e. at T0).
This study was conducted in two industrial rubber plantations in Ivory Coast (Fig. A). The Bongo plantation, belonging to the Société Africaine des Plantations d’Hévéas , is located in the Grand Bassam region and covers an area of 5700 ha in the South-East of the country (latitude 5°30′32.364′′ N, longitude 3°32′51.755′′ W). The Société des Caoutchoucs de Grand-Béréby is located in the South-West (latitude 4°43′9.696′′ N, longitude 7°6′41.795′′ W) and owns 16,300 ha of rubber plantations. Both sites are subjected to a sub-equatorial hot and humid climate, which is well-adapted to rubber tree plantations. The ranges provided (annual rainfall of 1700–1900 mm and annual average temperatures of 24–27 °C) are general references for the region and apply similarly to both sites. There are no significant differences in climate between the two sites. Soil physico-chemical properties have been described at both sites in a previous study (see Perron et al. ): the soil at the Bongo plantation is classified as a yellow ferralic Arenosol (FAO soil classification ) with a sandy loam texture (10% clay in the topsoil) and is characterized by an acidic pH (4.3). At the Grand-Béréby plantation, the soil is classified as a red Ferralsol with a clayey loam texture (23% clay in the topsoil) and is characterized by a slightly less acidic pH (around 4.7). Hereafter, the Bongo plantation will thus be referred as the sandy site, and the Grand-Béréby plantation as the clayey site . Total carbon and nitrogen content, and CEC are higher in the clayey site, and available phosphorus concentration in the topsoil layer is higher in the sandy site. The sandy site is characterized by slight slopes (< 5%), whereas the clayey site features hilly areas (slopes of 10–25%).
The experiment began before the logging of the old rubber trees (38–40 years old) by bulldozers, in November 2017. At both sites, the logged stands were the first cycle of rubber plantation replacing natural rainforest. Similar designs were applied at both sites, testing four agroecological practices combining tree logging residues and legume growth (hereafter referred to as treatments): no residues and the legume Pueraria phaseoloides (R0L1); fine residues (i.e. leaves, fine branches with a diameter < 20 cm and stumps) with the legume Pueraria phaseoloides (R1L1); all residues (i.e. including branches > 20 cm diameter and trunks) with the legume Pueraria phaseoloides (R2L1); and a control treatment without legume nor residues (R0L0). Each treatment was replicated into 4 randomized blocks, resulting in a total of 16 plots (40 × 40 m) per site (Fig. B). Logging residues were placed between each new rubber row. P . phaseoloides, was sown by broadcasting (10 kg ha −1 of humidified seeds) in February 2018. Fine logging residues composing the R1L1 treatment amounted to 36 and 51 t ha −1 of carbon per hectare in the sandy and the clayey sites, respectively. Input composed of all residues (i.e. both fine and coarse, R2L1 treatment) amounted to 97 and 245 t ha −1 of carbon per hectare in the sandy and the clayey sites, respectively. More details about the experimental design is available in Perron et al. and Kouakou et al. .
Ten soil samples were collected between rows at 0–10 cm depth in each plot and then pooled to form a composite soil sample (Fig. C). A first campaign was conducted in October 2017, before the old plantation logging, to assess initial soil properties before clear-cutting: one composite soil sample per block, i.e. 4 samples per site was collected. Sampling was then carried out every 6 months to monitor the temporal changes in soil microbial communities according to the application of logging residues and legume. To limit the influence of climate, especially rainfall, sampling was carried out in October and in April. These periods respectively correspond to the start of the dry and beginning of the wet seasons in Ivory Coast. A total of five sampling campaigns were carried out from October 2017 to October 2019.
We analysed 8 properties related to soil functioning from the Biofunctool® set of indicators [ – ]. Biofunctool® consists of a core set of expert-selected indicators, assessing carbon transformation, nutrient cycling and soil structure maintenance. Briefly, three indicators describe the soil nutrient status: available ammonium (NH 4 + ) and nitrate (NO 3 − ) from soil extraction with 1 M KCl, as well as NO 3 − dynamics with ion exchange membranes (AEMNO + ) . Permanganate oxidizable carbon (POXC) was assessed to estimate soil available carbon content. Four indicators evaluated the soil structure: aggregate stability at 0–2 cm depth (AggSurf) and at 2–10 cm depth (AggSoil) , water infiltration speed with the Beerkan method and the VESS method (Visual Evaluation of Soil Structure) . Soil moisture (H) and bulk density (BD) were also assessed to describe soil properties. For these analyses, soil samples were collected on the 0–10 cm soil layer, except for VESS that required a sampling at 0–25 cm depth.
Soil used for the DNA extraction was sampled simultaneously and nearby soil collected for Biofunctool® analysis. DNA extraction was performed using the “FastDNA™ SPIN kit for soil” extraction kit from MP Biomedicals™ following the manufacturer’s instructions. Extracted DNA extracted were then sent to the ADNid company (Qualtech groupe, Montpellier, France) for quantification, library preparation and sequencing. The purity and integrity of the DNA recovered was also verified after migration on a 1% agarose gel. Amplicons were generated by PCR amplification using the primer pair 338F/806R specific to the V3-V4 region of the 16S gene and the primer pair FF390/FR1specific to the V7-V8 region of the 18S RNA gene . PCR was conducted on 10 ng of template DNA into 15 μL total mix, submitted to an initial denaturation of 5 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 1 min at 55 °C and 30 s at 72 °C. A final extension of 30 min at 60 °C was also included to complete the reaction. Amplicons were verified on 1.5% agarose gel, purified using Agencourt® AMPure® XP kit (Beckman Coulter, Italy, Milano), and quantified by fluorimetry (Quant-iT™ Pico Green DNA Assay Kit). PCR products concentration was adjusted to 10 ng/μL. A second PCR step was performed using specific Illumina adapter, the index and the Illumina overhang adapter primers into 2 × KAPA Hifi Hotstart Ready Mix (Roche Molecular Systems, Inc). A volume of 5 μL of PCR products previously prepared was used into a 50 μL final volume of PCR mix. A 3 mn at 95 °C denaturation step was performed followed by 12 cycles of 30 s at 95 °C, 30 s at 55 °C, 30 s at 72 °C before a 5 mn at 72 °C final extension step. PCR2 products were purified and pooled together at 15 μg/μL in a final library. The library quality was controlled using a fragment analyzer. Sequencing runs, generating 2 × 250 bp, reads were performed on an Illumina MiSeq using V2 chemistry.
The raw sequencing data were processed using a custom script from the DADA2 pipeline , which is designed to resolve amplicon sequence variants (ASVs) . Raw sequences were first demultiplexed by comparing index reads with a key, and paired sequences were trimmed to uniform lengths. Sequences were dereplicated, and the unique sequence pairs were denoised using the dada function. Primers and adapters were screened and removed using the cutadapt script . Paired-end sequences were merged, and chimeras removed. Taxonomy assignments were determined with the IDtaxa function from the Decipher R package against the SILVA taxonomic database for both 16S and 18S sequences (release 132) . Sequence data were then manipulated using the phyloseq R package . ASVs affiliated to chloroplasts, mitochondria (16S rRNA gene dataset) were removed in order to keep only microbial sequences. The isContaminant function (default parameters) from the decontam package was used to identify contaminant sequences from reagents or introduced during the manipulation of the samples . A total of 9 prokaryote ASVs and 12 microeukaryote ASVs were identified as contaminants and removed from the datasets. Samples were rarefied to 5130 and 13,500 reads for 16S and 18S amplicon datasets, respectively, to account for differences in sequencing depth (rarefaction curves are presented in Fig. ). Two out of the 290 sample did not quite reach the asymptote but were kept because, as rare taxa were further filtered, the contribution did not introduce biais. ASVs present in only one sample and with less than 3 observations in the entire data set were excluded. The taxonomic affiliation of prevalent ASVs (i.e. observed in more than 5% of samples) was refined using nucleotide basic local alignment search tool (BLASTn) analyses on NCBI nr database based on the best hit or the last common ancestor between best hits. The FAPROTAX database was then used to assign prokaryote function to the filtered 16S data, and 51% of sequence variants (representing 36% of the total prokaryote abundance) were assigned to at least one group. We focused on key functions involved in nutrient cycling (i.e. nitrate denitrification, nitrogen fixation, nitrification, ureolysis), plant organic matter decomposition (i.e. aromatic compound degradation, chitinolysis, cellulolysis, xylanolysis, fermentation) and other key functions related with plant health (i.e. plant pathogens) and affected by soil compaction (i.e. dark sulphur oxidation).
To further describe structures of microbial communities, cross-domain co-occurrence networks were computed per site for each treatment first and then for each time point using the SPIEC-EASI (SParse InversE Covariance Estimation for Ecological Association Inference) pipeline . Before computation, 16S and 18S ASV datasets were filtered to keep taxa seen in at least 20% of the samples, as network inference requires sufficient data to estimate correlations. We identified hubs (i.e. the most connected ASVs that are potential keystone taxa) based on their node parameters (method developed by Berry and Widder ): a low betweenness centrality (lower quantile, < 0.9), and a high closeness centrality (higher quantile, > 0.8), transitivity (higher quantile, > 0.25) and degree (higher quantile, > 0.8). Microbial networks were then described based on key topological parameters: the node mean degree (i.e. connectivity), betweenness and closeness centralities, transitivity and the respective number of prokaryotic and microeukaryotic hubs and total nodes. We used the igraph R package to evaluate the topological properties of the co-occurrence network.
All bioinformatic and statistical analyses were performed using R version 4.2.0 . All graphical presentations have been created with the ggplot function of the ggplot2 package . Changes in β-diversity induced by the sites, time, treatments, and their interactions were assessed using a Permutational Multivariate ANalysis Of Variance (hereafter PERMANOVA), implemented in the adonis function from the vegan package on a Bray–Curtis dissimilarity matrix. To account for non-independence of samples collected from the same blocks, we included blocks as a random effect in the model using the strata option. Pairwise group comparisons were then tested using the pairwise.adonis2 function from the pairwiseAdonis package . Multivariate homogeneity of group dispersion was first tested with the betadisper function ( vegan package). Community structures were then presented with principal coordinates analysis (PCoA), and the envfit function from the vegan package was used to fit significant correlation with edaphic properties onto the ordination. The same procedure was used to describe prokaryote functional profiles. Processes at play in community assembly were described by partitioning the turnover and nestedness components of β-diversity from communities before perturbation (T0), measured respectively as Simpson pairwise dissimilarity and nestedness-fraction of Sorensen pairwise dissimilarity . Mixed linear models (using the lmer function from lme4 package ) were then used to assess the effect of time, sites and treatment on the Sorensen dissimilarity as well as on the true turnover (i.e. change in community composition) and the nestedness (i.e. differences in species assemblages due to one community being a subset of the more diverse community). When required, data were transformed using the bestNormalize R package to meet model assumption needs (homogeneity of variance and residuals normality). To account for non-independence of samples collected from the same blocks, we included blocks as a random effect in the model. Differences between groups were further determined by Tukey Post hoc tests (using the glht function of the multcomp package . Changes in α-diversity were addressed by calculating Hill numbers , which describe the total number of species ( q = 0), the Shannon–Wiener ( q = 1) and the Simpson ( q = 2) diversity indices . High values of q give much more weight to the most abundant species, whereas low values consider the diversity of all species. Hill numbers were computed using the entropart R package . Arithmetic mean was used to create diversity profiles for each combination of site, time, and treatment. Mixed linear models ( lmer function from lme4 R package ) accounting for block as a random effect were used to assess the effects of time, treatment and their interaction on hill numbers at q = 0, q = 1and q = 2. To describe the nature of diversity changes, differential abundances of microbial phyla were calculated (package DESeq2 ) across time, and across treatment at 24 months. Results with an adjusted p value < 0.05 and an absolute log2fold > 0.5 were considered as significant. In this study, resistance (disturbance rate) was approximated by comparisons between the pre- (i.e. at T0) and post-disturbance (i.e. after 6 months) soil microbial properties, and resilience (recovery rate) refers to the closeness between the final states (i.e. after 24 months) and the initial state (i.e. at T0).
Taxonomic Profiles of Soil Microbial Communities A total of 743,850 reads from 145 samples were present in the final 16S dataset, and 1,957,500 reads from 145 samples in the final 18S dataset. Thus, 2878 ASVs of 16S and 2401 ASVs of 18S, belonging to 19 prokaryotic phyla and 15 microeukaryotic order, were detected. Prokaryote communities were primarily composed of Proteobacteria (36% of 16S sequence reads), Firmicutes (24%) and Actinobacteria (23%). Fungi represented 71% of the 18S reads and were primarily composed of Ascomycota (Aspergillaceae representing 13% of the 18S sequence reads, Hypocreaceae 11%%, and Nectriaceae 3.5%). Site, Treatment and Time Shaping of Soil Microbial Community Structures A first global PERMANOVA analysis was performed to test for the effects of sites and agroecological practices on both prokaryotic and microeukaryotic community structures. Significant differences were revealed between sites for both prokaryotes and microeukaryotes (r 2 = 0.16; p < 0.001 and r 2 = 0.06; p < 0.001 respectively, data not shown). Therefore, we further subset both datasets to separately assess the relative influence of treatments and time in shaping communities in the sandy and clayey sites, respectively. Overall, PERMANOVA (Tables and ) revealed a significant and major effect of time (explaining around 30% of thevariance), and a smaller but still significant effect of treatment (explaining around 10% of the variance) in shaping soil communities at both sites. In the clayey site, distinct community structures were observed between treatments with tree residues (R1L1 and R2L1) and without (R0L0 and R0L1). In the sandy site, responses of soil microbial communities also varied between treatments over time (significant interaction effect, PERMANOVA, around 12% variation). There, after 24 months, each treatment has led to a new and distinct prokaryotic and microeukaryotic community. Differences in community structures to T0 (i.e. Sorensen dissimilarity, Fig. and Tables and ), which reflect community composition resilience, significantly decreased over time for prokaryotes at both sites, a trend much less pronounced for microeukaryotes, particularly in the clayey site where the observed decrease was not significant. The treatment with legume cover combined with fine residues (R1L1) induced a higher resilience for microeukaryotes at both sites, and for prokaryotes only in the sandy site. To further describe assembly processes at play over time after tree logging, Sorensen dissimilarity was partitioned into true turnover and nestedness turnover, i.e. change in community composition (i.e. in the identity of species between the two communities), and nestedness (i.e. differences in species assemblages due to one community being a subset of the more diverse community Fig. , Tables and ). At both sites and for both 16S and 18S datasets, the β Nestedness/Sorensen was continuously lower than 0.5 after rubber clear-cutting, indicating a prominent turnover effect in driving communities structuration. As for Sorensen dissimilarity, turnover was mainly modulated over time for prokaryotes, while the different treatments mainly drove microeukaryotes’ turnover. Treatments had no effect on nestedness, while time significantly impacted that of prokaryote at both sites, and of microeukaryote community only in the clayey site. Principal coordinates analysis of communities dissimilarity graphically reflects the shifts in community composition induced after clear-cutting between T0 and 6 months (Fig. , Table ). Communities at T0 were rather similar and then became more heterogeneous (dispersed) after soil perturbation. Resilience of communities was also perceptible as successive communities progressively became closer to T0 after 6, 12, 18 and 24 months, particularly evident for prokaryotes at both sites. To further describe the drivers of microbial community structuration, the influence of ten edaphic properties (i.e. soil ammonium, soil nitrate, nitrate plant availability, labile carbon, aggregate stability at two depths, water infiltration, soil structure, moisture, bulk density) on microbial patterns was studied. Most soil properties were found to be significantly correlated with microbial community structures, highlighting the close link between soil conditions and community assembly. At both sites, contrasting soil structures and humidity (higher soil aggregate stability and moisture at T0) and nutrient content (lower NH 4 + and NO 3 − at T0) discriminated community composition before and after tree-logging. Additionally, the intensity of soil structuring variables strongly differed between sites for prokaryote communities. In the sandy site, soil aggregate stability was the most structuring property, followed by NH 4 + , water infiltration, bulk density and NO 3 − . In the clayey site, soil moisture was the most structuring factor, followed by NH 4 + content and water infiltration. For microeukaryotes, similar patterns were observed at both sites, communities were mainly structured by NH 4 + , followed by soil moisture and aggregate stability, although the influence of these environmental filters was even more pronounced in the sandy site. Co-occurrence Networks for Describing Microbial Resistance and Resilience Dynamics For each time point and at each site, a cross-domain co-occurrence network was computed (Fig. and Fig. and , Table ). Tree clear-cutting deeply impacted microbial networks. We observed a net reduction in the total number of nodes and of core taxa, along with a decrease in networks connectivity, closeness centrality and transitivity, and an increase in betweenness centrality at both sites. However, the frequency of node degrees, betweenness and closeness centralities clearly indicated less robust network structures at 6 months in the sandy site. The lower 18S/16S node and hub ratios, and a net gain in 16S nodes, highlighted a higher sensitivity of microeukaryotes in the sandy site. The resilience of communities was noticeable by a gradual recovery of initial properties over time. Interestingly, a faster recovery of initial topological properties occurred in the sandy site, where connectivity even exceeded that of T0 at 18 months. This trend was observed together with a net increase in 18S/16S hub ratio after 18 months, suggesting a major role of microeukaryotes in microbiome resilience in the sandy site. Network topological properties were also computed to characterize the effect of the different treatments on soil communities. At both sites, the 18S/16S node ratio favored microeukaryote taxa where only legumes were grown (R0L1), while networks from the control treatments (R0L0) had fewer 18S nodes. The network derived from the treatment involving legume and fine residues (R1L1) was characterized by more 16S nodes. Differences in network topology induced by treatments were more pronounced in the sandy site. There, the two treatments including fine and all logging residues with legume (R1L1 and R2L1) exhibited high connectivity. The highest 18S/16S hub ratio was found in microbial network from soils treated with legume and all residues (R2L1). This trend did not reflect the 18S/16S network nodes ratio, suggesting a key role of microeukaryotes in this agroecological practice in a sandy pedological context. In the clayey site, the effect of treatment on networks properties was less perceptible. Drivers of Soil microbial α-Diversity and Taxonomic Composition Microbial α-diversity was studied using Hills number, and the effect of time and treatment was further tested on the diversity profiles, in each site (Fig. , Tables and ). The annotations q = 0, q = 1 and q = 2 respectively denote species richness, Shannon–Wiener diversity, and the distribution (Simpson) of individuals within each species. Diversity loss between T0 and 6 months illustrates the resistance of communities to the tree replacement process. Prokaryote diversity was resistant to soil perturbation, as no changes were observed between T0 and 6 months (except for q0 in specific treatments from the clayey site). Microeukaryote diversity was less resistant than prokaryote communities, as diversity losses were observed at both sites. Time significantly impacted the richness, Shannon diversity, and distribution of prokaryotic and microeukaryotic taxa at both sites, suggesting that tree-logging induced changes in the occurrence of both rare and prevalent microbial taxa. In the clayey site, prokaryote richness varied over time differently according to the various treatments (interaction effect). Surprisingly, 16S diversity from every treatment except control was higher at 6 months than at T0. Differences among treatments arose 18 months after perturbation, but then blurred after 24 months. For microeukaryotes, cover treatments did not modulate the impact of soil perturbation on diversity levels. Only time did, diversity indexes being lower at 6 months than at T0, then increasing towards values near the initial state for q1 and q2, and even higher for q0. In the sandy site, the effect of treatment on microbial α-diversity was more pronounced. Prokaryote communities from all treatments had similar richness at T0 and 6 months, then, 24 months after replanting, richness levels were distinct among treatments, with the highest in soil covered with legume and all residues (R2L1). With this treatment, prokaryote q1 tended to be generally higher than with other treatments. Hill numbers q1 and q2, which consider the most abundant 16S taxa, also increased over time in the sandy site. For microeukaryotes, total richness dropped after 6 months and then exceeded its initial level after 24 months, not being affected by the different treatments. However, q1 and q2 which consider the most prevalent taxa, were significantly influenced by treatments, being higher than at T0 in soil covered with legume only (R0L1), and with legume and all residues (R2L1), 24 months after tree replanting. Differential abundance testing confirmed that 9 out of the 19 prokaryotic phyla and 53 out of the 115 fungal families were highly sensitive to the tree replanting process (Fig. ). The soil perturbation reduced the abundance of Actinobacteriota , Firmicutes , Bacteroidota and Patescibacteria , while it stimulated the one of Acidobacteriota , Chloroflexi , Verrucomicrobiota , Nitrospirot a and Dependentiae . Then, all sensitive bacterial phyla tended towards a resilient trajectory after 24 months. The fungal families most affected by the tree replanting process were Choanephoraceae, Debaryomycetaceae, Rhynchogastremataceae, Sporiodiobolaceae, Trichosporonaceae and Xylariaceae. Aspergillaceae and Clavicipitaceae families abundances increased with soil perturbation. However, not all taxa followed a resilient trajectory, even after 2 years. Differential abundance revealed the sensitivity of certain microbial phyla to legume growth and residues input, compared to the control R0L0 (Fig. ). Treatment with legume only favored Tricosporonaceae and Sporidiobolaceae and reduced Cucurbitariaceae . Legume and fine residues (R1L1) increased the abundances of Actinobacteriota and Cyanobacteria , and reduced the abundance of Proteobacteria , Clavicipitaceae , Glomerellales and Venturiaceae . The input of coarse residues (R2L1) stimulated the abundance of Chloroflexi and Acidobacteriota , while reducing the abundances of Bacteroidota , Clavicipitaceae , Omphalotaceae and Venturiaceae . Resistance and Resilience of Soil Prokaryote Functioning Prokaryotes functions were determined by assigning metabolic pathways using the FAPROTAX database on identified taxa, and the effects of time and treatment on functional communities were further analyzed using PERMANOVA analysis (Fig. , Tables and and ). In the two sites, PERMANOVA results indicated a significant treatment effect with a low variation (r 2 ranged from 5 to 7%), while time was the most structuring driver, responsible for more than 50% of the total variation in microbial functions. The effect of treatments on functional profiles was not modulated over time (no interaction effect). PCoA described a functional shift between T0 and 6 months after tree-logging, and then a gradual return towards the initial state at both sites. To further describe how functional communities were impacted, key functions (i.e. nitrate denitrification, nitrogen fixation, nitrification, cellulolysis, dark sulfur oxidation, ureolysis, aromatic compound degradation, plant pathogen, chitinolysis) were projected onto the ordination space. The congruence towards T0 was mainly associated with an increase in denitrification potential (r 2 > 0.9 at both sites). Three N-related function (i.e. nitrogen fixation, denitrification and nitrification) pointed away from communities at 6 months, suggesting that these communities were less involved in N cycle. In the sandy site, a higher dark sulfur oxidation potential, which mostly relies on anoxygenic taxa, significantly discriminated functional profile communities at 6 months, while 6 months communities in the clayey site seemed mostly associated with higher ureolysis and aromatic compound degradation potentials. To compare patterns in prokaryote functional resistance and resilience after tree replanting, we computed functional Bray–Curtis distance to T0 and thus studied the effect of time and treatment on functional resilience (Fig. , Tables and ). At both sites, time had a significant effect on prokaryote functional distance to T0. We found a greater difference in functional communities between T0 and 6 months in the sandy than the clayey site, which again attests of a higher sensitivity of functional communities in the sandy site. After 24 months, the community from the sandy site also got closer to the initial state than communities from the clayey site, suggesting better functional resilience. Interestingly, treatment had an effect on functional distance to T0 only in the sandy site, where the treatment with legume and all residues (R2L1) led functional communities towards an equilibrium farther from the initial state.
A total of 743,850 reads from 145 samples were present in the final 16S dataset, and 1,957,500 reads from 145 samples in the final 18S dataset. Thus, 2878 ASVs of 16S and 2401 ASVs of 18S, belonging to 19 prokaryotic phyla and 15 microeukaryotic order, were detected. Prokaryote communities were primarily composed of Proteobacteria (36% of 16S sequence reads), Firmicutes (24%) and Actinobacteria (23%). Fungi represented 71% of the 18S reads and were primarily composed of Ascomycota (Aspergillaceae representing 13% of the 18S sequence reads, Hypocreaceae 11%%, and Nectriaceae 3.5%).
A first global PERMANOVA analysis was performed to test for the effects of sites and agroecological practices on both prokaryotic and microeukaryotic community structures. Significant differences were revealed between sites for both prokaryotes and microeukaryotes (r 2 = 0.16; p < 0.001 and r 2 = 0.06; p < 0.001 respectively, data not shown). Therefore, we further subset both datasets to separately assess the relative influence of treatments and time in shaping communities in the sandy and clayey sites, respectively. Overall, PERMANOVA (Tables and ) revealed a significant and major effect of time (explaining around 30% of thevariance), and a smaller but still significant effect of treatment (explaining around 10% of the variance) in shaping soil communities at both sites. In the clayey site, distinct community structures were observed between treatments with tree residues (R1L1 and R2L1) and without (R0L0 and R0L1). In the sandy site, responses of soil microbial communities also varied between treatments over time (significant interaction effect, PERMANOVA, around 12% variation). There, after 24 months, each treatment has led to a new and distinct prokaryotic and microeukaryotic community. Differences in community structures to T0 (i.e. Sorensen dissimilarity, Fig. and Tables and ), which reflect community composition resilience, significantly decreased over time for prokaryotes at both sites, a trend much less pronounced for microeukaryotes, particularly in the clayey site where the observed decrease was not significant. The treatment with legume cover combined with fine residues (R1L1) induced a higher resilience for microeukaryotes at both sites, and for prokaryotes only in the sandy site. To further describe assembly processes at play over time after tree logging, Sorensen dissimilarity was partitioned into true turnover and nestedness turnover, i.e. change in community composition (i.e. in the identity of species between the two communities), and nestedness (i.e. differences in species assemblages due to one community being a subset of the more diverse community Fig. , Tables and ). At both sites and for both 16S and 18S datasets, the β Nestedness/Sorensen was continuously lower than 0.5 after rubber clear-cutting, indicating a prominent turnover effect in driving communities structuration. As for Sorensen dissimilarity, turnover was mainly modulated over time for prokaryotes, while the different treatments mainly drove microeukaryotes’ turnover. Treatments had no effect on nestedness, while time significantly impacted that of prokaryote at both sites, and of microeukaryote community only in the clayey site. Principal coordinates analysis of communities dissimilarity graphically reflects the shifts in community composition induced after clear-cutting between T0 and 6 months (Fig. , Table ). Communities at T0 were rather similar and then became more heterogeneous (dispersed) after soil perturbation. Resilience of communities was also perceptible as successive communities progressively became closer to T0 after 6, 12, 18 and 24 months, particularly evident for prokaryotes at both sites. To further describe the drivers of microbial community structuration, the influence of ten edaphic properties (i.e. soil ammonium, soil nitrate, nitrate plant availability, labile carbon, aggregate stability at two depths, water infiltration, soil structure, moisture, bulk density) on microbial patterns was studied. Most soil properties were found to be significantly correlated with microbial community structures, highlighting the close link between soil conditions and community assembly. At both sites, contrasting soil structures and humidity (higher soil aggregate stability and moisture at T0) and nutrient content (lower NH 4 + and NO 3 − at T0) discriminated community composition before and after tree-logging. Additionally, the intensity of soil structuring variables strongly differed between sites for prokaryote communities. In the sandy site, soil aggregate stability was the most structuring property, followed by NH 4 + , water infiltration, bulk density and NO 3 − . In the clayey site, soil moisture was the most structuring factor, followed by NH 4 + content and water infiltration. For microeukaryotes, similar patterns were observed at both sites, communities were mainly structured by NH 4 + , followed by soil moisture and aggregate stability, although the influence of these environmental filters was even more pronounced in the sandy site.
For each time point and at each site, a cross-domain co-occurrence network was computed (Fig. and Fig. and , Table ). Tree clear-cutting deeply impacted microbial networks. We observed a net reduction in the total number of nodes and of core taxa, along with a decrease in networks connectivity, closeness centrality and transitivity, and an increase in betweenness centrality at both sites. However, the frequency of node degrees, betweenness and closeness centralities clearly indicated less robust network structures at 6 months in the sandy site. The lower 18S/16S node and hub ratios, and a net gain in 16S nodes, highlighted a higher sensitivity of microeukaryotes in the sandy site. The resilience of communities was noticeable by a gradual recovery of initial properties over time. Interestingly, a faster recovery of initial topological properties occurred in the sandy site, where connectivity even exceeded that of T0 at 18 months. This trend was observed together with a net increase in 18S/16S hub ratio after 18 months, suggesting a major role of microeukaryotes in microbiome resilience in the sandy site. Network topological properties were also computed to characterize the effect of the different treatments on soil communities. At both sites, the 18S/16S node ratio favored microeukaryote taxa where only legumes were grown (R0L1), while networks from the control treatments (R0L0) had fewer 18S nodes. The network derived from the treatment involving legume and fine residues (R1L1) was characterized by more 16S nodes. Differences in network topology induced by treatments were more pronounced in the sandy site. There, the two treatments including fine and all logging residues with legume (R1L1 and R2L1) exhibited high connectivity. The highest 18S/16S hub ratio was found in microbial network from soils treated with legume and all residues (R2L1). This trend did not reflect the 18S/16S network nodes ratio, suggesting a key role of microeukaryotes in this agroecological practice in a sandy pedological context. In the clayey site, the effect of treatment on networks properties was less perceptible.
Microbial α-diversity was studied using Hills number, and the effect of time and treatment was further tested on the diversity profiles, in each site (Fig. , Tables and ). The annotations q = 0, q = 1 and q = 2 respectively denote species richness, Shannon–Wiener diversity, and the distribution (Simpson) of individuals within each species. Diversity loss between T0 and 6 months illustrates the resistance of communities to the tree replacement process. Prokaryote diversity was resistant to soil perturbation, as no changes were observed between T0 and 6 months (except for q0 in specific treatments from the clayey site). Microeukaryote diversity was less resistant than prokaryote communities, as diversity losses were observed at both sites. Time significantly impacted the richness, Shannon diversity, and distribution of prokaryotic and microeukaryotic taxa at both sites, suggesting that tree-logging induced changes in the occurrence of both rare and prevalent microbial taxa. In the clayey site, prokaryote richness varied over time differently according to the various treatments (interaction effect). Surprisingly, 16S diversity from every treatment except control was higher at 6 months than at T0. Differences among treatments arose 18 months after perturbation, but then blurred after 24 months. For microeukaryotes, cover treatments did not modulate the impact of soil perturbation on diversity levels. Only time did, diversity indexes being lower at 6 months than at T0, then increasing towards values near the initial state for q1 and q2, and even higher for q0. In the sandy site, the effect of treatment on microbial α-diversity was more pronounced. Prokaryote communities from all treatments had similar richness at T0 and 6 months, then, 24 months after replanting, richness levels were distinct among treatments, with the highest in soil covered with legume and all residues (R2L1). With this treatment, prokaryote q1 tended to be generally higher than with other treatments. Hill numbers q1 and q2, which consider the most abundant 16S taxa, also increased over time in the sandy site. For microeukaryotes, total richness dropped after 6 months and then exceeded its initial level after 24 months, not being affected by the different treatments. However, q1 and q2 which consider the most prevalent taxa, were significantly influenced by treatments, being higher than at T0 in soil covered with legume only (R0L1), and with legume and all residues (R2L1), 24 months after tree replanting. Differential abundance testing confirmed that 9 out of the 19 prokaryotic phyla and 53 out of the 115 fungal families were highly sensitive to the tree replanting process (Fig. ). The soil perturbation reduced the abundance of Actinobacteriota , Firmicutes , Bacteroidota and Patescibacteria , while it stimulated the one of Acidobacteriota , Chloroflexi , Verrucomicrobiota , Nitrospirot a and Dependentiae . Then, all sensitive bacterial phyla tended towards a resilient trajectory after 24 months. The fungal families most affected by the tree replanting process were Choanephoraceae, Debaryomycetaceae, Rhynchogastremataceae, Sporiodiobolaceae, Trichosporonaceae and Xylariaceae. Aspergillaceae and Clavicipitaceae families abundances increased with soil perturbation. However, not all taxa followed a resilient trajectory, even after 2 years. Differential abundance revealed the sensitivity of certain microbial phyla to legume growth and residues input, compared to the control R0L0 (Fig. ). Treatment with legume only favored Tricosporonaceae and Sporidiobolaceae and reduced Cucurbitariaceae . Legume and fine residues (R1L1) increased the abundances of Actinobacteriota and Cyanobacteria , and reduced the abundance of Proteobacteria , Clavicipitaceae , Glomerellales and Venturiaceae . The input of coarse residues (R2L1) stimulated the abundance of Chloroflexi and Acidobacteriota , while reducing the abundances of Bacteroidota , Clavicipitaceae , Omphalotaceae and Venturiaceae .
Prokaryotes functions were determined by assigning metabolic pathways using the FAPROTAX database on identified taxa, and the effects of time and treatment on functional communities were further analyzed using PERMANOVA analysis (Fig. , Tables and and ). In the two sites, PERMANOVA results indicated a significant treatment effect with a low variation (r 2 ranged from 5 to 7%), while time was the most structuring driver, responsible for more than 50% of the total variation in microbial functions. The effect of treatments on functional profiles was not modulated over time (no interaction effect). PCoA described a functional shift between T0 and 6 months after tree-logging, and then a gradual return towards the initial state at both sites. To further describe how functional communities were impacted, key functions (i.e. nitrate denitrification, nitrogen fixation, nitrification, cellulolysis, dark sulfur oxidation, ureolysis, aromatic compound degradation, plant pathogen, chitinolysis) were projected onto the ordination space. The congruence towards T0 was mainly associated with an increase in denitrification potential (r 2 > 0.9 at both sites). Three N-related function (i.e. nitrogen fixation, denitrification and nitrification) pointed away from communities at 6 months, suggesting that these communities were less involved in N cycle. In the sandy site, a higher dark sulfur oxidation potential, which mostly relies on anoxygenic taxa, significantly discriminated functional profile communities at 6 months, while 6 months communities in the clayey site seemed mostly associated with higher ureolysis and aromatic compound degradation potentials. To compare patterns in prokaryote functional resistance and resilience after tree replanting, we computed functional Bray–Curtis distance to T0 and thus studied the effect of time and treatment on functional resilience (Fig. , Tables and ). At both sites, time had a significant effect on prokaryote functional distance to T0. We found a greater difference in functional communities between T0 and 6 months in the sandy than the clayey site, which again attests of a higher sensitivity of functional communities in the sandy site. After 24 months, the community from the sandy site also got closer to the initial state than communities from the clayey site, suggesting better functional resilience. Interestingly, treatment had an effect on functional distance to T0 only in the sandy site, where the treatment with legume and all residues (R2L1) led functional communities towards an equilibrium farther from the initial state.
Our study, based on a 24-month investigation of soil microbiota in two different experimental fields with distinct edaphic contexts, quantified and analyzed the resistance and resilience of prokaryotic and microeukaryotic communities following a disturbance induced by the tree replanting process with regards to different cover treatments (i.e. the use of logging residues and legume cover). Prokaryotes were generally more resistant to soil perturbation, while microeukaryotic communities were more affected. Prokaryotic recovery was also faster than that of microeukaryotes, the latter being deeply influenced by the cover treatments. These specific reassembly dynamics were particularly pronounced in the sandy site. Resistance of the Soil Microbiota to the Rubber Tree Replanting Process The rubber tree replanting process induced a sudden and deep reassembly of the soil microbiota at both sites. Indeed, 6 months after soil perturbations, net changes in microbial community structure, taxonomy and functioning, together with losses in network connectivity, were observed. The rubber tree replanting resulted in a shift in the taxonomic composition of both prokaryotic and microeukaryotic communities in both clayey and sandy pedological contexts after 6 months. We showed that communities at 6 months were concomitant with low soil moisture, weak aggregate stabilities, and high soil NH 4 + and NO 3 − contents. Wheeling with a heavy agricultural vehicle has been shown to increase bulk density in the topsoil and to substantially reduce air permeability and gas diffusion , which can thus lead to anoxic conditions . In addition, the removal of tree vegetation and subsequent opening of the canopy might also have led to shifts in soil microbial populations by altering the feedback system between plants and soil and changing soil microclimate . Soil perturbations promoted microbial taxa with lifestyle adapted to the observed harsh conditions (i.e. reduced moisture and soil compaction) such as facultative aerobic (i.e. Nitrospirota , parasites (i.e. Dependentiae and Clavicipitaceae ) or competitive bacteria (i.e. Acidobacteria ). Soil compaction can impact microbial metabolic activities by limiting or interrupting aerobic processes, such as nitrification and mineralisation . For instance, we observed that bacterial communities at 6 months were less susceptible to performing N-related functions (i.e. nitrogen fixation, nitrification, and denitrification). The observed functional changes could be explained by soil compaction, which shifts bacterial communities towards anaerobic bacteria , that might have inhibited N fixation and nitrification, and thus denitrification by substrate provision interruption. For prokaryote specifically, diversity levels of abundant taxa have not fallen after the tree replanting process and were sometimes even higher 6 months after soil perturbation. Similar findings have been reported by Hartmann and colleagues in response to forest soils’ compaction. Authors found an increase in bacterial diversity that they attributed to taxa with low oxygen availability and capable of anaerobic respiration in compacted soils. Conversely, microeukaryotic diversity was less resistant to the tree replanting-induced disturbances at both sites. Indeed, fungi are known to be particularly sensitive to soil compaction induced by deforestation . This can partly be explained by the generally higher sensitivity of microeukaryotes to low oxygen availability induced by soil compaction and other studies pointed such differential response to compaction . In our study, observations derived from co-occurrence network modelling also support the hypothesis of less resistant microeukaryote communities to the tree replanting process. At both sites, a net loss in network connectivity was observed, and this complexity reduction was mainly imputable to a loss of 18S nodes. While fungal network sensitivity to soil labor has been previously demonstrated , this is, to our knowledge, the first study to report lower resistance of the microeukaryote network over prokaryotes to soil compaction. Microbial Resilience Was Modulated by Soil Cover Treatments We observed that cover practices had only a weak impact on the resistance (observed 6 months after perturbation) of microbial communities to the tree replanting process. This was anticipated as neither legume growth nor tree residue decomposition had really started yet. The treatment effects were noticeable later in the resilience trajectory and varied between prokaryotes and microeukaryotes, with microeukaryotes being more responsive to logging residue inputs. This particular sensitivity may be related to the high potential of mycelia to actively colonize plant residues or to the niche modification induced by organisms from the tree phyllosphere that are inoculated with tree residues. These organisms are the early colonizers and decomposers of dead plant biomass and might play a key role in microbial succession during organic matter decomposition . This latter hypothesis is supported in our study by a higher complexity in networks from treatments that include tree residues. Soil properties improvement (higher C and N contents) in rubber-based diversified systems compared to monoculture was also evidenced in the study by . Microbial communities and the ratio between fungal and bacterial biomass are strongly influenced by the C/N ratio . Organic matter inputs used in our study had distinct C/N ratios (i.e. legumes only < legume with fine residues < legume with all residues) that we expected to affect soil microbial successions and community resilience. Pueraria phaseoloides is able to recruit a specific cohort of arbuscular mycorrhizal fungi (AMF) communities , and bacterial communities are dependent on mycorrhizae for their carbon resources . One could expect that legume cover should thus be an efficient strategy for soil properties recovery. However, in this study, the treatment involving only legume cover has led to intermediate resilience trajectories. An explanation could be that hampered N metabolism due to the anoxic soil conditions brought on by soil compaction has partially interrupted microbial successions. The treatment combining fine tree residues and legume cover has led to a better (though still incomplete) recovery of the Sorensen index at both sites for microeukaryotes and in the sandy site for prokaryotes after 24 months. The high C/N ratio of woody debris is well known to benefit to the fungal biomass. Indeed, fungi are the main decomposers of tree residues because of their ability to catabolize woody complex carbon molecules (i.e. cellulose and lignin) with the nutrient-mineralizing extracellular enzymes they produce and their hyphal extension capabilities . Carbon organic matter combined with sufficient N input might have favored the hyphal production. The latter is responsible for the formation of macro-aggregates that host most microbiological activities . It is thus likely that tree residues, by indirectly improving soil structure, favored microbial activities and speed up their succession. In the sandy site specifically, the treatment combining all tree residues with legume cover harbored the highest prokaryotic and microeukaryotic α-diversity levels 24 months after perturbation. This result is surprising as C:N stoichiometry has been globally shown to have some negative effects on microbial alpha diversity . It is possible that the high microbial richness observed after 24 months hides a diversity decline in the following months. Indeed, Dossa et al. observed fungal succession in decomposing woody debris across a tropical forest. They showed a decline in fungal diversity after 18 months that they explained by increasing competition through time for remaining resources. In this context, care needs to be taken in drawing any conclusions about a beneficial effect of this specific treatment; a longer diachronic study should provide more insights. A Leading Influence of Pedological Context in Driving Community Dynamics? The field site was the predominant driver of microbial recovery dynamics. The two studied field sites are subjected to a similar climate but harbor soil key textural and chemical differences. Briefly, both soils have acidic pH. The clayey site is more finely textured (clayey loam with 23% clay) than the sandy site (sandy loam with 10% clay) and is characterized by a higher N and C contents and lower available phosphorus, as fully described in Perron et al. . Soil texture, a well-known driver of microbial diversity , may explain the differences in microbial community structure across the two sites that already existed before the establishment of the experiment. Sandy soils exhibited less resistance but a better resilience than clayey soils to the soil tillage and compaction induced by the tree replantation process. This is not fully in line with Hartmann and colleagues , who observed less resistance and resilience in clayey than in sandy compacted soils. In their study, they exclusively focused on the effect of soil compaction, which contrasts with the subsoiling and windrowing that our studied soils underwent. We observed that the lowest resistance of sandy soils could be largely attributed to microeukaryotes. Fungi may better colonize sand-associated large pores, and recent studies have pointed out that fungal diversity was consistently promoted in coarse-textured soils . Altogether, this suggests that the particular sensitivity of sandy soils may thus be the consequence of soil tillage and successive compaction, known to disrupt the mycorrhizal network , and to particularly reduce fungal biomass . Due to its small particle size (< 2 µm), clay provides the largest surface area to bind organic material . Thus, soil organic matter associated with clay is more stabilized by chemical or physico-chemical binding to soil minerals , which could also partly explain the better resistance of the communities in the clayey site. A minor effect of treatment was observed on shaping microbial community structures and diversities in the clayey site. However, the effect of treatment varied over time for both prokaryotic and microeukaryotic communities in the sandy site, where, after 24 months, each treatment has led to new and distinct communities. It describes a study conducted by Neumann et al. , emphasizing the microbial responsiveness to organic matter inputs across different particle sizes, with a specific focus on the protective capacity of clay fractions against changes. The buffering effect of clay particles size could explain why we did not observe a clear effect of the various treatments in the clayey site. Our results suggest that differential impact of soil perturbation on the two textures might be responsible for the different resistance and resilience trajectories . However, as the amount of applied organic matter differed between the two sites and because only two sites were compared here, a proper dedicated empirical study should be conducted in order to validate such hypothesis.
The rubber tree replanting process induced a sudden and deep reassembly of the soil microbiota at both sites. Indeed, 6 months after soil perturbations, net changes in microbial community structure, taxonomy and functioning, together with losses in network connectivity, were observed. The rubber tree replanting resulted in a shift in the taxonomic composition of both prokaryotic and microeukaryotic communities in both clayey and sandy pedological contexts after 6 months. We showed that communities at 6 months were concomitant with low soil moisture, weak aggregate stabilities, and high soil NH 4 + and NO 3 − contents. Wheeling with a heavy agricultural vehicle has been shown to increase bulk density in the topsoil and to substantially reduce air permeability and gas diffusion , which can thus lead to anoxic conditions . In addition, the removal of tree vegetation and subsequent opening of the canopy might also have led to shifts in soil microbial populations by altering the feedback system between plants and soil and changing soil microclimate . Soil perturbations promoted microbial taxa with lifestyle adapted to the observed harsh conditions (i.e. reduced moisture and soil compaction) such as facultative aerobic (i.e. Nitrospirota , parasites (i.e. Dependentiae and Clavicipitaceae ) or competitive bacteria (i.e. Acidobacteria ). Soil compaction can impact microbial metabolic activities by limiting or interrupting aerobic processes, such as nitrification and mineralisation . For instance, we observed that bacterial communities at 6 months were less susceptible to performing N-related functions (i.e. nitrogen fixation, nitrification, and denitrification). The observed functional changes could be explained by soil compaction, which shifts bacterial communities towards anaerobic bacteria , that might have inhibited N fixation and nitrification, and thus denitrification by substrate provision interruption. For prokaryote specifically, diversity levels of abundant taxa have not fallen after the tree replanting process and were sometimes even higher 6 months after soil perturbation. Similar findings have been reported by Hartmann and colleagues in response to forest soils’ compaction. Authors found an increase in bacterial diversity that they attributed to taxa with low oxygen availability and capable of anaerobic respiration in compacted soils. Conversely, microeukaryotic diversity was less resistant to the tree replanting-induced disturbances at both sites. Indeed, fungi are known to be particularly sensitive to soil compaction induced by deforestation . This can partly be explained by the generally higher sensitivity of microeukaryotes to low oxygen availability induced by soil compaction and other studies pointed such differential response to compaction . In our study, observations derived from co-occurrence network modelling also support the hypothesis of less resistant microeukaryote communities to the tree replanting process. At both sites, a net loss in network connectivity was observed, and this complexity reduction was mainly imputable to a loss of 18S nodes. While fungal network sensitivity to soil labor has been previously demonstrated , this is, to our knowledge, the first study to report lower resistance of the microeukaryote network over prokaryotes to soil compaction.
We observed that cover practices had only a weak impact on the resistance (observed 6 months after perturbation) of microbial communities to the tree replanting process. This was anticipated as neither legume growth nor tree residue decomposition had really started yet. The treatment effects were noticeable later in the resilience trajectory and varied between prokaryotes and microeukaryotes, with microeukaryotes being more responsive to logging residue inputs. This particular sensitivity may be related to the high potential of mycelia to actively colonize plant residues or to the niche modification induced by organisms from the tree phyllosphere that are inoculated with tree residues. These organisms are the early colonizers and decomposers of dead plant biomass and might play a key role in microbial succession during organic matter decomposition . This latter hypothesis is supported in our study by a higher complexity in networks from treatments that include tree residues. Soil properties improvement (higher C and N contents) in rubber-based diversified systems compared to monoculture was also evidenced in the study by . Microbial communities and the ratio between fungal and bacterial biomass are strongly influenced by the C/N ratio . Organic matter inputs used in our study had distinct C/N ratios (i.e. legumes only < legume with fine residues < legume with all residues) that we expected to affect soil microbial successions and community resilience. Pueraria phaseoloides is able to recruit a specific cohort of arbuscular mycorrhizal fungi (AMF) communities , and bacterial communities are dependent on mycorrhizae for their carbon resources . One could expect that legume cover should thus be an efficient strategy for soil properties recovery. However, in this study, the treatment involving only legume cover has led to intermediate resilience trajectories. An explanation could be that hampered N metabolism due to the anoxic soil conditions brought on by soil compaction has partially interrupted microbial successions. The treatment combining fine tree residues and legume cover has led to a better (though still incomplete) recovery of the Sorensen index at both sites for microeukaryotes and in the sandy site for prokaryotes after 24 months. The high C/N ratio of woody debris is well known to benefit to the fungal biomass. Indeed, fungi are the main decomposers of tree residues because of their ability to catabolize woody complex carbon molecules (i.e. cellulose and lignin) with the nutrient-mineralizing extracellular enzymes they produce and their hyphal extension capabilities . Carbon organic matter combined with sufficient N input might have favored the hyphal production. The latter is responsible for the formation of macro-aggregates that host most microbiological activities . It is thus likely that tree residues, by indirectly improving soil structure, favored microbial activities and speed up their succession. In the sandy site specifically, the treatment combining all tree residues with legume cover harbored the highest prokaryotic and microeukaryotic α-diversity levels 24 months after perturbation. This result is surprising as C:N stoichiometry has been globally shown to have some negative effects on microbial alpha diversity . It is possible that the high microbial richness observed after 24 months hides a diversity decline in the following months. Indeed, Dossa et al. observed fungal succession in decomposing woody debris across a tropical forest. They showed a decline in fungal diversity after 18 months that they explained by increasing competition through time for remaining resources. In this context, care needs to be taken in drawing any conclusions about a beneficial effect of this specific treatment; a longer diachronic study should provide more insights.
The field site was the predominant driver of microbial recovery dynamics. The two studied field sites are subjected to a similar climate but harbor soil key textural and chemical differences. Briefly, both soils have acidic pH. The clayey site is more finely textured (clayey loam with 23% clay) than the sandy site (sandy loam with 10% clay) and is characterized by a higher N and C contents and lower available phosphorus, as fully described in Perron et al. . Soil texture, a well-known driver of microbial diversity , may explain the differences in microbial community structure across the two sites that already existed before the establishment of the experiment. Sandy soils exhibited less resistance but a better resilience than clayey soils to the soil tillage and compaction induced by the tree replantation process. This is not fully in line with Hartmann and colleagues , who observed less resistance and resilience in clayey than in sandy compacted soils. In their study, they exclusively focused on the effect of soil compaction, which contrasts with the subsoiling and windrowing that our studied soils underwent. We observed that the lowest resistance of sandy soils could be largely attributed to microeukaryotes. Fungi may better colonize sand-associated large pores, and recent studies have pointed out that fungal diversity was consistently promoted in coarse-textured soils . Altogether, this suggests that the particular sensitivity of sandy soils may thus be the consequence of soil tillage and successive compaction, known to disrupt the mycorrhizal network , and to particularly reduce fungal biomass . Due to its small particle size (< 2 µm), clay provides the largest surface area to bind organic material . Thus, soil organic matter associated with clay is more stabilized by chemical or physico-chemical binding to soil minerals , which could also partly explain the better resistance of the communities in the clayey site. A minor effect of treatment was observed on shaping microbial community structures and diversities in the clayey site. However, the effect of treatment varied over time for both prokaryotic and microeukaryotic communities in the sandy site, where, after 24 months, each treatment has led to new and distinct communities. It describes a study conducted by Neumann et al. , emphasizing the microbial responsiveness to organic matter inputs across different particle sizes, with a specific focus on the protective capacity of clay fractions against changes. The buffering effect of clay particles size could explain why we did not observe a clear effect of the various treatments in the clayey site. Our results suggest that differential impact of soil perturbation on the two textures might be responsible for the different resistance and resilience trajectories . However, as the amount of applied organic matter differed between the two sites and because only two sites were compared here, a proper dedicated empirical study should be conducted in order to validate such hypothesis.
Our diachronic monitoring emphasized a predominant role of the pedologic context in shaping the soil microbiome responses to rubber trees clear-cutting. The impact of tree-logging on soil communities was much more pronounced in the sandy site, particularly for microeukaryotes. The significant importance of soil texture in microbial response dynamics draws attention to the need to consider soil pedological context when designing agroecological practices that sustain soil functioning. The practice involving a combination of logging residues and legume cover in rubber plantation has shown certain beneficial effects on the resilience of microbial communities after tree-logging and associated soil perturbation in the sandy site. It thus appears as a promising agroecological practice that should be further studied. Despite the statistical power of our sampling design not allowing a deep description of the effect of treatments across time on microbial networks, cross-domain co-occurrence network modelling provided useful insights into the soil microbiome resistance and resilience. It not only supported the trend observed with alpha and beta diversities, but also highlighted the key role of microeukaryotes in the resilience dynamic. We argue that this tool should be more widely used to describe microbial community dynamics, and that microbial ecologists should thus anticipate the number of replicates required for its efficient use.
Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 1666 KB) Supplementary file2 (DOCX 83 KB)
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Molecular Imaging in Gynecology: Beyond Cancer | bf021ecd-1607-4bff-8887-bf3425b14183 | 11218728 | Gynaecology[mh] | Endometriosis is a chronic female reproductive disorder characterized by the growth of endometrial tissue outside the uterus . Most endometriosis lesions grow on the outer perimetrium, fallopian tubes, and ovaries. However, in some cases—known as deep infiltrating endometriosis—endometrial tissue may grow on the bowels, rectum, and bladder or even within the thoracic cavity . Approximately 10%–15% of all women of reproductive age have the condition, but it is most common in women with infertility (25%–50%) and chronic pelvic pain (70%–85%) . The most common symptoms include chronic pelvic pain, acute pelvic pain, and infertility, but women with endometriosis are susceptible to a wide variety of other comorbidities, including cancer, lupus, rheumatoid arthritis, and cardiovascular disease . The societal and economic costs of endometriosis are alarming: those afflicted experience stigma, diminished quality of life, and reduced work productivity, and its management is responsible for about $70 billion in yearly health expenditures in the United States alone . The current standard of care for the diagnosis of endometriosis is exploratory laparoscopy . This approach is invasive, painful, expensive, and prone to sampling and operator biases, factors that have combined to create long diagnostic delays for the condition . Anatomic imaging also plays an important role in the care of patients with the condition. Ultrasonography is frequently used as a prediagnostic tool to visualize lesions before surgical diagnosis and resection, but it can exhibit suboptimal sensitivity and accuracy in the context of smaller lesions . MRI has also been used to determine the size, location, and infiltrative stage of large lesions before surgical resection, especially in cases of deep infiltrating endometriosis . Unfortunately, MRI has proven suboptimal for the detection of small superficial lesions (i.e., <5 mm beneath the surface of the peritoneum) . In light of the clear limitations of extant methods for the detection of endometriosis, researchers have begun to explore the possibility of using nuclear imaging—and PET in particular—for the visualization of endometriosis. Silveira et al., for example, used a rat model of the disease to determine the uptake of [ 18 F]fluorocholine in superficial lesions. Although the activity concentration of the tracer in implanted endometriosis tissue was found to be 3-fold higher than that in the muscle and peritoneum, the biodistribution data revealed very high uptake in several healthy tissues, most notably (and problematically) the ovaries . More recently, Amartuvshin et al. probed the value of a 64 Cu-labeled variant of the vascular endothelial growth factor–targeting antibody bevacizumab for the delineation of subcutaneous endometriosis lesions. Unfortunately, in vivo experiments revealed low activity concentrations in the target tissue and high levels of uptake in healthy tissues . Shifting to the clinic, a handful of trials have focused on the potential of a trio of commonly used PET tracers—[ 18 F]FDG, 16α-[ 18 F]fluoro-17β-estradiol ([ 18 F]FES), and [ 68 Ga]Ga-DOTATATE—for the detection of endometriosis. Balogova et al., for example, performed [ 18 F]FDG PET/CT on 18 patients with known or suspected endometriosis and found that the radiotracer effectively delineated only 11 of 20 (55%) confirmed lesions . More recently, a team at the Université Libre de Bruxelles in Belgium performed [ 68 Ga]Ga-DOTATATE PET/CT on a cohort of patients ( n = 12) scheduled for exploratory laparoscopy for suspected endometriosis. Although the authors ultimately concluded that the radiotracer was effective for the visualization of deep infiltrating endometriosis (i.e., the sensitivity and specificity were 57% and 80%, respectively), no uptake was observed in superficial peritoneal endometriosis or ovarian endometriomas . Without question, the most promising clinical results have been obtained with [ 18 F]FES. In 2016, Cosma et al. performed [ 18 F]FES PET on a group of patients ( n = 4) with suspected endometriosis who also underwent MRI and laparoscopic excision coupled with histology. The data demonstrated that [ 18 F]FES PET/CT fully agreed with histology and showed greater accuracy than MRI for lesions: PET/CT correctly identified 9 of 9 lesions, whereas MRI produced 3 false negatives and 3 false positives . Molecular imaging could also play a key role in surgeries for endometriosis. Indeed, intraoperative imaging tools capable of visualizing endometriosis lesions could reduce operator bias during both laparoscopic diagnoses and surgical resections . Along these lines, a 2021 study by Al-Taher et al. compared intraoperative near-infrared fluorescence imaging with indocyanine green and conventional white-light laparoscopy for the delineation of endometriosis lesions in a cohort of 15 patients . The investigators determined that the positive predictive values for near-infrared fluorescence and white-light imaging were 69% and 64%, respectively, suggesting that the former could have modest value in the context of surgical resections. Interestingly, 2 other recent studies have sought to use intraoperative imaging to illuminate healthy tissues and thus differentiate endometriosis lesions. Aleksandrov et al. used indocyanine green to identify healthy rectum tissue when removing deep infiltrating endometriosis from the bowels , whereas Thigpen et al. used indocyanine green to differentiate between the ureters and endometriosis lesions during surgery . In both cases, the authors concluded that intraoperative near-infrared fluorescence imaging aided in the safe resection of the diseased tissue while reducing complications; however, the approximate location of the lesions still needs to be known before surgery. Taken together, the PET and near-infrared fluorescence studies described in this section clearly suggest that molecular imaging could play important roles in the diagnosis and treatment of endometriosis. However, it is impossible to deny that the results have been middling at best, a problem that we suspect is related to the reuse of established imaging agents (i.e., [ 18 F]FDG, [ 18 F]fluorocholine, and indocyanine green). It is likely that novel probes with specificity for endometrial tissue will offer the best chance for the development of clinically effective tools. Adenomyosis is a chronic disease similar to endometriosis that is characterized by the invasion and growth of endometrial tissue within the myometrium, the muscular middle layer of the uterus. Adenomyosis, although asymptomatic in almost a third of patients, can cause pelvic pain, infertility, and heavy menstrual bleeding . Ultrasonography and MRI are the primary diagnostic tools for the condition, but these modalities exhibit suboptimal accuracy, especially when adenomyosis is not suspected . For example, pelvic ultrasound has a sensitivity of 10.9% and a specificity of 98.3%. MRI fares slightly better, with a sensitivity of 29.7% and a specificity of 85.3%, numbers that improve slightly (though not overwhelmingly) during direct evaluations for the condition . New approaches to the diagnostic imaging of adenomyosis are thus needed, but there have been only a handful of reports on the use of nuclear imaging for the condition. And indeed, most of these have described the incidental uptake of radiopharmaceuticals in lesions. For example, a pair of reports that focused on cervical cancer and carcinomatosis has found that adenomyosis lesions can be [ 18 F]FDG-avid, complicating the identification of intrauterine metastases via [ 18 F]FDG PET . Furthermore, the aforementioned study that focused on the delineation of endometriosis with [ 68 Ga]Ga-DOTATATE also found that adenomyosis lesions exhibited increased accretion of the radiolabeled peptide as well . Finally, a single case report by Wu et al. from 2014 described the visualization of pyoadenomyosis—a rare complication of adenomyosis—via SPECT with [ 67 Ga]Ga 3+ . In sum, the data clearly underscore that the stage is set for new work in this area, particularly the development of dedicated probes specific for adenomyosis. Uterine fibroids, or leiomyomas, are benign clonal neoplasms of the uterus that occur in about 70% of women and disproportionately affect Black women . Though typically asymptomatic, these smooth muscle tumors can cause a wide range of symptoms—including pelvic pain, abnormal menstrual bleeding, and urinary and gastrointestinal issues—and can be associated with infertility and other adverse pregnancy outcomes . A variety of approaches has been used for the treatment of uterine fibroids, including myomectomy, radiofrequency ablation, uterine fibroid embolization, and hormonal birth control. Hysterectomy also remains a common treatment for the condition, with fibroids accounting for over one third of such procedures every year . Uterine fibroids are typically identified via ultrasonography, with postoperative histology providing a definitive diagnosis. Transvaginal sonography is inexpensive and exhibits high sensitivity (99%) and specificity (91%) that can be improved on even further with the addition of saline infusion sonohysterography for fibroids touching the uterine lining . MRI can also be used for diagnosis and offers sensitivity and specificity close to 100%, though it is significantly more expensive. That said, MRI can be useful in patients who are unsuitable for transvaginal sonography or saline infusion sonohysterography or who need preoperative mapping for myomectomy . Existing methods for the detection of uterine fibroids are clearly sufficient. However, clinical studies on the visualization of leiomyomas with extant and emerging PET tracers are critical, especially in the context of differentiating between fibroids and malignant lesions. For example, a handful of studies has combined to show that leiomyomas of younger and premenopausal women can be especially [ 18 F]FDG-avid, creating the possibility that these fibroids could be misidentified as leiomyosarcomas or endometrial carcinomas . A pair of clinical trials from Japan suggests that [ 18 F]FES and [ 18 F]FDG could play complementary roles in the differentiation of leiomyomas from endometrial carcinomas, leiomyosarcomas, and endometrial hyperplasia . More specifically, a malignant leiomyosarcoma showed positive [ 18 F]FDG accumulation (SUV, 10.5) and negative [ 18 F]FES accumulation (SUV, 1.0) in PET images, whereas a benign leiomyoma showed similarly positive uptake for both PET tracers . Finally, incidental uptake in uterine fibroids has been observed with several other PET tracers—i.e., [ 68 Ga]Ga-DOTATATE, Al[ 18 F]F-NOTA-FAPI, and [ 18 F]NaF—though more comprehensive follow-up studies have yet to be performed . In the end, it is unlikely that molecular imaging will play a key standalone role in the detection of uterine fibroids given the efficacy of existing diagnostic methods. That said, imaging—and PET in particular—could be a valuable tool for differentiating between benign fibroids and more dangerous leiomyosarcomas. More broadly, as the use of a library of radiopharmaceuticals applied in the clinic grows, it will be important to understand the uptake of common radiopharmaceuticals in leiomyomas to prevent false-positive cancer diagnoses. PCOS is an endocrine disorder that affects roughly 5%–15% of reproductive-aged women and can cause excess body hair, acne, weight gain, and infertility. Although the condition can be managed via hormone treatments and weight-loss drugs (as diabetes and insulin resistance are common comorbidities), accurate diagnoses are critical since many of these symptoms can be associated with other etiologies . Along these lines, PCOS is diagnosed when a patient satisfies 2 of the following 3 criteria: oligomenorrhea or anovulation, often leading to irregular periods; clinical or biochemical signs of hyperandrogenism; and polycystic ovaries on ultrasound. In light of this largely effective diagnostic paradigm, molecular imaging likely has a greater future role to play in understanding the condition rather than delineating it. Several studies have sought to use [ 18 F]FDG to image brown adipose tissue levels in women with PCOS, as brown adipose tissue is a highly energetic tissue that is often functionally abnormal in patients with PCOS. In a 2019 trial, for example, [ 18 F]FDG PET revealed that women with PCOS exhibited lower brown adipose tissue levels than those without the condition . A year later, similar results were obtained in a [ 18 F]FDG PET study that compared a cohort of women receiving metformin for PCOS and those without the condition, cementing low levels of brown adipose tissue as a common characteristic of PCOS and opening the door for [ 18 F]FDG as a tool for monitoring the treatment of the condition . Interestingly, this was not the first attempt at using nuclear imaging in the service of treating PCOS. In 2010, Priyadarshani et al. determined the biodistribution of a 99m Tc-labeled variant of noscapine in a rat model of PCOS, as the alkaloid was (at the time) being explored as a possible therapeutic for the condition . The results were reasonably promising—the 99m Tc-labeled noscapine produced 0.9 %ID/g in ovarian cysts compared with 0.06 %ID/g for a nonspecific control compound—but no follow-up studies have been published. In the future, it is unlikely that a nuclear imaging agent will be needed as a diagnostic tool for PCOS, but it is almost certain that both established tracers (e.g., [ 18 F]FDG) and novel cyst-specific probes could play important roles in improving our understanding of the disease. Adnexal masses are growths found around the ovaries, uterus, or fallopian tubes, including functional ovarian cysts, endometriomas, and teratomas. The accurate detection and identification of these lesions are critical, as they can interfere with cancer diagnoses and, in rare cases, become malignant themselves . Although most are asymptomatic, adnexal masses can cause pelvic pain on cyst enlargement, cyst rupture, or ovarian torsion . For patients who do experience symptomatic pain, diagnosis is typically performed via ultrasonography—most efficiently when performed by a sonographer using subjective pattern recognition—but other imaging modalities (e.g., pelvic MRI) may be leveraged for complementary information. Functional ovarian cysts are a class of adnexal masses that can develop on the ovaries, including follicular, corpus luteum, and theca lutein cysts. The most common approach to diagnosing these cysts is via transvaginal ultrasonography, an approach that offers a sensitivity of 93.5% and a specificity of 91.5% . Although these values are high, clinicians have nonetheless looked toward molecular imaging for complementary diagnostic tools. In a 2021 metaanalysis of 27 reports on the differentiation between benign and malignant adnexal masses via [ 18 F]FDG PET and MRI, the average sensitivity and specificity were 94% and 86%, respectively, for [ 18 F]FDG PET and 92% and 85%, respectively, for MRI . In a trial performed in Japan, an emergent type of MRI—amide proton transfer MRI—proved quite useful for the detection and classification of a variety of ovarian cysts. Indeed, amide proton transfer MRI provided correct diagnoses of each type of ovarian cyst, including serous cystadenomas, mucinous cystadenomas, and functional cysts, without additional follow-up studies . MRI has also been found effective for identifying other benign adnexal masses, such as teratomas. In a 2015 study of 26 patients, for example, MRI proved superior to [ 18 F]FDG PET for distinguishing between benign (mature) and malignant (immature) teratomas . A previous study with [ 67 Ga]Ga SPECT provided similar results . The dense and fibrous nature of Brenner tumors—an ovarian lesion that can be benign or malignant—facilitates ready visualization via MRI, making it the current diagnostic gold standard . There may be room for nuclear imaging, however. To wit, one recent study suggests that malignant Brenner tumors may exhibit higher uptake of [ 18 F]FDG than their benign cousins, aiding in the differentiation between the two . In assembling this review, we were taken aback (though not entirely surprised) by the degree to which benign gynecological pathologies remain understudied despite their global prevalence . We are heartened, however, to see that molecular imaging research in this area has increased over the last decade, as we believe that molecular imaging has the potential to improve patient outcomes. In conditions for which current diagnostic paradigms are inadequate—such as endometriosis and adenomyosis—molecular imaging could become a pivotal diagnostic tool. Yet molecular imaging could still have a part to play in cases for which standard-of-care diagnostic technologies are sufficient. In PCOS, for example, molecular imaging could help us better understand the syndrome’s diverse constellation of symptoms, whereas in the context of benign adnexal masses, it could be deployed to better differentiate between benign and malignant lesions. Going forward, we are eager to see what the next decade has in store for research at the intersection of these 2 areas. Although there are a plethora of viable paths for work, we believe that the development of novel imaging probes that specifically target these conditions will be especially critical. In the end, details aside, we are simply hopeful that advances in our field can help the millions of women with these conditions. No potential conflict of interest relevant to this article was reported. |
Human skeletal muscle fiber heterogeneity beyond myosin heavy chains | bd66304e-4c3a-4752-bf42-f582531dca15 | 11839989 | Biochemistry[mh] | Cellular heterogeneity is an inherent feature of all biological systems, allowing for cellular specialization to meet the diverse demands imposed upon tissues and cells . The classic view of skeletal muscle fiber heterogeneity is that motoneurons define the typology of the fibers within a motor unit, with fiber types (i.e., type 1, type 2A, and type 2X in humans) being defined by myosin heavy chain isoform (MYH) characteristics . This was first based on its ATPase pH lability , and later by its molecular MYH expression . However, skeletal muscle fibers are increasingly being viewed along a continuum, as opposed to discrete fiber types , fueled by the identification and subsequent acceptance of the existence of “hybrid” fibers that express varying proportions of multiple MYHs simultaneously. Nonetheless, the field still relies heavily on MYHs as primary classifiers of muscle fibers, a perspective that might be limited and heavily biased by early studies in rodents, displaying a different MYH expression profile and thus fiber type range than humans . The picture is further complicated by different skeletal muscles displaying specialized fiber type profiles within humans . The vastus lateralis is a mixed muscle type with average (and therefore representative) MYH expression profiles . Together with this, its accessibility for sampling makes it the most commonly studied muscle in humans. An unbiased exploration of skeletal muscle fiber diversity with powerful omics tools is thus heavily warranted, but challenging, in part owing to the multinucleated nature of skeletal muscle fibers. Nonetheless, both transcriptomics , and proteomics technologies have recently experienced a sensitivity revolution due to various technological advances, making profiling of skeletal muscle at a single fiber resolution now possible. As a result, notable progress has already been made in describing the single muscle fiber diversity and responses to atrophic stimuli and aging – . Importantly, these technological advances lend themselves to being advantageous in the clinical setting, helping to describe in greater detail and precision the dysregulation associated with disease. For example, the underlying pathophysiology of nemaline myopathy, one of the most prevalent genetic muscle disorders (MIM 605355 and MIM 161800), is complex and convoluted , , thus a deeper characterization of skeletal muscle fiber dysregulation may spur significant developments in our understanding of the disease. Our methodological development of transcriptome and proteome profiling of single skeletal muscle fibers manually isolated from human biopsy specimens, and their application to over a thousand fibers each, enables us to investigate the cellular heterogeneity of human skeletal muscle fibers. In doing so, we demonstrate the power of muscle fiber phenotyping at the transcriptomic and proteomic level, identifying that metabolic, ribosomal, and cell junction proteins are important sources of variation among muscle fibers. Furthermore, with our proteomics workflow we characterize the clinical implications of nemaline myopathies within single skeletal muscle fibers, revealing a coordinated shift towards non-oxidative fibers independently of MYH-based fiber type. Development of high-sensitivity and high-throughput single muscle fiber transcriptomic and proteomic pipelines To investigate the heterogeneity of human skeletal muscle fibers, we developed two workflows to enable transcriptome and proteome profiling of single skeletal muscle fibers (Figs. and Supplementary fig. ). Several methodological steps were developed and optimized, from sample storage and preservation of RNA and protein integrity to optimizing the throughput of each method. This was achieved for transcriptome analysis by inserting sample-specific molecular barcodes during the initial reverse transcription step, enabling pooling of 96 fibers for further efficient downstream processing. Rich transcriptome data was further obtained by deeper sequencing ( ± 1 M reads per fiber) compared to conventional single cell methods . For proteomics, we used a short chromatographic gradient (21 minutes) combined with DIA-PASEF data aquisition on timsTOF mass spectrometer to optimize proteome depth, whilst maintaining a high-throughput , . To investigate skeletal muscle fiber heterogeneity in the healthy state, the transcriptome was determined for 1050 individual fibers from 14 healthy adult donors, whilst the proteome was determined for 1038 fibers from 5 healthy adult donors (Supplementary Table ). These datasets will be referred to as the 1000 fiber transcriptome and proteome datasets, respectively, throughout this manuscript. Our approach detected a total of 27237 transcripts and 2983 proteins in the 1000 fiber transcriptomics and proteomics studies (Figs. , Supplementary Dataset – ). After filtering for > 1000 detected genes and for 50% valid values within each fiber in both transcriptomics and proteomics datasets, respectively, downstream bioinformatic analyses were performed on 925 and 974 fibers in the transcriptome and proteome, respectively. On average 4257 ± 1557 genes and 2015 ± 234 proteins (mean ± SD) were detected per fiber after filtering, with limited inter-individual variation (Supplementary figs. , Supplementary Dataset - ). The intra-individual variation within a participant was more substantial however, most likely due to differences in RNA/protein yield among fibers of different length and cross-sectional area. For the majority of proteins ( > 2000), the coefficient of variation was below 20% (Supplementary fig. ). Both methodologies captured a wide dynamic range of transcripts and proteins, with features known to be important for muscle contraction being highly expressed (e.g., ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Supplementary figs. ). A large part of the detected features were shared between the transcriptome and proteome datasets (Supplementary fig. ), alongside a reasonable correlation ( r = 0.52) in average UMI counts/LFQ intensities for these features (Supplementary fig. ). Type 2X is not a distinct fiber type We initially set out to define the MYH-based fiber type of each fiber using an optimized methodology, leveraging the high sensitivity and dynamic range of MYH expression in the omics datasets. Previous studies have used arbitrary cut-offs to assign a fiber as pure type 1, type 2A, type 2X, or hybrid, based on a fixed percentage of expression for the different MYHs , , . We employed a different approach, in which we ranked the expression of each fiber by the MYHs used for fiber typing: MYH7, MYH2 and MYH1, corresponding to type 1, type 2A and type 2X fibers, respectively. We then mathematically calculated the bottom knee for each of the resulting curves and used it as a threshold to assign a fiber as being positive (above threshold) or negative (below threshold) for each MYH (Figs. ). These data show that MYH7 (Fig. ) and MYH2 (Fig. ) have a more pronounced on/off expression profile on the RNA level, compared to the protein level. Indeed, at the protein level, very few fibers did not express MYH7 and no fibers had 100% MYH2 expression. Next, we used the determined expression thresholds to assign MYH-based fiber types for all fibers in each dataset. For example, a MYH7 + /MYH2 - /MYH1 - was assigned as type 1, and a MYH7 - /MYH2 + /MYH1 + was assigned as a hybrid type 2A/2X fiber (see Supplementary Table for full description). When combining all fibers, a very similar MYH-based fiber type distribution was observed at the RNA (Fig. ) and protein (Fig. ) levels, with an expected inter-individual variation in relative MYH-based fiber type composition (Supplementary fig. ). Most fibers were considered pure type 1 (34–35%) or type 2A (36–38%), although a substantial number of hybrid 2A/2X fibers (16–19%) were also detected. A striking discrepancy was that pure type 2X fibers could only be detected at the RNA but not at the protein level, indicating that fast MYH expression is, at least in part, post-transcriptionally regulated. We validated the MYH-based fiber typing from the proteomics data against an antibody-based dot blot technique, in which both methodologies were in 100% agreement on the identification of both pure type 1 and type 2A fibers (Supplementary fig. ). However, the increased sensitivity of the proteomics-based approach was superior in identifying hybrid fibers and in quantifying the proportion of each MYH within each fiber. These data demonstrate the efficacy of using an unbiased high-sensitivity omics-based approach for the characterization of skeletal muscle fiber types. We then utilized the full depth of information that transcriptomics and proteomics provide to classify fibers in an unbiased manner based on their whole transcriptome or proteome. Using uniform manifold approximation and projection (UMAP) for dimension reduction of 6 principal components (Supplementary figs. ), we were able to visualize the variation in fibers in the transcriptome (Fig. ) and proteome (Fig. ). Interestingly, fibers did not cluster by participant in either the transcriptomics or proteomics datasets (Supplementary figs. ), nor by test day (Supplementary fig. ), indicating that intra-individual variance in skeletal muscle fibers outweighs inter-individual variance. Two distinct clusters were apparent in the UMAP plots, which represented “fast” and “slow” fibers (Fig. ). MYH7 + (slow) fibers clustered to the positive side of UMAP1, and MYH2 + and MYH1 + (fast) fibers clustered to the negative side of UMAP1 (Fig. ). No distinction between the various fast MYH-based fiber types (i.e., type 2A, type 2X, or hybrid 2A/2X) could be identified, however, suggesting that when the whole transcriptome or proteome is taken into account, the expression of MYH1 (Fig. ), or other classical markers of type 2X fibers like ACTN3 or MYLK2 (Supplementary figs. ), does not discriminate between distinct fiber types. Furthermore, in contrast to MYH2 and MYH7, few transcripts or proteins positively correlate with MYH1 (Supplementary figs. ), suggesting that MYH1 abundance does not adequately reflect the muscle fiber transcriptome/proteome. Similar conclusions can be drawn when assessing the blended expression of the three MYH isoforms on a UMAP level (Supplementary figs. ). Thus, whilst type 2X fibers can be identified at the transcriptional level based solely on the quantification of MYHs, MYH1 + fibers are not distinct from other fast fibers when the whole transcriptome or proteome is considered. Considerable skeletal muscle fiber heterogeneity beyond myosin heavy chain isoforms As an initial exploration of fiber heterogeneity beyond MYHs, we assessed four established slow fiber type-specific proteins: TPM3, TNNT1, MYL3, and ATP2A2 . In both transcriptomics (Supplementary fig. ) and proteomics (Supplementary fig. ) approaches, the slow isoforms exhibited a high, although not perfect, Pearson correlation coefficient with MYH7. ~25 and 33% of the fibers in the transcriptomics (Supplementary fig. ) and proteomics (Supplementary fig. ) approaches, respectively, were not classified as pure slow fibers by all gene/protein isoforms. Thus, fiber typing based on multiple gene/protein isoforms introduces additional complexity, even with well-known proteins that are assumed to be fiber type-specific. This suggests that fiber typing based on isoforms of a single family of genes/proteins is likely inadequate to capture the true heterogeneity of skeletal muscle fibers. To further investigate the omics-wide phenotypical variability between human skeletal muscle fibers, we applied unbiased dimensionality reduction by principal component analysis (PCA) to our data (Fig. ). Similarly to the UMAP plot, neither participant nor test day influenced the clustering of fibers at the PCA level (Supplementary figs. ). MYH-based fiber type was explained by PC2 in both datasets, displaying clusters for slow type 1 fibers and a second cluster containing the fast type 2A, type 2X and hybrid type 2A/2X fibers (Fig. ). These two clusters were bridged in both datasets by a small number of hybrid type 1/2A fibers. As expected, over-representation analysis of the top PC drivers confirmed that PC2 is driven by contractile and metabolic features (Fig. & Supplementary fig. , Supplementary Dataset - ). In general, the MYH-based fiber types adequately explain the continual variance along PC2, except for the so-called type 2X fibers, which were spread across the entirety of the transcriptomic fast cluster. Unexpectedly, MYH-based fiber type explained only the second greatest degree of variability (PC2), indicating that other biological factors (PC1) independent of MYH-based fiber type, have a substantial role in regulating skeletal muscle fiber heterogeneity. Over-representation analysis of the top drivers in PC1 indicated that variance within PC1 was determined primarily by cell-cell adhesion and ribosomal content in the transcriptome, and costamere and ribosomal proteins in the proteome (Figs. & Supplementary fig. , Supplementary Dataset ). In skeletal muscle, the costamere connects the Z-disk to the sarcolemma and participates in force transmission and signaling . Annotating PCA plots with cell-cell adhesion (transcriptome, Fig. ) and costamere (proteome, Fig. ) features showed a strong shift to the left side of PC1, suggesting an enrichment for these features in some fibers. Closer inspection of fiber clustering at the UMAP level showed a MYH-based fiber type-independent gradient of expression for most features, rather than distinct subclusters of muscle fibers. This continuum holds true for several genes related to pathological conditions (Fig. ), such as CHCHD10 (neuromuscular disorders) , SLIT3 (muscle loss) and CTDNEP1 (muscle disease) . This same continuum was observed in the proteome, with proteins related to neurological diseases (UGDH) , insulin signaling (PHIP) and transcription (HIST1H2AB) (Fig. ). These data collectively show that slow/fast fiber type independent heterogeneity across fibers is of a continual nature. Fiber heterogeneity is post-transcriptionally regulated by ribosomal heterogeneity Interestingly, drivers of PC2 showed a good correlation ( r = 0.663) between the transcriptome and proteome (Fig. ), indicating that the slow and fast fiber types, particularly the contractile and the metabolic profile of skeletal muscle fibers, are transcriptionally regulated. However, there was no correlation between the drivers of PC1 in the transcriptome and the proteome (r = -0.027) (Fig. ). This suggests that slow/fast fiber type-independent variance is for a large part post-transcriptionally regulated. Since PC1 variance was largely explained by ribosomal gene ontology terms and given that ribosomes play a profound and specialized role in the cell by actively participating and influencing protein translation , we next set out to explore this unexpected ribosomal heterogeneity. We first colored the proteomics PCA plot based on the relative abundance of the proteins within the “cytosolic ribosome” GOCC term (Fig. ). Although the term was enriched on the positive side of PC1, and a slight gradient could be observed accordingly, ribosomal proteins were driving separation in both directions of PC1 (Fig. ). Amongst the ribosomal proteins enriched in the negative direction of PC1 were RPL18, RPS18 and RPS13 (Fig. ), whilst RPL31, RPL35 and RPL38 (Fig. ) were major drivers in the positive direction of PC1. Interestingly, RPL38 and RPS13 are highly expressed in skeletal muscle when compared to other tissues (Supplementary fig. ). These distinct ribosomal signatures across PC1 could not be observed in the transcriptome (Supplementary fig. ), indicating that this phenomenon is post-transcriptionally regulated. The concept of ribosomal heterogeneity and specialization was previously introduced, where the presence of distinct subpopulations of ribosomes (ribosomal heterogeneity) can directly influence protein translation in different tissues and cells by selectively translating specific mRNA transcript pools (ribosomal specialization). To identify sub-sets of ribosomal proteins that are co-expressed within skeletal muscle fibers, we performed an unsupervised hierarchical clustering analysis of ribosomal proteins within the proteome (Figs. , Supplementary Dataset ). As expected, ribosomal proteins did not cluster by MYH-based fiber type. However, we identified three distinct clusters of ribosomal proteins; the first of which (ribosomal_cluster_1) was coregulated alongside RPL38, therefore elevated in fibers in the positive direction of PC1. The second cluster (ribosomal_cluster_2) was coregulated alongside RPS13 and was elevated in fibers in the negative direction of PC1. A third cluster (ribosomal_cluster_3) displayed no coordinated differential expression within skeletal muscle fibers and could therefore be considered “core” ribosomal proteins within skeletal muscle. Both ribosomal_cluster_1 and ribosomal_cluster_2 contain ribosomal proteins previously demonstrated to regulate selective translation (e.g., RPL10A, RPL38, RPS19 and RPS25) and functionally influence development (e.g., RPL10A, RPL38) – . In line with the results from the PCA analysis, the observed heterogeneous abundance of these ribosomal proteins across fibers was also of a continual nature (Supplementary fig. ). To visualize the position of the ribosomal proteins that display heterogeneity within the ribosome, we utilized a structural model of the human 80S ribosome (Protein Data Bank: 4V6X) (Fig. ). Upon highlighting the ribosomal proteins that belong to the different ribosomal clusters, they were not located in close proximity, showing that our approach did not enrich for a particular area/section of the ribosome. Yet interestingly, ribosomal_cluster_2 contained a lower proportion of large ribosomal subunit proteins than ribosomal_cluster_1 and ribosomal_cluster_3 (Supplementary fig. ). We observed that the majority of the proteins that display variable stoichiometry within skeletal muscle fibers are located on the surface of the ribosome (Fig. ), which is consistent with an ability to interact with internal ribosome entry site (IRES) elements within distinct mRNA populations to coordinate selective translation , . Furthermore, numerous proteins that display variable stoichiometry within skeletal muscle fibers are located close to functional regions, such as the mRNA exit tunnel (Fig. ), which can selectively regulate translation elongation and arrest of specific nascent peptides . Overall, our data identifies that skeletal muscle ribosomal protein stoichiometry displays heterogeneity, driving variance between skeletal muscle fibers. Slow and fast fiber signatures and their transcriptional regulators We next set out to identify features of fast and slow skeletal muscle fibers and how these are transcriptionally regulated. Comparing the fast and slow clusters defined in the UMAPs of both datasets (Figs. & inlays Fig. ), transcriptome and proteome analysis yielded 1366 and 804 differentially abundant features, respectively (Figs. , Supplementary Dataset – ). Expected differences in sarcomeric (e.g., tropomyosin and troponin), excitation-contraction coupling (SERCA isoforms) and energy metabolism-related (e.g., ALDOA and CKB) features were observed. In addition, transcripts and proteins regulating protein ubiquitination displayed differences between fast and slow fibers (e.g., USP54, SH3RF2 , USP28 and USP48) (Fig. ). Furthermore, the microprotein-encoding gene RP11-451G4.2 (DWORF) , which has previously been shown to be differentially expressed between lamb muscle fiber types and to enhance SERCA activity in cardiac muscle , was significantly up-regulated in slow skeletal muscle fibers (Fig. ). Also at the single fiber level, clear differences could be observed for known features such as the metabolism-related isoforms of lactate dehydrogenase (LDHA and LDHB, Figs. and Supplementary fig. ) , , as well as previously unknown fiber type-specific features (e.g., IRX3 , USP54 , USP28, and DPYSL3) (Fig. ). There was a reasonable overlap of differentially expressed features (Supplementary fig. ) and correlation of fold changes between the transcriptomic and proteomic datasets, primarily driven by the large expression differences in sarcomeric features (Supplementary fig. ). Notably, some features (e.g., USP28, USP48, GOLGA4, AKAP13) showed strong post-transcriptional regulation with slow/fast fiber type-specific expression profiles only at the proteome level (Supplementary fig. ). We next performed over-representation analysis on the differentially abundant genes and proteins (Figs. , Supplementary Dataset ). Enriched pathways of features that were differential in both datasets showed expected differences such as fatty acid beta-oxidation and ketone metabolic process (slow fibers), myofilament/muscle contraction (fast and slow fibers, respectively) and carbohydrate catabolic process (fast fibers). Serine/Threonine protein phosphatase activity was also enriched in fast fibers, driven by features such as phosphatase regulatory and catalytic subunits (PPP3CB, PPP1R3D, and PPP1R3A), known to regulate glycogen metabolism (Supplementary figs. ). Other enriched pathways in fast fibers were Processing (P-) bodies (YTHDF3, TRIM21, LSM2) in the proteome (Supplementary fig. ), possibly related to post-transcriptional regulation , and transcription factor activity ( SREBF1 , RXRG , RORA ) in the transcriptome (Supplementary fig. ). Slow fibers showed enrichment for oxidoreductase activity ( BDH1 , DCXR , TXN2 ) (Supplementary fig. ), amide binding ( CPTP , PFDN2 , CRYAB ) (Supplementary fig. ), extracellular matrix ( CTSD , ADAMTSL4 , LAMC1 ) (Supplementary fig. ) and receptor-ligand activity ( FNDC5 , SPX , NENF ) (Supplementary fig. ). To get more insights into the transcriptional regulation of slow/fast fiber type signatures, we performed transcription factor enrichment analysis using SCENIC (Supplementary Dataset ). Many transcription factors were significantly enriched between fast and slow fibers (Fig. ). This included transcription factors such as MAFA , previously linked to the development of fast fibers , but also multiple transcription factors not previously linked to the fiber type specific gene program. These included PITX1 , EGR1 and MYF6 as the most enriched transcription factors within fast fibers (Fig. ). Conversely, ZSCAN30 and EPAS1 (also known as HIF2A ) were the most enriched transcription factors within slow fibers (Fig. ). In line with this, MAFA expression levels were higher in the UMAP area corresponding to fast muscle fibers, whereas an opposite expression pattern was observed for EPAS1 (Fig. ). Non-coding RNA and associated microproteins in human skeletal muscle fibers Alongside known protein-coding genes, there exists a multitude of non-coding RNA biotypes, potentially involved in the regulation of human development and disease , . Several non-coding RNAs displayed fiber type specificity in the transcriptomics dataset (Figs. & Supplementary Dataset ), including LINC01405 , which is very specific to slow fibers and is reported to be downregulated in muscle of mitochondrial myopathy patients . Conversely, RP11-255P5.3 , corresponding to the lnc-ERCC5-5 gene ( https://lncipedia.org/db/transcript/lnc-ERCC5-5:2 ) displayed fast fiber type-specificity. Both LINC01405 ( https://tinyurl.com/x5k9wj3h ) and RP11-255P5.3 ( https://tinyurl.com/29jmzder ) display specificity to skeletal muscle (Supplementary figs. ) and have very few or no known contractile genes within a 1 Mb genomic neighborhood, suggesting a specialized role in fiber type regulation rather than a regulatory role for a neighboring contractile gene. The slow/fast fiber type-specific specific expression profiles of LINC01405 and RP11-255P5.3 were independently validated using RNAscope (Fig. ). Recently, it has become apparent that numerous assumed non-coding transcripts code for transcribed microproteins, some of which regulate muscle function , . To identify microproteins with potential fiber type specificity, we searched our 1000 fiber proteomic dataset using a custom FASTA file containing sequences from the detected non-coding transcripts ( n = 305) from the 1000 fiber transcriptome dataset (Fig. ). This resulted in the identification of 197 microproteins arising from 22 distinct transcripts, of which 71 microproteins display differential regulation between slow and fast skeletal muscle fibers (Supplementary figs. and Supplementary Dataset ). Three microprotein products were identified for LINC01405 , of which one displays a slow-fiber type specificity similar to its transcript (Figs. and Supplementary fig. ). Thus, we identify LINC01405 as a microprotein-encoding gene, which displays specificity for slow skeletal muscle fibers. Nemaline myopathies induce a shift towards faster, less oxidative skeletal muscle fibers Having developed a comprehensive workflow to characterize the proteome of single muscle fibers at scale and discovered regulators of fiber heterogeneity in the healthy state, we applied this pipeline to understand how nemaline myopathy impacts skeletal muscle fiber heterogeneity. Nemaline myopathy is a genetic muscular disorder that causes muscle weakness, resulting in a range of complications for the affected children including respiratory difficulties, scoliosis, and physical immobility , . Typically, in nemaline myopathy, pathogenic variants in genes such as actin alpha 1 ( ACTA1 ) drive the fiber type composition towards slow fiber predominance, although this effect is heterogenous. The only clear exception is the troponin T1 ( TNNT1 ) nemaline myopathy, in which a fast fiber predominance is seen. Thus, a deeper characterization of the heterogeneity behind the skeletal muscle fiber dysregulation observed in nemaline myopathies may help untangle the complex relationship between these diseases and muscle fiber types. Muscle fibers isolated from patients with ACTA1 - and TNNT1 -mutation derived nemaline myopathies display substantial myofiber atrophy or hypotrophy, compared to healthy controls ( n = 3 per group) (Figs. , Supplementary Table ), which provides considerable technical challenge arising from the limited material for proteomic analysis. Nonetheless, we were able to detect 2485 proteins from 272 skeletal muscle fibers. After filtering for a minimum of 1000 quantified proteins per fiber, downstream bioinformatic analyses were performed on 250 fibers. On average of 1573 ± 359 proteins were quantified per fiber after filtering (Supplementary figs. , Supplementary Dataset – ). Importantly, only a modest reduction in proteome depth was apparent in samples from patients with nemaline myopathy, despite the markedly reduced fiber size. Furthermore, processing of this data with our custom FASTA file (including non-coding transcripts) identified five microproteins within the skeletal muscle fibers from nemaline myopathy patients (Supplementary Dataset ). A wide dynamic range was apparent in the proteome, whilst the shared proteins in the control participants correlated well with those in the previous analysis of the 1000 fiber proteome study (Supplementary fig. ). As nemaline myopathies influence the MYH-based fiber type proportions within skeletal muscle , , we first investigated the MYH-based fiber type of our nemaline myopathy patients and controls. Fiber type was determined using the unbiased approach described previously for the 1000 fiber studies (Supplementary figs. ) and once again pure type 2X fibers could not be identified (Fig. ). We observed a heterogeneous effect of nemaline myopathies on fiber type, as two patients with ACTA1 mutations displaying an increased proportion of type 1 fibers, whilst two patients with TNNT1 -nemaline myopathy displayed a reduced proportion of type 1 fibers (Fig. ). Indeed, MYH2 and the fast troponin isoforms were downregulated in ACTA1 -nemaline myopathy (TNNC2, TNNI2, and TNNT3), whilst MYH7 was downregulated in TNNT1 -nemaline myopathy (Supplementary fig. ). This is in line with previous reports of heterogeneous fiber type switching in nemaline myopathies , . We validated these findings using immunohistochemistry, finding type 1 fiber predominance in ACTA1 -nemaline myopathy patients, while the opposite was observed in TNNT1-nemaline myopathy patients (Fig. ). At the single-fiber proteome level, skeletal muscle fibers from ACTA1 - and TNNT1 -nemaline myopathy patients clustered away from the majority of control fibers, with TNNT1- nemaline myopathy fibers tending to be the most severely affected (Fig. ). This was particularly apparent when we produced a PCA plot of pseudo-bulked fibers for each patient, with TNNT1 -nemaline myopathy patients 2 & 3 lying furthest from the control samples (Supplementary figs. , Supplementary Dataset ). To further understand how fibers from myopathy patients compare to the healthy condition, we capitalized on the depth of information provided by our analysis in 1000 fiber proteome study from healthy adult participants. We projected the fibers from our myopathy data set (both ACTA1 - and TNNT1 -nemaline myopathy patients and controls) onto the PCA determined from the 1000 fiber proteome study (Fig. ). Control fibers displayed a similar distribution of MYH-based fiber type along PC2 as the 1000 fiber proteome study. However, the majority of fibers from nemaline myopathy patients shifted downwards along PC2, overlapping with the healthy fast fibers, irrespective of their own MYH-based fiber type. Thus, despite evidence for a fiber type shift towards type 1 fibers in ACTA1 -nemaline myopathy patients when quantified using MYH-based approaches, both ACTA1 - and TNNT1 -nemaline myopathies shift the skeletal muscle fiber proteome to resemble fast fibers. We next directly compared each patient group with the samples from healthy controls, resulting in 256 and 552 differentially abundant proteins in ACTA1 - and TNNT1 -nemaline myopathies, respectively (Figs. & Supplementary figs. , Supplementary Dataset ). Gene set enrichment analysis identified the coordinated reduction of mitochondrial proteins (Figs. , Supplementary Dataset ). Surprisingly, this was completely independent of MYH-based fiber type (Figs. & Supplementary fig. , Supplementary Dataset ), despite the divergent fiber type predominance between ACTA1 - and TNNT1 -nemaline myopathies. Three microproteins were also regulated by either ACTA1 - or TNNT1 -nemaline myopathies. Two of those microproteins, ENSG00000215483_TR14_ORF67 (also known as LINC00598 or Lnc-FOXO1 ) and ENSG00000229425_TR25_ORF40 ( lnc-NRIP1-2 ) displayed differential abundance only within type 1 fibers, with ENSG00000215483_TR14_ORF67 having previously reported to play a role in cell cycle regulation . On the other hand, ENSG00000232046_TR1_ORF437 (corresponding to LINC01798 ) was upregulated in both type 1 and type 2A fibers from ACTA1- nemaline myopathy when compared to healthy controls (Supplementary figs. , Supplementary Dataset ). Conversely, ribosomal proteins were largely unaffected by nemaline myopathies, although RPS17 was downregulated in ACTA1- nemaline myopathy (Fig. ). Enrichment analysis also identified the up-regulation of immune system process in both ACTA1 - and TNNT1 -nemaline myopathies, as well as cell adhesion in TNNT1 -nemaline myopathy (Fig. ). The enrichment of these extracellular terms, was reflected by extracellular matrix proteins driving the PCA in the negative direction in both PC1 and PC2, i.e., towards the most affected fibers (Fig. ). Both patient groups overexpressed extracellular proteins involved in immune response and the sarcolemma repair machinery, such as annexins (ANXA1, ANXA2, ANXA5) , and their interactor S100A11 (Supplementary figs. ). This process has been previously reported to be upregulated in muscle dystrophies , yet to our knowledge it has not been associated with nemaline myopathies before. A normal function of this molecular machinery is required for both sarcolemma membrane repair upon injury and for the fusion of new myoblasts to muscle fibers , . Therefore, an up-regulation of this process in both patient groups suggests a reparative response to damage caused by myofibril instability. The effects of each nemaline myopathy were well correlated (r = 0.736) and displayed reasonable overlap (Supplementary fig. ), indicating that ACTA1 - and TNNT1 -nemaline myopathies induce similar effects on the proteome. Nonetheless, a number of proteins displayed regulation in only ACTA1 - or TNNT1 -nemaline myopathies (Supplementary fig. ). MFAP4, a pro-fibrotic protein, was one of the most up-regulated proteins in TNNT1 -nemaline myopathy whilst unchanged in ACTA1 -nemaline myopathy. SKIC8, a component of the PAF1C complex, which regulates the transcription of HOX genes , was down-regulated in TNNT1 -nemaline myopathy but was unaffected in ACTA1 -nemaline myopathy (Supplementary fig. ). Direct comparisons between ACTA1 - and TNNT1 -nemaline myopathies identify a greater effect of TNNT1 -nemaline myopathies on the reduction of mitochondrial proteins and the increase of immune system proteins (Fig. & Supplementary figs. & Supplementary fig. ). These data are consistent with the greater degree of atrophy/hypotrophy apparent in TNNT1 - compared to ACTA1 -nemaline myopathies (Fig. ), indicating that TNNT1 -nemaline myopathy is the more severe form of the disease. In order to assess whether the observed effects of nemaline myopathy were also present at the whole muscle-level, we conducted a bulk proteome analysis on muscle biopsies from the same TNNT1- nemaline myopathy patients and compared them against control individuals ( n = 3 per group) (Supplementary fig. , Supplementary Dataset ). As expected, upon PCA, control individuals tightly clustered together whereas, similarly to the single fiber analysis, and TNNT1- nemaline myopathy patients displayed a higher inter-sample variance (Supplementary fig. ). The bulk analysis was able to recapitulate the differentially expressed proteins (Supplementary figs. , Supplementary Dataset ) and biological processes (Supplementary fig. , Supplementary Dataset ) highlighted in the single fiber comparison, although lost the potential to discriminate between fiber types, and did not account for the heterogeneous effect of the disease across different fibers. Together, these data demonstrate that single muscle fiber proteomics can illuminate clinical biology that is unapparent using targeted approaches such as immunoblotting. Furthermore, these data highlight the limitations of solely relying upon MYH-based fiber typing to describe phenotypic adaptations. Indeed, despite divergent MYH-based fiber type switching in actin and troponin nemaline myopathies, both nemaline myopathies uncouple the MYH-based fiber type from skeletal muscle fiber metabolism, shifting towards a faster, less oxidative muscle proteome. To investigate the heterogeneity of human skeletal muscle fibers, we developed two workflows to enable transcriptome and proteome profiling of single skeletal muscle fibers (Figs. and Supplementary fig. ). Several methodological steps were developed and optimized, from sample storage and preservation of RNA and protein integrity to optimizing the throughput of each method. This was achieved for transcriptome analysis by inserting sample-specific molecular barcodes during the initial reverse transcription step, enabling pooling of 96 fibers for further efficient downstream processing. Rich transcriptome data was further obtained by deeper sequencing ( ± 1 M reads per fiber) compared to conventional single cell methods . For proteomics, we used a short chromatographic gradient (21 minutes) combined with DIA-PASEF data aquisition on timsTOF mass spectrometer to optimize proteome depth, whilst maintaining a high-throughput , . To investigate skeletal muscle fiber heterogeneity in the healthy state, the transcriptome was determined for 1050 individual fibers from 14 healthy adult donors, whilst the proteome was determined for 1038 fibers from 5 healthy adult donors (Supplementary Table ). These datasets will be referred to as the 1000 fiber transcriptome and proteome datasets, respectively, throughout this manuscript. Our approach detected a total of 27237 transcripts and 2983 proteins in the 1000 fiber transcriptomics and proteomics studies (Figs. , Supplementary Dataset – ). After filtering for > 1000 detected genes and for 50% valid values within each fiber in both transcriptomics and proteomics datasets, respectively, downstream bioinformatic analyses were performed on 925 and 974 fibers in the transcriptome and proteome, respectively. On average 4257 ± 1557 genes and 2015 ± 234 proteins (mean ± SD) were detected per fiber after filtering, with limited inter-individual variation (Supplementary figs. , Supplementary Dataset - ). The intra-individual variation within a participant was more substantial however, most likely due to differences in RNA/protein yield among fibers of different length and cross-sectional area. For the majority of proteins ( > 2000), the coefficient of variation was below 20% (Supplementary fig. ). Both methodologies captured a wide dynamic range of transcripts and proteins, with features known to be important for muscle contraction being highly expressed (e.g., ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Supplementary figs. ). A large part of the detected features were shared between the transcriptome and proteome datasets (Supplementary fig. ), alongside a reasonable correlation ( r = 0.52) in average UMI counts/LFQ intensities for these features (Supplementary fig. ). We initially set out to define the MYH-based fiber type of each fiber using an optimized methodology, leveraging the high sensitivity and dynamic range of MYH expression in the omics datasets. Previous studies have used arbitrary cut-offs to assign a fiber as pure type 1, type 2A, type 2X, or hybrid, based on a fixed percentage of expression for the different MYHs , , . We employed a different approach, in which we ranked the expression of each fiber by the MYHs used for fiber typing: MYH7, MYH2 and MYH1, corresponding to type 1, type 2A and type 2X fibers, respectively. We then mathematically calculated the bottom knee for each of the resulting curves and used it as a threshold to assign a fiber as being positive (above threshold) or negative (below threshold) for each MYH (Figs. ). These data show that MYH7 (Fig. ) and MYH2 (Fig. ) have a more pronounced on/off expression profile on the RNA level, compared to the protein level. Indeed, at the protein level, very few fibers did not express MYH7 and no fibers had 100% MYH2 expression. Next, we used the determined expression thresholds to assign MYH-based fiber types for all fibers in each dataset. For example, a MYH7 + /MYH2 - /MYH1 - was assigned as type 1, and a MYH7 - /MYH2 + /MYH1 + was assigned as a hybrid type 2A/2X fiber (see Supplementary Table for full description). When combining all fibers, a very similar MYH-based fiber type distribution was observed at the RNA (Fig. ) and protein (Fig. ) levels, with an expected inter-individual variation in relative MYH-based fiber type composition (Supplementary fig. ). Most fibers were considered pure type 1 (34–35%) or type 2A (36–38%), although a substantial number of hybrid 2A/2X fibers (16–19%) were also detected. A striking discrepancy was that pure type 2X fibers could only be detected at the RNA but not at the protein level, indicating that fast MYH expression is, at least in part, post-transcriptionally regulated. We validated the MYH-based fiber typing from the proteomics data against an antibody-based dot blot technique, in which both methodologies were in 100% agreement on the identification of both pure type 1 and type 2A fibers (Supplementary fig. ). However, the increased sensitivity of the proteomics-based approach was superior in identifying hybrid fibers and in quantifying the proportion of each MYH within each fiber. These data demonstrate the efficacy of using an unbiased high-sensitivity omics-based approach for the characterization of skeletal muscle fiber types. We then utilized the full depth of information that transcriptomics and proteomics provide to classify fibers in an unbiased manner based on their whole transcriptome or proteome. Using uniform manifold approximation and projection (UMAP) for dimension reduction of 6 principal components (Supplementary figs. ), we were able to visualize the variation in fibers in the transcriptome (Fig. ) and proteome (Fig. ). Interestingly, fibers did not cluster by participant in either the transcriptomics or proteomics datasets (Supplementary figs. ), nor by test day (Supplementary fig. ), indicating that intra-individual variance in skeletal muscle fibers outweighs inter-individual variance. Two distinct clusters were apparent in the UMAP plots, which represented “fast” and “slow” fibers (Fig. ). MYH7 + (slow) fibers clustered to the positive side of UMAP1, and MYH2 + and MYH1 + (fast) fibers clustered to the negative side of UMAP1 (Fig. ). No distinction between the various fast MYH-based fiber types (i.e., type 2A, type 2X, or hybrid 2A/2X) could be identified, however, suggesting that when the whole transcriptome or proteome is taken into account, the expression of MYH1 (Fig. ), or other classical markers of type 2X fibers like ACTN3 or MYLK2 (Supplementary figs. ), does not discriminate between distinct fiber types. Furthermore, in contrast to MYH2 and MYH7, few transcripts or proteins positively correlate with MYH1 (Supplementary figs. ), suggesting that MYH1 abundance does not adequately reflect the muscle fiber transcriptome/proteome. Similar conclusions can be drawn when assessing the blended expression of the three MYH isoforms on a UMAP level (Supplementary figs. ). Thus, whilst type 2X fibers can be identified at the transcriptional level based solely on the quantification of MYHs, MYH1 + fibers are not distinct from other fast fibers when the whole transcriptome or proteome is considered. As an initial exploration of fiber heterogeneity beyond MYHs, we assessed four established slow fiber type-specific proteins: TPM3, TNNT1, MYL3, and ATP2A2 . In both transcriptomics (Supplementary fig. ) and proteomics (Supplementary fig. ) approaches, the slow isoforms exhibited a high, although not perfect, Pearson correlation coefficient with MYH7. ~25 and 33% of the fibers in the transcriptomics (Supplementary fig. ) and proteomics (Supplementary fig. ) approaches, respectively, were not classified as pure slow fibers by all gene/protein isoforms. Thus, fiber typing based on multiple gene/protein isoforms introduces additional complexity, even with well-known proteins that are assumed to be fiber type-specific. This suggests that fiber typing based on isoforms of a single family of genes/proteins is likely inadequate to capture the true heterogeneity of skeletal muscle fibers. To further investigate the omics-wide phenotypical variability between human skeletal muscle fibers, we applied unbiased dimensionality reduction by principal component analysis (PCA) to our data (Fig. ). Similarly to the UMAP plot, neither participant nor test day influenced the clustering of fibers at the PCA level (Supplementary figs. ). MYH-based fiber type was explained by PC2 in both datasets, displaying clusters for slow type 1 fibers and a second cluster containing the fast type 2A, type 2X and hybrid type 2A/2X fibers (Fig. ). These two clusters were bridged in both datasets by a small number of hybrid type 1/2A fibers. As expected, over-representation analysis of the top PC drivers confirmed that PC2 is driven by contractile and metabolic features (Fig. & Supplementary fig. , Supplementary Dataset - ). In general, the MYH-based fiber types adequately explain the continual variance along PC2, except for the so-called type 2X fibers, which were spread across the entirety of the transcriptomic fast cluster. Unexpectedly, MYH-based fiber type explained only the second greatest degree of variability (PC2), indicating that other biological factors (PC1) independent of MYH-based fiber type, have a substantial role in regulating skeletal muscle fiber heterogeneity. Over-representation analysis of the top drivers in PC1 indicated that variance within PC1 was determined primarily by cell-cell adhesion and ribosomal content in the transcriptome, and costamere and ribosomal proteins in the proteome (Figs. & Supplementary fig. , Supplementary Dataset ). In skeletal muscle, the costamere connects the Z-disk to the sarcolemma and participates in force transmission and signaling . Annotating PCA plots with cell-cell adhesion (transcriptome, Fig. ) and costamere (proteome, Fig. ) features showed a strong shift to the left side of PC1, suggesting an enrichment for these features in some fibers. Closer inspection of fiber clustering at the UMAP level showed a MYH-based fiber type-independent gradient of expression for most features, rather than distinct subclusters of muscle fibers. This continuum holds true for several genes related to pathological conditions (Fig. ), such as CHCHD10 (neuromuscular disorders) , SLIT3 (muscle loss) and CTDNEP1 (muscle disease) . This same continuum was observed in the proteome, with proteins related to neurological diseases (UGDH) , insulin signaling (PHIP) and transcription (HIST1H2AB) (Fig. ). These data collectively show that slow/fast fiber type independent heterogeneity across fibers is of a continual nature. Interestingly, drivers of PC2 showed a good correlation ( r = 0.663) between the transcriptome and proteome (Fig. ), indicating that the slow and fast fiber types, particularly the contractile and the metabolic profile of skeletal muscle fibers, are transcriptionally regulated. However, there was no correlation between the drivers of PC1 in the transcriptome and the proteome (r = -0.027) (Fig. ). This suggests that slow/fast fiber type-independent variance is for a large part post-transcriptionally regulated. Since PC1 variance was largely explained by ribosomal gene ontology terms and given that ribosomes play a profound and specialized role in the cell by actively participating and influencing protein translation , we next set out to explore this unexpected ribosomal heterogeneity. We first colored the proteomics PCA plot based on the relative abundance of the proteins within the “cytosolic ribosome” GOCC term (Fig. ). Although the term was enriched on the positive side of PC1, and a slight gradient could be observed accordingly, ribosomal proteins were driving separation in both directions of PC1 (Fig. ). Amongst the ribosomal proteins enriched in the negative direction of PC1 were RPL18, RPS18 and RPS13 (Fig. ), whilst RPL31, RPL35 and RPL38 (Fig. ) were major drivers in the positive direction of PC1. Interestingly, RPL38 and RPS13 are highly expressed in skeletal muscle when compared to other tissues (Supplementary fig. ). These distinct ribosomal signatures across PC1 could not be observed in the transcriptome (Supplementary fig. ), indicating that this phenomenon is post-transcriptionally regulated. The concept of ribosomal heterogeneity and specialization was previously introduced, where the presence of distinct subpopulations of ribosomes (ribosomal heterogeneity) can directly influence protein translation in different tissues and cells by selectively translating specific mRNA transcript pools (ribosomal specialization). To identify sub-sets of ribosomal proteins that are co-expressed within skeletal muscle fibers, we performed an unsupervised hierarchical clustering analysis of ribosomal proteins within the proteome (Figs. , Supplementary Dataset ). As expected, ribosomal proteins did not cluster by MYH-based fiber type. However, we identified three distinct clusters of ribosomal proteins; the first of which (ribosomal_cluster_1) was coregulated alongside RPL38, therefore elevated in fibers in the positive direction of PC1. The second cluster (ribosomal_cluster_2) was coregulated alongside RPS13 and was elevated in fibers in the negative direction of PC1. A third cluster (ribosomal_cluster_3) displayed no coordinated differential expression within skeletal muscle fibers and could therefore be considered “core” ribosomal proteins within skeletal muscle. Both ribosomal_cluster_1 and ribosomal_cluster_2 contain ribosomal proteins previously demonstrated to regulate selective translation (e.g., RPL10A, RPL38, RPS19 and RPS25) and functionally influence development (e.g., RPL10A, RPL38) – . In line with the results from the PCA analysis, the observed heterogeneous abundance of these ribosomal proteins across fibers was also of a continual nature (Supplementary fig. ). To visualize the position of the ribosomal proteins that display heterogeneity within the ribosome, we utilized a structural model of the human 80S ribosome (Protein Data Bank: 4V6X) (Fig. ). Upon highlighting the ribosomal proteins that belong to the different ribosomal clusters, they were not located in close proximity, showing that our approach did not enrich for a particular area/section of the ribosome. Yet interestingly, ribosomal_cluster_2 contained a lower proportion of large ribosomal subunit proteins than ribosomal_cluster_1 and ribosomal_cluster_3 (Supplementary fig. ). We observed that the majority of the proteins that display variable stoichiometry within skeletal muscle fibers are located on the surface of the ribosome (Fig. ), which is consistent with an ability to interact with internal ribosome entry site (IRES) elements within distinct mRNA populations to coordinate selective translation , . Furthermore, numerous proteins that display variable stoichiometry within skeletal muscle fibers are located close to functional regions, such as the mRNA exit tunnel (Fig. ), which can selectively regulate translation elongation and arrest of specific nascent peptides . Overall, our data identifies that skeletal muscle ribosomal protein stoichiometry displays heterogeneity, driving variance between skeletal muscle fibers. We next set out to identify features of fast and slow skeletal muscle fibers and how these are transcriptionally regulated. Comparing the fast and slow clusters defined in the UMAPs of both datasets (Figs. & inlays Fig. ), transcriptome and proteome analysis yielded 1366 and 804 differentially abundant features, respectively (Figs. , Supplementary Dataset – ). Expected differences in sarcomeric (e.g., tropomyosin and troponin), excitation-contraction coupling (SERCA isoforms) and energy metabolism-related (e.g., ALDOA and CKB) features were observed. In addition, transcripts and proteins regulating protein ubiquitination displayed differences between fast and slow fibers (e.g., USP54, SH3RF2 , USP28 and USP48) (Fig. ). Furthermore, the microprotein-encoding gene RP11-451G4.2 (DWORF) , which has previously been shown to be differentially expressed between lamb muscle fiber types and to enhance SERCA activity in cardiac muscle , was significantly up-regulated in slow skeletal muscle fibers (Fig. ). Also at the single fiber level, clear differences could be observed for known features such as the metabolism-related isoforms of lactate dehydrogenase (LDHA and LDHB, Figs. and Supplementary fig. ) , , as well as previously unknown fiber type-specific features (e.g., IRX3 , USP54 , USP28, and DPYSL3) (Fig. ). There was a reasonable overlap of differentially expressed features (Supplementary fig. ) and correlation of fold changes between the transcriptomic and proteomic datasets, primarily driven by the large expression differences in sarcomeric features (Supplementary fig. ). Notably, some features (e.g., USP28, USP48, GOLGA4, AKAP13) showed strong post-transcriptional regulation with slow/fast fiber type-specific expression profiles only at the proteome level (Supplementary fig. ). We next performed over-representation analysis on the differentially abundant genes and proteins (Figs. , Supplementary Dataset ). Enriched pathways of features that were differential in both datasets showed expected differences such as fatty acid beta-oxidation and ketone metabolic process (slow fibers), myofilament/muscle contraction (fast and slow fibers, respectively) and carbohydrate catabolic process (fast fibers). Serine/Threonine protein phosphatase activity was also enriched in fast fibers, driven by features such as phosphatase regulatory and catalytic subunits (PPP3CB, PPP1R3D, and PPP1R3A), known to regulate glycogen metabolism (Supplementary figs. ). Other enriched pathways in fast fibers were Processing (P-) bodies (YTHDF3, TRIM21, LSM2) in the proteome (Supplementary fig. ), possibly related to post-transcriptional regulation , and transcription factor activity ( SREBF1 , RXRG , RORA ) in the transcriptome (Supplementary fig. ). Slow fibers showed enrichment for oxidoreductase activity ( BDH1 , DCXR , TXN2 ) (Supplementary fig. ), amide binding ( CPTP , PFDN2 , CRYAB ) (Supplementary fig. ), extracellular matrix ( CTSD , ADAMTSL4 , LAMC1 ) (Supplementary fig. ) and receptor-ligand activity ( FNDC5 , SPX , NENF ) (Supplementary fig. ). To get more insights into the transcriptional regulation of slow/fast fiber type signatures, we performed transcription factor enrichment analysis using SCENIC (Supplementary Dataset ). Many transcription factors were significantly enriched between fast and slow fibers (Fig. ). This included transcription factors such as MAFA , previously linked to the development of fast fibers , but also multiple transcription factors not previously linked to the fiber type specific gene program. These included PITX1 , EGR1 and MYF6 as the most enriched transcription factors within fast fibers (Fig. ). Conversely, ZSCAN30 and EPAS1 (also known as HIF2A ) were the most enriched transcription factors within slow fibers (Fig. ). In line with this, MAFA expression levels were higher in the UMAP area corresponding to fast muscle fibers, whereas an opposite expression pattern was observed for EPAS1 (Fig. ). Alongside known protein-coding genes, there exists a multitude of non-coding RNA biotypes, potentially involved in the regulation of human development and disease , . Several non-coding RNAs displayed fiber type specificity in the transcriptomics dataset (Figs. & Supplementary Dataset ), including LINC01405 , which is very specific to slow fibers and is reported to be downregulated in muscle of mitochondrial myopathy patients . Conversely, RP11-255P5.3 , corresponding to the lnc-ERCC5-5 gene ( https://lncipedia.org/db/transcript/lnc-ERCC5-5:2 ) displayed fast fiber type-specificity. Both LINC01405 ( https://tinyurl.com/x5k9wj3h ) and RP11-255P5.3 ( https://tinyurl.com/29jmzder ) display specificity to skeletal muscle (Supplementary figs. ) and have very few or no known contractile genes within a 1 Mb genomic neighborhood, suggesting a specialized role in fiber type regulation rather than a regulatory role for a neighboring contractile gene. The slow/fast fiber type-specific specific expression profiles of LINC01405 and RP11-255P5.3 were independently validated using RNAscope (Fig. ). Recently, it has become apparent that numerous assumed non-coding transcripts code for transcribed microproteins, some of which regulate muscle function , . To identify microproteins with potential fiber type specificity, we searched our 1000 fiber proteomic dataset using a custom FASTA file containing sequences from the detected non-coding transcripts ( n = 305) from the 1000 fiber transcriptome dataset (Fig. ). This resulted in the identification of 197 microproteins arising from 22 distinct transcripts, of which 71 microproteins display differential regulation between slow and fast skeletal muscle fibers (Supplementary figs. and Supplementary Dataset ). Three microprotein products were identified for LINC01405 , of which one displays a slow-fiber type specificity similar to its transcript (Figs. and Supplementary fig. ). Thus, we identify LINC01405 as a microprotein-encoding gene, which displays specificity for slow skeletal muscle fibers. Having developed a comprehensive workflow to characterize the proteome of single muscle fibers at scale and discovered regulators of fiber heterogeneity in the healthy state, we applied this pipeline to understand how nemaline myopathy impacts skeletal muscle fiber heterogeneity. Nemaline myopathy is a genetic muscular disorder that causes muscle weakness, resulting in a range of complications for the affected children including respiratory difficulties, scoliosis, and physical immobility , . Typically, in nemaline myopathy, pathogenic variants in genes such as actin alpha 1 ( ACTA1 ) drive the fiber type composition towards slow fiber predominance, although this effect is heterogenous. The only clear exception is the troponin T1 ( TNNT1 ) nemaline myopathy, in which a fast fiber predominance is seen. Thus, a deeper characterization of the heterogeneity behind the skeletal muscle fiber dysregulation observed in nemaline myopathies may help untangle the complex relationship between these diseases and muscle fiber types. Muscle fibers isolated from patients with ACTA1 - and TNNT1 -mutation derived nemaline myopathies display substantial myofiber atrophy or hypotrophy, compared to healthy controls ( n = 3 per group) (Figs. , Supplementary Table ), which provides considerable technical challenge arising from the limited material for proteomic analysis. Nonetheless, we were able to detect 2485 proteins from 272 skeletal muscle fibers. After filtering for a minimum of 1000 quantified proteins per fiber, downstream bioinformatic analyses were performed on 250 fibers. On average of 1573 ± 359 proteins were quantified per fiber after filtering (Supplementary figs. , Supplementary Dataset – ). Importantly, only a modest reduction in proteome depth was apparent in samples from patients with nemaline myopathy, despite the markedly reduced fiber size. Furthermore, processing of this data with our custom FASTA file (including non-coding transcripts) identified five microproteins within the skeletal muscle fibers from nemaline myopathy patients (Supplementary Dataset ). A wide dynamic range was apparent in the proteome, whilst the shared proteins in the control participants correlated well with those in the previous analysis of the 1000 fiber proteome study (Supplementary fig. ). As nemaline myopathies influence the MYH-based fiber type proportions within skeletal muscle , , we first investigated the MYH-based fiber type of our nemaline myopathy patients and controls. Fiber type was determined using the unbiased approach described previously for the 1000 fiber studies (Supplementary figs. ) and once again pure type 2X fibers could not be identified (Fig. ). We observed a heterogeneous effect of nemaline myopathies on fiber type, as two patients with ACTA1 mutations displaying an increased proportion of type 1 fibers, whilst two patients with TNNT1 -nemaline myopathy displayed a reduced proportion of type 1 fibers (Fig. ). Indeed, MYH2 and the fast troponin isoforms were downregulated in ACTA1 -nemaline myopathy (TNNC2, TNNI2, and TNNT3), whilst MYH7 was downregulated in TNNT1 -nemaline myopathy (Supplementary fig. ). This is in line with previous reports of heterogeneous fiber type switching in nemaline myopathies , . We validated these findings using immunohistochemistry, finding type 1 fiber predominance in ACTA1 -nemaline myopathy patients, while the opposite was observed in TNNT1-nemaline myopathy patients (Fig. ). At the single-fiber proteome level, skeletal muscle fibers from ACTA1 - and TNNT1 -nemaline myopathy patients clustered away from the majority of control fibers, with TNNT1- nemaline myopathy fibers tending to be the most severely affected (Fig. ). This was particularly apparent when we produced a PCA plot of pseudo-bulked fibers for each patient, with TNNT1 -nemaline myopathy patients 2 & 3 lying furthest from the control samples (Supplementary figs. , Supplementary Dataset ). To further understand how fibers from myopathy patients compare to the healthy condition, we capitalized on the depth of information provided by our analysis in 1000 fiber proteome study from healthy adult participants. We projected the fibers from our myopathy data set (both ACTA1 - and TNNT1 -nemaline myopathy patients and controls) onto the PCA determined from the 1000 fiber proteome study (Fig. ). Control fibers displayed a similar distribution of MYH-based fiber type along PC2 as the 1000 fiber proteome study. However, the majority of fibers from nemaline myopathy patients shifted downwards along PC2, overlapping with the healthy fast fibers, irrespective of their own MYH-based fiber type. Thus, despite evidence for a fiber type shift towards type 1 fibers in ACTA1 -nemaline myopathy patients when quantified using MYH-based approaches, both ACTA1 - and TNNT1 -nemaline myopathies shift the skeletal muscle fiber proteome to resemble fast fibers. We next directly compared each patient group with the samples from healthy controls, resulting in 256 and 552 differentially abundant proteins in ACTA1 - and TNNT1 -nemaline myopathies, respectively (Figs. & Supplementary figs. , Supplementary Dataset ). Gene set enrichment analysis identified the coordinated reduction of mitochondrial proteins (Figs. , Supplementary Dataset ). Surprisingly, this was completely independent of MYH-based fiber type (Figs. & Supplementary fig. , Supplementary Dataset ), despite the divergent fiber type predominance between ACTA1 - and TNNT1 -nemaline myopathies. Three microproteins were also regulated by either ACTA1 - or TNNT1 -nemaline myopathies. Two of those microproteins, ENSG00000215483_TR14_ORF67 (also known as LINC00598 or Lnc-FOXO1 ) and ENSG00000229425_TR25_ORF40 ( lnc-NRIP1-2 ) displayed differential abundance only within type 1 fibers, with ENSG00000215483_TR14_ORF67 having previously reported to play a role in cell cycle regulation . On the other hand, ENSG00000232046_TR1_ORF437 (corresponding to LINC01798 ) was upregulated in both type 1 and type 2A fibers from ACTA1- nemaline myopathy when compared to healthy controls (Supplementary figs. , Supplementary Dataset ). Conversely, ribosomal proteins were largely unaffected by nemaline myopathies, although RPS17 was downregulated in ACTA1- nemaline myopathy (Fig. ). Enrichment analysis also identified the up-regulation of immune system process in both ACTA1 - and TNNT1 -nemaline myopathies, as well as cell adhesion in TNNT1 -nemaline myopathy (Fig. ). The enrichment of these extracellular terms, was reflected by extracellular matrix proteins driving the PCA in the negative direction in both PC1 and PC2, i.e., towards the most affected fibers (Fig. ). Both patient groups overexpressed extracellular proteins involved in immune response and the sarcolemma repair machinery, such as annexins (ANXA1, ANXA2, ANXA5) , and their interactor S100A11 (Supplementary figs. ). This process has been previously reported to be upregulated in muscle dystrophies , yet to our knowledge it has not been associated with nemaline myopathies before. A normal function of this molecular machinery is required for both sarcolemma membrane repair upon injury and for the fusion of new myoblasts to muscle fibers , . Therefore, an up-regulation of this process in both patient groups suggests a reparative response to damage caused by myofibril instability. The effects of each nemaline myopathy were well correlated (r = 0.736) and displayed reasonable overlap (Supplementary fig. ), indicating that ACTA1 - and TNNT1 -nemaline myopathies induce similar effects on the proteome. Nonetheless, a number of proteins displayed regulation in only ACTA1 - or TNNT1 -nemaline myopathies (Supplementary fig. ). MFAP4, a pro-fibrotic protein, was one of the most up-regulated proteins in TNNT1 -nemaline myopathy whilst unchanged in ACTA1 -nemaline myopathy. SKIC8, a component of the PAF1C complex, which regulates the transcription of HOX genes , was down-regulated in TNNT1 -nemaline myopathy but was unaffected in ACTA1 -nemaline myopathy (Supplementary fig. ). Direct comparisons between ACTA1 - and TNNT1 -nemaline myopathies identify a greater effect of TNNT1 -nemaline myopathies on the reduction of mitochondrial proteins and the increase of immune system proteins (Fig. & Supplementary figs. & Supplementary fig. ). These data are consistent with the greater degree of atrophy/hypotrophy apparent in TNNT1 - compared to ACTA1 -nemaline myopathies (Fig. ), indicating that TNNT1 -nemaline myopathy is the more severe form of the disease. In order to assess whether the observed effects of nemaline myopathy were also present at the whole muscle-level, we conducted a bulk proteome analysis on muscle biopsies from the same TNNT1- nemaline myopathy patients and compared them against control individuals ( n = 3 per group) (Supplementary fig. , Supplementary Dataset ). As expected, upon PCA, control individuals tightly clustered together whereas, similarly to the single fiber analysis, and TNNT1- nemaline myopathy patients displayed a higher inter-sample variance (Supplementary fig. ). The bulk analysis was able to recapitulate the differentially expressed proteins (Supplementary figs. , Supplementary Dataset ) and biological processes (Supplementary fig. , Supplementary Dataset ) highlighted in the single fiber comparison, although lost the potential to discriminate between fiber types, and did not account for the heterogeneous effect of the disease across different fibers. Together, these data demonstrate that single muscle fiber proteomics can illuminate clinical biology that is unapparent using targeted approaches such as immunoblotting. Furthermore, these data highlight the limitations of solely relying upon MYH-based fiber typing to describe phenotypic adaptations. Indeed, despite divergent MYH-based fiber type switching in actin and troponin nemaline myopathies, both nemaline myopathies uncouple the MYH-based fiber type from skeletal muscle fiber metabolism, shifting towards a faster, less oxidative muscle proteome. Cellular heterogeneity is important to enable tissues to meet a wide range of demands. In skeletal muscle, this has classically been described by fiber types characterized by varying degrees of force production and fatigability. It is apparent, however, that this explains just a fraction of the variation in skeletal muscle fibers, with far greater variability, complexity and nuance residing in skeletal muscle fibers than previously believed. With the advent and development in technologies, these elements that regulate skeletal muscle fibers are now able to be elucidated. Indeed, our data indicates that type 2X fibers may not be a distinct sub-classification of skeletal muscle fibers. Furthermore, we identify metabolic, ribosomal, and cell junction proteins as major determinants of skeletal muscle fiber heterogeneity. By applying our proteomics workflow to samples from nemaline myopathy patients, we further evidence that MYH-based fiber typing does not capture the complete heterogeneity of skeletal muscle, particularly when the system is perturbed. Indeed, nemaline myopathies induce a shift towards faster less oxidative fibers regardless of their MYH-based fiber type. Skeletal muscle fibers have been classified since the 19 th century , . Recent omics-based analyses have enabled us to start understanding expression profiles and responses to various stimuli specific to distinct MYH-based fiber types – , , , , . As demonstrated herein, omics approaches also have the advantage of increased sensitivity in the quantification of fiber type markers over traditional antibody-based approaches, whilst also not relying on the quantification of a single (or few) markers for determining skeletal muscle fiber type. We leveraged complementary transcriptomics and proteomics workflows, and integrated the results, to explore the transcriptional and post-transcriptional regulation of human skeletal muscle fiber heterogeneity. This pipeline resulted in the observation that pure type 2X fibers could not be identified at the protein level in vastus lateralis skeletal muscle of our cohort of young healthy males. This is in line with previous single fiber studies identifying < 1% pure type 2X fibers in healthy vastus lateralis , although this should be confirmed in other muscles in the future. The discrepancy between the identification of near pure type 2X fibers at the mRNA level but only hybrid type 2A/2X fibers at the protein level is puzzling. mRNA expression of MYH isoforms is not circadian , indicating that it is unlikely that we have simply “missed” the on-signal of MYH2 in the apparently pure type 2X fibers at the RNA level. One possible explanation may be differences in the protein and/or mRNA stability of MYH isoforms, though this is purely hypothetical. Indeed, no fast fiber was 100% pure for any MYH isoform, though whether levels of MYH1 mRNA expression in the range of 70–90% could result in similar MYH1 and MYH2 abundance at the protein level is unclear. Nonetheless, when the whole transcriptome or proteome is considered, clustering-based analyses could only confidently identify two distinct clusters, which represented slow and fast skeletal muscle fibers regardless of their exact MYH composition. This is consistent with analyses using single nuclei transcriptomics approaches which commonly identify only two distinct clusters of myonuclei – . Furthermore, whilst previous proteomics-based studies have identified type 2X fibers, these fibers did not cluster away from the rest of the fast fibers and only displayed a handful of differentially abundant proteins compared to other MYH-based fiber types . These findings indicate that we should revert to the view of muscle fiber classification from the early twentieth century and classify human skeletal muscle fibers not into three distinct classifications based on MYHs, but instead into just two clusters based on their metabolic and contractile properties . Better still, we should consider muscle fiber heterogeneity in multiple dimensions. Previous omics studies already pointed in this direction, by showing that skeletal muscle fibers do not form discrete clusters, yet fall along a continuum , , , , . Here, we found that over and above the variance within the contractile and metabolic signatures of skeletal muscle, fibers could also be separated by features related to the cell junction and translation machinery. Indeed, we identified ribosomal heterogeneity across skeletal muscle fibers that drives a slow/fast-fiber type independent heterogeneity. The underlying reason for such vast slow/fast-fiber type independent heterogeneity amongst fibers is not immediately obvious, though this could allude to a specialized spatial organization within a muscle fascicle to enable an optimal response to specific forces and loads , specialized cellular or organ communication with other cell types within the muscle microenvironment – , or differential ribosomal activity in individual fibers. Indeed, ribosomal heterogeneity, via paralog substitution of RPL3 and RPL3L or at an rRNA 2′O-methyl level, has been implicated in skeletal muscle hypertrophy , . Multi-omics and spatial applications in concert with functional characteristics of single muscle fibers will further advance our understanding of muscle biology on a multi-omics level . In analyzing the proteome of single muscle fibers from patients with nemaline myopathies, we also demonstrate the utility, power, and applicability of single muscle fiber proteomics in uncovering clinical pathophysiology in skeletal muscle. Furthermore, by comparing our workflow against a bulk proteome analysis, we were able to demonstrate that single muscle fiber proteomics is able to capture the same depth of information than bulk tissue omics and expand on it by including inter-fiber heterogeneity and muscle fiber type into consideration. In addition to observing expected, albeit divergent, differences in fiber type proportions in ACTA1 - and TNNT1 -nemaline myopathies compared to healthy controls , we also identified oxidative and extracellular remodeling, which is uncoupled from this MYH-based fiber type switch. Fibrosis has previously been reported for TNNT1 nemaline myopathy . However, our analysis builds upon this to also identify the up-regulation of stress-related secreted proteins within the extracellular space, such as annexins, involved in the sarcolemma repair machinery – in fibers from both ACTA1 - and TNNT1 -nemaline myopathy patients. Overall, the upregulation of annexins in muscle fibers from nemaline myopathy patients may represent a cellular response to rescue severely atrophying fibers. Whilst this study represents the largest omic analysis of intact human single muscle fibers to date, it is not without limitations. We isolated skeletal muscle fibers from a relatively small and homogenous sample of participants and from a single muscle ( vastus lateralis ). In this respect, it is impossible to rule out the existence of specific fiber populations in different muscle types and at the extremes of muscle physiology. For example, we cannot rule out that a sub type of ultra-fast fibers (e.g., pure type 2X fibers) may become apparent in highly trained sprint and/or power athletes or during muscle disuse , . Furthermore, our limited participant pool did not allow us to investigate sex differences in fiber heterogeneity, in which there are known differences in fiber type proportions between males and females. Furthermore, we were unable to perform transcriptomics and proteomics on the same muscle fibers or in samples from the same participants. As we and others continue to optimize single-cell and single muscle fiber omics-analyses towards ultra-low sample input (as demonstrated here with the analysis of fibers from nemaline myopathy patients), the possibility of combining multi-omics (and functional) approaches in a single muscle fiber is tantalizingly close. Collectively, our data identifies and explains transcriptional and post-transcriptional drivers of heterogeneity within skeletal muscle. In particular, we provide data to question long-standing dogmas within skeletal muscle physiology related to the classical definitions of MYH-based fiber types. In doing so we hope to reignite the debate and ultimately redefine our understanding of skeletal muscle fiber classifications and heterogeneity. 1000 fiber transcriptomics Participant information Fourteen participants (12 males / 2 females) of Caucasian origin volunteered to take part in this study, which was approved by the Ethical Committee of Ghent University Hospital (BC-10237), in agreement with the 2013 Declaration of Helsinki and registered on ClinicalTrials.gov (NCT05131555). General characteristics of the participants can be found in Supplementary Table . After oral and written informed consent, participants were medically screened before final inclusion. Participants were young (22-42 years old), healthy (no diseases and non-smoking) and moderately physically active. Maximal oxygen uptake was determined as marker of physical fitness during a graded incremental cycling test, as previously described . Muscle biopsy collection Muscle biopsies were collected in the rested and fasted state, on three different days, separated by 14 days. As these samples were collected as part of a larger study, on each of these days participants ingested a placebo (lactose), an H1-receptor antagonist (540 mg fexofenadine) or an H2-receptor antagonist (40 mg famotidine) 40 minutes before muscle biopsy collection. We have previously shown that these histamine receptor antagonists do not affect the resting skeletal muscle state , and no clustering was apparent based on condition in our quality control plots (Supplementary figs. & Supplementary fig. ). Dietary intake was standardized 48 hours before each experimental day (41.4 kcal/kg body weight, 5.1 g/kg body weight carbohydrates, 1.4 g/kg body weight protein and 1.6 g/kg body weight fat per day), followed by a standardized breakfast on the morning of the experimental day (1.5 g/kg bodyweight carbohydrates). Muscle biopsies of the m. vastus lateralis were then collected after local anesthesia (0.5 mL of 1% Xylocaïne without epinephrine) using the percutaneous Bergström technique with suction . The muscle samples were immediately submerged in RNA later and stored at 4 °C until manual fiber dissection (max. 3 days). Single fiber isolation Freshly excised muscle fiber bundles were transferred to fresh RNA later in a petri dish. Individual muscle fibers were then manually dissected using a stereomicroscope and fine forceps. Twenty-five fibers were dissected per biopsy, with special care to select fibers from different sections of the biopsy. After dissection, each fiber was carefully submerged in 3 µL of lysis buffer (SingleShot Cell Lysis kit, Bio-rad), containing proteinase K and DNase enzymes to remove unwanted proteins and DNA. Next, cell lysis and protein/DNA removal was initiated by short vortexing, spinning liquid down in a microcentrifuge and incubation at room temperature (10 min). Lysates were then incubated for 5 min at 37 °C and 5 min at 75 °C in a thermocycler (T100, Bio-Rad), immediately followed by storage at −80 °C until further processing. Sequencing library preparation Illumina-compatible libraries from polyadenylated RNA were prepared from 2 µL of the muscle fiber lysates using the QuantSeq-Pool 3’ mRNA-Seq library prep kit (Lexogen). Detailed methodology can be found in the manufacturer guidelines. The process was initiated by reverse transcription for first strand cDNA synthesis, during which Unique Molecular Identifiers (UMIs) and sample-specific i1 barcodes were introduced enabling sample pooling and reducing technical variability in the downstream process. Next, cDNA from 96 fibers was pooled and purified using magnetic beads, followed by RNA removal and second strand synthesis by random priming. Libraries were purified using magnetic beads, followed by addition of pool-specific i5/i7 indices and PCR amplification. A last purification step was performed, finalizing the Illumina-compatible libraries. A high sensitivity small DNA Fragment Analysis kit (Agilent Technologies, DNF-477-0500) was used to assess the quality of each library pool. Illumina sequencing The individual pools were further equimolarly (2 nM) pooled, based on Qubit-quantified concentrations. The final pool was subsequently sequenced with a NovaSeq S2 kit (1 × 100 nucleotides) with a loading of 2 nM (4% PhiX) in standard mode on a NovaSeq 6000 instrument. Primary data processing Our pipeline was based on the QuantSeq Pool data analysis pipeline from Lexogen ( https://github.com/Lexogen-Tools/quantseqpool_analysis ). First, the data was demultiplexed based on the i7/i5 indices with bcl2fastq2 (v2.20.0). The next demultiplexing step was performed via idemux (v0.1.6) according to the i1 sample-specific barcodes in read 2, followed by extraction of UMI sequences with umi_tools (v1.0.1). Trimming of the reads was then performed in multiple rounds with cutadapt (v3.4), with removal of too short reads (length <20) or reads consisting entirely of adapter sequences. Reads were then aligned to the human genome with STAR (v2.6.0c), followed by BAM file indexing with SAMtools (v1.11). Duplicates of reads were removed with umi_tools (v1.0.1). Finally, counting of the alignments was performed with featureCounts from Subread (v2.0.3). At several intermediate steps during the pipeline, quality control with performed with FastQC (v0.11.9). Initial Seurat processing All further bioinformatics processing and visualization was performed in R (v4.2.3), primarily with the Seurat (v 4.4.0) workflow . The individual UMI counts and metadata matrices were thus transformed into a Seurat object. Genes with expression in less than 30% of all fibers were removed. Low-quality samples were then removed based on a minimum threshold of 1000 UMI counts and 1000 detected genes. This resulted in a total of 925 fibers that passed all quality control filtering steps. Normalization of UMI counts was performed using the SCTransform v2 Seurat method , including all 7418 detected features and regressing out participant variation. All relevant metadata can be found in Supplementary Dataset . Proteomics Sample collection - 1000 fiber proteome Participant information Stored biobank muscle specimens were used for the purpose of the present study (Clinicaltrials.gov identifier: NCT04048993). The specimens were collected from five active and healthy male volunteers (aged 21–35 years) of Caucasian ancestry who gave their written and oral informed consent with approval from the Science Ethics Committee of the Capital Region in Denmark (H-1-2012-090) and complied with the guidelines of the 2013 Declaration of Helsinki. General characteristics of the participants can be found in Supplementary Table . Participants were young, healthy (no diseases and non-smoking) and moderately physically active. Muscle biopsy collection Participants arrived in the morning after an overnight fast and rested in the supine position for 1 hour. Then, local anesthesia (2-3 mL Xylocaine 2%; lidocaine without epinephrine, AstraZeneca, Denmark) was applied under the skin above the fascia at the belly of the m. vastus lateralis muscle. A muscle biopsy was sampled through a small 3–4 mm incision using a Bergström needle with suction. The muscle biopsy specimen was snap-frozen in liquid nitrogen and stored at −80 °C until analysis. Single muscle fiber isolation Muscle fibers were isolated from freeze-dried specimens as previously described . In brief, muscle biopsies were freeze-dried for 48 hours. Subsequently, fibers were isolated in a humidity- and temperature-controlled room (humidity of 25%) using fine forceps under a stereomicroscope. ~200 single muscle fibers were isolated for each biopsy, resulting in a total of 1038. To ensure the fibers settled at the bottom of the tube, each fiber-containing tube underwent centrifugation at 20,000 g using a small centrifuge. Next, fibers were resuspended in 15 µL of lysis buffer (1% sodium dodecyl sulfate (SDC), 40 mM chloroacetamide (CAA), 10 mM dithiothreitol (DTT) in 50 mM Tris pH 8.5). Sample collection – myopathy Participant information Six patients with severe nemaline myopathy were selected from our nemaline myopathy study cohort. Three patients (2 male and 1 female) had pathogenic variants in ACTA1 , representing the conventional severe form, and three patients had pathogenic variants in TNNT1 (3 male), resulting in a rare, progressive form of nemaline myopathy. Three healthy individuals with no history of neuromuscular disease were used as controls. All participants are of Caucasian ancestry (Supplementary Table ). Muscle biopsy collection Healthy control participant biopsies ( n = 3 males) were used from an original study , and were therefore collected, snap frozen in liquid nitrogen and stored in -80°C as per that originally described. For the present study, a fragment of this stored biopsy was dissected under sterile, frozen conditions before being prepared for single myofiber isolation (detailed below). Acquisition of biopsies of healthy control patients was approved by local ethics committee (Copenhagen and Frederiksberg) in Denmark (hs:h-15002266). Those of myopathy patients were consented, stored, and used in accordance with the Human Tissue Act under local ethical approval in United Kingdom (REC 13/NE/0373). All procedures were carried out in accordance to the Declaration of Helsinki. Single fiber isolation Dissected fragments of muscle biopsy were placed in ice-cold, 22 micron filtered relaxing solution (4 mM Mg-ATP, 1 mM free Mg 2+ , 10 -6.00 mM free Ca 2+ , 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate, KCl to an iconic strength of 180 mM and pH to 7.0) for ~3 minutes before being immersed in fresh relaxing solution on a sterile petri dish and mounted on ice under a dissection microscope for single fiber isolation. Fibers were cleaved from the tissue/biopsy ensuring a variety of sample/biopsy locations were used and that only single fibers were selected. Following isolation, fibers were manually moved to a sterile 96-well plate containing 15 µL of lysis buffer (identical to that detailed above) where the tweezers containing the fiber were submerged, spun and agitated in lysis buffer to ensure the fibers dissociated from the tweezers. To ensure fibers were settled to the bottom of the well, the 96-well plate was subjected to gentle vortexing and centrifugation (1,000 g). Proteomics analysis Sample preparation Samples from both proteomics studies followed the same sample preparation workflow. In order to extract the proteins, samples were boiled at 95 °C in a thermomixer with gentle shaking (800 rpm) and sonicated in a bioruptor instrument with 30 seconds on/off cycles for 15 minutes. A small 5 µL fraction of lysate from each sample was saved for antibody-based fiber typing of the 1000 fiber samples. Next, samples were processed following a modified version of the in-solution digestion sample preparation protocol. In brief, total volume was adjusted to 50 µl by addition of digestion buffer, containing 50 mM Tris PH 8.5 buffer, an enzyme to protein ratio of 1:500 LysC (Wako) and a 1:100 enzyme to protein ratio of trypsin (Promega). Single muscle fiber lysates were digested overnight in a thermomixer set to 37° C and 800 rpm. The next day, protein digestion was quenched by addition of 50 µl of 2% trifluoroacetic acid (TFA) in isopropanol. Peptides were desalted using in-house prepared single-use reverse-phase StageTips containing styrenedivinylbenzene reverse-phase sulfonate (SDB-RPS) disks. Then, desalted peptides were loaded in Evotips (Evosep) following manufacturer instructions prior LC-MS/MS analysis. Bulk tissue samples were prepared using the same protocol utilized for single fibers, with a few modifications to sample lysis. Tissue samples were first powdered using a tissue crusher over dry ice before resuspending the powder in the same lysis buffer described above. Then, the samples were homogenized using an IKA Turrax homogenizer for 2 minutes prior boiling and sonication. From there onwards the samples underwent the same protocol described above. Proteomics library preparation Fibers from the five healthy control individuals participating in the 1000 fiber study were carefully dissected and combined in order to create a pooled fiber lysate. Then, 200 µg of protein corresponding to each participant-specific lysate were pooled together into one final protein lysate that was processed following the same sample preparation workflow just described. 20 µg of desalted peptides were fractionated using High PH Reverse-Phase Chromatography (HpH-RP). Fractionation was carried out on a Kinetex 2.6 µm EVO C18 100 Å, 150 ×0.3 mm column manufactured by Phenomenex and using an EASY-nLC 1200 System (Thermo) operating at 1.5 µL/min. Separation was accomplished using a 62 min step-gradient starting from 3% to 60% solvent B (which consisted of 10 mM TEAB in 80% acetonitrile) and solvent A (containing 10 mM TEAB in water). The total run time was 98 min, which included wash and column equilibration. Throughout the fractionation, peptides were eluted and collected every 60 s, obtaining 96 single fractions without concatenation. Finally. 200 ng of HpH-RP fractionated peptides were loaded, concentrated and desalted on Evotips (Evosep) following the instructions provided by the manufacturer. Liquid chromatography tandem mass spectrometry Proteomics measurements were performed using LC-MS instrumentation consisting of an Evosep One HPLC system (Evosep) coupled via electrospray ionization to a timsTOF SCP mass spectrometer (Bruker). Peptides were separated utilizing 8 cm, 150 μM ID columns packed with C18 beads (1.5 μm) (Evosep). Chromatographic separation was achieved by the ‘60 samples per day’ method, followed by electrospray ionization through a CaptiveSpray ion source and a 10 μm emitter into the MS instrument. Single muscle fiber peptides were measured in DIA-PASEF mode following a previously described method , while library fractions were measured using DDA-PASEF. In brief, the DDA-PASEF scan range encompassed 100–1700 m/z for both MS and MS/MS, and TIMS mobility range was set to 0.6–1.6 (V cm −2 ). Both TIMS ramp and accumulation times were configured to 100 ms, and 10 PASEF ramps were recorded for a total cycle time of 1.17 s. The MS/MS target intensity and intensity threshold were defined as 20.000 and 1.000, respectively. An exclusion list of 0.4 min for precursors within 0.015 m/z and 0.015 V cm −2 width was also activated. For DIA-PASEF the scan range was established at 400-1000 (m/z), the TIMS mobility range to 0.64-1.37 (V cm −2 ), and ramp and accumulation times were both set to 100 ms. A short-gradient method was used, which included 8 DIA-PASEF scans with three 25 Da windows per ramp, resulting in an estimated cycle time of 0.95 sec. MS data processing Library files were processed using the MS-Fragger functionality within Fragpipe v19.0 under the SpecLib workflow with default settings, including a minimum peptide length of seven amino acids and maximum of two missed cleavages allowed . Spectra were searched against a human reviewed FASTA from Uniprot (March 2022, 20410 entries) and the output library contained a total of 5350 protein groups and 84383 precursors. Sample raw MS files were analyzed using DIA-NN version 1.8 in a library-based manner against the MS library just described. Protein groups quantification was based on proteotypic peptides, neural network was set to double-pass mode, quantification strategy was set to “Robust LC (high accuracy)” and the match between runs options was enabled, the rest of parameters remained as default, which included precursor FDR set to 1% and peptide length of 7-30 amino acids. Data processing Further data analysis was performed under the R environment (R version 4.22). Both a metadata data frame containing sample and participant information and the “PG_matrix.tsv” file from DIA-NN’s output were loaded in RStudio. The 1000 fiber data frame was filtered to remove samples with less than 50% valid protein intensity values, resulting in a total of 974 fibers. Next, rows were filtered to remove proteins with less than 30% valid values across samples, resulting in a total of 1685 proteins. Regarding the myopathy dataset, after filtering samples for 50% valid values, the number of samples was 250. We included in the analysis proteins that were quantified in 70% of the samples in at least one condition (conditions: control, actin myopathy and troponin myopathy), resulting in a total of 1545 proteins. Both data frames were then log2 transformed and normalized using the normalizeBetweenArrays function from the limma package (v 3.54.2), with the method argument set to quantile . Then, batch correction through the ComBat function from the sva package (v3.50.0) was applied to minimize the effect of the three technical batches originated during mass spectrometry measurement. Finally, missing values were replaced by random numbers from a Gaussian distribution with the default settings of the tImpute function from the PhosR package (v 1.12.0) . All relevant metadata for the 1000 fiber proteome and nemaline myopathy datasets can be found in Supplementary Data & , respectively. Bioinformatics analysis Transcriptome and proteome dynamic range The expression/intensity for each gene/protein was calculated relative to the total counts/intensity for each fiber. This value was then averaged across fibers in each dataset and log10-transformed. The overlap of detected features between both datasets were analyzed using the VennDiagram package (v 1.7.3). Coefficient of variation—proteomics For each of the 96 well plates used during the MS measurement of the 1000 fiber study, one technical control sample was included in A1 position to monitor total ion current intensity and quality control of the runs (a total of eleven technical controls). The coefficient of variation between proteins was calculated by dividing the standard deviation by the mean of the LFQ intensities from each protein across technical replicates and then multiplied by one hundred. Correlation analyzes Mean log2 transformed transcript counts, protein intensities, and/or fold change values across fibers were calculated, filtered for shared proteins/genes and Pearson correlation was calculated. Omics-based fiber typing Normalized counts and raw LFQ intensities were retrieved from well-described contractile proteins that have a slow (MYH7, TNNT1, TPM3, ATP2A2 and MYL3) and fast (MYH2, MYH1, TNNT3, TPM1, ATP2A1 and MYL1) isoforms . For each isoform combination, the relative expression of each was then calculated and samples were ordered from high to low. The mathematical bottom knee for each curve was then determined using the barcodeRanks function in the DropletUtils package (v 1.18.1). This threshold was used to assign fiber types as pure (type 1, type 2A or type 2X) or hybrid (hybrid 1/2A, hybrid 2A/2X or hybrid 1/2X) (Supplementary Table ). For the features with only two isoforms, fibers were assigned as ‘slow’, ‘fast’ or ‘hybrid’. To determine the overlap of the contractile features assigning a fiber as being slow, upset plots were generated using the upset function of the ComplexUpset package (v 1.3.3), and then simplified to bar plots. Principal component analysis (PCA) PCA was performed using the RunPCA function of the Seurat package. Scree plots were based on the fviz_eig function of the factoextra package (v 1.0.7), which worked after PCA with prcomp . Seurat clustering Uniform Manifold Approximation and Projection (UMAP) clustering was performed based on the K-nearest neighbor graph with the first 6 dimensions as input for both the transcriptome and proteome datasets (Supplementary fig. ). Feature plots were generated using the FeaturePlot function. UMAP plots were colored based on different criteria (MYH-based fiber types, participant, test day) stored in the metadata. Enrichment analysis Genes and protein sets were processed to obtain lists of features that were differentially expressed or, in the case of the top PCA drivers, in the top 5% drivers for the positive and negative direction of the first and second principal component. Over-representation analysis was then performed on these features with the enrichGO and simplify function of the clusterProfiler package (v 4.6.2) using all gene ontology terms. Obtained lists of significant terms were manually curated to extract interesting and relevant terms. Hierarchical clustering of ribosomal proteins Raw proteomics data was log2 transformed and filtered to contain proteins enlisted in the ‘cytosolic ribosome’ GO term, followed by Z-scoring prior heatmap visualization using the pheatmap function from the Pheatmap package (v 1.0.12). The number of clusters was determined by visual inspection and assigning a value of 3 to the cuttree function. Differential expression analysis To avoid artificially inflated p -values, which would arise from regarding every fiber as an independent replicate, we employed a pseudobulk differential expression analysis approach. We mathematically downsampled the total data points to one value per MYH-based fiber type per participant by aggregating (transcriptomics) or taking the median value (proteomics). Transcriptomics data were further processed using the DESeq2 pipeline (v 1.38.3) with a ‘~ participant + fiber type’ statistical model. 1000 fiber proteomics data was processed using the limma workflow, fitting the data to a linear model defined as: ‘~ 0 + fiber type + participant‘, whereas the myopathy dataset was fitted to ‘~ 0 + condition’ for the comparisons between conditions and ‘~ 0 + fiber type and condition’ for the comparisons including fiber type. Fitted models were then subjected to gene ranking using an empirical Bayes method using eBayes prior extracting the results through topTable , with p -value adjustment set to Benjamini-Hochberg, both functions from the limma package. Threshold for significantly different genes/proteins was defined as adjusted p- values smaller than 0.05 and a log fold change cut-off of 1 was applied. For the nemaline myopathy dataset, the Xiao significance score was applied, which combines expression fold change and statistical significance . Proteins with a Xiao score under 0.05 were regarded as differentially expressed between conditions. SCENIC Inference of active transcription factors in slow and fast fibers was performed using Single-Cell rEgulatory Network Inference and Clustering (SCENIC, pySCENIC version 0.12.1 with cisTarget v10 databases and annotations) . To prioritize fiber type specific transcription factors, both their fiber type specific expression at mRNA level and regulon activity were combined into a final prioritization score. This prioritization score was calculated as the sum of the z-score scaled differential expression score (logFC from pseudobulked data) and z-score scaled regulon specificity scores (RSS). Non-coding RNA For the transcriptomics data, the biotype of each gene was determined using the ‘GENEBIOTYPE’ column using the AnnotationDbi package (v 1.60.2) with the EnsDb.Hsapiens.v86 database. Genomic location interrogation was performed using the UCSC Human Genome Browser ( https://genome.ucsc.edu ). Tissue-specific gene expression of interesting long non-coding RNAs was explored using the GTEx Portal database. Microprotein identification Construction of putative lncRNA-encoded protein database RNA sequences of the non-coding transcripts were extracted using the getSequence function from the biomaRt package (v 2.56.1), with ‘transcript_exon_intron’ as the seqType . Both intergenic (lincRNA) and antisense long non-coding RNA (lncRNA) transcripts were utilized for database construction. A six-frame-translation was used to translate the corresponding RNA sequences into the proteins, as well as ORFfinder NCBI functionality ( https://www.ncbi.nlm.nih.gov/orffinder/ ) was used to extract open reading frames (ORFs) from the transcripts. Minimal ORF length was set to 75 nucleotides, genetic code was set to “Standard” and “Any sense codon” was used, as a start codon, to extract maximum number of open reading frames. The obtained protein fasta file contained multiple entries for each gene name, ensured by various combinations of: i) transcript identifiers, ii) ORF identifiers and iii) start:stop codons. Identification of lncRNA-encoded proteins DIA raw MS data were analyzed with Spectronaut v18 using an in-house generated sample-specific fasta, comprised of the reviewed human proteome (proteome ID: UP000005640, 20 426 proteins, downloaded Sep 2023) and lncRNA-encoded protein sequences (125 223 proteins), in directDIA mode. The default settings were used unless otherwise noted. Data filtering was set to “Qvalue”. False discovery rate (FDR) was set to 1% at peptide precursor level and 1% at protein level. Top3 peptide precursors were used for protein quantification. The downstream data analysis was performed using in-house developed R scripts. PCA projection The 1000 fiber and myopathy data sets were initially filtered to remove non-overlapping proteins. Then, they were combined and normalized using the normalizeBetweenArrays function from the limma package. The normalization method used was “quantile” to ensure that both data sets had the same distributions and were comparable. Subsequently, the merged data set was divided back into the two separate data sets, namely the 1000 fiber data set and the myopathy data set. For the 1000 fiber data set, PCA was calculated using the prcomp function. Moving on, the myopathy data set was multiplied by the PC loadings obtained from the 1000 fiber dataset to generate its PCA projection. Finally, the PCA projections from the myopathy samples were plotted on the top of the 1000 fiber PCA visualization. Muscle-specific ribosomal gene signature The skeletal muscle-specific ribosomal gene signature, consisting of log2 fold change values comparing the mRNA expression of ribosomal subunits in skeletal muscle compared to 52 other human tissues, was downloaded from Panda et al . Log2 fold change values were ranked to identify ribosomal proteins with the highest overexpression in human skeletal muscle. Structural analysis of ribosomal proteins The human 80S ribosome structural model (Protein Data Bank: 4V6X) was downloaded from the Protein Data Bank website (RCSB PDB). Visualization and editing of the ribosomal structure, and preparation of figures and movies were performed in UCSF ChimeraX . Antibody-based fiber typing Dot blot was conducted following a previously described protocol with a few modifications . Initially, two identical PVDF membranes were activated using 96% ethanol and washed with transfer buffer. Subsequently, the membranes were placed on wet filter paper with transfer buffer until they dried. Next, 1 µL of fiber lysate was spotted at the same position on both membranes, and the membranes were allowed to dry. Reactivation of the membranes was carried out using 96% ethanol, followed by gentle washing with TBST. The membranes were then blocked in TBST containing 5% skim milk for 15 minutes. After blocking, the membranes were washed three times with TBST and incubated with the primary antibody solution of either anti-MYH7 (A4.840) or anti-MYH2 (A4.74), both from Developmental Studies Hybridoma Bank (DSHB) at a dilution of 1:200 in TBST containing 1% skim milk for one hour. Subsequently, the membranes were gently washed three times with TBST and incubated with the secondary antibody (anti-mouse) at a dilution of 1:20,000 in TBST containing 1% skim milk for two hours. Finally, the membranes were washed three times for five minutes each with TBST and visualized using Immobilon Forte (Milipore) in a ChemiDoc XRS+ (Bio-Rad) imaging system. RNAscope 8 µm sections from three different fixed-frozen human muscle biopsies were used for RNAscope labeling and subsequent immunohistochemistry (IHC). For detection of RP11-255P5.3 and LINC01405 commercially available RNAscope Multiplex Fluorescent Assay V2 (Advanced Cell Diagnostics) and probes against Lnc-ERCC5-5-C1 (# 1276851-C1), and LINC01405-C2 (# 549201-C2) (Advanced Cell Diagnostics), were used according to the manufacturer’s protocols. To control tissue quality Positive 3-plex probe (# 320881) and negative 3-plex probe (# 320871) were used. To visualize different subtypes of muscle fibers, sections after RNAscope were blocked with 5% donkey serum and incubated with antibodies against MYH2 (A4.74-s (1:5); DSHB) and MYH7 (A4.840 (1:5); DSHB) overnight. After washing sections were incubated with Alexa Flour 488-conjugated Donkey Anti-Mouse IgG, Fcγ Subclass 1 and DyLightTM405-conjugated Donkey Anti-Mouse IgM secondary antibodies respectively and mounted with ProLong™ Diamond Antifade Mountant (Invitrogen). Slides were imaged using Zeiss Axio Observer microscope equipped with Axiocam 702 camera. Biopsies from three individuals were used for quantification, with 187 muscle fibers being counted in total. As in RNAscope each dot corresponds to one RNA molecule, the number of dots/mm 2 was used as a measure of RNA expression. We first determined the number of dots/mm 2 within each fiber for both probes, then averaged the results by fiber type and participant. The given average was then used as an input for a two sample t-test. Immunostaining for muscle sections Immunolabelling was performed on 10 μm cryosections, fixed in 4% PFA (10 min), permeabilized in 0.1% Triton X-100 (20 min) and blocked in 10% Normal Goat Serum (50062Z, Life Technologies) with 0.1% BSA (1 h). Sections were incubated o/n (4 o C) with primary antibodies against MYH7 (mouse monoclonal A4.951, Santa Cruz, sc-53090, diluted 1:25) or MYH2 (mouse monoclonal SC71, DSHB, 1:25), each combined with primary antibody against TNNT1 (rabbit polyclonal HPA058448, Sigma, diluted 1:500) in 5% goat serum with 0.1% BSA and 0.1% Triton X-100. Alexa Fluor Goat anti-Mouse 647 (A21237) was used as the secondary antibody for the MYHs and Alexa Fluor Donkey anti-Rabbit 488 (A11034) for the TNNT1 (Life Technologies, 1:500 each in 10% Normal Goat Serum). Fluorescent images were obtained with a 10x objective on a Zeiss Axio Observer 3 fluorescence microscope with a Colibri 5 led detector, combined with Zeiss Axiocam 705 mono camera, using Zen software (Zeiss). For visualization purposes, a selection of fibers were mounted on copper grids glued on a microscopy slide, and imaged under a stereomicroscope. Reporting summary Further information on research design is available in the linked to this article. Participant information Fourteen participants (12 males / 2 females) of Caucasian origin volunteered to take part in this study, which was approved by the Ethical Committee of Ghent University Hospital (BC-10237), in agreement with the 2013 Declaration of Helsinki and registered on ClinicalTrials.gov (NCT05131555). General characteristics of the participants can be found in Supplementary Table . After oral and written informed consent, participants were medically screened before final inclusion. Participants were young (22-42 years old), healthy (no diseases and non-smoking) and moderately physically active. Maximal oxygen uptake was determined as marker of physical fitness during a graded incremental cycling test, as previously described . Muscle biopsy collection Muscle biopsies were collected in the rested and fasted state, on three different days, separated by 14 days. As these samples were collected as part of a larger study, on each of these days participants ingested a placebo (lactose), an H1-receptor antagonist (540 mg fexofenadine) or an H2-receptor antagonist (40 mg famotidine) 40 minutes before muscle biopsy collection. We have previously shown that these histamine receptor antagonists do not affect the resting skeletal muscle state , and no clustering was apparent based on condition in our quality control plots (Supplementary figs. & Supplementary fig. ). Dietary intake was standardized 48 hours before each experimental day (41.4 kcal/kg body weight, 5.1 g/kg body weight carbohydrates, 1.4 g/kg body weight protein and 1.6 g/kg body weight fat per day), followed by a standardized breakfast on the morning of the experimental day (1.5 g/kg bodyweight carbohydrates). Muscle biopsies of the m. vastus lateralis were then collected after local anesthesia (0.5 mL of 1% Xylocaïne without epinephrine) using the percutaneous Bergström technique with suction . The muscle samples were immediately submerged in RNA later and stored at 4 °C until manual fiber dissection (max. 3 days). Single fiber isolation Freshly excised muscle fiber bundles were transferred to fresh RNA later in a petri dish. Individual muscle fibers were then manually dissected using a stereomicroscope and fine forceps. Twenty-five fibers were dissected per biopsy, with special care to select fibers from different sections of the biopsy. After dissection, each fiber was carefully submerged in 3 µL of lysis buffer (SingleShot Cell Lysis kit, Bio-rad), containing proteinase K and DNase enzymes to remove unwanted proteins and DNA. Next, cell lysis and protein/DNA removal was initiated by short vortexing, spinning liquid down in a microcentrifuge and incubation at room temperature (10 min). Lysates were then incubated for 5 min at 37 °C and 5 min at 75 °C in a thermocycler (T100, Bio-Rad), immediately followed by storage at −80 °C until further processing. Sequencing library preparation Illumina-compatible libraries from polyadenylated RNA were prepared from 2 µL of the muscle fiber lysates using the QuantSeq-Pool 3’ mRNA-Seq library prep kit (Lexogen). Detailed methodology can be found in the manufacturer guidelines. The process was initiated by reverse transcription for first strand cDNA synthesis, during which Unique Molecular Identifiers (UMIs) and sample-specific i1 barcodes were introduced enabling sample pooling and reducing technical variability in the downstream process. Next, cDNA from 96 fibers was pooled and purified using magnetic beads, followed by RNA removal and second strand synthesis by random priming. Libraries were purified using magnetic beads, followed by addition of pool-specific i5/i7 indices and PCR amplification. A last purification step was performed, finalizing the Illumina-compatible libraries. A high sensitivity small DNA Fragment Analysis kit (Agilent Technologies, DNF-477-0500) was used to assess the quality of each library pool. Illumina sequencing The individual pools were further equimolarly (2 nM) pooled, based on Qubit-quantified concentrations. The final pool was subsequently sequenced with a NovaSeq S2 kit (1 × 100 nucleotides) with a loading of 2 nM (4% PhiX) in standard mode on a NovaSeq 6000 instrument. Primary data processing Our pipeline was based on the QuantSeq Pool data analysis pipeline from Lexogen ( https://github.com/Lexogen-Tools/quantseqpool_analysis ). First, the data was demultiplexed based on the i7/i5 indices with bcl2fastq2 (v2.20.0). The next demultiplexing step was performed via idemux (v0.1.6) according to the i1 sample-specific barcodes in read 2, followed by extraction of UMI sequences with umi_tools (v1.0.1). Trimming of the reads was then performed in multiple rounds with cutadapt (v3.4), with removal of too short reads (length <20) or reads consisting entirely of adapter sequences. Reads were then aligned to the human genome with STAR (v2.6.0c), followed by BAM file indexing with SAMtools (v1.11). Duplicates of reads were removed with umi_tools (v1.0.1). Finally, counting of the alignments was performed with featureCounts from Subread (v2.0.3). At several intermediate steps during the pipeline, quality control with performed with FastQC (v0.11.9). Initial Seurat processing All further bioinformatics processing and visualization was performed in R (v4.2.3), primarily with the Seurat (v 4.4.0) workflow . The individual UMI counts and metadata matrices were thus transformed into a Seurat object. Genes with expression in less than 30% of all fibers were removed. Low-quality samples were then removed based on a minimum threshold of 1000 UMI counts and 1000 detected genes. This resulted in a total of 925 fibers that passed all quality control filtering steps. Normalization of UMI counts was performed using the SCTransform v2 Seurat method , including all 7418 detected features and regressing out participant variation. All relevant metadata can be found in Supplementary Dataset . Fourteen participants (12 males / 2 females) of Caucasian origin volunteered to take part in this study, which was approved by the Ethical Committee of Ghent University Hospital (BC-10237), in agreement with the 2013 Declaration of Helsinki and registered on ClinicalTrials.gov (NCT05131555). General characteristics of the participants can be found in Supplementary Table . After oral and written informed consent, participants were medically screened before final inclusion. Participants were young (22-42 years old), healthy (no diseases and non-smoking) and moderately physically active. Maximal oxygen uptake was determined as marker of physical fitness during a graded incremental cycling test, as previously described . Muscle biopsies were collected in the rested and fasted state, on three different days, separated by 14 days. As these samples were collected as part of a larger study, on each of these days participants ingested a placebo (lactose), an H1-receptor antagonist (540 mg fexofenadine) or an H2-receptor antagonist (40 mg famotidine) 40 minutes before muscle biopsy collection. We have previously shown that these histamine receptor antagonists do not affect the resting skeletal muscle state , and no clustering was apparent based on condition in our quality control plots (Supplementary figs. & Supplementary fig. ). Dietary intake was standardized 48 hours before each experimental day (41.4 kcal/kg body weight, 5.1 g/kg body weight carbohydrates, 1.4 g/kg body weight protein and 1.6 g/kg body weight fat per day), followed by a standardized breakfast on the morning of the experimental day (1.5 g/kg bodyweight carbohydrates). Muscle biopsies of the m. vastus lateralis were then collected after local anesthesia (0.5 mL of 1% Xylocaïne without epinephrine) using the percutaneous Bergström technique with suction . The muscle samples were immediately submerged in RNA later and stored at 4 °C until manual fiber dissection (max. 3 days). Freshly excised muscle fiber bundles were transferred to fresh RNA later in a petri dish. Individual muscle fibers were then manually dissected using a stereomicroscope and fine forceps. Twenty-five fibers were dissected per biopsy, with special care to select fibers from different sections of the biopsy. After dissection, each fiber was carefully submerged in 3 µL of lysis buffer (SingleShot Cell Lysis kit, Bio-rad), containing proteinase K and DNase enzymes to remove unwanted proteins and DNA. Next, cell lysis and protein/DNA removal was initiated by short vortexing, spinning liquid down in a microcentrifuge and incubation at room temperature (10 min). Lysates were then incubated for 5 min at 37 °C and 5 min at 75 °C in a thermocycler (T100, Bio-Rad), immediately followed by storage at −80 °C until further processing. Illumina-compatible libraries from polyadenylated RNA were prepared from 2 µL of the muscle fiber lysates using the QuantSeq-Pool 3’ mRNA-Seq library prep kit (Lexogen). Detailed methodology can be found in the manufacturer guidelines. The process was initiated by reverse transcription for first strand cDNA synthesis, during which Unique Molecular Identifiers (UMIs) and sample-specific i1 barcodes were introduced enabling sample pooling and reducing technical variability in the downstream process. Next, cDNA from 96 fibers was pooled and purified using magnetic beads, followed by RNA removal and second strand synthesis by random priming. Libraries were purified using magnetic beads, followed by addition of pool-specific i5/i7 indices and PCR amplification. A last purification step was performed, finalizing the Illumina-compatible libraries. A high sensitivity small DNA Fragment Analysis kit (Agilent Technologies, DNF-477-0500) was used to assess the quality of each library pool. The individual pools were further equimolarly (2 nM) pooled, based on Qubit-quantified concentrations. The final pool was subsequently sequenced with a NovaSeq S2 kit (1 × 100 nucleotides) with a loading of 2 nM (4% PhiX) in standard mode on a NovaSeq 6000 instrument. Our pipeline was based on the QuantSeq Pool data analysis pipeline from Lexogen ( https://github.com/Lexogen-Tools/quantseqpool_analysis ). First, the data was demultiplexed based on the i7/i5 indices with bcl2fastq2 (v2.20.0). The next demultiplexing step was performed via idemux (v0.1.6) according to the i1 sample-specific barcodes in read 2, followed by extraction of UMI sequences with umi_tools (v1.0.1). Trimming of the reads was then performed in multiple rounds with cutadapt (v3.4), with removal of too short reads (length <20) or reads consisting entirely of adapter sequences. Reads were then aligned to the human genome with STAR (v2.6.0c), followed by BAM file indexing with SAMtools (v1.11). Duplicates of reads were removed with umi_tools (v1.0.1). Finally, counting of the alignments was performed with featureCounts from Subread (v2.0.3). At several intermediate steps during the pipeline, quality control with performed with FastQC (v0.11.9). All further bioinformatics processing and visualization was performed in R (v4.2.3), primarily with the Seurat (v 4.4.0) workflow . The individual UMI counts and metadata matrices were thus transformed into a Seurat object. Genes with expression in less than 30% of all fibers were removed. Low-quality samples were then removed based on a minimum threshold of 1000 UMI counts and 1000 detected genes. This resulted in a total of 925 fibers that passed all quality control filtering steps. Normalization of UMI counts was performed using the SCTransform v2 Seurat method , including all 7418 detected features and regressing out participant variation. All relevant metadata can be found in Supplementary Dataset . Sample collection - 1000 fiber proteome Participant information Stored biobank muscle specimens were used for the purpose of the present study (Clinicaltrials.gov identifier: NCT04048993). The specimens were collected from five active and healthy male volunteers (aged 21–35 years) of Caucasian ancestry who gave their written and oral informed consent with approval from the Science Ethics Committee of the Capital Region in Denmark (H-1-2012-090) and complied with the guidelines of the 2013 Declaration of Helsinki. General characteristics of the participants can be found in Supplementary Table . Participants were young, healthy (no diseases and non-smoking) and moderately physically active. Muscle biopsy collection Participants arrived in the morning after an overnight fast and rested in the supine position for 1 hour. Then, local anesthesia (2-3 mL Xylocaine 2%; lidocaine without epinephrine, AstraZeneca, Denmark) was applied under the skin above the fascia at the belly of the m. vastus lateralis muscle. A muscle biopsy was sampled through a small 3–4 mm incision using a Bergström needle with suction. The muscle biopsy specimen was snap-frozen in liquid nitrogen and stored at −80 °C until analysis. Single muscle fiber isolation Muscle fibers were isolated from freeze-dried specimens as previously described . In brief, muscle biopsies were freeze-dried for 48 hours. Subsequently, fibers were isolated in a humidity- and temperature-controlled room (humidity of 25%) using fine forceps under a stereomicroscope. ~200 single muscle fibers were isolated for each biopsy, resulting in a total of 1038. To ensure the fibers settled at the bottom of the tube, each fiber-containing tube underwent centrifugation at 20,000 g using a small centrifuge. Next, fibers were resuspended in 15 µL of lysis buffer (1% sodium dodecyl sulfate (SDC), 40 mM chloroacetamide (CAA), 10 mM dithiothreitol (DTT) in 50 mM Tris pH 8.5). Sample collection – myopathy Participant information Six patients with severe nemaline myopathy were selected from our nemaline myopathy study cohort. Three patients (2 male and 1 female) had pathogenic variants in ACTA1 , representing the conventional severe form, and three patients had pathogenic variants in TNNT1 (3 male), resulting in a rare, progressive form of nemaline myopathy. Three healthy individuals with no history of neuromuscular disease were used as controls. All participants are of Caucasian ancestry (Supplementary Table ). Muscle biopsy collection Healthy control participant biopsies ( n = 3 males) were used from an original study , and were therefore collected, snap frozen in liquid nitrogen and stored in -80°C as per that originally described. For the present study, a fragment of this stored biopsy was dissected under sterile, frozen conditions before being prepared for single myofiber isolation (detailed below). Acquisition of biopsies of healthy control patients was approved by local ethics committee (Copenhagen and Frederiksberg) in Denmark (hs:h-15002266). Those of myopathy patients were consented, stored, and used in accordance with the Human Tissue Act under local ethical approval in United Kingdom (REC 13/NE/0373). All procedures were carried out in accordance to the Declaration of Helsinki. Single fiber isolation Dissected fragments of muscle biopsy were placed in ice-cold, 22 micron filtered relaxing solution (4 mM Mg-ATP, 1 mM free Mg 2+ , 10 -6.00 mM free Ca 2+ , 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate, KCl to an iconic strength of 180 mM and pH to 7.0) for ~3 minutes before being immersed in fresh relaxing solution on a sterile petri dish and mounted on ice under a dissection microscope for single fiber isolation. Fibers were cleaved from the tissue/biopsy ensuring a variety of sample/biopsy locations were used and that only single fibers were selected. Following isolation, fibers were manually moved to a sterile 96-well plate containing 15 µL of lysis buffer (identical to that detailed above) where the tweezers containing the fiber were submerged, spun and agitated in lysis buffer to ensure the fibers dissociated from the tweezers. To ensure fibers were settled to the bottom of the well, the 96-well plate was subjected to gentle vortexing and centrifugation (1,000 g). Proteomics analysis Sample preparation Samples from both proteomics studies followed the same sample preparation workflow. In order to extract the proteins, samples were boiled at 95 °C in a thermomixer with gentle shaking (800 rpm) and sonicated in a bioruptor instrument with 30 seconds on/off cycles for 15 minutes. A small 5 µL fraction of lysate from each sample was saved for antibody-based fiber typing of the 1000 fiber samples. Next, samples were processed following a modified version of the in-solution digestion sample preparation protocol. In brief, total volume was adjusted to 50 µl by addition of digestion buffer, containing 50 mM Tris PH 8.5 buffer, an enzyme to protein ratio of 1:500 LysC (Wako) and a 1:100 enzyme to protein ratio of trypsin (Promega). Single muscle fiber lysates were digested overnight in a thermomixer set to 37° C and 800 rpm. The next day, protein digestion was quenched by addition of 50 µl of 2% trifluoroacetic acid (TFA) in isopropanol. Peptides were desalted using in-house prepared single-use reverse-phase StageTips containing styrenedivinylbenzene reverse-phase sulfonate (SDB-RPS) disks. Then, desalted peptides were loaded in Evotips (Evosep) following manufacturer instructions prior LC-MS/MS analysis. Bulk tissue samples were prepared using the same protocol utilized for single fibers, with a few modifications to sample lysis. Tissue samples were first powdered using a tissue crusher over dry ice before resuspending the powder in the same lysis buffer described above. Then, the samples were homogenized using an IKA Turrax homogenizer for 2 minutes prior boiling and sonication. From there onwards the samples underwent the same protocol described above. Proteomics library preparation Fibers from the five healthy control individuals participating in the 1000 fiber study were carefully dissected and combined in order to create a pooled fiber lysate. Then, 200 µg of protein corresponding to each participant-specific lysate were pooled together into one final protein lysate that was processed following the same sample preparation workflow just described. 20 µg of desalted peptides were fractionated using High PH Reverse-Phase Chromatography (HpH-RP). Fractionation was carried out on a Kinetex 2.6 µm EVO C18 100 Å, 150 ×0.3 mm column manufactured by Phenomenex and using an EASY-nLC 1200 System (Thermo) operating at 1.5 µL/min. Separation was accomplished using a 62 min step-gradient starting from 3% to 60% solvent B (which consisted of 10 mM TEAB in 80% acetonitrile) and solvent A (containing 10 mM TEAB in water). The total run time was 98 min, which included wash and column equilibration. Throughout the fractionation, peptides were eluted and collected every 60 s, obtaining 96 single fractions without concatenation. Finally. 200 ng of HpH-RP fractionated peptides were loaded, concentrated and desalted on Evotips (Evosep) following the instructions provided by the manufacturer. Liquid chromatography tandem mass spectrometry Proteomics measurements were performed using LC-MS instrumentation consisting of an Evosep One HPLC system (Evosep) coupled via electrospray ionization to a timsTOF SCP mass spectrometer (Bruker). Peptides were separated utilizing 8 cm, 150 μM ID columns packed with C18 beads (1.5 μm) (Evosep). Chromatographic separation was achieved by the ‘60 samples per day’ method, followed by electrospray ionization through a CaptiveSpray ion source and a 10 μm emitter into the MS instrument. Single muscle fiber peptides were measured in DIA-PASEF mode following a previously described method , while library fractions were measured using DDA-PASEF. In brief, the DDA-PASEF scan range encompassed 100–1700 m/z for both MS and MS/MS, and TIMS mobility range was set to 0.6–1.6 (V cm −2 ). Both TIMS ramp and accumulation times were configured to 100 ms, and 10 PASEF ramps were recorded for a total cycle time of 1.17 s. The MS/MS target intensity and intensity threshold were defined as 20.000 and 1.000, respectively. An exclusion list of 0.4 min for precursors within 0.015 m/z and 0.015 V cm −2 width was also activated. For DIA-PASEF the scan range was established at 400-1000 (m/z), the TIMS mobility range to 0.64-1.37 (V cm −2 ), and ramp and accumulation times were both set to 100 ms. A short-gradient method was used, which included 8 DIA-PASEF scans with three 25 Da windows per ramp, resulting in an estimated cycle time of 0.95 sec. MS data processing Library files were processed using the MS-Fragger functionality within Fragpipe v19.0 under the SpecLib workflow with default settings, including a minimum peptide length of seven amino acids and maximum of two missed cleavages allowed . Spectra were searched against a human reviewed FASTA from Uniprot (March 2022, 20410 entries) and the output library contained a total of 5350 protein groups and 84383 precursors. Sample raw MS files were analyzed using DIA-NN version 1.8 in a library-based manner against the MS library just described. Protein groups quantification was based on proteotypic peptides, neural network was set to double-pass mode, quantification strategy was set to “Robust LC (high accuracy)” and the match between runs options was enabled, the rest of parameters remained as default, which included precursor FDR set to 1% and peptide length of 7-30 amino acids. Data processing Further data analysis was performed under the R environment (R version 4.22). Both a metadata data frame containing sample and participant information and the “PG_matrix.tsv” file from DIA-NN’s output were loaded in RStudio. The 1000 fiber data frame was filtered to remove samples with less than 50% valid protein intensity values, resulting in a total of 974 fibers. Next, rows were filtered to remove proteins with less than 30% valid values across samples, resulting in a total of 1685 proteins. Regarding the myopathy dataset, after filtering samples for 50% valid values, the number of samples was 250. We included in the analysis proteins that were quantified in 70% of the samples in at least one condition (conditions: control, actin myopathy and troponin myopathy), resulting in a total of 1545 proteins. Both data frames were then log2 transformed and normalized using the normalizeBetweenArrays function from the limma package (v 3.54.2), with the method argument set to quantile . Then, batch correction through the ComBat function from the sva package (v3.50.0) was applied to minimize the effect of the three technical batches originated during mass spectrometry measurement. Finally, missing values were replaced by random numbers from a Gaussian distribution with the default settings of the tImpute function from the PhosR package (v 1.12.0) . All relevant metadata for the 1000 fiber proteome and nemaline myopathy datasets can be found in Supplementary Data & , respectively. Participant information Stored biobank muscle specimens were used for the purpose of the present study (Clinicaltrials.gov identifier: NCT04048993). The specimens were collected from five active and healthy male volunteers (aged 21–35 years) of Caucasian ancestry who gave their written and oral informed consent with approval from the Science Ethics Committee of the Capital Region in Denmark (H-1-2012-090) and complied with the guidelines of the 2013 Declaration of Helsinki. General characteristics of the participants can be found in Supplementary Table . Participants were young, healthy (no diseases and non-smoking) and moderately physically active. Muscle biopsy collection Participants arrived in the morning after an overnight fast and rested in the supine position for 1 hour. Then, local anesthesia (2-3 mL Xylocaine 2%; lidocaine without epinephrine, AstraZeneca, Denmark) was applied under the skin above the fascia at the belly of the m. vastus lateralis muscle. A muscle biopsy was sampled through a small 3–4 mm incision using a Bergström needle with suction. The muscle biopsy specimen was snap-frozen in liquid nitrogen and stored at −80 °C until analysis. Single muscle fiber isolation Muscle fibers were isolated from freeze-dried specimens as previously described . In brief, muscle biopsies were freeze-dried for 48 hours. Subsequently, fibers were isolated in a humidity- and temperature-controlled room (humidity of 25%) using fine forceps under a stereomicroscope. ~200 single muscle fibers were isolated for each biopsy, resulting in a total of 1038. To ensure the fibers settled at the bottom of the tube, each fiber-containing tube underwent centrifugation at 20,000 g using a small centrifuge. Next, fibers were resuspended in 15 µL of lysis buffer (1% sodium dodecyl sulfate (SDC), 40 mM chloroacetamide (CAA), 10 mM dithiothreitol (DTT) in 50 mM Tris pH 8.5). Stored biobank muscle specimens were used for the purpose of the present study (Clinicaltrials.gov identifier: NCT04048993). The specimens were collected from five active and healthy male volunteers (aged 21–35 years) of Caucasian ancestry who gave their written and oral informed consent with approval from the Science Ethics Committee of the Capital Region in Denmark (H-1-2012-090) and complied with the guidelines of the 2013 Declaration of Helsinki. General characteristics of the participants can be found in Supplementary Table . Participants were young, healthy (no diseases and non-smoking) and moderately physically active. Participants arrived in the morning after an overnight fast and rested in the supine position for 1 hour. Then, local anesthesia (2-3 mL Xylocaine 2%; lidocaine without epinephrine, AstraZeneca, Denmark) was applied under the skin above the fascia at the belly of the m. vastus lateralis muscle. A muscle biopsy was sampled through a small 3–4 mm incision using a Bergström needle with suction. The muscle biopsy specimen was snap-frozen in liquid nitrogen and stored at −80 °C until analysis. Muscle fibers were isolated from freeze-dried specimens as previously described . In brief, muscle biopsies were freeze-dried for 48 hours. Subsequently, fibers were isolated in a humidity- and temperature-controlled room (humidity of 25%) using fine forceps under a stereomicroscope. ~200 single muscle fibers were isolated for each biopsy, resulting in a total of 1038. To ensure the fibers settled at the bottom of the tube, each fiber-containing tube underwent centrifugation at 20,000 g using a small centrifuge. Next, fibers were resuspended in 15 µL of lysis buffer (1% sodium dodecyl sulfate (SDC), 40 mM chloroacetamide (CAA), 10 mM dithiothreitol (DTT) in 50 mM Tris pH 8.5). Participant information Six patients with severe nemaline myopathy were selected from our nemaline myopathy study cohort. Three patients (2 male and 1 female) had pathogenic variants in ACTA1 , representing the conventional severe form, and three patients had pathogenic variants in TNNT1 (3 male), resulting in a rare, progressive form of nemaline myopathy. Three healthy individuals with no history of neuromuscular disease were used as controls. All participants are of Caucasian ancestry (Supplementary Table ). Muscle biopsy collection Healthy control participant biopsies ( n = 3 males) were used from an original study , and were therefore collected, snap frozen in liquid nitrogen and stored in -80°C as per that originally described. For the present study, a fragment of this stored biopsy was dissected under sterile, frozen conditions before being prepared for single myofiber isolation (detailed below). Acquisition of biopsies of healthy control patients was approved by local ethics committee (Copenhagen and Frederiksberg) in Denmark (hs:h-15002266). Those of myopathy patients were consented, stored, and used in accordance with the Human Tissue Act under local ethical approval in United Kingdom (REC 13/NE/0373). All procedures were carried out in accordance to the Declaration of Helsinki. Single fiber isolation Dissected fragments of muscle biopsy were placed in ice-cold, 22 micron filtered relaxing solution (4 mM Mg-ATP, 1 mM free Mg 2+ , 10 -6.00 mM free Ca 2+ , 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate, KCl to an iconic strength of 180 mM and pH to 7.0) for ~3 minutes before being immersed in fresh relaxing solution on a sterile petri dish and mounted on ice under a dissection microscope for single fiber isolation. Fibers were cleaved from the tissue/biopsy ensuring a variety of sample/biopsy locations were used and that only single fibers were selected. Following isolation, fibers were manually moved to a sterile 96-well plate containing 15 µL of lysis buffer (identical to that detailed above) where the tweezers containing the fiber were submerged, spun and agitated in lysis buffer to ensure the fibers dissociated from the tweezers. To ensure fibers were settled to the bottom of the well, the 96-well plate was subjected to gentle vortexing and centrifugation (1,000 g). Six patients with severe nemaline myopathy were selected from our nemaline myopathy study cohort. Three patients (2 male and 1 female) had pathogenic variants in ACTA1 , representing the conventional severe form, and three patients had pathogenic variants in TNNT1 (3 male), resulting in a rare, progressive form of nemaline myopathy. Three healthy individuals with no history of neuromuscular disease were used as controls. All participants are of Caucasian ancestry (Supplementary Table ). Healthy control participant biopsies ( n = 3 males) were used from an original study , and were therefore collected, snap frozen in liquid nitrogen and stored in -80°C as per that originally described. For the present study, a fragment of this stored biopsy was dissected under sterile, frozen conditions before being prepared for single myofiber isolation (detailed below). Acquisition of biopsies of healthy control patients was approved by local ethics committee (Copenhagen and Frederiksberg) in Denmark (hs:h-15002266). Those of myopathy patients were consented, stored, and used in accordance with the Human Tissue Act under local ethical approval in United Kingdom (REC 13/NE/0373). All procedures were carried out in accordance to the Declaration of Helsinki. Dissected fragments of muscle biopsy were placed in ice-cold, 22 micron filtered relaxing solution (4 mM Mg-ATP, 1 mM free Mg 2+ , 10 -6.00 mM free Ca 2+ , 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate, KCl to an iconic strength of 180 mM and pH to 7.0) for ~3 minutes before being immersed in fresh relaxing solution on a sterile petri dish and mounted on ice under a dissection microscope for single fiber isolation. Fibers were cleaved from the tissue/biopsy ensuring a variety of sample/biopsy locations were used and that only single fibers were selected. Following isolation, fibers were manually moved to a sterile 96-well plate containing 15 µL of lysis buffer (identical to that detailed above) where the tweezers containing the fiber were submerged, spun and agitated in lysis buffer to ensure the fibers dissociated from the tweezers. To ensure fibers were settled to the bottom of the well, the 96-well plate was subjected to gentle vortexing and centrifugation (1,000 g). Sample preparation Samples from both proteomics studies followed the same sample preparation workflow. In order to extract the proteins, samples were boiled at 95 °C in a thermomixer with gentle shaking (800 rpm) and sonicated in a bioruptor instrument with 30 seconds on/off cycles for 15 minutes. A small 5 µL fraction of lysate from each sample was saved for antibody-based fiber typing of the 1000 fiber samples. Next, samples were processed following a modified version of the in-solution digestion sample preparation protocol. In brief, total volume was adjusted to 50 µl by addition of digestion buffer, containing 50 mM Tris PH 8.5 buffer, an enzyme to protein ratio of 1:500 LysC (Wako) and a 1:100 enzyme to protein ratio of trypsin (Promega). Single muscle fiber lysates were digested overnight in a thermomixer set to 37° C and 800 rpm. The next day, protein digestion was quenched by addition of 50 µl of 2% trifluoroacetic acid (TFA) in isopropanol. Peptides were desalted using in-house prepared single-use reverse-phase StageTips containing styrenedivinylbenzene reverse-phase sulfonate (SDB-RPS) disks. Then, desalted peptides were loaded in Evotips (Evosep) following manufacturer instructions prior LC-MS/MS analysis. Bulk tissue samples were prepared using the same protocol utilized for single fibers, with a few modifications to sample lysis. Tissue samples were first powdered using a tissue crusher over dry ice before resuspending the powder in the same lysis buffer described above. Then, the samples were homogenized using an IKA Turrax homogenizer for 2 minutes prior boiling and sonication. From there onwards the samples underwent the same protocol described above. Proteomics library preparation Fibers from the five healthy control individuals participating in the 1000 fiber study were carefully dissected and combined in order to create a pooled fiber lysate. Then, 200 µg of protein corresponding to each participant-specific lysate were pooled together into one final protein lysate that was processed following the same sample preparation workflow just described. 20 µg of desalted peptides were fractionated using High PH Reverse-Phase Chromatography (HpH-RP). Fractionation was carried out on a Kinetex 2.6 µm EVO C18 100 Å, 150 ×0.3 mm column manufactured by Phenomenex and using an EASY-nLC 1200 System (Thermo) operating at 1.5 µL/min. Separation was accomplished using a 62 min step-gradient starting from 3% to 60% solvent B (which consisted of 10 mM TEAB in 80% acetonitrile) and solvent A (containing 10 mM TEAB in water). The total run time was 98 min, which included wash and column equilibration. Throughout the fractionation, peptides were eluted and collected every 60 s, obtaining 96 single fractions without concatenation. Finally. 200 ng of HpH-RP fractionated peptides were loaded, concentrated and desalted on Evotips (Evosep) following the instructions provided by the manufacturer. Liquid chromatography tandem mass spectrometry Proteomics measurements were performed using LC-MS instrumentation consisting of an Evosep One HPLC system (Evosep) coupled via electrospray ionization to a timsTOF SCP mass spectrometer (Bruker). Peptides were separated utilizing 8 cm, 150 μM ID columns packed with C18 beads (1.5 μm) (Evosep). Chromatographic separation was achieved by the ‘60 samples per day’ method, followed by electrospray ionization through a CaptiveSpray ion source and a 10 μm emitter into the MS instrument. Single muscle fiber peptides were measured in DIA-PASEF mode following a previously described method , while library fractions were measured using DDA-PASEF. In brief, the DDA-PASEF scan range encompassed 100–1700 m/z for both MS and MS/MS, and TIMS mobility range was set to 0.6–1.6 (V cm −2 ). Both TIMS ramp and accumulation times were configured to 100 ms, and 10 PASEF ramps were recorded for a total cycle time of 1.17 s. The MS/MS target intensity and intensity threshold were defined as 20.000 and 1.000, respectively. An exclusion list of 0.4 min for precursors within 0.015 m/z and 0.015 V cm −2 width was also activated. For DIA-PASEF the scan range was established at 400-1000 (m/z), the TIMS mobility range to 0.64-1.37 (V cm −2 ), and ramp and accumulation times were both set to 100 ms. A short-gradient method was used, which included 8 DIA-PASEF scans with three 25 Da windows per ramp, resulting in an estimated cycle time of 0.95 sec. MS data processing Library files were processed using the MS-Fragger functionality within Fragpipe v19.0 under the SpecLib workflow with default settings, including a minimum peptide length of seven amino acids and maximum of two missed cleavages allowed . Spectra were searched against a human reviewed FASTA from Uniprot (March 2022, 20410 entries) and the output library contained a total of 5350 protein groups and 84383 precursors. Sample raw MS files were analyzed using DIA-NN version 1.8 in a library-based manner against the MS library just described. Protein groups quantification was based on proteotypic peptides, neural network was set to double-pass mode, quantification strategy was set to “Robust LC (high accuracy)” and the match between runs options was enabled, the rest of parameters remained as default, which included precursor FDR set to 1% and peptide length of 7-30 amino acids. Data processing Further data analysis was performed under the R environment (R version 4.22). Both a metadata data frame containing sample and participant information and the “PG_matrix.tsv” file from DIA-NN’s output were loaded in RStudio. The 1000 fiber data frame was filtered to remove samples with less than 50% valid protein intensity values, resulting in a total of 974 fibers. Next, rows were filtered to remove proteins with less than 30% valid values across samples, resulting in a total of 1685 proteins. Regarding the myopathy dataset, after filtering samples for 50% valid values, the number of samples was 250. We included in the analysis proteins that were quantified in 70% of the samples in at least one condition (conditions: control, actin myopathy and troponin myopathy), resulting in a total of 1545 proteins. Both data frames were then log2 transformed and normalized using the normalizeBetweenArrays function from the limma package (v 3.54.2), with the method argument set to quantile . Then, batch correction through the ComBat function from the sva package (v3.50.0) was applied to minimize the effect of the three technical batches originated during mass spectrometry measurement. Finally, missing values were replaced by random numbers from a Gaussian distribution with the default settings of the tImpute function from the PhosR package (v 1.12.0) . All relevant metadata for the 1000 fiber proteome and nemaline myopathy datasets can be found in Supplementary Data & , respectively. Samples from both proteomics studies followed the same sample preparation workflow. In order to extract the proteins, samples were boiled at 95 °C in a thermomixer with gentle shaking (800 rpm) and sonicated in a bioruptor instrument with 30 seconds on/off cycles for 15 minutes. A small 5 µL fraction of lysate from each sample was saved for antibody-based fiber typing of the 1000 fiber samples. Next, samples were processed following a modified version of the in-solution digestion sample preparation protocol. In brief, total volume was adjusted to 50 µl by addition of digestion buffer, containing 50 mM Tris PH 8.5 buffer, an enzyme to protein ratio of 1:500 LysC (Wako) and a 1:100 enzyme to protein ratio of trypsin (Promega). Single muscle fiber lysates were digested overnight in a thermomixer set to 37° C and 800 rpm. The next day, protein digestion was quenched by addition of 50 µl of 2% trifluoroacetic acid (TFA) in isopropanol. Peptides were desalted using in-house prepared single-use reverse-phase StageTips containing styrenedivinylbenzene reverse-phase sulfonate (SDB-RPS) disks. Then, desalted peptides were loaded in Evotips (Evosep) following manufacturer instructions prior LC-MS/MS analysis. Bulk tissue samples were prepared using the same protocol utilized for single fibers, with a few modifications to sample lysis. Tissue samples were first powdered using a tissue crusher over dry ice before resuspending the powder in the same lysis buffer described above. Then, the samples were homogenized using an IKA Turrax homogenizer for 2 minutes prior boiling and sonication. From there onwards the samples underwent the same protocol described above. Fibers from the five healthy control individuals participating in the 1000 fiber study were carefully dissected and combined in order to create a pooled fiber lysate. Then, 200 µg of protein corresponding to each participant-specific lysate were pooled together into one final protein lysate that was processed following the same sample preparation workflow just described. 20 µg of desalted peptides were fractionated using High PH Reverse-Phase Chromatography (HpH-RP). Fractionation was carried out on a Kinetex 2.6 µm EVO C18 100 Å, 150 ×0.3 mm column manufactured by Phenomenex and using an EASY-nLC 1200 System (Thermo) operating at 1.5 µL/min. Separation was accomplished using a 62 min step-gradient starting from 3% to 60% solvent B (which consisted of 10 mM TEAB in 80% acetonitrile) and solvent A (containing 10 mM TEAB in water). The total run time was 98 min, which included wash and column equilibration. Throughout the fractionation, peptides were eluted and collected every 60 s, obtaining 96 single fractions without concatenation. Finally. 200 ng of HpH-RP fractionated peptides were loaded, concentrated and desalted on Evotips (Evosep) following the instructions provided by the manufacturer. Proteomics measurements were performed using LC-MS instrumentation consisting of an Evosep One HPLC system (Evosep) coupled via electrospray ionization to a timsTOF SCP mass spectrometer (Bruker). Peptides were separated utilizing 8 cm, 150 μM ID columns packed with C18 beads (1.5 μm) (Evosep). Chromatographic separation was achieved by the ‘60 samples per day’ method, followed by electrospray ionization through a CaptiveSpray ion source and a 10 μm emitter into the MS instrument. Single muscle fiber peptides were measured in DIA-PASEF mode following a previously described method , while library fractions were measured using DDA-PASEF. In brief, the DDA-PASEF scan range encompassed 100–1700 m/z for both MS and MS/MS, and TIMS mobility range was set to 0.6–1.6 (V cm −2 ). Both TIMS ramp and accumulation times were configured to 100 ms, and 10 PASEF ramps were recorded for a total cycle time of 1.17 s. The MS/MS target intensity and intensity threshold were defined as 20.000 and 1.000, respectively. An exclusion list of 0.4 min for precursors within 0.015 m/z and 0.015 V cm −2 width was also activated. For DIA-PASEF the scan range was established at 400-1000 (m/z), the TIMS mobility range to 0.64-1.37 (V cm −2 ), and ramp and accumulation times were both set to 100 ms. A short-gradient method was used, which included 8 DIA-PASEF scans with three 25 Da windows per ramp, resulting in an estimated cycle time of 0.95 sec. Library files were processed using the MS-Fragger functionality within Fragpipe v19.0 under the SpecLib workflow with default settings, including a minimum peptide length of seven amino acids and maximum of two missed cleavages allowed . Spectra were searched against a human reviewed FASTA from Uniprot (March 2022, 20410 entries) and the output library contained a total of 5350 protein groups and 84383 precursors. Sample raw MS files were analyzed using DIA-NN version 1.8 in a library-based manner against the MS library just described. Protein groups quantification was based on proteotypic peptides, neural network was set to double-pass mode, quantification strategy was set to “Robust LC (high accuracy)” and the match between runs options was enabled, the rest of parameters remained as default, which included precursor FDR set to 1% and peptide length of 7-30 amino acids. Further data analysis was performed under the R environment (R version 4.22). Both a metadata data frame containing sample and participant information and the “PG_matrix.tsv” file from DIA-NN’s output were loaded in RStudio. The 1000 fiber data frame was filtered to remove samples with less than 50% valid protein intensity values, resulting in a total of 974 fibers. Next, rows were filtered to remove proteins with less than 30% valid values across samples, resulting in a total of 1685 proteins. Regarding the myopathy dataset, after filtering samples for 50% valid values, the number of samples was 250. We included in the analysis proteins that were quantified in 70% of the samples in at least one condition (conditions: control, actin myopathy and troponin myopathy), resulting in a total of 1545 proteins. Both data frames were then log2 transformed and normalized using the normalizeBetweenArrays function from the limma package (v 3.54.2), with the method argument set to quantile . Then, batch correction through the ComBat function from the sva package (v3.50.0) was applied to minimize the effect of the three technical batches originated during mass spectrometry measurement. Finally, missing values were replaced by random numbers from a Gaussian distribution with the default settings of the tImpute function from the PhosR package (v 1.12.0) . All relevant metadata for the 1000 fiber proteome and nemaline myopathy datasets can be found in Supplementary Data & , respectively. Transcriptome and proteome dynamic range The expression/intensity for each gene/protein was calculated relative to the total counts/intensity for each fiber. This value was then averaged across fibers in each dataset and log10-transformed. The overlap of detected features between both datasets were analyzed using the VennDiagram package (v 1.7.3). Coefficient of variation—proteomics For each of the 96 well plates used during the MS measurement of the 1000 fiber study, one technical control sample was included in A1 position to monitor total ion current intensity and quality control of the runs (a total of eleven technical controls). The coefficient of variation between proteins was calculated by dividing the standard deviation by the mean of the LFQ intensities from each protein across technical replicates and then multiplied by one hundred. Correlation analyzes Mean log2 transformed transcript counts, protein intensities, and/or fold change values across fibers were calculated, filtered for shared proteins/genes and Pearson correlation was calculated. Omics-based fiber typing Normalized counts and raw LFQ intensities were retrieved from well-described contractile proteins that have a slow (MYH7, TNNT1, TPM3, ATP2A2 and MYL3) and fast (MYH2, MYH1, TNNT3, TPM1, ATP2A1 and MYL1) isoforms . For each isoform combination, the relative expression of each was then calculated and samples were ordered from high to low. The mathematical bottom knee for each curve was then determined using the barcodeRanks function in the DropletUtils package (v 1.18.1). This threshold was used to assign fiber types as pure (type 1, type 2A or type 2X) or hybrid (hybrid 1/2A, hybrid 2A/2X or hybrid 1/2X) (Supplementary Table ). For the features with only two isoforms, fibers were assigned as ‘slow’, ‘fast’ or ‘hybrid’. To determine the overlap of the contractile features assigning a fiber as being slow, upset plots were generated using the upset function of the ComplexUpset package (v 1.3.3), and then simplified to bar plots. Principal component analysis (PCA) PCA was performed using the RunPCA function of the Seurat package. Scree plots were based on the fviz_eig function of the factoextra package (v 1.0.7), which worked after PCA with prcomp . Seurat clustering Uniform Manifold Approximation and Projection (UMAP) clustering was performed based on the K-nearest neighbor graph with the first 6 dimensions as input for both the transcriptome and proteome datasets (Supplementary fig. ). Feature plots were generated using the FeaturePlot function. UMAP plots were colored based on different criteria (MYH-based fiber types, participant, test day) stored in the metadata. Enrichment analysis Genes and protein sets were processed to obtain lists of features that were differentially expressed or, in the case of the top PCA drivers, in the top 5% drivers for the positive and negative direction of the first and second principal component. Over-representation analysis was then performed on these features with the enrichGO and simplify function of the clusterProfiler package (v 4.6.2) using all gene ontology terms. Obtained lists of significant terms were manually curated to extract interesting and relevant terms. Hierarchical clustering of ribosomal proteins Raw proteomics data was log2 transformed and filtered to contain proteins enlisted in the ‘cytosolic ribosome’ GO term, followed by Z-scoring prior heatmap visualization using the pheatmap function from the Pheatmap package (v 1.0.12). The number of clusters was determined by visual inspection and assigning a value of 3 to the cuttree function. Differential expression analysis To avoid artificially inflated p -values, which would arise from regarding every fiber as an independent replicate, we employed a pseudobulk differential expression analysis approach. We mathematically downsampled the total data points to one value per MYH-based fiber type per participant by aggregating (transcriptomics) or taking the median value (proteomics). Transcriptomics data were further processed using the DESeq2 pipeline (v 1.38.3) with a ‘~ participant + fiber type’ statistical model. 1000 fiber proteomics data was processed using the limma workflow, fitting the data to a linear model defined as: ‘~ 0 + fiber type + participant‘, whereas the myopathy dataset was fitted to ‘~ 0 + condition’ for the comparisons between conditions and ‘~ 0 + fiber type and condition’ for the comparisons including fiber type. Fitted models were then subjected to gene ranking using an empirical Bayes method using eBayes prior extracting the results through topTable , with p -value adjustment set to Benjamini-Hochberg, both functions from the limma package. Threshold for significantly different genes/proteins was defined as adjusted p- values smaller than 0.05 and a log fold change cut-off of 1 was applied. For the nemaline myopathy dataset, the Xiao significance score was applied, which combines expression fold change and statistical significance . Proteins with a Xiao score under 0.05 were regarded as differentially expressed between conditions. SCENIC Inference of active transcription factors in slow and fast fibers was performed using Single-Cell rEgulatory Network Inference and Clustering (SCENIC, pySCENIC version 0.12.1 with cisTarget v10 databases and annotations) . To prioritize fiber type specific transcription factors, both their fiber type specific expression at mRNA level and regulon activity were combined into a final prioritization score. This prioritization score was calculated as the sum of the z-score scaled differential expression score (logFC from pseudobulked data) and z-score scaled regulon specificity scores (RSS). Non-coding RNA For the transcriptomics data, the biotype of each gene was determined using the ‘GENEBIOTYPE’ column using the AnnotationDbi package (v 1.60.2) with the EnsDb.Hsapiens.v86 database. Genomic location interrogation was performed using the UCSC Human Genome Browser ( https://genome.ucsc.edu ). Tissue-specific gene expression of interesting long non-coding RNAs was explored using the GTEx Portal database. The expression/intensity for each gene/protein was calculated relative to the total counts/intensity for each fiber. This value was then averaged across fibers in each dataset and log10-transformed. The overlap of detected features between both datasets were analyzed using the VennDiagram package (v 1.7.3). For each of the 96 well plates used during the MS measurement of the 1000 fiber study, one technical control sample was included in A1 position to monitor total ion current intensity and quality control of the runs (a total of eleven technical controls). The coefficient of variation between proteins was calculated by dividing the standard deviation by the mean of the LFQ intensities from each protein across technical replicates and then multiplied by one hundred. Mean log2 transformed transcript counts, protein intensities, and/or fold change values across fibers were calculated, filtered for shared proteins/genes and Pearson correlation was calculated. Normalized counts and raw LFQ intensities were retrieved from well-described contractile proteins that have a slow (MYH7, TNNT1, TPM3, ATP2A2 and MYL3) and fast (MYH2, MYH1, TNNT3, TPM1, ATP2A1 and MYL1) isoforms . For each isoform combination, the relative expression of each was then calculated and samples were ordered from high to low. The mathematical bottom knee for each curve was then determined using the barcodeRanks function in the DropletUtils package (v 1.18.1). This threshold was used to assign fiber types as pure (type 1, type 2A or type 2X) or hybrid (hybrid 1/2A, hybrid 2A/2X or hybrid 1/2X) (Supplementary Table ). For the features with only two isoforms, fibers were assigned as ‘slow’, ‘fast’ or ‘hybrid’. To determine the overlap of the contractile features assigning a fiber as being slow, upset plots were generated using the upset function of the ComplexUpset package (v 1.3.3), and then simplified to bar plots. PCA was performed using the RunPCA function of the Seurat package. Scree plots were based on the fviz_eig function of the factoextra package (v 1.0.7), which worked after PCA with prcomp . Uniform Manifold Approximation and Projection (UMAP) clustering was performed based on the K-nearest neighbor graph with the first 6 dimensions as input for both the transcriptome and proteome datasets (Supplementary fig. ). Feature plots were generated using the FeaturePlot function. UMAP plots were colored based on different criteria (MYH-based fiber types, participant, test day) stored in the metadata. Genes and protein sets were processed to obtain lists of features that were differentially expressed or, in the case of the top PCA drivers, in the top 5% drivers for the positive and negative direction of the first and second principal component. Over-representation analysis was then performed on these features with the enrichGO and simplify function of the clusterProfiler package (v 4.6.2) using all gene ontology terms. Obtained lists of significant terms were manually curated to extract interesting and relevant terms. Raw proteomics data was log2 transformed and filtered to contain proteins enlisted in the ‘cytosolic ribosome’ GO term, followed by Z-scoring prior heatmap visualization using the pheatmap function from the Pheatmap package (v 1.0.12). The number of clusters was determined by visual inspection and assigning a value of 3 to the cuttree function. To avoid artificially inflated p -values, which would arise from regarding every fiber as an independent replicate, we employed a pseudobulk differential expression analysis approach. We mathematically downsampled the total data points to one value per MYH-based fiber type per participant by aggregating (transcriptomics) or taking the median value (proteomics). Transcriptomics data were further processed using the DESeq2 pipeline (v 1.38.3) with a ‘~ participant + fiber type’ statistical model. 1000 fiber proteomics data was processed using the limma workflow, fitting the data to a linear model defined as: ‘~ 0 + fiber type + participant‘, whereas the myopathy dataset was fitted to ‘~ 0 + condition’ for the comparisons between conditions and ‘~ 0 + fiber type and condition’ for the comparisons including fiber type. Fitted models were then subjected to gene ranking using an empirical Bayes method using eBayes prior extracting the results through topTable , with p -value adjustment set to Benjamini-Hochberg, both functions from the limma package. Threshold for significantly different genes/proteins was defined as adjusted p- values smaller than 0.05 and a log fold change cut-off of 1 was applied. For the nemaline myopathy dataset, the Xiao significance score was applied, which combines expression fold change and statistical significance . Proteins with a Xiao score under 0.05 were regarded as differentially expressed between conditions. Inference of active transcription factors in slow and fast fibers was performed using Single-Cell rEgulatory Network Inference and Clustering (SCENIC, pySCENIC version 0.12.1 with cisTarget v10 databases and annotations) . To prioritize fiber type specific transcription factors, both their fiber type specific expression at mRNA level and regulon activity were combined into a final prioritization score. This prioritization score was calculated as the sum of the z-score scaled differential expression score (logFC from pseudobulked data) and z-score scaled regulon specificity scores (RSS). For the transcriptomics data, the biotype of each gene was determined using the ‘GENEBIOTYPE’ column using the AnnotationDbi package (v 1.60.2) with the EnsDb.Hsapiens.v86 database. Genomic location interrogation was performed using the UCSC Human Genome Browser ( https://genome.ucsc.edu ). Tissue-specific gene expression of interesting long non-coding RNAs was explored using the GTEx Portal database. Construction of putative lncRNA-encoded protein database RNA sequences of the non-coding transcripts were extracted using the getSequence function from the biomaRt package (v 2.56.1), with ‘transcript_exon_intron’ as the seqType . Both intergenic (lincRNA) and antisense long non-coding RNA (lncRNA) transcripts were utilized for database construction. A six-frame-translation was used to translate the corresponding RNA sequences into the proteins, as well as ORFfinder NCBI functionality ( https://www.ncbi.nlm.nih.gov/orffinder/ ) was used to extract open reading frames (ORFs) from the transcripts. Minimal ORF length was set to 75 nucleotides, genetic code was set to “Standard” and “Any sense codon” was used, as a start codon, to extract maximum number of open reading frames. The obtained protein fasta file contained multiple entries for each gene name, ensured by various combinations of: i) transcript identifiers, ii) ORF identifiers and iii) start:stop codons. Identification of lncRNA-encoded proteins DIA raw MS data were analyzed with Spectronaut v18 using an in-house generated sample-specific fasta, comprised of the reviewed human proteome (proteome ID: UP000005640, 20 426 proteins, downloaded Sep 2023) and lncRNA-encoded protein sequences (125 223 proteins), in directDIA mode. The default settings were used unless otherwise noted. Data filtering was set to “Qvalue”. False discovery rate (FDR) was set to 1% at peptide precursor level and 1% at protein level. Top3 peptide precursors were used for protein quantification. The downstream data analysis was performed using in-house developed R scripts. PCA projection The 1000 fiber and myopathy data sets were initially filtered to remove non-overlapping proteins. Then, they were combined and normalized using the normalizeBetweenArrays function from the limma package. The normalization method used was “quantile” to ensure that both data sets had the same distributions and were comparable. Subsequently, the merged data set was divided back into the two separate data sets, namely the 1000 fiber data set and the myopathy data set. For the 1000 fiber data set, PCA was calculated using the prcomp function. Moving on, the myopathy data set was multiplied by the PC loadings obtained from the 1000 fiber dataset to generate its PCA projection. Finally, the PCA projections from the myopathy samples were plotted on the top of the 1000 fiber PCA visualization. RNA sequences of the non-coding transcripts were extracted using the getSequence function from the biomaRt package (v 2.56.1), with ‘transcript_exon_intron’ as the seqType . Both intergenic (lincRNA) and antisense long non-coding RNA (lncRNA) transcripts were utilized for database construction. A six-frame-translation was used to translate the corresponding RNA sequences into the proteins, as well as ORFfinder NCBI functionality ( https://www.ncbi.nlm.nih.gov/orffinder/ ) was used to extract open reading frames (ORFs) from the transcripts. Minimal ORF length was set to 75 nucleotides, genetic code was set to “Standard” and “Any sense codon” was used, as a start codon, to extract maximum number of open reading frames. The obtained protein fasta file contained multiple entries for each gene name, ensured by various combinations of: i) transcript identifiers, ii) ORF identifiers and iii) start:stop codons. DIA raw MS data were analyzed with Spectronaut v18 using an in-house generated sample-specific fasta, comprised of the reviewed human proteome (proteome ID: UP000005640, 20 426 proteins, downloaded Sep 2023) and lncRNA-encoded protein sequences (125 223 proteins), in directDIA mode. The default settings were used unless otherwise noted. Data filtering was set to “Qvalue”. False discovery rate (FDR) was set to 1% at peptide precursor level and 1% at protein level. Top3 peptide precursors were used for protein quantification. The downstream data analysis was performed using in-house developed R scripts. PCA projection The 1000 fiber and myopathy data sets were initially filtered to remove non-overlapping proteins. Then, they were combined and normalized using the normalizeBetweenArrays function from the limma package. The normalization method used was “quantile” to ensure that both data sets had the same distributions and were comparable. Subsequently, the merged data set was divided back into the two separate data sets, namely the 1000 fiber data set and the myopathy data set. For the 1000 fiber data set, PCA was calculated using the prcomp function. Moving on, the myopathy data set was multiplied by the PC loadings obtained from the 1000 fiber dataset to generate its PCA projection. Finally, the PCA projections from the myopathy samples were plotted on the top of the 1000 fiber PCA visualization. The 1000 fiber and myopathy data sets were initially filtered to remove non-overlapping proteins. Then, they were combined and normalized using the normalizeBetweenArrays function from the limma package. The normalization method used was “quantile” to ensure that both data sets had the same distributions and were comparable. Subsequently, the merged data set was divided back into the two separate data sets, namely the 1000 fiber data set and the myopathy data set. For the 1000 fiber data set, PCA was calculated using the prcomp function. Moving on, the myopathy data set was multiplied by the PC loadings obtained from the 1000 fiber dataset to generate its PCA projection. Finally, the PCA projections from the myopathy samples were plotted on the top of the 1000 fiber PCA visualization. The skeletal muscle-specific ribosomal gene signature, consisting of log2 fold change values comparing the mRNA expression of ribosomal subunits in skeletal muscle compared to 52 other human tissues, was downloaded from Panda et al . Log2 fold change values were ranked to identify ribosomal proteins with the highest overexpression in human skeletal muscle. The human 80S ribosome structural model (Protein Data Bank: 4V6X) was downloaded from the Protein Data Bank website (RCSB PDB). Visualization and editing of the ribosomal structure, and preparation of figures and movies were performed in UCSF ChimeraX . Dot blot was conducted following a previously described protocol with a few modifications . Initially, two identical PVDF membranes were activated using 96% ethanol and washed with transfer buffer. Subsequently, the membranes were placed on wet filter paper with transfer buffer until they dried. Next, 1 µL of fiber lysate was spotted at the same position on both membranes, and the membranes were allowed to dry. Reactivation of the membranes was carried out using 96% ethanol, followed by gentle washing with TBST. The membranes were then blocked in TBST containing 5% skim milk for 15 minutes. After blocking, the membranes were washed three times with TBST and incubated with the primary antibody solution of either anti-MYH7 (A4.840) or anti-MYH2 (A4.74), both from Developmental Studies Hybridoma Bank (DSHB) at a dilution of 1:200 in TBST containing 1% skim milk for one hour. Subsequently, the membranes were gently washed three times with TBST and incubated with the secondary antibody (anti-mouse) at a dilution of 1:20,000 in TBST containing 1% skim milk for two hours. Finally, the membranes were washed three times for five minutes each with TBST and visualized using Immobilon Forte (Milipore) in a ChemiDoc XRS+ (Bio-Rad) imaging system. 8 µm sections from three different fixed-frozen human muscle biopsies were used for RNAscope labeling and subsequent immunohistochemistry (IHC). For detection of RP11-255P5.3 and LINC01405 commercially available RNAscope Multiplex Fluorescent Assay V2 (Advanced Cell Diagnostics) and probes against Lnc-ERCC5-5-C1 (# 1276851-C1), and LINC01405-C2 (# 549201-C2) (Advanced Cell Diagnostics), were used according to the manufacturer’s protocols. To control tissue quality Positive 3-plex probe (# 320881) and negative 3-plex probe (# 320871) were used. To visualize different subtypes of muscle fibers, sections after RNAscope were blocked with 5% donkey serum and incubated with antibodies against MYH2 (A4.74-s (1:5); DSHB) and MYH7 (A4.840 (1:5); DSHB) overnight. After washing sections were incubated with Alexa Flour 488-conjugated Donkey Anti-Mouse IgG, Fcγ Subclass 1 and DyLightTM405-conjugated Donkey Anti-Mouse IgM secondary antibodies respectively and mounted with ProLong™ Diamond Antifade Mountant (Invitrogen). Slides were imaged using Zeiss Axio Observer microscope equipped with Axiocam 702 camera. Biopsies from three individuals were used for quantification, with 187 muscle fibers being counted in total. As in RNAscope each dot corresponds to one RNA molecule, the number of dots/mm 2 was used as a measure of RNA expression. We first determined the number of dots/mm 2 within each fiber for both probes, then averaged the results by fiber type and participant. The given average was then used as an input for a two sample t-test. Immunolabelling was performed on 10 μm cryosections, fixed in 4% PFA (10 min), permeabilized in 0.1% Triton X-100 (20 min) and blocked in 10% Normal Goat Serum (50062Z, Life Technologies) with 0.1% BSA (1 h). Sections were incubated o/n (4 o C) with primary antibodies against MYH7 (mouse monoclonal A4.951, Santa Cruz, sc-53090, diluted 1:25) or MYH2 (mouse monoclonal SC71, DSHB, 1:25), each combined with primary antibody against TNNT1 (rabbit polyclonal HPA058448, Sigma, diluted 1:500) in 5% goat serum with 0.1% BSA and 0.1% Triton X-100. Alexa Fluor Goat anti-Mouse 647 (A21237) was used as the secondary antibody for the MYHs and Alexa Fluor Donkey anti-Rabbit 488 (A11034) for the TNNT1 (Life Technologies, 1:500 each in 10% Normal Goat Serum). Fluorescent images were obtained with a 10x objective on a Zeiss Axio Observer 3 fluorescence microscope with a Colibri 5 led detector, combined with Zeiss Axiocam 705 mono camera, using Zen software (Zeiss). For visualization purposes, a selection of fibers were mounted on copper grids glued on a microscopy slide, and imaged under a stereomicroscope. Further information on research design is available in the linked to this article. Supplementary information Description Of Additional Supplementary File Supplementary Dataset 1 Supplementary Dataset 2 Supplementary Dataset 3 Supplementary Dataset 4 Supplementary Dataset 5 Supplementary Dataset 6 Supplementary Dataset 7 Supplementary Dataset 8 Supplementary Dataset 9 Supplementary Dataset 10 Supplementary Dataset 11 Supplementary Dataset 12 Supplementary Dataset 13 Supplementary Dataset 14 Supplementary Dataset 15 Supplementary Dataset 16 Supplementary Dataset 17 Supplementary Dataset 18 Supplementary Dataset 19 Supplementary Dataset 20 Supplementary Dataset 21 Supplementary Dataset 22 Supplementary Dataset 23 Supplementary Dataset 24 Supplementary Dataset 25 Supplementary Dataset 26 Supplementary Dataset 27 Supplementary Dataset 28 Supplementary Dataset 29 Supplementary Dataset 30 Reporting Summary Transparent Peer Review file Source Data |
Digitally Guided Direct Composite Injection Technique with a Bi-layer Clear Mini-Index for the Management of Extensive Occlusal Caries in a Pediatric Patient: A Case Report | 26fd96c2-d3bb-4607-9267-9cbc81f453be | 11734273 | Dentistry[mh] | A 13-year-old female patient was referred to the Department of Operative Dentistry, Tokushima University Hospital, from an orthodontic clinic because of a high caries risk according to the CAMBRA (Caries Management by Risk Assessment) system. Upon examination, the patient presented with a swelling of the gingival tissue surrounding the mandibular right first molar (tooth 46), which had a defective RC restoration with secondary caries . The patient did not have any pain. Cold sensitivity testing was performed, and no response was elicited. Periodontal pocket depths were 3 mm around the tooth. Intraoral radiographs were taken with gutta-percha points in the sinus tracts , and chronic apical periodontitis was diagnosed. Furthermore, a deep carious lesion was also diagnosed in the left mandibular first molar (tooth 36) . Sensitivity testing gave a physiological result, and there were no symptoms. Root Canal Treatment (tooth 46) and Step-wise Excavation (Tooth 36) The previous RC restoration and secondary caries in tooth 46 were removed under rubber-dam isolation, without the need for local anesthesia. A caries detector dye solution (Caries Check, Nippon Shika Yakuhin; Shimonoseki, Japan) was used to verify complete caries removal. Subsequently, root canal treatment was carried out. A pre-mixed calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin) was applied into the root canals, and the access cavity was temporarily sealed with a glass-ionomer cement (Base Cement, Shofu; Kyoto, Japan). After 3 weeks, the sinus tract had healed and radiographic examination revealed a reduction in the periapical radiolucency, so the root canals of tooth 46 were obturated. This was followed by the provisional placement of a glass-ionomer cement (Base Cement, Shofu). For tooth 36, step-wise excavation was undertaken to manage the deep occlusal caries. While peripheral dentin was excavated non-selectively to ensure a durable seal of the lesion, selective caries removal of soft dentin using a spoon excavator was performed at the pulpal aspect of the cavity. Following a protocol by Momoi et al, a polycarboxylate cement containing a tannin/fluoride compound (HY-Bond Temporary Cement Soft, Shofu) was subsequently placed in the cavity, with re-entry planned after three months. The mesial incipient caries lesion found in the radiograph was only treated using a fluoride varnish, as it was not cavitated and the radiolucency was limited to the outer half of enamel. Finally, a full-mouth digital impression was taken using an intraoral scanner (Cerec Primescan, Dentsply Sirona; Bensheim, Germany). Given the sufficient amount of remaining dental hard tissues on teeth 36 and 46, it was planned to use RC for the final restoration in both teeth. Laboratory Preparation of an Improved, Bi-Layer Clear Mini-Index In the laboratory, a digital articulator (Dental System, 3Shape A/X; Copenhagen, Denmark) was used to create the diagnostic digital wax-ups and a mandibular cast was 3D-printed (Digital WAX 028D, DWS; Vincentza, Italy) . Bi-layer clear mini-indices consisting of an outer hard plastic shell and an inner elastic silicone material, were fabricated using the following process: A soft ethylene-vinyl acetate copolymer (EVA) thermoplastic sheet (Erkoflex 3mm, Erkodent; Pfalzgrafenweiler, Germany) was used as a spacer for the clear silicone part of the mini-index. The sheet was pressed against the 3D-printed model using a pressure molding machine (Erkopress, Erkodent) . The trimmed EVA sheet was repositioned on the 3D-printed model , and the rigid polyethylene terephthalate glycol (PETG) thermoplastic sheet (Erkodur, 2mm, Erkodent) of the outer layer was pressed onto it and trimmed ( and ). The rigid sheet served as the outer part of the mini-index. After separating the outer PETG layer from the inner EVA layer , the space in the rigid sheet was filled with a polyvinyl siloxane (PVS) impression material (Exaclear, GC; Tokyo, Japan) and pressed onto the 3D-printed model . For tooth 36, a less viscous silicone material (Exadenture, GC) was added to Exaclear to avoid air bubbles in pits and fissures. Polymerization was performed in a dental polymerizer (Palmat Elite, Heraeus Kulzer; Hanau, Germany) at +0.2 MPa for 10 minutes to prevent air voids inside the PVS inner layer. After polymerization, the mini-indices, consisting of a clear silicone interior and a rigid PETG exterior, were carefully removed from the 3D-printed model. A single hole with a 1-mm diameter penetrating the index was made using a round diamond bur at low speed and cleaned using micro-brushes . The margin of the mini-index was trimmed using a diamond bur at low speed above the gingival level to avoid interference with the rubber-dam clamp during the intraoral restorative procedures, resulting in the completed mini-indices . Restorative Procedure After the operative field was isolated using rubber-dam, the glass-ionomer cement was removed from tooth 46. The occlusal enamel margins were selectively acid etched using 37% phosphoric acid gel (K etchant, Kuraray Noritake; Tokyo, Japan) for 15 s , thoroughly rinsed with water and air dried. A 6% sodium hypochlorite solution was applied to the dentin for 30 s to deproteinize the smear layer, , followed by the application of an sulfinate-containing antioxidant (Clearfil DC Activator, Kuraray Noritake) for 10 s . , A two-step self-etching adhesive (Clearfil SE Bond 2, Kuraray Noritake) was applied according to the manufacturer’s instructions . A single-shade injectable RC (Clearfil SE Flow Universal, U shade, Kuraray Noritake) was incrementally placed in two 2-mm-thick layers and polymerized using an LED light-curing unit (Pencure 2000, J Morita; Saitama, Japan) for 20 s each . The prepared bi-layer mini-index was placed on the tooth, the same RC was injected through the access hole, and it was polymerized for 20 s . After removing the mini-index, light curing was repeated for an additional 20 s. A brown staining RC (Estelite Color, Tokuyama Dental; Tokyo, Japan) was placed and polymerized in the pits and fissures to mimic the natural appearance . A glycerin gel was placed on the occlusal surface, and light irradiation was performed again to promote a high polymerization degree of the staining composite. Finishing and polishing were performed chairside in a relatively short time, using a 2-step polishing system (DIACOMP PLUS DCP-OFm, DCP-OFf, EVE Ernst Vetter; Keltern, Germany) and a polishing brush in which polishing particles are embedded (OptiShine, Kerr; Orange, CA, USA). For tooth 36, the cavity was re-opened, and selective caries removal to firm dentin was performed . The cavity was then restored using the above-mentioned technique . Orthodontic treatment was performed after the restorative procedures. At a check-up after one year, the restorations presented marginal integrity without gaps and secondary caries. There were no cracks in the enamel, and no chipping of the RC was found. The surfaces were smooth, and no marginal discoloration was observed ( and ). The patient reported no dentin hypersensitivity. The radiographic examination did not reveal any abnormalities, such as periapical radiolucency in tooth 46 or pulp alteration in tooth 36 ( and ). The previous RC restoration and secondary caries in tooth 46 were removed under rubber-dam isolation, without the need for local anesthesia. A caries detector dye solution (Caries Check, Nippon Shika Yakuhin; Shimonoseki, Japan) was used to verify complete caries removal. Subsequently, root canal treatment was carried out. A pre-mixed calcium hydroxide paste (Calcipex II, Nippon Shika Yakuhin) was applied into the root canals, and the access cavity was temporarily sealed with a glass-ionomer cement (Base Cement, Shofu; Kyoto, Japan). After 3 weeks, the sinus tract had healed and radiographic examination revealed a reduction in the periapical radiolucency, so the root canals of tooth 46 were obturated. This was followed by the provisional placement of a glass-ionomer cement (Base Cement, Shofu). For tooth 36, step-wise excavation was undertaken to manage the deep occlusal caries. While peripheral dentin was excavated non-selectively to ensure a durable seal of the lesion, selective caries removal of soft dentin using a spoon excavator was performed at the pulpal aspect of the cavity. Following a protocol by Momoi et al, a polycarboxylate cement containing a tannin/fluoride compound (HY-Bond Temporary Cement Soft, Shofu) was subsequently placed in the cavity, with re-entry planned after three months. The mesial incipient caries lesion found in the radiograph was only treated using a fluoride varnish, as it was not cavitated and the radiolucency was limited to the outer half of enamel. Finally, a full-mouth digital impression was taken using an intraoral scanner (Cerec Primescan, Dentsply Sirona; Bensheim, Germany). Given the sufficient amount of remaining dental hard tissues on teeth 36 and 46, it was planned to use RC for the final restoration in both teeth. In the laboratory, a digital articulator (Dental System, 3Shape A/X; Copenhagen, Denmark) was used to create the diagnostic digital wax-ups and a mandibular cast was 3D-printed (Digital WAX 028D, DWS; Vincentza, Italy) . Bi-layer clear mini-indices consisting of an outer hard plastic shell and an inner elastic silicone material, were fabricated using the following process: A soft ethylene-vinyl acetate copolymer (EVA) thermoplastic sheet (Erkoflex 3mm, Erkodent; Pfalzgrafenweiler, Germany) was used as a spacer for the clear silicone part of the mini-index. The sheet was pressed against the 3D-printed model using a pressure molding machine (Erkopress, Erkodent) . The trimmed EVA sheet was repositioned on the 3D-printed model , and the rigid polyethylene terephthalate glycol (PETG) thermoplastic sheet (Erkodur, 2mm, Erkodent) of the outer layer was pressed onto it and trimmed ( and ). The rigid sheet served as the outer part of the mini-index. After separating the outer PETG layer from the inner EVA layer , the space in the rigid sheet was filled with a polyvinyl siloxane (PVS) impression material (Exaclear, GC; Tokyo, Japan) and pressed onto the 3D-printed model . For tooth 36, a less viscous silicone material (Exadenture, GC) was added to Exaclear to avoid air bubbles in pits and fissures. Polymerization was performed in a dental polymerizer (Palmat Elite, Heraeus Kulzer; Hanau, Germany) at +0.2 MPa for 10 minutes to prevent air voids inside the PVS inner layer. After polymerization, the mini-indices, consisting of a clear silicone interior and a rigid PETG exterior, were carefully removed from the 3D-printed model. A single hole with a 1-mm diameter penetrating the index was made using a round diamond bur at low speed and cleaned using micro-brushes . The margin of the mini-index was trimmed using a diamond bur at low speed above the gingival level to avoid interference with the rubber-dam clamp during the intraoral restorative procedures, resulting in the completed mini-indices . After the operative field was isolated using rubber-dam, the glass-ionomer cement was removed from tooth 46. The occlusal enamel margins were selectively acid etched using 37% phosphoric acid gel (K etchant, Kuraray Noritake; Tokyo, Japan) for 15 s , thoroughly rinsed with water and air dried. A 6% sodium hypochlorite solution was applied to the dentin for 30 s to deproteinize the smear layer, , followed by the application of an sulfinate-containing antioxidant (Clearfil DC Activator, Kuraray Noritake) for 10 s . , A two-step self-etching adhesive (Clearfil SE Bond 2, Kuraray Noritake) was applied according to the manufacturer’s instructions . A single-shade injectable RC (Clearfil SE Flow Universal, U shade, Kuraray Noritake) was incrementally placed in two 2-mm-thick layers and polymerized using an LED light-curing unit (Pencure 2000, J Morita; Saitama, Japan) for 20 s each . The prepared bi-layer mini-index was placed on the tooth, the same RC was injected through the access hole, and it was polymerized for 20 s . After removing the mini-index, light curing was repeated for an additional 20 s. A brown staining RC (Estelite Color, Tokuyama Dental; Tokyo, Japan) was placed and polymerized in the pits and fissures to mimic the natural appearance . A glycerin gel was placed on the occlusal surface, and light irradiation was performed again to promote a high polymerization degree of the staining composite. Finishing and polishing were performed chairside in a relatively short time, using a 2-step polishing system (DIACOMP PLUS DCP-OFm, DCP-OFf, EVE Ernst Vetter; Keltern, Germany) and a polishing brush in which polishing particles are embedded (OptiShine, Kerr; Orange, CA, USA). For tooth 36, the cavity was re-opened, and selective caries removal to firm dentin was performed . The cavity was then restored using the above-mentioned technique . Orthodontic treatment was performed after the restorative procedures. At a check-up after one year, the restorations presented marginal integrity without gaps and secondary caries. There were no cracks in the enamel, and no chipping of the RC was found. The surfaces were smooth, and no marginal discoloration was observed ( and ). The patient reported no dentin hypersensitivity. The radiographic examination did not reveal any abnormalities, such as periapical radiolucency in tooth 46 or pulp alteration in tooth 36 ( and ). Direct composite restorations have proven effective for the restorations of small cavities, such as teeth with Black’s class I-V cavities and non-carious cervical lesions. However, free-hand restoration of anatomical morphology in large cavities or endodontically treated teeth can be challenging. , , , Better outcomes can be achieved by planning and simulating the appropriate morphology before treatment and then accurately transferring it to the treated tooth. The composite injection technique, which uses a highly filled RC and a clear silicone index, can be beneficial from this clinical perspective. The highly filled flowable RCs, also known as injectable RCs, have been shown to possess mechanical strength, wear resistance, esthetics, and long-term durability similar to that of conventional RCs, with comparable clinical outcomes. , , , Single-shade injectable RCs are now also available, allowing acceptable shade-matching even without laborious shade selection and layering. To use the inverse injection technique with a single-tooth rubber-dam isolation, the silicone index needs to be trimmed and downsized. Given the elasticity of the clear silicone, this could hinder the dimensional accuracy of the restoration. To address this issue, a bi-layer clear mini-index was developed, using a rigid PETG thermoplastic sheet as an outer shell and a clear silicone as the inner layer. This bi-layer index enabled an accurate transfer of the final anatomical forms from the digital wax-up, including pits and fissures, thus reducing the time necessary for articulation and finishing compared to the free-hand placement. On the other hand, the fabrication of the bi-layer index is more expensive and laborious, and the workflow is not fully digital at this point. Given that the treatment is performed in two visits, this technique is applicable in the second appointment of an endodontic treatment or step-wise excavation, as presented in this case report. However, further development and optimization would be necessary to make the technique applicable in other clinical situations or class-II cavities. For the long-term success of direct RC restorations, a durable resin adhesive-dentin bond is essential. To enhance the durability of self-etch adhesives, the smear layer deproteinizing pretreatment (SLDP) using sodium hypochlorite or hypochlorous acid has recently been introduced. , , By applying a deproteinizing solution, the organic phase of the smear layer is removed, thus enhancing monomer infiltration and interaction with the underlying dentin. The deproteinizing pretreatment was followed by the application of a sulfinate-containing antioxidant, which counteracts the adverse effect of chloramine-derived radicals on the polymerization of adhesives and improves the resin adhesive-dentin bond. , In-vitro studies also suggest that the SLDP pretreatment can improve the durability of the resin adhesive-dentin bond by preventing the formation of the hybridized smear layer. However, clinical evidence supporting these findings is not available to date. Large occlusal cavities after endodontic treatment and step-wise caries removal were functionally and esthetically restored with digitally guided RC restorations using the inverse injection technique. The improved bi-layer clear mini-indices fitted the treated teeth even with single-tooth rubber-dam isolation, and transferred the anatomical form perfectly, thanks to the elastic inner layer and rigid outer shell. |
A transcriptomic atlas of mouse cerebellar cortex comprehensively defines cell types | 3ee0a0ce-8f06-465d-928c-1b743cd5225a | 8494635 | Physiology[mh] | The cerebellar cortex is composed of the same basic circuit replicated thousands of times. Mossy fibres from many brain regions excite granule cells that in turn excite Purkinje cells (PCs), the sole outputs of the cerebellar cortex. Powerful climbing fibre synapses, which originate in the inferior olive, excite PCs and regulate synaptic plasticity. Additional circuit elements include inhibitory interneurons such as molecular layer interneurons (MLIs), Purkinje layer interneurons (PLIs), Golgi cells, excitatory unipolar brush cells (UBCs) and supportive Bergmann glia. There is a growing recognition that cerebellar circuits exhibit regional specializations, such as a higher density of UBCs or more prevalent PC feedback to granule cells in some lobules. Molecular variation across regions has also been identified, such as the parasagittal banding pattern of alternating PCs with high and low levels of Aldoc expression . However, the extent to which cells are molecularly specialized in different regions is poorly understood. Achieving a comprehensive survey of cell types in the cerebellum poses some unique challenges. First, a large majority of the neurons are granule cells, making it difficult to accurately sample the rarer types. Second, for many of the morphologically and physiologically defined cell types—especially the interneuron populations—existing molecular characterization is extremely limited. Recent advances in single-cell RNA sequencing (scRNA-seq) technology – have increased the throughput of profiling to enable the systematic identification of cell types and states throughout the central nervous system – . Several recent studies have harnessed such techniques to examine some cell types in the developing mouse cerebellum – , but none has yet comprehensively defined mature cell types in the adult.
We developed a pipeline for high-throughput single-nucleus RNA-seq (snRNA-seq) with high transcript capture efficiency and nuclei yield, as well as consistent performance across regions of the adult mouse and post mortem human brain (10.17504/protocols.io.bck6iuze; Methods). To comprehensively sample cell types in the mouse cerebellum, we dissected and isolated nuclei from 16 different lobules, across both female and male replicates (Fig. , Extended Data Fig. , Methods). We recovered 780,553 nuclei profiles with a median transcript capture of 2,862 unique molecular identifiers (UMIs) per profile (Extended Data Fig. ), including 530,063 profiles from male donors, and 250,490 profiles from female donors, with minimal inter-individual batch effects (Extended Data Fig. ). To discover cell types, we used a previously developed clustering strategy (Methods) to partition 611,034 high-quality profiles into 46 clusters. We estimate that with this number of profiles, we can expect to sample even extremely rare cell types (prevalence of 0.15%) with a probability of greater than 90%, which suggests that we captured most transcriptional variation within the cerebellum (Extended Data Fig. ). We assigned each cluster to one of 18 known cell type identities on the basis of the expression of specific molecular markers that are known to correlate with defining morphological, histological and/or functional features (Fig. , Supplementary Table ). These annotations were also corroborated by the expected layer-specific localizations of marker genes in the Allen Brain Atlas (ABA) ( https://mouse.brain-map.org ) (Fig. ). Several cell types contained multiple clusters defined by differentially expressed markers, which suggests further heterogeneity within those populations (Extended Data Fig. , Supplementary Table ).
To quantify the regional specialization of cell types, we examined how our clusters distributed proportionally across each lobule. We found that eight of our nine PC clusters, as well as several granule cell clusters and one Bergmann glial cluster, showed the most significantly divergent lobule compositions (Pearson’s chi-squared test, false discovery rate (FDR) < 0.001; Methods) and exhibited greater than twofold enrichment in at least one lobule (Fig. ). There was high concordance in the regional composition of each of these types across replicates, which indicates consistent spatial enrichment patterns (Extended Data Fig. ). The nine PC clusters could be divided into two main groups on the basis of their expression of Aldoc , which defines parasagittal striping of Purkinje neurons across the cerebellum . Seven of the nine PC clusters were Aldoc -positive, indicating greater specialization in this population compared with the Aldoc -negative PCs. Combinatorial expression of Aldoc and at least one subtype-specific marker fully identified the Purkinje clusters (Fig. ). These Aldoc -positive and Aldoc- negative groups showed a regional enrichment pattern that was consistent with the known paths of parasagittal stripes across individual lobules (Fig. ). When characterizing the spatial variation of the PC subtypes, we found some with spatial patterns that were recently identified using Slide-seq technology (Aldoc_1 and Aldoc_7, marked by Gpr176 and Tox2 , respectively) , as well as several undescribed subtypes and patterns (Fig. , Extended Data Fig. ). Most of this PC diversity was concentrated in the posterior cerebellum, particularly the uvula and nodulus, consistent with these regions showing greater diversity in both function and connectivity , . We also observed regional specialization in excitatory interneurons and Bergmann glia. Among the five granule cell subtypes (Fig. ), three displayed cohesive spatial enrichment patterns (subtypes 1, 2 and 3) (Fig. , Extended Data Fig. ). In addition, and consistent with previous work , the UBCs as a whole were highly enriched in the posterior lobules (Extended Data Fig. ). Finally, we identified a Bergmann glial subtype that expressed the marker genes Mybpc1 and Wif1 (Fig. ), with high enrichment in lobule VI, the uvula and nodulus (Fig. , Extended Data Fig. ). The regional specialization of interneuron and glial populations is in contrast to the cerebral cortex, where molecular heterogeneity across regions is largely limited to projection neurons , .
Molecularly defined cell populations can be highly discrete—such as the distinctions between chandelier and basket interneuron types in the cerebral cortex —or they can vary more continuously, such as the cross-regional differences among principal cells of the striatum , and cortex , . The cerebellum is known to contain several canonical cell types that exist as morphological and functional continua, such as the basket and stellate interneurons of the molecular layer . To examine continuous features of molecular variation in greater detail within interneuron types, we created a metric to quantify and visualize the continuity of gene expression between two cell clusters. In brief, we fit a logistic curve for differentially expressed genes along the dominant expression trajectory , extracting the maximum slope ( m ) of the curve (Methods, Fig. ). We expect m values to be smaller for genes that are representative of more continuous expression variation (Fig. ). Our cluster analysis initially identified three populations of UBCs, similar in number to the two to four discrete types suggested by previous immunohistochemistry studies – . However, comparing m values across 200 highly variable genes within the UBC, Golgi cell and MLI populations suggested that in UBCs, many genes showed continuous variation (Fig. ), including Grm1 , Plcb4 , Calb2 and Plcb1 (Fig. ). Cross-species, integrative analysis with cerebellar cells derived from two post mortem human donors (Methods) revealed evolutionary conservation of the continuum (Extended Data Fig. ), with graded expression of many of the same genes, including Grm1 and Grm2 (Extended Data Fig. ). Functionally, UBCs have been classified on the basis of their response to mossy fibre activation. Discrete ON and OFF categories have previously been emphasized, although some properties of UBCs do not readily conform to these distinct categories , , . Here we focused on whether the molecular gradients in the expression of metabotropic receptors readily translated to a continuum of functional properties. We pressure-applied glutamate and measured the spiking responses of UBCs with on-cell recordings, and then measured glutamate-evoked currents in the cell (Methods). In some cells, glutamate rapidly and transiently increased spiking and evoked a long-lasting inward current (Fig. , top left). For other cells, glutamate transiently suppressed spontaneous firing and evoked an outward current (Fig. , bottom left). Many UBCs, however, had more complex, mixed responses to glutamate; we refer to these as ‘biphasic’ cells. In one cell, for example, glutamate evoked a delayed increase in firing, caused by an initial outward current followed by a longer lasting inward current (Fig. , middle left). A summary of the glutamate-evoked currents (Fig. , right) suggests that the graded nature of the molecular properties of UBCs may lead to graded electrical response properties. To link the functional and molecular continua more directly, we recorded from cells treated with agonists of mGluR1 ( Grm1 ) or mGluR2 ( Grm2 ) (Fig. ). Responses were graded across the UBC population, with a significant number of cells that responded to both agonists (Fig. , Extended Data Fig. ). This suggests that the biphasic response profile probably corresponds to the molecular continuum defined by snRNA-seq. Further studies are needed to determine the relationship between these diverse responses to applied agonists, and the responses of the cells to mossy fibre activation.
MLIs are spontaneously active interneurons that inhibit PCs as well as other MLIs. MLIs are canonically subdivided into stellate cells located in the outer third of the molecular layer, and basket cells located in the inner third of the molecular layer that synapse onto PC somata and form specialized contacts known as pinceaus, which ephaptically inhibit PCs. Many MLIs, particularly those in the middle of the molecular layer, share morphological features with both basket and stellate cells . Thus, MLIs are thought to represent a single functional and morphological continuum. Our clustering analysis of MLIs and PLIs, by contrast, identified two discrete populations of MLIs. The first population, ‘MLI1’, uniformly expressed Lypd6 , Sorcs3 and Ptprk (Figs. c, ). The second population, ‘MLI2’, was highly molecularly distinct from MLI1, and expressed numerous markers that are also found in PLIs, such as Nxph1 and Cdh22 (Fig. ). Single-molecule fluorescence in situ hybridization (smFISH) experiments with Sorcs3 and Nxph1 showed that the markers were entirely mutually exclusive (Fig. ). A cross-species analysis with 14,971 human MLI and PLI profiles demonstrated that the MLI1 and MLI2 distinction is evolutionarily conserved (Extended Data Fig. ). To examine the developmental specification of these two populations, we clustered 79,373 total nuclei from peri- and postnatal mice across several time points (ranging from embryonic day (E) 18 to postnatal day (P) 16). From a cluster of 5,519 GABA (γ-aminobutyric acid)-producing neuron progenitors, marked by the expression of canonical markers Tfap2b , Ascl1 and Pax2 , (Methods, Extended Data Fig. ), we were able to distinguish developmental trajectories that corresponded to the MLI1 ( Sorcs3 -positive) and MLI2 ( Nxph1 -positive, Klhl1- negative) populations, with differentiation of the two types beginning at P4 and largely complete by P16 (Fig. , Extended Data Fig. ). Although both populations originate from a single group of progenitors, trajectory analysis revealed several lineage-specific markers (Extended Data Fig. ). Among the MLI2 trajectory markers, we identified genes such as Fam135b , the expression of which persisted into adulthood, and Fos , which is only transiently differentially expressed between the MLI1 and MLI2 trajectories (Fig. , Extended Data Fig. ). This high expression of several immediate early genes (Extended Data Fig. ) selectively in early MLI2 cells could indicate that differential activity is associated with MLI2 specification. MLI1s and MLI2s were present throughout the entire molecular layer, which indicates that the distinction between MLI1 and MLI2 does not correspond to the canonical basket and stellate distinction (Extended Data Fig. ). To understand the morphological, physiological and molecular characteristics of the MLI populations better, we developed a pipeline to record from individual MLIs in brain slices, image their morphologies, and then ascertain their molecular MLI1 and MLI2 identities by smFISH (Methods, Fig. ). Consistent with the marker analysis (Fig. ), MLI1s had a stellate morphology in the distal third of the molecular layer, whereas MLI1s located near the PC layer had a basket morphology, with contacts near PC initial segments (Fig. , Extended Data Fig. ). We next examined whether MLI2s, in which we could not identify systematic molecular heterogeneity, had graded morphological properties. MLI2s in the distal third of the molecular layer also had stellate cell morphology, whereas MLI2s near the PC layer had a distinct morphology and appeared to form synapses preferentially near the PC layer (Extended Data Fig. ). Although further studies are needed to determine whether MLI2s form pinceaus, it is clear that both MLI1 and MLI2 showed a similar continuum in their morphological properties. The electrical characteristics of MLI1s and MLI2s also showed numerous distinctions. The average spontaneous firing rate was significantly higher for MLI1s than for MLI2s (Mann–Whitney test, P = 0.0015) (Fig. ), and the membrane resistance ( R m ) of MLI1s was lower than that of MLI2s (Fig. ). In addition, we found that MLI2s were more excitable than MLI1s (Fig. ), and displayed a stronger hyperpolarization-activated current (Extended Data Fig. ). MLIs are known to be electrically coupled via gap junctions , but it is not clear whether this is true for both MLI1s and MLI2s. In the cerebral cortex and some other brain regions, interneurons often electrically couple selectively to neurons of the same type, but not other types , . We therefore examined whether this also applies to MLI1s and MLI2s. The expression of Gjd2 , the gene encoding the dominant gap junction protein in MLIs , was found in MLI1s but not MLI2s, both in our single-nucleus data (Fig. ) and by smFISH (Fig. ), which suggests potential differences in electrical coupling. Notably, the two clusters of Golgi cells, another interneuron type known to be electrically coupled , , differentially expressed many of the same markers, including Sorcs3 , Gjd2 and Nxph1 in both human and mouse (Extended Data Figs. e, f, ). Action potentials in coupled MLIs produce small depolarizations known as spikelets that are thought to promote synchronous activity between MLIs . We therefore investigated whether spikelets are present in MLI1s and absent in MLI2s. Consistent with the gene expression profile, we observed spikelets in 71% of MLI1s and not in MLI2s (Fig. ; P < 0.001, Fisher’s exact test). These findings suggest that most MLI1s are coupled to other MLI1s by gap junctions, whereas MLI2s show no electrical coupling to other MLIs.
In this Article, we used high-throughput, region-specific transcriptome sampling to build a comprehensive taxonomy of cell types in the mouse cerebellar cortex, and quantify spatial variation across individual regions. Our joint analyses with post mortem human samples indicated that the neuronal populations defined in mouse were generally conserved in human (Extended Data Fig. ), consistent with a recent comparative analysis in the cerebral cortex . We find considerably more regional specialization in PCs—especially in posterior lobules—than was previously recognized. These PC subtypes overlap with greater local abundances in UBCs and in distinct specializations in granule cells, which indicates a higher degree of regional circuit heterogeneity than previously thought. Our dataset is freely available to the neuroscience community ( https://portal.nemoarchive.org/ ; https://singlecell.broadinstitute.org ), facilitating functional characterization of these populations, many of which are entirely novel. One of the biggest challenges facing the comprehensive cell typing of the brain is the correspondence problem : how to integrate definitions of cell types on the basis of the many modalities of measurement used to characterize brain cells. We found success by first defining populations using systematic molecular profiling, and then relating these populations to physiological and morphological features using targeted, joint analyses of individual cells. We were surprised that the cerebellar MLIs—one of the first sets of neurons to be characterized more than 130 years ago —are in fact composed of two molecularly and physiologically discrete populations, that each shows a similar morphological continuum along the depth axis of the molecular layer. As comprehensive cell typing proceeds across other brain regions, we expect the emergence of similar basic discoveries that challenge and extend our understanding of cellular specialization in the nervous system.
Animals Nuclei suspensions for mouse (C57BL/6J, Jackson Labs) cerebellum profiles were generated from 2 female and 4 male adult mice (60 days old), 1 male E18 mouse, 1 male P0 (newborn) mouse, 1 female P4 (4 days old) mouse, 1 female P8, 2 male P12 and 2 female P16 mice. Adult mice were group-housed with a 12-h light-dark schedule and allowed to acclimate to their housing environment for two weeks after arrival. Timed pregnant mice were received and euthanized to yield E18 mice 6 days after arrival. Newborn mice were housed as individual litters for up to 16 days. All experiments were approved by and in accordance with Broad IACUC protocol number 012-09-16. Brain preparation At E18, P0, P4, P8, P12, P16 and P60, C57BL/6J mice were anaesthetized by administration of isoflurane in a gas chamber flowing 3% isoflurane for 1 min. Anaesthesia was confirmed by checking for a negative tail and paw pinch response. Mice were moved to a dissection tray and anaesthesia was prolonged via a nose cone flowing 3% isoflurane for the duration of the procedure. Transcardial perfusions were performed on adult, pregnant (E18), P8, P12 and P16 mice with ice-cold pH 7.4 HEPES buffer containing 110 mM NaCl, 10 mM HEPES, 25 mM glucose, 75 mM sucrose, 7.5 mM MgCl 2 , and 2.5 mM KCl to remove blood from the brain. P0 and P4 mice were unperfused. The brain was removed from P60, P8, P12 and P16 mice and frozen for 3 min in liquid nitrogen vapour. E18, P0 and P4 mice were sagittally bisected after similarly freezing their brains in situ. All tissue was moved to −80 °C for long-term storage. A detailed protocol is available at protocols.io (10.17504/protocols.io.bcbrism6). Generation of cerebellar nuclei profiles Frozen adult mouse brains were securely mounted by the frontal cortex onto cryostat chucks with OCT embedding compound such that the entire posterior half including the cerebellum and brainstem were left exposed and thermally unperturbed. Dissection of each of 16 cerebellar vermal and cortical lobules was performed by hand in the cryostat using an ophthalmic microscalpel (Feather safety Razor P-715) pre-cooled to −20 °C and donning four surgical loupes. Whole E18, P0, P4, P8, P12 and P16 mouse cerebella were similarly curated by dissecting rhombomeric cerebellar rudiments from sagittal frozen brain hemispheres using a pre-cooled 1-mm disposable biopsy punch (Integra Miltex). Each excised tissue dissectate was placed into a pre-cooled 0.25 ml PCR tube using pre-cooled forceps and stored at −80 °C. Nuclei were extracted from this frozen tissue using gentle, detergent-based dissociation, according to a protocol available at protocols.io (10.17504/protocols.io.bck6iuze) adapted from one provided by the McCarroll laboratory (Harvard Medical School), and loaded into the 10x Chromium V3 system. Reverse transcription and library generation were performed according to the manufacturer’s protocol. Floating slice hybridization chain reaction on acute slices Acute cerebellar slices containing Alexa 594-filled patched cells were fixed as described and stored in 70% ethanol at 4 °C until hybridization chain reaction (HCR). They were then subjected to a ‘floating slice HCR’ protocol in which the recorded cells could be simultaneously re-imaged in conjunction with HCR expression analysis in situ and catalogued as to their positions in the cerebellum. A detailed protocol (10.17504/protocols.io.bck7iuzn) was performed using the following HCR probes and matching hairpins purchased from Molecular Instruments: glutamate metabotropic receptor 8 ( Grm8 ) lot number PRC005, connexin 36 ( Gjd2 ) lot number PRD854 and PRA673, cadherin22 ( Cdh22 ) lot number PRC011, neurexophilin 1 ( Nxph1 ) lot number PRC675 and PRC466, leucine-rich glioma-inactivated protein 2 ( Lgi2 ) lot number PRC012, somatostatin ( Sst ) lot number PRA213 and sortilin related VPS10 domain containing receptor 3 ( Sorcs3 ) lot number PRC004. Amplification hairpins used were type B1, B2 and B3 in 488 nm, 647 nm and 546 nm respectively. Patch fill and HCR co-imaging After floating-slice HCR, slices were mounted between no.1 coverslips with antifade compound (ProLong Glass, Invitrogen) and images were collected on an Andor CSU-X spinning disk confocal system coupled to a Nikon Eclipse Ti microscope equipped with an Andor iKon-M camera. The images were acquired with an oil immersion objective at 60×. The Alexa 594 patched cell backfill channel (561 nm) plus associated HCR probe/hairpin channels (488 nm and 647 nm) were projected through a 10–20-μm thick z -series so that an unambiguous determination of the association between the patch-filled cell and its HCR gene expression could be made. Images were processed using Nikon NIS Elements 4.4 and Nikon NIS AR. Human brain and nuclei processing Human donor tissue was supplied by the Human Brain and Spinal Fluid Resource Center at UCLA, through the NIH NeuroBioBank. This work was determined by the Office of Research Subjects Protection at the Broad Institute not to meet the definition of human subjects research (project ID NHSR-4235). Nuclei suspensions from human cerebellum were generated from two neuropathologically normal control cases—one female tissue donor, aged 35, and one male tissue donor, aged 36. These fresh frozen tissues had post mortem intervals of 12 and 13.5 h respectively, and were provided as whole cerebella cut into four coronal slabs. A sub-dissection of frozen cerebellar lobules was performed on dry ice just before 10x processing and nuclei were extracted from this frozen tissue using gentle, detergent-based dissociation, according to a protocol available at protocols.io (10.17504/protocols.io.bck6iuze). Electrophysiology experiments Acute parasagittal slices were prepared at 240-μm thickness from wild-type mice aged P30–P50. Mice were anaesthetized with an intraperitoneal injection of ketamine (10 mg kg −1 ), perfused transcardially with an ice-cold solution containing (in mM): 110 choline chloride, 7 MgCl 2 , 2.5 KCl, 1.25 NaH 2 PO 4 , 0.5 CaCl 2 , 25 glucose, 11.5 sodium ascorbate, 3 sodium pyruvate, 25 NaHCO 3 , 0.003 ( R )-CPP, equilibrated with 95% O 2 and 5% CO 2 . Slices were cut in the same solution and were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 1.5 CaCl 2 and 25 glucose equilibrated with 95% O 2 and 5% CO 2 at approximately 34 °C for 30 min. Slices were then kept at room temperature until recording. All UBC recordings were done at 34–36 °C with (in μM) 2 ( R )-CPP, 5 NBQX, 1 strychnine, 10 SR95531 (gabazine) and 1.5 CGP in the bath to isolate metabotropic currents. Loose cell-attached recordings were made with ACSF-filled patch pipettes of 3–5 MΩ resistance. Whole-cell voltage-clamp recordings were performed while holding the cell at −70 mV with an internal containing (in mM): 140 KCl, 4 NaCl, 0.5 CaCl 2 , 10 HEPES, 4 MgATP, 0.3 NaGTP, 5 EGTA 5, and 2 QX-314, pH adjusted to 7.2 with KOH. Brief puffs of glutamate (1 mM for 50 ms at 5 psi) were delivered using a Picospritzer II (General Valve Corp.) in both cell-attached and whole-cell configuration to assure consistent responses. The heat map of current traces from all cells are sorted by the score over the first principal axis after singular value decomposition (SVD) of recordings over all cells. For whole-cell recordings with pharmacology, we used an K-methanesulfonate internal containing (in mM): 122 K-methanesulfonate, 9 NaCl, 9 HEPES, 0.036 CaCl 2 , 1.62 MgCl 2 , 4 MgATP, 0.3 GTP (Tris salt), 14 creatine phosphate (Tris salt), and 0.18 EGTA, pH 7.4. A junction potential of −8 mV was compensated for during recording. 300nM TTX was added to the ACSF in conjunction with the synaptic blockers listed above. Three pipettes filled with ACSF containing 1 mM glutamate, 100 μM DHPG or 1 μM LY354740 were positioned within 20 μm of the recorded cell. Pressure applications of each agonist were delivered at 10 psi with durations of 40–50 ms. Agonist applications were separated by 30 s. Two to three trials were collected for each agonist. MLI recordings were performed at approximately 32 °C with an internal solution containing (in mM) 150 K-gluconate, 3 KCl, 10 HEPES, 3 MgATP, 0.5 GTP, 5 phosphocreatine-tris 2 and 5 phosphocreatine-Na 2 , 2 mg ml −1 biocytin and 0.1 Alexa 594 (pH adjusted to 7.2 with KOH, osmolality adjusted to 310 mOsm kg −1 ). Visually guided whole-cell recordings were obtained with patch pipettes of around 4 MΩ resistance pulled from borosilicate capillary glass (BF150-86-10, Sutter Instrument). Electrophysiology data was acquired using a Multiclamp 700B amplifier (Axon Instruments), digitized at 20 kHz and filtered at 4 kHz. For isolating spikelets in MLI recordings, cells were held at −65 mV in voltage clamp and the following receptor antagonists were added to the solution (in μM) to block synaptic currents: 2 ( R )-CPP, 5 NBQX, 1 strychnine, 10 SR95531 (gabazine), 1.5 CGP. All drugs were purchased from Abcam and Tocris. To obtain an input-output curve, MLIs were maintained at 60–65 mV with a constant hyperpolarizing current, and 250 ms current steps ranging from −30 pA to +100 pA were injected in 10 pA increments. To activate the hyperpolarization-evoked current ( I h ), MLIs were held at −65 mV and a 30 pA hyperpolarizing current step of 500 ms duration was injected. The amplitude of I h was calculated as the difference between the maximal current evoked by the hyperpolarizing current step and the average steady-state current at the end (480–500 ms) of the current step. Capacitance and input resistance ( R i ) were determined using a 10 pA, 50 ms hyperpolarizing current step. To prevent excessive dialysis and to ensure successful detection of mRNAs in the recorded cells, the total duration of recordings did not exceed 10 min. Acquisition and analysis of electrophysiological data were performed using custom routines written in MATLAB (Mathworks), IgorPro (Wavemetrics), or AxoGraphX. Data are reported as median ± interquartile range, and statistical analysis was carried out using the Mann–Whitney or Fisher’s exact test, as indicated. Statistical significance was assumed at P < 0.05. To determine the presence of spikelets, peak detection was used to generate event-triggered average waveforms with thresholds based on the mean absolute deviation (MAD) of the raw trace. Spikelet recordings were scored for the presence of spikelets blind to the molecular identity of the cells. The analysis was restricted to cells recorded in the presence of synaptic blockers. Imaging and analysis MLIs were filled with 100 μM Alexa-594 via patch pipette to visualize their morphology using two-photon imaging. After completion of the electrophysiological recordings the patch electrode was retracted slowly and the cell resealed. We used a custom-built two-photon laser-scanning microscope with a 40×, 0.8 numerical aperture (NA) objective (Olympus Optical) and a pulsed two-photon laser (Chameleon or MIRA 900, Coherent, 800 nm excitation). DIC images were acquired at the end of each experiment and locations of each cell within the slice were recorded. Two-photon images were further processed in ImageJ. Tissue fixation of acute slices After recording and imaging, cerebellar slices were transferred to a well-plate and submerged in 2–4% PFA in PBS (pH 7.4) and incubated overnight at 4 °C. Slices were then washed in PBS (3 × 5 min) and then kept in 70% ethanol in RNase-free water until HCR was performed. Preprocessing of sequencing reads Sequencing reads from mouse cerebellum experiments were demultiplexed and aligned to a mouse (mm10) premrna reference using CellRanger v3.0.2 with default settings. Digital gene expression matrices were generated with the CellRanger count function. Sequencing reads from human cerebellum experiments were demultiplexed and aligned to a human (hg19) premrna reference using the Drop-seq alignment workflow , which was also used to generate the downstream digital gene expression matrices. Estimation of adequate rare cell type detection To estimate the probability of sufficiently sampling rare cell types in the cerebellum as a function of total number of nuclei sampled, we used the approach proposed by the Satija laboratory ( https://satijalab.org/howmanycells ), with the assumption of at most 10 very rare cell types, each with a prevalence of 0.15%. We derived this minimum based on the observed prevalences of the two rarest cell types we identified (OPC_4, Purkinje_Aldoc_2). We set 70 cells as the threshold for sufficient sampling, and calculated the overall probability as a negative binomial (NB) density: [12pt]{minimal}
$${}{(k;n,p)}^{m}$$ NB ( k ; n , p ) m in which k = 70, P = 0.0015, m = 10, and n represents the total number of cells sampled. Cell type clustering and annotation After generation of digital gene expression matrices as described above, we filtered out nuclei with fewer than 500 UMIs. We then performed cell type annotation iteratively through a number of rounds of dimensionality reduction, clustering, and removal of putative doublets and cells with high mitochondrial expression. For the preliminary clustering step, we performed standard preprocessing (UMI normalization, highly variable gene selection, scaling) with Seurat v2.3.4 as previously described . We used principal component analysis (PCA) with 30 components and Louvain community detection with resolution 0.1 to identify major clusters (resulting in 34 clusters). At this stage, we merged several clusters (primarily granule cell clusters) based on shared expression of canonical cell type markers, and removed one cluster whose top differentially expressed genes were mitochondrial (resulting in 11 clusters). For subsequent rounds of cluster annotation within these major cell type clusters, we applied a variation of the LIGER workflow previously described , using integrative non-negative matrix factorization (iNMF) to limit the effects of sample- and sex-specific gene expression. In brief, we normalized each cell by the number of UMIs, selected highly variable genes and spatially variable genes (see section below), performed iNMF, and clustered using Louvain community detection (omitting the quantile normalization step). Clusters whose top differentially expressed genes indicated contamination from a different cell type or high expression of mitochondrial genes were removed during the annotation process, and not included in subsequent rounds of annotation. This iterative annotation process was repeated until no contaminating clusters were identified in a round of clustering. Differential expression analysis within rounds of annotation was performed with the Wilcoxon rank sum test using Seurat’s FindAllMarkers function. Comprehensive differential expression analysis across all 46 final annotated clusters was performed using the wilcoxauc function from the presto package . A full set of parameters used in the LIGER annotation steps and further details can be found in Supplementary Table . For visualization as in Fig. , we merged all annotated high-quality nuclei and repeated preliminary preprocessing steps before performing UMAP using 25 principal components. Integrated analysis of human and mouse data After generation of digital gene expression matrices for the human nuclei profiles, we filtered out nuclei with fewer than 500 UMIs. We then performed a preliminary round of cell type annotation using the standard LIGER workflow (integrating across batches) to identify the primary human interneuron populations (UBCs, MLIs and PLIs, Golgi cells, granule cells; based on the same markers as in Supplementary Table ). We repeated an iteration of the same workflow for the four cell populations specified above (with an additional quantile normalization step) in order to identify and remove putative doublet and artefactual populations. Finally, we performed iNMF metagene projection as previously described to project the human datasets into latent spaces derived from the corresponding mouse cell type datasets. We then performed quantile normalization and Louvain clustering, assigning joint clusters based on the previously annotated mouse data clusters. For the granule cell joint analysis, we first limited the mouse data to include only the five cerebellar regions sampled in human data collection (lobules II, VII, VIII, IX and X). For the Golgi cell joint analysis, we performed iNMF (integrating across species), instead of metagene projection. Spatially variable gene selection To identify genes with high regional variance, we first computed the log of the index of dispersion (log variance-to-mean ratio, logVMR) for each gene, across each of the 16 lobular regions. Next, we simulated a Gaussian null distribution whose centre was the logVMR mode, found by performing a kernel density estimation of the logVMRs (using the density function in R, followed by the turnpoints function). The standard deviation of the Gaussian was computed by reflecting the values less than the mode across the centre. Genes whose logVMRs were in the upper tail with P < 0.01 (Benjamini–Hochberg adjusted) were ruled as spatially variable. For the granule cell and PC cluster analyses, adjusted P -value thresholds were set to 0.001 and 0.002, respectively. Cluster regional composition testing and lobule enrichment To determine whether the lobule composition of a cluster differs significantly from the corresponding outer level cell type lobule distribution, we used a multinomial test approximated by Pearson’s chi-squared test with k − 1 degrees of freedom, in which k was the total number of lobules sampled (16). The expected number of nuclei for a cluster i and lobule j was estimated as follows: [12pt]{minimal}
$${E}_{ij}={N}_{i} _{j}}{{ }_{j}{N}_{j}}$$ E i j = N i × N j ∑ j N j where N i is the total number of nuclei in cluster i and N j is the number of nuclei in lobule j (across all clusters in the outer level cell type, as defined below). The resulting P values were FDR-adjusted (Benjamini–Hochberg) using the p.adjust function in R. Lobule enrichment (LE) scores for each cluster i and each lobule j were calculated by: [12pt]{minimal}
$${{}}_{ij}=\,_{ij}}{{ }_{j}{n}_{ij}}}{_{j}}{{ }_{j}{N}_{j}}}$$ LE i j = n i j ∑ j n i j N j ∑ j N j in which n ij is the observed number of nuclei in cluster i and lobule j , and N j is the number of nuclei in lobule j (across all clusters in the outer level cell type). For this analysis, we used coarse cell type definitions shown coloured in the Fig. , and merged the PLI clusters. For lobule composition testing and replicate consistency analysis below, we downsampled granule cells to 60,000 nuclei (the next most numerous cell type were the MLI and PLI clusters with 45,767 nuclei). To determine the consistency of lobule enrichment scores across replicates in each region, we designated two sets of replicates by assigning nuclei from the most represented replicate in each region and cluster analysis to ‘replicate 1’ and nuclei from the second most represented replicate in each region to ‘replicate 2’. This assignment was used because not all regions had representation from all individuals profiled, and some had representation from only two individuals. We calculated lobule enrichment scores for each cluster using each of the replicate sets separately; we then calculated the Pearson correlation between the two sets of lobule enrichment scores for each cluster. We would expect correlation to be high for clusters when lobule enrichment is biologically consistent. We note that one cluster (Purkinje_Aldoc_2), was excluded from the replicate consistency analysis as under this design, it had representation from only a single aggregated replicate. However, we confirmed that lobule enrichment for this cluster was strongly consistent with Allen Brain Atlas expression staining (Extended Data Fig. ). Continuity of gene expression To characterize molecular variation across cell types, we attempted to quantify the continuity of scaled gene expression across a given cell type pair, ordered by pseudotime rank (calculated using Monocle2). For each gene, we fit a logistic curve to the scaled gene expression values and calculated the maximum slope ( m ) of the resulting curve, after normalizing for both the number of cells and dynamic range of the logistic fit. To limit computational complexity, we downsampled cell type pairs to 5,000 total nuclei. We fit curves and computed m values for the most significantly differentially expressed genes across five cell type pairs (Fig. ). Differentially expressed genes were determined using Seurat’s FindMarkers function. We then plotted the cumulative distribution of m values for the top 200 genes for each cell type pair; genes were selected based on ordering by absolute Spearman correlation between scaled gene expression and pseudotime rank. Trajectory analysis of peri- and postnatal mouse cerebellum data After generation of digital gene expression matrices for the peri- and postnatal mouse profiles, we filtered out nuclei with fewer than 500 UMIs. We applied the LIGER workflow (similarly to the adult mouse data analysis), to identify clusters corresponding to major developmental pathways. We then isolated the cluster corresponding to GABAergic progenitors (marked by expression of Tfap2b and other canonical markers). We performed a second iteration of LIGER iNMF and Louvain clustering on this population and generated a UMAP representation. Using this UMAP representation, we calculated pseudotime ordering and a corresponding trajectory graph with Monocle3 . To identify modules of genes which varied along the computed trajectory, we used the graph_test and find_gene_modules functions from Monocle3. Reporting summary Further information on research design is available in the linked to this paper.
Nuclei suspensions for mouse (C57BL/6J, Jackson Labs) cerebellum profiles were generated from 2 female and 4 male adult mice (60 days old), 1 male E18 mouse, 1 male P0 (newborn) mouse, 1 female P4 (4 days old) mouse, 1 female P8, 2 male P12 and 2 female P16 mice. Adult mice were group-housed with a 12-h light-dark schedule and allowed to acclimate to their housing environment for two weeks after arrival. Timed pregnant mice were received and euthanized to yield E18 mice 6 days after arrival. Newborn mice were housed as individual litters for up to 16 days. All experiments were approved by and in accordance with Broad IACUC protocol number 012-09-16.
At E18, P0, P4, P8, P12, P16 and P60, C57BL/6J mice were anaesthetized by administration of isoflurane in a gas chamber flowing 3% isoflurane for 1 min. Anaesthesia was confirmed by checking for a negative tail and paw pinch response. Mice were moved to a dissection tray and anaesthesia was prolonged via a nose cone flowing 3% isoflurane for the duration of the procedure. Transcardial perfusions were performed on adult, pregnant (E18), P8, P12 and P16 mice with ice-cold pH 7.4 HEPES buffer containing 110 mM NaCl, 10 mM HEPES, 25 mM glucose, 75 mM sucrose, 7.5 mM MgCl 2 , and 2.5 mM KCl to remove blood from the brain. P0 and P4 mice were unperfused. The brain was removed from P60, P8, P12 and P16 mice and frozen for 3 min in liquid nitrogen vapour. E18, P0 and P4 mice were sagittally bisected after similarly freezing their brains in situ. All tissue was moved to −80 °C for long-term storage. A detailed protocol is available at protocols.io (10.17504/protocols.io.bcbrism6).
Frozen adult mouse brains were securely mounted by the frontal cortex onto cryostat chucks with OCT embedding compound such that the entire posterior half including the cerebellum and brainstem were left exposed and thermally unperturbed. Dissection of each of 16 cerebellar vermal and cortical lobules was performed by hand in the cryostat using an ophthalmic microscalpel (Feather safety Razor P-715) pre-cooled to −20 °C and donning four surgical loupes. Whole E18, P0, P4, P8, P12 and P16 mouse cerebella were similarly curated by dissecting rhombomeric cerebellar rudiments from sagittal frozen brain hemispheres using a pre-cooled 1-mm disposable biopsy punch (Integra Miltex). Each excised tissue dissectate was placed into a pre-cooled 0.25 ml PCR tube using pre-cooled forceps and stored at −80 °C. Nuclei were extracted from this frozen tissue using gentle, detergent-based dissociation, according to a protocol available at protocols.io (10.17504/protocols.io.bck6iuze) adapted from one provided by the McCarroll laboratory (Harvard Medical School), and loaded into the 10x Chromium V3 system. Reverse transcription and library generation were performed according to the manufacturer’s protocol.
Acute cerebellar slices containing Alexa 594-filled patched cells were fixed as described and stored in 70% ethanol at 4 °C until hybridization chain reaction (HCR). They were then subjected to a ‘floating slice HCR’ protocol in which the recorded cells could be simultaneously re-imaged in conjunction with HCR expression analysis in situ and catalogued as to their positions in the cerebellum. A detailed protocol (10.17504/protocols.io.bck7iuzn) was performed using the following HCR probes and matching hairpins purchased from Molecular Instruments: glutamate metabotropic receptor 8 ( Grm8 ) lot number PRC005, connexin 36 ( Gjd2 ) lot number PRD854 and PRA673, cadherin22 ( Cdh22 ) lot number PRC011, neurexophilin 1 ( Nxph1 ) lot number PRC675 and PRC466, leucine-rich glioma-inactivated protein 2 ( Lgi2 ) lot number PRC012, somatostatin ( Sst ) lot number PRA213 and sortilin related VPS10 domain containing receptor 3 ( Sorcs3 ) lot number PRC004. Amplification hairpins used were type B1, B2 and B3 in 488 nm, 647 nm and 546 nm respectively.
After floating-slice HCR, slices were mounted between no.1 coverslips with antifade compound (ProLong Glass, Invitrogen) and images were collected on an Andor CSU-X spinning disk confocal system coupled to a Nikon Eclipse Ti microscope equipped with an Andor iKon-M camera. The images were acquired with an oil immersion objective at 60×. The Alexa 594 patched cell backfill channel (561 nm) plus associated HCR probe/hairpin channels (488 nm and 647 nm) were projected through a 10–20-μm thick z -series so that an unambiguous determination of the association between the patch-filled cell and its HCR gene expression could be made. Images were processed using Nikon NIS Elements 4.4 and Nikon NIS AR.
Human donor tissue was supplied by the Human Brain and Spinal Fluid Resource Center at UCLA, through the NIH NeuroBioBank. This work was determined by the Office of Research Subjects Protection at the Broad Institute not to meet the definition of human subjects research (project ID NHSR-4235). Nuclei suspensions from human cerebellum were generated from two neuropathologically normal control cases—one female tissue donor, aged 35, and one male tissue donor, aged 36. These fresh frozen tissues had post mortem intervals of 12 and 13.5 h respectively, and were provided as whole cerebella cut into four coronal slabs. A sub-dissection of frozen cerebellar lobules was performed on dry ice just before 10x processing and nuclei were extracted from this frozen tissue using gentle, detergent-based dissociation, according to a protocol available at protocols.io (10.17504/protocols.io.bck6iuze).
Acute parasagittal slices were prepared at 240-μm thickness from wild-type mice aged P30–P50. Mice were anaesthetized with an intraperitoneal injection of ketamine (10 mg kg −1 ), perfused transcardially with an ice-cold solution containing (in mM): 110 choline chloride, 7 MgCl 2 , 2.5 KCl, 1.25 NaH 2 PO 4 , 0.5 CaCl 2 , 25 glucose, 11.5 sodium ascorbate, 3 sodium pyruvate, 25 NaHCO 3 , 0.003 ( R )-CPP, equilibrated with 95% O 2 and 5% CO 2 . Slices were cut in the same solution and were then transferred to artificial cerebrospinal fluid (ACSF) containing (in mM) 125 NaCl, 26 NaHCO 3 , 1.25 NaH 2 PO 4 , 2.5 KCl, 1 MgCl 2 , 1.5 CaCl 2 and 25 glucose equilibrated with 95% O 2 and 5% CO 2 at approximately 34 °C for 30 min. Slices were then kept at room temperature until recording. All UBC recordings were done at 34–36 °C with (in μM) 2 ( R )-CPP, 5 NBQX, 1 strychnine, 10 SR95531 (gabazine) and 1.5 CGP in the bath to isolate metabotropic currents. Loose cell-attached recordings were made with ACSF-filled patch pipettes of 3–5 MΩ resistance. Whole-cell voltage-clamp recordings were performed while holding the cell at −70 mV with an internal containing (in mM): 140 KCl, 4 NaCl, 0.5 CaCl 2 , 10 HEPES, 4 MgATP, 0.3 NaGTP, 5 EGTA 5, and 2 QX-314, pH adjusted to 7.2 with KOH. Brief puffs of glutamate (1 mM for 50 ms at 5 psi) were delivered using a Picospritzer II (General Valve Corp.) in both cell-attached and whole-cell configuration to assure consistent responses. The heat map of current traces from all cells are sorted by the score over the first principal axis after singular value decomposition (SVD) of recordings over all cells. For whole-cell recordings with pharmacology, we used an K-methanesulfonate internal containing (in mM): 122 K-methanesulfonate, 9 NaCl, 9 HEPES, 0.036 CaCl 2 , 1.62 MgCl 2 , 4 MgATP, 0.3 GTP (Tris salt), 14 creatine phosphate (Tris salt), and 0.18 EGTA, pH 7.4. A junction potential of −8 mV was compensated for during recording. 300nM TTX was added to the ACSF in conjunction with the synaptic blockers listed above. Three pipettes filled with ACSF containing 1 mM glutamate, 100 μM DHPG or 1 μM LY354740 were positioned within 20 μm of the recorded cell. Pressure applications of each agonist were delivered at 10 psi with durations of 40–50 ms. Agonist applications were separated by 30 s. Two to three trials were collected for each agonist. MLI recordings were performed at approximately 32 °C with an internal solution containing (in mM) 150 K-gluconate, 3 KCl, 10 HEPES, 3 MgATP, 0.5 GTP, 5 phosphocreatine-tris 2 and 5 phosphocreatine-Na 2 , 2 mg ml −1 biocytin and 0.1 Alexa 594 (pH adjusted to 7.2 with KOH, osmolality adjusted to 310 mOsm kg −1 ). Visually guided whole-cell recordings were obtained with patch pipettes of around 4 MΩ resistance pulled from borosilicate capillary glass (BF150-86-10, Sutter Instrument). Electrophysiology data was acquired using a Multiclamp 700B amplifier (Axon Instruments), digitized at 20 kHz and filtered at 4 kHz. For isolating spikelets in MLI recordings, cells were held at −65 mV in voltage clamp and the following receptor antagonists were added to the solution (in μM) to block synaptic currents: 2 ( R )-CPP, 5 NBQX, 1 strychnine, 10 SR95531 (gabazine), 1.5 CGP. All drugs were purchased from Abcam and Tocris. To obtain an input-output curve, MLIs were maintained at 60–65 mV with a constant hyperpolarizing current, and 250 ms current steps ranging from −30 pA to +100 pA were injected in 10 pA increments. To activate the hyperpolarization-evoked current ( I h ), MLIs were held at −65 mV and a 30 pA hyperpolarizing current step of 500 ms duration was injected. The amplitude of I h was calculated as the difference between the maximal current evoked by the hyperpolarizing current step and the average steady-state current at the end (480–500 ms) of the current step. Capacitance and input resistance ( R i ) were determined using a 10 pA, 50 ms hyperpolarizing current step. To prevent excessive dialysis and to ensure successful detection of mRNAs in the recorded cells, the total duration of recordings did not exceed 10 min. Acquisition and analysis of electrophysiological data were performed using custom routines written in MATLAB (Mathworks), IgorPro (Wavemetrics), or AxoGraphX. Data are reported as median ± interquartile range, and statistical analysis was carried out using the Mann–Whitney or Fisher’s exact test, as indicated. Statistical significance was assumed at P < 0.05. To determine the presence of spikelets, peak detection was used to generate event-triggered average waveforms with thresholds based on the mean absolute deviation (MAD) of the raw trace. Spikelet recordings were scored for the presence of spikelets blind to the molecular identity of the cells. The analysis was restricted to cells recorded in the presence of synaptic blockers.
MLIs were filled with 100 μM Alexa-594 via patch pipette to visualize their morphology using two-photon imaging. After completion of the electrophysiological recordings the patch electrode was retracted slowly and the cell resealed. We used a custom-built two-photon laser-scanning microscope with a 40×, 0.8 numerical aperture (NA) objective (Olympus Optical) and a pulsed two-photon laser (Chameleon or MIRA 900, Coherent, 800 nm excitation). DIC images were acquired at the end of each experiment and locations of each cell within the slice were recorded. Two-photon images were further processed in ImageJ.
After recording and imaging, cerebellar slices were transferred to a well-plate and submerged in 2–4% PFA in PBS (pH 7.4) and incubated overnight at 4 °C. Slices were then washed in PBS (3 × 5 min) and then kept in 70% ethanol in RNase-free water until HCR was performed.
Sequencing reads from mouse cerebellum experiments were demultiplexed and aligned to a mouse (mm10) premrna reference using CellRanger v3.0.2 with default settings. Digital gene expression matrices were generated with the CellRanger count function. Sequencing reads from human cerebellum experiments were demultiplexed and aligned to a human (hg19) premrna reference using the Drop-seq alignment workflow , which was also used to generate the downstream digital gene expression matrices.
To estimate the probability of sufficiently sampling rare cell types in the cerebellum as a function of total number of nuclei sampled, we used the approach proposed by the Satija laboratory ( https://satijalab.org/howmanycells ), with the assumption of at most 10 very rare cell types, each with a prevalence of 0.15%. We derived this minimum based on the observed prevalences of the two rarest cell types we identified (OPC_4, Purkinje_Aldoc_2). We set 70 cells as the threshold for sufficient sampling, and calculated the overall probability as a negative binomial (NB) density: [12pt]{minimal}
$${}{(k;n,p)}^{m}$$ NB ( k ; n , p ) m in which k = 70, P = 0.0015, m = 10, and n represents the total number of cells sampled.
After generation of digital gene expression matrices as described above, we filtered out nuclei with fewer than 500 UMIs. We then performed cell type annotation iteratively through a number of rounds of dimensionality reduction, clustering, and removal of putative doublets and cells with high mitochondrial expression. For the preliminary clustering step, we performed standard preprocessing (UMI normalization, highly variable gene selection, scaling) with Seurat v2.3.4 as previously described . We used principal component analysis (PCA) with 30 components and Louvain community detection with resolution 0.1 to identify major clusters (resulting in 34 clusters). At this stage, we merged several clusters (primarily granule cell clusters) based on shared expression of canonical cell type markers, and removed one cluster whose top differentially expressed genes were mitochondrial (resulting in 11 clusters). For subsequent rounds of cluster annotation within these major cell type clusters, we applied a variation of the LIGER workflow previously described , using integrative non-negative matrix factorization (iNMF) to limit the effects of sample- and sex-specific gene expression. In brief, we normalized each cell by the number of UMIs, selected highly variable genes and spatially variable genes (see section below), performed iNMF, and clustered using Louvain community detection (omitting the quantile normalization step). Clusters whose top differentially expressed genes indicated contamination from a different cell type or high expression of mitochondrial genes were removed during the annotation process, and not included in subsequent rounds of annotation. This iterative annotation process was repeated until no contaminating clusters were identified in a round of clustering. Differential expression analysis within rounds of annotation was performed with the Wilcoxon rank sum test using Seurat’s FindAllMarkers function. Comprehensive differential expression analysis across all 46 final annotated clusters was performed using the wilcoxauc function from the presto package . A full set of parameters used in the LIGER annotation steps and further details can be found in Supplementary Table . For visualization as in Fig. , we merged all annotated high-quality nuclei and repeated preliminary preprocessing steps before performing UMAP using 25 principal components.
After generation of digital gene expression matrices for the human nuclei profiles, we filtered out nuclei with fewer than 500 UMIs. We then performed a preliminary round of cell type annotation using the standard LIGER workflow (integrating across batches) to identify the primary human interneuron populations (UBCs, MLIs and PLIs, Golgi cells, granule cells; based on the same markers as in Supplementary Table ). We repeated an iteration of the same workflow for the four cell populations specified above (with an additional quantile normalization step) in order to identify and remove putative doublet and artefactual populations. Finally, we performed iNMF metagene projection as previously described to project the human datasets into latent spaces derived from the corresponding mouse cell type datasets. We then performed quantile normalization and Louvain clustering, assigning joint clusters based on the previously annotated mouse data clusters. For the granule cell joint analysis, we first limited the mouse data to include only the five cerebellar regions sampled in human data collection (lobules II, VII, VIII, IX and X). For the Golgi cell joint analysis, we performed iNMF (integrating across species), instead of metagene projection.
To identify genes with high regional variance, we first computed the log of the index of dispersion (log variance-to-mean ratio, logVMR) for each gene, across each of the 16 lobular regions. Next, we simulated a Gaussian null distribution whose centre was the logVMR mode, found by performing a kernel density estimation of the logVMRs (using the density function in R, followed by the turnpoints function). The standard deviation of the Gaussian was computed by reflecting the values less than the mode across the centre. Genes whose logVMRs were in the upper tail with P < 0.01 (Benjamini–Hochberg adjusted) were ruled as spatially variable. For the granule cell and PC cluster analyses, adjusted P -value thresholds were set to 0.001 and 0.002, respectively.
To determine whether the lobule composition of a cluster differs significantly from the corresponding outer level cell type lobule distribution, we used a multinomial test approximated by Pearson’s chi-squared test with k − 1 degrees of freedom, in which k was the total number of lobules sampled (16). The expected number of nuclei for a cluster i and lobule j was estimated as follows: [12pt]{minimal}
$${E}_{ij}={N}_{i} _{j}}{{ }_{j}{N}_{j}}$$ E i j = N i × N j ∑ j N j where N i is the total number of nuclei in cluster i and N j is the number of nuclei in lobule j (across all clusters in the outer level cell type, as defined below). The resulting P values were FDR-adjusted (Benjamini–Hochberg) using the p.adjust function in R. Lobule enrichment (LE) scores for each cluster i and each lobule j were calculated by: [12pt]{minimal}
$${{}}_{ij}=\,_{ij}}{{ }_{j}{n}_{ij}}}{_{j}}{{ }_{j}{N}_{j}}}$$ LE i j = n i j ∑ j n i j N j ∑ j N j in which n ij is the observed number of nuclei in cluster i and lobule j , and N j is the number of nuclei in lobule j (across all clusters in the outer level cell type). For this analysis, we used coarse cell type definitions shown coloured in the Fig. , and merged the PLI clusters. For lobule composition testing and replicate consistency analysis below, we downsampled granule cells to 60,000 nuclei (the next most numerous cell type were the MLI and PLI clusters with 45,767 nuclei). To determine the consistency of lobule enrichment scores across replicates in each region, we designated two sets of replicates by assigning nuclei from the most represented replicate in each region and cluster analysis to ‘replicate 1’ and nuclei from the second most represented replicate in each region to ‘replicate 2’. This assignment was used because not all regions had representation from all individuals profiled, and some had representation from only two individuals. We calculated lobule enrichment scores for each cluster using each of the replicate sets separately; we then calculated the Pearson correlation between the two sets of lobule enrichment scores for each cluster. We would expect correlation to be high for clusters when lobule enrichment is biologically consistent. We note that one cluster (Purkinje_Aldoc_2), was excluded from the replicate consistency analysis as under this design, it had representation from only a single aggregated replicate. However, we confirmed that lobule enrichment for this cluster was strongly consistent with Allen Brain Atlas expression staining (Extended Data Fig. ).
To characterize molecular variation across cell types, we attempted to quantify the continuity of scaled gene expression across a given cell type pair, ordered by pseudotime rank (calculated using Monocle2). For each gene, we fit a logistic curve to the scaled gene expression values and calculated the maximum slope ( m ) of the resulting curve, after normalizing for both the number of cells and dynamic range of the logistic fit. To limit computational complexity, we downsampled cell type pairs to 5,000 total nuclei. We fit curves and computed m values for the most significantly differentially expressed genes across five cell type pairs (Fig. ). Differentially expressed genes were determined using Seurat’s FindMarkers function. We then plotted the cumulative distribution of m values for the top 200 genes for each cell type pair; genes were selected based on ordering by absolute Spearman correlation between scaled gene expression and pseudotime rank.
After generation of digital gene expression matrices for the peri- and postnatal mouse profiles, we filtered out nuclei with fewer than 500 UMIs. We applied the LIGER workflow (similarly to the adult mouse data analysis), to identify clusters corresponding to major developmental pathways. We then isolated the cluster corresponding to GABAergic progenitors (marked by expression of Tfap2b and other canonical markers). We performed a second iteration of LIGER iNMF and Louvain clustering on this population and generated a UMAP representation. Using this UMAP representation, we calculated pseudotime ordering and a corresponding trajectory graph with Monocle3 . To identify modules of genes which varied along the computed trajectory, we used the graph_test and find_gene_modules functions from Monocle3.
Further information on research design is available in the linked to this paper.
Any methods, additional references, Nature Research 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/s41586-021-03220-z.
Supplementary Information This file contains Supplementary Note for Extended Data Fig. 8, legends for Supplementary Tables 1-4, and Supplementary References. Reporting Summary Peer Review File Supplementary Tables This file contains Supplementary Tables 1-4 (see Supplementary Information for full legends). Supplementary Table 1: Established cell type markers. Supplementary Table 2: Highly differentially expressed genes across all clusters. Supplementary Table 3: Summary of electrophysiological data. Supplementary Table 4: Function parameters used during iterative cluster annotation.
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Comparison of the safety of esophagojejunal overlap and π-shaped anastomosis in totally laparoscopic total gastrectomy | 67f81706-6e7f-479f-b2d0-b8ddc9f1313b | 11730524 | Laparoscopy[mh] | The first laparoscopy-assisted gastric surgery was performed by Kitano more than 20 years ago. Laparoscopic gastric surgery is becoming increasingly popular and advanced. Totally laparoscopic surgery has gradually become the mainstream because of the advantages of small abdominal incision and less abdominal tissue trauma. The change in surgical methods has also changed the anastomotic methods of gastric cancer surgery. Although the circular stapling method can be used for anastomosis in gastroesophageal junction cancer with a higher location, surgeons must have higher surgical skills. This approach is associated with larger abdominal wounds and higher rates of postoperative anastomotic stenosis . However, it is not suitable for totally laparoscopic operation. Linear staplers are gradually being used because they can pass through the trocar and have a more flexible operating angle. It has irreplaceable advantages, particularly in anastomosis for totally laparoscopic surgery. Total gastrectomy is still the main radical treatment for advanced gastric cancer and gastroesophageal junction cancer . Linear stapled anastomosis has become the most important anastomosis method after totally laparoscopic total gastrectomy (TLTG). Linear stapled anastomosis includes isoperistaltic and antiperistaltic anastomosis. In recent years, the overlap method is one of the representative types of isoperistaltic anastomosis, and π-shaped anastomosis is the most popular type of antiperistaltic anastomosis. The safety and population suitable for these two anastomotic methods are still unclear, and relevant studies are lacking. Each center chooses the anastomosis method according to its center’s experience and habit. Therefore, this study intends to explore the early postoperative complications of the two anastomosis methods and the population suitable for each method to provide a basis for the selection of anastomosis methods for subsequent TLTG.
Patients Patients with gastric cancer who underwent TLTG in the Gastrointestinal Cancer Center of Peking University Cancer Hospital between June 2020 and May 2023 were retrospectively collected. Patients with (1) overlap or π-shaped anastomosis, (2) pTNM stages I–III, and (3) postoperative pathologically confirmed adenocarcinoma were included. Patients with a (1) history of abdominal surgery and (2) intraoperative combined organ resection were excluded. All patients provided informed consent, and the study was approved by the Research Ethics Committee of Peking University Cancer Hospital. Main surgical and anastomotic procedures Five trocars were routinely used in our center, and gastric mobilization and lymph node dissection were performed according to the Gastric Cancer Treatment Guidelines formulated by the Japan Gastric Cancer Association . π-shaped anastomosis The jejunal mesentery was severed at a site of 50 cm from the Treitz ligament. The jejunum was lifted, the opposite side of the mesangial margin and the right side wall of the cardia were opened, and a linear stapler was placed for esophagojejunum side-to-side anastomosis. The esophagus and jejunum were closed and severed using a linear stapler. The anastomosis and closed edge were strengthened by suturing. The jejunum was opened at a site 40 cm from the distal end of the anastomosis, and the contralateral mesenteric margin was opened at a site 40 cm from the Treitz ligament. Jejune-jejunal side-to-side anastomosis was performed. The linear cutter stapler closes the common opening. The anastomosis and closed margin were strengthened by suturing (Fig. , Additional file 1). Overlap anastomosis The jejunum and mesangium were severed 50 cm away from the Treitz ligament, and the closed margin was sutured continuously. The distal jejunum was raised, and the opposite side of the mesangial margin and the right lateral wall of the esophagus were opened. The right lateral wall of the esophagus and the jejunum were anastomosed with a linear stapler. The common opening was closed using barbed threads, and the closing edge is stitched continuously. The jejunum was opened at a site 50 cm from the distal end of the anastomosis and 40 cm from the Treitz ligament. Jejune-jejunal side-to-side anastomosis was performed by using a linear cutter stapler. The linear cutter stapler closes the common opening. The anastomosis and closed margin were sutured (Fig. , Additional file 2). The two types of anastomosis, overlap anastomosis and π-shaped anastomosis, are decided by the patient according to his inclination. If the patient has no inclination, there is an attending surgeon who decides the anastomosis based on the intraoperative situation. All chief surgeons have more than 10 years of experience in laparoscopic surgery and perform at least 200 abdominal surgeries per year. Data collection and statistical analysis Age, sex, history of neoadjuvant chemotherapy, length of postoperative hospital stay, anastomotic leakage, postoperative lung infection, and other relevant data were collected from the course records. The operative time and intraoperative blood loss were collected from surgical records. The location of the tumor center, number of lymph nodes removed, pathological TNM stage, and esophageal resection margin were recorded from the postoperative pathological examination. Categorical variables were presented as frequency or percentages and compared using χ2 or Fisher’s exact tests. Continuous variables were expressed as means ± standard deviations or median (interquartile range [IQR]) and were compared between groups using the independent t-test or Wilcoxon rank sum test if the data were not normally distributed. A p-value of < 0.05 was considered statistically significant. IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA) was used for data analysis.
Patients with gastric cancer who underwent TLTG in the Gastrointestinal Cancer Center of Peking University Cancer Hospital between June 2020 and May 2023 were retrospectively collected. Patients with (1) overlap or π-shaped anastomosis, (2) pTNM stages I–III, and (3) postoperative pathologically confirmed adenocarcinoma were included. Patients with a (1) history of abdominal surgery and (2) intraoperative combined organ resection were excluded. All patients provided informed consent, and the study was approved by the Research Ethics Committee of Peking University Cancer Hospital.
Five trocars were routinely used in our center, and gastric mobilization and lymph node dissection were performed according to the Gastric Cancer Treatment Guidelines formulated by the Japan Gastric Cancer Association . π-shaped anastomosis The jejunal mesentery was severed at a site of 50 cm from the Treitz ligament. The jejunum was lifted, the opposite side of the mesangial margin and the right side wall of the cardia were opened, and a linear stapler was placed for esophagojejunum side-to-side anastomosis. The esophagus and jejunum were closed and severed using a linear stapler. The anastomosis and closed edge were strengthened by suturing. The jejunum was opened at a site 40 cm from the distal end of the anastomosis, and the contralateral mesenteric margin was opened at a site 40 cm from the Treitz ligament. Jejune-jejunal side-to-side anastomosis was performed. The linear cutter stapler closes the common opening. The anastomosis and closed margin were strengthened by suturing (Fig. , Additional file 1). Overlap anastomosis The jejunum and mesangium were severed 50 cm away from the Treitz ligament, and the closed margin was sutured continuously. The distal jejunum was raised, and the opposite side of the mesangial margin and the right lateral wall of the esophagus were opened. The right lateral wall of the esophagus and the jejunum were anastomosed with a linear stapler. The common opening was closed using barbed threads, and the closing edge is stitched continuously. The jejunum was opened at a site 50 cm from the distal end of the anastomosis and 40 cm from the Treitz ligament. Jejune-jejunal side-to-side anastomosis was performed by using a linear cutter stapler. The linear cutter stapler closes the common opening. The anastomosis and closed margin were sutured (Fig. , Additional file 2). The two types of anastomosis, overlap anastomosis and π-shaped anastomosis, are decided by the patient according to his inclination. If the patient has no inclination, there is an attending surgeon who decides the anastomosis based on the intraoperative situation. All chief surgeons have more than 10 years of experience in laparoscopic surgery and perform at least 200 abdominal surgeries per year.
The jejunal mesentery was severed at a site of 50 cm from the Treitz ligament. The jejunum was lifted, the opposite side of the mesangial margin and the right side wall of the cardia were opened, and a linear stapler was placed for esophagojejunum side-to-side anastomosis. The esophagus and jejunum were closed and severed using a linear stapler. The anastomosis and closed edge were strengthened by suturing. The jejunum was opened at a site 40 cm from the distal end of the anastomosis, and the contralateral mesenteric margin was opened at a site 40 cm from the Treitz ligament. Jejune-jejunal side-to-side anastomosis was performed. The linear cutter stapler closes the common opening. The anastomosis and closed margin were strengthened by suturing (Fig. , Additional file 1).
The jejunum and mesangium were severed 50 cm away from the Treitz ligament, and the closed margin was sutured continuously. The distal jejunum was raised, and the opposite side of the mesangial margin and the right lateral wall of the esophagus were opened. The right lateral wall of the esophagus and the jejunum were anastomosed with a linear stapler. The common opening was closed using barbed threads, and the closing edge is stitched continuously. The jejunum was opened at a site 50 cm from the distal end of the anastomosis and 40 cm from the Treitz ligament. Jejune-jejunal side-to-side anastomosis was performed by using a linear cutter stapler. The linear cutter stapler closes the common opening. The anastomosis and closed margin were sutured (Fig. , Additional file 2). The two types of anastomosis, overlap anastomosis and π-shaped anastomosis, are decided by the patient according to his inclination. If the patient has no inclination, there is an attending surgeon who decides the anastomosis based on the intraoperative situation. All chief surgeons have more than 10 years of experience in laparoscopic surgery and perform at least 200 abdominal surgeries per year.
Age, sex, history of neoadjuvant chemotherapy, length of postoperative hospital stay, anastomotic leakage, postoperative lung infection, and other relevant data were collected from the course records. The operative time and intraoperative blood loss were collected from surgical records. The location of the tumor center, number of lymph nodes removed, pathological TNM stage, and esophageal resection margin were recorded from the postoperative pathological examination. Categorical variables were presented as frequency or percentages and compared using χ2 or Fisher’s exact tests. Continuous variables were expressed as means ± standard deviations or median (interquartile range [IQR]) and were compared between groups using the independent t-test or Wilcoxon rank sum test if the data were not normally distributed. A p-value of < 0.05 was considered statistically significant. IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA) was used for data analysis.
Clinicopathological characteristics of patients In total, 162 patients met the criteria, including 96 patients with π-shaped anastomosis and 66 with overlap anastomosis, and underwent R0 resection. The tumor center location was different between the π-shaped anastomosis group and the overlap anastomosis group ( p < 0.05), i.e., the overlap anastomosis group was dominated by the gastroesophageal junction, whereas the π-shaped anastomosis was dominated by the gastric fundus and stomach body. No significant differences were found in the postoperative tumor pathological stage, preoperative neoadjuvant chemotherapy, and other general clinical data between the two groups (Table ). Operation and postoperative complications No significant differences were found in surgical contents, including the operation time and intraoperative blood loss ( p > 0.05). The incidence of anastomotic leakage and the length of postoperative hospital stay were higher after π-shaped anastomosis than after overlap anastomosis ( p < 0.05). No significant differences were noted in other postoperative complications and pathological conditions ( p > 0.05). Postoperative esophageal margins were negative in both groups (Table ).
In total, 162 patients met the criteria, including 96 patients with π-shaped anastomosis and 66 with overlap anastomosis, and underwent R0 resection. The tumor center location was different between the π-shaped anastomosis group and the overlap anastomosis group ( p < 0.05), i.e., the overlap anastomosis group was dominated by the gastroesophageal junction, whereas the π-shaped anastomosis was dominated by the gastric fundus and stomach body. No significant differences were found in the postoperative tumor pathological stage, preoperative neoadjuvant chemotherapy, and other general clinical data between the two groups (Table ).
No significant differences were found in surgical contents, including the operation time and intraoperative blood loss ( p > 0.05). The incidence of anastomotic leakage and the length of postoperative hospital stay were higher after π-shaped anastomosis than after overlap anastomosis ( p < 0.05). No significant differences were noted in other postoperative complications and pathological conditions ( p > 0.05). Postoperative esophageal margins were negative in both groups (Table ).
This study compared overlap anastomosis with π-shaped anastomosis in TLTG using a linear stapler. No significant difference in operation time and intraoperative blood loss was found between the overlap anastomosis and π-shaped anastomosis; however, the π-shaped anastomosis had a higher incidence of anastomotic leakage. Compared with overlap anastomosis, π-shaped anastomosis has no significant advantage in operation time. This finding is consistent with the results of most current studies [ – ]. For example, Guo et al. believed that with the proficiency of surgical techniques, the overlap anastomosis time would gradually become shorter . In a retrospective study, Gao concluded that the operation time was shorter with the π-shaped anastomosis compared to the overlap anastomosis . This finding may be related to not reinforcing the anastomosis or simply reinforcing it. Although π-shaped anastomosis saves time by closing the common opening and separating the esophagus and jejunum simultaneously, the exact reinforcement of the anastomosis is necessary, particularly at the corners of the π-anastomosis, and is often tricky and time-consuming. Specifically, for gastroesophageal junction tumors, the anastomosis often enters the mediastinum and is difficult to reinforce. Kwon, the inventor of π-anastomosis, also emphasized the importance of reinforcing the anastomosis . Thus, there may be no significant time difference between the two anastomosis methods. π-anastomosis is conducted with anastomosis first followed by transection. This procedure may cause residual lesions at the resection margin of the esophageal stump. In this study, π-shaped anastomosis did not show statistically significant margin problems, which may be related to the use of intraoperative gastroscopy or preoperative carbon nanoparticles for lesion localization in our center. As an advantage, overlap anastomosis ensures that anastomosis can be performed after complete tumor resection. Relieving the tension after esophagojejunoanastomosis is one of the important reasons for the initial invention of overlap anastomosis . This study showed that the incidence of anastomotic leakage was significantly higher in the π-anastomosis group than in the overlap anastomosis group. Considering this issue, previous studies also have different conclusions. Some studies have suggested that overlap anastomosis significantly reduces the incidence of anastomotic leakage compared with functional methods [ , – ]. However, others did not find a significant difference in postoperative complications between the two anastomosis methods [ – , ]. This may be due to the antiperistalsis of π-shaped anastomosis. In the corner, intestinal peristalsis leads to greater tension here; however, with a tight suture, it is still prone to anastomotic leakage. The tension was more uniform following overlap anastomosis. In addition, for peristaltic anastomosis, no two opposing pulling forces exist at the anastomotic site . Based on the intraoperative conditions and postoperative complications of the two anastomosis methods, overlap anastomosis is the preferred choice for TLTG. π-shaped anastomosis is not recommended for patients with carcinoma of gastroesophageal junction. After anastomosis, the anastomosis site was difficult to access through the thoracic cavity, a large tension of the esophagus pulling upward may occur, and suturing is difficult. In this study, patients with π-shaped anastomosis had a lower tumor location than those with overlap anastomosis. This still did not prevent more complications of anastomotic leakage. Kwon also pointed out that π-anastomosis is not suitable for patients with gastroesophageal junction . Ko et al. and Miura et al. also believed that overlap anastomosis should be selected for patients with esophageal invasion through comparative study and surgical experience . This study has some limitations. First, this was a retrospective study, and the study design may have been biased. Therefore, randomized, multicenter clinical trials are needed to confirm the safety and efficacy of the two anastomosis techniques. Second, this study only assessed short-term complications with the two anastomosis methods. Long-term complications, such as anastomotic stenosis, of the two anastomosis methods were not analyzed; thus, long-term follow-up is needed.
Overlap anastomosis is recommended for TLTG, and it has a lower incidence of postoperative anastomotic leakage. Moreover, π-shaped anastomosis is not suitable for gastroesophageal junction cancer.
Supplementary Material 1. π-shaped anastomosis. Surgery demonstration of π-shaped anastomosis. Supplementary Material 2. Overlap anastomosis. Surgery demonstration of overlap anastomosis.
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Routine Clinic Surveillance on Arteriovenous Graft Patency in Hemodialysis Patients with Previous Access Complications | c2203691-60ba-4b54-89f5-10eeb1d1b997 | 11866542 | Surgical Procedures, Operative[mh] | Hemodialysis (HD) is the primary renal replacement therapy for patients with end-stage renal disease . This treatment necessitates the use of a vascular access (VA) such as arteriovenous fistulas (AVFs), arteriovenous grafts (AVGs), and both cuffed and non-cuffed catheters, to effectively remove metabolic waste and excess fluid from the patient's bloodstream. The maintenance of a well-functioning VA is crucial for the optimal management of patients undergoing HD. AVFs are preferred due to their lower thrombosis rates, longer patency, and superior patient outcomes compared to other modalities . However, for patients with poor vascular anatomy or advanced age, AVGs may be a suitable alternative , despite their significantly lower patency rates compared to AVFs - . The importance of clinical evaluation of arteriovenous access (AVA) during HD sessions is well-supported by current guidelines . The potential benefit of routine surveillance is the ability to preserve AVA patency by identifying dysfunction before full occlusion occurs. Routine surveillance includes monitoring intra-dialysis venous pressure and access flow, allowing for timely preemptive interventions such as percutaneous transluminal angioplasty (PTA), stenting, thrombectomy, bypass surgery, and ultimately access reconstruction. However, studies on AVA surveillance have yielded inconsistent results, with some indicating a beneficial impact on patency - , whereas the outcomes of routine surveillance for AVGs remain ambiguous , . In addition, the economic impact of these surveillance measures on healthcare systems remains unclear - . The objective of this study was to assess the impact of routine surveillance at a multidisciplinary clinic in a tertiary teaching hospital on the clinical and economic outcomes of AVGs in HD patients with previous access complications.
Statement of ethical approval This study was approved by the Institutional Review Board of Ditmanson Medical Foundation Chia-Yi Christian Hospital (approval number: IRB2023067). The need for informed consent was waived due to the retrospective nature of the study. All methods were conducted in compliance with applicable guidelines and regulations . Data source To enhance the quality of dialysis VA care, a specialized outpatient clinic was established in our hospital on May 1, 2020, through the collaborative efforts of nephrology and cardiology teams . This clinic was designed to address the needs of HD patients with VA complications, such as maturation failure, challenging cannulation, elevated intra-dialysis venous pressure, reduced intra-dialysis blood flow, prolonged post-dialysis hemostasis, unexplained swelling of the limb on the side of the AVA, and other warning signs identified through guideline-directed physical examinations performed by dialysis staff. Nephrologists and dialysis staff actively encourage patients with previous VA complications undergoing interventions to attend routine surveillance at the clinic, which is predominantly led by cardiologists receiving specialized training in vascular access assessment. Following referral, the clinic provides comprehensive and objective access monitoring through physical examinations, supplemented by ultrasound when necessary. Salvage interventions are performed for patients whose VA demonstrates complications or imaging abnormalities, such as inadequate flow to achieve dialysis adequacy or stenosis exceeding 50% as detected by ultrasound. In addition, cardiologists at the clinic engage in periodic discussions with nephrologists and dialysis staff to address complex cases and optimize management plans. We gathered demographic data including sex, age, HD vintage, and characteristics of AVGs. All AVGs in this study were constructed using expanded polytetrafluoroethylene grafts. We also collected information on comorbidities, history of parathyroidectomy, use of far-infrared radiation therapy, administration of antiplatelet and antihypotensive agents, and laboratory data at the time of recruitment. Furthermore, data on episodes and costs associated with AVG interventions, including AVA reconstruction, graft-anastomosis stenting, PTA, ultrasound for the AVG interventions and clinic visits were extracted from the hospital's electronic medical records system. The datasets generated or analyzed in this study are available from the corresponding author upon reasonable request. Study design We recruited patients undergoing HD thrice weekly from the clinic's launch date. The exclusion criteria were patients undergoing dialysis for less than 6 months ( n = 7), those using catheters or AVFs ( n = 134) for dialysis, those who had not undergone salvage interventions for their AVAs ( n = 181), those receiving interventions at other facilities ( n = 14), and those with incomplete data ( n = 2). The recruited patients were then categorized into two groups: those receiving routine clinic surveillance and those without. Routine clinic surveillance was defined as regular clinic visits scheduled at intervals ranging from 3 to 6 months, commencing from the establishment date of the clinic. Patients in the routine clinic surveillance group who experienced VA complications outside of the scheduled visit intervals could arrange additional appointments as needed. The follow-up period ended at the patient's death, kidney transplant, transfer to another medical facility, or December 31, 2022, whichever occurred first. Figure depicts the recruitment, allocation, and follow-up process. In total, the study analyzed 87 patients, with 22 (25.3%) receiving routine clinic surveillance and 65 (74.7%) not receiving routine clinic surveillance. The primary outcomes of this study were the rates of different AVG interventions among the recruited patients throughout the follow-up period. AVA reconstruction was defined as the creation of a new VA after the previous one failed to support adequate dialysis treatment. Sensitivity analysis was performed to validate our findings. We also compared the secondary patency of AVGs between groups, defined as the duration from recruitment to AVG abandonment, and analyzed the costs associated with AVG interventions. Furthermore, we evaluated correlations between patient demographic and AVA reconstruction. Statistical analysis Statistical analyses were conducted using MedCalc Statistical Software (version 23.0.6, MedCalc Software Ltd., Ostend, Belgium). Categorical variables were expressed as frequencies or percentages, while continuous variables were presented as means and standard deviations. The chi-square test or Mann-Whitney test was used for comparisons between variables, as appropriate. A Cox proportional hazards regression model was used to assess the impact of routine clinic follow-up on time to AVA reconstruction. The model estimated hazard ratios after adjusting for potential confounders and significant variables between groups, to provide robust estimates of the association between routine follow-up and the risk of AVA reconstruction. Logistic regression analysis was used to determine the crude odds ratios between categorical variables and outcomes, while Spearman's rank correlation coefficients were calculated to assess correlations between continuous variables and outcomes. Statistical significance was set at a two-tailed p value of less than 0.05.
This study was approved by the Institutional Review Board of Ditmanson Medical Foundation Chia-Yi Christian Hospital (approval number: IRB2023067). The need for informed consent was waived due to the retrospective nature of the study. All methods were conducted in compliance with applicable guidelines and regulations .
To enhance the quality of dialysis VA care, a specialized outpatient clinic was established in our hospital on May 1, 2020, through the collaborative efforts of nephrology and cardiology teams . This clinic was designed to address the needs of HD patients with VA complications, such as maturation failure, challenging cannulation, elevated intra-dialysis venous pressure, reduced intra-dialysis blood flow, prolonged post-dialysis hemostasis, unexplained swelling of the limb on the side of the AVA, and other warning signs identified through guideline-directed physical examinations performed by dialysis staff. Nephrologists and dialysis staff actively encourage patients with previous VA complications undergoing interventions to attend routine surveillance at the clinic, which is predominantly led by cardiologists receiving specialized training in vascular access assessment. Following referral, the clinic provides comprehensive and objective access monitoring through physical examinations, supplemented by ultrasound when necessary. Salvage interventions are performed for patients whose VA demonstrates complications or imaging abnormalities, such as inadequate flow to achieve dialysis adequacy or stenosis exceeding 50% as detected by ultrasound. In addition, cardiologists at the clinic engage in periodic discussions with nephrologists and dialysis staff to address complex cases and optimize management plans. We gathered demographic data including sex, age, HD vintage, and characteristics of AVGs. All AVGs in this study were constructed using expanded polytetrafluoroethylene grafts. We also collected information on comorbidities, history of parathyroidectomy, use of far-infrared radiation therapy, administration of antiplatelet and antihypotensive agents, and laboratory data at the time of recruitment. Furthermore, data on episodes and costs associated with AVG interventions, including AVA reconstruction, graft-anastomosis stenting, PTA, ultrasound for the AVG interventions and clinic visits were extracted from the hospital's electronic medical records system. The datasets generated or analyzed in this study are available from the corresponding author upon reasonable request.
We recruited patients undergoing HD thrice weekly from the clinic's launch date. The exclusion criteria were patients undergoing dialysis for less than 6 months ( n = 7), those using catheters or AVFs ( n = 134) for dialysis, those who had not undergone salvage interventions for their AVAs ( n = 181), those receiving interventions at other facilities ( n = 14), and those with incomplete data ( n = 2). The recruited patients were then categorized into two groups: those receiving routine clinic surveillance and those without. Routine clinic surveillance was defined as regular clinic visits scheduled at intervals ranging from 3 to 6 months, commencing from the establishment date of the clinic. Patients in the routine clinic surveillance group who experienced VA complications outside of the scheduled visit intervals could arrange additional appointments as needed. The follow-up period ended at the patient's death, kidney transplant, transfer to another medical facility, or December 31, 2022, whichever occurred first. Figure depicts the recruitment, allocation, and follow-up process. In total, the study analyzed 87 patients, with 22 (25.3%) receiving routine clinic surveillance and 65 (74.7%) not receiving routine clinic surveillance. The primary outcomes of this study were the rates of different AVG interventions among the recruited patients throughout the follow-up period. AVA reconstruction was defined as the creation of a new VA after the previous one failed to support adequate dialysis treatment. Sensitivity analysis was performed to validate our findings. We also compared the secondary patency of AVGs between groups, defined as the duration from recruitment to AVG abandonment, and analyzed the costs associated with AVG interventions. Furthermore, we evaluated correlations between patient demographic and AVA reconstruction.
Statistical analyses were conducted using MedCalc Statistical Software (version 23.0.6, MedCalc Software Ltd., Ostend, Belgium). Categorical variables were expressed as frequencies or percentages, while continuous variables were presented as means and standard deviations. The chi-square test or Mann-Whitney test was used for comparisons between variables, as appropriate. A Cox proportional hazards regression model was used to assess the impact of routine clinic follow-up on time to AVA reconstruction. The model estimated hazard ratios after adjusting for potential confounders and significant variables between groups, to provide robust estimates of the association between routine follow-up and the risk of AVA reconstruction. Logistic regression analysis was used to determine the crude odds ratios between categorical variables and outcomes, while Spearman's rank correlation coefficients were calculated to assess correlations between continuous variables and outcomes. Statistical significance was set at a two-tailed p value of less than 0.05.
The baseline demographic and clinical characteristics of the recruited patients are presented in Table . No significant difference was observed in sex distribution between the two groups, and there were also no significant differences in age, HD vintage, AVG characteristics, comorbidities, history of parathyroidectomy, use of far-infrared radiation therapy, or administration of antiplatelet or antihypotensive agents. Regarding laboratory data, the patients undergoing routine clinic surveillance had a significantly higher hemoglobin level compared to those not receiving routine follow-up (10.8 g/dL vs. 10.1 g/dL, p < 0.01). Other laboratory parameters were comparable between the two groups. The primary outcomes of the patients with and without routine clinic surveillance are detailed in Table . The rates of AVG interventions were expressed as occurrence per 100 patient-months, adjusted for HD vintage. No significant difference in the rate of AVA reconstruction was observed between the patients with and without routine clinic surveillance (0.46 / 100 patient-months vs. 0.5 / 100 patient-months, p = 0.99). Conversely, the rates of graft-anastomosis stenting (0.66 / 100 patient-months vs. 0.2 / 100 patient-months, p = 0.02) and PTA (30.19 / 100 patient-months vs. 14.17 / 100 patient-months, p < 0.01) were significantly higher in the patients with routine clinic surveillance. Sensitivity analysis, including patients matched for hemoglobin level, patients aged over 65 years, and those receiving antiplatelet agents, corroborated our primary findings. The secondary patency of AVGs between the patients with and without routine clinic surveillance is depicted in Figure , using the Cox proportional hazards regression model. After adjusting for hemoglobin level, the survival probability did not differ significantly between the two groups (adjusted hazard ratio: 0.83, p = 0.79). The costs of AVG interventions expressed in New Taiwan Dollars (NTD) per patient during the follow-up period in both groups are detailed in Table . The cost of AVA reconstruction was lower in the patients with routine clinic surveillance compared to those without, although the difference was not significant (9414 NTD per patient vs. 10620 NTD per patient, p = 0.96). However, the costs of graft-anastomosis stenting (2563 NTD per patient vs. 434 NTD per patient, p = 0.02) and PTA (91309 NTDs per patient vs. 38548 NTD per patient, p < 0.01) were significantly higher in the patients with routine clinic surveillance. Overall, the total costs of AVG interventions, including the costs of ultrasound and clinic visits (shown in Table ), were significantly higher in the routine surveillance group compared to the group without routine surveillance (110672 NTD per patient vs. 51874 NTD per patient, p < 0.01). The correlations between patient demographic information and AVA reconstruction are presented in and 2. Analysis revealed no significant correlations between categorical variables or continuous variables and AVA reconstruction in the patients with routine clinic surveillance.
This study investigated the outcomes of routine clinic surveillance on AVG patency in HD patients with previous access complications. The results showed no significant differences in AVA reconstruction or secondary patency rates, but significant increases in graft-anastomosis stenting and PTA rates in the patients undergoing routine clinic surveillance compared to those without. Furthermore, the patients with routine clinic surveillance incurred significantly higher costs to maintain AVG patency. These findings underscore the need for more detailed evaluations of the cost-effectiveness of routine AVG monitoring in this patient cohort. AVA reconstruction is typically required when an access fails to maintain adequate function despite salvage interventions , and routine surveillance may help reduce its occurrence. In this study, we observed no significant difference in AVA reconstruction rate between the patients undergoing routine clinic surveillance and those without, a finding validated by sensitivity analysis involving matched hemoglobin level, as well as subgroups of older patients and those on antiplatelet therapy. McCarley et al. evaluated the clinical and financial outcomes of different VA surveillance methods in a cohort of 132 HD patients . Their results showed variable costs in the patients with AVGs, reflecting differences in the rates of access reconstruction or revision across surveillance methods, compared to a control group without surveillance . The discrepancies between our findings and those of McCarley et al. may be attributed to differences in the definition of AVA reconstruction. Several salvage interventions are available to restore the patency of AVGs, with graft-anastomosis stenting and PTA being the primary approaches in our hospital. Graft-anastomosis stenting is indicated for cases requiring frequent PTA at the stenotic graft-anastomosis site. In our study, both graft-anastomosis stenting and PTA rates were significantly higher in the routine clinic surveillance group, a finding that was further supported by sensitivity analysis. Moist et al. conducted a randomized controlled trial (RCT) involving 112 HD patients to assess the effect of monthly AVG flow monitoring on thrombosis and access loss, and found that the intervention rates in the treatment group were 1.65 times higher than those in the control group . Similarly, a prospective RCT by Robbin et al. assessed the impact of regular ultrasound surveillance on stenosis in 126 HD patients with AVGs, and found that the frequency of preemptive PTA was 64% higher in the ultrasound surveillance group compared to the control group . In addition, Hoeben et al. assessed the impact of routine surveillance on intervention rates in a cohort of 86 patients, and observed that the frequency of interventions was significantly higher in the group receiving regular surveillance compared to those without . Moreover, in a cohort of 363 patients, Plantinga et al. observed that those undergoing more frequent monitoring were 1.4 times more likely to require an intervention compared to those with less frequent monitoring . Furthermore, national data from the Netherlands reported by Tordoir et al. indicated that multidisciplinary discussions of AVA problems increased the rate of preemptive endovascular interventions . In a cohort of 60 patients, Mauro et al. assessed AVG secondary patency between patients in a surveillance program and those receiving clinical assessment . In the surveillance group, 15 AVG malfunctions were detected and treated with graft-anastomosis stenting and PTA, while no malfunctions were observed in the historical control group . Despite these findings, some studies have reported minimal differences in intervention rates. The Hemodialysis Access Surveillance Evaluation Study, a multicenter RCT by Salman et al. compared monthly ultrasound AVA flow surveillance with standard care in 436 HD patients, and found no statistically significant difference in the total number of procedures between the groups . Similarly, Schuman et al. compared AVA outcomes between ultrasound-based flow measurements and clinical criteria, and reported a modest 1.17-fold increase in intervention rate in the ultrasound group only . These discrepancies may be due to differences in study populations and design. Maintaining AVA functionality is a crucial issue in HD-related research. In our study, secondary patency of AVGs was defined as the period from the initiation of the clinic to the date of AVG abandonment. Cox proportional hazards regression was used to compare secondary patency between the patients with and without routine clinic surveillance. After adjusting for baseline demographic and clinical variables, no significant difference in AVG secondary patency was identified between the two groups. Ram et al. conducted an RCT involving 101 patients, and applied criteria including clinical symptoms, AVG flow measurements, and ultrasound findings to guide referrals for PTA, and they observed no significant difference in 2-year AVG survival rate between the groups . Similarly, Dember et al. performed an RCT of 64 patients to compare the prophylactic repair of AVG stenosis with repair at the time of thrombosis . Over the 3.5-year study period, no significant differences were observed in AVG abandonment rates or time to abandonment between the intervention and observation groups . These findings are consistent with other RCTs , . In addition, Lumsden et al. compared a surveillance program involving prophylactic PTA for stenoses greater than 50% with a non-interventional approach in 65 patients, and found no significant differences in patency rates at 6 and 12 months between the groups . These outcomes align with similar findings from other cohort studies , . In contrast, Mauro et al. compared AVG secondary patency between patients in a surveillance program and those undergoing clinical assessment, and found that the 5-year patency rate was significantly higher in the surveillance group . However, it is important to acknowledge that the comparison groups were analyzed in different temporal and geographic contexts. Overall, our findings align with the existing literature, including subgroup analysis in systematic reviews and meta-analyses by Tonelli et al. and Casey et al. which found no significant difference in AVG abandonment when comparing AVA flow surveillance with standard care. The economic burden of HD places a significant strain on healthcare systems , and the costs associated with AVA interventions further exacerbate this challenge . The costs related to graft-anastomosis stenting and PTA were significantly higher in the routine surveillance group compared to those without surveillance in this study. Consequently, the total costs of AVG-related interventions were more than twice as high in the routine surveillance group compared to the patients without routine surveillance. In the RCT by Ram et al. mentioned above, subgroup analysis revealed that costs related to monthly AVG flow monitoring, quarterly stenosis evaluations, and total PTAs were higher in the surveillance group . These findings align with the cost outcomes observed in our study. In contrast, McCarley et al. reported a 49% reduction in the total costs of managing thrombosis-related events in AVGs with ultrasound-assisted flow monitoring compared to no monitoring, and a 54% reduction compared to venous pressure monitoring . Given that AVG interventions were primarily managed on an outpatient basis and the demographic and clinical characteristics were comparable between the surveillance and non-surveillance groups, our study specifically examined the costs associated with outpatient interventions, excluding expenses associated with dialysis catheters and hospitalizations. Further investigations are warranted to evaluate the clinical benefits and cost-effectiveness of routine surveillance, including an analysis of potential long-term benefits, such as reduced hospitalizations and complication rates. The Kidney Disease Outcomes Quality Initiative guidelines recommend regular physical examinations of AVGs by experienced practitioners to identify clinical signs of flow dysfunction. However, routine surveillance methods, including AVG flow measurement, pressure monitoring, or imaging for stenosis beyond standard clinical monitoring, are not advised for improving AVG patency . We have established a multidisciplinary clinic with bidirectional feedback, involving nephrologists, dialysis staff, and trained cardiologists, in conjunction with guideline-directed assessments in the dialysis unit, to optimize AVA patency in our HD patients. This collaborative approach offers a fresh perspective on AVG follow-up. However, our surveillance strategy did not significantly improve AVG secondary patency and was associated with higher intervention rates and increased costs of AVG-related care. This study also has several limitations. First, being a retrospective analysis from a single tertiary teaching hospital, the findings may not be widely generalizable. Future research involving larger sample sizes or multicenter data is recommended to validate these results and improve their applicability. Additionally, the follow-up period for the recruited patients was limited to a maximum of 1.5 years, and longer follow-up durations may be necessary to fully assess long-term outcomes. Second, the collaborative clinic adopted an individualized approach rather than a standardized protocol for the assessment and management of AVG. Furthermore, the multidisciplinary team lacked the inclusion of vascular surgeons, who play a critical role in AVA reconstruction. Third, critical variables such as AVG flow, intra-dialysis venous pressure, and outcomes such as dialysis catheter use and associated hospitalization data were not recorded. Fourth, the reasons for AVA reconstruction are not limited to AVG occlusion , however they were not specified in our analysis. Lastly, despite multivariate analysis was performed to account for known variables, the influence of unmeasured confounding factors, such as smoking status, on the outcomes cannot be completely excluded.
We observed no significant differences in AVA reconstruction or secondary patency rate between HD patients with AVGs receiving routine collaborative clinic surveillance and those without. However, routine clinic surveillance was associated with a marked increase in graft-anastomosis stenting and PTA, resulting in significantly higher costs for maintaining AVG patency. These findings highlight the need for further assessment of the cost-effectiveness of routine AVG surveillance in this patient population.
Supplementary tables.
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Dynamics of blood microsatellite instability (bMSI) burden predicts outcome of a patient treated with immune checkpoint inhibitors: a case report of hyperprogressive disease | 10a2fd69-a195-40b9-b555-8664c0b14bfa | 11836019 | Biopsy[mh] | Introduction Microsatellite instability (MSI) is a phenomenon of hypermutation occurring at microsatellite loci, which are tracts of short repetitive DNA motifs. MSI comes as the shortening of the mononucleotide tracts due to failure to correct DNA polymerase slippage mistakes due to defects in the mismatch repair system (dMMR) ( , ). MSI/dMMR occurs among various tumor types ( ), and is associated with improved outcomes of patients treated with immune checkpoint inhibitors (ICI) ( – ). The effectiveness of ICI has been well demonstrated for patients with MSI-positive colorectal cancer (CRC) in numerous clinical studies ( – ). Standard diagnostic methods such as immunohistochemistry (IHC) or polymerase chain reaction (PCR) are routinely used in clinical practice for the assessment of MSI/dMMR and provide a binary assessment of MSI/dMMR (positive or negative) ( ). Next generation sequencing (NGS) is an emerging method for assessing MSI ( – ). The advantages of assessing MSI status using NGS include the ability to study a larger number of short tandem repeats (STRs), as well as to simultaneously analyze the mutational profile and mutational burden (TMB) of the tumor ( ). Much like routine methods, standard NGS pipelines classify tumors as stable or unstable based on the percentage of unstable STRs ( ). Recent studies have demonstrated excellent sensitivity of NGS-based assays for MSI analysis in liquid biopsies ( ). However, current binary classification of STRs only allows to analyze the percentage of unstable STRs. This approach does not allow for a quantitative assessment of MSI burden as the percentage of unstable DNA fragments in the sample ( ). Similarly to the analysis of variant allele frequencies of mutations, the quantitative assessment of MSI might reflect the clonality of the tumor, as well as can be utilized to measure tumor dynamics when liquid biopsy is used. The use of liquid biopsy makes it possible to detect early changes in response to therapy that cannot be detected by standard imaging methods such as computer tomography (CT) ( – ). Typically, somatic mutations are used for tracing the dynamics of the tumor. However, the assessment of DNA signatures such as MSI is not currently used for these purposes. Here, we report a clinical case of a colorectal cancer patient with MSI confirmed by PCR and NGS. The patient was treated with ICI, eventually demonstrating hyperprogression. An algorithm for qualitative measurement of MSI burden was used for tracking its changes in serial on-treatment plasma samples (bMSI). Here, for the first time, we demonstrate that the dynamics of bMSI correlate with driver mutational burden and disease progression.
Case presentation A 40-year-old male was diagnosed with sigmoid cancer in November 2021. He underwent right-sided hemicolectomy on 23 November 2021. Histologically, colon adenocarcinoma pT3N1bM0 was then confirmed. CT scan in December 2021 revealed no signs of continued tumor growth and a lesion in upper lobe (S4) of the right lung up to 18 mm with no enlargement in size compared to previous CT scans. The patient then underwent 6 cycles of adjuvant chemotherapy with XELOX and 3 cycles of capecitabine monotherapy from December 2021 to July 2022. In January 2022, in between treatment cycles, the patient underwent PET/CT scan, which did not confirm a lesion in the lung as metastatic due to low 18FDG uptake. In April 2022, no KRAS/NRAS/BRAF alterations were found via PCR, and the IHC for HER2 was negative. However, following the results of a 5-loci PCR, the tumor was found to be MSI-positive. In line with the patient’s decision, no subsequent germline testing to rule out Lynch syndrome was performed. Following the completion of adjuvant chemotherapy, the patient underwent PET/CT in August 2022, which revealed the enlargement of size and contrast accumulation in S4 of the right lung (up to 5 cm) and the appearance of a new lesion, measuring up to 3 cm in the right hepatic lobe (S8). As it was not clear whether an inflamed retention cyst or metastatic lesion was observed in the lung, a bronchoscopy with brush-biopsy was performed. Consequent cytology did not reveal any atypical cells, and the lesion was determined a cyst. The patient was offered to undergo a liver biopsy to confirm the progression of the disease, but he declined and decided to remain under observation. The next follow-up visit was in February 2023. The patient underwent CT that revealed the same lesions: 55x36 mm in S4 of the right lung and 34x27 mm in the S8 of the liver. Due to the enlarged size of the lesions, the patient was offered to undergo systemic therapy first. As the primary tumor was MSI-positive, monotherapy with nivolumab was recommended. The patient was then enrolled in an observational clinical trial BLOOMSI (NCT06414304). As part of the trial, the pre-treatment blood plasma sample was collected on 27.03.2023. On March 28th 2023, the patient started nivolumab. During the time on treatment, two serial plasma samples were collected, 17 and 31 days after therapy initiation (on 13.04.2023 and 27.04.2023, respectively). On May 2nd 2023, while receiving the 3rd cycle, the patient complained of limb weakness and mild joint pain. Following the recommendation of a general practitioner, he started to take еtoricoxib with short term pain relief. The patient received a total of 4 cycles of nivolumab, and the symptoms were increasing. After the discontinuation of nivolumab, a post-treatment blood plasma sample was obtained (on 11.05.2023, 45 days after the start of treatment). In the beginning of June, patient underwent radiography of the hip joints and CT of the brain, but no abnormalities were found. Further CT scan of the lumbosacral region of the spine revealed metastases of the vertebrae in Th11-S5 and pathological fractures of the L2, L4 vertebrae bodies. Metastases were also found in pelvic bones leading to their destruction, with the spread of the pathological process to soft tissue at the level of the iliac crests and lumbar lymph nodes. Furthermore, adrenal glands and liver were affected by metastatic lesions. Decompression laminectomy at the level of L4 vertebrae with tumor removal with microsurgical reconstruction of the L4 root nerve with posterior stabilization was performed on July 7th. Subsequent histology confirmed the colorectal origin of the metastatic lesion. Following the surgery, the patient did not receive any systemic treatment. In August 2023, he received treatment for lumbosacral abscess after laminectomy and died a month later ( , ).
Genomic testing and bMSI burden dynamics Consistent with current treatment guidelines, MSI was evaluated via standard 5-loci PCR panel, uncovering MSI positivity in the patient’s primary tumor sample. Based on the results of PCR, the patient was included in the local observational clinical trial evaluating the dynamics of MSI burden in serial samples from patients receiving ICI. As part of the trial, confirmatory MSI/dMMR testing was performed using a 4-antibody IHC (loss of two proteins was observed) and NGS via Solo test Atlas Pro panel covering 34 commonly altered cancer-related genes and 30 MSI mononucleotide tandem repeats. NGS was performed on primary tumor and serial plasma (liquid biopsy, LB) samples received prior to the start of ICI therapy, on the 17th, 31st days on therapy, as well as after the discontinuation of ICI. NGS revealed point mutations in KRAS, PIK3CA, PTEN and TP53 (the latter was only observed in the FFPE sample) in the pre-treatment FFPE and LB samples ( ). Of note, initial PCR testing for common CRC alterations in colorectal cancer found no KRAS mutations, whereas NGS testing of a tumor sample found a rare hotspot KRAS p.Lys117Asn mutation ( ) with a VAF of 22%. MSIsensor2 ( ) revealed a slight decrease unstable STRs by 3% at the 14th day after therapy initiation and gradual increase by 7% and 12% in the subsequent liquid biopsy samples during ICI treatment (73%, 71%, 76% and 85% for baseline, 14th day, 28th day and 1st control LB, respectively). KRAS, PIK3CA and PTEN driver mutations were identified in LB samples with almost the same variant allele frequency (13.9, 15.6 and 17.3% respectively) ( ), similarly to the molecular profile seen in primary FFPE sample (22.1%, 18.2% and 18.7%, respectively) alluding to its homogeneous representation across tumor clones in primary tumor. Across serial LB samples driver mutations demonstrated positive dynamics at 14th day on therapy (decrease by 41%, 35% and 8% for KRAS, PIK3CA and PTEN mutations, respectively), and negative dynamics afterwards, although PTEN mutation demonstrated dissimilar dynamics contrasting to ones of PIK3CA and KRAS hinting at the positive selection of PTEN-mutated clone during the treatment ( ). To assess MSI quantitatively in FFPE sample, i.e. to estimate the percentage of tumor cells exhibiting MSI, we calculated the ratio of total amount of sequencing reads in NGS data supporting DNA fragments with altered STR sequences (i.e. demonstrating shortage of reference STR length by 4b.p. and more) to the total amount of sequencing reads supporting any STR sequences. The calculated percentage was close to the VAF of KRAS, PIK3CA and PTEN mutations ( ), suggesting that quantitatively estimated MSI burden was correlated with tumor clonality. Similarly to the FFPE sample, MSI burden was in line with VAFs of driver mutations in pre-treatment LB sample. Further tracing of bMSI burden across serial LB samples demonstrated a gradual increase without significant drop-outs seen for KRAS and PIK3CA mutations hinting at co-evolution of tumor clones exhibiting PTEN mutation and MSI.
Discussion MSI/dMMR is a known biomarker of ICI response across tumor types, including colorectal cancer ( , ). Gold standard methods for MSI/dMMR detection include PCR and IHC, however in recent years NGS has been recognized as a potentially decisive tool for selecting candidates for ICI ( ). Although tumor samples are considered preferential for MSI/dMMR testing, various issues, including insufficient tumor purity and overall suboptimal tissue quality might result in ambiguous results ( ). Large-scale studies suggest that liquid biopsy might perform as well as tumor samples for identifying MSI-positive patients who are candidates for ICI ( , , ). A high concordance was observed between baseline tissue and plasma mutations ( ). Evaluation of MSI dynamics in liquid biopsies has been reported to correlate with response to immunotherapy or lack thereof ( , ). Additionally, serial ctDNA analysis has been shown to be prognostic of tumor dynamics, with an increase of ctDNA indicating progressive disease ( – ). In our patient, while an increase in MSI burden was immediate and persistent, the increase of driver and passenger mutational burden was delayed in both pre- and on-treatment serial samples. Moreover, bMSI increase preceded the first clinical signs of disease progression, whereas an increase in mutational burden coincided with disease progression ( ). Since no CT imaging was done in the course of nivolumab treatment, we could not compare the results of imaging studies to LB analysis, however, the results of the latter are consistent with the patient’s symptoms, which were later confirmed to have originated from progressive disease. Previous studies have suggested that analysis of LB has the potential to outperform standard radiographic imaging for predicting treatment outcomes ( ), however no studies have reported the utility of MSI burden as a screening tool for monitoring tumor dynamics in the course of ICI. While these findings are interesting, they require further validation in prospective clinical trials to validate the role of bMSI for tumor response monitoring in MSI-positive patients. It is worth mentioning that initial tumor testing with PCR, in contrast with NGS, did not reveal a KRAS p.Lys117Asn hotspot mutation ( ). Precise determination of KRAS mutational status is crucial for tailoring therapy for patients with advanced MSS/pMMR colorectal cancer ( ). Although seen at lower rates than mutations affecting codons 12, 13 and 61, the KRAS codon 117 mutation is considered a ‘standard’ KRAS mutation, indicating that all standard testing methodologies used in the clinic should be able to detect this mutation. For instance, in KEYNOTE-177, no benefit from pembrolizumab monotherapy was observed for patients with MSI and RAS mutations ( ). However, as seen in the CheckMate 8HW trial, the presence of RAS mutations does not interfere with the efficacy of anti-PD-1 and anti-CTLA-4 combination therapy for patients with MSI-positive CRC ( ), suggesting that dual ICI might have been preferential for this patient. Results of these studies suggest that oncogenic KRAS mutations have a potential to influence the patient’s outcome following nivolumab monotherapy. However, this was not observed in our case, as we saw significant degression of KRAS-mutated clones at day 14 after therapy initiation in contrast to tumor clones possessing PTEN-mutation and MSI. The term hyperprogression has been a topic of debate among the scientific community, however since it indicates the state of rapid progression of disease in the course of immunotherapy, it seems applicable for our patient’s case ( ). Hyperprogression in response to immunotherapy is often linked to MDM2 or MDM4 amplification, however, targeted NGS sequencing performed for our patient did not allow for MDM2/4 analysis, since these genes were not included in the panel design ( ). Furthermore, the small panel size did not allow for the evaluation of tumor mutational burden (TMB). TMB is an important predictive biomarker of ICI benefit, reflecting a number of mutations occurring in tumor cells. Tumors with high TMB tend to be more immunogenic and more responsive to ICI therapy ( ). Even in the presence of MSI, patients with low TMB demonstrate poor outcomes when treated with ICI ( ). However, given the relatively low amount of passenger mutations observed in our patient’s samples, ultra-high TMB could be potentially ruled out, as in samples with ultra-high TMB harbor passenger mutations in at least one of the genes analyzed by our panel. For instance, 60% and 76% of patients included in the MSK MetTropism study with TMB>20 Mut/Mb and >40 Mut/Mb, respectively, had at least one passenger mutation in the analyzed genes ( ). Additionally, some authors have linked EGFR amplifications to the state of hyperprogression in response to immunotherapy ( ), however, our patient was EGFR amplification-negative. However, since the information on MDM2/4/EGFR amplifications was unavailable due to the panel design, the question of whether the patient’s hyperprogressive disease was associated with molecular correlates. Although high TMB could be potentially ruled out in this case, precise TMB estimation could not be performed, which could be considered a significant limitation of the study, since TMB is an important biomarker of ICI benefit. Moreover, the activation of T-helpers has gained attention as another mechanism of ICI resistance ( ). Finally, 30-40% of patients treated with ICI fail to respond to therapy ( ). Overall, our patient’s case highlights the potential of ctDNA MSI burden as a tool for monitoring tumor response and clonal evolution in the course of ICI in patients with MSI-positive colorectal cancer. Further study of the clinical application of MSI burden by liquid biopsy could potentially allow timely assessment of treatment effectiveness in patients before visible signs of progression over time among patients with colorectal and other MSI-positive solid tumors.
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Modified titanium post with polished sidewalls and prefabricated shoulders can effectively preserve the teeth with subgingival defects | 37331f01-b78e-4bc2-ad5c-48a90d634128 | 11754749 | Dentistry[mh] | Caries or fractures often result in a subgingival dental defect, requiring crown lengthening or orthodontic traction to expose the defect margin above the gingiva, followed by post-core crown restoration for long-term and effective preservation – . However, crown lengthening surgery leads to significant alterations in clinical attachment level and probing depth at both treated and adjacent sites . Crown lengthening has a significant impact on the long-term survival rates of the teeth. The 10-year survival rates of teeth with crown lengthening (51–54%) were statistically lower compared to those without crown lengthening (74.5–76%) , ; and the risk of tooth extraction following crown lengthening was found to be 2.3 times higher compared to teeth without crown lengthening. Additionally, it was observed that teeth with an inadequate crown-root ratio (1:1) exhibited the lowest survival rate (40%) in comparison to teeth with a sufficient crown-root ratio (< 1:1) . Furthermore, implant restoration after extraction is commonly recommended for teeth with deep defects beneath the gingiva. Although dental implants are considered as an optimal restoration choice for Missing teeth, they lack sensory function and buffer adjustment ability comparable to natural teeth due to the absence of surrounding periodontal membrane , . Nowadays, preserving every natural tooth as much as possible has gradually gained attention from an expanding cohort of dental professionals. Numerous studies have endeavored to achieve an equilibrium by employing modified crown lengthening, a minimally invasive therapeutic approach that involves the precise removal of a small quantity of soft and hard tissue while reconstructing the biological width; and according to the report, this technique has exhibited promising outcomes in facilitating periodontal and periapical healing – . Nevertheless, it should be noted that the modified crown lengthening still leading to a less favorable long-term prognosis: within 9 years after undergoing modified crown lengthening, the survival rate of treated teeth decreased from 97.8 to 40.4%, with an average clinical success time of 6.2 years; risk factors such as smoking and poor dental plaque control significantly exacerbate the failure rate , . In order to avoid damage to the soft and hard tissues around the tooth caused by crown lengthening and optimize the preservation of teeth with deep subgingival defects, we modified the conventional titanium post, with finely polished side walls in directly contact with the gingival and a prefabricated shoulder flush with the gingival margin, and tried to utilize it for restoring the subgingival tooth defect. This study was to evaluate the clinical effectiveness of the modified titanium post and analyze the stress distribution in both the tooth and periodontal tissue after restoration with the titanium post using three-dimensional finite element analysis.
The present study adhered to the Ethical Review Measures for Biomedical Research involving Human Subjects of the Ministry of Health of China and complied with the relevant provisions of the Declaration of Helsinki regarding biological experiments in human subjects. Additionally, it obtained approval from the Ethics Committee of Tianjin Stomatological Hospital (Approval Number: PH2022-B-027). The purpose of the study, treatment process, and unconditional right to withdraw were explained to each participant by the dentist prior to their participation in this clinical study. The study was conducted on the basis that each participant understood and signed an informed consent form. Clinical research Premolars and molars with defects extending 2 mm or more below the gingiva were included in this study. These teeth underwent either fiber post restoration after crown lengthening or modified titanium post restoration without crown lengthening. For teeth with the undergingival defect of 2–4 mm (≥ 2 mm and < 4 mm), we will arrange them into the Fiber post group and the Titanium post group respectively according to the order of the patient’s visit. Patients enrolled in the study with odd numbers were enrolled in the fiber post group, and patients enrolled in the study with even numbers were enrolled in the titanium post group. Teeth with the undergingival defect of 4 mm or more (≥ 4 mm) were included in the titanium post group. Inclusion criteria: (1) The defect involved a minimum of one wall but no more than three walls of the tooth, located at a subgingival level of ≥ 2 mm. The remaining walls exhibited a height above the gingiva of ≥ 2 mm and a thickness after tooth preparation of ≥ 2 mm. The pulp floor remained intact, and the abutment teeth demonstrated sufficient resistance. (2) The teeth had undergone successful root canal treatment. Periapical conditions were favorable, with no percussion pain, no loosening, or only mild loosening. The patient was required to undergo periodontal therapy to eliminate supragingival calculus and alleviate gingival inflammation. We required that the patient familiarize themselves with and consistently apply the Pasteur brushing technique: Use a small-headed, soft-bristled toothbrush and position it at a 45-degree angle on the labial/lingual surface of the teeth. Direct the bristles towards the gums, ensuring that the tips enter the gingival sulcus. Perform horizontal vibrations with moderate force and small amplitude for approximately 10 times before moving the toothbrush to the next adjacent area. Ensure that there is an overlap between two consecutive brushing areas, covering 2–3 teeth each time. The bristles should then be directed over the occlusal surfaces of the teeth, brushing back and forth. It is essential to ensure that all tooth surfaces are thoroughly cleaned. for thorough tooth brushing thrice a day, with each session lasting 4 min. Furthermore, we underscored the importance of incorporating dental floss and interdental brushes into daily oral hygiene routines, while also emphasizing the necessity of visiting the dentist once every 6 months. During regular check-ups, the efficacy of the patient’s dental cleaning practices was rigorously evaluated, and any signs of gingivitis were promptly addressed through targeted instructions for improved cleansing in affected areas. The details of the patients who provided teeth and the grouping are recorded in Table . Sixteen teeth exhibiting subgingival defects measuring ≥ 2 mm and < 4 mm were randomly allocated to either group Crown Lengthening (group CL) or group Titanium Post- shallow defect (group TP-s). Teeth in group CL underwent crown lengthening followed by fiber post and all-porcelain crown restoration, while teeth in group TP-s received titanium post and all-porcelain crown restoration without undergoing crown lengthening. Additionally, eight teeth with subgingival defects measuring ≥ 4 mm that were deemed unsuitable for crown lengthening and recommended for extraction were included in group Titanium Post - deep defect (group TP-d), where modified titanium post and all-porcelain crowns were utilized to preserve the tooth as much as possible. The purpose of this study was to verify the effectiveness and rationality of titanium post in treating subgingival defects. Although we expected that modified titanium post would achieve a therapeutic effect that was not inferior to the crown lengthening, due to a lack of sufficient clinical effectiveness and safety data, and the absence of similar studies to refer to. For risk control purposes, we conducted the study within a limited scope and did not enroll a large number of subjects. The sample size was estimated using repeated measure ANOVA. Five repeated measurements were conducted for each groups, with each group necessitating eight samples. Among the 16 patients who received titanium posts (Group TP-s and TP-d), all achieved stable and healthy periodontal conditions, with better or comparable indicators to those of crown lengthening. Crown lengthening: The defect edge in group CL was exposed 2 mm above the gingival margin through crown lengthening. Eight weeks after crown lengthening, quartz fiber post and cores and full zirconia crowns were used for tooth restoration. (2) For group TP-s and group TP-d, tooth preparations included a 2 mm occlusal restorative space, a 90° rounded shoulder located 0.5 mm below the buccal gingival margin, and post channel preparation. (3) An impression was taken using silicone rubber material, then casted a plaster model. The initial follow-up visit involved the process of trying on and adjusting the post until it was accurately positioned, ensuring a precise fit between the edge of the post as well as the tooth defect edge, while also achieving appropriate placement of the prefabricated shoulder. The post was cemented using polycarboxylate cement. Excess adhesive was meticulously removed and the area, especially the subgingival defect, was thoroughly cleaned and rinsed. The crown impression, bite record, and temporary crown were obtained. During the second follow-up appointment, the permanent crown was cemented in place and any excess adhesive surrounding the crown was meticulously removed. The dentist provided instructions on oral health maintenance education after the crown restoration. The design features of the modified titanium post were illustrated in Fig. . Specifically, the side wall contacting gingival of the defect region was polished to achieve a high level of smoothness (3). A prefabricated shoulder (2) at a height corresponding to gingival margin in defect area that aligns seamlessly with the shoulder prepared on natural teeth (4). The indexes of observation were as follows: ① Gingival Index was recorded for evaluating the gingival status in subgingival defect area, which involved assessing changes in gingival color, texture, and bleeding tendency using a blunt periodontal probe. Scoring criteria included: 0 = healthy gums; 1 = mild inflammation with slight gum color change and mild edema without probing bleeding; 2 = moderate gingival inflammation characterized by redness, bright edema, and probing bleeding; 3 = severe gum inflammation presenting obvious redness or swelling with ulcers and spontaneous bleeding tendency. ② Probing Depth in subgingival defect area was measured by using a graduated periodontal probe inserted into gingival sulcus along the major axis of tooth from margin to bottom of furrow. ③ Modified gingival sulcus bleeding index in subgingival defect area was assessed by gently probing buccal/tongue side of gingiva below it at a depth of approximately 1 mm within defect area for observing any bleeding after sliding parallelly for about 30 s. Scoring criteria included: 0 = no bleeding; 1 = spot-like scattered bleeding in gingival sulcus; 2 = linear bleeding in gingival sulcus; and finally, 3 = spontaneous bleeding. ④ Tooth Mobility was recorded based on following scale : TM = 0 represented non-mobility or physiological mobility; TM = 1 indicated only buccolingual mobility or loosening displacement range < 1 mm; TM = 2 referred to both buccolingual and proximal-distal mobility or loosening displacement range between 1 and 2 mm; TM = 3 denoted buccolingual, proximal-distal and vertical combined mobility or loosening displacement > 2 mm. ⑤ Gingival papillary height was recorded. Jemt classification of gingival papilla height loss was used to measure the gingival papilla height by referring to the highest point of gingival curve between the prosthesis and the adjacent permanent teeth. The gingival papilla height was divided into 5 degrees: 0 = no gingival papilla. 1 = less than half the height of the gingival papilla; 2 = the height of the gingival papilla is at least half, but does not reach the contact point of the two teeth; 3 = The gingival papilla completely fills the adjacent space and is consistent with the gingival papilla of the adjacent teeth; 4 = gingival papillary hyperplasia. The probe used for the gingival index and modified gingival sulcus bleeding index is a blunt probe (Hu-Friedy PCP11.5B7, American). The probe used for probing Depth detection is a graduated probe (Kangqiao KP-C2 002-0613, Shanghai, China). The periodontal indexes were assessed at the initial visit prior to treatment and at 3, 6, 12 and 18 months following crown repair completion. These time points were recorded as 0, 3, 6, 12, 18 respectively. Finite element analysis The CT scan data in DICOM format of adult male maxillary first premolars with normal morphology, absence of caries and periodontal disease were selected. The Mimics 21.0 software (Materialise) was utilized to construct a precise model representing the normal structure of maxillary first premolars (STL format), encompassing cortical and cancellous bone of alveolar bone, dentin, enamel, gingiva, periodontal ligament, dental pulp. The Geomagic Studio 2014 software (Raindrop) was utilized for the repair, noise reduction, surface enhancement, and assembly of the structures to generate a normal model of the first premolar. Based on this normal model, three different groups were performed: normal crown restoration (Control group), titanium post restoration without implementation of crown lengthening (Titanium post group), and crown lengthening + fiber post restoration (Fiber post group). Figure shows the finite element model of the teeth and the structure of each part. The Hypermesh 14.0 software (Altair) was utilized for meshing the models. Each structural organization employed a solid cell mesh that was subdivided into tetrahedral Tet4 Element units. The number of grid nodes and cells was presented in Table . The finite element pre-processing software MSC. Patran 2019 (NASA) and MSC. Nastran 2019 (NASA) were utilized for configuring the finite element mesh properties of the model. The structures are assumed to possess isotropic, uniform, and continuous linear elastic materials – , with their corresponding material parameters presented in Table . Subsequently, boundary condition constraints and load applications were implemented (Fig. ), followed by subsequent calculations and analyses. Data analysis. Using one-way analysis of variance or independent sample t test to examine the differences among various observation time points within the same group, as well as the disparities between different groups at a same time point.
Premolars and molars with defects extending 2 mm or more below the gingiva were included in this study. These teeth underwent either fiber post restoration after crown lengthening or modified titanium post restoration without crown lengthening. For teeth with the undergingival defect of 2–4 mm (≥ 2 mm and < 4 mm), we will arrange them into the Fiber post group and the Titanium post group respectively according to the order of the patient’s visit. Patients enrolled in the study with odd numbers were enrolled in the fiber post group, and patients enrolled in the study with even numbers were enrolled in the titanium post group. Teeth with the undergingival defect of 4 mm or more (≥ 4 mm) were included in the titanium post group. Inclusion criteria: (1) The defect involved a minimum of one wall but no more than three walls of the tooth, located at a subgingival level of ≥ 2 mm. The remaining walls exhibited a height above the gingiva of ≥ 2 mm and a thickness after tooth preparation of ≥ 2 mm. The pulp floor remained intact, and the abutment teeth demonstrated sufficient resistance. (2) The teeth had undergone successful root canal treatment. Periapical conditions were favorable, with no percussion pain, no loosening, or only mild loosening. The patient was required to undergo periodontal therapy to eliminate supragingival calculus and alleviate gingival inflammation. We required that the patient familiarize themselves with and consistently apply the Pasteur brushing technique: Use a small-headed, soft-bristled toothbrush and position it at a 45-degree angle on the labial/lingual surface of the teeth. Direct the bristles towards the gums, ensuring that the tips enter the gingival sulcus. Perform horizontal vibrations with moderate force and small amplitude for approximately 10 times before moving the toothbrush to the next adjacent area. Ensure that there is an overlap between two consecutive brushing areas, covering 2–3 teeth each time. The bristles should then be directed over the occlusal surfaces of the teeth, brushing back and forth. It is essential to ensure that all tooth surfaces are thoroughly cleaned. for thorough tooth brushing thrice a day, with each session lasting 4 min. Furthermore, we underscored the importance of incorporating dental floss and interdental brushes into daily oral hygiene routines, while also emphasizing the necessity of visiting the dentist once every 6 months. During regular check-ups, the efficacy of the patient’s dental cleaning practices was rigorously evaluated, and any signs of gingivitis were promptly addressed through targeted instructions for improved cleansing in affected areas. The details of the patients who provided teeth and the grouping are recorded in Table . Sixteen teeth exhibiting subgingival defects measuring ≥ 2 mm and < 4 mm were randomly allocated to either group Crown Lengthening (group CL) or group Titanium Post- shallow defect (group TP-s). Teeth in group CL underwent crown lengthening followed by fiber post and all-porcelain crown restoration, while teeth in group TP-s received titanium post and all-porcelain crown restoration without undergoing crown lengthening. Additionally, eight teeth with subgingival defects measuring ≥ 4 mm that were deemed unsuitable for crown lengthening and recommended for extraction were included in group Titanium Post - deep defect (group TP-d), where modified titanium post and all-porcelain crowns were utilized to preserve the tooth as much as possible. The purpose of this study was to verify the effectiveness and rationality of titanium post in treating subgingival defects. Although we expected that modified titanium post would achieve a therapeutic effect that was not inferior to the crown lengthening, due to a lack of sufficient clinical effectiveness and safety data, and the absence of similar studies to refer to. For risk control purposes, we conducted the study within a limited scope and did not enroll a large number of subjects. The sample size was estimated using repeated measure ANOVA. Five repeated measurements were conducted for each groups, with each group necessitating eight samples. Among the 16 patients who received titanium posts (Group TP-s and TP-d), all achieved stable and healthy periodontal conditions, with better or comparable indicators to those of crown lengthening. Crown lengthening: The defect edge in group CL was exposed 2 mm above the gingival margin through crown lengthening. Eight weeks after crown lengthening, quartz fiber post and cores and full zirconia crowns were used for tooth restoration. (2) For group TP-s and group TP-d, tooth preparations included a 2 mm occlusal restorative space, a 90° rounded shoulder located 0.5 mm below the buccal gingival margin, and post channel preparation. (3) An impression was taken using silicone rubber material, then casted a plaster model. The initial follow-up visit involved the process of trying on and adjusting the post until it was accurately positioned, ensuring a precise fit between the edge of the post as well as the tooth defect edge, while also achieving appropriate placement of the prefabricated shoulder. The post was cemented using polycarboxylate cement. Excess adhesive was meticulously removed and the area, especially the subgingival defect, was thoroughly cleaned and rinsed. The crown impression, bite record, and temporary crown were obtained. During the second follow-up appointment, the permanent crown was cemented in place and any excess adhesive surrounding the crown was meticulously removed. The dentist provided instructions on oral health maintenance education after the crown restoration. The design features of the modified titanium post were illustrated in Fig. . Specifically, the side wall contacting gingival of the defect region was polished to achieve a high level of smoothness (3). A prefabricated shoulder (2) at a height corresponding to gingival margin in defect area that aligns seamlessly with the shoulder prepared on natural teeth (4). The indexes of observation were as follows: ① Gingival Index was recorded for evaluating the gingival status in subgingival defect area, which involved assessing changes in gingival color, texture, and bleeding tendency using a blunt periodontal probe. Scoring criteria included: 0 = healthy gums; 1 = mild inflammation with slight gum color change and mild edema without probing bleeding; 2 = moderate gingival inflammation characterized by redness, bright edema, and probing bleeding; 3 = severe gum inflammation presenting obvious redness or swelling with ulcers and spontaneous bleeding tendency. ② Probing Depth in subgingival defect area was measured by using a graduated periodontal probe inserted into gingival sulcus along the major axis of tooth from margin to bottom of furrow. ③ Modified gingival sulcus bleeding index in subgingival defect area was assessed by gently probing buccal/tongue side of gingiva below it at a depth of approximately 1 mm within defect area for observing any bleeding after sliding parallelly for about 30 s. Scoring criteria included: 0 = no bleeding; 1 = spot-like scattered bleeding in gingival sulcus; 2 = linear bleeding in gingival sulcus; and finally, 3 = spontaneous bleeding. ④ Tooth Mobility was recorded based on following scale : TM = 0 represented non-mobility or physiological mobility; TM = 1 indicated only buccolingual mobility or loosening displacement range < 1 mm; TM = 2 referred to both buccolingual and proximal-distal mobility or loosening displacement range between 1 and 2 mm; TM = 3 denoted buccolingual, proximal-distal and vertical combined mobility or loosening displacement > 2 mm. ⑤ Gingival papillary height was recorded. Jemt classification of gingival papilla height loss was used to measure the gingival papilla height by referring to the highest point of gingival curve between the prosthesis and the adjacent permanent teeth. The gingival papilla height was divided into 5 degrees: 0 = no gingival papilla. 1 = less than half the height of the gingival papilla; 2 = the height of the gingival papilla is at least half, but does not reach the contact point of the two teeth; 3 = The gingival papilla completely fills the adjacent space and is consistent with the gingival papilla of the adjacent teeth; 4 = gingival papillary hyperplasia. The probe used for the gingival index and modified gingival sulcus bleeding index is a blunt probe (Hu-Friedy PCP11.5B7, American). The probe used for probing Depth detection is a graduated probe (Kangqiao KP-C2 002-0613, Shanghai, China). The periodontal indexes were assessed at the initial visit prior to treatment and at 3, 6, 12 and 18 months following crown repair completion. These time points were recorded as 0, 3, 6, 12, 18 respectively.
The CT scan data in DICOM format of adult male maxillary first premolars with normal morphology, absence of caries and periodontal disease were selected. The Mimics 21.0 software (Materialise) was utilized to construct a precise model representing the normal structure of maxillary first premolars (STL format), encompassing cortical and cancellous bone of alveolar bone, dentin, enamel, gingiva, periodontal ligament, dental pulp. The Geomagic Studio 2014 software (Raindrop) was utilized for the repair, noise reduction, surface enhancement, and assembly of the structures to generate a normal model of the first premolar. Based on this normal model, three different groups were performed: normal crown restoration (Control group), titanium post restoration without implementation of crown lengthening (Titanium post group), and crown lengthening + fiber post restoration (Fiber post group). Figure shows the finite element model of the teeth and the structure of each part. The Hypermesh 14.0 software (Altair) was utilized for meshing the models. Each structural organization employed a solid cell mesh that was subdivided into tetrahedral Tet4 Element units. The number of grid nodes and cells was presented in Table . The finite element pre-processing software MSC. Patran 2019 (NASA) and MSC. Nastran 2019 (NASA) were utilized for configuring the finite element mesh properties of the model. The structures are assumed to possess isotropic, uniform, and continuous linear elastic materials – , with their corresponding material parameters presented in Table . Subsequently, boundary condition constraints and load applications were implemented (Fig. ), followed by subsequent calculations and analyses. Data analysis. Using one-way analysis of variance or independent sample t test to examine the differences among various observation time points within the same group, as well as the disparities between different groups at a same time point.
Clinical examination Figure illustrated the periodontal health monitoring of a tooth with a lingual subgingival defect of 4–5 mm, which was repaired with titanium post. The periodontium was healthy and the gingival were not inflamed. Indexs of clinical study including gingival index, modification sulcus bleeding index, probing depth, tooth mobility, and the scores of the gingival papilla height were presented in Tables , , , and . One-way analysis of variance was used to compare the difference among 3 groups at each observation point. After restoration, there was a gradual decrease in the gingival index and sulcus bleeding index of the defect area in groups CL, TP-s, and TP-d; but no statistically significant differences were observed between the three groups at each observation point (Tables and ). The probing depth were presented in Table . The probing depth of group TP-d was significantly higher than that of group CL and group TP-s before restoration and 3 months post-restoration; however, there were no statistically significant differences observed between group TP-d and groups CL or TP-s at 6, 12 and 18 months post-restoration. The probing depth in both groups CL and TP-s showed a slight reduction after restoration compared to pre-restoration levels, but these changes were not statistically significant. Tooth mobility was recorded in Table . There were no significant differences in tooth mobility among the three groups before restoration. However, after crown lengthening, the tooth mobility of group CL was consistently and significantly higher than that of group TP-s and group TP-d throughout the follow-up observation periods, despite a gradual decrease over time. The tooth mobility of group TP-s and group TP-d remained unchanged before and after restoration. Gingival papilla height scores are presented in Table . The difference in gingival papilla height among 3 groups were not statistically significant prior to restoration. Following crown lengthening, the gingival papilla height of group CL exhibited a significant decrease and significantly lower than that of group TP-s and group TP-d throughout the follow-up period. There were no significant changes in gingival papilla height for group TP-s and group TP-d before and after restoration. Results of finite element analysis The stresses in the alveolar bone, dentin, periodontal membrane, and post were analyzed under both vertical and horizontal loads. One stress value was recorded from each of eight uniformly symmetrical locations within a 0.5 mm range around the maximum stress point. As a result, a total of nine stress values were obtained, including the maximum stress. One-way analysis of variance or independent sample t test were used to compare the difference among groups. The stress distribution under vertical loads When subjected to a vertical load, as shown in Fig. , the internal stresses in the alveolar bone were primarily concentrated in the apical region of the buccal root. In both the Control group and Fiber post group, dentin stresses were concentrated in the crest of lingual alveolar, while in the Titanium post group, they were focused on the lateral wall of the root canal. The stress in the periodontal membrane was predominantly concentrated on the crest of lingual alveolar. The stress analysis in each structure record in Table . The mean stress in the alveolar bone, dentin, and periodontal membrane was significantly higher in the Fiber post group compared to both the Control group and the Titanium post group. There was no significant difference in stress between the Titanium post group and the Control group in the alveolar bone and dentin, while in the periodontal membrane, the stress of the Titanium post group was significantly lower than that of the Control group. Furthermore, stress in titanium posts exhibited significantly higher levels compared to the stress of fiber posts. The stress distribution under horizontal loads When subjected to a horizontal load (Fig. ), internal stresses in the alveolar bone and the periodontal membrane were primarily concentrated in the crest of lingual alveolar. In both the Control group and Fiber post group, stresses in dentin were concentrated in the lingual alveolar crest, while in the Titanium post group, the stresses were focused on the lateral wall of the lingual root canal. It can be seen from Table , the mean stress in the alveolar bone of the Fiber post group exhibited a significantly higher level compared to both the stress of the Control group and the Titanium post group; however, there was no statistically significant distinction observed between the Control group and the Titanium post group. The mean stress in the dentin of the Control group was significantly lower compared to both the Titanium post group and Fiber post groups, while the stress in the Fiber post group exhibited a statistically higher level than that in the Titanium post group. The stresses in the periodontal membrane of both the Fiber post group and Titanium post group were significantly higher compared to the Control group. Additionally, the mean stress in the Titanium post was found to be significantly greater than that in the Fiber post.
Figure illustrated the periodontal health monitoring of a tooth with a lingual subgingival defect of 4–5 mm, which was repaired with titanium post. The periodontium was healthy and the gingival were not inflamed. Indexs of clinical study including gingival index, modification sulcus bleeding index, probing depth, tooth mobility, and the scores of the gingival papilla height were presented in Tables , , , and . One-way analysis of variance was used to compare the difference among 3 groups at each observation point. After restoration, there was a gradual decrease in the gingival index and sulcus bleeding index of the defect area in groups CL, TP-s, and TP-d; but no statistically significant differences were observed between the three groups at each observation point (Tables and ). The probing depth were presented in Table . The probing depth of group TP-d was significantly higher than that of group CL and group TP-s before restoration and 3 months post-restoration; however, there were no statistically significant differences observed between group TP-d and groups CL or TP-s at 6, 12 and 18 months post-restoration. The probing depth in both groups CL and TP-s showed a slight reduction after restoration compared to pre-restoration levels, but these changes were not statistically significant. Tooth mobility was recorded in Table . There were no significant differences in tooth mobility among the three groups before restoration. However, after crown lengthening, the tooth mobility of group CL was consistently and significantly higher than that of group TP-s and group TP-d throughout the follow-up observation periods, despite a gradual decrease over time. The tooth mobility of group TP-s and group TP-d remained unchanged before and after restoration. Gingival papilla height scores are presented in Table . The difference in gingival papilla height among 3 groups were not statistically significant prior to restoration. Following crown lengthening, the gingival papilla height of group CL exhibited a significant decrease and significantly lower than that of group TP-s and group TP-d throughout the follow-up period. There were no significant changes in gingival papilla height for group TP-s and group TP-d before and after restoration.
The stresses in the alveolar bone, dentin, periodontal membrane, and post were analyzed under both vertical and horizontal loads. One stress value was recorded from each of eight uniformly symmetrical locations within a 0.5 mm range around the maximum stress point. As a result, a total of nine stress values were obtained, including the maximum stress. One-way analysis of variance or independent sample t test were used to compare the difference among groups. The stress distribution under vertical loads When subjected to a vertical load, as shown in Fig. , the internal stresses in the alveolar bone were primarily concentrated in the apical region of the buccal root. In both the Control group and Fiber post group, dentin stresses were concentrated in the crest of lingual alveolar, while in the Titanium post group, they were focused on the lateral wall of the root canal. The stress in the periodontal membrane was predominantly concentrated on the crest of lingual alveolar. The stress analysis in each structure record in Table . The mean stress in the alveolar bone, dentin, and periodontal membrane was significantly higher in the Fiber post group compared to both the Control group and the Titanium post group. There was no significant difference in stress between the Titanium post group and the Control group in the alveolar bone and dentin, while in the periodontal membrane, the stress of the Titanium post group was significantly lower than that of the Control group. Furthermore, stress in titanium posts exhibited significantly higher levels compared to the stress of fiber posts. The stress distribution under horizontal loads When subjected to a horizontal load (Fig. ), internal stresses in the alveolar bone and the periodontal membrane were primarily concentrated in the crest of lingual alveolar. In both the Control group and Fiber post group, stresses in dentin were concentrated in the lingual alveolar crest, while in the Titanium post group, the stresses were focused on the lateral wall of the lingual root canal. It can be seen from Table , the mean stress in the alveolar bone of the Fiber post group exhibited a significantly higher level compared to both the stress of the Control group and the Titanium post group; however, there was no statistically significant distinction observed between the Control group and the Titanium post group. The mean stress in the dentin of the Control group was significantly lower compared to both the Titanium post group and Fiber post groups, while the stress in the Fiber post group exhibited a statistically higher level than that in the Titanium post group. The stresses in the periodontal membrane of both the Fiber post group and Titanium post group were significantly higher compared to the Control group. Additionally, the mean stress in the Titanium post was found to be significantly greater than that in the Fiber post.
When subjected to a vertical load, as shown in Fig. , the internal stresses in the alveolar bone were primarily concentrated in the apical region of the buccal root. In both the Control group and Fiber post group, dentin stresses were concentrated in the crest of lingual alveolar, while in the Titanium post group, they were focused on the lateral wall of the root canal. The stress in the periodontal membrane was predominantly concentrated on the crest of lingual alveolar. The stress analysis in each structure record in Table . The mean stress in the alveolar bone, dentin, and periodontal membrane was significantly higher in the Fiber post group compared to both the Control group and the Titanium post group. There was no significant difference in stress between the Titanium post group and the Control group in the alveolar bone and dentin, while in the periodontal membrane, the stress of the Titanium post group was significantly lower than that of the Control group. Furthermore, stress in titanium posts exhibited significantly higher levels compared to the stress of fiber posts.
When subjected to a horizontal load (Fig. ), internal stresses in the alveolar bone and the periodontal membrane were primarily concentrated in the crest of lingual alveolar. In both the Control group and Fiber post group, stresses in dentin were concentrated in the lingual alveolar crest, while in the Titanium post group, the stresses were focused on the lateral wall of the lingual root canal. It can be seen from Table , the mean stress in the alveolar bone of the Fiber post group exhibited a significantly higher level compared to both the stress of the Control group and the Titanium post group; however, there was no statistically significant distinction observed between the Control group and the Titanium post group. The mean stress in the dentin of the Control group was significantly lower compared to both the Titanium post group and Fiber post groups, while the stress in the Fiber post group exhibited a statistically higher level than that in the Titanium post group. The stresses in the periodontal membrane of both the Fiber post group and Titanium post group were significantly higher compared to the Control group. Additionally, the mean stress in the Titanium post was found to be significantly greater than that in the Fiber post.
The clinical restoration effect of titanium posts on teeth with subgingival defects is superior to crown lengthening. Although both treatment methods can result in healthy periodontal status for the teeth, titanium posts do not lead to increased tooth mobility and reduced interproximal papilla height, which are unavoidable consequences of crown lengthening. Under both vertical and horizontal loading conditions, the stress within the dental and periodontal tissues of teeth restored with titanium posts is lower than that of teeth restored with fiber posts after crown lengthening. This is advantageous for maintaining long-term periodontal health. Crown lengthening has been used in clinical practice for a long time, and it has a definite therapeutic effect on teeth with subgingival defects. However, the complications of increased tooth mobility, loss of gingival papilla height, and reduced long-term success rate cannot be ignored – . Although researchers have tried to improve crown lengthening by minimizing the amount of bone and gingival tissue resection while appropriately exposing the defect area and reducing the depth of the gingival pocket, the problem of reduced long-term success rate cannot be avoided , . Using titanium posts to repair damaged teeth avoids resection of the alveolar bone and gingival tissue, and the height of the bone around the root and the gingival papilla height are maintained, significantly reducing the incidence of post-treatment tooth mobility and interproximal spaces and promoting better oral health maintenance. Additionally, titanium post restoration is more patient-friendly, eliminating the pain of crown lengthening and reducing treatment costs. In this study, the deep periodontal pockets originally located under the gingiva of the teeth treated with titanium posts all became shallow to the normal level. We speculated that the gingival might attach to the smooth surface of the titanium post, which was similar to the attachment of the gingival epithelium to the implant through hemi-desmosomes and internal basal lamina – . This may also be the reason why the gingival index, sulcus bleeding index and especially probing depth gradually returned to normal. While it still remains uncertain whether there is bonding between the gingival tissue and the polished surface of the titanium post; this warrants further confirmation through animal experiments. The results of clinical studies have confirmed that excessive bite force or occlusal trauma can result in an increase in probing depth, exacerbation of the bleeding index upon probing, widening of the periodontal ligament space, deepening of sub osseous pockets, and intensification of the occurrence and progression of periodontitis , . Reducing occlusal force has been demonstrated to decrease probing depth, improve periodontal bleeding status, decrease periodontal purulent discharge, and alter the composition of subgingival plaque biofilm . The reason is that excessive external force upregulate proinflammatory cytokines in human periodontal ligament-derived fibroblasts (HPDF), such as monocyte chemotactic protein 1 (MCP-1), TNF α/β, PTGS2, PGE2, IL-1β/IL6 expression and RANKL/M-CSF levels to cause or aggravate inflammation within the periodontal tissue , . Clearly, external forces significantly influence the health of periodontal tissues. When subjected to external loads, stress is generated within dental and periodontal tissues. Finite element analysis technique is widely utilized in dental research for applications like implantation procedures orthodontics treatment planning jaw trauma assessment – to evaluate internal stresses within tissues or structures. Therefore we employed finite element analysis to investigate stress distribution within the periodontium after fiber post restoration following crown lengthening procedure with titanium post restoration. It was observed that internal stresses within dental and periodontal tissues were significantly lower after titanium post restoration compared to those after crown lengthening. The aforementioned statement may explain the potential contribution to the consistent preservation of periodontal health after titanium post restoration. It is noteworthy that, although there was no significant difference in the stress in the jaws between the titanium post group and the normal crown group under horizontal loading, the stress in the dentin and the peridental membrane of the titanium post group were significantly higher than those of the normal crown group. It is suggested that when applying titanium post, the cusp inclination of the crown should be designed reasonably to eliminate the lateral force during chewing. The stress in the titanium post is significantly higher than that in the fiber post due to a notable discrepancy in their elastic moduli. Specifically, while the elastic modulus of the fiber post (15000 MPa) closely approximates that of dentin (18600 MPa), the elastic modulus of the titanium post (110000 MPa) greatly surpasses that of dentin, resulting in considerably elevated internal stress within the titanium post. However, our primary focus lies not on the stress within the post but rather on evaluating and understanding the stresses in dentin, periodontal membrane, and bone tissue health, which are crucial factors affecting overall dental and periodontal well-being. The limitation of this study lies in its conservative approach towards subject selection, resulting in a small sample size for clinical trials and a short observation period. This somewhat compromises the persuasive power of the conclusions. However, it is evident that titanium posts have a significant and effective clinical effect on repairing subgingival defect teeth. A eighteen-month observation period is sufficient for the teeth to achieve a stable periodontal state, indicating that regular oral cleaning and maintenance by subjects can ensure restoration stability. The longest observation period among subjects has been nearly 4 years, during which the teeth have remained remarkably healthy. Therefore, we have robust confidence in the high long-term success rate following restoration with titanium post.
The efficacy of utilizing a modified titanium post for the restoration of subgingival defects surpasses that of crown lengthening significantly. The gingiva exhibits a state of health, with normal probing depth, and the implementation of a modified titanium post does not induce tooth mobility or atroph of gingival papilla. The stress exerted in periodontal tissues in teeth restored using a modified titanium post was reduced compared to those treated with crown lengthening which maybe why the tooth could maintain a healthy and stable periodontal state.
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Response of Soil Microbiota, Enzymes, and Plants to the Fungicide Azoxystrobin | 02819eaf-e9ef-4866-b930-73d7225be67b | 11311602 | Microbiology[mh] | Pesticides underpin the maintenance of plant quality and health through their key role in eradicating diseases, pests, and weeds . Azoxystrobin is a fungicide from the group of strobilurins, extensively used in agricultural production due to its broad spectrum of effects and high efficacy against fungal pathogens of crops . Strobilurin was originally isolated from the fungus Strobilurus tenacellus in 1977, whereas azoxystrobin was introduced on the German market in 1996 . Edwards et al. have pointed out that the half-life of azoxystrobin in soil spans from 14 days to 6 months, which is related to the activity of soil microorganisms and enzymes. In addition, azoxystrobin is a fungicide from the group of external quinone inhibitors that inhibit mitochondrial respiration by blocking the transfer of electrons in the cytochrome bc1 complex. Moreover, it inhibits the oxidation of nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP) and exerts multifaceted effects on Ascomycetes , Basidiomycetes , and Oomycetes fungi. Its EC 50 (half-maximal effective concentration) was reported to range from 0.003 to 0.031 µg mm −3 of the liquid culture medium against Cercospora zeae-maydis and from 0.12 to 297.22 µg mm −3 of the liquid culture medium against Aspergillus flavus . Azoxystrobin has often been detected in different ecosystems at higher than acceptable concentrations and therefore may pose a severe threat to organisms found therein . Pesticides are effective in protecting plants and boosting their yield. However, when used in non-observance of good agricultural practice, they may elicit adverse effects on animal health, as well as water and food quality . Improper and long-term use of fungicides can lead to changes in soil ecosystems as they disturb the abundance, activity, and functioning of the soil microbiota, and in biogeochemical cycles of nitrogen, carbon, phosphorus, and sulfur. This, in turn, may deteriorate the quality and fertility of the soil, which plays a huge role in the environment, by, for instance, providing many nutrients to organisms, increasing plant production, or maintaining biodiversity of the environment . Soil organisms contribute to its proper functioning by influencing its physicochemical and biological properties, which ultimately affect crop productivity. Soil microorganisms are essential for maintaining proper ecological balance, soil fertility, plant growth, and pesticide degradation . By secreting various types of enzymes, lipids, and other biologically active macromolecules, they can affect the fate of pesticides in the soil environment . Therefore, the assessment of the impact of pesticides, including fungicides, on soil microbiota and soil biochemical processes is a sound action taken to maintain the sustainable development of soil ecosystems . Fungicides and their metabolites can be a major stressor for soil microorganisms, which can lead to both a reduction in their diversity and function in the soil, ultimately affecting ecological functionality . Continuous use of fungicides also has detrimental effects on microbial metabolism, soil nutrient cycling, and plant function. Thus, excessive use of these chemicals can lead to high accumulation and prolonged persistence in the soil system, which adversely affects the soil environment . An example is the study by Verdenelli et al. , who noted a significant reduction in the abundance and diversity of gram-positive and gram-negative bacteria and arbuscular fungi under the influence of carbendazim and iprodione applied at the highest dose (4.50 mg kg −1 d.m. soil and 8.30 mg kg −1 d.m. soil, respectively). Another example is difenoconazole introduced into the soil at 0.04 mg kg −1 d.m. soil, which led to a decrease in microbial biomass in loamy-sandy soil, as the microorganisms used more energy to detoxify the environment than for their growth . According to Chamberlain et al. , the composition and diversity of microorganisms in the soil affect the regulation of the main functions of the soil, i.e., the circulation of elements, and these functions in turn indirectly affect the growth and yield of plants by, among other things, providing them with nutrients. Soil enzymes are believed to originate mainly from microorganisms, but also from plant and animal remains entering the soil. They accumulate in the soil as free enzymes or are stabilized on soil organic matter. Soil enzymes are essential for microbial life functions, as they participate in all biochemical processes occurring in the soil, as well as increase the rate of organic matter decomposition reactions, resulting in the release of nutrients into the soil environment. Due to their stability and sensitivity, they are used as indicators of soil health . A study by Filimon et al. showed that difenoconazole introduced into chernozem soil at doses of 37, 75, and 150 mg kg −1 d.m. soil under both field and laboratory conditions of all enzymes tested (dehydrogenases, urease, protease, and acid phosphatase) inhibited enzyme activity, with the most inhibitory effect on dehydrogenase activity. The application of another fungicide myclobutanil at a dose of 0.1 mg kg −1 d.m. soil contributed to an increase in dehydrogenase activity, while already higher doses (1.0 and 10 mg kg −1 ) significantly inhibited the activity of these enzymes . In a study conducted by Satapute et al. determining the effect of propiconazole (doses of 1.0, 15.0, and 20 kg ha −1 ) on enzymes in red sandy-loam black-earth soil, it was found that the tested fungicide stimulated the activity of urease and phosphatases in the first 2 weeks, while after 3 weeks the activity of these enzymes significantly decreased in sites containing doses of 15.0 and 20.0 mg kg −1 of propiconazole. However, the activity of these enzymes was higher in deep-black soil than in red sandy loam. Fungicides can also be taken up by plants, which can lead to an increase in the production of reactive oxygen species, with subsequent inhibition of normal physiological and biochemical processes in plants and disruption of photosynthesis, thereby reducing yields. Examples of the adverse effects of these chemicals on plants include, for example, that of pendimethalin, which significantly inhibits the germination of Zea mays L. seeds as its concentration in the soil increases, or that of fipronil, which significantly reduced the germination of rice seeds compared to control soil . The use of fungicides can cause biochemical and physiological changes in antioxidants, which has an initial effect on plant germination, subsequent growth and development, and ultimately on yield . However, the inactivation of fungicides and their elimination from the soil environment is carried out mainly through microbial processes, as microorganisms are capable of producing enzymes that carry out catabolic processes . It was observed that microorganisms belonging to the Actinomycetota, Proteobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Basidiomycota phylum are more abundant in soil contaminated with fungicides than in non-contaminated soil (control soil). Tremendous abilities to degrade fungicides are demonstrated by microorganisms of the genera: Acinetobacter , Achromobacter , Agrobacterium , Alcaligenes , Arthrobacter , Azospirillum , Enterobacter , Bacillus , Burkholderia , Cupriavidus , Flavimonas , Brevibacterium , Flavobacterium , Klebsiella , Micrococcus , Methylobacterium , Mesorhizobium , Ochrobactrum , Peanibacillus , Pseudomonas , Pseudaminobacter, Rhizobium , Ralstonia , Serratia , Shinella , Sphingomonas Streptomyces , Xanthomonas , and Yersinia . The aim of this study was, therefore, to assess the effect of soil amendment with two doses of azoxystrobin (field and contaminating doses) on microbiota, enzymes, and plants 30, 60, and 90 days after their application. The study results will allow for a broader understanding of changes in soil microbial populations and biochemical processes taking place in the soil environment. In addition, they may indicate differences in the sensitivity of microorganisms, enzymes, and plants to azoxystrobin. The research hypotheses formulated have assumed that the accumulation of azoxystrobin in soil causes (a) severe disorders in the microbiome, (b) destabilization of enzyme activity, and (c) inhibition of plant growth and development.
2.1. Response of Soil Microbiota to Azoxystrobin Statistical analysis showed that the number of organotrophic bacteria was most affected by the interaction of the studied factors (η 2 = 42.28%); that of actinobacteria, by soil incubation time (η 2 = 59.00%); and that of fungi, by azoxystrobin doses (η 2 = 51.30%) . Compared to the control soil (soil C), the number of organotrophic bacteria in soil F (field dose) increased 1.2-fold and 1.3-fold on days 30 and 90 of the experiment, respectively, and that of actinobacteria increased 1.9-fold, 1.1-fold, and 1.2-fold on days 30, 60, and 90, respectively, whereas a 1.2-fold decrease was noted in the number of fungi on day 30, a 2.2-fold decrease on day 60, and a 1.4-fold decrease on day 90 . In the case of soil P (polluting dose), on day 30 of the experiment, the numbers of organotrophic bacteria and actinobacteria were 1.6-fold and 1.3-fold higher compared to the control, respectively. Analyses conducted on days 60 and 90 demonstrate the inhibition of soil microbiota proliferation in this soil, as evidenced by a 1.3-fold and 1.2-fold decrease in the abundance of organotrophic bacteria, respectively, and a 1.1-fold decrease in that of actinobacteria on both dates. Fungi responded to the highest dose of azoxystrobin with a decrease in their numbers at all dates of analyses. And so, a 1.6-fold decrease was noted in their number in soil P on day 30, a 4.0-fold decrease on day 60, and a 2.6-fold decrease on day 90, compared to the control soil. Regardless of azoxystrobin dose, the highest mean number of organotrophic bacteria (3.069 × 10 9 cfu kg −1 d.m. soil) and actinobacteria (2.105 × 10 9 cfu kg −1 d.m. soil) was observed on day 90, whereas that of fungi was on day 30 (1.350 × 10 7 cfu kg −1 d.m. soil). The most intensive proliferation in the soil was observed in the case of organotrophic bacteria and was the least intensive one—in the case of fungi. Diversified effects of azoxystrobin over time are confirmed by changes in the population numbers of microorganisms . The inhibition in the counts of organotrophic bacteria ranged from 9.50% (soil F, day 60) to 37.98% (soil P, day 60), in those of actinobacteria from 10.46% (soil P, day 60) to 11.66% (soil P, day 90), and in those of fungi from 14.11% (soil F, day 30) to 74.82% (soil P, day 60 day). In the F and P soil samples, the growth of organotrophic bacteria was stimulated on day 30 and inhibited on day 60. In the case of actinobacteria, an increase in their number was recorded at all test dates in soil F, whereas there was a decrease in soil P on days 60 and 90. In turn, the number of fungi decreased in soil F and P in all terms of analyses (days 30, 60, and 90 of the experiment). The colony development index (CD) of microorganisms was affected to the greatest extent by the incubation time of soil (from 52.75% to 78.63%), to a lesser extent by the interaction of factors (from 8.58% to 12.39%), and to the least extent by azoxystrobin dose (from 1.54% to 11.79%). Taking into account the incubation time of the soil, the highest CD values of organotrophic bacteria (mean CD = 62.758) and actinobacteria (mean CD = 24.623) were recorded on day 90, and the highest CD of fungi (mean CD = 24.832) on day 30. The CD of organotrophic bacteria was the highest in soil C on day 90 (CD = 66.617), that of actinobacteria in soil F on day 90 (CD = 24.861), and that of fungi in soil F on day 30 (CD = 25.645). Of all groups of microorganisms, the highest CD value was computed for organotrophic bacteria, and the lowest one for fungi. Statistical analysis of the observed variance showed that the incubation time of soil had the strongest impact on the ecophysiological diversity index (EP) of organotrophic bacteria (η 2 = 64.3%) and actinobacteria (η 2 = 70.10%), whereas the azoxystrobin dose on EP of fungi was (η 2 = 51.62%). The organotrophic bacteria, actinobacteria, and fungi had the highest EP values in soil C on day 60 (i.e., 0.833, 0.844, and 0.875, respectively). Regardless of azoxystrobin dose applied, the highest EP values were computed for organotrophic bacteria (mean EP = 0.762), actinobacteria (mean EP = 0.825), and fungi (mean EP = 0.761) on day 60 of the experiment. Azoxystrobin also contributed to the soil imbalance, as evidenced by the index of soil return to the equilibrium state—resilience index (RL) . The greatest changes occurred in the fungal population, because the RL values computed on days 60 and 90 were negative (mean RL values were RL = −0.734 and RL = −0.471, respectively). In the case of organotrophic bacteria and actinobacteria, the mean RL values were positive in all terms of analyses. shows a phylogenetic tree of bacteria, and shows a phylogenetic tree of fungi. Soil C was most heavily populated by PP952050.1 Bacillaceae bacterium strain (C), PP952049.1 Bacillus cereus strain (C); and by PP952060.1 Talaromyces pinophilus isolate KF751644.1 PP955260.1; and PP952061.1 Trichoderma pnophilus isolate (C) fungi. In turn, soil F was colonized by PP952047.1 Prestia megaterium strain (F), PP952048.1 Peribacillus simplex (F), PP952058.1 Penicillium chrysogenum isolate (F), and PP952059.1 Talaromyces piinophilus isolate (F), whereas soil P was colonized by PP952052.1 Prestia megaterium strain (P), PP952051.1 Bacillus mycoides strain (P), and PP952062.1 Keratinophyton terreum isolate (P), which may tolerate and degrade azoxystrobin. 2.2. Response of Soil Enzymes to Azoxystrobin In this study, soil incubation time had the greatest impact on soil enzyme activity (η 2 ranged from 87.86% to 99.76%), while the other variables analyzed, namely azoxystrobin dose and interaction of factors, had little effect on soil enzymes . Regardless of the fungicide dose, the highest dehydrogenases activity (mean 28.361 µmol TPF kg −1 d.m. soil h −1 ) and urease activity (mean 2.129 mmol N-NH 4 kg −1 d.m. soil h −1 ) were determined on day 30, whereas the highest catalase activity (mean 0.536 mol O 2 kg −1 d.m. soil h −1 ) and alkaline phosphatase activity (mean 2.369 mmol PNP kg −1 d.m. soil h −1 ) were determined on day 60, and the highest acidic phosphatase activity (mean 1.969 mmol PNP kg −1 d.m. soil h −1 ) was determined on day 90. In soil F, an increase was observed in all terms of analyses (30, 60, and 90 days) in the activity of dehydrogenases, catalase, and alkaline phosphatase compared to soil C, whereas on day 30 there was an increase in alkaline phosphatase activity, and on day 90 there was a decrease in urease activity. In soil P, dehydrogenases activity decreased compared to soil C in all terms of analyses, alkaline phosphatase and acid phosphatase activities on days 30 and 90, and urease activity on day 90 of the experiment. In the same soil, an increase in catalase activity was observed on days 30 and 60, and an increase in alkaline phosphatase on day 60 . Dehydrogenases activity decreased by 0.45% (soil P, day 90) to 3.75% (soil P, day 60), catalase activity by 1.36% (soil P, day 90), alkaline phosphatase activity by 5.39% (soil P, day 90) to 11.45% (soil P day 30), acid phosphatase activity by 0.98% (soil F, day 90) to 8.55% (soil P, day 90), and urease activity by 0.84% (soil P, day 30) to 18.88% (soil P, day 90) . In all analytical terms, the activity of dehydrogenases, catalase, and alkaline phosphatase was stimulated in soil F, whereas dehydrogenases and urease activity were inhibited in soil P. In addition, in soil P, the activity of catalase increased significantly on days 30 and 60, whereas activities of alkaline phosphatase and acid phosphatase were suppressed on days 30 and 90. Azoxystrobin caused significant changes in sandy clay, which is confirmed by the values of the soil resilience index . The greatest disturbance in the soil was noted based on dehydrogenases activity (mean RL = −0.144 on day 60 and RL = −0.037 on day 90) and acid phosphatase activity (mean RL = −0.594 on day 60 and RL = −0.461 on day 90). Adverse changes were also observed in urease activity on day 60 (mean RL = −0.544). The highest mean RL values were determined for catalase activity followed by alkaline phosphatase activity. Their positive RL values indicate that these enzymes are able to return to a state of biochemical equilibrium. 2.3. Pearson’s Simple Correlation Coefficients between Microbiological and Biochemical Soil Parameters The activity of dehydrogenases, alkaline phosphatase, and urease were significantly positively correlated with the EP of organotrophic bacteria and actinobacteria, while negatively correlated with the number of actinobacteria and CD of organotrophic bacteria and actinobacteria. An opposite correlation was found for acid phosphatase. Catalase activity was significantly positively correlated with the abundance of organotrophic bacteria, fungi, and actinobacteria and with the CD of actinobacteria. In addition, the activities of dehydrogenases and alkaline phosphatase were significantly negatively correlated with the count of organotrophic bacteria . 2.4. Response of Plants to Azoxystrobin The percentage of the observed variability (η 2 ) of the factors examined showed that the azoxystrobin dose elicited the greatest changes in plant growth and development . Its dose affected seed germination in 64.89% ( S. saccharatum L.) to 87.57% ( S. alba L.), while it affected root growth in 65.01% ( S. saccharatum L.) to 70.68% ( S. alba L.). The incubation time of the soil modified the seed germination in 2.23% ( S. alba L.) to 23.97% ( S. saccharatum L.) and the root growth in 18.95% ( S. alba L.) to 26.14% ( L. sativum L.). The dose of azoxystrobin and duration of its retention in the soil significantly affected the germination of seeds and the elongation of plant roots . In soil P, the greatest inhibition of the germination of L. sativum L. and S. saccharatum L. seeds occurred on day 90 (by 58.43% and 54.23%, respectively) and of S. alba seeds on day 60 (by 63.92%). The greatest inhibition of root elongation of plants was recorded on day 90. Compared to the control soil, the root elongation decreased by 54.90% for L. sativum L., by 50.92% for S. alba L., and by 53.78% for S. saccharatum L. A significant inhibition of seed germination and extension of plant roots compared to soil C was also observed in the soil F; however, this inhibition was still less than in the soil P.
Statistical analysis showed that the number of organotrophic bacteria was most affected by the interaction of the studied factors (η 2 = 42.28%); that of actinobacteria, by soil incubation time (η 2 = 59.00%); and that of fungi, by azoxystrobin doses (η 2 = 51.30%) . Compared to the control soil (soil C), the number of organotrophic bacteria in soil F (field dose) increased 1.2-fold and 1.3-fold on days 30 and 90 of the experiment, respectively, and that of actinobacteria increased 1.9-fold, 1.1-fold, and 1.2-fold on days 30, 60, and 90, respectively, whereas a 1.2-fold decrease was noted in the number of fungi on day 30, a 2.2-fold decrease on day 60, and a 1.4-fold decrease on day 90 . In the case of soil P (polluting dose), on day 30 of the experiment, the numbers of organotrophic bacteria and actinobacteria were 1.6-fold and 1.3-fold higher compared to the control, respectively. Analyses conducted on days 60 and 90 demonstrate the inhibition of soil microbiota proliferation in this soil, as evidenced by a 1.3-fold and 1.2-fold decrease in the abundance of organotrophic bacteria, respectively, and a 1.1-fold decrease in that of actinobacteria on both dates. Fungi responded to the highest dose of azoxystrobin with a decrease in their numbers at all dates of analyses. And so, a 1.6-fold decrease was noted in their number in soil P on day 30, a 4.0-fold decrease on day 60, and a 2.6-fold decrease on day 90, compared to the control soil. Regardless of azoxystrobin dose, the highest mean number of organotrophic bacteria (3.069 × 10 9 cfu kg −1 d.m. soil) and actinobacteria (2.105 × 10 9 cfu kg −1 d.m. soil) was observed on day 90, whereas that of fungi was on day 30 (1.350 × 10 7 cfu kg −1 d.m. soil). The most intensive proliferation in the soil was observed in the case of organotrophic bacteria and was the least intensive one—in the case of fungi. Diversified effects of azoxystrobin over time are confirmed by changes in the population numbers of microorganisms . The inhibition in the counts of organotrophic bacteria ranged from 9.50% (soil F, day 60) to 37.98% (soil P, day 60), in those of actinobacteria from 10.46% (soil P, day 60) to 11.66% (soil P, day 90), and in those of fungi from 14.11% (soil F, day 30) to 74.82% (soil P, day 60 day). In the F and P soil samples, the growth of organotrophic bacteria was stimulated on day 30 and inhibited on day 60. In the case of actinobacteria, an increase in their number was recorded at all test dates in soil F, whereas there was a decrease in soil P on days 60 and 90. In turn, the number of fungi decreased in soil F and P in all terms of analyses (days 30, 60, and 90 of the experiment). The colony development index (CD) of microorganisms was affected to the greatest extent by the incubation time of soil (from 52.75% to 78.63%), to a lesser extent by the interaction of factors (from 8.58% to 12.39%), and to the least extent by azoxystrobin dose (from 1.54% to 11.79%). Taking into account the incubation time of the soil, the highest CD values of organotrophic bacteria (mean CD = 62.758) and actinobacteria (mean CD = 24.623) were recorded on day 90, and the highest CD of fungi (mean CD = 24.832) on day 30. The CD of organotrophic bacteria was the highest in soil C on day 90 (CD = 66.617), that of actinobacteria in soil F on day 90 (CD = 24.861), and that of fungi in soil F on day 30 (CD = 25.645). Of all groups of microorganisms, the highest CD value was computed for organotrophic bacteria, and the lowest one for fungi. Statistical analysis of the observed variance showed that the incubation time of soil had the strongest impact on the ecophysiological diversity index (EP) of organotrophic bacteria (η 2 = 64.3%) and actinobacteria (η 2 = 70.10%), whereas the azoxystrobin dose on EP of fungi was (η 2 = 51.62%). The organotrophic bacteria, actinobacteria, and fungi had the highest EP values in soil C on day 60 (i.e., 0.833, 0.844, and 0.875, respectively). Regardless of azoxystrobin dose applied, the highest EP values were computed for organotrophic bacteria (mean EP = 0.762), actinobacteria (mean EP = 0.825), and fungi (mean EP = 0.761) on day 60 of the experiment. Azoxystrobin also contributed to the soil imbalance, as evidenced by the index of soil return to the equilibrium state—resilience index (RL) . The greatest changes occurred in the fungal population, because the RL values computed on days 60 and 90 were negative (mean RL values were RL = −0.734 and RL = −0.471, respectively). In the case of organotrophic bacteria and actinobacteria, the mean RL values were positive in all terms of analyses. shows a phylogenetic tree of bacteria, and shows a phylogenetic tree of fungi. Soil C was most heavily populated by PP952050.1 Bacillaceae bacterium strain (C), PP952049.1 Bacillus cereus strain (C); and by PP952060.1 Talaromyces pinophilus isolate KF751644.1 PP955260.1; and PP952061.1 Trichoderma pnophilus isolate (C) fungi. In turn, soil F was colonized by PP952047.1 Prestia megaterium strain (F), PP952048.1 Peribacillus simplex (F), PP952058.1 Penicillium chrysogenum isolate (F), and PP952059.1 Talaromyces piinophilus isolate (F), whereas soil P was colonized by PP952052.1 Prestia megaterium strain (P), PP952051.1 Bacillus mycoides strain (P), and PP952062.1 Keratinophyton terreum isolate (P), which may tolerate and degrade azoxystrobin.
In this study, soil incubation time had the greatest impact on soil enzyme activity (η 2 ranged from 87.86% to 99.76%), while the other variables analyzed, namely azoxystrobin dose and interaction of factors, had little effect on soil enzymes . Regardless of the fungicide dose, the highest dehydrogenases activity (mean 28.361 µmol TPF kg −1 d.m. soil h −1 ) and urease activity (mean 2.129 mmol N-NH 4 kg −1 d.m. soil h −1 ) were determined on day 30, whereas the highest catalase activity (mean 0.536 mol O 2 kg −1 d.m. soil h −1 ) and alkaline phosphatase activity (mean 2.369 mmol PNP kg −1 d.m. soil h −1 ) were determined on day 60, and the highest acidic phosphatase activity (mean 1.969 mmol PNP kg −1 d.m. soil h −1 ) was determined on day 90. In soil F, an increase was observed in all terms of analyses (30, 60, and 90 days) in the activity of dehydrogenases, catalase, and alkaline phosphatase compared to soil C, whereas on day 30 there was an increase in alkaline phosphatase activity, and on day 90 there was a decrease in urease activity. In soil P, dehydrogenases activity decreased compared to soil C in all terms of analyses, alkaline phosphatase and acid phosphatase activities on days 30 and 90, and urease activity on day 90 of the experiment. In the same soil, an increase in catalase activity was observed on days 30 and 60, and an increase in alkaline phosphatase on day 60 . Dehydrogenases activity decreased by 0.45% (soil P, day 90) to 3.75% (soil P, day 60), catalase activity by 1.36% (soil P, day 90), alkaline phosphatase activity by 5.39% (soil P, day 90) to 11.45% (soil P day 30), acid phosphatase activity by 0.98% (soil F, day 90) to 8.55% (soil P, day 90), and urease activity by 0.84% (soil P, day 30) to 18.88% (soil P, day 90) . In all analytical terms, the activity of dehydrogenases, catalase, and alkaline phosphatase was stimulated in soil F, whereas dehydrogenases and urease activity were inhibited in soil P. In addition, in soil P, the activity of catalase increased significantly on days 30 and 60, whereas activities of alkaline phosphatase and acid phosphatase were suppressed on days 30 and 90. Azoxystrobin caused significant changes in sandy clay, which is confirmed by the values of the soil resilience index . The greatest disturbance in the soil was noted based on dehydrogenases activity (mean RL = −0.144 on day 60 and RL = −0.037 on day 90) and acid phosphatase activity (mean RL = −0.594 on day 60 and RL = −0.461 on day 90). Adverse changes were also observed in urease activity on day 60 (mean RL = −0.544). The highest mean RL values were determined for catalase activity followed by alkaline phosphatase activity. Their positive RL values indicate that these enzymes are able to return to a state of biochemical equilibrium.
The activity of dehydrogenases, alkaline phosphatase, and urease were significantly positively correlated with the EP of organotrophic bacteria and actinobacteria, while negatively correlated with the number of actinobacteria and CD of organotrophic bacteria and actinobacteria. An opposite correlation was found for acid phosphatase. Catalase activity was significantly positively correlated with the abundance of organotrophic bacteria, fungi, and actinobacteria and with the CD of actinobacteria. In addition, the activities of dehydrogenases and alkaline phosphatase were significantly negatively correlated with the count of organotrophic bacteria .
The percentage of the observed variability (η 2 ) of the factors examined showed that the azoxystrobin dose elicited the greatest changes in plant growth and development . Its dose affected seed germination in 64.89% ( S. saccharatum L.) to 87.57% ( S. alba L.), while it affected root growth in 65.01% ( S. saccharatum L.) to 70.68% ( S. alba L.). The incubation time of the soil modified the seed germination in 2.23% ( S. alba L.) to 23.97% ( S. saccharatum L.) and the root growth in 18.95% ( S. alba L.) to 26.14% ( L. sativum L.). The dose of azoxystrobin and duration of its retention in the soil significantly affected the germination of seeds and the elongation of plant roots . In soil P, the greatest inhibition of the germination of L. sativum L. and S. saccharatum L. seeds occurred on day 90 (by 58.43% and 54.23%, respectively) and of S. alba seeds on day 60 (by 63.92%). The greatest inhibition of root elongation of plants was recorded on day 90. Compared to the control soil, the root elongation decreased by 54.90% for L. sativum L., by 50.92% for S. alba L., and by 53.78% for S. saccharatum L. A significant inhibition of seed germination and extension of plant roots compared to soil C was also observed in the soil F; however, this inhibition was still less than in the soil P.
3.1. Response of Soil Microbiota to Azoxystrobin Pesticides, including fungicides and their metabolites, can exert an immediate effect on soil microorganisms, triggering changes in their population and diversity . One such pesticide is azoxystrobin, which, 21 and 28 days after application to the soil in a dose of 10 mg kg −1 , significantly reduced the numbers of bacteria and actinobacteria compared to the control soil but had no significant effect on the fungi population . In the present study, azoxystrobin applied in the field dose stimulated the growth of organotrophic bacteria and actinobacteria, but it inhibited the growth of fungi. In turn, its contaminating dose reduced the population numbers of all analyzed groups of microorganisms. A small amount of the fungicide in the soil could have been used by organotrophic bacteria and actinobacteria as a source of nutrients; therefore, the recommended dose of azoxystrobin could increase their numbers . However, a fungicide dose being few or several times higher than the agronomic dose may directly affect the survival of microorganisms by disrupting the metabolic pathways in their cells . The sensitivity of microorganisms to increased amounts of azoxystrobin may be due to oxidative stress generated upon the release of electrons from the respiratory chain in the form of reactive oxygen . Fungicides affect not only the proliferation of microbial populations but also their diversity . They modify the composition of the microbial community because of their effects on non-target organisms . The present study demonstrated differences in the colony development index (CD) and the ecophysiological diversity index (EP) of microorganisms. In the soil treated with azoxystrobin, there was an increase in the CD values computed for organotrophic bacteria, actinobacteria, and fungi compared to the control soil. The r-strategies (fast-growing microorganisms) prevailed among organotrophic bacteria, whereas the k-strategists (slow-growing microorganisms) prevailed among actinobacteria and fungi. This was evidenced by the CDs, the mean values of which were CD = 50.27 for organotrophic bacteria and CD = 23.49 and CD = 24.38 for actinobacteria and fungi, respectively. Therefore, it can be concluded that the addition of chemical compounds to the soil may determine the proportions between r-strategists and k-strategists . Azoxystrobin exerted diverse effects on the EP of microorganisms, which was also caused by the time of its retention in the soil. However, the greatest changes caused by azoxystrobin presence in the soil, which were manifested by EP decrease, were observed in the case of fungi. The adaptation of microorganisms to adverse conditions is largely dependent on their activity, as well as on the degree of severity of the stress factor occurring in the soil environment . Fungicides are degraded in the soil environment by various microorganisms that produce specific enzymes capable of their degradation . For example, Alexandrino et al. demonstrated that bacteria of the genera Pseudomonas , Rhodobacter, Ochobacterum , Comamonas , Hydrogenophaga , Azospirillum , Methylbacillus , and Acinetobacter had high degradation potential against epoxiconazole and fludioxonil, as they degraded 10 mg dm −3 of these fungicides within 21 days. In turn, Feng et al. have reported that Arthrobacter , Bacillus , Cupriavidus , Pseudomonas , Klebsiella , Rhodanobacter , Stenothrofomonas , and Aphanoascus are microorganisms that break down strobilurin compounds. Clinton et al. isolated two species of bacteria from the soil contaminated with trifloxystrobin, namely Bacillus flexus and Bacillus amyloliquefaciens , whereas Howell et al. observed that Cuprividus spp. and Rhodobacter spp. exerted a degrading effect against azoxystrobin. Actinomyces spp. and Ochrabactrum spp. are also capable of degrading azoxystrobin . In our study, we identified microorganisms, i.e., bacteria PP952052.1 Prestia megaterium strain (P) and PP952051.1 Bacillus mycoides strain (P), and fungi PP952062.1 Keratinophyton terreum isolate (P), which may show high tolerance to azoxystrobin and the potential for its degradation. In turn, Feng et al. isolated a strain of bacteria Chrobacrum anthropi SH14 from soil contaminated with azoxystrobin, which was able to degrade 86.30% of the 50 µg cm −3 medium dose of this pesticide within 5 days. 3.2. Response of Soil Enzymes to Azoxystrobin The activity of soil enzymes is closely related to the quality and fertility of the soil. These biological parameters of the soil respond quickly to the effects of high pesticide doses . Wang et al. reported that azoxystrobin, used at doses of 0.1 to 10 mg kg −1 , inhibited dehydrogenases activity in all terms of analyses (i.e., on days 7, 14, 21, and 28) and urease activity up to day 14 while stimulating catalase activity and not significantly affecting protease activity. In the experiment described in this manuscript, the contaminating dose of azoxystrobin inhibited the activity of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease, while its agronomic dose enhanced activities of all analyzed soil enzymes. The inactivating effect of azoxystrobin on soil enzymes may have been due to the inhibition of microbial population multiplication, which indirectly affected the secretion of enzymes whose activity is strongly dependent on the number and biomass of microorganisms . Adverse effects of azoxystrobin (doses: 2.90, 14.65, and 35.00 mg kg −1 ) on the activity of soil enzymes such as dehydrogenases, urease, alkaline phosphatase, acid phosphatase, arylsulfatase, and β -glucosidase were noted in the study by Boteva et al. , with dehydrogenases and arylsulfatase being the most sensitive, and urease being the most resistant to soil treatment with this pesticide. In the present study, catalase was the most resistant to azoxystrobin, as evidenced by its enhanced activity in the soil contaminated with this compound. The increase in its activity may suggest that some microorganisms used azoxystrobin as a source of nutrients and energy necessary for their growth, which contributed to boosted catalase secretion by their cells . The increased catalase production by microorganisms probably caused fungicide degradation and strengthened the protective barrier of microorganisms against oxidizing compounds . The positive or negative effects of azoxystrobin on soil enzymes are mainly related to its dose and duration of its retention in the soil . 3.3. Response of Plants to Azoxystrobin In addition to their antifungal activity, strobilurins improve plant quality by intensifying photosynthesis; increasing contents of nitrogen, chlorophyll, and protein; and delaying the aging of plants . Amaro et al. and Chiu-Yueh et al. have pointed to a very strong effect of fungicides from the group of strobilurin compounds on the physiology and growth of plants. In the present study, azoxystrobin added to soil both in the recommended field dose and the contaminating dose, inhibited seed germination and elongation of L. sativum L., S. alba L., and S. saccharatum L. shoots in all analytical terms (days 30, 60, and 90). Eman et al. determined the effect of azoxystrobin applied at the recommended dose and also at doses 0.5-fold and 2-fold higher than the recommended dose on the germination of Triticum aestivum L. and Raphanus sativus L. seeds. They found that azoxystrobin significantly reduced their seed germination percentage and the length of their roots and shoots. Particularly significant reduction in the length of roots and shoots of Triticum aestivum L. and Raphanus sativus L. was reported at a 2-fold-higher dose than the recommended one, which reduced the length of roots and shoots in wheat by 13.20% and 26.02%, respectively and in radish by 17.67% and 51.67%, respectively. In the present study, an azoxystrobin dose of 32.92 mg kg −1 caused, on average, 50.31% and 45.26% reduction in the length of shoots and roots of L. sativum L., respectively; 57.52% and 48.29% reductions in S. alba L.; and 45.32% and 44.65% reductions in these traits in S. saccharatum L., respectively. The impaired plant growth could have been due to the blocking of the cytochrome bc1 complex, which inhibited cell division and water uptake by plants . Amaro et al. , who assessed the effect of an azoxystrobin dose of 60 g ha −1 , found a reduction in the rate of CO 2 assimilation, transpiration, stomata conductivity, and carbon concentration in cucumber plants.
Pesticides, including fungicides and their metabolites, can exert an immediate effect on soil microorganisms, triggering changes in their population and diversity . One such pesticide is azoxystrobin, which, 21 and 28 days after application to the soil in a dose of 10 mg kg −1 , significantly reduced the numbers of bacteria and actinobacteria compared to the control soil but had no significant effect on the fungi population . In the present study, azoxystrobin applied in the field dose stimulated the growth of organotrophic bacteria and actinobacteria, but it inhibited the growth of fungi. In turn, its contaminating dose reduced the population numbers of all analyzed groups of microorganisms. A small amount of the fungicide in the soil could have been used by organotrophic bacteria and actinobacteria as a source of nutrients; therefore, the recommended dose of azoxystrobin could increase their numbers . However, a fungicide dose being few or several times higher than the agronomic dose may directly affect the survival of microorganisms by disrupting the metabolic pathways in their cells . The sensitivity of microorganisms to increased amounts of azoxystrobin may be due to oxidative stress generated upon the release of electrons from the respiratory chain in the form of reactive oxygen . Fungicides affect not only the proliferation of microbial populations but also their diversity . They modify the composition of the microbial community because of their effects on non-target organisms . The present study demonstrated differences in the colony development index (CD) and the ecophysiological diversity index (EP) of microorganisms. In the soil treated with azoxystrobin, there was an increase in the CD values computed for organotrophic bacteria, actinobacteria, and fungi compared to the control soil. The r-strategies (fast-growing microorganisms) prevailed among organotrophic bacteria, whereas the k-strategists (slow-growing microorganisms) prevailed among actinobacteria and fungi. This was evidenced by the CDs, the mean values of which were CD = 50.27 for organotrophic bacteria and CD = 23.49 and CD = 24.38 for actinobacteria and fungi, respectively. Therefore, it can be concluded that the addition of chemical compounds to the soil may determine the proportions between r-strategists and k-strategists . Azoxystrobin exerted diverse effects on the EP of microorganisms, which was also caused by the time of its retention in the soil. However, the greatest changes caused by azoxystrobin presence in the soil, which were manifested by EP decrease, were observed in the case of fungi. The adaptation of microorganisms to adverse conditions is largely dependent on their activity, as well as on the degree of severity of the stress factor occurring in the soil environment . Fungicides are degraded in the soil environment by various microorganisms that produce specific enzymes capable of their degradation . For example, Alexandrino et al. demonstrated that bacteria of the genera Pseudomonas , Rhodobacter, Ochobacterum , Comamonas , Hydrogenophaga , Azospirillum , Methylbacillus , and Acinetobacter had high degradation potential against epoxiconazole and fludioxonil, as they degraded 10 mg dm −3 of these fungicides within 21 days. In turn, Feng et al. have reported that Arthrobacter , Bacillus , Cupriavidus , Pseudomonas , Klebsiella , Rhodanobacter , Stenothrofomonas , and Aphanoascus are microorganisms that break down strobilurin compounds. Clinton et al. isolated two species of bacteria from the soil contaminated with trifloxystrobin, namely Bacillus flexus and Bacillus amyloliquefaciens , whereas Howell et al. observed that Cuprividus spp. and Rhodobacter spp. exerted a degrading effect against azoxystrobin. Actinomyces spp. and Ochrabactrum spp. are also capable of degrading azoxystrobin . In our study, we identified microorganisms, i.e., bacteria PP952052.1 Prestia megaterium strain (P) and PP952051.1 Bacillus mycoides strain (P), and fungi PP952062.1 Keratinophyton terreum isolate (P), which may show high tolerance to azoxystrobin and the potential for its degradation. In turn, Feng et al. isolated a strain of bacteria Chrobacrum anthropi SH14 from soil contaminated with azoxystrobin, which was able to degrade 86.30% of the 50 µg cm −3 medium dose of this pesticide within 5 days.
The activity of soil enzymes is closely related to the quality and fertility of the soil. These biological parameters of the soil respond quickly to the effects of high pesticide doses . Wang et al. reported that azoxystrobin, used at doses of 0.1 to 10 mg kg −1 , inhibited dehydrogenases activity in all terms of analyses (i.e., on days 7, 14, 21, and 28) and urease activity up to day 14 while stimulating catalase activity and not significantly affecting protease activity. In the experiment described in this manuscript, the contaminating dose of azoxystrobin inhibited the activity of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease, while its agronomic dose enhanced activities of all analyzed soil enzymes. The inactivating effect of azoxystrobin on soil enzymes may have been due to the inhibition of microbial population multiplication, which indirectly affected the secretion of enzymes whose activity is strongly dependent on the number and biomass of microorganisms . Adverse effects of azoxystrobin (doses: 2.90, 14.65, and 35.00 mg kg −1 ) on the activity of soil enzymes such as dehydrogenases, urease, alkaline phosphatase, acid phosphatase, arylsulfatase, and β -glucosidase were noted in the study by Boteva et al. , with dehydrogenases and arylsulfatase being the most sensitive, and urease being the most resistant to soil treatment with this pesticide. In the present study, catalase was the most resistant to azoxystrobin, as evidenced by its enhanced activity in the soil contaminated with this compound. The increase in its activity may suggest that some microorganisms used azoxystrobin as a source of nutrients and energy necessary for their growth, which contributed to boosted catalase secretion by their cells . The increased catalase production by microorganisms probably caused fungicide degradation and strengthened the protective barrier of microorganisms against oxidizing compounds . The positive or negative effects of azoxystrobin on soil enzymes are mainly related to its dose and duration of its retention in the soil .
In addition to their antifungal activity, strobilurins improve plant quality by intensifying photosynthesis; increasing contents of nitrogen, chlorophyll, and protein; and delaying the aging of plants . Amaro et al. and Chiu-Yueh et al. have pointed to a very strong effect of fungicides from the group of strobilurin compounds on the physiology and growth of plants. In the present study, azoxystrobin added to soil both in the recommended field dose and the contaminating dose, inhibited seed germination and elongation of L. sativum L., S. alba L., and S. saccharatum L. shoots in all analytical terms (days 30, 60, and 90). Eman et al. determined the effect of azoxystrobin applied at the recommended dose and also at doses 0.5-fold and 2-fold higher than the recommended dose on the germination of Triticum aestivum L. and Raphanus sativus L. seeds. They found that azoxystrobin significantly reduced their seed germination percentage and the length of their roots and shoots. Particularly significant reduction in the length of roots and shoots of Triticum aestivum L. and Raphanus sativus L. was reported at a 2-fold-higher dose than the recommended one, which reduced the length of roots and shoots in wheat by 13.20% and 26.02%, respectively and in radish by 17.67% and 51.67%, respectively. In the present study, an azoxystrobin dose of 32.92 mg kg −1 caused, on average, 50.31% and 45.26% reduction in the length of shoots and roots of L. sativum L., respectively; 57.52% and 48.29% reductions in S. alba L.; and 45.32% and 44.65% reductions in these traits in S. saccharatum L., respectively. The impaired plant growth could have been due to the blocking of the cytochrome bc1 complex, which inhibited cell division and water uptake by plants . Amaro et al. , who assessed the effect of an azoxystrobin dose of 60 g ha −1 , found a reduction in the rate of CO 2 assimilation, transpiration, stomata conductivity, and carbon concentration in cucumber plants.
4.1. Soil Materials Soil material was collected from the humus-horizontal soil depth of 0 to 20 cm from Tomaszkowo, located in the north-eastern part of Poland (53.71610° N, 20.41670° E). This was soil belonging to the Eutric Cambisols subtype, which was formed on sandy loam (69.41% sand fraction, 27.71% clay fraction, and 2.88% silt fraction) . Selected physicochemical and chemical properties of the soil (soil granulometric composition; pH, hydrolytic acidity; sum of base exchangeable cations bases; organic carbon content; total nitrogen content; and total exchangeable cations K + , Na + , Ca 2+ , and Mg 2+ ) can be found in . The analyses were performed in 3 replicates according to the methodology described in the study by Borowik et al. . 4.2. Azoxystrobin The experiment conducted introduced azoxystrobin into the soil in the form of Amistar 250 SC (azoxystrobin amounts to 250 g dm −3 of the formulation) as a pure substance at rates of 0.110 mg kg −1 (field dose) and 32.92 mg kg −1 (polluting dose). The formulation was manufactured by Syngenta Crop Protection AG (Stein, Switzerland). The formulation was marketed in Poland in 2011, and the distribution authorization was granted to Syngenta (Warsaw, Poland). The expiry date of the authorization of the preparation of Amistar 250 SC by the company Syngenta is 31 December 2025 . The single dose recommended by the manufacturer amounts to 0.5 to 3.0 dm 3 ha −1 . This preparation is used in the protection of crops (winter wheat, spring wheat, rye, winter barley, spring barley, winter triticale, spring triticale, and winter oilseed rape) and vegetable crops (potato, onion, green bean, green pea, head cabbage, Chinese cabbage, cauliflower, carrot, lettuce, tomato, leek, celery, and pepper). The selected physicochemical properties of azoxystrobin are presented in . The structural formula of azoxystrobin was made using ISIS Draw 2.3 . 4.3. Establishment of the Experiment and Procedure for Conducting the Experiment The procedure for setting up an experiment under strictly controlled conditions (laboratory experiment) in 3 replicates for each combination and each test date (27 beakers in total). The procedure for setting up the experiment consisted of weighing 100 g each of air-dried soil put through a sieve (2 mm diameter) into glass beakers (150 cm 3 capacity). In the respective sites, azoxystrobin in the form of an aqueous emulsion was applied once in the following amounts (mg kg −1 d.m. soil): 0.00 mg (soil without added fungicide), 0.110 mg (field dose), and 32.92 mg (polluting dose). The literature generally describes studies on the impact of small doses of azoxystrobin on soil properties and plant development . Therefore, our research aimed to assess the impact of this active substance in contaminating amounts on the biological parameters of the soil. The soil material was thoroughly homogenized and brought to a moisture content of 50% of the capillary water capacity using distilled water. The soil in the beaker was covered with perforated foil and incubated in a thermostat maintaining a constant temperature (25 °C) for 30, 60, and 90 days. Soil moisture was monitored throughout the experiment, and soil losses were replenished. Soil microbiological and enzymatic analyses were performed on three test dates. For the Phytotoxkit tests, a separate batch of the experiment was set up (9 replicates for each combination and each test date, resulting in a total of 81 beakers). A total of 150 g of soil was weighed into each beaker. The conditions for setting up and running the experiment were identical to those for the soil used for microbiological and enzymatic analysis. 4.4. Conducting Microbiological Analysis of Soil At three study dates (30, 60, and 90 days), soil microbiological analysis was carried out using the serial dilution method. Into 90 cm 3 of sterile saline (0.85% NaCl) were weighed 10 g of soil of each sample analyzed; then, the whole was mixed on a shaker (120 rpm for 30 min), and a series of dilutions were made. An amount of 1 cm 3 of the specified dilution (organotrophic bacteria and actinobacteria—10 −5 , fungi—10 −3 ) and 17 cm 3 of selective medium were introduced into sterile Petri dishes in parallel. Bunt and Rovira medium for organotrophic bacteria, Küster and Williams medium for actinobacteria, and Martin medium for fungi were used for culture. The microbial material was incubated for 10 days in a thermostat at 28 °C; the grown colonies of microorganisms were counted each day. The composition of the microbial media is presented in . The exact procedure for performing the microbiological analysis is described according to Kucharski et al. and Wyszkowska et al. . These analyses were performed in 9 replicates for each combination. Each day, the grown colonies of microorganisms were counted. The number of microorganisms was expressed in colony-forming units per kg of soil (cfu kg −1 d.m. soil). 4.5. Isolation of Microorganisms from Soil and Their Identification On day 90 of the experiment, bacteria and fungi were isolated from the control soil and the soil containing azoxystrobin in the amounts of 0.110 mg kg −1 and 32.92 mg kg −1 by serial dilution. Isolation of bacteria and fungi was carried out by making serial dilutions by suspending 10 g of each of the soil samples analyzed in sterile saline (0.85% NaCl) (1:10 ratio). The prepared dilutions (bacteria—10 −5 and fungi—by 10 −3 ) were introduced at a rate of 1 cm 3 into a Petri dish (3 repetitions). PCA medium was used to grow bacteria, while fungi were grown in Sabouraud medium, the composition of which is presented in . The prepared microbial material was incubated at 37 °C (from 24 to 48 h). Serial passaging of characteristic colonies of microorganisms was performed to obtain pure cultures. Genomic DNA was isolated using a Bead-Beat Micro Gravity kit (A&A Biotechnology, Gdansk, Poland), which separated DNA by electrophoresis in a 1.0% agarose gel (5 mm 3 sample per gel). For the PCR reaction, a reaction mixture of the following composition was used: 5 mm 3 (~50 ng) of genomic DNA, 25 mm 3 of 2× PCR Master Mix Plus High GC (A&A Biotechnology, Gdansk, Poland), 0.2 mm 3 of each primer at 100 μM, and 19.6 mm 3 of sterile water. B-all For (GAG TTT GAT CCT GGC TCA G) and B-all Rev (ACG GCT ACC TTA CGA CTT) primers were used to isolate the 16S rRNA gene of bacteria, while ITS1 (TTC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) primers were used to isolate the ITS region of fungi. Conditions for the PCR reaction can be found in . After the PCR reaction on an agarose gel (2.0%), the reaction mixture was separated (2 mm 3 of sample per gel), and the amplified DNA fragments were purified using the Clean-Up AX kit (A&A Biotechnology, Poland). The resulting PCR products were resuspended in 10.0 mM Tris-HCl pH 8.0 and diluted to a concentration of 100 ng mm −3 . DNA sequencing was performed by Macrogen (Amsterdam, Netherlands) on a 3730 XL Analyzer DNA analyzer (Life Technologies Holding Pte Ltd., Singapore) . The DNA sequences obtained were compared with GenBank (National Center of Biotechnology Information) data. The DNA sequences of the 16S rRNA subunit of bacteria were compared using BLAST (Basic Local Alignment Search Tool) software [ https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 1 July 2024)], while the ITS regions of fungi were compared using Internal Transcribed Spacer software [ https://www.applied-maths.com/download/software (accessed on 1 July 2024)]. The access in the GenBank database for the nucleotide sequences of bacteria are under numbers ranging from PP952047 to PP952052 [ https://www.ncbi.nlm.nih.gov/nuccore/PP952047.1:PP952052 (accessed on 1 July 2024), https://www.ncbi.nlm.nih.gov/nuccore (accessed on 1 July 2024)], while those of fungi are under numbers ranging from PP952058 to PP952062 [ https://www.ncbi.nlm.nih.gov/nuccore/PP952058.1:PP52062.1 (accessed on 1 July 2024)]. Based on the obtained nucleotide sequences of the identified microorganisms, a phylogenetic tree was created using the neighbor-joining method with the MEGA 11 software [ https://www.megasoftware.net/show_eua (accessed on 1 July 2024)] . The conditions for creating the phylogenetic tree in MEGA 11 software were as follows: statistical method—neighbor-joining (NJ); scope—all selected taxa; test phylogeny—bootstrap method (no. of bootstrap reconstruction—500 for bacteria and fungi); substitution type—nucleotide; model/methods—Maximum Composite Likelihood; substitution of include—d: transitions and transversion; rates among sites—uniform rates; pattern among lineages—same (homogenous); gaps/missing data treatment—partial deletion; select codon positions—1st + 2nd + 3rd + noncoding; number of threads—1; searching NJ tree—100%, by sequence difference—0.066 for bacteria and 0.036 for fungi. 4.6. Conducting Enzymatic Analysis of Soil Enzymatic analyses of the soil were performed at 30, 60, and 90 days (9 replicates) determining the activity of dehydrogenases, catalase, alkaline phosphatase, acid phosphatase, and urease. Then, 2,3,5-triphenyl tetrazolium chloride was used to determine dehydrogenases activity, hydrogen peroxide—catalase, 4-nitrophenyl phosphate disodium—alkaline phosphatase, acid phosphatase, and urea—urease. Dehydrogenase activity was determined by the Öhlinger method, catalase by the Johnson and Temple method, and alkaline phosphatase and urease by the Alef and Nannpieri method. These analyses were performed according to the procedure given in the studies by Wyszkowska et al. and Wyszkowska et al. . 4.7. The Effect of Azoxystrobin on Seed Germination and Plant Root Elongation The effects of azoxystrobin on the growth and development of plants ( Lepidium sativum L., Sinapsis alba L., and Sorgum saccharatum L.) were assessed at specific test dates (30, 60, and 90 days) using the Phytotoxkit test. The soil (110 g) from the control (soil without added fungicide) and with azoxystrobin at 0.110 mg kg −1 and 32.92 mg kg −1 was introduced onto plastic plates. Then, 10 seeds of each plant were placed on moist filter paper in 3 replicates. The material thus prepared was incubated for 72 h (temperature 25 °C). The shoot and root lengths of the test plants were then measured. 4.8. Calculation of Results The following soil biological indices were calculated at each test date (30, 60, and 90 days): ▪ Colony development index (CD) of microorganisms : CD values range from 0 to 100. CD values close to 100 indicate rapid growth of the microorganism population in a short period. (1) C D = N 1 1 + N 2 2 + N 3 3 + ⋯ + N 10 10 × 100 where N 1 , N 2 , N 3 , …, N 10 —is the ratio of the total number of microbial colonies grown (1, 2, 3, …, 10th day of incubation) to the total number of microbial colonies grown over the entire incubation period (10 days); ▪ The ecophysiological diversity index (EP) of microorganisms takes values from 0 to 1, measuring the stability and homogeneity of microorganisms over time. EP values close to 1 indicate steady growth of microorganisms in the environment. (2) E P = − Ʃ ( p i × l o g 10 p i ) where p i is the ratio of the sum of the number of microbial colonies grown per incubation day to the total number of microbial colonies grown over the entire incubation period (10 days); ▪ Changes (Ch a ) of microbial abundance, enzyme activity, seed germination, and root elongation in soil caused by azoxystrobin: A positive Ch a value indicates stimulation of the analyzed parameters under the influence of azoxystrobin, while a negative value indicates inhibition. (3) C h a = ( A − C ) C × 100 % where A represents values of analyzed parameters in the soil with azoxystrobin, and C represents values of analyzed parameters in the control soil; ▪ The resilience index (RL) of azoxystrobin-treated soil is determined by microbial abundance and enzyme activity . RL values range from −1 to 1. A RL value close to −1 indicates that the soil is not returning to equilibrium. A RL value close to 1 indicates that the soil is returning to equilibrium. A RL value close to 0 indicates that the soil is out of or slightly out of equilibrium. (4) RLat t x = 2 | D 0 | ( | C 0 | + | D x | ) where D 0 is the difference in soil microbial numbers and enzyme activity between a control soil sample (C 0 ) and an azoxystrobin-treated soil sample (t 0 ), and D x is the difference in soil microbial numbers and enzyme activity between a control and an azoxystrobin-treated sample after 60 and 90 days of soil incubation. 4.9. Statistical Analyses of Results The results obtained were statistically processed with a two-factor (factor 1: azoxystrobin dose, factor 2: soil incubation time) ANOVA analysis of variance ( p ≤ 0.01) using Statistica 13.3 software . The percentage of observed variability in the soil parameters studied was determined using the η 2 coefficient. Homogeneous groups were calculated using Tukey’s test at p ≤ 0.01 to determine the most significant differences between mean values. Pearson’s simple correlation coefficients between microbiological and biochemical soil parameters were presented as a heat map.
Soil material was collected from the humus-horizontal soil depth of 0 to 20 cm from Tomaszkowo, located in the north-eastern part of Poland (53.71610° N, 20.41670° E). This was soil belonging to the Eutric Cambisols subtype, which was formed on sandy loam (69.41% sand fraction, 27.71% clay fraction, and 2.88% silt fraction) . Selected physicochemical and chemical properties of the soil (soil granulometric composition; pH, hydrolytic acidity; sum of base exchangeable cations bases; organic carbon content; total nitrogen content; and total exchangeable cations K + , Na + , Ca 2+ , and Mg 2+ ) can be found in . The analyses were performed in 3 replicates according to the methodology described in the study by Borowik et al. .
The experiment conducted introduced azoxystrobin into the soil in the form of Amistar 250 SC (azoxystrobin amounts to 250 g dm −3 of the formulation) as a pure substance at rates of 0.110 mg kg −1 (field dose) and 32.92 mg kg −1 (polluting dose). The formulation was manufactured by Syngenta Crop Protection AG (Stein, Switzerland). The formulation was marketed in Poland in 2011, and the distribution authorization was granted to Syngenta (Warsaw, Poland). The expiry date of the authorization of the preparation of Amistar 250 SC by the company Syngenta is 31 December 2025 . The single dose recommended by the manufacturer amounts to 0.5 to 3.0 dm 3 ha −1 . This preparation is used in the protection of crops (winter wheat, spring wheat, rye, winter barley, spring barley, winter triticale, spring triticale, and winter oilseed rape) and vegetable crops (potato, onion, green bean, green pea, head cabbage, Chinese cabbage, cauliflower, carrot, lettuce, tomato, leek, celery, and pepper). The selected physicochemical properties of azoxystrobin are presented in . The structural formula of azoxystrobin was made using ISIS Draw 2.3 .
The procedure for setting up an experiment under strictly controlled conditions (laboratory experiment) in 3 replicates for each combination and each test date (27 beakers in total). The procedure for setting up the experiment consisted of weighing 100 g each of air-dried soil put through a sieve (2 mm diameter) into glass beakers (150 cm 3 capacity). In the respective sites, azoxystrobin in the form of an aqueous emulsion was applied once in the following amounts (mg kg −1 d.m. soil): 0.00 mg (soil without added fungicide), 0.110 mg (field dose), and 32.92 mg (polluting dose). The literature generally describes studies on the impact of small doses of azoxystrobin on soil properties and plant development . Therefore, our research aimed to assess the impact of this active substance in contaminating amounts on the biological parameters of the soil. The soil material was thoroughly homogenized and brought to a moisture content of 50% of the capillary water capacity using distilled water. The soil in the beaker was covered with perforated foil and incubated in a thermostat maintaining a constant temperature (25 °C) for 30, 60, and 90 days. Soil moisture was monitored throughout the experiment, and soil losses were replenished. Soil microbiological and enzymatic analyses were performed on three test dates. For the Phytotoxkit tests, a separate batch of the experiment was set up (9 replicates for each combination and each test date, resulting in a total of 81 beakers). A total of 150 g of soil was weighed into each beaker. The conditions for setting up and running the experiment were identical to those for the soil used for microbiological and enzymatic analysis.
At three study dates (30, 60, and 90 days), soil microbiological analysis was carried out using the serial dilution method. Into 90 cm 3 of sterile saline (0.85% NaCl) were weighed 10 g of soil of each sample analyzed; then, the whole was mixed on a shaker (120 rpm for 30 min), and a series of dilutions were made. An amount of 1 cm 3 of the specified dilution (organotrophic bacteria and actinobacteria—10 −5 , fungi—10 −3 ) and 17 cm 3 of selective medium were introduced into sterile Petri dishes in parallel. Bunt and Rovira medium for organotrophic bacteria, Küster and Williams medium for actinobacteria, and Martin medium for fungi were used for culture. The microbial material was incubated for 10 days in a thermostat at 28 °C; the grown colonies of microorganisms were counted each day. The composition of the microbial media is presented in . The exact procedure for performing the microbiological analysis is described according to Kucharski et al. and Wyszkowska et al. . These analyses were performed in 9 replicates for each combination. Each day, the grown colonies of microorganisms were counted. The number of microorganisms was expressed in colony-forming units per kg of soil (cfu kg −1 d.m. soil).
On day 90 of the experiment, bacteria and fungi were isolated from the control soil and the soil containing azoxystrobin in the amounts of 0.110 mg kg −1 and 32.92 mg kg −1 by serial dilution. Isolation of bacteria and fungi was carried out by making serial dilutions by suspending 10 g of each of the soil samples analyzed in sterile saline (0.85% NaCl) (1:10 ratio). The prepared dilutions (bacteria—10 −5 and fungi—by 10 −3 ) were introduced at a rate of 1 cm 3 into a Petri dish (3 repetitions). PCA medium was used to grow bacteria, while fungi were grown in Sabouraud medium, the composition of which is presented in . The prepared microbial material was incubated at 37 °C (from 24 to 48 h). Serial passaging of characteristic colonies of microorganisms was performed to obtain pure cultures. Genomic DNA was isolated using a Bead-Beat Micro Gravity kit (A&A Biotechnology, Gdansk, Poland), which separated DNA by electrophoresis in a 1.0% agarose gel (5 mm 3 sample per gel). For the PCR reaction, a reaction mixture of the following composition was used: 5 mm 3 (~50 ng) of genomic DNA, 25 mm 3 of 2× PCR Master Mix Plus High GC (A&A Biotechnology, Gdansk, Poland), 0.2 mm 3 of each primer at 100 μM, and 19.6 mm 3 of sterile water. B-all For (GAG TTT GAT CCT GGC TCA G) and B-all Rev (ACG GCT ACC TTA CGA CTT) primers were used to isolate the 16S rRNA gene of bacteria, while ITS1 (TTC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) primers were used to isolate the ITS region of fungi. Conditions for the PCR reaction can be found in . After the PCR reaction on an agarose gel (2.0%), the reaction mixture was separated (2 mm 3 of sample per gel), and the amplified DNA fragments were purified using the Clean-Up AX kit (A&A Biotechnology, Poland). The resulting PCR products were resuspended in 10.0 mM Tris-HCl pH 8.0 and diluted to a concentration of 100 ng mm −3 . DNA sequencing was performed by Macrogen (Amsterdam, Netherlands) on a 3730 XL Analyzer DNA analyzer (Life Technologies Holding Pte Ltd., Singapore) . The DNA sequences obtained were compared with GenBank (National Center of Biotechnology Information) data. The DNA sequences of the 16S rRNA subunit of bacteria were compared using BLAST (Basic Local Alignment Search Tool) software [ https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 1 July 2024)], while the ITS regions of fungi were compared using Internal Transcribed Spacer software [ https://www.applied-maths.com/download/software (accessed on 1 July 2024)]. The access in the GenBank database for the nucleotide sequences of bacteria are under numbers ranging from PP952047 to PP952052 [ https://www.ncbi.nlm.nih.gov/nuccore/PP952047.1:PP952052 (accessed on 1 July 2024), https://www.ncbi.nlm.nih.gov/nuccore (accessed on 1 July 2024)], while those of fungi are under numbers ranging from PP952058 to PP952062 [ https://www.ncbi.nlm.nih.gov/nuccore/PP952058.1:PP52062.1 (accessed on 1 July 2024)]. Based on the obtained nucleotide sequences of the identified microorganisms, a phylogenetic tree was created using the neighbor-joining method with the MEGA 11 software [ https://www.megasoftware.net/show_eua (accessed on 1 July 2024)] . The conditions for creating the phylogenetic tree in MEGA 11 software were as follows: statistical method—neighbor-joining (NJ); scope—all selected taxa; test phylogeny—bootstrap method (no. of bootstrap reconstruction—500 for bacteria and fungi); substitution type—nucleotide; model/methods—Maximum Composite Likelihood; substitution of include—d: transitions and transversion; rates among sites—uniform rates; pattern among lineages—same (homogenous); gaps/missing data treatment—partial deletion; select codon positions—1st + 2nd + 3rd + noncoding; number of threads—1; searching NJ tree—100%, by sequence difference—0.066 for bacteria and 0.036 for fungi.
Enzymatic analyses of the soil were performed at 30, 60, and 90 days (9 replicates) determining the activity of dehydrogenases, catalase, alkaline phosphatase, acid phosphatase, and urease. Then, 2,3,5-triphenyl tetrazolium chloride was used to determine dehydrogenases activity, hydrogen peroxide—catalase, 4-nitrophenyl phosphate disodium—alkaline phosphatase, acid phosphatase, and urea—urease. Dehydrogenase activity was determined by the Öhlinger method, catalase by the Johnson and Temple method, and alkaline phosphatase and urease by the Alef and Nannpieri method. These analyses were performed according to the procedure given in the studies by Wyszkowska et al. and Wyszkowska et al. .
The effects of azoxystrobin on the growth and development of plants ( Lepidium sativum L., Sinapsis alba L., and Sorgum saccharatum L.) were assessed at specific test dates (30, 60, and 90 days) using the Phytotoxkit test. The soil (110 g) from the control (soil without added fungicide) and with azoxystrobin at 0.110 mg kg −1 and 32.92 mg kg −1 was introduced onto plastic plates. Then, 10 seeds of each plant were placed on moist filter paper in 3 replicates. The material thus prepared was incubated for 72 h (temperature 25 °C). The shoot and root lengths of the test plants were then measured.
The following soil biological indices were calculated at each test date (30, 60, and 90 days): ▪ Colony development index (CD) of microorganisms : CD values range from 0 to 100. CD values close to 100 indicate rapid growth of the microorganism population in a short period. (1) C D = N 1 1 + N 2 2 + N 3 3 + ⋯ + N 10 10 × 100 where N 1 , N 2 , N 3 , …, N 10 —is the ratio of the total number of microbial colonies grown (1, 2, 3, …, 10th day of incubation) to the total number of microbial colonies grown over the entire incubation period (10 days); ▪ The ecophysiological diversity index (EP) of microorganisms takes values from 0 to 1, measuring the stability and homogeneity of microorganisms over time. EP values close to 1 indicate steady growth of microorganisms in the environment. (2) E P = − Ʃ ( p i × l o g 10 p i ) where p i is the ratio of the sum of the number of microbial colonies grown per incubation day to the total number of microbial colonies grown over the entire incubation period (10 days); ▪ Changes (Ch a ) of microbial abundance, enzyme activity, seed germination, and root elongation in soil caused by azoxystrobin: A positive Ch a value indicates stimulation of the analyzed parameters under the influence of azoxystrobin, while a negative value indicates inhibition. (3) C h a = ( A − C ) C × 100 % where A represents values of analyzed parameters in the soil with azoxystrobin, and C represents values of analyzed parameters in the control soil; ▪ The resilience index (RL) of azoxystrobin-treated soil is determined by microbial abundance and enzyme activity . RL values range from −1 to 1. A RL value close to −1 indicates that the soil is not returning to equilibrium. A RL value close to 1 indicates that the soil is returning to equilibrium. A RL value close to 0 indicates that the soil is out of or slightly out of equilibrium. (4) RLat t x = 2 | D 0 | ( | C 0 | + | D x | ) where D 0 is the difference in soil microbial numbers and enzyme activity between a control soil sample (C 0 ) and an azoxystrobin-treated soil sample (t 0 ), and D x is the difference in soil microbial numbers and enzyme activity between a control and an azoxystrobin-treated sample after 60 and 90 days of soil incubation.
The results obtained were statistically processed with a two-factor (factor 1: azoxystrobin dose, factor 2: soil incubation time) ANOVA analysis of variance ( p ≤ 0.01) using Statistica 13.3 software . The percentage of observed variability in the soil parameters studied was determined using the η 2 coefficient. Homogeneous groups were calculated using Tukey’s test at p ≤ 0.01 to determine the most significant differences between mean values. Pearson’s simple correlation coefficients between microbiological and biochemical soil parameters were presented as a heat map.
Azoxystrobin was observed to induce changes in soil microbiome and enzymatic activity and also in plant growth and development over time. Its field dose (0.110 mg kg −1 ) increased the numbers of organotrophic bacteria and actinobacteria, the CD values, and the activity of soil enzymes. In turn, it reduced the number of fungi, decreased the EP values, and inhibited seed germination and root elongation of the tested plants. In turn, its contaminating dose (32.92 mg kg −1 ) reduced the number of fungi; suppressed activities of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease; and decreased the EP values, while increasing the CD values and enhancing catalase activity. In addition, it significantly inhibited seed germination and root elongation of Lepidium sativum L., Sinapsis alba L., and Sorgum saccharatum . It was observed that control soil (soil not contaminated with the fungicide) was most heavily populated by the genera bacteria PP952050.1 Bacillaceae bacterium strain (C), PP952049.1 Bacillus cereus strain (C), fungi PP952060.1 Talaromyces pinophilus isolate (C), and PP952061.1 Trichoderma viride isolate (C), while the contaminated soil was most heavily populated by bacteria PP952052.1 Prestia megaterium strain (P), PP952051.1 Bacillus mycoides strain (P), and fungi PP952062.1 Keratinophyton terreum isolate (P). The effects of azoxystrobin on the microbiota, enzymes, and plants varied over time, depending on dose, the species of microorganisms and plants, and enzyme type. The study results indicate that azoxystrobin can trigger significant changes in soil biological parameters, particularly when applied in the contaminating dose. It caused permanent disorders in the growth of fungi and the activity of dehydrogenases, acid phosphatase, and urease, as evidenced by negative values of the RL. The identification of bacteria and fungi in the soil containing azoxystrobin can be harnessed to restore soils contaminated with this fungicide by their bioaugmentation with resistant and degrading strains.
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Reassessment of SST4 Somatostatin Receptor Expression Using SST4-eGFP Knockin Mice and the Novel Rabbit Monoclonal Anti-Human SST4 Antibody 7H49L61 | 9f07d5c0-dbbf-464a-9c78-69fcc26a771d | 8657703 | Anatomy[mh] | Somatostatin is a cyclic neuropeptide that regulates the release of various neurotransmitters in the central and peripheral nervous system and the secretion of a large number of hormones from the anterior pituitary, pancreas, and endocrine cells within the gastrointestinal tract. Additionally, it causes a reduction of gastrointestinal tract motility and gallbladder contractility. Finally, somatostatin exerts antiproliferative effects and plays a regulatory role in the immune system . The biological functions of somatostatin are mediated via a family of five G-protein-coupled receptors, termed SST1 to SST5, that are expressed in varying patterns and density throughout the body, including brain, pituitary gland, neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, pancreatic islets, adrenal glands, and the immune system. Somatostatin receptors have also been found in pituitary adenomas and in neuroendocrine tumors of different origins where they mediate inhibitory effects on both hormone secretion and tumor growth. Apart from the tissue distribution, SSTs also differ in their affinity for synthetic somatostatin analogs, intracellular signaling pathways, and biological functions . Little is known about SST4 compared to other SSTs, for which highly specific monoclonal antibodies have been available for years and which, as a result, could be examined more closely in terms of their occurrence in tissues and their mode of action. The existing knowledge is based on mRNA expression analyses, receptor autoradiography studies, immunostaining with polyclonal antibodies, SST4 knockout mouse models, and SST4-transfected cell lines . Regarding signaling pathways, it is known that SST4, by coupling to G i/o proteins, inhibits adenylate cyclase, thus leading to reduced cAMP production . In rat cortical neurons and retinal and dorsal root ganglion cells, SST4 reduces intracellular Ca 2+ concentrations by inhibiting voltage-dependent Ca 2+ channels and activates inwardly rectifying potassium (GIRK) and M channels. This results in membrane hyperpolarization and a subsequent reduction of Ca 2+ and Na + influx through voltage-dependent Ca 2+ and TRPV1 channels [ , , , , , , , ]. SST4 seems to be the only family member that not only inhibits cell proliferation via complex regulatory mechanisms, such as tyrosine phosphatase SHP-2 activation and upregulation of the cyclin-dependent kinase inhibitor p21 , but also stimulates cell proliferation via an activation signal to protein kinase C and by MAP kinase-mediated serine phosphorylation leading to activation of STAT3 . With respect to SST4 trafficking, observations of an instant dissociation of receptor and ligand after ligand binding, subsequent internalization, and rapid recycling in cells transfected with the human SST4 contrast with those that could not find any internalization of the receptor in rat tissue or in cells transfected with rat SST4 . Therefore, species-specific differences in behavior and possibly also in signaling mechanisms of SST4 are to be assumed . Concerning function, SST4 knockout mice have a higher number of spontaneous epileptic seizures than controls, suggesting an important role for SST4 in the hippocampus , consistent with the observation of reduced somatostatin binding in the hippocampal CA1 area in these mice . SST4 may also be important in memory, cognition, and learning performance and has an impact on behavior in stressful situations [ , , ]. Because it protects against inflammation and hyperalgesia, SST4 is being evaluated as a therapeutic target for a novel class of anti-inflammatory and/or analgesic drugs [ , , , , , ]. There is also evidence that SST4 may be a pharmacological target for Alzheimer’s disease . Using mRNA analysis and polyclonal antibodies, SST4 has been previously localized in rats in diverse brain areas, such as in layers I–VI of the neocortex, the hippocampus formation, the hilar region of the dentate gyrus, the amygdala, the hypothalamus, the striatum, the nucleus accumbens, the globus pallidus, the olfactory bulb, and other structures of the olfactory system [ , , , ]. It has also been shown to be expressed in retinal ganglion cells and in dorsal root ganglia . Compared to the CNS, the presence of SST4 in the peripheral organs in rats is less well characterized. Here, SST4 mRNA and protein expression have been found in the lung, heart, and placenta . Using polyclonal antibodies, SST4 receptors have been discovered in the human cortex, hippocampus, nucleus ruber, globus pallidus, cerebellum, and medulla , as well as in the exocrine pancreas , the parathyroid glands, the bronchial glands, the fundic glands of the stomach, the Brunner’s glands in the duodenum, and the distal tubules of the kidney . Additionally, very recently, in a SST4 humanized mouse line, SST4 expression has been disclosed in the cerebral cortex, the olfactory bulb, the hippocampus, the amygdala, and in the trigeminal ganglia . In human tumors, SST4 expression was often recorded along with other SST family members [ , , , , , , , , , , , , , , , , , , , , ]. However, the specificity of the polyclonal antibodies used for SST4 staining was also questioned by some of the authors (e.g., [ , , ]). Most of these studies revealed that SST4 was either not expressed in the tumors investigated or at least present to a significantly lower degree compared to the other SSTs. In all cases, only cytoplasmic staining of the cells was observed. To address the lack of suitable specific monoclonal anti-SST4 antibodies for the localization of SST4 in mouse or human tissues, we generated an SST4-eGFP knockin mouse line and a novel rabbit monoclonal anti-human SST4 antibody as novel tools to facilitate unequivocal SST4 receptor localization. Using anti-GFP antibodies and mRNA in situ hybridization, we show that the novel mouse line enables a detailed localization of SST4 in different mouse tissues. We also demonstrate that the novel anti-human SST4 antibody 7H49L61 is well suited both for immunoblot analysis in basic research and for immunohistochemical staining of routine clinical pathology samples. This antibody was then used for the evaluation of SST4 expression in a large set of formalin-fixed paraffin-embedded human normal and neoplastic tissue samples in order to obtain a broad expression profile both for the human body and for human tumors.
2.1. SST4-eGFP Expression in the Knockin Mouse Line In C57BL/6-wild-type mice, in situ hybridization experiments revealed high levels of Sstr4 mRNA expression in the infragranular cortical layers but very low levels in the supragranular cortical layers ( A,C). Sstr4 -positive neurons were also observed in trigeminal ganglia ( A). Similarly, eGFP fluorescence immunostaining in SST4-eGFP knockin mice revealed strong SST4 expression in cortical pyramidal cells and in the pyramidal cells of the hippocampal areas CA1–CA3 ( E,G). In the granule cells of the dentate gyrus, in contrast, only low SST4 expression was noticed. In trigeminal ganglia, mainly small- to medium-diameter neurons were SST4 positive ( C). No immunostaining was observed in wild-type mice, which lack SST4-eGFP expression ( F,H and D). In the subsequent eGFP stainings of paraffin sections of different tissues from SST4-eGFP knockin mice, a strong immunosignal was noted in the pyramidal cells of the cortex and of the hippocampal areas CA1–CA3, in neurons of the amygdala, in fibers of the posterior pituitary, in excretory ducts of the salivary glands and the pancreas, and in the adrenal cortex. A slight staining was also observed in some cell populations of the anterior pituitary, in the acinar cells of the exocrine pancreas, and in the distal tubules of the kidney. In contrast, no noticeable SST4 expression could be detected in the lung, liver, pancreatic islets, adrenal medulla, thymus, spleen, or lymph nodes ( ). 2.2. Specificity of the Monoclonal Rabbit Anti-Human SST4 Antibody 7H49L61 The SST4 expression pattern for eGFP immunostaining in the cortex of SST4-GFP knockin mice was virtually identical to the staining pattern for the monoclonal rabbit anti-human SST4 antibody 7H49L61 in human cortex samples with clear staining of the cell bodies and the axons of the cortical pyramidal cells ( A,B). Pre-incubation of 7H49L61 with its immunizing peptide resulted in a complete extinction of the immunostaining ( C, +Peptide). In immunoblots of membrane preparations from SST4 -transfected HEK-293 cells, the antibody recognized a broad band migrating at 50–60 kDa, the expected molecular weight of the glycosylated receptor . In mock-transfected HEK-293 cells, in contrast, no immunosignal could be observed ( D). 2.3. SST4 Expression in Normal Human Tissues The rabbit monoclonal anti-SST4 antibody 7H49L61 was then applied in immunohistochemical staining of various human normal tissues. In most cases, immunostaining was localized to the plasma membrane of the cells, but cytoplasmic staining was also observed. As depicted in , very strong immunostaining of SST4 was observed in the pyramidal cells of the cortex, in neurons as well as in some satellite cells of the trigeminal ganglia, in nerve fibers within the posterior pituitary gland, in the acinar cells and in the excretory ducts of the exocrine pancreas, in all three layers of the adrenal cortex, and in syncytiotrophoblasts of the placenta. SST4 was also present in the intestinal ganglia, fundic glands of the stomach, Brunner’s glands, epithelium and Paneth cells of the duodenum, bile duct epithelial cells of the liver, and distal and (to a significantly smaller extent) proximal tubules as well as in the glomerular mesangial cells of the kidney. No noticeable staining was observed in the anterior pituitary gland, lung, thyroid gland, liver hepatocytes, pancreatic islet cells, adrenal medulla, spleen, lymph nodes, or bone marrow. 2.4. SST4 Expression in Human Tumors The patterns of SST4 expression in human tumor samples are summarized in . Representative examples of immunostaining are shown in . Again, both membranous and cytoplasmic staining of cells was observed. Notably, SST4 expression in the tumor samples displayed substantial inter- and intra-individual variability. Pronounced SST4 expression associated with a high number of SST4-positive samples (immunoreactivity score (IRS) ≥ 3) and higher IRS values was seen in glioblastomas, parathyroid adenomas, gastric cancer, pancreatic adenocarcinomas, pheochromocytomas, and lymphomas. However, for most other tumor entities, single samples with high levels of SST4 expression were noted. Interestingly, no noticeable immunostaining was found in small cell lung cancer tissues.
In C57BL/6-wild-type mice, in situ hybridization experiments revealed high levels of Sstr4 mRNA expression in the infragranular cortical layers but very low levels in the supragranular cortical layers ( A,C). Sstr4 -positive neurons were also observed in trigeminal ganglia ( A). Similarly, eGFP fluorescence immunostaining in SST4-eGFP knockin mice revealed strong SST4 expression in cortical pyramidal cells and in the pyramidal cells of the hippocampal areas CA1–CA3 ( E,G). In the granule cells of the dentate gyrus, in contrast, only low SST4 expression was noticed. In trigeminal ganglia, mainly small- to medium-diameter neurons were SST4 positive ( C). No immunostaining was observed in wild-type mice, which lack SST4-eGFP expression ( F,H and D). In the subsequent eGFP stainings of paraffin sections of different tissues from SST4-eGFP knockin mice, a strong immunosignal was noted in the pyramidal cells of the cortex and of the hippocampal areas CA1–CA3, in neurons of the amygdala, in fibers of the posterior pituitary, in excretory ducts of the salivary glands and the pancreas, and in the adrenal cortex. A slight staining was also observed in some cell populations of the anterior pituitary, in the acinar cells of the exocrine pancreas, and in the distal tubules of the kidney. In contrast, no noticeable SST4 expression could be detected in the lung, liver, pancreatic islets, adrenal medulla, thymus, spleen, or lymph nodes ( ).
The SST4 expression pattern for eGFP immunostaining in the cortex of SST4-GFP knockin mice was virtually identical to the staining pattern for the monoclonal rabbit anti-human SST4 antibody 7H49L61 in human cortex samples with clear staining of the cell bodies and the axons of the cortical pyramidal cells ( A,B). Pre-incubation of 7H49L61 with its immunizing peptide resulted in a complete extinction of the immunostaining ( C, +Peptide). In immunoblots of membrane preparations from SST4 -transfected HEK-293 cells, the antibody recognized a broad band migrating at 50–60 kDa, the expected molecular weight of the glycosylated receptor . In mock-transfected HEK-293 cells, in contrast, no immunosignal could be observed ( D).
The rabbit monoclonal anti-SST4 antibody 7H49L61 was then applied in immunohistochemical staining of various human normal tissues. In most cases, immunostaining was localized to the plasma membrane of the cells, but cytoplasmic staining was also observed. As depicted in , very strong immunostaining of SST4 was observed in the pyramidal cells of the cortex, in neurons as well as in some satellite cells of the trigeminal ganglia, in nerve fibers within the posterior pituitary gland, in the acinar cells and in the excretory ducts of the exocrine pancreas, in all three layers of the adrenal cortex, and in syncytiotrophoblasts of the placenta. SST4 was also present in the intestinal ganglia, fundic glands of the stomach, Brunner’s glands, epithelium and Paneth cells of the duodenum, bile duct epithelial cells of the liver, and distal and (to a significantly smaller extent) proximal tubules as well as in the glomerular mesangial cells of the kidney. No noticeable staining was observed in the anterior pituitary gland, lung, thyroid gland, liver hepatocytes, pancreatic islet cells, adrenal medulla, spleen, lymph nodes, or bone marrow.
The patterns of SST4 expression in human tumor samples are summarized in . Representative examples of immunostaining are shown in . Again, both membranous and cytoplasmic staining of cells was observed. Notably, SST4 expression in the tumor samples displayed substantial inter- and intra-individual variability. Pronounced SST4 expression associated with a high number of SST4-positive samples (immunoreactivity score (IRS) ≥ 3) and higher IRS values was seen in glioblastomas, parathyroid adenomas, gastric cancer, pancreatic adenocarcinomas, pheochromocytomas, and lymphomas. However, for most other tumor entities, single samples with high levels of SST4 expression were noted. Interestingly, no noticeable immunostaining was found in small cell lung cancer tissues.
3.1. SST4-eGFP Expression in the Knockin Mouse Line To enable visualization of SST4 expression at the protein level in mice, we generated a SST4-eGFP knockin mouse line. In the present study, we provide strong evidence that the immunosignals obtained with this mouse line indeed reflect SST4 receptor expression. First, the mRNA expression data obtained by in situ hybridization experiments in wild-type mice correlated well with SST4-eGFP protein expression in the knockin mice. Second, with the novel monoclonal SST4 antibody 7H49L61 virtually the same staining pattern was observed in humans, although there may be some species differences, especially with respect to the pituitary and the pancreas, which need to be further evaluated. Third, both staining patterns correspond well to the published mRNA and protein expression data for mice, rats, and humans [ , , , , , , ]. Given that monoclonal anti-mouse SST4 antibodies are currently not available, the SST4-eGFP knockin mouse line can serve as a useful tool for the detection of SST4 in mice at the cellular and subcellular level by direct fluorescence microscopy or immunohistochemical techniques using anti-GFP antibodies. 3.2. Specificity of the Monoclonal Rabbit Anti-human SST4 Antibody 7H49L61 Unlike polyclonal antibodies, monoclonal antibodies are directed against a single epitope, generally leading to greater specificity, and they are available indefinitely in unlimited amounts with consistent quality. Therefore, we developed a monoclonal anti-SST4 antibody suitable for immunoblots in basic research and for immunohistochemical staining of formalin-fixed paraffin-embedded tissues during routine histopathological examinations. In the present study, we demonstrated that the carboxyl-terminal tail of human SST4 can serve as an epitope for generating a rabbit monoclonal antibody that can be effectively used for both applications. We provided strong evidence that the anti-SST4 antibody 7H49L61 specifically detects its target receptor and does not cross-react with other proteins. First, in immunoblots, 7H49L61 specifically detected its cognate receptor in crude extracts from SST4-transfected HEK-293 cells but not in extracts from mock-transfected cells. Second, 7H49L61 yielded highly efficient staining of distinct cell populations known to express SST4 [ , , ] in formalin-fixed paraffin-embedded human tissue samples. Third, the SST4-immunosignals were completely abolished after pre-adsorption of the antibody with its immunizing peptide. Finally, virtually identical staining patterns were observed in the human cortical and trigeminal ganglion samples stained with 7H49L61 and in the corresponding tissues of the SST4-eGFP knockin mice. Most notably, however, 7H49L61 is the first SST4 antibody that facilitates detection of bona fide plasma membrane receptors in human formalin-fixed paraffin-embedded tissues. 3.3. SST4 Expression in Normal Human Tissues In the present investigation, a distinct immunostaining for SST4 was found in the cell bodies and the axons of cortical pyramidal cells, consistent with published data obtained using polyclonal antibodies in humans and in rats [ , , ] as well as in SST4 humanized mice . SST4 has also been identified in neurons and satellite cells of trigeminal and dorsal root ganglia of mice and rats, respectively, by mRNA analysis or with polyclonal antibodies, and it was proposed that the analgesic effects of SST4 agonists are partially mediated by these receptors [ , , ]. We also found SST4 expression in the intramural ganglia of the gastrointestinal tract, indicating broad expression of SST4 in the peripheral nervous system. Similar to published data both at the mRNA and at the protein level , no SST4 expression was observed in the anterior pituitary; hence, it is believed that SST4 agonists may represent good analgesics without exerting any major endocrine side effects . In contrast, a strong staining of nerve fibers within the posterior pituitary was detected, which has not been reported previously and suggests a possible involvement of SST4 in the regulation of oxytocin and adiuretin secretion from these fibers. While pancreatic islet cells were SST4 negative, in the present study, a distinct staining of acinar cells of the exocrine pancreas was observed. Similar observations have been made in the literature using polyclonal antibodies and an involvement of SST4 in the well-known inhibitory role of somatostatin on pancreatic juice excretion but not in insulin or glucagon release was suggested. As reported previously , the presence of SST4 was also noted in the glands of the gastric fundus and in the epithelium, Brunner’s glands, and Paneth cells of the duodenum. At these sites, SST4 (and other SSTs) may mediate the inhibitory effects of somatostatin on gastric acid and duodenal bicarbonate secretion . The additional presence of SST4 in bile duct epithelia suggests that it also participates in the reduction of bile production by somatostatin [ , , ]. Consistent with previous reports , SST4 expression was further observed in the distal tubules and, to a lesser extent, in the proximal tubules and the glomerular mesangial cells of the kidneys. Here, SST4 may be involved in the effects of somatostatin on glomerular vascular tone and on water and electrolyte transport . While SST4 could not be detected in adrenal medulla, in the current study, strong SST4 expression was noticed in all three layers of the adrenal cortex. This finding, which has not been reported before, suggests an involvement of SST4 in the regulation of adrenal cortex hormone secretion. Finally, confirming mRNA expression data , in the present investigation, strong SST4 expression was noted in the syncytiotrophoblasts of the human placenta. This suggests a regulatory effect of SST4 on the release of hormones like progesterone, human chorionic gonadotropin, human placental lactogen, or leptin by these cells. All in all, our studies revealed a unique expression profile and function for SST4 compared to the other SSTs in humans. SST4, unlike the other SSTs, is not expressed in endocrine or neuroendocrine cells and tissues, such as the anterior pituitary, islet cells of the pancreas, neuroendocrine cells of the bronchopulmonary or gastrointestinal tract, or in the adrenal medulla. 3.4. SST4 Expression in Human Tumors For the 26 different tumor entities evaluated for SST4 expression, high expression was noted in glioblastomas, parathyroid adenomas, gastric and pancreatic adenocarcinomas, pheochromocytomas, and lymphomas, but also in the other entities, single cases with high IRS values were observed. In most cases, our results confirmed previous data obtained with polyclonal antibodies on the frequency and intensity of expression in tumors. These entities were papillary and follicular thyroid carcinomas [ , , ], parathyroid adenomas , squamous cell carcinomas of the lung , small cell lung cancer , hepatocellular and cholangiocellular carcinomas , urinary bladder cancer , prostate adenocarcinomas , breast cancer [ , , ], endometrial and cervical cancer , and ovarian cancer . In some instances, previously published expression levels were lower than found in the present investigation, which may be due to the higher sensitivity and specificity of the novel monoclonal antibody 7H49L61. These included medullary thyroid carcinomas , adenocarcinomas of the lung , gastrointestinal stromal tumors , pancreatic adenocarcinomas , pheochromocytomas , and lymphomas . Among the 26 tumor entities that were investigated in the present study, 7 tumor types (glioblastomas, anaplastic thyroid carcinomas, gastric cancer, colon cancer, renal clear cell carcinomas, testicular cancer, and malignant melanomas) were evaluated for SST4 expression at the protein level for the first time. Overall, SST4 shows a completely different expression pattern compared to the other SST family members in human tumors. While the other SSTs are predominantly found in tumors of (neuro)endocrine origin, SST4 is more prominently present in adenocarcinomas, probably because the SST4 (but not the other SSTs) is already present in the respective normal tissues. Especially in the tumor types with a large number of SST4-positive samples and with high expression intensities in the individual tumors, SST4 may be of diagnostic or therapeutic value. Particularly for glioblastomas, gastric cancer, or pancreatic adenocarcinomas, all with very low 5-year survival rates, new diagnostic or therapeutic options are urgently needed. In these tumor entities, further investigations with larger sample numbers and taking into account the clinical data of the patients are warranted.
To enable visualization of SST4 expression at the protein level in mice, we generated a SST4-eGFP knockin mouse line. In the present study, we provide strong evidence that the immunosignals obtained with this mouse line indeed reflect SST4 receptor expression. First, the mRNA expression data obtained by in situ hybridization experiments in wild-type mice correlated well with SST4-eGFP protein expression in the knockin mice. Second, with the novel monoclonal SST4 antibody 7H49L61 virtually the same staining pattern was observed in humans, although there may be some species differences, especially with respect to the pituitary and the pancreas, which need to be further evaluated. Third, both staining patterns correspond well to the published mRNA and protein expression data for mice, rats, and humans [ , , , , , , ]. Given that monoclonal anti-mouse SST4 antibodies are currently not available, the SST4-eGFP knockin mouse line can serve as a useful tool for the detection of SST4 in mice at the cellular and subcellular level by direct fluorescence microscopy or immunohistochemical techniques using anti-GFP antibodies.
Unlike polyclonal antibodies, monoclonal antibodies are directed against a single epitope, generally leading to greater specificity, and they are available indefinitely in unlimited amounts with consistent quality. Therefore, we developed a monoclonal anti-SST4 antibody suitable for immunoblots in basic research and for immunohistochemical staining of formalin-fixed paraffin-embedded tissues during routine histopathological examinations. In the present study, we demonstrated that the carboxyl-terminal tail of human SST4 can serve as an epitope for generating a rabbit monoclonal antibody that can be effectively used for both applications. We provided strong evidence that the anti-SST4 antibody 7H49L61 specifically detects its target receptor and does not cross-react with other proteins. First, in immunoblots, 7H49L61 specifically detected its cognate receptor in crude extracts from SST4-transfected HEK-293 cells but not in extracts from mock-transfected cells. Second, 7H49L61 yielded highly efficient staining of distinct cell populations known to express SST4 [ , , ] in formalin-fixed paraffin-embedded human tissue samples. Third, the SST4-immunosignals were completely abolished after pre-adsorption of the antibody with its immunizing peptide. Finally, virtually identical staining patterns were observed in the human cortical and trigeminal ganglion samples stained with 7H49L61 and in the corresponding tissues of the SST4-eGFP knockin mice. Most notably, however, 7H49L61 is the first SST4 antibody that facilitates detection of bona fide plasma membrane receptors in human formalin-fixed paraffin-embedded tissues.
In the present investigation, a distinct immunostaining for SST4 was found in the cell bodies and the axons of cortical pyramidal cells, consistent with published data obtained using polyclonal antibodies in humans and in rats [ , , ] as well as in SST4 humanized mice . SST4 has also been identified in neurons and satellite cells of trigeminal and dorsal root ganglia of mice and rats, respectively, by mRNA analysis or with polyclonal antibodies, and it was proposed that the analgesic effects of SST4 agonists are partially mediated by these receptors [ , , ]. We also found SST4 expression in the intramural ganglia of the gastrointestinal tract, indicating broad expression of SST4 in the peripheral nervous system. Similar to published data both at the mRNA and at the protein level , no SST4 expression was observed in the anterior pituitary; hence, it is believed that SST4 agonists may represent good analgesics without exerting any major endocrine side effects . In contrast, a strong staining of nerve fibers within the posterior pituitary was detected, which has not been reported previously and suggests a possible involvement of SST4 in the regulation of oxytocin and adiuretin secretion from these fibers. While pancreatic islet cells were SST4 negative, in the present study, a distinct staining of acinar cells of the exocrine pancreas was observed. Similar observations have been made in the literature using polyclonal antibodies and an involvement of SST4 in the well-known inhibitory role of somatostatin on pancreatic juice excretion but not in insulin or glucagon release was suggested. As reported previously , the presence of SST4 was also noted in the glands of the gastric fundus and in the epithelium, Brunner’s glands, and Paneth cells of the duodenum. At these sites, SST4 (and other SSTs) may mediate the inhibitory effects of somatostatin on gastric acid and duodenal bicarbonate secretion . The additional presence of SST4 in bile duct epithelia suggests that it also participates in the reduction of bile production by somatostatin [ , , ]. Consistent with previous reports , SST4 expression was further observed in the distal tubules and, to a lesser extent, in the proximal tubules and the glomerular mesangial cells of the kidneys. Here, SST4 may be involved in the effects of somatostatin on glomerular vascular tone and on water and electrolyte transport . While SST4 could not be detected in adrenal medulla, in the current study, strong SST4 expression was noticed in all three layers of the adrenal cortex. This finding, which has not been reported before, suggests an involvement of SST4 in the regulation of adrenal cortex hormone secretion. Finally, confirming mRNA expression data , in the present investigation, strong SST4 expression was noted in the syncytiotrophoblasts of the human placenta. This suggests a regulatory effect of SST4 on the release of hormones like progesterone, human chorionic gonadotropin, human placental lactogen, or leptin by these cells. All in all, our studies revealed a unique expression profile and function for SST4 compared to the other SSTs in humans. SST4, unlike the other SSTs, is not expressed in endocrine or neuroendocrine cells and tissues, such as the anterior pituitary, islet cells of the pancreas, neuroendocrine cells of the bronchopulmonary or gastrointestinal tract, or in the adrenal medulla.
For the 26 different tumor entities evaluated for SST4 expression, high expression was noted in glioblastomas, parathyroid adenomas, gastric and pancreatic adenocarcinomas, pheochromocytomas, and lymphomas, but also in the other entities, single cases with high IRS values were observed. In most cases, our results confirmed previous data obtained with polyclonal antibodies on the frequency and intensity of expression in tumors. These entities were papillary and follicular thyroid carcinomas [ , , ], parathyroid adenomas , squamous cell carcinomas of the lung , small cell lung cancer , hepatocellular and cholangiocellular carcinomas , urinary bladder cancer , prostate adenocarcinomas , breast cancer [ , , ], endometrial and cervical cancer , and ovarian cancer . In some instances, previously published expression levels were lower than found in the present investigation, which may be due to the higher sensitivity and specificity of the novel monoclonal antibody 7H49L61. These included medullary thyroid carcinomas , adenocarcinomas of the lung , gastrointestinal stromal tumors , pancreatic adenocarcinomas , pheochromocytomas , and lymphomas . Among the 26 tumor entities that were investigated in the present study, 7 tumor types (glioblastomas, anaplastic thyroid carcinomas, gastric cancer, colon cancer, renal clear cell carcinomas, testicular cancer, and malignant melanomas) were evaluated for SST4 expression at the protein level for the first time. Overall, SST4 shows a completely different expression pattern compared to the other SST family members in human tumors. While the other SSTs are predominantly found in tumors of (neuro)endocrine origin, SST4 is more prominently present in adenocarcinomas, probably because the SST4 (but not the other SSTs) is already present in the respective normal tissues. Especially in the tumor types with a large number of SST4-positive samples and with high expression intensities in the individual tumors, SST4 may be of diagnostic or therapeutic value. Particularly for glioblastomas, gastric cancer, or pancreatic adenocarcinomas, all with very low 5-year survival rates, new diagnostic or therapeutic options are urgently needed. In these tumor entities, further investigations with larger sample numbers and taking into account the clinical data of the patients are warranted.
4.1. Monoclonal Antibody A rabbit monoclonal antibody (7H49L61) that recognizes the carboxyl-terminal tail of human SST4 was generated in collaboration with and obtained from Thermo Fisher Scientific (Waltham, MA, USA). The peptide used for immunizations of the rabbits was CQQEALQPEPGRKRIPLTRTTTF, corresponding to residues 366–388 of human SST4. This sequence is unique to human SST4; therefore, 7H49L61 does not cross-react with rat or mouse SST4. 4.2. Immunoblot Analysis Human embryonic kidney 293 cells (HEK-293; DMSZ, Braunschweig, Germany), either mock-transfected or transfected with human Sst4 , were seeded onto poly-L-lysine-coated 60-mm dishes, and grown to 80% confluency. Cells were lysed in detergent buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL aprotinin, and 10 mg/mL bacitracin]. SST4 was enriched using wheat germ lectin agarose beads. Proteins were separated on a 7.5% SDS-polyacrylamide gel by electrophoresis and immunoblotted onto polyvinylidene fluoride (PVDF) membranes. Blots were incubated with the rabbit monoclonal anti-SST4 antibody 7H49L61 (1:500) followed by a peroxidase-conjugated secondary anti-rabbit antibody (1:5000) (Santa Cruz Biotechnology, Dallas, TX, USA) and detected using enhanced chemiluminescence (Amersham, Braunschweig, Germany). 4.3. SST4-eGFP Knockin Mice For the examination of SST4 expression in mice, a SST4-eGFP knockin mouse line was developed in a C57BL/6 background (Cyagen, Santa Clara, CA, USA). In these mice, the SST4 receptor is expressed as a fusion protein with a green fluorescent protein (eGFP) attached to its carboxyl terminus. Animals were housed in plastic cages under standardized conditions (light-dark cycle 12/12 h, temperature 22 ± 2 °C, humidity 50 ± 10%, pellet diet Altromin 1316 (Altromin, Lage, Germany), water ad libitum). The principles of good laboratory animal care and the German Law on the Protection of Animals, as well as Directive 2010/63/EU, were followed. 4.4. Immunohistochemistry on SST4-eGFP Knockin Mouse Tissues SST4-eGFP knockin mice as well as wild-type littermates of either sex were sacrificed using 5% isoflurane and transcardially perfused with PBS followed by 4% formaldehyde/PBS (pH 7.4). For immunofluorescence stainings, brain and trigeminal ganglia were rapidly dissected and post-fixed in the same fixative for 24 h at room temperature. After immersion in 30% sucrose in TPBS (10 mmol/L Tris, 10 mmol/L phosphate, 155 mmol/L NaCl, pH 7.4) for 48 h at 4 °C, 40-µm cryosections were prepared and subjected to free-floating immunohistochemistry as described previously . Briefly, free-floating sections were successively incubated with 50% methanol in TPBS and with 5% BSA/0.3% Triton X-100 in TPBS before applying the rabbit anti-GFP antibody (ab290, Abcam, Cambridge, UK; 1:7500) at 4 °C overnight. For amplification, the biotin/tyramine method was used: samples were incubated for 2 h with the biotinylated goat anti-rabbit antibody (Dianova, Hamburg, Germany; 1:400) before applying ABC Elite Kit peroxidase (PK-6100, Vector Laboratories, Burlingame, CA, USA) and working buffer containing 0.015% H 2 O 2 and 7.5 nmol/L biotinylated tyramine. Streptavidin-coupled Cy3 (S11223, Invitrogen, Waltham, MA, USA) was used at 1:500 in working buffer for detection. For double-labeling immunofluorescence experiments, samples were incubated additionally with monoclonal mouse anti-NeuN antibody (MAB377, Millipore, Burlington, MA, USA; 1:300) followed by donkey anti-mouse Cy5 (Dianova, Hamburg, Germany; 1:500). Specimens were analyzed using an LSM Meta 510 confocal microscope (Carl Zeiss, Jena, Germany). For immunohistochemical stainings on paraffin sections, organs were quickly removed, fixed for 72 h in 4% formaldehyde/PBS (pH 7.4), dehydrated, and embedded in paraffin blocks. The further procedure was as described below for the human tissue samples. As primary antibody, a rabbit anti-GFP antibody (ab290, Abcam, Cambridge, UK; 1:1000) was used. 4.5. In Situ Hybridization Experiments In situ hybridization was carried out on brain and trigeminal ganglion tissues from C57BL/6 wild-type mice as described previously using 35 S-labeled riboprobes. The Sstr4 probe corresponded to nucleotides 262–1790 of the coding sequence of the receptor gene and was controlled using the sense strand probe. 4.6. Human Tissue Samples For the evaluation of SST4 expression in different human tumors, 230 formalin-fixed and paraffin-embedded tumor samples ( ) were obtained from the Department of Pathology of the University Hospital, Otto-von-Guericke-University, Magdeburg, Germany. Many of the tumor specimens contained adjacent non-malignant tissue that was also analyzed. Tumor-free human tissue samples from the cortex, trigeminal ganglia, pituitary, lung, heart, liver, stomach, duodenum, colon, pancreas, kidneys, adrenals, spleen, lymph nodes, placenta, and bone marrow (n = 3–6 each), obtained from the Department of Pathology of the University Hospital, Otto-von-Guericke-University, Magdeburg, Germany, were also evaluated. Staining patterns were compared to the non-malignant tissues surrounding the tumors. 4.7. Immunohistochemistry on Human Tissue Samples From the paraffin blocks, 4-µm sections were prepared and floated onto positively charged slides. Immunostaining was performed by an indirect peroxidase labeling method as described previously . Briefly, sections were dewaxed, microwaved in 10 mM citric acid (pH 6.0) for 16 min at 600 W, and incubated with 7H49L61 (1:500) overnight at 4 °C. The primary antibody was detected using biotinylated anti-rabbit IgG followed by incubation with peroxidase-conjugated avidin (Vector ABC “Elite” kit; Vector Laboratories, Burlingame, CA, USA). The binding of the primary antibody was visualized using 3-amino-9-ethyl carbazole in acetate buffer (BioGenex, San Ramon, CA, USA). Sections were rinsed, counterstained with Mayer’s hematoxylin, and mounted in Vectamount™ mounting medium (Vector Laboratories, Burlingame, CA, USA). For immunohistochemical controls, 7H49L61 was either omitted or adsorbed for 2 h at room temperature with 10 µg/mL of the peptide used for immunizations. The staining of SST4 in the tumors was scored with the semiquantitative immunoreactivity score (IRS) according to Remmele and Stegner (1987) . The percentage of positive tumor cells categorized in five grades (no positive cells [0], <10% positive cells [1], 10–50% positive cells [2], 51–80% positive cells [3], and >80% positive cells [4]) was multiplied by the staining intensity categorized in four grades (no staining [0], mild staining [1], moderate staining [2], and strong staining [3]). Thus, IRS values ranging from 0 to 12 were obtained. Only tumor samples with an IRS value ≥3 were considered SST4 positive.
A rabbit monoclonal antibody (7H49L61) that recognizes the carboxyl-terminal tail of human SST4 was generated in collaboration with and obtained from Thermo Fisher Scientific (Waltham, MA, USA). The peptide used for immunizations of the rabbits was CQQEALQPEPGRKRIPLTRTTTF, corresponding to residues 366–388 of human SST4. This sequence is unique to human SST4; therefore, 7H49L61 does not cross-react with rat or mouse SST4.
Human embryonic kidney 293 cells (HEK-293; DMSZ, Braunschweig, Germany), either mock-transfected or transfected with human Sst4 , were seeded onto poly-L-lysine-coated 60-mm dishes, and grown to 80% confluency. Cells were lysed in detergent buffer [20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL aprotinin, and 10 mg/mL bacitracin]. SST4 was enriched using wheat germ lectin agarose beads. Proteins were separated on a 7.5% SDS-polyacrylamide gel by electrophoresis and immunoblotted onto polyvinylidene fluoride (PVDF) membranes. Blots were incubated with the rabbit monoclonal anti-SST4 antibody 7H49L61 (1:500) followed by a peroxidase-conjugated secondary anti-rabbit antibody (1:5000) (Santa Cruz Biotechnology, Dallas, TX, USA) and detected using enhanced chemiluminescence (Amersham, Braunschweig, Germany).
For the examination of SST4 expression in mice, a SST4-eGFP knockin mouse line was developed in a C57BL/6 background (Cyagen, Santa Clara, CA, USA). In these mice, the SST4 receptor is expressed as a fusion protein with a green fluorescent protein (eGFP) attached to its carboxyl terminus. Animals were housed in plastic cages under standardized conditions (light-dark cycle 12/12 h, temperature 22 ± 2 °C, humidity 50 ± 10%, pellet diet Altromin 1316 (Altromin, Lage, Germany), water ad libitum). The principles of good laboratory animal care and the German Law on the Protection of Animals, as well as Directive 2010/63/EU, were followed.
SST4-eGFP knockin mice as well as wild-type littermates of either sex were sacrificed using 5% isoflurane and transcardially perfused with PBS followed by 4% formaldehyde/PBS (pH 7.4). For immunofluorescence stainings, brain and trigeminal ganglia were rapidly dissected and post-fixed in the same fixative for 24 h at room temperature. After immersion in 30% sucrose in TPBS (10 mmol/L Tris, 10 mmol/L phosphate, 155 mmol/L NaCl, pH 7.4) for 48 h at 4 °C, 40-µm cryosections were prepared and subjected to free-floating immunohistochemistry as described previously . Briefly, free-floating sections were successively incubated with 50% methanol in TPBS and with 5% BSA/0.3% Triton X-100 in TPBS before applying the rabbit anti-GFP antibody (ab290, Abcam, Cambridge, UK; 1:7500) at 4 °C overnight. For amplification, the biotin/tyramine method was used: samples were incubated for 2 h with the biotinylated goat anti-rabbit antibody (Dianova, Hamburg, Germany; 1:400) before applying ABC Elite Kit peroxidase (PK-6100, Vector Laboratories, Burlingame, CA, USA) and working buffer containing 0.015% H 2 O 2 and 7.5 nmol/L biotinylated tyramine. Streptavidin-coupled Cy3 (S11223, Invitrogen, Waltham, MA, USA) was used at 1:500 in working buffer for detection. For double-labeling immunofluorescence experiments, samples were incubated additionally with monoclonal mouse anti-NeuN antibody (MAB377, Millipore, Burlington, MA, USA; 1:300) followed by donkey anti-mouse Cy5 (Dianova, Hamburg, Germany; 1:500). Specimens were analyzed using an LSM Meta 510 confocal microscope (Carl Zeiss, Jena, Germany). For immunohistochemical stainings on paraffin sections, organs were quickly removed, fixed for 72 h in 4% formaldehyde/PBS (pH 7.4), dehydrated, and embedded in paraffin blocks. The further procedure was as described below for the human tissue samples. As primary antibody, a rabbit anti-GFP antibody (ab290, Abcam, Cambridge, UK; 1:1000) was used.
In situ hybridization was carried out on brain and trigeminal ganglion tissues from C57BL/6 wild-type mice as described previously using 35 S-labeled riboprobes. The Sstr4 probe corresponded to nucleotides 262–1790 of the coding sequence of the receptor gene and was controlled using the sense strand probe.
For the evaluation of SST4 expression in different human tumors, 230 formalin-fixed and paraffin-embedded tumor samples ( ) were obtained from the Department of Pathology of the University Hospital, Otto-von-Guericke-University, Magdeburg, Germany. Many of the tumor specimens contained adjacent non-malignant tissue that was also analyzed. Tumor-free human tissue samples from the cortex, trigeminal ganglia, pituitary, lung, heart, liver, stomach, duodenum, colon, pancreas, kidneys, adrenals, spleen, lymph nodes, placenta, and bone marrow (n = 3–6 each), obtained from the Department of Pathology of the University Hospital, Otto-von-Guericke-University, Magdeburg, Germany, were also evaluated. Staining patterns were compared to the non-malignant tissues surrounding the tumors.
From the paraffin blocks, 4-µm sections were prepared and floated onto positively charged slides. Immunostaining was performed by an indirect peroxidase labeling method as described previously . Briefly, sections were dewaxed, microwaved in 10 mM citric acid (pH 6.0) for 16 min at 600 W, and incubated with 7H49L61 (1:500) overnight at 4 °C. The primary antibody was detected using biotinylated anti-rabbit IgG followed by incubation with peroxidase-conjugated avidin (Vector ABC “Elite” kit; Vector Laboratories, Burlingame, CA, USA). The binding of the primary antibody was visualized using 3-amino-9-ethyl carbazole in acetate buffer (BioGenex, San Ramon, CA, USA). Sections were rinsed, counterstained with Mayer’s hematoxylin, and mounted in Vectamount™ mounting medium (Vector Laboratories, Burlingame, CA, USA). For immunohistochemical controls, 7H49L61 was either omitted or adsorbed for 2 h at room temperature with 10 µg/mL of the peptide used for immunizations. The staining of SST4 in the tumors was scored with the semiquantitative immunoreactivity score (IRS) according to Remmele and Stegner (1987) . The percentage of positive tumor cells categorized in five grades (no positive cells [0], <10% positive cells [1], 10–50% positive cells [2], 51–80% positive cells [3], and >80% positive cells [4]) was multiplied by the staining intensity categorized in four grades (no staining [0], mild staining [1], moderate staining [2], and strong staining [3]). Thus, IRS values ranging from 0 to 12 were obtained. Only tumor samples with an IRS value ≥3 were considered SST4 positive.
We developed an SST4-eGFP knockin mouse line that enables SST4 localization in mice in great detail either by direct imaging of the receptor under a fluorescent microscope or by immunohistochemistry using commercially available anti-GFP antibodies. We also generated a rabbit monoclonal anti-human SST4 antibody, 7H49L61, that is well suited for immunoblots in basic research and for visualizing human SST4 in formalin-fixed paraffin-embedded tissues during routine histopathological examinations. Notably, 7H49L61 is the first antibody that detects membrane-bound SST4 receptors in human tissues. Using 7H49L61 enabled us to generate the first unequivocal SST4 expression profile in a wide variety of normal and neoplastic human tissues.
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Integrated health Services for Children: a qualitative study of family perspectives | a1169f93-9840-4976-b22e-c7a3940ffb18 | 7901188 | Pediatrics[mh] | Design A qualitative interview study of participants who had experienced the early phase of a new integrated service was undertaken from June 2018–March 2019. Setting The Evelina CYPHP approach is being trialled for children with ongoing conditions in south London, a highly diverse inner-city setting. The early phase of this integrated care project includes children with one or more of the following conditions: constipation, eczema, asthma, or epilepsy. The roll out of CYPHP was phased, allowing an opportunistic randomised control trial, and nested implementation and economic evaluation studies, to be conducted, prior to the approach being adopted as part of routine care. CYPHP aims to strengthen the local health system through better integrated care. At the organisational level, a novel population health management (PHM) system is used to identify eligible children from primary care records, then invite children and families to complete a biopsychosocial pre-assessment, via a cloud-based portal. The information is used to triage and tailor early intervention care which includes health promotion and, when required, holistic care specifically for the needs of the child and family. Care is provided by a multidisciplinary health team consisting of specially trained Children’s Nurses, General Practitioners, Paediatricians, and mental health specialists. Most care is delivered by Children’s Nurses who provide a first point of contact for families, act as care navigators, coordinate and deliver care across primary, community, and hospital settings. The entire multidisciplinary team is available when needed, working closely with primary and community services, and standard referral pathways are used for specialist care. All clinical information regarding services delivered by CYPHP is held on shared electronic health records. Further details of the service can be found in recent publications . Recruitment and sample The PHM system enables all eligible children to be contacted proactively and, together with those actively referred to the service, these children and their parents or carers are invited to complete the biopsychosocial pre-assessment and participate in ongoing research. Preliminary analyses of those completing the preassessment ( n = 219) indicate that a substantial proportion of children (60–76%) reported uncontrolled symptoms for asthma, constipation or eczema, and 28% reported high to very high scores on mental health symptom questionnaires . Additionally, 68% of the cohort were from ethnic minorities. For this nested study, children and caregivers receiving the CYPHP service, who had identified that they were interested in participating in research, were eligible for recruitment. A researcher contacted families, via phone, once they had received care for at least 2 weeks. The researcher was independent of the clinical service and had no previous contact with families. Families were fully informed about the aims of the study and were invited to take part in an interview on their experiences of the service. Out of the initial 40 parents contacted, thirty-seven caregivers gave written consent; children and young people over 12 years of age provided written assent. Recruitment continued until data saturation, which was established when additional new information from interviews was no longer obtained. Caregivers could take part in interviews without their child present; all children took part with their caregiver present. Maximum variation sampling was used to ensure a range of child age, sex, condition, and socioeconomic status. Data collection Demographic data relating to the child’s age, condition, time in service, and index of multiple deprivation (IMD) of home address was obtained. The IMD is the official measure of deprivation across England describing relative deprivation for small areas or neighbourhoods (containing on average 650 households) called lower-super-output areas . IMD is calculated from seven distinct domains of deprivation, encompassing a wide range of living conditions; areas are ranked and assigned deciles ranging from the most deprived areas (1) to least deprived (10). Interviews lasted between 40 and 60 min and were completed at a local clinic ( N = 2) or in the family home ( N = 35). Interview guides were developed with involvement from public and patient involvement groups and healthcare providers and adapted for different age groups; questions focussed on experiences of the integrated care service and symptom management. The main interview guide for this study is provided in Additional file . A range of art-based methods were used to engage younger children in the discussions, asking children to construct a pictorial timeline or collage of how they felt before, during and after involvement in the integrated service . Children spoke to the researchers as they created their work, and once finished, children were asked to talk about their artwork. Analysis Interviews were audio recorded and transcribed verbatim. Analysis was directed at qualitative description, a method which stays close to participants’ own accounts , using thematic content analysis drawing on techniques from grounded theory traditions with the aid of qualitative software (NVivo Version11). This entailed three researchers separately coding transcripts in an iterative process that combined deductive coding (from themes in integrated care literature) and inductive coding from comparisons within and across the data set. After coding a subset of the data, an initial set of codes was discussed by the research team, and compared within and across interviews, to identify patterns and amalgamate into themes. The research team met regularly to discuss and revise the emergent themes and ensure analytical rigour, with any discrepancies discussed to ensure analysis captured all perspectives. Ethical approval Ethical approval was granted by the Health Research Authority (Reference: 17/SW/0275). Findings Thirty-seven families completed interviews (19 patients, 40 caregivers and 8 siblings). Children and young people were between 29 weeks and 15 years of age (48.6% male). Sociodemographic characteristics of participants are detailed in Table : of the sample, 35.1% were from the 20% most deprived areas within the UK. Exposure to the new service varied widely, depending on condition and needs (ranging between 3 and 71 weeks). Despite this, families described important commonalities of integrated care in combination with aspects of perceived quality and effectiveness of the service. Most families valued the integrated service they received, commenting on advantages over prior experiences of traditional services, their willingness to recommend the service to others, and support of continued funding for the service. “To the people in charge; you need to expand this service to everybody, everybody who has children with medical conditions, who need the help, who require the help.” [Caregiver 14; Constipation] Below, we describe these as four key themes (Table ) defined by summary phrases derived from participants’ accounts. The first theme, The Nurse Understands Our World, comprised of four subthemes, demonstrates the importance of addressing broader determinants of health. The second theme was entitled, Professionals Involve Us in Treatment, and again was formed of four subthemes. This theme highlights the importance of relationships with key providers, developed through physical presence and supplemented by technology. The third theme, Professionals Support All Our Needs, comprised of three subthemes, speaks to the importance of holistic care provided by a key professional but supported by the wider health system. The final theme entitled The System is Glued Together, formed of five subthemes, speaks to the value of a health system that is perceived as coordinated, across health, social and education sectors. The following sections describe these themes and associated subthemes in detail. Themes and Subthemes are presented in Table ; the thematic analysis developed integrates subthemes to create a coherent narrative. The nurse understands our world A key component of integrated care that families reported as important was care that addresses their personal and social needs. Families in this study reported a variety of social needs including damp housing that exacerbated asthma, difficulties attending school due to ongoing symptoms, and challenges maintaining social relationships when supporting a child with an ongoing condition. Despite families emphasising the importance of discussing these needs, several acknowledged an initial reticence, describing questions around personal and social needs as “ intrusive ”. For example, one mother living in a socioeconomically deprived neighbourhood, reported fear of being “ penalized ” by the Nurse when asked about her home environment, considering these questions to be unrelated to her child’s health (Caregiver 23). “At first, I was a bit like is she [Nurse] was going to penalize us?... She’s coming in here to fix the eczema, why should she want to know this?” (Caregiver 23; Eczema) The consensus among families interviewed was that time with the Nurse was needed to facilitate the disclosure of personal information. Most families reported that they developed a level of “ trust ” with their Nurse. For example, one mother describes how, despite her initial concern around disclosing personal information, she felt able to disclose her needs once she had developed a relationship with her Nurse (Caregiver 30). However, in direct contrast, a second mother raised ongoing concerns about disclosing personal information (Caregiver 25). Caregiver 25 was the only participant to raise ongoing concerns around disclosing personal circumstances to the Nurse. These concerns surrounding the discussion of personal and social circumstances in a consultation about a health condition may reflect the traditional separation of health and social services. “ It was weird at first, but I made us a cuppa tea, had a general chit-chat. And by the end of my first cup, I felt I could open up to her. Like I knew her more as a person .” (Caregiver 30; Constipation) “O ur problems [is] her seizures, fix that … if she don’t want to speak about school, fine … if I don’t want to talk about my job, then leave it. What you do with that stuff anyway … I don’t want it on her record. ” (Caregiver 25; Epilepsy) Most participants expressed views that discussion around the broader determinants of health, and time spent with providers, enabled them to feel understood by the Nurse. This understanding then facilitated the delivery of interventions suited to the family’s needs and contextualized to their social circumstances. A typical example is provided below, whereby the child’s Nurse supported him in his desire to play football, as an important health goal or outcome the child wanted to achieve (Child 27). Evidence that this support was a key component of integrated care comes from families’ descriptions of prior experiences with health providers whose advice felt inappropriately narrow and had not considered their wider needs. “One time they said it was running in my football and I should stop. But football is everything, I want to be a professional footballer and you’ve basically said I can’t do that [referring to previous experiences of health care]. But [Nurse] really understood and took that on board and said like how we can work with asthma and football.” (Child 27; Asthma) For some families, material benefits resulted from these discussions, including housing support, food vouchers, services, and goods. However, on occasion, expectations were raised about needs that could not be met. An example of this is provided by Caregiver 31, who described their Nurse’s challenges in obtaining the resources required to support the family in the context of a stretched housing sector. “So [Nurse] tried to push Council property, but they won’t give [a property to] me because they said oh too many people are in the queue, they won’t give me. [Nurse] tried hard and pushed, he tried.” (Caregiver 31; Asthma) Whether discussion of needs resulted in changes in social circumstances or not, the key was that families felt understood by their Nurse, and that this social contextualising of their child’s health was welcomed and valued by most families. Professionals involve us in treatment Connected to the capacity of the Nurse to understand families’ worlds, the second theme relates to the nature of relationships that this connection engenders within the health care system. Most families, including those who were initially reticent, described a mutual trust that developed between themselves and the Nurse over time. This reciprocal trust appeared key to family engagement with the service. This manifested in participants feeling confident in asking questions about care, feeling able to suggest alternatives as well as receiving advice, and a sense of both parties (families and professionals) learning together. Numerous families reported a two-way process with the Nurse that enabled different understandings of conditions to be discussed, including beliefs around the urgency of treatment and appropriateness of medication. There was also a sense that the Nurse’s availability to families facilitated this collaborative approach (Caregiver 4). “If something happened, I could text her or call her and say, “this has happened, do you think I should do x, y or z?” We did that last week, texted her and we spoke together and came with a plan together.” (Caregiver 4; Eczema) Reciprocal relationships with professionals were also highlighted by older children as an essential component of their care. That this was attributed to integrated care was evidenced in their comparisons with previous services, in which children recalled feeling less involved in decision making. Older children reported that being involved in treatment allowed them to feel more agency and engagement with their care, highlighting the importance of respecting young people’s autonomy in treatment decisions (Child 34). “[Nurse and Mental Health Provider] actually talk to me before they talked to my parents, which I really liked because it meant that they got my permission first … I felt I was actually in control. I want to do this.” (Child 34; Asthma) These relationships with professionals and trust in the Nurse appeared to be built through experiences of professionals being transparent about mutual learning. Caregiver 32 provides a typical example of this. “[The Nurse] is continually learning, she was telling me about conferences she’d been to … and that kind of reassured me and let me trust her.” (Caregiver 32; Constipation) Physical presence, at least early in the relationship, was important. All families spoke of the importance of face-to-face consultations in facilitating the development of a relationship with their Nurse (Caregiver 22). Families reported that communications technologies (email, SMS, phone calls) maintained this relationship over time, but, importantly, these were only positively rated once carers had established a physical face-to-face relationship. The uncertainties recounted by one father (Caregiver 5), whose partner had more contact with health providers, gives an example of the importance of relationship-building. “[ The Nurse] got to know us, like as people. She could see us and understand us. But she knows us now, knows what we need … so now we do it on the phone more. ” (Caregiver 22; Eczema) “ I don’t have the rapport that [Partner] has because she’s the one that sees [The Nurse], so I don’t know how good [The Nurse] is in terms of that, you know, in terms of her overall knowledge and also what she’s allowed to advise on. ” (Caregiver 5; Epilepsy) Most families described increasing independence in managing children’s health over time, where they could rely on ‘ remote ’, rather than in-person, consultancy from their Nurse: “I would only ever go to A&E now if either I couldn’t get hold of [The Nurse], or it was a horrendous one, other than that we’re kind of happy to have sort of remote consultancy.” (Caregiver 5; Epilepsy) However, as discharge from the service approached, families’ expectations varied, with half expressing readiness for independent self-management and others reticent about discharge from the service. As one child says, “ I know what I’m doing now, I’ve got control” (Child 35; Asthma). By contrast, Caregiver 29 questioned the fairness of discharge from the service; despite her child’s needs being met, this mother wished to stay in contact with the service. This desire appeared prominent among families who were referred to the service with high levels of need as indicated by frequent use of emergency department services and concurrent mental health needs. For some families, the prospective withdrawal of the Nurse was framed as a withdrawal of care, rather than a marker of growing independence. “We’re doing everything anyway, we haven’t been to hospital … I just felt like … just stay around for a little bit just in case … to have all this and then get it taken away. That’s just not right.” (Caregiver 29; Constipation) Relationships with Nurses were facilitated through open communication, availability to families, shared learning, and face to face relationships. Ensuring active family involvement in decision making processes was key to success, supporting engagement with the service, independent self-management, and readiness for discharge. Professionals support all our needs One of the key principles of integrated care is considering physical, social, and psychological needs. Most families described the integrated service as providing holistic care, within a family, rather than just focusing on a condition. One mother used the metaphor of the service creating a “ whole circle ” supporting her family. “It’s a support system, it is not only about the child or about the person who is having the issue, it’s about the whole circle that is surrounding them, so, which is not going to improve the life of only the person who is sick, but only, even the people that are surrounded in that circle.” (Caregiver 18; Constipation) Of note, this mother speaks of a “ system ”, highlighting the importance of a wider network of professionals who support the family. References to this “ system ” are voiced by another mother who describes her Nurse talking with a paediatrician about her child’s care, highlighting again the role of the multidisciplinary team in facilitating holistic care (Caregiver 2). Caregiver 32 highlights the perceived crucial role of the Nurse as the empathetic interface for the multidisciplinary team. Both listening to families and identifying and advising on a broad range of health-related issues, including health promotion, disease and complication prevention, and the Nurse provided early intervention, coordinated care and onward referral within the multidisciplinary team when required. Other families detailed the ways Nurses discussed and identified mental health needs, and successfully brought in the wider team for onward referral to specialist services. “Sometimes [Nurse] said she’s wasn’t sure. And so it’s good to know that there’s people she can talk to, things like when she needs a paediatrician, there’s even that.” (Caregiver 2; Eczema) “[Nurse] was helping with all the anxieties, doing exercise, eating well, the anger but it got quite bad … She spoke to her team and that’s how [Mental Health Nurse] became involved … And what helped her most is that she [Mental Health Nurse] works alongside [Nurse], so she was aware of [CYP]’s problems.” (Caregiver 32; Constipation) Most interviewees were positive about these holistic aspects of integrated care and welcomed support for the whole family. However, for two families, this aspect of integrated care was perceived as unhelpful, and even unnecessary or time wasting. Both these families described considerable use of health services prior to referral to the integrated service, and their views are resonant with a biomedical focus, in which physical and mental health are separate domains: “ You’ve got this mental health thing, oh my God. Great but not for us … I just wanted to get onto the point with asthma and that’s it .” (Caregiver 26; Asthma) Holistic care is, then, a core component of integrated care from families’ perspectives: rejected by a few who preferred a more biomedical approach but emphasised and valued by most families who welcome additional support beyond the presenting condition, reassurance for caregivers, and improving the skills of the whole family in condition management. A system that is glued together The fourth theme is that of encountering a system which did not just address holistic needs, but also responded in a co-ordinated and coherent way. This is summarised as ‘a system that is glued together’, which reflects the ability of the integrated service to coordinate across primary and secondary care, as well as health, social, and educational sectors. Interviewees frequently described their Nurse’s ability to coordinate across providers, pulling information together, and enabling all professionals in the patient’s life to provide seamless support. “I think the service helped us socially by being present with the school and being a common point of contact with school, GP and hospital.” (Caregiver 6; Epilepsy) Most families described good communication between their Nurse, primary and secondary care, and other non-health sector services, which enabled coherent and coordinated care. Again, evidence that this was a feature of the integrated service was typically provided by comparisons of previous encounters with health and other providers (Caregiver 2). Families typically described the Nurse as ‘ talking ’ to other professionals to achieve this, however some families reported not quite knowing how communication was achieved. Several mothers described how this communication enabled efficient access to secondary care services, highlighting the Nurse’s role as patient advocate (Caregiver 4). “I don’t know how [Nurse] can manage to see what the GP is writing down because I’ve had so many problems where one doctor can see [Child’s] notes but the other can’t.” (Caregiver 2; Eczema) “[ Nurse] actually trying to get us seen by dermatology again this week … so she said she’s just talk with them [dermatology] and that’s it, done.” (Caregiver 4; Eczema) As a core component of integrated care, coordination was on occasion flagged because of its absence. Two examples of missing coordination were in the context of their children undergoing further diagnostic tests with multiple providers. This highlights the importance of Nurses with expertise in sharing information across providers, particularly during times of clinical uncertainty, which may require input from a range of providers across primary and secondary care. In addition, this demonstrates the value of coordination across sectors and providers for effective integrated care. “I was a bit baffled by her last consultant’s and GP appointment, they was saying different thing to me and I was saying to [the Nurse], it doesn’t make sense, that is confusing, it doesn’t go with what you said.” (Caregiver 19; Asthma) However, more commonly, coordination was recognised as more present in the integrated system than it had been in previous health services. Regarding the new service, families reported enhanced coordination across health providers, and between health and social sectors. One frequently reported example of the difference between an integrated service and a conventional service, was coordination between the health and education sectors, which enabled patients to follow treatment plans outside the home or hospital setting. This was facilitated by the Nurse supporting schools to take on a meaningful role in the child’s health, which appeared to be achieved through the provision of school and student training, enabling coordination across sectors (Child 33). “My teachers and friends all know what to do if something happens. So, I don’t worry about it. My mum and dad don’t worry about it. Because everyone knows what to do if something did happen. And that’s probably because they’ve all been taught about epilepsy and about my epilepsy.” (Child 33; Epilepsy) Despite families valuing coordination across sectors, some caregivers highlighted the challenges they, and their Nurse, had in coordinating care with both the education sector and across the primary-secondary interface. These difficulties highlight the challenges in working within the context of school and healthcare workforce constraints (Caregiver 19). “[ Nurse ] was waiting for school to get back about [Child] … I went in and said, oh by the way, the [Nurse] has been trying to contact to try and speak to you about [Child] and has had no response … they say they’re too busy.” (Caregiver 19; Asthma) Whether families experienced a coordinated service or experienced challenges with coordination across the system largely depended on how well health, social and education sectors were updated with children’s health records: a key aspect of integrated care.
A qualitative interview study of participants who had experienced the early phase of a new integrated service was undertaken from June 2018–March 2019.
The Evelina CYPHP approach is being trialled for children with ongoing conditions in south London, a highly diverse inner-city setting. The early phase of this integrated care project includes children with one or more of the following conditions: constipation, eczema, asthma, or epilepsy. The roll out of CYPHP was phased, allowing an opportunistic randomised control trial, and nested implementation and economic evaluation studies, to be conducted, prior to the approach being adopted as part of routine care. CYPHP aims to strengthen the local health system through better integrated care. At the organisational level, a novel population health management (PHM) system is used to identify eligible children from primary care records, then invite children and families to complete a biopsychosocial pre-assessment, via a cloud-based portal. The information is used to triage and tailor early intervention care which includes health promotion and, when required, holistic care specifically for the needs of the child and family. Care is provided by a multidisciplinary health team consisting of specially trained Children’s Nurses, General Practitioners, Paediatricians, and mental health specialists. Most care is delivered by Children’s Nurses who provide a first point of contact for families, act as care navigators, coordinate and deliver care across primary, community, and hospital settings. The entire multidisciplinary team is available when needed, working closely with primary and community services, and standard referral pathways are used for specialist care. All clinical information regarding services delivered by CYPHP is held on shared electronic health records. Further details of the service can be found in recent publications .
The PHM system enables all eligible children to be contacted proactively and, together with those actively referred to the service, these children and their parents or carers are invited to complete the biopsychosocial pre-assessment and participate in ongoing research. Preliminary analyses of those completing the preassessment ( n = 219) indicate that a substantial proportion of children (60–76%) reported uncontrolled symptoms for asthma, constipation or eczema, and 28% reported high to very high scores on mental health symptom questionnaires . Additionally, 68% of the cohort were from ethnic minorities. For this nested study, children and caregivers receiving the CYPHP service, who had identified that they were interested in participating in research, were eligible for recruitment. A researcher contacted families, via phone, once they had received care for at least 2 weeks. The researcher was independent of the clinical service and had no previous contact with families. Families were fully informed about the aims of the study and were invited to take part in an interview on their experiences of the service. Out of the initial 40 parents contacted, thirty-seven caregivers gave written consent; children and young people over 12 years of age provided written assent. Recruitment continued until data saturation, which was established when additional new information from interviews was no longer obtained. Caregivers could take part in interviews without their child present; all children took part with their caregiver present. Maximum variation sampling was used to ensure a range of child age, sex, condition, and socioeconomic status.
Demographic data relating to the child’s age, condition, time in service, and index of multiple deprivation (IMD) of home address was obtained. The IMD is the official measure of deprivation across England describing relative deprivation for small areas or neighbourhoods (containing on average 650 households) called lower-super-output areas . IMD is calculated from seven distinct domains of deprivation, encompassing a wide range of living conditions; areas are ranked and assigned deciles ranging from the most deprived areas (1) to least deprived (10). Interviews lasted between 40 and 60 min and were completed at a local clinic ( N = 2) or in the family home ( N = 35). Interview guides were developed with involvement from public and patient involvement groups and healthcare providers and adapted for different age groups; questions focussed on experiences of the integrated care service and symptom management. The main interview guide for this study is provided in Additional file . A range of art-based methods were used to engage younger children in the discussions, asking children to construct a pictorial timeline or collage of how they felt before, during and after involvement in the integrated service . Children spoke to the researchers as they created their work, and once finished, children were asked to talk about their artwork.
Interviews were audio recorded and transcribed verbatim. Analysis was directed at qualitative description, a method which stays close to participants’ own accounts , using thematic content analysis drawing on techniques from grounded theory traditions with the aid of qualitative software (NVivo Version11). This entailed three researchers separately coding transcripts in an iterative process that combined deductive coding (from themes in integrated care literature) and inductive coding from comparisons within and across the data set. After coding a subset of the data, an initial set of codes was discussed by the research team, and compared within and across interviews, to identify patterns and amalgamate into themes. The research team met regularly to discuss and revise the emergent themes and ensure analytical rigour, with any discrepancies discussed to ensure analysis captured all perspectives.
Ethical approval was granted by the Health Research Authority (Reference: 17/SW/0275).
Thirty-seven families completed interviews (19 patients, 40 caregivers and 8 siblings). Children and young people were between 29 weeks and 15 years of age (48.6% male). Sociodemographic characteristics of participants are detailed in Table : of the sample, 35.1% were from the 20% most deprived areas within the UK. Exposure to the new service varied widely, depending on condition and needs (ranging between 3 and 71 weeks). Despite this, families described important commonalities of integrated care in combination with aspects of perceived quality and effectiveness of the service. Most families valued the integrated service they received, commenting on advantages over prior experiences of traditional services, their willingness to recommend the service to others, and support of continued funding for the service. “To the people in charge; you need to expand this service to everybody, everybody who has children with medical conditions, who need the help, who require the help.” [Caregiver 14; Constipation] Below, we describe these as four key themes (Table ) defined by summary phrases derived from participants’ accounts. The first theme, The Nurse Understands Our World, comprised of four subthemes, demonstrates the importance of addressing broader determinants of health. The second theme was entitled, Professionals Involve Us in Treatment, and again was formed of four subthemes. This theme highlights the importance of relationships with key providers, developed through physical presence and supplemented by technology. The third theme, Professionals Support All Our Needs, comprised of three subthemes, speaks to the importance of holistic care provided by a key professional but supported by the wider health system. The final theme entitled The System is Glued Together, formed of five subthemes, speaks to the value of a health system that is perceived as coordinated, across health, social and education sectors. The following sections describe these themes and associated subthemes in detail. Themes and Subthemes are presented in Table ; the thematic analysis developed integrates subthemes to create a coherent narrative.
A key component of integrated care that families reported as important was care that addresses their personal and social needs. Families in this study reported a variety of social needs including damp housing that exacerbated asthma, difficulties attending school due to ongoing symptoms, and challenges maintaining social relationships when supporting a child with an ongoing condition. Despite families emphasising the importance of discussing these needs, several acknowledged an initial reticence, describing questions around personal and social needs as “ intrusive ”. For example, one mother living in a socioeconomically deprived neighbourhood, reported fear of being “ penalized ” by the Nurse when asked about her home environment, considering these questions to be unrelated to her child’s health (Caregiver 23). “At first, I was a bit like is she [Nurse] was going to penalize us?... She’s coming in here to fix the eczema, why should she want to know this?” (Caregiver 23; Eczema) The consensus among families interviewed was that time with the Nurse was needed to facilitate the disclosure of personal information. Most families reported that they developed a level of “ trust ” with their Nurse. For example, one mother describes how, despite her initial concern around disclosing personal information, she felt able to disclose her needs once she had developed a relationship with her Nurse (Caregiver 30). However, in direct contrast, a second mother raised ongoing concerns about disclosing personal information (Caregiver 25). Caregiver 25 was the only participant to raise ongoing concerns around disclosing personal circumstances to the Nurse. These concerns surrounding the discussion of personal and social circumstances in a consultation about a health condition may reflect the traditional separation of health and social services. “ It was weird at first, but I made us a cuppa tea, had a general chit-chat. And by the end of my first cup, I felt I could open up to her. Like I knew her more as a person .” (Caregiver 30; Constipation) “O ur problems [is] her seizures, fix that … if she don’t want to speak about school, fine … if I don’t want to talk about my job, then leave it. What you do with that stuff anyway … I don’t want it on her record. ” (Caregiver 25; Epilepsy) Most participants expressed views that discussion around the broader determinants of health, and time spent with providers, enabled them to feel understood by the Nurse. This understanding then facilitated the delivery of interventions suited to the family’s needs and contextualized to their social circumstances. A typical example is provided below, whereby the child’s Nurse supported him in his desire to play football, as an important health goal or outcome the child wanted to achieve (Child 27). Evidence that this support was a key component of integrated care comes from families’ descriptions of prior experiences with health providers whose advice felt inappropriately narrow and had not considered their wider needs. “One time they said it was running in my football and I should stop. But football is everything, I want to be a professional footballer and you’ve basically said I can’t do that [referring to previous experiences of health care]. But [Nurse] really understood and took that on board and said like how we can work with asthma and football.” (Child 27; Asthma) For some families, material benefits resulted from these discussions, including housing support, food vouchers, services, and goods. However, on occasion, expectations were raised about needs that could not be met. An example of this is provided by Caregiver 31, who described their Nurse’s challenges in obtaining the resources required to support the family in the context of a stretched housing sector. “So [Nurse] tried to push Council property, but they won’t give [a property to] me because they said oh too many people are in the queue, they won’t give me. [Nurse] tried hard and pushed, he tried.” (Caregiver 31; Asthma) Whether discussion of needs resulted in changes in social circumstances or not, the key was that families felt understood by their Nurse, and that this social contextualising of their child’s health was welcomed and valued by most families.
Connected to the capacity of the Nurse to understand families’ worlds, the second theme relates to the nature of relationships that this connection engenders within the health care system. Most families, including those who were initially reticent, described a mutual trust that developed between themselves and the Nurse over time. This reciprocal trust appeared key to family engagement with the service. This manifested in participants feeling confident in asking questions about care, feeling able to suggest alternatives as well as receiving advice, and a sense of both parties (families and professionals) learning together. Numerous families reported a two-way process with the Nurse that enabled different understandings of conditions to be discussed, including beliefs around the urgency of treatment and appropriateness of medication. There was also a sense that the Nurse’s availability to families facilitated this collaborative approach (Caregiver 4). “If something happened, I could text her or call her and say, “this has happened, do you think I should do x, y or z?” We did that last week, texted her and we spoke together and came with a plan together.” (Caregiver 4; Eczema) Reciprocal relationships with professionals were also highlighted by older children as an essential component of their care. That this was attributed to integrated care was evidenced in their comparisons with previous services, in which children recalled feeling less involved in decision making. Older children reported that being involved in treatment allowed them to feel more agency and engagement with their care, highlighting the importance of respecting young people’s autonomy in treatment decisions (Child 34). “[Nurse and Mental Health Provider] actually talk to me before they talked to my parents, which I really liked because it meant that they got my permission first … I felt I was actually in control. I want to do this.” (Child 34; Asthma) These relationships with professionals and trust in the Nurse appeared to be built through experiences of professionals being transparent about mutual learning. Caregiver 32 provides a typical example of this. “[The Nurse] is continually learning, she was telling me about conferences she’d been to … and that kind of reassured me and let me trust her.” (Caregiver 32; Constipation) Physical presence, at least early in the relationship, was important. All families spoke of the importance of face-to-face consultations in facilitating the development of a relationship with their Nurse (Caregiver 22). Families reported that communications technologies (email, SMS, phone calls) maintained this relationship over time, but, importantly, these were only positively rated once carers had established a physical face-to-face relationship. The uncertainties recounted by one father (Caregiver 5), whose partner had more contact with health providers, gives an example of the importance of relationship-building. “[ The Nurse] got to know us, like as people. She could see us and understand us. But she knows us now, knows what we need … so now we do it on the phone more. ” (Caregiver 22; Eczema) “ I don’t have the rapport that [Partner] has because she’s the one that sees [The Nurse], so I don’t know how good [The Nurse] is in terms of that, you know, in terms of her overall knowledge and also what she’s allowed to advise on. ” (Caregiver 5; Epilepsy) Most families described increasing independence in managing children’s health over time, where they could rely on ‘ remote ’, rather than in-person, consultancy from their Nurse: “I would only ever go to A&E now if either I couldn’t get hold of [The Nurse], or it was a horrendous one, other than that we’re kind of happy to have sort of remote consultancy.” (Caregiver 5; Epilepsy) However, as discharge from the service approached, families’ expectations varied, with half expressing readiness for independent self-management and others reticent about discharge from the service. As one child says, “ I know what I’m doing now, I’ve got control” (Child 35; Asthma). By contrast, Caregiver 29 questioned the fairness of discharge from the service; despite her child’s needs being met, this mother wished to stay in contact with the service. This desire appeared prominent among families who were referred to the service with high levels of need as indicated by frequent use of emergency department services and concurrent mental health needs. For some families, the prospective withdrawal of the Nurse was framed as a withdrawal of care, rather than a marker of growing independence. “We’re doing everything anyway, we haven’t been to hospital … I just felt like … just stay around for a little bit just in case … to have all this and then get it taken away. That’s just not right.” (Caregiver 29; Constipation) Relationships with Nurses were facilitated through open communication, availability to families, shared learning, and face to face relationships. Ensuring active family involvement in decision making processes was key to success, supporting engagement with the service, independent self-management, and readiness for discharge.
One of the key principles of integrated care is considering physical, social, and psychological needs. Most families described the integrated service as providing holistic care, within a family, rather than just focusing on a condition. One mother used the metaphor of the service creating a “ whole circle ” supporting her family. “It’s a support system, it is not only about the child or about the person who is having the issue, it’s about the whole circle that is surrounding them, so, which is not going to improve the life of only the person who is sick, but only, even the people that are surrounded in that circle.” (Caregiver 18; Constipation) Of note, this mother speaks of a “ system ”, highlighting the importance of a wider network of professionals who support the family. References to this “ system ” are voiced by another mother who describes her Nurse talking with a paediatrician about her child’s care, highlighting again the role of the multidisciplinary team in facilitating holistic care (Caregiver 2). Caregiver 32 highlights the perceived crucial role of the Nurse as the empathetic interface for the multidisciplinary team. Both listening to families and identifying and advising on a broad range of health-related issues, including health promotion, disease and complication prevention, and the Nurse provided early intervention, coordinated care and onward referral within the multidisciplinary team when required. Other families detailed the ways Nurses discussed and identified mental health needs, and successfully brought in the wider team for onward referral to specialist services. “Sometimes [Nurse] said she’s wasn’t sure. And so it’s good to know that there’s people she can talk to, things like when she needs a paediatrician, there’s even that.” (Caregiver 2; Eczema) “[Nurse] was helping with all the anxieties, doing exercise, eating well, the anger but it got quite bad … She spoke to her team and that’s how [Mental Health Nurse] became involved … And what helped her most is that she [Mental Health Nurse] works alongside [Nurse], so she was aware of [CYP]’s problems.” (Caregiver 32; Constipation) Most interviewees were positive about these holistic aspects of integrated care and welcomed support for the whole family. However, for two families, this aspect of integrated care was perceived as unhelpful, and even unnecessary or time wasting. Both these families described considerable use of health services prior to referral to the integrated service, and their views are resonant with a biomedical focus, in which physical and mental health are separate domains: “ You’ve got this mental health thing, oh my God. Great but not for us … I just wanted to get onto the point with asthma and that’s it .” (Caregiver 26; Asthma) Holistic care is, then, a core component of integrated care from families’ perspectives: rejected by a few who preferred a more biomedical approach but emphasised and valued by most families who welcome additional support beyond the presenting condition, reassurance for caregivers, and improving the skills of the whole family in condition management.
The fourth theme is that of encountering a system which did not just address holistic needs, but also responded in a co-ordinated and coherent way. This is summarised as ‘a system that is glued together’, which reflects the ability of the integrated service to coordinate across primary and secondary care, as well as health, social, and educational sectors. Interviewees frequently described their Nurse’s ability to coordinate across providers, pulling information together, and enabling all professionals in the patient’s life to provide seamless support. “I think the service helped us socially by being present with the school and being a common point of contact with school, GP and hospital.” (Caregiver 6; Epilepsy) Most families described good communication between their Nurse, primary and secondary care, and other non-health sector services, which enabled coherent and coordinated care. Again, evidence that this was a feature of the integrated service was typically provided by comparisons of previous encounters with health and other providers (Caregiver 2). Families typically described the Nurse as ‘ talking ’ to other professionals to achieve this, however some families reported not quite knowing how communication was achieved. Several mothers described how this communication enabled efficient access to secondary care services, highlighting the Nurse’s role as patient advocate (Caregiver 4). “I don’t know how [Nurse] can manage to see what the GP is writing down because I’ve had so many problems where one doctor can see [Child’s] notes but the other can’t.” (Caregiver 2; Eczema) “[ Nurse] actually trying to get us seen by dermatology again this week … so she said she’s just talk with them [dermatology] and that’s it, done.” (Caregiver 4; Eczema) As a core component of integrated care, coordination was on occasion flagged because of its absence. Two examples of missing coordination were in the context of their children undergoing further diagnostic tests with multiple providers. This highlights the importance of Nurses with expertise in sharing information across providers, particularly during times of clinical uncertainty, which may require input from a range of providers across primary and secondary care. In addition, this demonstrates the value of coordination across sectors and providers for effective integrated care. “I was a bit baffled by her last consultant’s and GP appointment, they was saying different thing to me and I was saying to [the Nurse], it doesn’t make sense, that is confusing, it doesn’t go with what you said.” (Caregiver 19; Asthma) However, more commonly, coordination was recognised as more present in the integrated system than it had been in previous health services. Regarding the new service, families reported enhanced coordination across health providers, and between health and social sectors. One frequently reported example of the difference between an integrated service and a conventional service, was coordination between the health and education sectors, which enabled patients to follow treatment plans outside the home or hospital setting. This was facilitated by the Nurse supporting schools to take on a meaningful role in the child’s health, which appeared to be achieved through the provision of school and student training, enabling coordination across sectors (Child 33). “My teachers and friends all know what to do if something happens. So, I don’t worry about it. My mum and dad don’t worry about it. Because everyone knows what to do if something did happen. And that’s probably because they’ve all been taught about epilepsy and about my epilepsy.” (Child 33; Epilepsy) Despite families valuing coordination across sectors, some caregivers highlighted the challenges they, and their Nurse, had in coordinating care with both the education sector and across the primary-secondary interface. These difficulties highlight the challenges in working within the context of school and healthcare workforce constraints (Caregiver 19). “[ Nurse ] was waiting for school to get back about [Child] … I went in and said, oh by the way, the [Nurse] has been trying to contact to try and speak to you about [Child] and has had no response … they say they’re too busy.” (Caregiver 19; Asthma) Whether families experienced a coordinated service or experienced challenges with coordination across the system largely depended on how well health, social and education sectors were updated with children’s health records: a key aspect of integrated care.
Despite the wealth of calls for integration of complex systems to address the gaps in healthcare delivery for child health, there has been little evidence on what integrated approaches look like for children and young people . This is the first study using robust qualitative research to document family perspectives on a new integrated service and to identify essential components of integrated care for children and young people. The four components identified as contributing to the quality of integrated care are: 1) that the nurse understood their world 2) that professionals involved the family in treatment; 3) that professionals provided holistic support to the family; and 4) that the system was ‘glued together’, in that professionals coordinated the existing health, social, and education systems. A growing body of work speaks to the importance of positive social aspects of system integration . The nurses’ role was key to the implementation of the CYPHP model. Within this it was trust between the family and the practitioner that seemed to be central. Trust is important in both effective relationships between patient and provider, and across the health system . In their realist review, Tyler et al. highlight the importance of “bridgers” or “connectors” in enabling trust between families and the health system to foster integrated care . Similarly, families in this study highly valued the relational aspects of integrated care; relationships with providers, and trust, were central to families’ descriptions of good care. Like Tyler’s findings, trust was embodied to some extent within the Nurse and the role he/she played in engaging the child and wider family in treatment, providing holistic care, and facilitating access to specialist care. Clinical processes that facilitated these relational aspects of care included increased time with, and ease of access to, the Nurse. Face-to-face consultations with the Nurse were important; once established, relationships could then be supplemented using communications technology. These findings support Vassilev’s argument that technology is one component of an integrated system that only delivers benefits to patients when it supports the development of established patient-provider relationships . This work expands upon the work of Plochg and colleagues, who question whether integration can improve the performance of healthcare without breaking down current silos within the health system . In the model studied here, such structural integration across primary and secondary care seemed manifest in families’ accounts of the value of their Nurse’s actions in care coordination. Families spoke of the Nurse advocating for their needs, working as part of a multidisciplinary team, across primary-secondary healthcare boundaries and across interfaces of health, social and educational services. Clinical processes enacted by the Nurse went beyond navigating or signposting to services or resources, for example Nurses advocated and through closer inter-sectoral collaboration enabled referral to additional services. Further work is needed to understand the complexities of implementing this way of working in which building family relationships and coordination with the wider health system seem crucial, to negate unintended consequences, for example on discharge from care. Finally, this work highlights the contextual and organisational processes that contribute to perceptions of high-quality integrated care . Families highlighted the importance of delivering care which takes their holistic needs and social contexts into account, thereby also addressing the wider determinants of their health. However, the identification of additional needs, in a financially stretched health, educational, and social care environment meant that identified needs outside the health service could not always be met. Care navigation may be a necessary condition for quality care from family perspectives, but it is not a sufficient condition. Challenges reflect difficulties in integrating across sectors, whilst operating in constrained economic conditions. Integrated care cannot, therefore, necessarily mitigate shortcomings elsewhere in-service provision and policy; broader system integration and whole society change is essential to improve the quality of life for children with long term conditions. For child health, inter-sectoral collaboration across health and education sectors was critical and facilitated through the family’s Nurse. These findings resonate with the wider literature, highlighting the importance of inter-sectoral collaboration as a key mechanism in ensuring the success of integrated child health programmes . Strengths and limitations The inclusion of family perspectives from diverse socio-economic areas provides valuable insight into the essential components of integrated care for children and young people with ongoing conditions. A considerable strength of this study was the inclusion of families who have traditionally been thought of as experiencing greater barriers to accessing healthcare, including those from the 20% most deprived areas across the UK. However, as the sample was self-selecting, findings may represent the views of those who were more articulate and willing to come forward. In addition, the intervention described here was implemented across one large health setting; it is anticipated that integration at the broader system-level may be more challenging. Furthermore, caregivers interviewed were largely female, with only two fathers participating in interviews.
The inclusion of family perspectives from diverse socio-economic areas provides valuable insight into the essential components of integrated care for children and young people with ongoing conditions. A considerable strength of this study was the inclusion of families who have traditionally been thought of as experiencing greater barriers to accessing healthcare, including those from the 20% most deprived areas across the UK. However, as the sample was self-selecting, findings may represent the views of those who were more articulate and willing to come forward. In addition, the intervention described here was implemented across one large health setting; it is anticipated that integration at the broader system-level may be more challenging. Furthermore, caregivers interviewed were largely female, with only two fathers participating in interviews.
Families value specific elements of integrated care, identifying key components that make integrated services high quality. A diverse sample of families with a range of experiences shared important common views about the components needed to integrate health services for children and young people with ongoing conditions. Drawing on our analyses, we make the following recommendations for integrating child health services in ways that are resonant with families’ perspectives: Patient empowerment is delivered through personal relationships. Communication technologies can then subsequently supplement the therapeutic relationship. Co-ordination between health, social, and education services for children is vital. Assessment and understanding of families’ social needs and supporting young people’s autonomy are areas where integrated care may be enhanced. Nurses working across organisational boundaries, working as care providers and navigators, may mitigate, but not overcome poorly funded, fragmented services.
Additional file 1.
|
Elaboration, validation and reliability of the safety protocol for
pediatric thirst management | 30069b9f-c334-4aa6-af67-8d614fd091f2 | 7365611 | Pediatrics[mh] | The perioperative period brings innumerable coping challenges for the child. In the
preoperative period, preparations inherent to the procedure, such as fasting, bring
anxiety and discomfort . Fasting is indicated in order to avoid adverse events such as
bronchoaspiration for gastric contents . Although its indication is recognized and the literature currently
recommends reduced fasting times , excessive periods are identified in practice . Recent evidence shows that abbreviating the fasting time not increase
adverse events incidence . Fasting is extended to the immediate postoperative period (IPP) and fluids are
usually released in the first three hours for most children . However, a clinical trial revealed that fluid intake even more
precociously in the Post Anesthesia Care Unit (PACU) did not increase the incidence
of nausea and vomiting . The benefits of early fluid release in the IPP are: More parental
satisfaction, happier and less uncomfortable children with pain, reduced use of
medication for nausea, reduced length of stay in PACU, and reduced
thirst . Anesthetic recovery is characterized by the return of consciousness and during
awakening, the child may experience pain, being confused and agitation. Thirst also
influences the child’s mode of awakening and recovering from anesthesia, being one
of the factors responsible for the anguish they experience in this
period . The surgical child is at high risk for developing thirst due to hydroelectrolytic
imbalance, endotracheal intubation, use of medications, among others . The nursing team working in the PACU therefore needs to consider
thirst as an object of care intentionally, identifying, measuring, assessing safety
and using effective strategies to reduce the child’s thirst . The team, however, usually feels insecure to treat thirst in the anesthetic recovery phase, as it does not have systematic
instruments that assess safety to offer a method of relieving pediatric thirst,
prolonging the suffering of the child and his family . To support the team in the decision to use a thirst relief strategy, the Safety
Protocol for Thirst Management (SPTM) of adult patients in PACU was
elaborated . The team has also used this instrument for the infant patient, even
without proving that the proposed evaluation criteria for the adult are also
relevant for the child. The instrument validation process is essential for the results to be significant,
reliable, precise and accurate . Validity and reliability are the main aspects in the process. Validity
verifies whether the instrument measures exactly what it proposes to measure and
reliability represents the degree of coherence with which the instrument measures
the attribute . The need to develop and validate a safety protocol for the management of thirst in
children in the IPP is justified by its high prevalence and intensity . In addition, no instrument was found to support the practice of PACU
professionals in the assessment of adequate criteria that allow the effective use of
effective strategies to relieve the child’s thirst in this period. The objective of
this study was, therefore, to elaborate, validate and evaluate the reliability of
the Safety Protocol for Pediatric Thirst Management (SPPTM) in the IPP.
Methodological, quantitative research, carried out between July 2017 and April 2018.
In view of the difficulty in finding specific methodologies for the elaboration of
protocols that presuppose decision-making for care and aiming to follow a rigorous
methodological process, an adaptation of the steps of the Pasquali model was
used . This model is based on psychometry that measures subjective phenomena
and was used by another protocol validation study as a guide to its
steps . This model consists of three procedures - theoretical, experimental
and analytical , whose steps are summarized in . In the theoretical procedures stage, it is recommended to search the literature,
clustering the knowledge of specialists and observation extracted from practical
experience . The psychological system was defined as safety for pediatric
thirst management in the immediate postoperative period, and assessment criteria as the property of the psychological system
(attributes), whose evaluation is the object of this study. The elaboration of the
protocol was carefully based on scientific literature, interviews with specialists
and systematic observation of the child’s anesthetic recovery . A literature search was conducted in the: Literatura Latino-Americana e do Caribe em
Ciências da Saúde (LILACS), Cumulative Index to Nursing and Allied Health Literature
(CINAHL), National Library of Medicine (PubMed) e The Cochrane Library, using the
following descriptors: child, pre-school, students, hospitalized child, recovery
period from anesthesia, post-operative period, caring for the child, thirst,
recovery room, scales, respiration, awareness state, cough, general anesthesia,
nausea and vomiting, swallowing, postoperative complications, gastrointestinal
content, aspiration pneumonia e oral hydration. The criteria for inclusion were the
following: Publications in books and articles indexed in the selected databases with
descriptors in Portuguese, Spanish and English, from 1960 onwards, since it was the
decade when the first descriptions of the child’s arousal upon awakening from
general anesthesia were found. Anesthesiology, child development and growth, and
surgical child care books were also examined . Eighteen experts were consulted under the following inclusion criteria: having
experience in assisting hospitalized children or in the IPP, working in large public
and/or private hospitals in the city of Londrina. The invitation was made
electronically and later the interviews were scheduled in accessible places for the
professional. The interviews took place in person, and the professionals answered a
script made up by six guiding questions: What should be observed in the emergency of
pediatric anesthesia?; What instruments to use to assess the child’s anesthetic
recovery?; Which protective reflexes are most important to be evaluated in the
child?; How should the assessment of children recovering from anesthesia be? What
needs to be considered to manage a thirst relief strategy in children who recover
from anesthesia?, and Is there a difference in age? Responses were recorded and
tabulated in an Excel 2010 ® spreadsheet and analyzed according to the
frequency of citations. The main researcher made a period of systematic observation on the children’s
anesthetic recovery. During August and September de 2017 the main behaviors
presented when they awoke from the general anesthesia were recorded. Seventeen
children submitted to general anesthesia and older than three years were evaluated,
selected by convenience according to the researcher’s availability during this
period. The results of the literature search were organized in a table listing the main
surgical anesthetic complications, scales for assessing the child’s awareness and
criteria for allowing early fluid intake in the IPP. In the second column, the
responses of the 18 specialists were systematized, considering the criteria
considered relevant for the release of oral liquids during the child’s anesthetic
recovery. The third column consisted of the main behaviors of the children observed
when they woke up from anesthesia. After extensive analysis by the main researchers,
the most relevant common criteria were selected among the three stages. Next, the constructs were defined, which consists of a clear and precise
conceptualization of each criterion selected to assess safety for the management of
pediatric thirst . In the protocol, they are the items to be evaluated and are described
in detail below each criterion. For example, what to evaluate in the criterion
“level of consciousness”, what to evaluate in “movement” and so on. Subsequently, the behavioral representation of the constructs was
established and the actions that the nurse must take to assess safety for the
management of thirst were defined . Finally, the operational manual was
prepared, which presents the theoretical basis of the protocol. Theoretical analysis was performed by specialists through the apparent
validation , semantic analysis and content validation . Two Ph.D. nurses specialized in children were invited to perform the
apparent validation in September 2017. The criterion for choosing these
professionals was expertise in the pediatric area and in instrument validation, who
were not part of the thirst study group. The semantic analysis occurred in a tertiary-level university hospital in northern Paraná, in
October 2017. Eight participants were invited, divided into two distinct groups. The
first with four Ph.D. nurses with experience in child care in PACU; the second with
four students from the last year of the Nursing undergraduate course. The protocol
was presented verbally to the two groups in separate meetings, item by item, later
on, the participants were asked to reproduce their understanding on the exposed
content. The content validation took place in November and December 2017 through the Delphi
Technic . Thirteen professionals were invited, two did not accept to participate
and two did not return the instruments in the appointed period. Therefore, nine
judges participated, including nurses (n = five), anesthesiologists (n = three) and
a speech therapist (n = one). There was a concern to include judges from different
academic backgrounds so that the contributions to the instrument could include a
multiprofessional look. The judges were chosen according to their experience in
child care in PACU in different institutional realities, and one, for her experience
in validating instruments. The judges worked professionally in Londrina (PR) and São
Paulo (SP), and all had postgraduate education, being the doctorate the most
frequent (n = four, 45%). Professional experience was over five years for all the
specialists. The judges who participated in this stage were present at other moments
in elaborating and validating the protocol: Interview stage (n = five) and apparent
validation (n=two). The invitation was made by telephone, informing the research objectives and how
participation would be. Upon agreement, they were asked for the email address for
subsequent shipment of the validation instruments. In the first contact via e-mail,
a letter was sent with the research objectives, validation procedures and invitation
to participate as a judge. In the annex, the protocol, four validation instruments,
instrument of characterization of the judge and the Free and Informed Consent Form
(FICF). The reply to that email was considered acceptance to participate in the
survey. The instruments for validation were adapted from other studies . The Content Validity Index was used ( Content Validity Index - CVI),
based on the proportion of judges who considered the item valid . The CVI was estimated for each protocol evaluation criterion, set of
items, operational procedures and operating manual. The individual CVI was
calculated from the ratio of the number of specialists who scored three or four for
the item on an ordinal scale of one to four (from does not contemplate to
contemplate), or on a dichotomous scale (yes and no), by the total number of
experts. The total CVI was calculated from the average of the CVIs of the
items . The minimum agreement established between the judges was
0.80 . Microsoft Office Excel 2010 ® was used for the
calculations. Four assessment tools were sent to the judges. First one, the judges evaluated the
safety criteria according to the requirements: Attributable (reflects quality aspect
for nursing care), Accessible (data is accessed quickly, with minimal extra effort
and cost), Communicable (the relevance of the measure can be easily communicated and
understood), Effective/accurate (measures what it is proposed to measure), Feasible
(the measure is applicable) and Objective (the measure allows clear and precise
measurement action, without subjective judgment). The judges indicated points
ranging from one to four, with one = does not consider security for thirst
management; two = unable to contemplate security for thirst management without
review; three = includes security for the thirst management, but needs a minimum
change; four = includes security for the management of the thirst. The second assessed the set of items, ticking yes or no on the following
requirements: Behavioral (allows clear and precise assessment), Objectivity (allows
punctual response), Clarity (spelled out in a clear, simple and unambiguous way),
Relevance (evaluates safety for the management of thirst), and Precision (each
evaluation item is distinct from the others, do not elicit confused). The third assessed the operating procedures using the same requirements as instrument
two. The fourth instrument evaluated the validity of the operational manual, indicating
yes or no in the requirements of Descriptor (it is clear and objective in what it
proposes to measure) and Scientific Basis (it is sufficient to evidence the
indicator). Within the experimental procedures, a pilot test was carried out with six children in
January 2018 to adjust the collection procedures. Initially, the researchers
evaluated the child 30 minutes after arriving at the PACU, but it was observed that
a longer time was needed to start the evaluation, as they were still sleepy, with
limitations to participate in the process. Then, the first assessment was
established 45 minutes after arrival at the PACU, and the second, 15 minutes after
the first. There was no need for changes in the collection instrument. Pilot test
participants were not included in the sample. The analytical procedures consisted of assessing the protocol’s reliability by
inter-rater agreement. The kappa coefficient was used to estimate
the agreement among the evaluators, calculated by the ratio of the proportion of
times the observers agreed (corrected by agreement due to chance) to the maximum
proportion of times they could agree . The determination of the agreement strength of the kappa values followed the following recommendation: Less than
zero, poor agreement; from zero to 0.20, negligible agreement; 0.21 to 0.40, smooth
agreement; 0.41 to 0.60, moderate agreement; from 0.61 a 0.80, substantial
agreement, from 0.81 to one, almost perfect agreement . Reliability was assessed in the PACU of a tertiary-level teaching hospital in the
State of Paraná, with the participation of two pairs of nurses. The first was made
up by the researcher and a resident in perioperative nursing; the second, by the
researcher and a nurse from the PACU. The pairs were chosen for their availability
to participate in the research and for their experience in child care in the PACU,
conditioned they first participated in the training on the SPPTM. The sample was determined by collection time, totaling four months. The criteria for
inclusion were the following: Surgical children aged between three and 12 years old,
of both sexes, to be recovering from anesthesia in the PACU, undergoing procedures
of any specialty and anesthetic technique, elective or emergency procedures
performed from Monday to Friday, from 7:00 am to 7:00 pm, conditioned to
availability of the two evaluators. The exclusion criterion was a child with
neurological disorders and mental disorders, as they might not be able to express
the necessary answers for the assessment. The minimum age for inclusion was three
years old, because, from then on, the child is able to speak his own name, name
objects, show ability to move, has more precise movements and can handle
objects . The pair of nurses applied the protocol independently and simultaneously, without
communication among the evaluators. While one professional applied the SPPTM, the
other just followed and recorded the considerations; in the next evaluation, the
professionals reversed the order. The pair waited for the child’s arrival at the
PACU and the researcher talked to their parents, asking for authorization to carry
out the research. At this moment, they were informed on the goals of the study and
how the child participation would take place. After having accepted, the primary
guardian signed the FICF, the children were asked about their willingness to
participate in the research, and no child refused. A 12-year-old child participated
in the study and signed the FICF. After arriving at the PACU, the researcher
explained the objectives of the research and assessed their intention to take part
in it. It was determined that each child could be assessed twice: the first
assessment 45 minutes after arriving at the PACU; the second, 15 minutes after the
first one. If the child was agitated, tearful or with pain, a longer time was
observed to begin collection. One used the program Statistical Package for
Social Sciences - SPSS ® (version 20.0) to calculate the kappa coefficient and carry out the descriptive analysis.
In the literature search, a specific evaluation scale was found when the child
awakens from sedation and regains consciousness, with the following items: eye
response, appearance and function, and body movement . Regarding the main complications in the IPP, pain, nausea, vomiting
and emergency delirium (ED) stand out. It is a common condition in children in the
IPP, defined as a disturbance in the child’s awareness and attention to his
environment, with disorientation and perceptual changes , with the presence of restlessness, crying, moaning or irritating
speech and screams . Few evaluation criteria were found for early fluid release:
Spontaneous verbalization of the child , appears to be awake enough and receive a score that is greater than or equal to four on the Face, Legs, Activity, Cry, Consolability scale
(FLACC), a scale that assesses the child’s pain. In the interview stage, the experts pointed out the following safety criteria:
assessment of level of consciousness (n = 12), airway protection reflexes (cough n =
17, swallowing n = 12, crying n = three), absence of nausea and vomiting (n =
three), movement evaluation (n = five), consider the participation of the main
caregiver (n = four), child’s will (n = nine), medical criterion (n = two), surgical
time and size (n = one). During the observation period of the child’s recovery, the main findings were the
following: Variability in the time of emergence of anesthesia, bodily behaviors such
as movement of limbs and eyes, type of verbalization and presence of crying. Based on the analysis of the previous steps, the following evaluation criteria were
then selected to compose the SPPTM: level of consciousness, movement, airway
protection (coughing and swallowing) and absence of nausea and vomiting. The
selection sought to meet the maximum requirements for safety, simplicity and ease of
application in clinical practice. Then, it was defined with the consulted
specialists that the protocol can be used in children aged between three and 12
years old. Apparent validation resulted in the inclusion of the respiratory standard assessment
criteria, which had not been proposed. In its final version, the protocol consisted
of five evaluation criteria, arranged in a graphic algorithm , in which it is necessary to approve the child in all
the evaluated criteria. The identification of any clinical condition that shows
failure in the evaluated criterion represents interruption in the use of the
protocol. Then, a new assessment should be started after a period that allows a
change in the child’s clinical status. The five evaluation criteria are described in capital letters in the protocol and
identified in dark gray. Below each one of them there are the items to be evaluated
and in the right column are the operational procedures. The sentences highlighted in
light gray represent approval in the criterion. Additionally, the operational manual
was elaborated, which contains the theoretical basis for the protocol. The manual
can be obtained in full in the author’s master’s dissertation. As for the semantic analysis, the group of students and the group of Ph.D. nurses did
not have any difficulty to understand the items. They only made some editorial
adjustments and changes to the evaluation orders. A single round of content evaluation by the experts was sufficient to overcome the
minimum agreement of 0.80 . shows the CVI values of
the evaluation criteria and their representative items. displays the CVI values of the
operating procedures and the operating manual for each criterion. During the reliability assessment, SPPTM was applied 116 times in 58 children. The
mean age was 7.2 years old (sd 2.6). Children of all ages for whom the protocol was
developed took part: three years (n = seven), four years (n = three), five years (n
= eight), six years (n = seven), seven years (n = six), eight years (n = six), nine
(n = seven), ten years (n = five), 11 years (n = eight), and 12 years (n = one). Male children predominated 42 (73%); the frequency of procedures by surgical clinics
was: infant and pediatric surgery 32 (55%), otorhinolaryngology 15 (26%),
orthopedics eight (14%), ophthalmology two (3%), head and neck one (2%). The
anesthetic technique with the greatest use was general anesthesia 51 (88%). As for
the classification of surgical risk, according to ASA, most were classified as ASA
I, 49 (84%), followed by classification II, nine (16%). The majority of the
procedures was of elective nature 50 (86%), being eight (14%) of urgency. shows the values of kappa calculated for each SPPTM evaluation item, with almost
perfect agreement for all items .
The contribution of this study consists of making available an unprecedented,
judicious, objective, valid and accurate instrument that allows assessing safety to
manage strategies for relieving thirst for infant patients in the IPP. For the
elaboration, validation and evaluation of the protocol’s reliability, high
scientific rigor followed . The interviews with specialists made it possible to observe how diverse and
subjective the criteria used by the professionals responsible for authorizing
methods to relieve thirst in the IPP are. Professionals reported that, most of the
time, they look at the child in the PACU and assess whether, apparently, they are
awake enough and without complaints, then they allow the intake of liquid orally.
However, this assessment is not standardized or based on criteria and varies
according to the determination of “being well awake” by each professional. It was
also observed that, when liquid intake is authorized, there is no consensus as to
the type and volume to offer. There were reports on the limitation of specific
literature for the child, resulting in adapted evaluations, which consider criteria
of adult patients. Currently, the anesthesiologist is responsible for the
authorization for liquid oral ingest in the PACU, which explains the greater number
of them in the interview stage. The “level of consciousness” criterion was one of the most frequently suggested by
professionals, considered an essential item to determine the emergence of the
anesthetic state during the IPP. When asked about the scales used to assess
children’s awareness, the answers were varied: Glasgow comma scale , Comfort-Behavior , Index Steward scale of Aldrete and Kroulik . However, the Glasgow and Comfort-B scales do not apply to children in
the IPP, because they assess the level of sedation and have been validated for
children in the intensive care unit. The Steward Index and the scale of Aldrete and Kroulik , although targeted at patients in the PACU, may not be adequate to be
used with a child . A scale for assessing the child’s consciousness after sedation was found in the
literature . This is the Vancouver Sedative Recovery Scale (VSRS),
a scale made up by 12 items covering three categories of indicators: Response,
appearance and function of the eyes, and body movement. Reliability was assessed in
82 children aged between nine months and 17 years old. The internal consistency
measured by Cronbach’s alpha was 0.85, interobserver agreement 0.90, and values of kappa for the individual items ranged from 0.65 to
0.89 , values similar to those found in this study. Some items on this scale
are similar to those of the SPPTM: The child is alert, sleepy, able to make eye
contact, presence of spontaneous and intentional movements. The other scales found in the literature consist of ED measurement scales. One of the
most used scales to measure this condition is the Pediatric Anesthesia Emergence
Delirium (PAED), made up by the following items: the child makes eye contact with
the caregiver; the child’s actions are purposeful; the child is aware of the
surroundings; the child is restless, and the child is inconsolable. This scale was
evaluated on 46 children aged between 18 months and six years and displayed an
internal consistency of 0.89 and a reliability of 0.84 . Therefore, for selecting the items for evaluating the SPPTM awareness
level criterion, the presence of these behaviors was considered. When evaluating the item “is oriented” in the behavioral requirement, some experts
indicated that children aged between three and five years could possibly not answer
their name and age because they are in an unknown environment and regaining
consciousness. There was no such difficulty during the application of the protocol
in practice. However, this study employed a convenience sample, and a larger number
of this population would be needed to assess this issue in depth. It is more difficult to assess the child’s level of consciousness than that of the
adult, and it is challenging to identify the child’s inability to
communicate . When assessing reliability, the evaluators disagreed on the items “is
alert” and “is sleepy”, confirming the difficulty and subjectivity in assessing the
child’s level of consciousness. The need for a period of interaction with the child
was identified before starting up the assessment. Two judges considered the criterion “movement” as not relevant in measuring safety
for the thirst management. For others (n=three), it represents an evaluation
criterion complementary to the level of consciousness, measured by the ability to
perform intentional movements and keep the head firm and aligned with the trunk.
Additionally, the presence of voluntary and purposeful movements is part of the
scales for assessing the child’s consciousness , justifying the choice to keep this item in the protocol. In addition,
the ability to move with intentionality may indicate reversal of general inhaled
anesthetics and neuromuscular blockers. The evaluation of criterion “airway protection” ensures the verification of the
return of protective cough and swallowing reflexes. These reflexes indicate that the
patient is able to defend himself against a possible bronchopulmonary
aspiration . The incidence of perioperative pulmonary aspiration in pediatric
patients varies from one to ten in 10,000. Additionally, when there is a
consequence, it is considered mild and, to date, there have been no reports of
mortality from pulmonary aspiration in children . Evaluating the protective reflexes in the SPPTM presupposes the
evaluation of cough and swallowing. Two experts pointed out in the content validation that the assessment of protective
reflexes (coughing and swallowing) could encounter some difficulty with younger
children. However, they considered this item as of extreme relevance in order to
determine the safety for oral liquid release in the IPP. Therefore, a prior approach
to the child is recommended, in order to reduce the anxiety and fear present in this
period, so that there is a bond and trust in the moment of assessment. During interviews with specialists, it was mentioned that crying could be considered
a protective reflex, indicating that the child’s airway would be free. But crying
can represent several situations, such as pain, discomfort, irritation, agitation
and ED. Differentiating their presence is difficult and subjective, therefore, in
the protocol, the presence of crying characterizes the child’s failure to receive a
method of relieving thirst. The “breathing pattern” consists of the assessment of respiratory frequency and
respiratory effort, when signs of accessory muscle contraction, intercostal,
subcostal and wishbone retraction, and nose wing beats must be absent . For some professionals, the evaluation of this criterion signals the
main changes in the child’s clinical status. Furthermore, adverse perioperative
respiratory events represent one of the main reasons for morbidity and mortality in
children . The absence of “nausea and vomiting” is paramount for administering methods for
relieving thirst. The presence of vomiting is still a complication feared by the
team due to the possibility of subsequent pulmonary aspiration, although recently,
its incidence is between 25% and 30% in children undergoing general
anesthesia . The absence of these complications indicates reversion of anesthetic
agents. Clinical trials have evaluated whether post-operative fasting would reduce the
incidence of nausea and vomiting in children. One study found no statistically
significant difference between the two groups observed, with incidence of 15% in the
liberal group and 22% in the fasting group (p = 0.39) . Another study revealed an association between early postoperative oral
fluid intake and a reduction in the incidence of vomiting, which was 11.4% in the
liberal group and 23.9% in the fasting group . In both studies cited, the child’s willingness to receive liquid and
food was considered. When the child is forced to drink fluid early, there is
increased vomiting incidence . The experts considered the child’s willingness to drink and the
child’s verbalization as relevant evaluation criteria. Therefore, when questioning
the presence of thirst in the child, it is also necessary to question his
willingness to receive any strategy to relieve thirst and only then begin the SPPTM
assessment. The application of SPPTM by nurses showed a high overall value of the kappa coefficient. This means that this instrument has
inter-rater agreement, indicating that it can be reproduced in other realities.
Thus, there is an indication that this instrument is a useful tool for the nursing
care in the PACU, minimizing the presence of a prevalent and intense symptom such as
thirst, especially for the infant patients. One of the obstacles encountered in conducting this study was the scarcity of
instruments to assess the child’s anesthetic recovery, resulting in the difficulty
of structuring the criteria to direct the child’s assessment in this period in
relation to the release of liquid orally by the professionals. This study,
therefore, has come to fill a gap in the literature and to subsidize the care
provided to the surgical child with thirst. Assessing safety for thirst management, using relevant selected criteria, allows
nurses to look intentionally at a frequent symptom and to safely intervene safely in
its management. It is noteworthy that the protocol was designed for children who do
not have communication limitations and children without contraindications to
receiving oral fluids in the IPP. The limitation of this study was centered on the convenience sample. It is suggested,
therefore, that the protocol be applied to a larger number of children, in other
institutions and with stratification by age. Further studies are needed to assess
factors associated with approval of the protocol, as well as the most suitable
moments for its use in the child’s anesthetic recovery. Even so, the reliability
values of the SPPTM were high, indicating the accuracy of this instrument.
The SPPTM was elaborated based on the relevant signs and symptoms in determining
safety for administering methods to relieve pediatric thirst in the IPP. The safety
criteria and their representative items were identified after a rigorous scientific
basis, interviews with specialists and a period of systematic observation of the
child’s anesthetic recovery. This unprecedented protocol proposes five evaluation criteria: Level of
consciousness, movement, airway protection (coughing and swallowing), breathing
pattern (respiratory rate and respiratory effort), and nausea and vomiting. The judges performed theoretical analyzes through apparent, semantic and content
validity. The SPPTM is easy to understand, has relevant and relevant content, with a
high level of agreement among the judges on all the items evaluated. This indicates
that the evaluation criteria proposed by the protocol measure with satisfaction the
safety for the management of pediatric thirst. When evaluating the reliability of the protocol in its practical application with
surgical children aged between 3 and 12 years in the IPP, it was possible to observe
an almost perfect agreement between the evaluators. The SPPTM is, therefore, a valid and accurate instrument, indicating that it is a
useful tool for use in clinical practice in the PACU, enabling the safe management
of pediatric thirst.
|
HBV Transmission Knowledge Among Korean-American Chronic Hepatitis B Patients in the United States | 6f6f47a6-d3b0-4b61-8236-504b1a0ba93b | 11937223 | Pathologic Processes[mh] | Chronic hepatitis B (CHB) is a condition that disproportionately affects Asian Americans in the United States (US). Although Asian Americans only make up 6% of the US population, this group makes up approximately 58% of all hepatitis B cases . Many of these cases can be attributed to immigrants arriving from Asian HBV-endemic countries . In the US, hepatitis B virus (HBV) screening rates among Asian Americans have previously been found to be low (< 50%) . Delays in treatment from undiagnosed HBV can lead to hepatocellular carcinoma and other hepatitis B-related morbidity and mortality . Knowledge and awareness of HBV are associated with better healthcare-seeking behaviors . Sociodemographic factors associated with better knowledge include younger age, higher English proficiency, education attainment, and socioeconomic status [ , , ]. Even after diagnosis, having knowledge of transmission is crucial for patients to continue practicing prevention strategies to limit the spread of HBV. The most common route of transmission for HBV is through perinatal transmission, sexual intercourse, and contact with infected blood . Among Asian CHB patients, several misperceptions about HBV transmission have been identified, such as spreading HBV through the sharing of food, eating and drinking utensils, and breastfeeding [ , , ]. These misperceptions can lead to increased social distancing and stigma against people with CHB . Moreover, it can cause heightened stress and anxiety for those living with CHB, and unnecessary lifestyle restrictions associated with fears of spreading HBV to others [ , , ]. Previous studies have measured HBV knowledge among Asian Americans [ , , , ], but few have explored HBV knowledge among a subset of Korean-American CHB patients who receive specialized hepatology care from a Korean clinician. Cultural concordance in care is thought to remove specific barriers, such as language, that Asian American patients would face otherwise . By having a healthcare provider who speaks the same language and has the same cultural background, communication between patients and providers may be more effective, potentially leading to improved understanding of CHB among patients . The objectives of this study are to assess the current state of HBV transmission knowledge among Korean-American CHB patients and to see whether language preference impacts their knowledge of Hepatitis B transmission.
Study Design and Participant Sample This is an explanatory mixed-methods study using survey data and in-depth interviews . This analysis is part of a larger prospective longitudinal cohort study that aims to look at disparities in liver disease progression among Korean Americans with CHB. Patients were recruited from two clinical sites in Philadelphia and Los Angeles, where they receive care from bilingual Korean-American hepatologists. The cohort enrollment criteria include individuals who are over 18 years old, have the cognitive ability to provide informed consent and complete study procedures, have been a recipient of care from the participating clinical site since at least 2016, and have a diagnosis of CHB, but without a prior diagnosis of hepatocellular cancer, or co-infection with hepatitis C or HIV. Recruitment occurred between August 2021 and January 2023. Participants could complete the survey and interview in Korean or English. More information about the study design for the overall study can be found here . Medical record review identified 552 eligible patients, of whom 140 (25.4%) did not return for care nor respond to multiple contact attempts. Of 412 patients contacted, 4 (1%) declined to return to the clinic during enrollment, and 16 (3.8%) were ineligible after further screening. Of 392 eligible patients, 27 (6.8%) refused to participate, and 2 did not complete the enrollment questionnaire; therefore, the final analytic cohort includes 363 patients. Baseline Survey Measures Outcome Measures Transmission knowledge was measured with our previously validated index listing ten potential transmission modes with response choices of yes, no, or don’t know . Each item was recoded (1 = correct, 0 = incorrect/don’t know), and summed scores of 0–10 were used to measure respondents’ knowledge, with higher scores indicating greater knowledge about HBV transmission. The summed score showed good reliability (alpha = 0.87), suggesting that the individual items reliably captured underlying knowledge of HBV transmission . Independent Measures Sociodemographic variables used in these analyses included respondent gender, age, country of birth, education, survey language preference, use of five possible sources for HBV-related information, having a primary care provider (PCP), years since HBV diagnosis, event precipitating HBV testing and diagnosis and awareness of infected family members. Qualitative Interviews Approximately one year after enrollment, qualitative in-depth interviews were conducted with a subsample of participants. These interviews explored baseline findings to provide contextual understanding of participants’ lived experience with CHB. Participants were selected using a quota-sampling strategy to balance gender, geographic location, and language, and purposive sampling to capture cohort diversity in age, socioeconomic background, immigration stories, and health histories. A sample size of 30 participants was selected a priori, based on the focused nature of the interview topic and the potential for richly descriptive interview content from each participant. Of the 363 participants in the total cohort, 49 participants were contacted sequentially as potential interview participants. Among the 49 contacted participants, 7 declined, 5 agreed but did not complete scheduling, and 7 did not respond despite repeated contact attempts. Two bilingual research assistants conducted audio-recorded interviews over the phone in the participant’s preferred language. The interview guide used a phenomenological approach to explore each participant’s lived experience from diagnosis to the present. Participants provided verbal consent and received a $40 gift card. Each interview was transcribed verbatim, and Korean interviews were translated into English. The first iteration of the codebook included deductive and inductive themes. Reliability was established by having four co-authors independently code two transcripts, adding additional themes and codes as needed, and discussing coding differences to reach consensus. Using the finalized codebook, two research assistants who conducted the interviews coded the remaining interviews, using NVivo14 for coding and thematic analysis. Analysis All survey data were cleaned and analyzed using SPSS. Descriptive statistics were used to examine the sample’s baseline characteristics and distribution of responses to HBV knowledge questions. Bivariate associations were explored between the total HBV knowledge score and all independent variables. For categorical variables, mean group scores were compared using t-tests and p-values. For continuous variables, correlations between the HBV knowledge score and each variable were presented with p-values. A final multivariable linear regression was selected using a stepwise backward selection process. Thematic analysis of participants’ experience talking about HBV transmission with others was conducted for all qualitative in-depth interviews. Central themes with example quotes from participants are presented.
This is an explanatory mixed-methods study using survey data and in-depth interviews . This analysis is part of a larger prospective longitudinal cohort study that aims to look at disparities in liver disease progression among Korean Americans with CHB. Patients were recruited from two clinical sites in Philadelphia and Los Angeles, where they receive care from bilingual Korean-American hepatologists. The cohort enrollment criteria include individuals who are over 18 years old, have the cognitive ability to provide informed consent and complete study procedures, have been a recipient of care from the participating clinical site since at least 2016, and have a diagnosis of CHB, but without a prior diagnosis of hepatocellular cancer, or co-infection with hepatitis C or HIV. Recruitment occurred between August 2021 and January 2023. Participants could complete the survey and interview in Korean or English. More information about the study design for the overall study can be found here . Medical record review identified 552 eligible patients, of whom 140 (25.4%) did not return for care nor respond to multiple contact attempts. Of 412 patients contacted, 4 (1%) declined to return to the clinic during enrollment, and 16 (3.8%) were ineligible after further screening. Of 392 eligible patients, 27 (6.8%) refused to participate, and 2 did not complete the enrollment questionnaire; therefore, the final analytic cohort includes 363 patients.
Outcome Measures Transmission knowledge was measured with our previously validated index listing ten potential transmission modes with response choices of yes, no, or don’t know . Each item was recoded (1 = correct, 0 = incorrect/don’t know), and summed scores of 0–10 were used to measure respondents’ knowledge, with higher scores indicating greater knowledge about HBV transmission. The summed score showed good reliability (alpha = 0.87), suggesting that the individual items reliably captured underlying knowledge of HBV transmission . Independent Measures Sociodemographic variables used in these analyses included respondent gender, age, country of birth, education, survey language preference, use of five possible sources for HBV-related information, having a primary care provider (PCP), years since HBV diagnosis, event precipitating HBV testing and diagnosis and awareness of infected family members.
Transmission knowledge was measured with our previously validated index listing ten potential transmission modes with response choices of yes, no, or don’t know . Each item was recoded (1 = correct, 0 = incorrect/don’t know), and summed scores of 0–10 were used to measure respondents’ knowledge, with higher scores indicating greater knowledge about HBV transmission. The summed score showed good reliability (alpha = 0.87), suggesting that the individual items reliably captured underlying knowledge of HBV transmission .
Sociodemographic variables used in these analyses included respondent gender, age, country of birth, education, survey language preference, use of five possible sources for HBV-related information, having a primary care provider (PCP), years since HBV diagnosis, event precipitating HBV testing and diagnosis and awareness of infected family members.
Approximately one year after enrollment, qualitative in-depth interviews were conducted with a subsample of participants. These interviews explored baseline findings to provide contextual understanding of participants’ lived experience with CHB. Participants were selected using a quota-sampling strategy to balance gender, geographic location, and language, and purposive sampling to capture cohort diversity in age, socioeconomic background, immigration stories, and health histories. A sample size of 30 participants was selected a priori, based on the focused nature of the interview topic and the potential for richly descriptive interview content from each participant. Of the 363 participants in the total cohort, 49 participants were contacted sequentially as potential interview participants. Among the 49 contacted participants, 7 declined, 5 agreed but did not complete scheduling, and 7 did not respond despite repeated contact attempts. Two bilingual research assistants conducted audio-recorded interviews over the phone in the participant’s preferred language. The interview guide used a phenomenological approach to explore each participant’s lived experience from diagnosis to the present. Participants provided verbal consent and received a $40 gift card. Each interview was transcribed verbatim, and Korean interviews were translated into English. The first iteration of the codebook included deductive and inductive themes. Reliability was established by having four co-authors independently code two transcripts, adding additional themes and codes as needed, and discussing coding differences to reach consensus. Using the finalized codebook, two research assistants who conducted the interviews coded the remaining interviews, using NVivo14 for coding and thematic analysis.
All survey data were cleaned and analyzed using SPSS. Descriptive statistics were used to examine the sample’s baseline characteristics and distribution of responses to HBV knowledge questions. Bivariate associations were explored between the total HBV knowledge score and all independent variables. For categorical variables, mean group scores were compared using t-tests and p-values. For continuous variables, correlations between the HBV knowledge score and each variable were presented with p-values. A final multivariable linear regression was selected using a stepwise backward selection process. Thematic analysis of participants’ experience talking about HBV transmission with others was conducted for all qualitative in-depth interviews. Central themes with example quotes from participants are presented.
Quantitative Findings Table : Cohort Demographics The cohort was 56% male, with an average age of 60 years, and an age range from 19 to 84 years. Most participants were first-generation (97%), college-educated (56%), preferred completing the survey in Korean (82%), and had a PCP (94%). Patients who preferred English were significantly younger, more likely to be born in the US, and more educated than patients who preferred Korean. Patients received their HBV diagnosis on average 27 years ago, 45% of participants were diagnosed through a regular checkup, and 67% knew that a family member also had HBV infection. Doctors and medical caregivers, newspapers and magazines, and the internet were the most common sources of information used to learn about HBV. Approximately 27% of participants used three sources of information to get HBV information, and 5% did not use any sources of information. Table : Knowledge Question Responses The average knowledge score among all participants was 6.3 out of 10. Looking at each question of the knowledge scale, only 30% of all participants correctly identified that HBV is not transmitted through pre-chewed food or breastfeeding, and only 61% correctly answered that HBV could be transmitted through sexual intercourse. Most participants correctly identified that HBV cannot be transmitted through holding hands (90%) and that transmission could occur perinatally (82%). However, for any given question, 8.3–20.1% of the entire cohort responded “don’t know.” There were significant differences in responses by language for six transmission questions: sharing a toothbrush, eating pre-chewed food from an infected person, being coughed/sneezed on, sexual intercourse, holding hands, and breastfeeding. English-language participants were more likely to correctly answer that HBV could not be transmitted by eating pre-chewed food (46.2% versus 26.8%, p = 0.003), being coughed or sneezed on (78.5% versus 61.7%, p = 0.033), holding hands (98.5% versus 87.6%, p = 0.034), and a better understanding that HBV could be transmitted through sexual intercourse (76.9% versus 57.0%, p = 0.004). Korean-language participants were better able to identify that HBV can be transmitted by sharing a toothbrush (66.8% versus 47.7%, p = 0.012). Half of the Korean-language participants incorrectly responded that HBV could be transmitted through breastfeeding, while 32% of English-language participants did not know the answer. Table : Bivariate Associations Knowledge scores did not differ by gender, country of birth, or having a PCP. Between different languages, English-language participants scored significantly higher than Korean-language participants (6.9 versus 6.2, p = 0.033) (Fig. ). Participants who used the internet to seek HBV information had significantly higher knowledge scores than those who did not (6.6 versus 5.9, p = 0.003). Those with a known family history of HBV had higher knowledge scores compared to those who did not (6.5 versus 5.9, p = 0.019). Older age was negatively correlated with knowledge score (-0.28, p < 0.001). Higher education level (0.16, p = 0.003) and number of information sources used (0.15, p = 0.005) were positively correlated with knowledge score. Table : Multivariable Linear Regression The final model contains three covariates that remained significantly associated with HBV transmission knowledge score: age, education level, and total number of information sources used. Older age remained negatively associated with knowledge score (β=-0.25, p < 0.001). More years of formal education (β = 0.09, p = 0.076) and a greater number of information sources used to seek HBV information (β = 0.12, p = 0.023) were positively associated with higher knowledge scores. Qualitative Findings (Table ) The 30 in-depth interview participants were approximately balanced by gender (16 men/14 women), clinical site (15 Philadelphia/15 Los Angeles), and language (15 English/15 Korean). Average knowledge score among interview participants did not differ significantly from the overall cohort (7.0 versus 6.3, p = 0.09). A similar pattern is observed within language groups. Among all English participants, knowledge scores did not significantly differ between those who did and did not participate in the qualitative interview (7.4 versus 6.7, p = 0.189). Similarly, with Korean participants, knowledge scores did not significantly differ between those who did and did not participate in the qualitative interview (6.5 versus 6.2, p = 0.628). Doctor-Patient Communication Many participants referred to conversations they had with their healthcare providers, which had proven beneficial by helping them address fears or misperceptions about their condition. One woman recounts her experience with her OBGYN: What I had my son, my first kid, I was concerned about the transmission of it, but my OBGYN reassured me…there’s vaccination now … I felt a little more comfortable after finding that out. These conversations also educated patients about preventing further spread of infection: But [name of doctor] told me that I shouldn’t share toothpaste and toothbrushes … we live in the same house, but we don’t share razors or anything. Now it’s become a habit, but I still am careful. Although talking to providers was described as informative and beneficial, one patient has also expressed difficulty applying the knowledge they learned from their providers. I understand quickly when the doctor explains it, but when I try to recall and use that knowledge later, I can’t. Another patient expressed not feeling the need to comprehend the education they were receiving from their provider. Instead, they felt it was just a matter of adhering to their provider’s recommendation. I actually don’t think I really tried to understand. I believe that ‘it’s not about whether I understand or not, but rather that the doctor is an expert, and their judgment is correct.’ If the doctor says something needed to be done, I just did it. Misperceptions About Transmission Through Shared Food Although HBV cannot be transmitted through saliva and sharing food, patients expressed general worry about eating with others. The doctor told me that I had to be careful with my relationships with others to prevent the spread … especially with food. There were also instances where patients knew that they could not spread HBV to others during shared meals but still felt the need to be cautious in front of others. For example, when I eat from my plate, I can share food with my daughter …But people think that you can’t do that if you have hepatitis B – that it’s something you shouldn’t do … people think it’s a contagious behavior. So, I have no choice but to be careful. Typically, beliefs held by others caused patients to be more cautious about their behavior, even if they knew it was unnecessary. In addition, some misinformed patients spoke about promoting excessive restrictions within their social circles. It’s not like it’s some contagious disease. But there are precautions to be taken, like when drinking alcohol, I always tell my friends not to share glasses… and if food is served in an open manner, scoop out your portion and place it on your plate, and avoid clashing chopsticks. At the most extreme, some participants have reduced social interactions out of fear of transmitting the virus to others. Yeah, if the articles that I read at the time was true, I could actually transmit through my saliva. So, I don’t want to share my utensils or meals, anything like that. So, I started having less meals with friends or other people.
Table : Cohort Demographics The cohort was 56% male, with an average age of 60 years, and an age range from 19 to 84 years. Most participants were first-generation (97%), college-educated (56%), preferred completing the survey in Korean (82%), and had a PCP (94%). Patients who preferred English were significantly younger, more likely to be born in the US, and more educated than patients who preferred Korean. Patients received their HBV diagnosis on average 27 years ago, 45% of participants were diagnosed through a regular checkup, and 67% knew that a family member also had HBV infection. Doctors and medical caregivers, newspapers and magazines, and the internet were the most common sources of information used to learn about HBV. Approximately 27% of participants used three sources of information to get HBV information, and 5% did not use any sources of information. Table : Knowledge Question Responses The average knowledge score among all participants was 6.3 out of 10. Looking at each question of the knowledge scale, only 30% of all participants correctly identified that HBV is not transmitted through pre-chewed food or breastfeeding, and only 61% correctly answered that HBV could be transmitted through sexual intercourse. Most participants correctly identified that HBV cannot be transmitted through holding hands (90%) and that transmission could occur perinatally (82%). However, for any given question, 8.3–20.1% of the entire cohort responded “don’t know.” There were significant differences in responses by language for six transmission questions: sharing a toothbrush, eating pre-chewed food from an infected person, being coughed/sneezed on, sexual intercourse, holding hands, and breastfeeding. English-language participants were more likely to correctly answer that HBV could not be transmitted by eating pre-chewed food (46.2% versus 26.8%, p = 0.003), being coughed or sneezed on (78.5% versus 61.7%, p = 0.033), holding hands (98.5% versus 87.6%, p = 0.034), and a better understanding that HBV could be transmitted through sexual intercourse (76.9% versus 57.0%, p = 0.004). Korean-language participants were better able to identify that HBV can be transmitted by sharing a toothbrush (66.8% versus 47.7%, p = 0.012). Half of the Korean-language participants incorrectly responded that HBV could be transmitted through breastfeeding, while 32% of English-language participants did not know the answer. Table : Bivariate Associations Knowledge scores did not differ by gender, country of birth, or having a PCP. Between different languages, English-language participants scored significantly higher than Korean-language participants (6.9 versus 6.2, p = 0.033) (Fig. ). Participants who used the internet to seek HBV information had significantly higher knowledge scores than those who did not (6.6 versus 5.9, p = 0.003). Those with a known family history of HBV had higher knowledge scores compared to those who did not (6.5 versus 5.9, p = 0.019). Older age was negatively correlated with knowledge score (-0.28, p < 0.001). Higher education level (0.16, p = 0.003) and number of information sources used (0.15, p = 0.005) were positively correlated with knowledge score. Table : Multivariable Linear Regression The final model contains three covariates that remained significantly associated with HBV transmission knowledge score: age, education level, and total number of information sources used. Older age remained negatively associated with knowledge score (β=-0.25, p < 0.001). More years of formal education (β = 0.09, p = 0.076) and a greater number of information sources used to seek HBV information (β = 0.12, p = 0.023) were positively associated with higher knowledge scores.
: Cohort Demographics The cohort was 56% male, with an average age of 60 years, and an age range from 19 to 84 years. Most participants were first-generation (97%), college-educated (56%), preferred completing the survey in Korean (82%), and had a PCP (94%). Patients who preferred English were significantly younger, more likely to be born in the US, and more educated than patients who preferred Korean. Patients received their HBV diagnosis on average 27 years ago, 45% of participants were diagnosed through a regular checkup, and 67% knew that a family member also had HBV infection. Doctors and medical caregivers, newspapers and magazines, and the internet were the most common sources of information used to learn about HBV. Approximately 27% of participants used three sources of information to get HBV information, and 5% did not use any sources of information.
: Knowledge Question Responses The average knowledge score among all participants was 6.3 out of 10. Looking at each question of the knowledge scale, only 30% of all participants correctly identified that HBV is not transmitted through pre-chewed food or breastfeeding, and only 61% correctly answered that HBV could be transmitted through sexual intercourse. Most participants correctly identified that HBV cannot be transmitted through holding hands (90%) and that transmission could occur perinatally (82%). However, for any given question, 8.3–20.1% of the entire cohort responded “don’t know.” There were significant differences in responses by language for six transmission questions: sharing a toothbrush, eating pre-chewed food from an infected person, being coughed/sneezed on, sexual intercourse, holding hands, and breastfeeding. English-language participants were more likely to correctly answer that HBV could not be transmitted by eating pre-chewed food (46.2% versus 26.8%, p = 0.003), being coughed or sneezed on (78.5% versus 61.7%, p = 0.033), holding hands (98.5% versus 87.6%, p = 0.034), and a better understanding that HBV could be transmitted through sexual intercourse (76.9% versus 57.0%, p = 0.004). Korean-language participants were better able to identify that HBV can be transmitted by sharing a toothbrush (66.8% versus 47.7%, p = 0.012). Half of the Korean-language participants incorrectly responded that HBV could be transmitted through breastfeeding, while 32% of English-language participants did not know the answer.
: Bivariate Associations Knowledge scores did not differ by gender, country of birth, or having a PCP. Between different languages, English-language participants scored significantly higher than Korean-language participants (6.9 versus 6.2, p = 0.033) (Fig. ). Participants who used the internet to seek HBV information had significantly higher knowledge scores than those who did not (6.6 versus 5.9, p = 0.003). Those with a known family history of HBV had higher knowledge scores compared to those who did not (6.5 versus 5.9, p = 0.019). Older age was negatively correlated with knowledge score (-0.28, p < 0.001). Higher education level (0.16, p = 0.003) and number of information sources used (0.15, p = 0.005) were positively correlated with knowledge score.
: Multivariable Linear Regression The final model contains three covariates that remained significantly associated with HBV transmission knowledge score: age, education level, and total number of information sources used. Older age remained negatively associated with knowledge score (β=-0.25, p < 0.001). More years of formal education (β = 0.09, p = 0.076) and a greater number of information sources used to seek HBV information (β = 0.12, p = 0.023) were positively associated with higher knowledge scores.
) The 30 in-depth interview participants were approximately balanced by gender (16 men/14 women), clinical site (15 Philadelphia/15 Los Angeles), and language (15 English/15 Korean). Average knowledge score among interview participants did not differ significantly from the overall cohort (7.0 versus 6.3, p = 0.09). A similar pattern is observed within language groups. Among all English participants, knowledge scores did not significantly differ between those who did and did not participate in the qualitative interview (7.4 versus 6.7, p = 0.189). Similarly, with Korean participants, knowledge scores did not significantly differ between those who did and did not participate in the qualitative interview (6.5 versus 6.2, p = 0.628). Doctor-Patient Communication Many participants referred to conversations they had with their healthcare providers, which had proven beneficial by helping them address fears or misperceptions about their condition. One woman recounts her experience with her OBGYN: What I had my son, my first kid, I was concerned about the transmission of it, but my OBGYN reassured me…there’s vaccination now … I felt a little more comfortable after finding that out. These conversations also educated patients about preventing further spread of infection: But [name of doctor] told me that I shouldn’t share toothpaste and toothbrushes … we live in the same house, but we don’t share razors or anything. Now it’s become a habit, but I still am careful. Although talking to providers was described as informative and beneficial, one patient has also expressed difficulty applying the knowledge they learned from their providers. I understand quickly when the doctor explains it, but when I try to recall and use that knowledge later, I can’t. Another patient expressed not feeling the need to comprehend the education they were receiving from their provider. Instead, they felt it was just a matter of adhering to their provider’s recommendation. I actually don’t think I really tried to understand. I believe that ‘it’s not about whether I understand or not, but rather that the doctor is an expert, and their judgment is correct.’ If the doctor says something needed to be done, I just did it. Misperceptions About Transmission Through Shared Food Although HBV cannot be transmitted through saliva and sharing food, patients expressed general worry about eating with others. The doctor told me that I had to be careful with my relationships with others to prevent the spread … especially with food. There were also instances where patients knew that they could not spread HBV to others during shared meals but still felt the need to be cautious in front of others. For example, when I eat from my plate, I can share food with my daughter …But people think that you can’t do that if you have hepatitis B – that it’s something you shouldn’t do … people think it’s a contagious behavior. So, I have no choice but to be careful. Typically, beliefs held by others caused patients to be more cautious about their behavior, even if they knew it was unnecessary. In addition, some misinformed patients spoke about promoting excessive restrictions within their social circles. It’s not like it’s some contagious disease. But there are precautions to be taken, like when drinking alcohol, I always tell my friends not to share glasses… and if food is served in an open manner, scoop out your portion and place it on your plate, and avoid clashing chopsticks. At the most extreme, some participants have reduced social interactions out of fear of transmitting the virus to others. Yeah, if the articles that I read at the time was true, I could actually transmit through my saliva. So, I don’t want to share my utensils or meals, anything like that. So, I started having less meals with friends or other people.
Many participants referred to conversations they had with their healthcare providers, which had proven beneficial by helping them address fears or misperceptions about their condition. One woman recounts her experience with her OBGYN: What I had my son, my first kid, I was concerned about the transmission of it, but my OBGYN reassured me…there’s vaccination now … I felt a little more comfortable after finding that out. These conversations also educated patients about preventing further spread of infection: But [name of doctor] told me that I shouldn’t share toothpaste and toothbrushes … we live in the same house, but we don’t share razors or anything. Now it’s become a habit, but I still am careful. Although talking to providers was described as informative and beneficial, one patient has also expressed difficulty applying the knowledge they learned from their providers. I understand quickly when the doctor explains it, but when I try to recall and use that knowledge later, I can’t. Another patient expressed not feeling the need to comprehend the education they were receiving from their provider. Instead, they felt it was just a matter of adhering to their provider’s recommendation. I actually don’t think I really tried to understand. I believe that ‘it’s not about whether I understand or not, but rather that the doctor is an expert, and their judgment is correct.’ If the doctor says something needed to be done, I just did it.
Although HBV cannot be transmitted through saliva and sharing food, patients expressed general worry about eating with others. The doctor told me that I had to be careful with my relationships with others to prevent the spread … especially with food. There were also instances where patients knew that they could not spread HBV to others during shared meals but still felt the need to be cautious in front of others. For example, when I eat from my plate, I can share food with my daughter …But people think that you can’t do that if you have hepatitis B – that it’s something you shouldn’t do … people think it’s a contagious behavior. So, I have no choice but to be careful. Typically, beliefs held by others caused patients to be more cautious about their behavior, even if they knew it was unnecessary. In addition, some misinformed patients spoke about promoting excessive restrictions within their social circles. It’s not like it’s some contagious disease. But there are precautions to be taken, like when drinking alcohol, I always tell my friends not to share glasses… and if food is served in an open manner, scoop out your portion and place it on your plate, and avoid clashing chopsticks. At the most extreme, some participants have reduced social interactions out of fear of transmitting the virus to others. Yeah, if the articles that I read at the time was true, I could actually transmit through my saliva. So, I don’t want to share my utensils or meals, anything like that. So, I started having less meals with friends or other people.
Health education about ways to reduce the transmission of HBV is important for patients to reduce the spread of the infection. Knowledge empowers individuals to have more agency to make informed decisions. Our findings show that knowledge is still inadequate, and opportunities exist to improve education in this population. Gaps in HBV Transmission Knowledge The breakdown of knowledge score shows interesting patterns in gaps of knowledge in this population. Only 30% of the cohort knew that HBV could not be transmitted through breastfeeding or prechewed food from an infected person. Concerns may arise because HBV DNA can be detected in breast milk and the possibility of blood from cracked nipples . If a nursing mother’s nipple is cracked and bleeding, the virus could be transmitted to the infant during nursing. However, this could only occur in the unlikely situation that the child also had an open wound which was exposed to the mother’s blood, leading to percutaneous transmission. Population-based studies in both the U.S. and China found that breastfeeding did not increase the risk of HBV transmission if the child received their first HBV vaccine at birth . Regardless of this information, providers may still be overly cautious of this risk and may not encourage HBV-infected mothers to breastfeed . This is consistent with one woman’s account of her OBGYN’s recommendation against breastfeeding. However, breastfeeding provides many benefits to the infant and is encouraged for women with CHB . There are also concerns about prechewed food coming in contact with infected blood through a cut in the mouth. Transmission through this method is less documented for HBV, and few studies have shown clinical evidence to prove otherwise. Prechewing food for young children is a cultural practice not commonly practiced within Korean culture, so most of our participants may not be familiar with this. There were also significant differences in understanding transmission routes between language groups. Sexual intercourse is one prominent route of transmission, but only 57% of Korean-language patients correctly answered that compared to 77% of English-language patients. A previous study of Korean American parents in New York found that only 23% of participants knew that HBV could be transmitted through sexual contact . Although this was much lower than the current study population, one explanation may be attributed to differences in education regarding sexual health in Korea compared to the US. A survey conducted in Korea to assess the public’s general understanding of liver-related diseases found that approximately 60% of their sample did not know that HBV could be transmitted through sexual contact . This is consistent with our results. Since our patients were diagnosed with HBV on average 27 years ago, many of them may have received their diagnoses while they were still in Korea. Results for our Korean language patients’ understanding of transmission through sexual intercourse may reflect cultural norms. Sociodemographic factors contributing to health literacy may also contribute to higher scores among English-language participants. Health literacy is the ability to acquire, process, and understand basic health information . Although language was not significant in the final model, education attainment and the number of information sources used may have a more proximal impact on the pathway of effects. Suggesting that regardless of language, persons with CHB can acquire the knowledge they need if they can access information from multiple sources. Regardless of language, many participants were unable to answer questions about HBV transmission. This is concerning as these patients have received specialized care for at least five years. The responses show a gap in health education and suggest an opportunity to improve knowledge about HBV transmission routes. Predictors of High HBV Transmission Knowledge Scores It is also important to speculate as to why HBV transmission knowledge was lower among older respondents despite their tending to have more years of experience with the disease. This finding is consistent with other studies and may be explained in part by the life course trajectory of patients’ experiences of CHB as a chronic condition . Participants most commonly named “talking to my doctor” as their source of HBV information. Many patients would have received their initial CHB-related health education from their physician during diagnosis and treatment initiation. Although we do not know what information those clinicians provided, for older patients, whose diagnosis occurred decades ago, earlier information may now be outdated. Furthermore, as time progresses, the focus of clinical visits may shift to treatment and maintenance. Older patients may not be able to recall the health education they previously received, and clinicians may not see it as relevant to re-educate older patients about transmission via routes such as breastfeeding or intercourse. Print media and the Internet were other sources of information used. This reflects other passive and active ways patients can acquire HBV knowledge outside their regular medical appointments. It suggests that having multiple points of opportunity for patients to learn about HBV throughout their disease journey can increase their knowledge about transmission routes. Provider Influence on HBV Transmission Knowledge Language and cultural differences are barriers that can hinder medical care for CHB among Asian American populations . Asian American patients who felt that their doctor did not understand their culture and values were more likely to report dissatisfaction with their quality of care and less likely to trust their clinician . All patients in this study received specialized liver care from Korean-American hepatologists fluent in English and Korean. This model of ethnically concordant care reduces barriers by making communication and sharing information easier. Participants also shared in their qualitative interviews how their hepatologist and OBGYN were influential in teaching them about HBV transmission. Our results underscore the importance of healthcare providers as trusted sources of information and emotional support. However, qualitative interviews revealed concerns about patients’ comprehension and application of the information they receive. Culturally, physicians, especially specialists, are highly respected and are perceived to have absolute authority over a patient’s treatment plan . This hierarchical relationship may make it difficult for patients to inquire freely and not appear rude to an authority Figs. . Although patient adherence is a positive attribute, it is also important for patients to feel agency in comprehending medical advice given by their clinicians. Unless they can comprehend it, they are unlikely to be able to recall any knowledge in the future and apply it. Misinformation and Stigma among CHB Patients Our qualitative results show that misperceptions about spreading HBV through food remain prevalent in this population. In Korean culture, sharing food, such as soups and stews, during meals is very common. Participants mentioned avoiding sharing food and utensils to reduce HBV transmission, consistent with other studies of Asian Americans with similar eating practices [ , , ]. In our analysis, some participants believe HBV can spread through saliva and sharing food, leading to social restrictions. Others understand transmission routes but avoid sharing to prevent stigma. HBV-related stigma is a persistent issue that can cause fear, anxiety, and worry among CHB patients, and impact health-seeking behaviors and disease management [ – ]. Lack of education and misinformation about HBV transmission is a driver of HBV-related stigma [ – ]. Proactively educating the general population is important in reducing misinformation and will help foster more positive environments for CHB patients. Strengths and Limitations This study utilizes a sequential mixed-methods design using data from structured surveys and in-depth interviews. This design allows for a more comprehensive understanding of knowledge in this population of CHB patients . A strength of this study is that the population was specific to Korean-American CHB patients receiving care from Korean hepatologists. Previous studies about HBV among Korean-Americans have been limited to those in the community or those receiving care in a care system [ , , ]. Prior studies have not looked at the effects of racial concordance of care in this population. Findings from this study provide insight into existing barriers to knowledge within this care model. One limitation is that the survey was cross-sectional, so we must be cautious when considering associations between knowledge scores and independent predictors. For example, it is possible that greater knowledge of HBV could motivate a patient to seek additional sources of information, that these relationships are bidirectional, and that the association between older age and less knowledge is both an aging and a cohort effect. The interviews partially mediate this limitation by allowing patients to recount their life managing CHB and offer insight into their lived experiences. A second limitation is that our survey responses were all self-reported. Patients diagnosed decades ago may have difficulty responding to questions that require them to think back to earlier parts of their disease journey. Finally, we did not ask direct questions about transmission knowledge in the qualitative interviews. However, many participants did share about their knowledge acquisition experience and strategies they used to manage HBV.
The breakdown of knowledge score shows interesting patterns in gaps of knowledge in this population. Only 30% of the cohort knew that HBV could not be transmitted through breastfeeding or prechewed food from an infected person. Concerns may arise because HBV DNA can be detected in breast milk and the possibility of blood from cracked nipples . If a nursing mother’s nipple is cracked and bleeding, the virus could be transmitted to the infant during nursing. However, this could only occur in the unlikely situation that the child also had an open wound which was exposed to the mother’s blood, leading to percutaneous transmission. Population-based studies in both the U.S. and China found that breastfeeding did not increase the risk of HBV transmission if the child received their first HBV vaccine at birth . Regardless of this information, providers may still be overly cautious of this risk and may not encourage HBV-infected mothers to breastfeed . This is consistent with one woman’s account of her OBGYN’s recommendation against breastfeeding. However, breastfeeding provides many benefits to the infant and is encouraged for women with CHB . There are also concerns about prechewed food coming in contact with infected blood through a cut in the mouth. Transmission through this method is less documented for HBV, and few studies have shown clinical evidence to prove otherwise. Prechewing food for young children is a cultural practice not commonly practiced within Korean culture, so most of our participants may not be familiar with this. There were also significant differences in understanding transmission routes between language groups. Sexual intercourse is one prominent route of transmission, but only 57% of Korean-language patients correctly answered that compared to 77% of English-language patients. A previous study of Korean American parents in New York found that only 23% of participants knew that HBV could be transmitted through sexual contact . Although this was much lower than the current study population, one explanation may be attributed to differences in education regarding sexual health in Korea compared to the US. A survey conducted in Korea to assess the public’s general understanding of liver-related diseases found that approximately 60% of their sample did not know that HBV could be transmitted through sexual contact . This is consistent with our results. Since our patients were diagnosed with HBV on average 27 years ago, many of them may have received their diagnoses while they were still in Korea. Results for our Korean language patients’ understanding of transmission through sexual intercourse may reflect cultural norms. Sociodemographic factors contributing to health literacy may also contribute to higher scores among English-language participants. Health literacy is the ability to acquire, process, and understand basic health information . Although language was not significant in the final model, education attainment and the number of information sources used may have a more proximal impact on the pathway of effects. Suggesting that regardless of language, persons with CHB can acquire the knowledge they need if they can access information from multiple sources. Regardless of language, many participants were unable to answer questions about HBV transmission. This is concerning as these patients have received specialized care for at least five years. The responses show a gap in health education and suggest an opportunity to improve knowledge about HBV transmission routes.
It is also important to speculate as to why HBV transmission knowledge was lower among older respondents despite their tending to have more years of experience with the disease. This finding is consistent with other studies and may be explained in part by the life course trajectory of patients’ experiences of CHB as a chronic condition . Participants most commonly named “talking to my doctor” as their source of HBV information. Many patients would have received their initial CHB-related health education from their physician during diagnosis and treatment initiation. Although we do not know what information those clinicians provided, for older patients, whose diagnosis occurred decades ago, earlier information may now be outdated. Furthermore, as time progresses, the focus of clinical visits may shift to treatment and maintenance. Older patients may not be able to recall the health education they previously received, and clinicians may not see it as relevant to re-educate older patients about transmission via routes such as breastfeeding or intercourse. Print media and the Internet were other sources of information used. This reflects other passive and active ways patients can acquire HBV knowledge outside their regular medical appointments. It suggests that having multiple points of opportunity for patients to learn about HBV throughout their disease journey can increase their knowledge about transmission routes.
Language and cultural differences are barriers that can hinder medical care for CHB among Asian American populations . Asian American patients who felt that their doctor did not understand their culture and values were more likely to report dissatisfaction with their quality of care and less likely to trust their clinician . All patients in this study received specialized liver care from Korean-American hepatologists fluent in English and Korean. This model of ethnically concordant care reduces barriers by making communication and sharing information easier. Participants also shared in their qualitative interviews how their hepatologist and OBGYN were influential in teaching them about HBV transmission. Our results underscore the importance of healthcare providers as trusted sources of information and emotional support. However, qualitative interviews revealed concerns about patients’ comprehension and application of the information they receive. Culturally, physicians, especially specialists, are highly respected and are perceived to have absolute authority over a patient’s treatment plan . This hierarchical relationship may make it difficult for patients to inquire freely and not appear rude to an authority Figs. . Although patient adherence is a positive attribute, it is also important for patients to feel agency in comprehending medical advice given by their clinicians. Unless they can comprehend it, they are unlikely to be able to recall any knowledge in the future and apply it.
Our qualitative results show that misperceptions about spreading HBV through food remain prevalent in this population. In Korean culture, sharing food, such as soups and stews, during meals is very common. Participants mentioned avoiding sharing food and utensils to reduce HBV transmission, consistent with other studies of Asian Americans with similar eating practices [ , , ]. In our analysis, some participants believe HBV can spread through saliva and sharing food, leading to social restrictions. Others understand transmission routes but avoid sharing to prevent stigma. HBV-related stigma is a persistent issue that can cause fear, anxiety, and worry among CHB patients, and impact health-seeking behaviors and disease management [ – ]. Lack of education and misinformation about HBV transmission is a driver of HBV-related stigma [ – ]. Proactively educating the general population is important in reducing misinformation and will help foster more positive environments for CHB patients.
This study utilizes a sequential mixed-methods design using data from structured surveys and in-depth interviews. This design allows for a more comprehensive understanding of knowledge in this population of CHB patients . A strength of this study is that the population was specific to Korean-American CHB patients receiving care from Korean hepatologists. Previous studies about HBV among Korean-Americans have been limited to those in the community or those receiving care in a care system [ , , ]. Prior studies have not looked at the effects of racial concordance of care in this population. Findings from this study provide insight into existing barriers to knowledge within this care model. One limitation is that the survey was cross-sectional, so we must be cautious when considering associations between knowledge scores and independent predictors. For example, it is possible that greater knowledge of HBV could motivate a patient to seek additional sources of information, that these relationships are bidirectional, and that the association between older age and less knowledge is both an aging and a cohort effect. The interviews partially mediate this limitation by allowing patients to recount their life managing CHB and offer insight into their lived experiences. A second limitation is that our survey responses were all self-reported. Patients diagnosed decades ago may have difficulty responding to questions that require them to think back to earlier parts of their disease journey. Finally, we did not ask direct questions about transmission knowledge in the qualitative interviews. However, many participants did share about their knowledge acquisition experience and strategies they used to manage HBV.
These results suggest that despite receiving culturally concordant care from expert hepatologists, some Korean-American CHB patients have an inadequate understanding of transmission. Misperceptions about transmission pathways still exist, and older age, less formal education, and fewer sources of HBV information may result in lower knowledge. Continued health education after the point of diagnosis is important in maintaining accurate knowledge and reducing misperceptions about HBV transmission. Although there is no clinical risk associated with patients being cautious about the spread of HBV, being overly cautious can negatively impact the patient’s quality of life as they navigate the already challenging task of managing a chronic condition. Results from this study and existing literature also highlight the role of the provider as a trusted source of information in providing health education and emotional support. Further research should be done to identify additional factors that influence knowledge acquisition and retention. Doing so will be important for developing culturally appropriate health education for this population.
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Health care providers’ perspectives on providing end-of-life psychiatric care in cardiology and oncology hospitals: a cross-sectional questionnaire survey | a03ee174-e5a3-4644-9b87-6705e9d6f6f9 | 10014396 | Internal Medicine[mh] | Heart failure (HF) is potentially fatal, unless a heart transplantation is performed, and it is a serious healthcare and economic burden on patients and their caregivers. The World Health Organization estimated the worldwide mortality from cardiovascular disease at 15.2 million in 2016 , making it the most common cause of death (40%) among middle-aged and older adults . Despite the recent rapid progress in medical treatments, the median survival rate after patients’ first hospitalization is low in severe HF (2.1 years) . In addition, HF has inflicted a burden of $180 million on the global health system . Patients with advanced HF commonly experience psychological symptoms, the most common of which are depression and anxiety, as well as physical symptoms, such as dyspnea, pain, or fatigue . Severe clinical depression is diagnosed in 12 to 33% of all patients with heart disease and in 38 to 42% of those with severe HF, featuring New York Heart Association class III-IV symptoms . Among patients with HF, 29% exhibit severe and clinically significant anxiety symptoms, and 9% have anxiety disorders, including generalized anxiety disorders . In addition, psychological symptoms have a highly negative impact on the quality of life and are associated with poor treatment adherence, severe physical symptoms, long-term hospitalization, and a reduced survival rate . Therefore, psychological symptoms, such as depression or anxiety, are particularly challenging problems for patients with end-stage HF . Psychiatric care, including pharmacotherapy and psychotherapy, can be of benefit for patients with HF who have psychological symptoms. However, there is inadequate evidence for the efficacy of pharmacotherapy in patients with HF , and psychiatric pharmacotherapy, such as antidepressants, increases the risk of all-cause death among HF patients . Nevertheless, psychotherapy has received attention among patients with HF in recent years, and cognitive behavioral therapy in particular has been shown to improve psychological symptoms . Relaxation, meditation, and mindfulness-based psychoeducation can also alleviate these symptoms . However, there is limited evidence and guidance on the efficacy of such psychiatric care among patients with terminal HF . In patients with end-stage cancer, many of whom experience psychological symptoms similar to patients with end-stage HF, many studies have demonstrated the effectiveness of pharmacotherapy and psychotherapy . Workshops or guidelines for oncologists can also enhance their practical skills in providing end-of-life psychiatric care . A comparison between the difficulties in providing psychiatric care for patients with end-stage HF versus those with cancer could provide useful insights into potential barriers to providing psychiatric care for patients with end-stage HF. However, to date, no study has examined the barriers to providing psychiatric care for patients with HF. In addition, we believe that a qualitative study design, examining thee difficulties faced by health care providers in pain management, would be also helpful in investigating the difficulties with psychiatric management and identifying the barriers to providing psychiatric care . The aims of this study were to identify and compare the barriers faced by health care providers of cardiology and oncology hospitals in providing psychiatric care to end-of-life patients.
Design and participants This was a national, cross-sectional survey conducted among Japanese health care providers of cardiology and oncology hospitals using self-completed questionnaires. We mailed the questionnaires to the departments of cardiovascular internal medicine of 427 implantable cardioverter defibrillators (ICD) specialized hospitals and to the departments of respiratory medicine of 347 designated cancer hospitals; we asked them to deliver the questionnaires directly to the chief physicians and the chief nurses in each department in March 2018. ICD specialized hospitals are equipped to perform implantation of ICDs and are the center of cardiovascular medicine in Japan. Additionally, designated cancer hospitals, recommended by the prefectural governments, can provide high-quality cancer treatment, as guaranteed by the Ministry of Health, Labour and Welfare in Japan. These medical facilities provide palliative care by a team of medical professional, provide specialized cancer treatments, establish local cooperation systems for cancer treatments, and provide consultation, support, and information for cancer patients. Demographic and clinical characteristics We collected demographic and clinical information from the self-completed questionnaires. First, we included the following data: sex, age, and medical license of the staff of each health care provider. Second, we included the following data: area (Hokkaido/Tohoku, Kanto/Koshinetsu, Chubu/Hokuriku, Kinki, Chugoku/Shikoku, and Kyushu/Okinawa area), hospital type (national medical center, academic medical center, general hospital except academic medical center, specialized hospital), the number of hospital beds, and the presence of a palliative care unit, palliative care team, liaison psychiatry team, palliative care physicians, psychiatrists, and psychologists at hospitals. Outcome measures Difficulty in providing palliative care The Palliative Care Difficulties Scale—a 15-item self-reported scale—was developed in Japan . The responses are scored in the format of a 4-point Likert-type scale ranging from 0 to 3 (overall score range: 0–42). The scale contains of the following five factors, each having three items: (1) alleviating symptoms, (2) expert support, (3) multidisciplinary communication, (4) communication with patient/family, and (5) community coordination. The reliability and validity of this measure were sufficiently supported in an earlier study . Difficulty in providing end-of-life psychiatric care We developed the following original question (Sup.1) for assessing the difficulty in providing end-of-life psychiatric care: “Do you face challenges in providing psychiatric care for patients at their end of life?” The possible answers were “yes” or “no.” Barriers to providing end-of-life psychiatric care To identify the barriers to providing end-of-life psychological care, we asked the following original question (Sup.1) to participants who answered “yes” to the above question: “What challenges do you face in providing psychological care to patients at their end of life?” Participants could respond freely to this open-ended question. Qualitative analyses Content analysis was used to analyze the responses to the open ended question answered freely. Content analysis is an objective and systematic procedure used to draw conclusions by creating categories of data from verbatim or unstructured data . We conducted a quantitative content analysis according to previous studies in palliative care settings . Our content analysis procedure was conducted as follows: (1) all text data were divided into thematic units, which are units of words with one logical meaning; (2) two researchers, a clinical psychologist (KI), and a cardiovascular nurse (SM) extracted all statements from the free descriptions related to the study topic, such as the barriers to providing end-of-life psychiatric care; (3) a clinical psychologist (KI), a cardiovascular nurse (SM), and two psychiatrists in the palliative care team (EM and TT) carefully conceptualized similarities and differences in the content, and defined all categories; and (4) two coders, a student of psychology, and a psychiatric clinical nurse independently determined how each thematic unit that was identified corresponded with any category. The concordance rate and kappa coefficient of the determinations of the categories were used as reliability indicators. The kappa coefficient was calculated using 20% of the data and random sampling was conducted based on the data from a standard set derived from a previous study, with more than 10% or 50 units of data . Statistical analyses First, we summarized the characteristics of the participants and hospitals using standard descriptive statistics. Second, the mean difference in difficulties in providing palliative care was compared between oncological and cardiovascular hospitals using a t test, and the frequency of difficulties in providing end-of-life psychiatric care was compared between oncological and cardiovascular hospitals using χ 2 test. Third, the frequency of the thematic units that were categorized in the above content analysis was compared between health care providers in oncological and cardiovascular hospitals using χ 2 test. The significance level was set at 5%. All data were analyzed using IBM SPSS Statistics for Windows, version 24 (IBM Corp., NY, USA).
This was a national, cross-sectional survey conducted among Japanese health care providers of cardiology and oncology hospitals using self-completed questionnaires. We mailed the questionnaires to the departments of cardiovascular internal medicine of 427 implantable cardioverter defibrillators (ICD) specialized hospitals and to the departments of respiratory medicine of 347 designated cancer hospitals; we asked them to deliver the questionnaires directly to the chief physicians and the chief nurses in each department in March 2018. ICD specialized hospitals are equipped to perform implantation of ICDs and are the center of cardiovascular medicine in Japan. Additionally, designated cancer hospitals, recommended by the prefectural governments, can provide high-quality cancer treatment, as guaranteed by the Ministry of Health, Labour and Welfare in Japan. These medical facilities provide palliative care by a team of medical professional, provide specialized cancer treatments, establish local cooperation systems for cancer treatments, and provide consultation, support, and information for cancer patients.
We collected demographic and clinical information from the self-completed questionnaires. First, we included the following data: sex, age, and medical license of the staff of each health care provider. Second, we included the following data: area (Hokkaido/Tohoku, Kanto/Koshinetsu, Chubu/Hokuriku, Kinki, Chugoku/Shikoku, and Kyushu/Okinawa area), hospital type (national medical center, academic medical center, general hospital except academic medical center, specialized hospital), the number of hospital beds, and the presence of a palliative care unit, palliative care team, liaison psychiatry team, palliative care physicians, psychiatrists, and psychologists at hospitals.
Difficulty in providing palliative care The Palliative Care Difficulties Scale—a 15-item self-reported scale—was developed in Japan . The responses are scored in the format of a 4-point Likert-type scale ranging from 0 to 3 (overall score range: 0–42). The scale contains of the following five factors, each having three items: (1) alleviating symptoms, (2) expert support, (3) multidisciplinary communication, (4) communication with patient/family, and (5) community coordination. The reliability and validity of this measure were sufficiently supported in an earlier study . Difficulty in providing end-of-life psychiatric care We developed the following original question (Sup.1) for assessing the difficulty in providing end-of-life psychiatric care: “Do you face challenges in providing psychiatric care for patients at their end of life?” The possible answers were “yes” or “no.” Barriers to providing end-of-life psychiatric care To identify the barriers to providing end-of-life psychological care, we asked the following original question (Sup.1) to participants who answered “yes” to the above question: “What challenges do you face in providing psychological care to patients at their end of life?” Participants could respond freely to this open-ended question.
The Palliative Care Difficulties Scale—a 15-item self-reported scale—was developed in Japan . The responses are scored in the format of a 4-point Likert-type scale ranging from 0 to 3 (overall score range: 0–42). The scale contains of the following five factors, each having three items: (1) alleviating symptoms, (2) expert support, (3) multidisciplinary communication, (4) communication with patient/family, and (5) community coordination. The reliability and validity of this measure were sufficiently supported in an earlier study .
We developed the following original question (Sup.1) for assessing the difficulty in providing end-of-life psychiatric care: “Do you face challenges in providing psychiatric care for patients at their end of life?” The possible answers were “yes” or “no.”
To identify the barriers to providing end-of-life psychological care, we asked the following original question (Sup.1) to participants who answered “yes” to the above question: “What challenges do you face in providing psychological care to patients at their end of life?” Participants could respond freely to this open-ended question.
Content analysis was used to analyze the responses to the open ended question answered freely. Content analysis is an objective and systematic procedure used to draw conclusions by creating categories of data from verbatim or unstructured data . We conducted a quantitative content analysis according to previous studies in palliative care settings . Our content analysis procedure was conducted as follows: (1) all text data were divided into thematic units, which are units of words with one logical meaning; (2) two researchers, a clinical psychologist (KI), and a cardiovascular nurse (SM) extracted all statements from the free descriptions related to the study topic, such as the barriers to providing end-of-life psychiatric care; (3) a clinical psychologist (KI), a cardiovascular nurse (SM), and two psychiatrists in the palliative care team (EM and TT) carefully conceptualized similarities and differences in the content, and defined all categories; and (4) two coders, a student of psychology, and a psychiatric clinical nurse independently determined how each thematic unit that was identified corresponded with any category. The concordance rate and kappa coefficient of the determinations of the categories were used as reliability indicators. The kappa coefficient was calculated using 20% of the data and random sampling was conducted based on the data from a standard set derived from a previous study, with more than 10% or 50 units of data .
First, we summarized the characteristics of the participants and hospitals using standard descriptive statistics. Second, the mean difference in difficulties in providing palliative care was compared between oncological and cardiovascular hospitals using a t test, and the frequency of difficulties in providing end-of-life psychiatric care was compared between oncological and cardiovascular hospitals using χ 2 test. Third, the frequency of the thematic units that were categorized in the above content analysis was compared between health care providers in oncological and cardiovascular hospitals using χ 2 test. The significance level was set at 5%. All data were analyzed using IBM SPSS Statistics for Windows, version 24 (IBM Corp., NY, USA).
Demographic and clinical characteristics From the 347 oncology and 427 cardiology hospitals, 130 oncological physicians (37.5%), 94 oncological nurses (27.1%), 120 cardiovascular physicians (28.1%), and 93 cardiovascular nurses (21.8%) were included in the analysis (Fig. ). The characteristics of the study participants and hospitals are listed in Table . More than 90% of physicians were specialists, such as lung cancer or cardiovascular specialists, and approximately half of the nurses were certified in a specialized field, including cancer nursing or palliative care. The sex ratio (men:women) was 1.4:1. Regarding both oncology and cardiology hospitals, more than 90% were general hospitals, approximately 60% were large-scale facilities (≥ 500 hospital beds), more than 80% had palliative care teams, and approximately 70% had psychiatric or psychological care specialists. Difficulty in providing end-of-life palliative and psychiatric care We found that the Palliative Care Difficulties Scale scores were significantly higher in health care providers among cardiology hospitals compared to that of oncology hospitals for “alleviating symptoms” and “expert support” ( F [423] = 8.63, p = 0.00 and F [414] = 18.96, p = 0.00, respectively), whereas no significant differences were found for any other factor ( F [426] = 3.50, p = 0.06 for multidisciplinary communication; F [424] = 2.82, p = 0.09 for communication with patient/family; F [423] = 1.11, p = 0.29 for community coordination) (Fig. ). The frequency of difficulties in providing end-of-life psychiatric care according to the χ 2 test and exact probability test is shown in Fig. . A total of 135 (62.2%) oncological and 125 (59.8%) cardiovascular health care providers had difficulties in providing end-of-life psychiatric care. There was no significant difference in the frequency of difficulties faced by healthcare providers of oncology and cardiology hospitals ( χ 2 = 0.26, p = 0.62). Barrier to providing end-of-life psychiatric care using qualitative methods We extracted 52 attributes from the content analysis, 40 of which were classified by the semantic content into “patients’ personal problems,” “family members’ problems,” “professionals’ personal problems,” “communication problems between professionals and patients,” “problems specific to end-of-life care,” “problems specific to psychiatric care,” “problems of institution or system,” and “problems specific to non-cancer patients” (Table ). The Kappa coefficient derived by the two independent coders was 0.54 in the random 20% data of this study. The frequency of barriers to providing psychiatric end-of-life care is shown in Table . We found that the “problems specific to non-cancer patients” occurred more frequently in health care providers of cardiology than that of oncology hospitals ( χ 2 = 22.475, p = 0.00). There was no significant difference between the frequencies of any other barrier between health care providers of oncology and cardiology hospitals.
From the 347 oncology and 427 cardiology hospitals, 130 oncological physicians (37.5%), 94 oncological nurses (27.1%), 120 cardiovascular physicians (28.1%), and 93 cardiovascular nurses (21.8%) were included in the analysis (Fig. ). The characteristics of the study participants and hospitals are listed in Table . More than 90% of physicians were specialists, such as lung cancer or cardiovascular specialists, and approximately half of the nurses were certified in a specialized field, including cancer nursing or palliative care. The sex ratio (men:women) was 1.4:1. Regarding both oncology and cardiology hospitals, more than 90% were general hospitals, approximately 60% were large-scale facilities (≥ 500 hospital beds), more than 80% had palliative care teams, and approximately 70% had psychiatric or psychological care specialists.
We found that the Palliative Care Difficulties Scale scores were significantly higher in health care providers among cardiology hospitals compared to that of oncology hospitals for “alleviating symptoms” and “expert support” ( F [423] = 8.63, p = 0.00 and F [414] = 18.96, p = 0.00, respectively), whereas no significant differences were found for any other factor ( F [426] = 3.50, p = 0.06 for multidisciplinary communication; F [424] = 2.82, p = 0.09 for communication with patient/family; F [423] = 1.11, p = 0.29 for community coordination) (Fig. ). The frequency of difficulties in providing end-of-life psychiatric care according to the χ 2 test and exact probability test is shown in Fig. . A total of 135 (62.2%) oncological and 125 (59.8%) cardiovascular health care providers had difficulties in providing end-of-life psychiatric care. There was no significant difference in the frequency of difficulties faced by healthcare providers of oncology and cardiology hospitals ( χ 2 = 0.26, p = 0.62).
We extracted 52 attributes from the content analysis, 40 of which were classified by the semantic content into “patients’ personal problems,” “family members’ problems,” “professionals’ personal problems,” “communication problems between professionals and patients,” “problems specific to end-of-life care,” “problems specific to psychiatric care,” “problems of institution or system,” and “problems specific to non-cancer patients” (Table ). The Kappa coefficient derived by the two independent coders was 0.54 in the random 20% data of this study. The frequency of barriers to providing psychiatric end-of-life care is shown in Table . We found that the “problems specific to non-cancer patients” occurred more frequently in health care providers of cardiology than that of oncology hospitals ( χ 2 = 22.475, p = 0.00). There was no significant difference between the frequencies of any other barrier between health care providers of oncology and cardiology hospitals.
This is the first study that investigated the barriers to providing psychiatric care for end-stage HF patients compared to end-stage cancer patients. Although we found no significant difference in the frequency of those who perceive barriers to providing end-of-life psychiatric care between the cardiology and oncology settings, there can be a difference in the context in which they perceive barriers. A particularly important result was that the cardiovascular health care providers faced problems with psychiatric care, which were specific to non-cancer patients, such as obtaining professional support, useful guidelines, or training opportunities. This study was useful in exploring solutions for providing sufficient psychiatric care for end-stage HF patients, by eliminating barriers using a bottom-up qualitative approach. Our results indicated that there were three challenges faced by health care providers in providing psychiatric care to end-of-life patients. First, knowledge of mental health issues specific to the end-of-life is necessary for health care providers to provide psychiatric care. Cardiovascular health care providers found it particularly difficult to improve their knowledge and skills for performing psychiatric assessments and for treating psychological and cardiac symptoms. In particular, depression, in addition to fatigue or pain, is one of the most common symptoms and imposes a heavy burden on patients with advanced HF . Some clinical practice guidelines on HF emphasize the need for psychiatric care for HF patients with depression as part of symptom management in Western countries . However, even these guidelines have insufficient information about a specific psychiatric assessment and treatment for patients with HF. Participants in this study also described that they had little access to information needed to improve their knowledge and skills in psychiatric care. For cancer patients, lack of knowledge and training among health care providers is a barrier to providing psychiatric care , and therefore some Japanese academic societies have held seminars or workshops to promote psychiatric care knowledge for oncologists or any other health care providers in the last few decades. Taken together, we recommend an expansion of the existing training and education system and provision of detailed guidelines as a way to provide access to methods of psychiatric assessment and treatment for psychological symptoms in patients with advanced HF. Furthermore, physical symptom management was also identified as a difficulty for cardiovascular health care providers compared with oncological health care providers in this study. Interventions directed at alleviating physical symptoms related to HF can lead to a reduction in psychological symptoms in palliative care . In the future, we recommend the development of a training system for end-of-life care professionals aimed at providing training for both physical and psychiatric care. Second, cooperation among health care providers with different specialties is important in providing psychiatric care for end-stage patients. Many health care providers felt that it was difficult to coordinate professional-patient relationships in both cardiovascular and oncological settings. Interventions to enhance communication between professionals and patients can improve the latter’s psychological well-being . Professional-patient relationship and communication are also important for the quality and outcome of medical treatment . Particularly in palliative settings, a lack of communication between professionals and patients can lead to the inhibition of critical decisions such as ICD deactivations . Practically, general education and specialized education can improve communication skills among health care providers and facilitate professional-patient communication . Advanced care planning can also encourage effective communication between professionals and patients with HF . Therefore, we conclude that a useful tool or training system for improving communication skills as well as psychiatric care skills among health care providers could enhance end-of-life care in cardiovascular settings. Third, health care providers’ own difficulties and distresses can be resolved to implement psychiatric care smoothly for end-stage patients. A professional’s personal psychological or physical distress could be a barrier to providing psychiatric care. Professional participants in this study described that many cardiovascular and oncological hospitals do not have sufficient staff and are consequently overwhelmed by the workload, leading to unsatisfactory psychiatric care for palliative patients. Health care providers also feel unable to provide sufficient spiritual psychiatric care for end-of-life patients . Reducing the workload and ensuring adequate time management for health care providers remain critical goals in modern Japanese medical settings. Limitations Our study has three major limitations. First, recall bias may have occurred because of the self-reported nature of the questionnaires. However, we conducted a content analysis by two researchers independently and ensured objectivity. Second, although the study conducted on a nation-wide level in Japan, the data may not be generalizable to other populations of the world. Therefore, future studies investigating the same research questions in other countries will be essential to validate our findings and to add to the evidence database. Third, as this study was conducted before the COVID-19 pandemic, our findings may not be consistent with the current situation in the Japanese medical field. Although it is noteworthy that the medical field is constantly overwhelmed with achieving a level of infection control, and the perception of health care providers regarding the significance of providing psychiatric care at the end of life is also changing.
Our study has three major limitations. First, recall bias may have occurred because of the self-reported nature of the questionnaires. However, we conducted a content analysis by two researchers independently and ensured objectivity. Second, although the study conducted on a nation-wide level in Japan, the data may not be generalizable to other populations of the world. Therefore, future studies investigating the same research questions in other countries will be essential to validate our findings and to add to the evidence database. Third, as this study was conducted before the COVID-19 pandemic, our findings may not be consistent with the current situation in the Japanese medical field. Although it is noteworthy that the medical field is constantly overwhelmed with achieving a level of infection control, and the perception of health care providers regarding the significance of providing psychiatric care at the end of life is also changing.
Our results demonstrated that (1) both cardiovascular and oncological health care providers perceive the barriers to providing end-of-life psychiatric care; (2) both of them faced challenges in terms of patients’ personal problems, family members’ problems, professionals’ personal problems, communication problems between professionals and patients, problems specific to end-of-life care, problems specific to psychiatric care, problems of institution or system, and problems specific to non-cancer patients; and (3) cardiovascular providers particularly faced challenges specific to non-cancer patients, compared to oncology providers. These results suggest that health care providers in cardiovascular hospitals, in contrast to those in oncological hospitals, experience problems in obtaining useful guidelines or training opportunities. We recommend the staffing to provide adequate psychiatric care for end-stage HF patients, and the provision of continuous educational opportunities for health care providers involved with psychiatric and palliative care for patients with HF. However, our study also indicates that both oncological and cardiovascular health care providers face challenges in providing end-of-life psychiatric care, which stem from patients’ or health care providers’ personal problems, among others. Therefore, we should also develop strategies to overcome not only the understaffing situation in medical services but also a lack of professionals’ psychiatric care skills.
Below is the link to the electronic supplementary material. Sup. 1 The questionnaire English translated version
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Assessing the Occurrence of Host-Specific Faecal Indicator Markers in Water Systems as a Function of Water, Sanitation and Hygiene Practices: A Case Study in Rural Communities of Vhembe District Municipality, South Africa | 1c4fe687-20b8-44bb-8ddc-a17b68aa1ee8 | 10819538 | Microbiology[mh] | There are many ways in which people become exposed to water that is contaminated with faecal matter, bearing disease-causing microbes. To detect this potential risk, host-associated markers have been used to identify faecal pollution in water sources and in drinking water , with significant gains. Bacteroides spp. exhibit a strong host specificity and are therefore a reliable way of detecting faecal contamination in water bodies . Their association with diarrhoea-causing pathogens in both humans and animals make them a reliable indicator of more serious source-specific and zoonotic enteric pathogens. A study of the wildlife–livestock interface that took place in the Vhembe district revealed that transboundary disease transfer and livestock depredation by wild animals have been happening in the area and its neighbouring countries such as Zimbabwe and Mozambique and particularly in the South African Kruger National Park (KNP) . Some wildlife of the KNP, including buffaloes, were found to be permanently infected with some diseases such as ticks, bovine tuberculosis and foot-and-mouth, any of which may be easily transmissible to the domesticated cow, other domestic animals and humans, should an escape happen . This is particularly alarming given the geographic morphology of water sources running through the villages before emptying into the Luvuvhu River and eventually the Limpopo River, along the KNP. Additionally, domestic animals pose health risks for their owners in rural environments. The efforts to cut back on costs, while ensuring security for their stock forces farmers to keep animals in close proximity, with very close contact daily . Domesticated animals that are fed household wastes or neglected to become scavengers may increase the risk of zoonosis in a household. Ironically, in these rural areas, clinics and laboratories for animals are non-existent . Small-scale farmers make up a significant portion of rural communities. There is a huge reliance on the already stressed freshwater sources for domestic use, including irrigation and husbandry . It is important for the crops that are cultivated to be of good quality, as this could be a pathway for faecal–oral pathogens. Subsistence farming is often undertaken in schools as part of feeding schemes, raising funds through sales or even engaging learners in agricultural education as part of their curriculum. Previous studies have found that younger children in primary schools are often carriers of enteric parasites . The chain from contaminated soil, fomites, hands, pets and vectors needs to be broken. This is especially true for households with members as young as the age of five years and below. Death by diarrhoea is a global concern in this age range, and an elevated risk exists . Penakalapati et al. share findings gathered through their review that flies have been associated with trachoma and poor hygiene along with animal ownership. Habitual behaviours remain a great risk to public health . It has been argued that having access to clean water and improved sanitation facilities does not necessarily equate to a diarrhoeal-disease-free community . Basic habits such as the regular washing of hands can significantly reduce WASH-related conditions . This is true when soap is used. Paradoxically, the basis remains that clean water and soap need to be available to achieve that end. Behaviour can either be looked at as the more cost-effective intervention for recognisable improvements, or as one of the hardest aspects to change when deeply rooted within a population , rendering it even more costly to uproot. There is a constant shift in migration from rural to urban settings or the urbanisation of existing homelands for benefits offered by the state. This inevitable pursuit of better living standards leads to an increased demand for service delivery, which the treatment plants are not able to meet. Wastewater treatment plants (WWTPs) become overloaded due to receiving elevated volumes of wastewater beyond the original design capacity. With a large percentage of household members in rural areas earning less than ZAR 500 (USD ±27) per month , the capacity to afford cleaning agents is low. Many households run without a consistent or fixed basic income. Members scramble to make ends meet, with the situation aggravated in child-headed households. If there is not enough money for meals to get by, spending on sanitation products is not a reality. This includes cleaning agents and handwashing soap, with a direct impact on hygiene. Studies have shown that treating water at the point of use (POU) greatly reduced the abundance of faecal indicator bacteria and diarrhoea-causing pathogens in a village in Limpopo [ , , ]. In another study conducted in KwaZulu-Natal, Khabo-Mmekoa et al. confirmed the contamination of water that was consumed at household taps with enteric pathogenic microorganisms; the extent of the contamination was less than that observed in household container-stored water of rural dwellers, but contaminated nonetheless. When the impact of sanitation intervention on pathogen manifestation in a household was investigated , no significant difference was observed between the control arm and the sanitation (intervention) arm with regard to faecal contamination. This study had introduced some hygiene improvements in rural household compounds but found minimal impact. The need for a wider coverage was established to be one of the reasons, because pathogens are transported universally. This emphasises the gravity of breaking habitual behaviour. It was reported that densely populated communities experience more frequent water contamination issues . In addition to this is the lack of safe handling of wastes and excreta and, lastly, the occurrence of intensive farming. Previous investigations in South Africa have underscored the critical issue of non-compliance of small-scale WWTPs and drinking water treatment plants (DWTPs) in Limpopo Province posing a threat to public health . The deficiency was notable when the compliance with microbiological and chemical parameters was not met; even more so when pertinent data could not be managed or projected. This lapse shed light on the quality of operational performance of such plants. In 2016, Limpopo Province recorded the highest lack of safe and reliable water supply . While there has been progress over subsequent years, a substantial effort is still necessary to meet the Sustainable Development Goals (SDGs) by 2030 . Active and competent authorities are essential for the success of the goal to reach full coverage of water and sanitation in South Africa. The aim of this study was to assess the occurrence of host-specific faecal indicator markers in water systems in order to pinpoint the most prevalent contributor to faecal contamination from the catchment area to the household level in some rural areas in the Vhembe District Municipality, Limpopo Province, South Africa. We sought to answer the following questions: Is the presence of Bacteroides in water samples reflected in household practices? Is faecal contamination of water introduced where animals are found? Does animal ownership have an effect on the contamination of water? Are natural water sources more contaminated by domestic animals or by humans?
2.1. Science Ethics and Informed Consent Permission from the Vhembe District Municipality (VDM) was granted prior to visiting the study sites and conducting the study. Thereafter, an application was submitted to the Tshwane University of Technology (TUT) faculty committee for research ethics, and it was approved. Consent was sought from the tribal offices, which serve as the gatekeepers to clusters of villages, and this was granted with the condition that the headman or chief in each village be visited personally to give their own informed consent. Lastly, the purpose of the study was explained to each interviewee at household level before being asked for permission to partake in the study. Simple random sampling of villages and households was undertaken. Consequently, the study included only adult participants who were full-time residents of their respective households and possessed a basic understanding of at least one of the languages, namely, Tshivenda, Xitsonga, English, Sepedi or Setswana. 2.2. Site Description The study was conducted in the VDM, which is located in the northern part of Limpopo Province, South Africa. It shares borders with the Southern African Economic Development Community (SADEC) and countries such as Mozambique, Zimbabwe and Botswana. It has an average annual rainfall of 600 mm, filling the perennial rivers in the wet season. The Luvuvhu catchment formed the central part of this study. It is the drainage point of all rivers in the VDM before emptying into the largest river, known as the Limpopo River, which flows into the Indian ocean via Mozambique. The average annual temperature in South Africa is 17.5 °C with the mean temperature in the warmer months (December and January) at 22 °C and 11 °C in the cooler season (June and July). The VDM is in the warmest parts of the country and maintains annual temperatures in the higher range of the scale at around 25 °C and higher in most months . The most prevalent spoken languages are Tshivenda and Xitsonga. The population is estimated to be 1,402,779 and is made up of four local municipalities, as depicted in . These are the Makhado, Collins Chabane, Thulamela and Musina local municipalities. A survey was conducted in three of these local municipalities. Musina Local Municipality was excluded from the study due to predominantly using groundwater as the main water source and additionally being the least populated. The municipal profile analysis reports household users of boreholes making up approximately 9% of the district municipality. It was hence decided that the selected three local municipalities would represent the district. Thulamela is the most populated, at a head count of 497,237 . 2.3. Questionnaire Design The questionnaire was designed in a semi-structured format, incorporating both closed-ended and open-ended questions, to accommodate diverse responses and provide valuable insights. In this manner, the gathering of data was accurate, yet flexible enough to give the respondents and the interviewer freedom to converse freely in order to assure security and confidence, and therefore source more honest answers. Community engagement was carried out for a general understanding of the locals, as they are directly affected by the state of WASH and their immediate social economic context as depicted in . The design was aimed at evaluating the objective of WASH strategies that are already in place being effective in waterborne disease prevention according to the current daily practices and circumstances. 2.4. Study Survey on WASH The study was conducted using a cross-sectional design, and the sample size was determined to be 5% of the population in each village based on the regional statistics from the local municipality. A questionnaire was used to gather information determining the socio-demographic stature and WASH-related factors within the communities, potentially elevating risks to human health. Male and female respondents were not discriminated. Three DWTPs supplying the selected villages and the only WWTP linked to the water network were included in the study ( ). The interviews were conducted from Monday to Friday in the daytime during the period of March 2020 to March 2021, while adhering to COVID-19 restrictions in place at the time. A total of 133 interviews were conducted with 51 residents in Ka-Mhinga, 40 in Ha-Mutsha and 42 in Maniini villages. 2.5. Water Sample Collection A total of 360 water samples were collected ( ) from March to August 2021. In each of the three villages located within the respective local municipalities, a distinct distribution network was observed from the catchment to the DWTP and to the end user. Water was sampled at various points in the three cycles, both up- and downstream of the water sources supplying the DWTP, raw and finished water inside the DWTP and at the point of use. The only WWTP that was functional in the water network was also sampled. The DWTPs fed directly from natural surface water bodies which formed part of the study site. Water samples at the households were collected from the yard taps, most of which were located a few metres from the house. The taps were run for a few seconds before collection. Most houses stored water owing to the periodic water shortages. On the days that sample collection took place without running water, the participants would offer the stored water. The samples were treated the same. 2.6. Isolation of Bacteroides This study ran concurrently with a study on protozoan parasites, where water samples were collected in 25 L polycan drums and filtered using Pall Envirochek TM 1 μm HV filter capsule (Pall South Africa (Pty) Ltd., Midrand, South Africa). The supernatant solution (500 mL) used from protozoa recovery was filtered through a 0.2 µM mixed cellulose ester filter . The bacteria trapped in the membrane were washed with Tween ® 20 and antifoam before being centrifuged at 1500× g for 20 min. The supernatant was discarded, and the pellet stored at −80 °C before being further processed. The DNA was extracted using Zymo Research Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa) following the manufacturer’s instructions. Briefly, ≤250 mg of the pellet was added to a ZR BashingBead™ Lysis with buffer and then centrifuged in a microcentrifuge for 1 min; this was followed by precipitation and purification using provided wash solutions, and then finally eluted with 50 µL of elution buffer. 2.7. Molecular Identification of Isolates For Bacteroides , the extracted DNA was subjected to qPCR for the detection of faecal pollution sources, using host-specific (human, cow, pig, chicken and dog) marker assays. The primers and probes used for each marker are depicted in . The qPCR assay was performed with the total reaction volume of 20 µL, containing 10 µL of iQ™ Multiplex Powermix (Bio-Rad, Hercules, CA, USA), 2 µL of primer/probe mix, 6 µL of PCR-grade water and 2.0 µL of template DNA . The assay was run in a Bio-Rad CFX96 Touch™ Real-Time PCR Detection System. The following cycling conditions applied: 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s. No-template controls were used in every run, along with negative controls and the plasmid DNA containing the genetic markers specific to Bacteroides for the PCR assay. The specificity, sensitivity and accuracy of the marker assays for the geographic region were tested in a separate and similar study in our laboratory. The DNA used was from faecal samples from the selected hosts . The study considered the number of samples that were either true negatives or true positives and those that were either false negatives or false positives to formulate equations to calculate the sensitivity, specificity and accuracy. Briefly, the specificity of BacHum marker for humans was 97.00%, the BacCow marker for cattle was 92.00%, Cytb for poultry was 95.00%, and lastly, the swine marker, Pig-2-Bac was 100.00%. The canine marker BacCan was 97.00% specific for dog faecal samples. The sensitivity was determined to be 100.00% for both human and cow markers, 71.00% for pig, 80.00% for chicken and 75.00% for the dog markers. The accuracy was also determined to be between 93.00% to 98.00% across the markers, with the highest values assigned to human and cow markers and the lowest to chicken and dog markers. The limit of detection for the primer sets were determined to fall within 26.17 and 31.65 gene copies per µL whereas the cut-off values ranged from 37.88 to 39.94 gene copies per µL for all markers. 2.8. Statistical Analysis Results were entered into Stata/SE Version 14.1. software (StataCorp 2015). Bivariate linear regression analysis was used to represent the association between the presence or absence of markers of host-specific faecal contamination (binary outcome variable/y) in households against WASH practices (quantified predictive variable/x). Descriptive statistics were used to interpret the smaller observations of the catchment and treatment plants.
Permission from the Vhembe District Municipality (VDM) was granted prior to visiting the study sites and conducting the study. Thereafter, an application was submitted to the Tshwane University of Technology (TUT) faculty committee for research ethics, and it was approved. Consent was sought from the tribal offices, which serve as the gatekeepers to clusters of villages, and this was granted with the condition that the headman or chief in each village be visited personally to give their own informed consent. Lastly, the purpose of the study was explained to each interviewee at household level before being asked for permission to partake in the study. Simple random sampling of villages and households was undertaken. Consequently, the study included only adult participants who were full-time residents of their respective households and possessed a basic understanding of at least one of the languages, namely, Tshivenda, Xitsonga, English, Sepedi or Setswana.
The study was conducted in the VDM, which is located in the northern part of Limpopo Province, South Africa. It shares borders with the Southern African Economic Development Community (SADEC) and countries such as Mozambique, Zimbabwe and Botswana. It has an average annual rainfall of 600 mm, filling the perennial rivers in the wet season. The Luvuvhu catchment formed the central part of this study. It is the drainage point of all rivers in the VDM before emptying into the largest river, known as the Limpopo River, which flows into the Indian ocean via Mozambique. The average annual temperature in South Africa is 17.5 °C with the mean temperature in the warmer months (December and January) at 22 °C and 11 °C in the cooler season (June and July). The VDM is in the warmest parts of the country and maintains annual temperatures in the higher range of the scale at around 25 °C and higher in most months . The most prevalent spoken languages are Tshivenda and Xitsonga. The population is estimated to be 1,402,779 and is made up of four local municipalities, as depicted in . These are the Makhado, Collins Chabane, Thulamela and Musina local municipalities. A survey was conducted in three of these local municipalities. Musina Local Municipality was excluded from the study due to predominantly using groundwater as the main water source and additionally being the least populated. The municipal profile analysis reports household users of boreholes making up approximately 9% of the district municipality. It was hence decided that the selected three local municipalities would represent the district. Thulamela is the most populated, at a head count of 497,237 .
The questionnaire was designed in a semi-structured format, incorporating both closed-ended and open-ended questions, to accommodate diverse responses and provide valuable insights. In this manner, the gathering of data was accurate, yet flexible enough to give the respondents and the interviewer freedom to converse freely in order to assure security and confidence, and therefore source more honest answers. Community engagement was carried out for a general understanding of the locals, as they are directly affected by the state of WASH and their immediate social economic context as depicted in . The design was aimed at evaluating the objective of WASH strategies that are already in place being effective in waterborne disease prevention according to the current daily practices and circumstances.
The study was conducted using a cross-sectional design, and the sample size was determined to be 5% of the population in each village based on the regional statistics from the local municipality. A questionnaire was used to gather information determining the socio-demographic stature and WASH-related factors within the communities, potentially elevating risks to human health. Male and female respondents were not discriminated. Three DWTPs supplying the selected villages and the only WWTP linked to the water network were included in the study ( ). The interviews were conducted from Monday to Friday in the daytime during the period of March 2020 to March 2021, while adhering to COVID-19 restrictions in place at the time. A total of 133 interviews were conducted with 51 residents in Ka-Mhinga, 40 in Ha-Mutsha and 42 in Maniini villages.
A total of 360 water samples were collected ( ) from March to August 2021. In each of the three villages located within the respective local municipalities, a distinct distribution network was observed from the catchment to the DWTP and to the end user. Water was sampled at various points in the three cycles, both up- and downstream of the water sources supplying the DWTP, raw and finished water inside the DWTP and at the point of use. The only WWTP that was functional in the water network was also sampled. The DWTPs fed directly from natural surface water bodies which formed part of the study site. Water samples at the households were collected from the yard taps, most of which were located a few metres from the house. The taps were run for a few seconds before collection. Most houses stored water owing to the periodic water shortages. On the days that sample collection took place without running water, the participants would offer the stored water. The samples were treated the same.
This study ran concurrently with a study on protozoan parasites, where water samples were collected in 25 L polycan drums and filtered using Pall Envirochek TM 1 μm HV filter capsule (Pall South Africa (Pty) Ltd., Midrand, South Africa). The supernatant solution (500 mL) used from protozoa recovery was filtered through a 0.2 µM mixed cellulose ester filter . The bacteria trapped in the membrane were washed with Tween ® 20 and antifoam before being centrifuged at 1500× g for 20 min. The supernatant was discarded, and the pellet stored at −80 °C before being further processed. The DNA was extracted using Zymo Research Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Inqaba Biotechnical Industries (Pty) Ltd., Pretoria, South Africa) following the manufacturer’s instructions. Briefly, ≤250 mg of the pellet was added to a ZR BashingBead™ Lysis with buffer and then centrifuged in a microcentrifuge for 1 min; this was followed by precipitation and purification using provided wash solutions, and then finally eluted with 50 µL of elution buffer.
For Bacteroides , the extracted DNA was subjected to qPCR for the detection of faecal pollution sources, using host-specific (human, cow, pig, chicken and dog) marker assays. The primers and probes used for each marker are depicted in . The qPCR assay was performed with the total reaction volume of 20 µL, containing 10 µL of iQ™ Multiplex Powermix (Bio-Rad, Hercules, CA, USA), 2 µL of primer/probe mix, 6 µL of PCR-grade water and 2.0 µL of template DNA . The assay was run in a Bio-Rad CFX96 Touch™ Real-Time PCR Detection System. The following cycling conditions applied: 95 °C for 30 s, followed by 45 cycles at 95 °C for 5 s and 60 °C for 30 s. No-template controls were used in every run, along with negative controls and the plasmid DNA containing the genetic markers specific to Bacteroides for the PCR assay. The specificity, sensitivity and accuracy of the marker assays for the geographic region were tested in a separate and similar study in our laboratory. The DNA used was from faecal samples from the selected hosts . The study considered the number of samples that were either true negatives or true positives and those that were either false negatives or false positives to formulate equations to calculate the sensitivity, specificity and accuracy. Briefly, the specificity of BacHum marker for humans was 97.00%, the BacCow marker for cattle was 92.00%, Cytb for poultry was 95.00%, and lastly, the swine marker, Pig-2-Bac was 100.00%. The canine marker BacCan was 97.00% specific for dog faecal samples. The sensitivity was determined to be 100.00% for both human and cow markers, 71.00% for pig, 80.00% for chicken and 75.00% for the dog markers. The accuracy was also determined to be between 93.00% to 98.00% across the markers, with the highest values assigned to human and cow markers and the lowest to chicken and dog markers. The limit of detection for the primer sets were determined to fall within 26.17 and 31.65 gene copies per µL whereas the cut-off values ranged from 37.88 to 39.94 gene copies per µL for all markers.
Results were entered into Stata/SE Version 14.1. software (StataCorp 2015). Bivariate linear regression analysis was used to represent the association between the presence or absence of markers of host-specific faecal contamination (binary outcome variable/y) in households against WASH practices (quantified predictive variable/x). Descriptive statistics were used to interpret the smaller observations of the catchment and treatment plants.
3.1. Survey Findings 3.1.1. Socio-Economic Status of the VDM The respondents consisted of 39 males and 94 females. The oldest interviewee was 84, with the youngest being 22. The average age of the respondents was 45.70, with a standard deviation of 17.48. Households had on average 4.5 members in a family, with the largest family having 12 members. The highest number of children in a household was six (from 0 to 18 years). The households were largely made up of women and children. Of the 132 households, 71 had at least one person working, leaving 46.22% of the population without basic employment. The population showed a peak of young male adults and a steep decrease in females compared to males in the older age ranges ( ). 3.1.2. Water Systems All three communities used unprotected municipal piped water to the yard. In addition, communal taps were present at designated street corners in Ka-Mhinga. Only groundwater sources, mostly as boreholes, in individual properties were protected. In all three communities, some respondents made use of untreated raw water from the river for daily duties, not limited to domestic use. Nearly 89.00% of the respondents stored water owing to the erratic supply ( ). From the population, 124 of the 133 households (approximately 93.23%) used piped municipal supply (improved drinking water sources) from unprotected water bodies ( ). Only a few households were recorded as direct users of untreated water from the surface water sources. 3.1.3. Sanitation, Hygiene and Health The survey revealed that 99.25% of household members use sanitation facilities, with the remainder practising open defecation. A few (six) of the participants agreed to the usage of the facility by children under five years, yet disclosed safety concerns for children of less than five years of age to use the household sanitation facility. Those who answered “no” either have no children of that age (106) or have infants using diapers (21) ( ). Wastewater created within households was often disposed of in pit toilets or in the household backyard dumping site, where wastes would usually be buried or burnt ( ). When asked about frequently occurring illnesses in the household, the majority of the replies (74.44%) were none. Following this, the incidence of diarrhoea was reported as 14.29%, then body rash, other diseases, and lastly, trachoma. Only 10 respondents disclosed that children less than five years old suffered from diarrhoea in their households. It was also discovered that many of those cases lasted 2 or 3 days with largely watery diarrhoea rather than mucopurulent stools. Stools with blood were not reported. Among the age group of 50–65 years, diarrhoea was reported by 3.01% of respondents, and 3.76% acknowledged an incident with family members above 65 years. The last known occurrence of diarrhoea was reported as being mainly a week before. Members of the household presenting with diarrhoea are generally taken to the clinic and not anywhere else. All the respondents mentioned that no measures were taken to isolate the ill person until they recovered. 3.1.4. Animals Found in Villages Observations during the survey indicated the presence of wild birds and primates around water sources along with domesticated animals. The interviews gave more insight on animals within households ( ). 3.1.5. Water Treatment Plants The treatment works that formed part of the study made use of a municipal laboratory for sample testing, which was centrally located at the VDM site. Potable water samples were sent there weekly or bi-weekly. It was also found that all the treatment plants had chlorine readily available all the time. The plants did not report any kind of vandalism. Some of the pumps and sludge treatment tanks at the Thohoyandou WWTP required repairs to function optimally ( ). The WWTP discharges its treated wastewater into the Mvudi River, which is a tributary of the Luvuvhu River, upstream of the Nandoni Dam. Maturation ponds were part of the treatment system on site, becoming more useful in events of mechanical breakdowns. 3.2. Sources of Faecal Pollution The interviews revealed that three households in Ka-Mhinga had some animals when no others did. These households also cultivated land within their yards. Animal ownership was the lowest in Ha-Mutsha and the highest in Maniini. In terms of detecting host-specific faecal markers, cow faecal matter was not detected in Ha-Mutsha households, while pig faecal matter was detected there only once, followed by incremental increases in the detection of human and dog faecal matter, and lastly, the chicken-specific faecal marker was detected most frequently. illustrates the source of faecal contamination at the POU in the study sites as determined by qPCR. Overall, in Ka-Mhinga households, the dog-associated marker at a detection rate of 33.33% was the most prevalent, while in Maniini, it was 18.42%. Moreover, Ka-Mhinga exhibited the highest poultry source of faecal contamination at 28.89%, followed by Ha-Mutsha at 15.00% and lastly Maniini at half of the contamination found in Ha-Mutsha. Ka-Mhinga shows a higher occurrence of faecal contamination and Ha-Mutsha the lowest. The sources of faecal contamination at treatment plants—drinking water and wastewater—are presented in . The Vondo WTP supplying Ha-Mutsha village presented the lowest faecal contamination, with none of the markers detected in the final water. A similar observation was noted in Nandoni WTP, supplying Maniini village, showing the human-associated marker in raw water only. A similar but higher detection rate was observed for Mhinga final water. The highest faecal contamination is recorded in the wastewater effluents as well as in Ka-Mhinga raw water (abstraction point). The human-associated marker was detected most frequently across the sites. Overall, the surface water sources were found to be polluted with faecal matter, with all the target markers detected, except for the Vondo Dam ( ). The Nandoni Dam displayed all five faecal markers, with the pig-associated marker being the most prevalent at 87.50%. The Vondo Dam, on the other hand, presented a lower detection rate of the target markers; the human-associated marker was the most prevalent at 25.00%. The regression analysis ( ) reflected overall varying relationships from no relationship (0.0) to moderate association (0.4). Out of 55 observations, where 11 WASH variables were each run against the 5 target markers, 14 (25.50%) presented a moderate relationship (R 2 = 0.3–0.5) with a p value that was smaller than alpha (0.00–0.01). The highest of these was variable (e), water treatment in household (R 2 = 0.414; p = 0.00), followed by (f), presence of wash basin (R 2 = 0.413; p = 0.00), both associated with chicken faecal contamination. Weak relationships (R 2 = 0.1–0.2) were observed for 30/55 (54.50%) observations. From those, only 20.00% were found not to be significant, with a p value that was greater than alpha (0.05). The remaining 80.00% were significant ( p = 0.00–0.049).
3.1.1. Socio-Economic Status of the VDM The respondents consisted of 39 males and 94 females. The oldest interviewee was 84, with the youngest being 22. The average age of the respondents was 45.70, with a standard deviation of 17.48. Households had on average 4.5 members in a family, with the largest family having 12 members. The highest number of children in a household was six (from 0 to 18 years). The households were largely made up of women and children. Of the 132 households, 71 had at least one person working, leaving 46.22% of the population without basic employment. The population showed a peak of young male adults and a steep decrease in females compared to males in the older age ranges ( ). 3.1.2. Water Systems All three communities used unprotected municipal piped water to the yard. In addition, communal taps were present at designated street corners in Ka-Mhinga. Only groundwater sources, mostly as boreholes, in individual properties were protected. In all three communities, some respondents made use of untreated raw water from the river for daily duties, not limited to domestic use. Nearly 89.00% of the respondents stored water owing to the erratic supply ( ). From the population, 124 of the 133 households (approximately 93.23%) used piped municipal supply (improved drinking water sources) from unprotected water bodies ( ). Only a few households were recorded as direct users of untreated water from the surface water sources. 3.1.3. Sanitation, Hygiene and Health The survey revealed that 99.25% of household members use sanitation facilities, with the remainder practising open defecation. A few (six) of the participants agreed to the usage of the facility by children under five years, yet disclosed safety concerns for children of less than five years of age to use the household sanitation facility. Those who answered “no” either have no children of that age (106) or have infants using diapers (21) ( ). Wastewater created within households was often disposed of in pit toilets or in the household backyard dumping site, where wastes would usually be buried or burnt ( ). When asked about frequently occurring illnesses in the household, the majority of the replies (74.44%) were none. Following this, the incidence of diarrhoea was reported as 14.29%, then body rash, other diseases, and lastly, trachoma. Only 10 respondents disclosed that children less than five years old suffered from diarrhoea in their households. It was also discovered that many of those cases lasted 2 or 3 days with largely watery diarrhoea rather than mucopurulent stools. Stools with blood were not reported. Among the age group of 50–65 years, diarrhoea was reported by 3.01% of respondents, and 3.76% acknowledged an incident with family members above 65 years. The last known occurrence of diarrhoea was reported as being mainly a week before. Members of the household presenting with diarrhoea are generally taken to the clinic and not anywhere else. All the respondents mentioned that no measures were taken to isolate the ill person until they recovered. 3.1.4. Animals Found in Villages Observations during the survey indicated the presence of wild birds and primates around water sources along with domesticated animals. The interviews gave more insight on animals within households ( ). 3.1.5. Water Treatment Plants The treatment works that formed part of the study made use of a municipal laboratory for sample testing, which was centrally located at the VDM site. Potable water samples were sent there weekly or bi-weekly. It was also found that all the treatment plants had chlorine readily available all the time. The plants did not report any kind of vandalism. Some of the pumps and sludge treatment tanks at the Thohoyandou WWTP required repairs to function optimally ( ). The WWTP discharges its treated wastewater into the Mvudi River, which is a tributary of the Luvuvhu River, upstream of the Nandoni Dam. Maturation ponds were part of the treatment system on site, becoming more useful in events of mechanical breakdowns.
The respondents consisted of 39 males and 94 females. The oldest interviewee was 84, with the youngest being 22. The average age of the respondents was 45.70, with a standard deviation of 17.48. Households had on average 4.5 members in a family, with the largest family having 12 members. The highest number of children in a household was six (from 0 to 18 years). The households were largely made up of women and children. Of the 132 households, 71 had at least one person working, leaving 46.22% of the population without basic employment. The population showed a peak of young male adults and a steep decrease in females compared to males in the older age ranges ( ).
All three communities used unprotected municipal piped water to the yard. In addition, communal taps were present at designated street corners in Ka-Mhinga. Only groundwater sources, mostly as boreholes, in individual properties were protected. In all three communities, some respondents made use of untreated raw water from the river for daily duties, not limited to domestic use. Nearly 89.00% of the respondents stored water owing to the erratic supply ( ). From the population, 124 of the 133 households (approximately 93.23%) used piped municipal supply (improved drinking water sources) from unprotected water bodies ( ). Only a few households were recorded as direct users of untreated water from the surface water sources.
The survey revealed that 99.25% of household members use sanitation facilities, with the remainder practising open defecation. A few (six) of the participants agreed to the usage of the facility by children under five years, yet disclosed safety concerns for children of less than five years of age to use the household sanitation facility. Those who answered “no” either have no children of that age (106) or have infants using diapers (21) ( ). Wastewater created within households was often disposed of in pit toilets or in the household backyard dumping site, where wastes would usually be buried or burnt ( ). When asked about frequently occurring illnesses in the household, the majority of the replies (74.44%) were none. Following this, the incidence of diarrhoea was reported as 14.29%, then body rash, other diseases, and lastly, trachoma. Only 10 respondents disclosed that children less than five years old suffered from diarrhoea in their households. It was also discovered that many of those cases lasted 2 or 3 days with largely watery diarrhoea rather than mucopurulent stools. Stools with blood were not reported. Among the age group of 50–65 years, diarrhoea was reported by 3.01% of respondents, and 3.76% acknowledged an incident with family members above 65 years. The last known occurrence of diarrhoea was reported as being mainly a week before. Members of the household presenting with diarrhoea are generally taken to the clinic and not anywhere else. All the respondents mentioned that no measures were taken to isolate the ill person until they recovered.
Observations during the survey indicated the presence of wild birds and primates around water sources along with domesticated animals. The interviews gave more insight on animals within households ( ).
The treatment works that formed part of the study made use of a municipal laboratory for sample testing, which was centrally located at the VDM site. Potable water samples were sent there weekly or bi-weekly. It was also found that all the treatment plants had chlorine readily available all the time. The plants did not report any kind of vandalism. Some of the pumps and sludge treatment tanks at the Thohoyandou WWTP required repairs to function optimally ( ). The WWTP discharges its treated wastewater into the Mvudi River, which is a tributary of the Luvuvhu River, upstream of the Nandoni Dam. Maturation ponds were part of the treatment system on site, becoming more useful in events of mechanical breakdowns.
The interviews revealed that three households in Ka-Mhinga had some animals when no others did. These households also cultivated land within their yards. Animal ownership was the lowest in Ha-Mutsha and the highest in Maniini. In terms of detecting host-specific faecal markers, cow faecal matter was not detected in Ha-Mutsha households, while pig faecal matter was detected there only once, followed by incremental increases in the detection of human and dog faecal matter, and lastly, the chicken-specific faecal marker was detected most frequently. illustrates the source of faecal contamination at the POU in the study sites as determined by qPCR. Overall, in Ka-Mhinga households, the dog-associated marker at a detection rate of 33.33% was the most prevalent, while in Maniini, it was 18.42%. Moreover, Ka-Mhinga exhibited the highest poultry source of faecal contamination at 28.89%, followed by Ha-Mutsha at 15.00% and lastly Maniini at half of the contamination found in Ha-Mutsha. Ka-Mhinga shows a higher occurrence of faecal contamination and Ha-Mutsha the lowest. The sources of faecal contamination at treatment plants—drinking water and wastewater—are presented in . The Vondo WTP supplying Ha-Mutsha village presented the lowest faecal contamination, with none of the markers detected in the final water. A similar observation was noted in Nandoni WTP, supplying Maniini village, showing the human-associated marker in raw water only. A similar but higher detection rate was observed for Mhinga final water. The highest faecal contamination is recorded in the wastewater effluents as well as in Ka-Mhinga raw water (abstraction point). The human-associated marker was detected most frequently across the sites. Overall, the surface water sources were found to be polluted with faecal matter, with all the target markers detected, except for the Vondo Dam ( ). The Nandoni Dam displayed all five faecal markers, with the pig-associated marker being the most prevalent at 87.50%. The Vondo Dam, on the other hand, presented a lower detection rate of the target markers; the human-associated marker was the most prevalent at 25.00%. The regression analysis ( ) reflected overall varying relationships from no relationship (0.0) to moderate association (0.4). Out of 55 observations, where 11 WASH variables were each run against the 5 target markers, 14 (25.50%) presented a moderate relationship (R 2 = 0.3–0.5) with a p value that was smaller than alpha (0.00–0.01). The highest of these was variable (e), water treatment in household (R 2 = 0.414; p = 0.00), followed by (f), presence of wash basin (R 2 = 0.413; p = 0.00), both associated with chicken faecal contamination. Weak relationships (R 2 = 0.1–0.2) were observed for 30/55 (54.50%) observations. From those, only 20.00% were found not to be significant, with a p value that was greater than alpha (0.05). The remaining 80.00% were significant ( p = 0.00–0.049).
There remains a need to support and capacitate rural communities to monitor their daily drinking water quality and better manage the sanitation process . Based on this premise, we sought to investigate the link between WASH practices and the presence of faecal contamination in drinking water supplies. The survey revealed that more women than men were found to be available in the households ( ), thus confirming their role as primary caregivers, as described by WHO and other researchers . This factor reiterates the need for focused interventions that will make a direct impact on the lives of those who carry much of the care and the domestic burden, giving women and children a voice in their communities. In this study population, more males were of a young age, pointing to the burden on frail elderly women for ensuring that there is sufficient water for domestic uses in households. The findings concur with the percentage of women (91.84%) fetching water far outnumbering the percentage of men (3.06%) ( ). The WHO/UNICEF outlines the dire need to move away from the distinctive effects of water scarcity on gender roles, as women are often disproportionately affected by water scarcity. It seems a distant reality in this study population with such polar numbers of males or females fetching water. Unless serious intervention is effectively implemented, the female members of the community will continue to be negatively impacted by the lack of WASH. The depiction of the gender ratio ( ) illustrates a sharp peak of males in the age range of 22–31 years and a subsequent sharp drop in the 32–41-year age group. The authors Stecklove and Menashe-Oren elaborate on gender ratios being driven by determinants such as rural–urban migration and employment. Males in their thirties migrate to find work, while their counterparts stay at home to provide care for the young. The authors elaborate on the trajectories that rural youth would follow, suggesting an increase in sub-Saharan African youth (15–24-year age group) up to the year 2050; this was also evident in our findings. This projection speaks to the large presence of under-five children there today, as well as the high fertility rate among young couples, and the emphasis to protect these vulnerable young from enteric pathogens. When considering the cost of reagents ( ), a majority (40.60%) of the respondents alluded to purchasing at least one or two cleaning agents per month, spending over ZAR 10–49 (USD ±0.5 to 2). Following this were those spending ZAR 0 and 14.29% spending more than ZAR 100 (USD ±5) monthly. It was further found that close to all participants admitted to the capacity of purchasing cleaning agents and soap for washing hands. There was a weak association between the presence of markers in a household and the washing with soap practice, all having R 2 values ranging between 0.089 and 0.257. The highest association in this range was the relationship with the chicken marker of faecal contamination, with a p value of 0.004 ( ). A study carried out on the knowledge and practices of caregivers on household water treatment in Ethiopia found that application of best practice was low even when the caregiver had knowledge of and a positive attitude towards safe drinking water in the household . In our study, the knowledge of household water treatment methods was not investigated as a factor of educational level, because the information sought was not specifically from the primary caregiver. Although a larger proportion of the respondents were lacking higher education (figures withheld) it suggests little impact on the burden of faecal contamination at the household level. The overall water coverage was high, finding 93.23% of respondents making use of improved drinking water sources abstracted from various natural water sources ( ). Almost all the respondents (39/40) in Ha-Mutsha were satisfied with the supply or availability of water in their households ( ). This satisfaction could be attributed to the mixing of the municipal supply with personal boreholes. However, residents in villages like Maniini and Ka-Mhinga, relying more on municipal supply, expressed their grief about the availability of water in their communities. Additionally, there are worrying numbers of respondents storing water for everyday use (118/133). This not only applies to users of boreholes using untreated water but becomes riskier for users of household storage containers using utensils to draw water. The previously safe water loses integrity and becomes a breeding ground for potentially harmful pathogens [ , , , ]. To exacerbate matters, 87.30% of respondents do not practise any form of household water disinfection methods. Many respondents stored water for periods of a week and longer ( ). This ultimately leads to the need for water treatment at the household level, which in this case is barely practised. When water is treated and stored for prolonged periods, the risk of reinfection increases. Better sanitation in urban areas could be achieved by connecting households to sewer lines as well as through the basic hygiene practice of handwashing with soap . It was established during the survey that even the water collection point itself, yard tap or communal tap, could be described as unsanitary, thus posing the irony of clean water from a dirty tap. Nonetheless, having the option of several water sources is an advantage for the villages. Our findings reveal an applaudable 99.00% sanitation coverage. The use of pit latrines was the most popular at 63.00%, and open defaecation was the least popular at 1.00%. These impressive figures in a rural setting give hope for full coverage and show that the disregard for sanitation may be a thing of the past, as was found by Sibiya and Gumbo . However, findings in show 107/133 people without a handwash basin in the sanitation facility. A recent study revealed the risks at play after toilet use when hands are not immediately cleansed . The authors also reveal the improvements to hygiene when a flush toilet is used. Exposure to faecal pathogens moves from latrines to fomites. It is, therefore, essential to immediately wash hands with soap after using the toilets. The use of manure on farms necessitates good husbandry practices with proper veterinary care. This, in turn, would alleviate zoonosis . Not only does bad manure management introduce pathogens into the water course, but Font-Palma mentions the heavy metals, antibiotics, and the release of gases contributing towards greenhouse gases and salt toxicity. Above all is the critical need for water when farming. If the farmers were to use contaminated water without prior treatment for crop production, it may be a source of pathogens and the produce may be contaminated. The application of manure to agricultural fields must also be done with care; to disinfect manure for safer use requires resources that may not be readily available for local farmers. In our study, participants spend an average of ZAR 50 (USD > 2) monthly on cleaning agents. This is not nearly enough for maintenance of healthy disease-free livestock. However, in this regard, several methods described by Parihar et al. may be applicable at affordable costs for local farmers. These are not limited to composting of faeces using handmade trenches, composting of animal carcasses and the various approaches to achieving good management of livestock wastes. The results revealed that 76/133 respondents were not owners of animals. In addition, 55/133 observations found no animals around water sources ( ). The most apparent indication of faecal contamination are cows, donkeys, goats and pigs around water sources. The mixed animal variable represents instances where the individual observations were made in combination with one or more of the other animals. The subsequent qPCR assays would determine that the dominant sources of faecal contamination were chickens and pigs in Nandoni Dam and the Luvuvhu River in Ka-Mhinga ( ). Moreover, chickens, dogs and cats were not commonly observed around surface water sources but showed a higher presence at households in that order. Similarly, Cabral showed dogs to have the highest faecal bacterial load compared to other domestic animals such as chickens, pigs and cats. This author found dogs to have 9.0 and 8.4 log 10 cells/g of wet weight faeces of faecal streptococci and Clostridium perfringens , respectively. Among warm-blooded animals and humans, the highest count for faecal indicator bacteria was reported for human faeces, and the lowest counts for wild animals with faecal indicator bacteria ratios that are 10-fold lower than those of domestic animals. Many domesticated animals roam around the village drinking water from leaking taps, puddles and storage containers in the households if not provided with a drinking bowl or trough. Increasing urbanisation and the strive for a better life raise security concerns, therefore necessitating higher measures of protection against crime. The high presence of dog markers may stem from this reason. Dogs are also often used by farmers to assist in guarding livestock while grazing. Similarly, livestock that are kept passively and allowed to forage randomly often have little veterinary care. This allows them to be reservoirs for disease-causing zoonotic parasites and pose a risk to public health . Surveying the water treatment plants revealed them to be in operational condition. Only one (Mhinga DWTP) of the three DWTPs was experiencing some challenges, especially in the wet season when the pipes would be blocked or broken after storms, with no repairs being carried out. The only WWTP in the study was found to have problems as well, with some infrastructure needing replacement or repairs. The staff confirmed their satisfaction in their positions and duties, as well as the ability to calculate the dosage for the disinfection agent in secondary treatment in the DWTP. Work carried out by Dunkin et al. suggests the contributing factor of biofilm interactions with water microorganisms in distribution systems. Holding this as true would mean that the water retention time inside the pipes would consequentially be a factor as well. The longer the water is held in the system, the higher the chances of variability of microconsortia . The negligible act of regularly opening the faucet, or not, in this regard would be significant. In addition, the volume of water used from a faucet as a function of time could contribute towards water quality. However small the change in the quality of water, it can be significant when the focus is on drinking water. Although the water pressure in this system was not evaluated, the risk of infiltration exists due to an old infrastructure and common leaks, exacerbated by an erratic supply. In terms of the overall sources of contamination as seen in , Ka-Mhinga was observed as the most contaminated, with four markers detected, and Ha-Mutsha as the least contaminated. Although no indicators of faecal contamination were detected in Ha-Mutsha and Nandoni WTP finished water, the raw water abstracted for the Nandoni DWTP (25.00%) showed possible human faecal contamination ( ). The Mhinga DWTP finished water showed the presence of human faecal contamination at a detection rate of 28.60%, marking the highest contamination among the finished drinking water samples, suggestive of insufficient processing of the water in the DWTP before distribution. The Mhinga DWTP raw water was found to be dominated by the cow marker at a detection rate of 37.50%, followed by 25.00% for the human and chicken markers. We assigned the high detection rates to the many activities that are constantly taking place at the abstraction point, as it is easily accessible to humans and animals, as well as the short distance between the abstraction point and the small treatment plant. The presence of host-specific faecal contamination is seen with the pig-associated marker being the lowest among surface water sources and peaking in the Nandoni Dam. The Nandoni Dam displays the highest faecal contamination in this group. This goes hand in hand with the abundant anthropogenic activities taking place in and around the dam at the easily accessible shallow ends lying at the feet of several villages. Natural water sources have proven of more importance to rural dwellers than communities in urban areas in the Venda area, especially because locals rely heavily on agriculture as a form of income generation . However, it is the combination of rural livelihoods and urban developments that contributes to water source deterioration and consequentially has a negative impact on the rural communities compared to urban residents. Our study found that although the study areas had a municipal water supply connection to the yard or in their street, water supply was inconsistent and there was a need for either an alternate supply or for storage. Surveying revealed Ka-Mhinga to be more rural than the other two villages, with Ha-Mutsha being the more urbanised among them. The WASH variables analysed showed few to moderate relationships with one or more MST markers ( ). The chicken marker had significant associations with all but three WASH variables. This means that the presence of chicken faecal contamination was significant across the household standpipes in relation to those variables, whether they kept chickens or not. However, frequent cases of diarrhoea, crops around water sources and total number of members in a household were not significant relative to the chicken marker. The WASH variable of different household animals wassignificant for the human, pig and chicken markers, meaning that the presence of these markers was of notable importance in households with or without animals, whereas cow and dog were not significant. The WASH variables duration of water storage, method of drawing water, water treatment in household and presence of wash basin were found to show a significant relationship with all five markers. This reflects some of the basic and daily practices in a household. Simply storing water for use during a scheduled water cut could negatively impact the water quality and put lives at risk of faecal pollution, right in the home. The dog-associated marker was the most detected across all sites, followed by human and chicken, with pig being the least detected. Moreover, Penakalapati et al. found that inconsistencies are common when linking animal presence in a household with the prevalence of diarrhoea amongst children in the household. In our study, frequent diarrhoea was significantly linked to the occurrence of human and pig markers, showing a weak relationship ( ). Illnesses often occurring in the household showed a moderate relation to cow, dog, human and chicken markers. Cow and dog markers were further found to have a weak association with crops grown around water sources. It has been reported that humans that live in close proximity to animals are at an increased risk of zoonotic infection . Contrary to this, the high detection rate of markers indicated widespread faecal contamination, even in households without livestock or pets. Animal faecal wastes need to be managed properly to lower the chances of sickness. Roaming of stray animals needs a form of control as well. The Venda area generates money through farming and ecotourism and is the fastest growing in the province . Waste stabilisation ponds as a control measure in WWTPs can pose risks in terms of performance. Ponds can be easily overloaded, especially if the design was poor to begin with, or short-circuited, leading to reduced retention times and the discharge of inadequately treated effluent with higher microbial loads. It was observed that the specified personal protective equipment was not used by staff, thereby increasing the chances of aerosol or dermal contact with wastewater potentially harbouring diarrhoeic pathogens, among other harmful agents . Due to Thulamela being overpopulated, the WWTP was overloaded . One DWTP and the WWTP have reported increased challenges during storm events. This leaves the end-user at the mercy of climate change . Flood waters introduce a broad range of pathogens into natural water sources. Floods also cause interruptions in the daily operations and consequently increase the chances of more water supply interruptions for users. This only exaggerates the risk of waterborne diseases, as communities would turn to storage as one of their options. Alternatively, water could be purchased from neighbours, in which case the distance to access safe drinking water increases, thus regressing to unimproved water sources, as defined by the World Health Organization (WHO). Unfortunately, this was found to hold true in the study. In our study, animal ownership was not a contributing factor to disease prevalence within households. It is possible to find a marker in a household where there are no animals, but the neighbouring households keep animals. It is not uncommon to find animals existing nomadically between households that are located close together or in a community. This could be a possible pathway for pathogens to spread, especially in settings where sanitation or hygiene are lacking. The overall MST profile matched what was expected after the general observations carried out during the survey phase of the project. These findings should advise existing water and sanitation safety strategies to strengthen community accountability and drive action towards high-quality public health. Drinking Water Safety Plans and Sanitation Safety Plans proposed by WHO and global projects such as the Global Water Pathogen Project encourage community members to have a sense of ownership for this precious resource and work together with authorities in the efforts to protect water bodies from pathogens .
The target hosts investigated as sources of faecal contamination in water sources were found with varying prevalence in the catchments and at the household level. The detection of dog faecal matter being the most prolific in household water samples goes hand in hand with the general understanding of human relationships with companion animals and their presence around homes for general safety or for guarding grazing livestock in agricultural settings. Similar to that are the levels of cow markers in surface waters, where most of the time is spent grazing. Above all, human faecal matter is known to pose greater risks to public health than that from animals. Our study detected high levels of the human-associated marker in all the water samples, indicating the possibility of pathogenic microorganisms that would be especially detrimental to those with weaker immune systems like babies under the age of five years and the elderly above 65 years. The main source of water in the villages was found to be piped municipal water to the yard tap, as well as privately owned boreholes. The absence of alternative water sources leaves users at the mercy of purchasing water from those with boreholes in the event of water supply interruptions. The presence of livestock other than those targeted here needs to be investigated as contributors to water pollution. The reason for keeping animals was not investigated. Perhaps if it had, there would be insights into reasons for keeping animals, such as for commercial purposes, or keeping dogs for security, cats for rodent control or simply keeping animals for food. The reason would allude to steps that are necessary for animal movement control and to whose responsibility it might be to ensure that the appropriate measures are taken. Better management of animal wastes is imperative, as is safer handling of diapers, toys, utensils, and good quality hygiene practices after animal contact. We recommend fencing and bridging of water sources to limit animal access. Additionally, on-farm treatment of water for consumption by animals and better management including treatment of animal faecal deposits before use may ameliorate zoonosis. There was inconsistent agreement between the microbial source tracking results and household surveys, suggestive of widespread sanitation issues, even in households without domesticated animals. The heavy reliance of this study on interviews rather than on observations skewed the true outcome, in that participants were in some instances unable to recall, for example, the last diarrhoeal episode. In fact, the bias was observed quite often when participants became uneasy with some sensitive information. We recommend future studies that better manage this aspect. The data gathered here, which should be useful to environmentalists, water engineers and the likes of town planners, managers and economists, can be used to troubleshoot and then develop new tactics for addressing the issues at hand. It would be beneficial to make use of more than one marker for each assay to close the window of doubt on the absence of faecal pollution upon the undetectable presence of one marker. We recommend the use of microarray cards or the coupling of MST with chemical source tracking (CST) for faecal pollution of water sources.
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Extract from | ee694e10-3601-4b34-bd23-a1aed907e792 | 8492811 | Anatomy[mh] | With the popularization of Chinese herbal medicines, the interaction between Chinese herbal medicines and other drugs has become an increasingly important safety concern in the clinical application of traditional medicines . Research in this area is expanding rapidly. The interactions between Chinese herbal medicines and drugs are complex, involving many chemical components and many types of pharmacological activity. The currently known herb-drug interactions (HDIs) occur mainly at the pharmacokinetic and pharmacodynamic levels; in particular, a great deal of research has focused on the pharmacokinetic interactions, in particular, on the absorption, distribution, metabolism and excretion , . However, studies on the pharmacodynamic interactions between herbs and drugs are still very limited. Herbal medicines can affect drug metabolism by altering the activity of liver enzymes, such as cytochrome P450 (CYP450), and membrane transporters, such as P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (Mrp2), which may change the pharmacodynamic process of drugs and thus cause drug concentrations to change in vivo , . Pirarubicin (THP) is an anthracycline that interferes with the synthesis of DNA in tumor cells and is widely used in the treatment of several kinds of tumors (Fig. A) . However, the clinical use of THP has been severely limited due to its severe cardiotoxicity, which is characterized by irreversibility and drug accumulation . Liver metabolism and excretion, especially via the efflux transporter P-gp, Mrp2, play an important role in the metabolism of anthracycline; therefore, special attention should be paid to the occurrence of cardiotoxicity when THP is taken alongside drugs that can inhibit cardiac metabolism and drug excretion , . Dioscorea bulbifera L. is a Chinese herbal medicine that is commonly used in the clinic (Fig. B). This herb has a wide range of pharmacological activity. In recent years, the antitumor effect of extract from Dioscorea bulbifera L. rhizomes (DB) has received increasing attention; for example, it can inhibit cervical cancer and hepatocarcinoma . DB extract contains a variety of components, including diterpene lactones, steroidal saponins, flavonoids, alkaloids, and micronutrients, among others . DB extract can lead to liver injury and downregulation of the intrahepatic expression of P-gp. These effects may be an important contributor to abnormal biotransformation in the process of drug metabolism . Thus, we hypothesize that DB may increase the cardiotoxicity risk of THP when the two are co-administered. In our study, we explored liver and heart injury in mice and assessed the accumulation of THP, the localization and expression of P-gp and Mrp2 to investigate the mechanisms of the HDI between DB and THP.
Effect of DB extract on the cardiotoxicity induced by THP Heart biopsies from mice treated with THP and DB extract showed homogeneous red stain of cardiac myocytes, loose arrangement of cardiac fibers, muscle fiber breakage and obviously light staining (Fig. B). Only cardiac muscle light staining was observed in the THP group, and normal myocardial cell morphology was observed in both of the control group and DB group. Effect of DB extract on the accumulation of THP The precision and accuracy were evaluated at three concentration levels (0.8, 5.0 and 15.0 μg/mL) in 3 replicates. Intraday precisions were analyzed 6 times in one day and interday precisions were analyzed for 6 consecutive days. Intra-day and inter-day precisions used the relative standard deviation (RSD) as indicator (Supplementary Table ). We detected the THP concentrations in THP and THP + DB groups. In THP + DB group, the concentrations of THP in both tissues were significantly elevated ( p < 0.05) (Fig. ). Effect of DB extract on the intrahepatic expression of P-gp and Mrp2 P-gp and Mrp2 membrane localization was found in hepatocytes after THP treatment, which was eliminated by coadministration of THP and DB. As expected, there was no P-gp and Mrp2 membrane localization observed in mice of the DB and control groups (Figs. A, A). Similarly, compared to the control group, the levels of P-gp and Mrp2 expressions in mouse livers of THP group were increased by 3.76- and 4.36-fold, respectively ( p < 0.01). However, there was absent when THP and DB were co-administered ( p < 0.01) (Figs. B, B). Effect of DB extract on the hepatocytes In the control and THP groups, the hepatic lobules of the liver tissue were clear, the hepatocytes were arranged in an orderly manner, and the nuclei of the hepatocytes were clearly visible. In the THP and THP + DB groups, the normal structure of the hepatic lobules had disappeared, the hepatic sinusoids were absent, the hepatic cells surrounding the portal vein showed flaky necrosis, there were many infiltrating inflammatory cells, the hepatocytes were obviously edematous, the cytoplasm was loose and empty, and balloon-like degeneration was visible (Fig. ). The damage of hepatocytes caused by DB extract was mainly embodied by the change of cytoplasm, which could not be reflected in the result of immunohistochemistry (Figs. , ) due to the staining site of hematoxylin.
Heart biopsies from mice treated with THP and DB extract showed homogeneous red stain of cardiac myocytes, loose arrangement of cardiac fibers, muscle fiber breakage and obviously light staining (Fig. B). Only cardiac muscle light staining was observed in the THP group, and normal myocardial cell morphology was observed in both of the control group and DB group.
The precision and accuracy were evaluated at three concentration levels (0.8, 5.0 and 15.0 μg/mL) in 3 replicates. Intraday precisions were analyzed 6 times in one day and interday precisions were analyzed for 6 consecutive days. Intra-day and inter-day precisions used the relative standard deviation (RSD) as indicator (Supplementary Table ). We detected the THP concentrations in THP and THP + DB groups. In THP + DB group, the concentrations of THP in both tissues were significantly elevated ( p < 0.05) (Fig. ).
P-gp and Mrp2 membrane localization was found in hepatocytes after THP treatment, which was eliminated by coadministration of THP and DB. As expected, there was no P-gp and Mrp2 membrane localization observed in mice of the DB and control groups (Figs. A, A). Similarly, compared to the control group, the levels of P-gp and Mrp2 expressions in mouse livers of THP group were increased by 3.76- and 4.36-fold, respectively ( p < 0.01). However, there was absent when THP and DB were co-administered ( p < 0.01) (Figs. B, B).
In the control and THP groups, the hepatic lobules of the liver tissue were clear, the hepatocytes were arranged in an orderly manner, and the nuclei of the hepatocytes were clearly visible. In the THP and THP + DB groups, the normal structure of the hepatic lobules had disappeared, the hepatic sinusoids were absent, the hepatic cells surrounding the portal vein showed flaky necrosis, there were many infiltrating inflammatory cells, the hepatocytes were obviously edematous, the cytoplasm was loose and empty, and balloon-like degeneration was visible (Fig. ). The damage of hepatocytes caused by DB extract was mainly embodied by the change of cytoplasm, which could not be reflected in the result of immunohistochemistry (Figs. , ) due to the staining site of hematoxylin.
The use of herbal medicines as an important component of multicomponent therapy has increased steadily over the past decade. Eighty percent of people in Asian countries use herbs to promote health and treat common diseases, such as inflammation, pain, heart disease, cirrhosis, and central nervous system disease. However, with the increasing use of herbal medicines, the risk of HDIs has increased as well , . DB polysaccharides have antitumor properties in mice bearing U14 cervical carcinoma and strengthen the antitumor effect of cyclophosphamide . However, DB extract can enhance the cardiotoxicity of THP, according to our study. Cardiotoxicity is the main adverse effect of THP. K S Sridhar et al. had reported that there was no cardiotoxicity with a cumulative dose of 300 mg/m 2 THP . The drug administration plan was based on the methods in literature and preliminary work of our group , . The dose of pirarubicin was not the clinical equivalent. In order to speed up the formation of cardiotoxicity induced by THP, mice were administrated with a doubled-dosage of THP single high dose which could develop cardiotoxicity after two days of administration. The muscle mass heart biopsies of the THP group showed extensive cardiovascular damage, muscle fiber breakage and necrosis in the THP + DB group according to the histopathology results. Combined DB extract and THP treatment showed a trend suggesting that cardiotoxicity could be strengthened by DB supplementation. Next, we explored the mechanisms by which DB extract causes increased THP cardiotoxicity. Since the cardiotoxicity induced by THP is dose dependent, we hypothesize that the increased cardiotoxicity after combined use is due to the accumulation of THP in the body. The concentrations of THP in serum and heart were significantly elevated by the co-administration of DB extract and THP. THP efflux is mainly involved in P-gp, and the use of THP will lead to an increase in P-gp, which is also the reason for THP resistance. When a drug induces the function of efflux transporters, which accelerates the transport of the drug itself or a combination of other drugs out of the cell to reduce the toxic side effects of the drug on the cell, but it also reduces the efficacy of the drug. When a drug appears to inhibit efflux transporters, it will stay in cells and organs for a long time, enhancing the efficacy of the drug will also increase toxic side effects. P-gp and Mrp2 plays an important role in anthracycline efflux which can lead to drug accumulation if suppressed . Because of the reduction of P-gp and Mrp2 in DB group, we hypothesized that THP accumulation was due to the inhibition of P-gp and Mrp2 expression by DB extract, which resulted in the accumulation of THP in vivo and increased cardiotoxicity. There is high expression of P-gp in liver, kidney, brain and placenta , which suggests that P-gp plays an important role in the excretion of exogenous substances and metabolites into the intestinal cavity, bile and urine and in preventing their accumulation in the body. Its high expression in the normal liver may influence the absorption and transport of drugs and affect their efficacy, which is one of the reasons for drug-drug interactions. Improper use of xanthate can cause multiple organ damage, such as liver damage and kidney damage, especially hepatotoxicity . Niu et al. found that ethyl acetate extract could cause serious liver injury in ICR mice . Similar to P-gp, Mrp2 is also one of the transporters involved in metabolism in the liver. Mrp2 can transport various substrates and is an important role in drug resistance in cancer – . The expression of Mrp2 by THP has not been reported in the literature. We hypothesized that DB extract may lead to THP accumulation in the body via intrahepatic expression change of P-gp and Mrp2. According to the liver immunohistochemical results, THP significantly enhanced P-gp and Mrp2 -positive staining in the membrane and cytoplasm of mouse liver cells, and the P-gp and Mrp2 expression as detected by Western blot also increased. The expression of P-gp and Mrp2 in the DB group was lower than that in the control group, which indicated that DB could inhibit the intrahepatic P-gp and Mrp2 expression. In the THP + DB group, the positive staining of P-gp and Mrp2 decreased significantly, and the protein expression also decreased significantly. These results suggested that DB extract can significantly reduce the high expression of P-gp and Mrp2 induced by THP. As an efflux transporter of THP, the decreased expression of P-gp and Mrp2 lead to accumulation of THP, which increased the cardiotoxicity of THP. Thus, the downregulated intrahepatic expression of P-gp and Mrp2 led to a decrease in extracellular THP and then the accumulation of THP, which led to severer cardiotoxicity. To explore the mechanism by which DB extract inhibits the intrahepatic protein expression of P-gp and Mrp2, we studied the morphological changes in liver cells in mice by immunohistochemistry. Many inflammatory cells infiltrated the hepatic tissue. It is suggested that the decrease in P-gp and Mrp2 expression by DB extract may be caused by the destruction of hepatocytes after liver injury. P-gp and Mrp2 may play an important role in HDIs between DB extract and THP. However, it is not clear whether DB extract is the substrate of P-gp and Mrp2 and further research of effect between DB extract and P-gp and Mrp2 is needed. One limitation of this study is that we merely studied the effects of DB water extract on the cardiotoxicity of THP, instead of specific component of DB. However, DB water extract contains a number of components, including diterpene lactones, steroidal saponins, flavonoids, alkaloids, and micronutrients. Diterpene lactones are a large class of active and toxic components in DB extract that have anti-inflammatory, antibacterial and antitumor effects but also lead to hepatotoxicity. The component-induced HDI and the molecular mechanisms of DB extract on P-gp and Mrp2 activity and expression should be verified in the next phase. In conclusion, the mechanism of HDI between DB extract and THP may be the hepatotoxicity of DB, leading to liver cell damage, downregulating the expression of P-gp and Mrp2, leading to a reduction in THP exocytosis, accumulation and aggravating cardiotoxicity (Fig. ). In clinical it is necessary to avoid or monitor cardiotoxicity when DB extract and THP are co-administration.
Animals Male Kunming mice (20–25 g) were provided by the Laboratory Animal Center of Jilin University, and the environment temperature was 22 °C and the light and dark were alternated. Every five mice were in one cage and were given plenty of feed and water. There was a week for the mice to adjust to the environment before the experiments. All experiment procedures were approved by the Experimental Animal Ethics Committee of the First Hospital of Jilin University (No. 2019-238). Drugs Crude dried DB were purchased from Anhui Province, China. THP for injection was purchased from HISUN PHARMACEUTICAL (Taizhou, China). Methods All methods described in this work were performed in accordance with relevant guidelines and regulations of Laboratory Animal Center of the First Hospital of Jilin University. Preparation of DB water extract The dried DB were crushed, and 100 g of DB pieces were soaked in water for 12 h. Then the pieces were extracted with water at 90 °C for 120 min. The extraction process was repeated for three times. All of the extracted solution was blended and filtered, and then inspissated at − 80 °C. Ultimately, each 300 mL of water contained the dissolved extract of 100 g of crude DB . The quantification of DB water extract and marker ingredient Diosbulbin B was analyzed by high-performance liquid chromatography (HPLC). The concentration of Diosbulbin B in DB solution sample was 1.26 mg/mL . Drug administration and tissue collection in mice Male Kunming mice (22–25 g) were randomly divided into four groups (control group, THP group, THP + DB group and DB group) with 10 mice in each group. The THP + DB and DB groups were given aqueous DB solution (0.1 mL/10 g) orally once daily for 14 consecutive days. Control (Con) and THP groups were given vehicle (water). On Day 12, the mice in THP and THP + DB groups were injected with THP (20 mg/kg) intraperitoneally. Control and DB groups were intraperitoneally injected with 0.9% NaCl on Day 12 . The mice that died during the experiment period were excluded and there was no mouse excluded in the experiment. On the 14th day, blood was collected from mouse eye sockets. The heart and liver tissues were collected and partially fixed in 4% paraformaldehyde for morphology observation and immunohistochemistry. The rest of tissues were stored at − 80 °C (Fig. A). Morphology observation The livers and hearts of mice were washed and placed in 4% formaldehyde and immersed in paraffin, then cut into 5-micron-thick slides to stain with hematoxylin and eosin (HE) and examined with an optical microscope (Olympus, Japan) . High performance liquid chromatography-mass spectrometer (HPLC–MS) The concentrations of THP in serum and heart were detected by HPLC–MS analysis using a 1200 HPLC (Agilent, USA) and an API4000Q MS (AB Sciex, USA) equipped with an electrospray ionization (ESI) source. Chromatographic column was SB C18 columns. The composition of mobile phase was acetonitrile and water with 0.1% formic acid and the flow rate was 0.25 mL/min. The column temperature was 30 °C (Table ) . The internal standard was daunorubicin. The MS was accomplished in positive ionizational multiple reaction monitoring (MRM) mode. The optimum values of MS were as follows: ionspray voltage, 5.5 kV; desolvation gas flow pressure, 0.75 MPa; nebulizer gas flow pressure, 0.625 MPa; source temperature, 550.0 °C. Immunohistochemistry Livers of mice were embedded in paraffin and cut into sections. The sections were incubated with rabbit anti-P-gp, rabbit anti-Mrp2 (Santa Cruz Biotechnology, Texas, USA) at 4 °C overnight, then incubated with horseradish peroxidase-labelled secondary antibody. After coloration the sections were observed with a BX51 optical microscope (Olympus, Japan). Western blot Mouse livers were homogenized in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS) and centrifuged at 12,000 g for 5 min at 4 °C. The supernatant was collected, and total protein was quantified using the Bradford method. Cell lysates contained 50 μg of protein per lane were separated by 10% SDS-PAGE and electrotransferred onto PVDF membranes. The membranes were blocked with TBST buffer for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies for 2 h. Then the membranes were washed and incubated with secondary antibodies for 1 h at room temperature. Immunoreactive proteins were visualized using an electro-chemi-luminescence (ECL) system, and the band density was analyzed using ImageJ software (Bethesda, MD). Statistical analysis All data were described as the mean values ± standard deviation (SD) and the analysis was done with SPSS 18.0 software ( https://dl.pconline.com.cn/download/1118212-1.html and the version number is 18.0.0). Differences between groups of HPLC–MS experiments were assessed by Student’s t -test after the data were confirmed to meet the assumptions for those tests. And statistics of data in the Western blot were performed with Two-way ANOVA.
Male Kunming mice (20–25 g) were provided by the Laboratory Animal Center of Jilin University, and the environment temperature was 22 °C and the light and dark were alternated. Every five mice were in one cage and were given plenty of feed and water. There was a week for the mice to adjust to the environment before the experiments. All experiment procedures were approved by the Experimental Animal Ethics Committee of the First Hospital of Jilin University (No. 2019-238).
Crude dried DB were purchased from Anhui Province, China. THP for injection was purchased from HISUN PHARMACEUTICAL (Taizhou, China).
All methods described in this work were performed in accordance with relevant guidelines and regulations of Laboratory Animal Center of the First Hospital of Jilin University.
The dried DB were crushed, and 100 g of DB pieces were soaked in water for 12 h. Then the pieces were extracted with water at 90 °C for 120 min. The extraction process was repeated for three times. All of the extracted solution was blended and filtered, and then inspissated at − 80 °C. Ultimately, each 300 mL of water contained the dissolved extract of 100 g of crude DB . The quantification of DB water extract and marker ingredient Diosbulbin B was analyzed by high-performance liquid chromatography (HPLC). The concentration of Diosbulbin B in DB solution sample was 1.26 mg/mL .
Male Kunming mice (22–25 g) were randomly divided into four groups (control group, THP group, THP + DB group and DB group) with 10 mice in each group. The THP + DB and DB groups were given aqueous DB solution (0.1 mL/10 g) orally once daily for 14 consecutive days. Control (Con) and THP groups were given vehicle (water). On Day 12, the mice in THP and THP + DB groups were injected with THP (20 mg/kg) intraperitoneally. Control and DB groups were intraperitoneally injected with 0.9% NaCl on Day 12 . The mice that died during the experiment period were excluded and there was no mouse excluded in the experiment. On the 14th day, blood was collected from mouse eye sockets. The heart and liver tissues were collected and partially fixed in 4% paraformaldehyde for morphology observation and immunohistochemistry. The rest of tissues were stored at − 80 °C (Fig. A).
The livers and hearts of mice were washed and placed in 4% formaldehyde and immersed in paraffin, then cut into 5-micron-thick slides to stain with hematoxylin and eosin (HE) and examined with an optical microscope (Olympus, Japan) .
The concentrations of THP in serum and heart were detected by HPLC–MS analysis using a 1200 HPLC (Agilent, USA) and an API4000Q MS (AB Sciex, USA) equipped with an electrospray ionization (ESI) source. Chromatographic column was SB C18 columns. The composition of mobile phase was acetonitrile and water with 0.1% formic acid and the flow rate was 0.25 mL/min. The column temperature was 30 °C (Table ) . The internal standard was daunorubicin. The MS was accomplished in positive ionizational multiple reaction monitoring (MRM) mode. The optimum values of MS were as follows: ionspray voltage, 5.5 kV; desolvation gas flow pressure, 0.75 MPa; nebulizer gas flow pressure, 0.625 MPa; source temperature, 550.0 °C.
Livers of mice were embedded in paraffin and cut into sections. The sections were incubated with rabbit anti-P-gp, rabbit anti-Mrp2 (Santa Cruz Biotechnology, Texas, USA) at 4 °C overnight, then incubated with horseradish peroxidase-labelled secondary antibody. After coloration the sections were observed with a BX51 optical microscope (Olympus, Japan).
Mouse livers were homogenized in RIPA buffer (1% Triton X-100, 1% deoxycholate, 0.1% SDS) and centrifuged at 12,000 g for 5 min at 4 °C. The supernatant was collected, and total protein was quantified using the Bradford method. Cell lysates contained 50 μg of protein per lane were separated by 10% SDS-PAGE and electrotransferred onto PVDF membranes. The membranes were blocked with TBST buffer for 1 h at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies for 2 h. Then the membranes were washed and incubated with secondary antibodies for 1 h at room temperature. Immunoreactive proteins were visualized using an electro-chemi-luminescence (ECL) system, and the band density was analyzed using ImageJ software (Bethesda, MD).
All data were described as the mean values ± standard deviation (SD) and the analysis was done with SPSS 18.0 software ( https://dl.pconline.com.cn/download/1118212-1.html and the version number is 18.0.0). Differences between groups of HPLC–MS experiments were assessed by Student’s t -test after the data were confirmed to meet the assumptions for those tests. And statistics of data in the Western blot were performed with Two-way ANOVA.
Supplementary Information.
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Risk analysis for radiotherapy at the Universitätsklinikum Erlangen | 0a2cef6a-4d86-412a-9299-58645cbacaa3 | 9948825 | Internal Medicine[mh] | Introduction Although there have been requirements for quality standards , , for the implementation of radiation therapy for decades, there are legal requirements for additional requirements. Additionally, various laws and ordinances require hospitals to have operational risk analysis. That applies not only to radiation therapy but to all areas of the clinic. Using Germany as an example, the commercial department of a clinic is obliged to carry out risk analysis by legal regulations such as Law on Control and Transparency in Business ( KonTraG) (all italic written abbreviations are explained with an English translation and in some detail in in ). Above a certain size, a clinic is also regarded as a critical infrastructure and therefore the IT infrastructure must meet the requirements of the IT Security Law , , . The Medical Device Operator Ordinance requires the operator of medical devices to carry out a risk analysis of the IT infrastructure before putting new medical devices into operation in heterogeneous medical device networks. For departments that treat with ionizing radiation, the Radiation Protection Ordinance also applies, which requires a risk analysis before introducing new treatment methods and before changes to existing treatment methods. provides an overview of the applicable laws and regulations. Several methods exist to perform risk analysis. Failure mode and effect analysis (FMEA), is widely used in industry and recommended, e.g., by the German Federal Office for Radiation Protection (BfS) or the TG 100 of the American Association for Physicists in Medicine (AAPM) . There are already many publications describing this method , , . The basic idea of FMEA is to analyze the processes and divide them into sub-processes. For each process step, one can then assign failure modes. For each failure mode, the likelihood of occurrence (O), its severity (S), and the likelihood of going undetected (D) is determined. TG 100 suggests a 10-fold scale to classify O, S, and D. Using these metrics, the risk priority number (RPN) is calculated by multiplying the three values of O, S, D, respectively . Since, as far as we know, there is no publication with an overview of all the regulations concerning risk analysis that must be followed by a clinic in Germany in general and by a radiation oncology department in particular, this work aims to provide such an overview and implications for fulfilling it. The Department of Radiation Oncology is a pilot clinic within the Universitätsklinikum Erlangen (UK Erlangen) for risk analysis. We present our experiences with risk assessment using only severity and occurrence scales and a two-dimensional criticality matrix in addition to the software systems that support these processes. The reason why we have chosen severity and occurrence as risk scales will be explained in the following.
Materials and methods 2.1 Relevant laws and regulations Commercial risk analysis must be performed by hospitals depending on their legal form. The legal basis for this is the KonTraG . This part of risk analysis is usually carried out by the commercial department , , , , . For hospitals with more than 30,000 in-patients per year, IT security risk analysis must be done as regulated by the IT security law along with BSI Guideline for Critical Infrastructures . This task is typically performed by the IT department . The tasks of radiation therapy include clinical risk analysis through the requirements of the G-BA and risk analysis according to the new Radiation Protection Ordinance . These will be explained in more detail in the following. The G-BA is the highest decision-making body of the joint self-administration of physicians, dentists, psychotherapists, hospitals, and health insurance funds in Germany . Among other things, it decides on quality assurance measures for the outpatient and inpatient areas of the health system . According to the G-BA , a risk analysis system, a system where patients can report complaints, and a critical incident and reporting system (CIRS) must be introduced across clinics, §135a SGB Sec. (3) . For clinics that work with ionizing radiation, §126 of the Radiation Protection Ordinance also applies, which obliges those responsible for radiation protection to carry out a risk analysis before the introduction of a new treatment method or before changing an existing treatment method . The legal basis for this is §86 No. 14 of the Radiation Protection Law . This applies to external beam therapy, brachytherapy, nuclear medicine, and orthovoltage radiation therapy . The BfS suggests renewing risk analysis every three years . The Radiation Protection Ordinance demands a retention period of ten years . 2.2 Overview of UK Erlangen and the Department of Radiation Oncology The UK Erlangen currently has 1394 beds, 8063 employees, and 1268 physicians . These work in 25 different medical departments, covering the full medical spectrum . A separate department is responsible for supporting the IT infrastructure of the clinic including three separate data centers. As UK Erlangen exceeds the limit of 30,000 inpatients per year, the clinic also falls under the KRITIS Ordinance. Thus, the rules of the BSI law apply. The Department of Radiation Oncology performs percutaneous radiotherapy using five medical linear accelerators, brachytherapy (PDR/HDR), orthovoltage radiation therapy and hyperthermia. It is utilizing a planning CT, a ward with approximately 50 beds, an own outpatient section for chemotherapy, and the required software infrastructure to operate the above-mentioned medical devices. Per year about 2700 patients are treated, of which ∼500 receive brachytherapy, ∼600 orthovoltage radiation therapy, and ∼1600 external beam therapy. 2.3 Implementation of risk analysis at UK Erlangen The central information hub in the intranet of UK Erlangen is the wiki software Confluence (v7.1, Atlassian, London). Risk analysis is implemented using JIRA (v8.14, Atlassian, London) and the extension Risk Register (ProjectBalm, Australia). Furthermore, Confluence is used for the documentation of the risk analysis meetings. shows the dependencies between JIRA and Confluence with the features of both. Various adjustments were programmed in-house to meet requirements not covered in the original implementation. As displayed in , the GUI is split into tabs covering risk identification, risk assessment, and planning of measures. Each tab includes multiple entries (e.g. risk category, cause, configurable naming) of different field types which can be chosen from the library JIRA offers. Fields can be linked to the active directory of Windows (Microsoft, Redmond, USA) allowing pre-filtered entry options, e.g., only measures of a certain department rather than all measures of the university clinic. Technically, this is implemented using the “Jira Query Language” (JQL ) which was also used for the creation of user-specific lists. In JIRA, the process types “risk” and “measure” were developed according to the in-house needs, i.e., the input mask for “risk” was mapped according to the ISO 31000 risk analysis process. The reciprocal m : n linking of n risks and m measures was also configured independently. The risk analysis portal is based on comprehensive authorization management, i.e., the centralized and decentralized views of risks and measures are regulated by different authorization roles. All risks and measures can be tracked thanks to the risk analysis matrix and the key wording for resubmission. Risks entries are created in JIRA in four steps with screenshots of a representative risk shown in . First, the risk identification takes place, in which the risk is specified exactly. This is followed by the gross risk assessment specifying the risk implications without any measures ( b). Third, the net risk assessment must be carried out. Here, the risk is assessed considering the measures currently in place ( c and d). Gross and net risks are quantified by their occurrence O and severity S using the scales defined in , , respectively. First, the risks in the workflow steps that may have a direct impact on patient health are considered. The system can be extended with other scales for other risk areas such as occupational safety or for the assessment of economic risks. Last, all measures, i.e., existing ones reducing gross to net risk, but potentially also new measures need to be added and linked to the risk. Based on occurrence and severity, all risks (e.g., of a department) can be attributed in one of three categories (see ) and displayed in a risk matrix, .
Relevant laws and regulations Commercial risk analysis must be performed by hospitals depending on their legal form. The legal basis for this is the KonTraG . This part of risk analysis is usually carried out by the commercial department , , , , . For hospitals with more than 30,000 in-patients per year, IT security risk analysis must be done as regulated by the IT security law along with BSI Guideline for Critical Infrastructures . This task is typically performed by the IT department . The tasks of radiation therapy include clinical risk analysis through the requirements of the G-BA and risk analysis according to the new Radiation Protection Ordinance . These will be explained in more detail in the following. The G-BA is the highest decision-making body of the joint self-administration of physicians, dentists, psychotherapists, hospitals, and health insurance funds in Germany . Among other things, it decides on quality assurance measures for the outpatient and inpatient areas of the health system . According to the G-BA , a risk analysis system, a system where patients can report complaints, and a critical incident and reporting system (CIRS) must be introduced across clinics, §135a SGB Sec. (3) . For clinics that work with ionizing radiation, §126 of the Radiation Protection Ordinance also applies, which obliges those responsible for radiation protection to carry out a risk analysis before the introduction of a new treatment method or before changing an existing treatment method . The legal basis for this is §86 No. 14 of the Radiation Protection Law . This applies to external beam therapy, brachytherapy, nuclear medicine, and orthovoltage radiation therapy . The BfS suggests renewing risk analysis every three years . The Radiation Protection Ordinance demands a retention period of ten years .
Overview of UK Erlangen and the Department of Radiation Oncology The UK Erlangen currently has 1394 beds, 8063 employees, and 1268 physicians . These work in 25 different medical departments, covering the full medical spectrum . A separate department is responsible for supporting the IT infrastructure of the clinic including three separate data centers. As UK Erlangen exceeds the limit of 30,000 inpatients per year, the clinic also falls under the KRITIS Ordinance. Thus, the rules of the BSI law apply. The Department of Radiation Oncology performs percutaneous radiotherapy using five medical linear accelerators, brachytherapy (PDR/HDR), orthovoltage radiation therapy and hyperthermia. It is utilizing a planning CT, a ward with approximately 50 beds, an own outpatient section for chemotherapy, and the required software infrastructure to operate the above-mentioned medical devices. Per year about 2700 patients are treated, of which ∼500 receive brachytherapy, ∼600 orthovoltage radiation therapy, and ∼1600 external beam therapy.
Implementation of risk analysis at UK Erlangen The central information hub in the intranet of UK Erlangen is the wiki software Confluence (v7.1, Atlassian, London). Risk analysis is implemented using JIRA (v8.14, Atlassian, London) and the extension Risk Register (ProjectBalm, Australia). Furthermore, Confluence is used for the documentation of the risk analysis meetings. shows the dependencies between JIRA and Confluence with the features of both. Various adjustments were programmed in-house to meet requirements not covered in the original implementation. As displayed in , the GUI is split into tabs covering risk identification, risk assessment, and planning of measures. Each tab includes multiple entries (e.g. risk category, cause, configurable naming) of different field types which can be chosen from the library JIRA offers. Fields can be linked to the active directory of Windows (Microsoft, Redmond, USA) allowing pre-filtered entry options, e.g., only measures of a certain department rather than all measures of the university clinic. Technically, this is implemented using the “Jira Query Language” (JQL ) which was also used for the creation of user-specific lists. In JIRA, the process types “risk” and “measure” were developed according to the in-house needs, i.e., the input mask for “risk” was mapped according to the ISO 31000 risk analysis process. The reciprocal m : n linking of n risks and m measures was also configured independently. The risk analysis portal is based on comprehensive authorization management, i.e., the centralized and decentralized views of risks and measures are regulated by different authorization roles. All risks and measures can be tracked thanks to the risk analysis matrix and the key wording for resubmission. Risks entries are created in JIRA in four steps with screenshots of a representative risk shown in . First, the risk identification takes place, in which the risk is specified exactly. This is followed by the gross risk assessment specifying the risk implications without any measures ( b). Third, the net risk assessment must be carried out. Here, the risk is assessed considering the measures currently in place ( c and d). Gross and net risks are quantified by their occurrence O and severity S using the scales defined in , , respectively. First, the risks in the workflow steps that may have a direct impact on patient health are considered. The system can be extended with other scales for other risk areas such as occupational safety or for the assessment of economic risks. Last, all measures, i.e., existing ones reducing gross to net risk, but potentially also new measures need to be added and linked to the risk. Based on occurrence and severity, all risks (e.g., of a department) can be attributed in one of three categories (see ) and displayed in a risk matrix, .
Initial experiences Risk analysis has been carried out at the Department of Radiation Oncology since March 2020, and 41 1 h-meetings of an expert panel have taken place till July 2021. The first month was required to get familiar with risk analysis. The panel is led by the quality assurance officer of the clinic and further constituted by senior physicians, physicists, radiotherapy technologists and additional representatives of the department in case the discussed topic requires their expertise. Risk assessment is based on the workflow underlying the treatment of patients that is displayed in (details in ) and was defined by the panel in the first meetings. Based on this process chain of 13 steps, the individual failure modes resulting in risk entries are discussed in the weekly meeting. A total of n = 38 risks and m = 50 measures were discussed in the period mentioned above. No explicit use of failure modes reported in literature was made. In this initial phase of risk analysis only those risks and respective measures are discussed that are most important to the clinical process. Low-priority risks are currently documented in Confluence for future discussion. Deviations from the workflow-based assessment are accepted in case every day's problems or incidences trigger discussions within the panel. shows the evolution of risk matrices as a function of measures introduced over time. Measures can be either in progress or implemented. Measures in progress refer to those that require writing a Standard Operating Procedure (SOP) or implementing additional equipment (e.g., biometrical validation of patients). If a measure has SOPs and these have been communicated, it is considered to have been implemented. a shows the risk matrix in the case that no measures are taken (Gross Risk Assessment) with the total number for each risk category listed in b. At the time risk analysis was initiated, assessment of the net risk resulted in the risk matrix shown in c. The number of implemented measures to reach the net situation is listed in d. e shows the risk matrix resulting from risk analysis by introduction of further measures (see f) that were initiated in the discussions of the expert panel. Not all those very recent measures were already implemented and thus some risks currently remain in the red/critical category. Comparison of a and c further shows that risks mainly varied in occurrence. After introducing new measures risks can also vary in severity ( e). Overall, the experience has shown that one can discuss around 1 risk per hour. In addition, the QA officer works again for 15–20 min documenting the risks and measures properly. If it is required, additional time is needed for creation of SOPs which can be multiple hours especially if several experts need to be involved. This gives a total of at least 6–7 working hours per week for risk analysis if the meeting takes place. So far, risks have not been re-evaluated as the suggested 3-year period is not yet reached.
Discussion Risk analysis is required by the laws described for almost all areas of a radiation oncology department. Following the amendment of the Radiation Protection Ordinance by §126 enacted in 2019, at least a decided risk analysis now also applies to radiation protection. This paper presents a methodology and an implementation of risk analysis of a large Department of Radiation Oncology in Germany, which considers both the guidelines of the G-BA and the new Radiation Protection Ordinance . After getting a clear overview of the currently applicable laws and regulations, risk analysis was introduced to the RM team by the QA officer. It then took around four weeks for everybody to familiarize themselves with risk analysis with reference to quantification of severity and occurrence scales. Part of the familiarization were literature studies , , , , , , , , and participation of dedicated meetings e.g., of the professional societies. An important issue is the amount of time required to perform risk analysis. Perks et al. required about 100 h for the analysis of an SBRT process . Ford et al. reported less than a month and 75 h for a broader FMEA that included the entire treatment process . To date, it has taken us about 270 h for implementing a risk analysis team and addressing most of the external beam treatment workflow. The limited progress compared to Ford et al. can also be attributed to the fact that we had no experience in dealing with FMEA and risk analysis at the beginning and that our core team consisted of seven clinical specialists and one QM officer, i.e., a large team. This large team had the advantage that two employees from each professional group were involved. In case individuals could not make it to the weekly meeting, e.g., due to other clinical tasks, at least a core team remained and could continue the risk analysis. In addition, further colleagues are meanwhile involved and thus familiar with risk analysis, so that if one employee from the core team is unable to attend, a substitute is available. Having more employee's leads to better results in the discussion, even if this means that more time must be invested. Despite this substantial time only the main workflow was discussed. Risk assessments and evaluations for special and rare treatment techniques such as total skin irradiation and/or the introduction of new treatment concepts/devices were handled separately . Software supporting risk analysis is commercially available from several vendors also dedicated for clinical purposes. Despite that fact, we decided to build our risk analysis software based on JIRA so that the integration into the Confluence-based internal workspace can be ensured. In addition, solutions dedicated to radiation oncology are still sparse . Frequently used alternatives are also in-house options based on office tools that do not allow user management, revise, and other similar essentials when designing the solution for a large university clinic , , . JIRA has been shown to help with risk analysis. After a short training period, many users could operate the tool and create risks. With the current methodology, the probability of occurrence and severity of a failure mode is subjectively estimated by the expert panel. Ideally, quantitative feedback, e.g., from an incident reporting system (CIRS or alike) would be included in the specifications . Part of the estimation and discussion process is the identification of measures that mitigate the risk once completed. Measures are described and attributed to person(s) with a due date for fulfillment. Each measure can be linked to one or multiple risks. Finding the cause of a failure would require a Fault Tree Analysis (FTA), which is currently not implemented. In addition, JIRA cannot display a process chain, which is essential for an FMEA. In addition, many risks are caused by the IT equipment used. Thus, in a further step, the risk analysis of the Department for Radiation Oncology should also incorporate the relevant IT infrastructure. Since a risk analysis of the entire IT of the university clinic is anyway conducted in accordance with the requirements of the BSI law synergistic effects can be exploited. Similarly, a specific risk analysis of the devices of the medical equipment of the Department of Radiation Oncology has not yet been performed. For this purpose, it would be desirable to map the workflows in the form of a process diagram that implements the requirements of the Radiation Protection Ordinance and the G-BA . Improvements of the software infrastructure are thus currently discussed internally. Another aspect is the scales used to evaluate the risk entries. The scales should allow a meaningful but also comprehensive graduation of occurrences (O), severities (S), and (if applicable) detectability (D). Care must be taken to ensure that the scale is understood and accepted by all parties involved and that it can be used to assess risks within a reasonable time. Many publications use a risk scale based on the TG-100 report for risk analysis , , , . This provides for a ten-level subdivision of the risk levels. In addition, a risk priority number (RPN) is formed from S, D, and O. This method has the advantage that it allows a very fine-grained risk assessment. The disadvantage is that due to the many intermediate levels in the subdivision, it can take a long time in the discussion of the risks until all participants have agreed on a value. A recent publication describes a risk scale that is specifically adapted to risk analysis in radiation oncology using questionnaires . The method we use, with a five-stage scale and only two dimensions by integrating detectability into occurrence, has the advantage that risks can be classified quickly, and all risks are displayable in a risk matrix. Performing a risk analysis using a risk matrix is also described in . The implementation of measures can in principle shift risks in occurrence and severity as shown in a radiation oncology setting . In our experience, for most failure modes the introduction of measures only reduced the occurrence in particular for the transition from gross to net risks ( a,c). Measures that also reduce severity were established within the process of risk analysis ( d and e). The risks from the risk analysis are re-evaluated after three years at the latest, with the aim of getting the risks into increasingly harmless risk regions through new measures. It is noticeable that most of the risks are critical risks or risks that need to be monitored. This can be explained by the fact that the FMEA is based on the clinical experience of the staff involved and not yet by quantitative measures such as incidence levels of e.g., near misses. It is thus more likely but also desirable to note critical risks than the less critical ones. Contributing to this trend is the already mentioned internal decision of focusing on the risks most important to the clinical process leading to the fact that we have not yet identified all possible risks that occur in a workflow step with the missing ones likely being conditionally acceptable risks. Research by Terezakis et al. has shown that about 42% of all risks are not identified by an FMEA . Therefore, FMEA should be complemented by incident learning systems. A Critical Incident and Reporting System (CIRS) implements this in our clinic.
Conclusion Risk analysis is required in multiple laws and regulations , , , , , and thus affecting essentially each department of a large clinic. This is in particular true for radiation oncology since recently also radiation protection law requires risk assessments. An interdisciplinary group was formed for risk assessment meetings nominally weekly. Risk identification is carried out using the process chain of the treatment workflow. Confluence, JIRA, and Risk Register are used for risk documentation as these tools could be integrated seamlessly into the intranet portal used at UKER. Identification of 38 risks, development of 50 measures to overcome those risks, and the required documentation took ∼260 person hours in 41 meetings. Additional time is required for writing e.g., SOPs foreseen in the developed measures.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors have no relevant conflicts of interest to disclose.
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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. |
Action-driven remapping of hippocampal neuronal populations in jumping rats | 2f1b2490-dce0-45c1-a103-6b3af62e1382 | 9245695 | Physiology[mh] | We trained rats to walk back and forth on a linear track (1.8 m) for water reward, available at a platform at each end of the track . After they moved comfortably and steadily on the track, the rats were trained to jump a gap within the track. During this shaping period, the gap distance was gradually increased from 5 to 30 cm. Following these pretraining sessions (5 to 15 d in different rats), the formal jump sessions started. A 30-cm gap between two parts of the track were created at one of three fixed locations on the track . After 7 to 20 prejump control trials (no gap), a gap was introduced, requiring the rat to jump across it in both directions of travel. After 20 trials, the position of the gap position was shifted, and after the second block of 20 trials, a third gap position was introduced. The order of the gap positions varied randomly across sessions. After the jump trials (a trial equaled travel in one direction), the rat ran post-jump control trials without a gap until it was satiated. Each animal was implanted with silicon probes above the dorsal hippocampal CA1 pyramidal layer. The recording headstage also contained an accelerometer, which allowed continuous monitoring of the x , y , and z positions; velocity; and acceleration of the rat’s head . In addition, head position was tracked with an OptiTrack system including six infrared cameras that allowed for the three-dimensional reconstruction of the animals’ head position and head orientation to within 1 mm at 120 Hz ( Materials and Methods ). Behavioral Observations. Traversing the track on control trials took a median of 2.0 s at a median speed of 76 cm/s ( n = 2,212) ( SI Appendix , Fig. 1 ). Rats learned to jump the gaps quickly at high efficiency. One rat fell once during training, but never during recordings, over the course of more than 3,000 jumps in 28 sessions across the 4 rats (1 m) from track to floor. Wild rats can fall from a height of 50 feet (∼15 m) without getting hurt, corresponding to >99.9% efficacy . The act of jumping can be divided into four distinguishable phases: preparation, takeoff, flight, and landing . During preparation, the rat aligned its four feet to the edge of the track, lowered its head steadily, or moved its head up and down a few times ( Movie S1 ). Such self-generated head bobbing produces retinal motion of the image of the landing platform and is critical to determine the desired distance of the jump . The time of takeoff was determined from the accelerometer reading as the peak of the second derivative of the accelerometer reading of the horizontal direction . Similarly, the location and time of landing on the track were determined from the minimum of the second derivative of the accelerometer reading of the vertical direction ( and SI Appendix , Fig. 1 ). The duration of preparation phase was longest at the middle gap (gap 2) in both directions of travel ( ; mean ± S.D. wait time: gap 1 = 1.04 ± 0.52 s, gap 2 = 1.31 ± 0.49 s, and gap 3 = 1.14 ± 0.67). Flight time (i.e., time in air) was consistent across sessions and rats ( ; mean ± S.D. flight time: jump 1= 0.17 ± 0.03 s [ n = 1,536]; jump 2 = 0.18 ± 0.04 s; jump 3 = 0.19 ± 0.05 s). During the flight, the peak velocity exceeded 1.7 m/s, and the velocity and acceleration profiles varied stereotypically across trials ( and SI Appendix , Fig. 1 ; mean ± S.D. velocity: jump 1 = 176 ± 33 cm/s; jump 2 = 171 ± 48 cm/s; jump 3 = 164 ± 51 cm/s). The rat walked relatively slowly prior to jump and maintained momentum after jumping by galloping after landing ( ; before jump = 57 m/s; after jump = 120 m/s; P = 0 by Kruskal-Wallis test). Galloping was relatively stereotyped at ∼5 Hz, as illustrated by the vertical bands in . This difference in running patterns likely reflected the animal’s increased confidence after the jump, since it did not depend on the available travel distance before or after the jump. Local Field Potential and Population Firing-Rate Correlates of Gap Jumping. The theta frequency in the local field potential (LFP) increased in frequency and power during jumping, reaching a maximum frequency (9.3 ± 1.0 Hz) during the flight phase and after landing, in time with the rat’s velocity and firing rates of interneurons . Interestingly, the rate changes of interneurons and pyramidal cells in CA1 preceded those in CA3 ( SI Appendix , Fig. 2 ), implying that CA1 activity was not inherited from CA3 neurons. Theta frequency during postjump galloping was significantly higher than during prejump walking . The firing rates of interneurons showed a relatively linear relationship with speed during running , but this relationship became nonlinear at very high speeds , associated with jumping . Jumping reset the phase of theta waves. The reliability of theta-phase reset was quantified by the phase consistency across trials ( SI Appendix , Materials and Methods and Fig. 10 ), referenced to the moment of takeoff ( and ). Theta-phase reset was also visible by the increased phase consistence of interneuron spiking . The consequence of theta-phase reset was the persisting phase consistency for several theta cycles, although significant phase consistency was present only for three cycles, surrounding the jump . Given this short duration and the presence of only one or two theta cycles during the jump itself, we could not determine precisely whether the reset occurred during the takeoff or the flight. Neuronal Population-Measure Correlates of Gap Jumping. We examined encoding of the jump by computing population vector correlations, which tell us how hippocampal activity is correlated in space. The population vector correlations were computed by first z -scoring the trial-averaged firing rate for a given position of each neuron with mean activity above a noise threshold in at least one jump or control condition and then calculating the correlation across neurons between their activities at each combination of position bins . Average population vector correlation decreased to near zero within <40 cm of separation , as reported previously in similar situations . The population vector correlation analysis includes all active neurons and, therefore, is not biased by the definition of a place cell. In contrast to the high spatial correlation across control trials, spatial correlations of CA1 population activity between control and jump trials showed a strong reduction, except at the two extreme ends of the track. Importantly, the decreased spatial correlation was not restricted to the gap area; it was already low prior to the jump and persisted after the jump . Comparison of jump trials across different gap conditions also yielded low spatial correlations , indicating remapping of neuronal activity across jump trials as well. Yet, in contrast to the steady near-zero correlations between control and jump trials , comparison of jump trials revealed a visible increase of population vector correlations at the jump location across gap positions . This observation implies the possibility of the presence of neurons, which represent different gaps similarly (see remarks about jump cells in Discussion ). Population representation and changes across conditions were remarkably similar between CA1 and CA3 populations . These findings are in agreement with previous analyses showing that population vector correlation is a more sensitive measure than population firing-rate analysis (compare and ) . Single Neuron-Firing Correlates of Gap Jumping. The population analysis suggested that the act of jumping resulted in a combination of rate and global remapping of place fields of individual hippocampal pyramidal neurons in virtually the entire track, even though the distant spatial cues and motor behavior remained similar before and, to a great extent, after the jump itself. In a typical session, only a small fraction of neurons displayed stable place fields with unaltered rates during jump trials, whereas the overwhelming majority of place fields were modified one way or another. In addition to the minority stable neurons (group 1), neurons with a modified firing pattern could be classified by subjective criteria into the following four major groups: group 2, novel place fields; group 3, neurons whose rates were decreased (truncated or attenuated); group 4, neurons with increased (amplified) rates during jump trials; and group 5, jump cells. Each of these changes could occur before, during, or after the jump (gap). Since hippocampal pyramidal cells form different place fields and sequences during opposite direction runs on linear tracks , we evaluated place fields separately on left and right travels. Stable place fields were found only at the start and end locations of the track ( and SI Appendix , Fig. 3 ). Neurons with novel place fields (group 2) did not have a place field on prejump control trials, and the new field could occur anywhere on the track, including areas before, after, and even during the jump ( and SI Appendix , Fig. 3 ). Neurons in group 3 had an existing place field in control trials, which was suppressed in jump trials. Spike suppression was typically largest when the place field coincided with the gap, but suppression was also present when the gap was either before or after the location of the place field ( and SI Appendix , Fig. 4 ). When the gap coincided with the beginning of the place field, the remaining part of the place field was still expressed ( , fourth graph down in column). Firing rates of group 4 place cells increased on jump trials. Similar to the attenuated group, enhanced spiking could occur not only during gap jumping itself but also before or after the gap, and the center of the place field moved either toward or away from the gap ( and SI Appendix , Fig. 5 ). The distinction of the attenuated and amplified groups is somewhat arbitrary since spike rate decrease and increase could be observed within the same place cell with different jump locations ( SI Appendix , Figs. 4 and 5 ). Neurons classified as jump cells (group 5) also meet the criterion of a novel place field, because firing at the future place field was absent during control trials. However, criteria for jump cells also included the requirements that they fired in relationship with two or three gaps and their fields moved with the gap on different trial types. Jumps cells occurred not only during jumping but could be present either before or after the gap ( and SI Appendix , Fig. 6 ), suggesting that the motor action of jumping was not the necessary driver of spiking. When the jump cell occurred prior to departure, its duration lasted throughout the 1- to 1.5-s wait time, during which its spikes underwent a gradual phase precession . With the exception of one neuron ( SI Appendix , Fig. 7 ), jump fields occurred only during either left or right, but not both, direction of travel. Therefore, a more appropriate description of these neurons would be “conjunction cells of gap and head direction.” Although the error-prone nature of subjective classification of firing pattern types are acknowledged, the distribution of these five groups was similar in both CA1 and CA3 regions , implying a relatively random redistribution of the neuron pool in the context of a modified map. Overall, the analysis of individual neurons supports the remapping conclusion by the population vector analysis. In addition to the aforementioned five groups, the remaining putative pyramidal neurons either had very few spikes to quantify place fields or displayed scattered and nonpatterned firing throughout the track (group 6: unclassified or “other” neurons; SI Appendix , Fig. 8 ). Yet, firing fields of some of these unclassified neurons were also modified in jump trials ( SI Appendix , Fig. 8 ). Fast-firing, putative interneurons showed a correlation with running speed , but the relationship between speed and interneuron firing rate was also modified by context, as demonstrated by the different interneuron firing rates at different gaps and the difference between left and right travels ( SI Appendix , Fig. 9 ). Remapping and the Internal Temporal/Theta-Phase Structure of Hippocampal Populations. Despite the large changes of firing patterns of individual neurons, the population structure of the neurons remained similar. First, we investigated the distance relationship between place fields and theta timescale-related timing of place-cell pairs by calculating the compression index . The compression index quantifies the relationship between travel distances (or travel time) between the peaks of overlapping place fields and the time (phase) offset of their spikes within theta cycles. When the time and phase offsets of the theta timescale cross-correlograms were plotted as a function of the distance between the peaks of their firing fields, we found that despite the extensive firing-rate modification of individual place fields, the compression index remained similar between control and jump trials ( ; for detection of theta phase, see SI Appendix , Fig. 10 ), indicating that fine-timescale properties of modified spike sequences within place-coding cell assemblies were preserved despite jump perturbation. Another measure of place coding is the relationship between the animal’s instantaneous position and the theta phase at which the neuron spikes [i.e., phase precession ]. Phase precession describes the association between a linear variable (position on track) and a circular phase (theta cycle) variable . Despite the prominent spike-rate modulation and moderate place-field shape distortions during jump trials, the position–theta-phase spike relationship remained invariant , implying that theta phase is a more reliable “code” of position than a firing rate code . This invariance was a result of reduced place-field size and altered slope [radian or field width ] during jump trials . The average reduction of place-field size was likely a result of the several place fields silenced midfield due to the presence of the gap . In a few examples, the major part of the place field coincided with the gap, and spiking was completely suppressed during the jump. Despite the absence of spikes for the major part of the place field, when firing resumed at the end of the field, the theta-phase assignment of spikes was the same as during control trials ( and SI Appendix , Fig. 10 ) .
Traversing the track on control trials took a median of 2.0 s at a median speed of 76 cm/s ( n = 2,212) ( SI Appendix , Fig. 1 ). Rats learned to jump the gaps quickly at high efficiency. One rat fell once during training, but never during recordings, over the course of more than 3,000 jumps in 28 sessions across the 4 rats (1 m) from track to floor. Wild rats can fall from a height of 50 feet (∼15 m) without getting hurt, corresponding to >99.9% efficacy . The act of jumping can be divided into four distinguishable phases: preparation, takeoff, flight, and landing . During preparation, the rat aligned its four feet to the edge of the track, lowered its head steadily, or moved its head up and down a few times ( Movie S1 ). Such self-generated head bobbing produces retinal motion of the image of the landing platform and is critical to determine the desired distance of the jump . The time of takeoff was determined from the accelerometer reading as the peak of the second derivative of the accelerometer reading of the horizontal direction . Similarly, the location and time of landing on the track were determined from the minimum of the second derivative of the accelerometer reading of the vertical direction ( and SI Appendix , Fig. 1 ). The duration of preparation phase was longest at the middle gap (gap 2) in both directions of travel ( ; mean ± S.D. wait time: gap 1 = 1.04 ± 0.52 s, gap 2 = 1.31 ± 0.49 s, and gap 3 = 1.14 ± 0.67). Flight time (i.e., time in air) was consistent across sessions and rats ( ; mean ± S.D. flight time: jump 1= 0.17 ± 0.03 s [ n = 1,536]; jump 2 = 0.18 ± 0.04 s; jump 3 = 0.19 ± 0.05 s). During the flight, the peak velocity exceeded 1.7 m/s, and the velocity and acceleration profiles varied stereotypically across trials ( and SI Appendix , Fig. 1 ; mean ± S.D. velocity: jump 1 = 176 ± 33 cm/s; jump 2 = 171 ± 48 cm/s; jump 3 = 164 ± 51 cm/s). The rat walked relatively slowly prior to jump and maintained momentum after jumping by galloping after landing ( ; before jump = 57 m/s; after jump = 120 m/s; P = 0 by Kruskal-Wallis test). Galloping was relatively stereotyped at ∼5 Hz, as illustrated by the vertical bands in . This difference in running patterns likely reflected the animal’s increased confidence after the jump, since it did not depend on the available travel distance before or after the jump.
The theta frequency in the local field potential (LFP) increased in frequency and power during jumping, reaching a maximum frequency (9.3 ± 1.0 Hz) during the flight phase and after landing, in time with the rat’s velocity and firing rates of interneurons . Interestingly, the rate changes of interneurons and pyramidal cells in CA1 preceded those in CA3 ( SI Appendix , Fig. 2 ), implying that CA1 activity was not inherited from CA3 neurons. Theta frequency during postjump galloping was significantly higher than during prejump walking . The firing rates of interneurons showed a relatively linear relationship with speed during running , but this relationship became nonlinear at very high speeds , associated with jumping . Jumping reset the phase of theta waves. The reliability of theta-phase reset was quantified by the phase consistency across trials ( SI Appendix , Materials and Methods and Fig. 10 ), referenced to the moment of takeoff ( and ). Theta-phase reset was also visible by the increased phase consistence of interneuron spiking . The consequence of theta-phase reset was the persisting phase consistency for several theta cycles, although significant phase consistency was present only for three cycles, surrounding the jump . Given this short duration and the presence of only one or two theta cycles during the jump itself, we could not determine precisely whether the reset occurred during the takeoff or the flight.
We examined encoding of the jump by computing population vector correlations, which tell us how hippocampal activity is correlated in space. The population vector correlations were computed by first z -scoring the trial-averaged firing rate for a given position of each neuron with mean activity above a noise threshold in at least one jump or control condition and then calculating the correlation across neurons between their activities at each combination of position bins . Average population vector correlation decreased to near zero within <40 cm of separation , as reported previously in similar situations . The population vector correlation analysis includes all active neurons and, therefore, is not biased by the definition of a place cell. In contrast to the high spatial correlation across control trials, spatial correlations of CA1 population activity between control and jump trials showed a strong reduction, except at the two extreme ends of the track. Importantly, the decreased spatial correlation was not restricted to the gap area; it was already low prior to the jump and persisted after the jump . Comparison of jump trials across different gap conditions also yielded low spatial correlations , indicating remapping of neuronal activity across jump trials as well. Yet, in contrast to the steady near-zero correlations between control and jump trials , comparison of jump trials revealed a visible increase of population vector correlations at the jump location across gap positions . This observation implies the possibility of the presence of neurons, which represent different gaps similarly (see remarks about jump cells in Discussion ). Population representation and changes across conditions were remarkably similar between CA1 and CA3 populations . These findings are in agreement with previous analyses showing that population vector correlation is a more sensitive measure than population firing-rate analysis (compare and ) .
The population analysis suggested that the act of jumping resulted in a combination of rate and global remapping of place fields of individual hippocampal pyramidal neurons in virtually the entire track, even though the distant spatial cues and motor behavior remained similar before and, to a great extent, after the jump itself. In a typical session, only a small fraction of neurons displayed stable place fields with unaltered rates during jump trials, whereas the overwhelming majority of place fields were modified one way or another. In addition to the minority stable neurons (group 1), neurons with a modified firing pattern could be classified by subjective criteria into the following four major groups: group 2, novel place fields; group 3, neurons whose rates were decreased (truncated or attenuated); group 4, neurons with increased (amplified) rates during jump trials; and group 5, jump cells. Each of these changes could occur before, during, or after the jump (gap). Since hippocampal pyramidal cells form different place fields and sequences during opposite direction runs on linear tracks , we evaluated place fields separately on left and right travels. Stable place fields were found only at the start and end locations of the track ( and SI Appendix , Fig. 3 ). Neurons with novel place fields (group 2) did not have a place field on prejump control trials, and the new field could occur anywhere on the track, including areas before, after, and even during the jump ( and SI Appendix , Fig. 3 ). Neurons in group 3 had an existing place field in control trials, which was suppressed in jump trials. Spike suppression was typically largest when the place field coincided with the gap, but suppression was also present when the gap was either before or after the location of the place field ( and SI Appendix , Fig. 4 ). When the gap coincided with the beginning of the place field, the remaining part of the place field was still expressed ( , fourth graph down in column). Firing rates of group 4 place cells increased on jump trials. Similar to the attenuated group, enhanced spiking could occur not only during gap jumping itself but also before or after the gap, and the center of the place field moved either toward or away from the gap ( and SI Appendix , Fig. 5 ). The distinction of the attenuated and amplified groups is somewhat arbitrary since spike rate decrease and increase could be observed within the same place cell with different jump locations ( SI Appendix , Figs. 4 and 5 ). Neurons classified as jump cells (group 5) also meet the criterion of a novel place field, because firing at the future place field was absent during control trials. However, criteria for jump cells also included the requirements that they fired in relationship with two or three gaps and their fields moved with the gap on different trial types. Jumps cells occurred not only during jumping but could be present either before or after the gap ( and SI Appendix , Fig. 6 ), suggesting that the motor action of jumping was not the necessary driver of spiking. When the jump cell occurred prior to departure, its duration lasted throughout the 1- to 1.5-s wait time, during which its spikes underwent a gradual phase precession . With the exception of one neuron ( SI Appendix , Fig. 7 ), jump fields occurred only during either left or right, but not both, direction of travel. Therefore, a more appropriate description of these neurons would be “conjunction cells of gap and head direction.” Although the error-prone nature of subjective classification of firing pattern types are acknowledged, the distribution of these five groups was similar in both CA1 and CA3 regions , implying a relatively random redistribution of the neuron pool in the context of a modified map. Overall, the analysis of individual neurons supports the remapping conclusion by the population vector analysis. In addition to the aforementioned five groups, the remaining putative pyramidal neurons either had very few spikes to quantify place fields or displayed scattered and nonpatterned firing throughout the track (group 6: unclassified or “other” neurons; SI Appendix , Fig. 8 ). Yet, firing fields of some of these unclassified neurons were also modified in jump trials ( SI Appendix , Fig. 8 ). Fast-firing, putative interneurons showed a correlation with running speed , but the relationship between speed and interneuron firing rate was also modified by context, as demonstrated by the different interneuron firing rates at different gaps and the difference between left and right travels ( SI Appendix , Fig. 9 ).
Despite the large changes of firing patterns of individual neurons, the population structure of the neurons remained similar. First, we investigated the distance relationship between place fields and theta timescale-related timing of place-cell pairs by calculating the compression index . The compression index quantifies the relationship between travel distances (or travel time) between the peaks of overlapping place fields and the time (phase) offset of their spikes within theta cycles. When the time and phase offsets of the theta timescale cross-correlograms were plotted as a function of the distance between the peaks of their firing fields, we found that despite the extensive firing-rate modification of individual place fields, the compression index remained similar between control and jump trials ( ; for detection of theta phase, see SI Appendix , Fig. 10 ), indicating that fine-timescale properties of modified spike sequences within place-coding cell assemblies were preserved despite jump perturbation. Another measure of place coding is the relationship between the animal’s instantaneous position and the theta phase at which the neuron spikes [i.e., phase precession ]. Phase precession describes the association between a linear variable (position on track) and a circular phase (theta cycle) variable . Despite the prominent spike-rate modulation and moderate place-field shape distortions during jump trials, the position–theta-phase spike relationship remained invariant , implying that theta phase is a more reliable “code” of position than a firing rate code . This invariance was a result of reduced place-field size and altered slope [radian or field width ] during jump trials . The average reduction of place-field size was likely a result of the several place fields silenced midfield due to the presence of the gap . In a few examples, the major part of the place field coincided with the gap, and spiking was completely suppressed during the jump. Despite the absence of spikes for the major part of the place field, when firing resumed at the end of the field, the theta-phase assignment of spikes was the same as during control trials ( and SI Appendix , Fig. 10 ) .
Jumping produced a stereotypic behavior associated with consistent electrophysiological patterns, including phase reset of LFP theta, global firing-rate changes, and population vector shifts of hippocampal neurons. These collective neuronal patterns were associated with modified firing of individual hippocampal neurons, appearance of novel place fields, and the emergence of jump-specific cells. Despite large changes in firing rates, the theta phase versus animal-position relationships of place cells remained stable. Thus, planning and executions of actions are as effective in altering hippocampal neuronal organization as are environmental cues. Resetting Theta Oscillations. A reliable relationship between various motor actions and the phase of the theta cycle has previously been reported . In our experiments, jumping was associated with LFP theta-phase reset. The flight time itself was <0.2 s, typically one or two theta cycles, but the trial-to-trial phase consistency persisted for several theta cycles after the jump. The phase reset coincided with an increase of both theta amplitude and frequency, as also reported during vertical jumping . The phase reset was also evident from the session average of interneuron firing, essentially tracking the phase-shifted LFP theta cycles. Finally, when the gap coincided with the place field of a pyramidal neuron, a sudden phase shift in spike–theta-phase preference was detected. This sudden shift may have occurred because in one or two theta cycles, the rat’s head was displaced by 30 cm, thus maintaining the relationship between spatial cues and spike–theta-phase relationship . Alternatively, the phase shift might reflect the altered relationship between spiking of the jump cell and the phase-shifted reference LFP theta. Another issue that has remained ambiguous is whether jumping induced phase resetting of theta oscillations or whether the timing of jumping was biased by the phase of the theta cycle . The theta reset might have been brought about the corollary discharge from the motor command signal or from the sensory feedback of muscle activity . Reorganization of the Hippocampal Map by Jumping. Firing sequences of hippocampal neurons during exploration in one- or two-dimensional environments have been assumed to be driven by internal mechanisms . For each environment, a new map with different firing patterns is retrieved, due to shifting from one neuronal “attractor” to another , known as remapping or as an altered manifold . Under some conditions, reorganization is manifested as a change in firing rates at the same locations (“rate remapping”), whereas between distinct environments, new place fields appear and old ones disappear or move to a new location [“global remapping” ]. Remapping can be induced not only by spatial manipulations and changes in sensory inputs but also by motivational state , experience , and memory load . In our experiments, nearly all place fields, with the exception of a small fraction of stable place fields at the two ends of the track, likely anchored to the start, or goal, platforms , were modified one way or another. In jump trials, firing pattern reorganization occurred as a combination of global and rate remapping. To explain these changes, one can make the argument that the absence of a piece of the track is a local sensory cue and, thus, this experimenter-induced change is the sole explanation of remapping . However, local cues are not expected to induce such a strong remapping as we have observed in our experiments. Several previous studies have examined the impact of overt or hidden local cues on hippocampal neuronal firing. These experiments vary from reporting no effect to reporting moderate effects on place-field activity, including place-field enrichment, smaller place fields, increased spatial tuning (i.e., firing-rate increase), altered phase precession, and increased or reduced spatial encoding . Common to these previous experiments (but see refs. and ) is that the described firing-pattern alterations were confined to the particular segment of the environment where the density of visual and tactile cues was enriched. In contrast, in our experiments, remapping occurred virtually on the entire track. In fact, firing-pattern changes were similar at, near, and far from the gap, implying that, in jump trials, the brain regarded the track differently from the same track in control trials without a gap. The implication is that from the beginning of each trial, a unique attractor, containing partially the same neurons, was retrieved, depending on whether it was a control trial or a jump trial with different gap locations. This hypothesis is supported by the relatively equal probability distribution of the five groups of place fields of both CA1 and CA3 neurons on jump trials, including persisting place fields, novel place fields, place fields with increased or decreased firing rates, and jump cells. These findings suggest that the expected different action plan (i.e., preparation for jumping) from the beginning of the trial exerts a stronger internal impact on the hippocampus than do changes in local cues. Neurons whose firing rates were attenuated (group 3) or amplified (group 4) during jump trials may be related (or identical) to “object-location memory” cells . When objects are removed from an environment or repositioned, object-location memory cells, located in both CA1 and CA3 regions, increase their firing at the locations where the objects used to be . Similar to previous interpretations , rate remapping is an indication of conjunctive features of neurons, the ability to simultaneously encode multiple types of information . An explicit mechanism offered for such conjunctive coding is that the spike phase of theta is primarily responsible for locating the animal in the environment , whereas the independent firing rate is available to code for other features, such as speed, objects, goals, or motivation . This hypothesis is supported by our observations that despite the strong changes in firing rates, the theta-spike phase versus animal-position relationship remained unaltered during jump trials. According to the classic theory, place fields are induced and maintained by a combination of sensory inputs and intracellular mechanisms . An alternative view is that neuronal sequences are self-organized at the circuit levels and such preexisting sequences are matched to particular environments . Our observation that the compression of distance to theta-phase (time) offset between place-cell pairs remained unaltered during jump trials, despite changes in firing patterns of individual place cells, supports the internally organized hippocampal model. Previous experiments also found that theta time offset of spikes between place-cell pairs remains fixed, despite increasing distances between place-field peaks in larger environments , running speed differences , or different temporal requirements . Yet, action- and environment-anchored reference frames can coexist in the hippocampus . Cases where the preexisting place field coincided with the location of jumping provide insight into the triggering mechanisms and maintenance of place fields. In a few group 3 neurons, the beginning or a large part of a place field was completely obliterated by the jump. Despite the absence of spikes in a large portion of the place field, spiking resumed after the jump and at the expected theta phases. These findings also support the internally organized model, which assumes that that spiking of hippocampal place cells within their fields is an assembly product . From this assembly point of view, we hypothesize that the place field in group 3 neurons was not abolished but suppressed by inhibition, and when the neuron was released from inhibition after the jump, it continued to fire together with its less-affected recorded and nonrecorded assembly peers. This hypothesis can explain how neurons that were silenced during the jump continued to fire at the same theta phase as during control trials. Sentinel Function of the Hippocampus. Remapping of place fields is often interpreted as an example of an operation in a dynamically changing circuit capable of updating neuronal assembly sequences and needed to support episodic memory . One can assume that jumping a gap and traveling back and forth on a linear track do not need the hippocampus (although we have not tested this directly); therefore, the observed sequential changes are not relevant to behavior. Yet, in line with our findings, previous studies have already shown that hippocampal neuron firing patterns do change in response to various environmental, motivation, and motor variables, even in situations which do not require the hippocampus or its allied structures . These studies are compatible with the hypothesis that a main function of the hippocampal system is to continuously monitor the activity of the neocortex and respond selectively to unexpected changes with appropriate selection of assembly sequences. In this sentinel function, the hippocampus perpetually compares the difference between neocortical neuronal messages and the reconstructed, predicted versions of those messages by the hippocampus . Only when the discrepancy between inputs and planned actions (“error”) is large does the hippocampal circuit induce new neuronal trajectories . Jump Cells. In a previous study, rats were trained to avoid an electric shock by jumping up onto the rim of a box with 33-cm walls . A fraction of hippocampal pyramidal neurons fired selectively during the vertical jump, and the authors suggested that these jump cells corresponded to place cells in the z (i.e., vertical) dimension. This contention is further supported by a report showing that during rearing, specific cells may become active at particular locations . Our findings allow for a different interpretation. First, in our experiments, the rats jumped horizontally and the elevation of their head during the flight was less than the length of the rat . Second, while jumps cells were active at the same z distance from the track, their x coordinate was different, yet neurons repeatedly fired at two or three gap locations and at different rates. Third, the majority of our jump cells did not coincide with the jump itself but were active during running either before or after the gap. Finally, virtually all jump cells were active in only one travel direction. These findings eliminate the possibility that jump cells were linked strictly to motoric actions. Such direction (or “context”) and location specificity suggests that jump cells are not fundamentally different from place cells but require the conjunction of a cue and an appropriate action plan. In the few jump cells whose fields coincided with the act of jumping itself, the phase preference of spikes moved from the peak to the trough of the theta waves in just one or two cycles. This observation is consistent with the hypothesis that jump cells possess the essential features of place cells , since the magnitude of a full theta-cycle phase precession corresponds to the length of the place field , irrespective how many theta cycles it takes for the animal to traverse the field [i.e., speed ]. Neurons designated as jump-specific cells in our study share several features of a related (or identical) class of neurons known as landmark-vector cells . Landmark-vector cells fire at a similar distance and direction from a landmark and may follow the landmark when moved. Similarly, our jump cells also displayed vectorial features, since they fired selectively at the gap or at a constant distance from the gap but only during either left- or right-bound travels.
A reliable relationship between various motor actions and the phase of the theta cycle has previously been reported . In our experiments, jumping was associated with LFP theta-phase reset. The flight time itself was <0.2 s, typically one or two theta cycles, but the trial-to-trial phase consistency persisted for several theta cycles after the jump. The phase reset coincided with an increase of both theta amplitude and frequency, as also reported during vertical jumping . The phase reset was also evident from the session average of interneuron firing, essentially tracking the phase-shifted LFP theta cycles. Finally, when the gap coincided with the place field of a pyramidal neuron, a sudden phase shift in spike–theta-phase preference was detected. This sudden shift may have occurred because in one or two theta cycles, the rat’s head was displaced by 30 cm, thus maintaining the relationship between spatial cues and spike–theta-phase relationship . Alternatively, the phase shift might reflect the altered relationship between spiking of the jump cell and the phase-shifted reference LFP theta. Another issue that has remained ambiguous is whether jumping induced phase resetting of theta oscillations or whether the timing of jumping was biased by the phase of the theta cycle . The theta reset might have been brought about the corollary discharge from the motor command signal or from the sensory feedback of muscle activity .
Firing sequences of hippocampal neurons during exploration in one- or two-dimensional environments have been assumed to be driven by internal mechanisms . For each environment, a new map with different firing patterns is retrieved, due to shifting from one neuronal “attractor” to another , known as remapping or as an altered manifold . Under some conditions, reorganization is manifested as a change in firing rates at the same locations (“rate remapping”), whereas between distinct environments, new place fields appear and old ones disappear or move to a new location [“global remapping” ]. Remapping can be induced not only by spatial manipulations and changes in sensory inputs but also by motivational state , experience , and memory load . In our experiments, nearly all place fields, with the exception of a small fraction of stable place fields at the two ends of the track, likely anchored to the start, or goal, platforms , were modified one way or another. In jump trials, firing pattern reorganization occurred as a combination of global and rate remapping. To explain these changes, one can make the argument that the absence of a piece of the track is a local sensory cue and, thus, this experimenter-induced change is the sole explanation of remapping . However, local cues are not expected to induce such a strong remapping as we have observed in our experiments. Several previous studies have examined the impact of overt or hidden local cues on hippocampal neuronal firing. These experiments vary from reporting no effect to reporting moderate effects on place-field activity, including place-field enrichment, smaller place fields, increased spatial tuning (i.e., firing-rate increase), altered phase precession, and increased or reduced spatial encoding . Common to these previous experiments (but see refs. and ) is that the described firing-pattern alterations were confined to the particular segment of the environment where the density of visual and tactile cues was enriched. In contrast, in our experiments, remapping occurred virtually on the entire track. In fact, firing-pattern changes were similar at, near, and far from the gap, implying that, in jump trials, the brain regarded the track differently from the same track in control trials without a gap. The implication is that from the beginning of each trial, a unique attractor, containing partially the same neurons, was retrieved, depending on whether it was a control trial or a jump trial with different gap locations. This hypothesis is supported by the relatively equal probability distribution of the five groups of place fields of both CA1 and CA3 neurons on jump trials, including persisting place fields, novel place fields, place fields with increased or decreased firing rates, and jump cells. These findings suggest that the expected different action plan (i.e., preparation for jumping) from the beginning of the trial exerts a stronger internal impact on the hippocampus than do changes in local cues. Neurons whose firing rates were attenuated (group 3) or amplified (group 4) during jump trials may be related (or identical) to “object-location memory” cells . When objects are removed from an environment or repositioned, object-location memory cells, located in both CA1 and CA3 regions, increase their firing at the locations where the objects used to be . Similar to previous interpretations , rate remapping is an indication of conjunctive features of neurons, the ability to simultaneously encode multiple types of information . An explicit mechanism offered for such conjunctive coding is that the spike phase of theta is primarily responsible for locating the animal in the environment , whereas the independent firing rate is available to code for other features, such as speed, objects, goals, or motivation . This hypothesis is supported by our observations that despite the strong changes in firing rates, the theta-spike phase versus animal-position relationship remained unaltered during jump trials. According to the classic theory, place fields are induced and maintained by a combination of sensory inputs and intracellular mechanisms . An alternative view is that neuronal sequences are self-organized at the circuit levels and such preexisting sequences are matched to particular environments . Our observation that the compression of distance to theta-phase (time) offset between place-cell pairs remained unaltered during jump trials, despite changes in firing patterns of individual place cells, supports the internally organized hippocampal model. Previous experiments also found that theta time offset of spikes between place-cell pairs remains fixed, despite increasing distances between place-field peaks in larger environments , running speed differences , or different temporal requirements . Yet, action- and environment-anchored reference frames can coexist in the hippocampus . Cases where the preexisting place field coincided with the location of jumping provide insight into the triggering mechanisms and maintenance of place fields. In a few group 3 neurons, the beginning or a large part of a place field was completely obliterated by the jump. Despite the absence of spikes in a large portion of the place field, spiking resumed after the jump and at the expected theta phases. These findings also support the internally organized model, which assumes that that spiking of hippocampal place cells within their fields is an assembly product . From this assembly point of view, we hypothesize that the place field in group 3 neurons was not abolished but suppressed by inhibition, and when the neuron was released from inhibition after the jump, it continued to fire together with its less-affected recorded and nonrecorded assembly peers. This hypothesis can explain how neurons that were silenced during the jump continued to fire at the same theta phase as during control trials.
Remapping of place fields is often interpreted as an example of an operation in a dynamically changing circuit capable of updating neuronal assembly sequences and needed to support episodic memory . One can assume that jumping a gap and traveling back and forth on a linear track do not need the hippocampus (although we have not tested this directly); therefore, the observed sequential changes are not relevant to behavior. Yet, in line with our findings, previous studies have already shown that hippocampal neuron firing patterns do change in response to various environmental, motivation, and motor variables, even in situations which do not require the hippocampus or its allied structures . These studies are compatible with the hypothesis that a main function of the hippocampal system is to continuously monitor the activity of the neocortex and respond selectively to unexpected changes with appropriate selection of assembly sequences. In this sentinel function, the hippocampus perpetually compares the difference between neocortical neuronal messages and the reconstructed, predicted versions of those messages by the hippocampus . Only when the discrepancy between inputs and planned actions (“error”) is large does the hippocampal circuit induce new neuronal trajectories .
In a previous study, rats were trained to avoid an electric shock by jumping up onto the rim of a box with 33-cm walls . A fraction of hippocampal pyramidal neurons fired selectively during the vertical jump, and the authors suggested that these jump cells corresponded to place cells in the z (i.e., vertical) dimension. This contention is further supported by a report showing that during rearing, specific cells may become active at particular locations . Our findings allow for a different interpretation. First, in our experiments, the rats jumped horizontally and the elevation of their head during the flight was less than the length of the rat . Second, while jumps cells were active at the same z distance from the track, their x coordinate was different, yet neurons repeatedly fired at two or three gap locations and at different rates. Third, the majority of our jump cells did not coincide with the jump itself but were active during running either before or after the gap. Finally, virtually all jump cells were active in only one travel direction. These findings eliminate the possibility that jump cells were linked strictly to motoric actions. Such direction (or “context”) and location specificity suggests that jump cells are not fundamentally different from place cells but require the conjunction of a cue and an appropriate action plan. In the few jump cells whose fields coincided with the act of jumping itself, the phase preference of spikes moved from the peak to the trough of the theta waves in just one or two cycles. This observation is consistent with the hypothesis that jump cells possess the essential features of place cells , since the magnitude of a full theta-cycle phase precession corresponds to the length of the place field , irrespective how many theta cycles it takes for the animal to traverse the field [i.e., speed ]. Neurons designated as jump-specific cells in our study share several features of a related (or identical) class of neurons known as landmark-vector cells . Landmark-vector cells fire at a similar distance and direction from a landmark and may follow the landmark when moved. Similarly, our jump cells also displayed vectorial features, since they fired selectively at the gap or at a constant distance from the gap but only during either left- or right-bound travels.
All experiments were approved by the Institutional Animal Care and Use Committee at New York University Medical Center. Details about of surgery, locations of the probes, and recordings are available in ref. and in the SI Appendix, Materials and Methods . The takeoff during jumping was determined by the peak of the horizontal acceleration, and the landing was determined by the peak of the negative horizontal acceleration. Wait time was determined by the time spent at a speed less than 9 cm/s before the jump takeoff. Theta phase was extracted by filtering the LFP signal with a third order Butterworth bandpass filter (6 to 13 Hz), then applying the Hilbert transform to extract just the phase. Circular deviance, the circular analog of SD, was measured across each time bin. Place fields were determined as described in ref. . Jump-specific cells were identified by eye. Circular–linear correlations, circular deviance, and the Rayleigh test were performed using the CircStat toolbox for circular statistics .
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Managing mental health in chronic care in general practice: a feasibility study of the Healthy Mind intervention | cf036f74-29da-43f2-b176-fc189f9e8ae1 | 10851809 | Family Medicine[mh] | Type 2 diabetes (T2D) and chronic ischaemic heart disease (IHD) are among the most prevalent chronic diseases worldwide . In these patient groups, the prevalence of depression is almost ten times higher than in the background population . The mechanisms mediating the association between reduced mental health and chronic somatic conditions are complex and include a number of bio-psycho-social factors, like symptom burden, adverse effects of medication, and the psychological burden of living with chronic disease. Also, chronic physical conditions can increase the risk of reduced mental health and vice versa . In addition, worse somatic health outcomes and increased mortality rates are reported in patients with comorbid physical and mental conditions, partly due to reduced self-care activities and poor medication adherence . International guidelines for T2D and IHD management emphasise the importance of attending to mental health issues in addition to somatic and biomedical aspects of the disease . Nevertheless, the primary focus remains on the biomedical aspects . General practice is responsible for providing the care for these patients in most developed countries. Typically, these patients are offered 1–2 annual chronic care consultations with the general practitioner (GP) and/or practice staff, usually a practice nurse (PN). The continuity of care facilitates a patient-centred approach and offers confidentiality and knowledge about psycho-social aspects of the individual patient’s life. Therefore, the chronic care consultations in general practice could be a highly relevant setting for facilitating early detection and treatment of mental health issues. Previous studies have found that non-pharmacological interventions based in primary care to target mild depression are effective, acceptable and preferred over medical treatments by patients . One such intervention is problem-solving therapy (PST), which is a well-established psychological treatment that can be tailored to a primary care setting and has proven an effective treatment for depression and other mood disorders . In recent years, several national guidelines have recommended PST for management of depression in primary care . PST is an easily accessible and tangible treatment for both patients and providers, and PST can be delivered by trained healthcare providers within the time constraints of general practice . Thus, PST seems to fit within the setting of general practice and may be a valuable treatment for patients with poor mental well-being and T2D and/or IHD. However, PST is not widely applied in Danish general practice, and most healthcare providers are unfamiliar with the approach. Moreover, to the authors’ knowledge, PST has never been offered to patients with a somatic disease based on screen-detected poor mental well-being. The Healthy Mind intervention was designed as a complex intervention in the general practice setting to offer PST to patients with poor mental well-being and T2D and/or IHD . Patients are screened for poor mental well-being at the annual chronic care consultation, and PST sessions are offered to patients on indication of screen-detected poor mental well-being. Feasibility testing of interventions is highly recommended prior to a full-scale evaluation, in order to explore whether the intervention can be delivered in the specific setting . Important outcomes to assess include the fit of the intervention to the setting and the patient group (appropriateness), receivers’ and deliverers’ perception of the intervention (acceptability), and the degree to which the intervention is delivered as intended (fidelity) . The aim of this study was to evaluate the feasibility of the Healthy Mind intervention by assessing the appropriateness, acceptability and fidelity.
Design and setting This single-arm feasibility study was conducted in Danish general practice from March to May 2022. Danish health care is mainly tax funded, and all residents have free and equal access to general practice services. Reimbursement and services offered in general practice are dictated by the collective agreement between the Danish Organization of General Practitioners and Danish Regions. GPs are independent contractors, who are free to organise their clinics within the framework of the collective agreement. Practice nurses are often employed in general practice and conduct independent consultations, including chronic care consultations under GP supervision. According to the collective agreement in Denmark, reimbursement can be provided for up to seven 30-minute talk therapy sessions per patient over a 12-month period . Intervention PST aims to enhance the patient’s problem-solving skills, coping and self-care through empowerment and behavioural activation . According to proposed behavioural models, effective problem-solving skills are the operating principle determining whether negative life events and problems develop into reduced mental health . In PST, training of these skills is essential, and the patient must actively engage in problem-solving, whereas the PST provider takes the role as facilitator and guide. In the Healthy Mind intervention, patients used worksheets as a central part of PST. The worksheets guided the problem-solving process through five sequential steps (one worksheet per step): (1) Identifying the problem, (2) Defining the problem, (3) Generating solutions, (4) Analysing advantages and disadvantages for each generated solution, and (5) Implementing the most appropriate solution. Additional supporting material was developed for the healthcare provider to facilitate the PST sessions. Issues addressed during the PST sessions could be related to all aspects of the patient’s life. To match the Danish general practice setting, patients were offered up to seven PST sessions (each of a duration of 30 min). This was in accordance with previous recommendations of offering up to 6–12 sessions of a duration of 30–60 min each . The number of sessions offered to each individual patient was based on the patient’s preferences and the healthcare provider’s assessment of need. PST training The PST training course was based on insights from a previous study in which 10 Danish GPs received PST training and offered PST to patients with various emotional issues . In the present study, healthcare providers participated in a two-day PST course, which was hosted by two psychologists with special PST expertise. The course consisted of 1.5 days of PST training followed by a half-day training session, which was conducted one month later. Thereby, the healthcare providers had the opportunity to share and draw on each other’s experiences between the first and second session. The training covered the theoretical basis of PST and consisted of theoretical education and practical exercises, including role play, based on the four stages of Kolb’s learning cycle: concrete experience, reflective observation, abstract conceptualisation and active experimentation . Furthermore, participants discussed how the intervention could be delivered in their individual general practices, and they developed plans for introducing and implementing PST in their general practices. Participants Recruitment of healthcare providers Three general practices were recruited through e-mails sent directly to the clinics. The research group recruited these particular general practices since they differed in size, staff composition and location (rural/urban), and their personnel had previously expressed interest in working with mental health to members of the research group. At least one GP and one PN from each individual practice were mandated to participate in the study, and a total of three GPs and five PNs agreed to participate. All included healthcare providers were obliged to participate in PST training, but the individual general practice could decide who was to deliver PST to their patients. shows the affiliation of healthcare providers and location of general practices. Recruitment of patients Eligible patients were adults diagnosed with T2D and/or IHD who attended an annual chronic care consultation in general practice. After giving informed consent, eligible patients were screened with the World Health Organization Well-being Index (WHO-5). The WHO-5 index is a psychometrically reliable and valid questionnaire that comprises five positively phrased questions measuring the subjective mental well-being of the responder. It yields a total score ranging from 0 to 100 points, with scores below 50 points indicating reduced mental well-being . Patients with a score below 50 points were offered PST sessions provided by the GP or PN. Patients were excluded if they did not speak or understand Danish, were in a psychotic or suicidal mental state, had severely impaired cognitive function or underwent other simultaneous psychological treatments. Support to intervention practices To support implementation and uptake of the intervention, weekly updates were provided by email to the general practices. This included information about the number of recruited patients and the number of completed PST sessions in each practice. During the study period, each practice was expected to include 2–4 patients. Theoretical framework Implementation issues can be explored through specific implementation outcomes that enable systematic assessment of implementation and aids the understanding of implementation processes . These outcomes can be studied during active implementation phases but are also common in studies conducted during the early phases of exploration and preparation, such as feasibility studies . Inspired by Proctor et al. this study assessed three early-stage implementation outcomes regarded as key for evaluating the feasibility of the Healthy Mind intervention: appropriateness, acceptability and fidelity. The operationalisation of the implementation outcomes is described in . Further, in pilot and feasibility studies, one may assess the feasibility of the intervention and/or the evaluation design . In this study, we focused on the feasibility of the intervention. Data collection During the study period, one semi-structured group interview was conducted with healthcare providers from each of the participating general practice. A focus group interview with all participating healthcare providers was conducted at the end of the study period. Two PNs were unable to participate in the focus group interview, and they participated subsequently in individual telephone interviews. Group interviews were conducted in the respective general practices and the focus group interview was conducted in general practice 2. Before the interviews, all participants gave informed consent after receiving information about the study. Patients participated in individual telephone interviews after their final PST session. An overview of the qualitative data collection is provided in . All interviews were completed by AM and/or AS and were audio-recorded. Interviews were based on semi-structured interview guides with open-ended questions aiming to explore the implementation of the intervention by assessing implementation outcomes, as described in . The participants were asked about their experiences with delivering (healthcare providers) or receiving (patients) the intervention, how well the intervention fitted the general practice setting and the patient group, and their general perception of PST. Overall, the interviewers encouraged the participants to freely express their thoughts and opinions. The interview guides were developed during multiple discussion rounds in the multidisciplinary research group and are available on request. Analyses Data were coded deductively based on operationalisations of the implementation outcomes: appropriateness, acceptability and fidelity . As suggested by Nevedal et al. , the analyses were conducted directly from the audio-recordings, thereby deriving detailed notes and captured quotes. To validate the coding, this was done independently by two authors (AS and SER), and any disagreements were negotiated until agreement was reached. A third author (AM) validated the analysis, and it was condensed into one joint narrative describing both healthcare providers’ and patients’ perspectives on appropriateness, acceptability and fidelity. Inspired by the concept of information power, we continuously considered the study aim, sample specificity, use of theory, quality of dialogue, and analysis strategy to further validate the analysis . Ethical considerations The study complied with the Declaration of Helsinki, which outlines some general ethical principles for good medical research practice . Patients were informed, orally and in writing, about the study. They were informed that participation was voluntary and that they could withdraw from the study at any point in time. Written consent to participate was obtained from the patients. General practices were remunerated in accordance with their time spent on study-related activities. Data storage and access complied with the General Data Protection Regulation (GDPR) of the European Union, and the project was listed in the record of processing activities at the Research Unit for General Practice, Aarhus . According to the Danish National Committee on Health Research Ethics, no ethical approval was required for this study, since it did not include any human material .
This single-arm feasibility study was conducted in Danish general practice from March to May 2022. Danish health care is mainly tax funded, and all residents have free and equal access to general practice services. Reimbursement and services offered in general practice are dictated by the collective agreement between the Danish Organization of General Practitioners and Danish Regions. GPs are independent contractors, who are free to organise their clinics within the framework of the collective agreement. Practice nurses are often employed in general practice and conduct independent consultations, including chronic care consultations under GP supervision. According to the collective agreement in Denmark, reimbursement can be provided for up to seven 30-minute talk therapy sessions per patient over a 12-month period .
PST aims to enhance the patient’s problem-solving skills, coping and self-care through empowerment and behavioural activation . According to proposed behavioural models, effective problem-solving skills are the operating principle determining whether negative life events and problems develop into reduced mental health . In PST, training of these skills is essential, and the patient must actively engage in problem-solving, whereas the PST provider takes the role as facilitator and guide. In the Healthy Mind intervention, patients used worksheets as a central part of PST. The worksheets guided the problem-solving process through five sequential steps (one worksheet per step): (1) Identifying the problem, (2) Defining the problem, (3) Generating solutions, (4) Analysing advantages and disadvantages for each generated solution, and (5) Implementing the most appropriate solution. Additional supporting material was developed for the healthcare provider to facilitate the PST sessions. Issues addressed during the PST sessions could be related to all aspects of the patient’s life. To match the Danish general practice setting, patients were offered up to seven PST sessions (each of a duration of 30 min). This was in accordance with previous recommendations of offering up to 6–12 sessions of a duration of 30–60 min each . The number of sessions offered to each individual patient was based on the patient’s preferences and the healthcare provider’s assessment of need. PST training The PST training course was based on insights from a previous study in which 10 Danish GPs received PST training and offered PST to patients with various emotional issues . In the present study, healthcare providers participated in a two-day PST course, which was hosted by two psychologists with special PST expertise. The course consisted of 1.5 days of PST training followed by a half-day training session, which was conducted one month later. Thereby, the healthcare providers had the opportunity to share and draw on each other’s experiences between the first and second session. The training covered the theoretical basis of PST and consisted of theoretical education and practical exercises, including role play, based on the four stages of Kolb’s learning cycle: concrete experience, reflective observation, abstract conceptualisation and active experimentation . Furthermore, participants discussed how the intervention could be delivered in their individual general practices, and they developed plans for introducing and implementing PST in their general practices.
The PST training course was based on insights from a previous study in which 10 Danish GPs received PST training and offered PST to patients with various emotional issues . In the present study, healthcare providers participated in a two-day PST course, which was hosted by two psychologists with special PST expertise. The course consisted of 1.5 days of PST training followed by a half-day training session, which was conducted one month later. Thereby, the healthcare providers had the opportunity to share and draw on each other’s experiences between the first and second session. The training covered the theoretical basis of PST and consisted of theoretical education and practical exercises, including role play, based on the four stages of Kolb’s learning cycle: concrete experience, reflective observation, abstract conceptualisation and active experimentation . Furthermore, participants discussed how the intervention could be delivered in their individual general practices, and they developed plans for introducing and implementing PST in their general practices.
Recruitment of healthcare providers Three general practices were recruited through e-mails sent directly to the clinics. The research group recruited these particular general practices since they differed in size, staff composition and location (rural/urban), and their personnel had previously expressed interest in working with mental health to members of the research group. At least one GP and one PN from each individual practice were mandated to participate in the study, and a total of three GPs and five PNs agreed to participate. All included healthcare providers were obliged to participate in PST training, but the individual general practice could decide who was to deliver PST to their patients. shows the affiliation of healthcare providers and location of general practices. Recruitment of patients Eligible patients were adults diagnosed with T2D and/or IHD who attended an annual chronic care consultation in general practice. After giving informed consent, eligible patients were screened with the World Health Organization Well-being Index (WHO-5). The WHO-5 index is a psychometrically reliable and valid questionnaire that comprises five positively phrased questions measuring the subjective mental well-being of the responder. It yields a total score ranging from 0 to 100 points, with scores below 50 points indicating reduced mental well-being . Patients with a score below 50 points were offered PST sessions provided by the GP or PN. Patients were excluded if they did not speak or understand Danish, were in a psychotic or suicidal mental state, had severely impaired cognitive function or underwent other simultaneous psychological treatments. Support to intervention practices To support implementation and uptake of the intervention, weekly updates were provided by email to the general practices. This included information about the number of recruited patients and the number of completed PST sessions in each practice. During the study period, each practice was expected to include 2–4 patients.
Three general practices were recruited through e-mails sent directly to the clinics. The research group recruited these particular general practices since they differed in size, staff composition and location (rural/urban), and their personnel had previously expressed interest in working with mental health to members of the research group. At least one GP and one PN from each individual practice were mandated to participate in the study, and a total of three GPs and five PNs agreed to participate. All included healthcare providers were obliged to participate in PST training, but the individual general practice could decide who was to deliver PST to their patients. shows the affiliation of healthcare providers and location of general practices.
Eligible patients were adults diagnosed with T2D and/or IHD who attended an annual chronic care consultation in general practice. After giving informed consent, eligible patients were screened with the World Health Organization Well-being Index (WHO-5). The WHO-5 index is a psychometrically reliable and valid questionnaire that comprises five positively phrased questions measuring the subjective mental well-being of the responder. It yields a total score ranging from 0 to 100 points, with scores below 50 points indicating reduced mental well-being . Patients with a score below 50 points were offered PST sessions provided by the GP or PN. Patients were excluded if they did not speak or understand Danish, were in a psychotic or suicidal mental state, had severely impaired cognitive function or underwent other simultaneous psychological treatments.
To support implementation and uptake of the intervention, weekly updates were provided by email to the general practices. This included information about the number of recruited patients and the number of completed PST sessions in each practice. During the study period, each practice was expected to include 2–4 patients.
Implementation issues can be explored through specific implementation outcomes that enable systematic assessment of implementation and aids the understanding of implementation processes . These outcomes can be studied during active implementation phases but are also common in studies conducted during the early phases of exploration and preparation, such as feasibility studies . Inspired by Proctor et al. this study assessed three early-stage implementation outcomes regarded as key for evaluating the feasibility of the Healthy Mind intervention: appropriateness, acceptability and fidelity. The operationalisation of the implementation outcomes is described in . Further, in pilot and feasibility studies, one may assess the feasibility of the intervention and/or the evaluation design . In this study, we focused on the feasibility of the intervention.
During the study period, one semi-structured group interview was conducted with healthcare providers from each of the participating general practice. A focus group interview with all participating healthcare providers was conducted at the end of the study period. Two PNs were unable to participate in the focus group interview, and they participated subsequently in individual telephone interviews. Group interviews were conducted in the respective general practices and the focus group interview was conducted in general practice 2. Before the interviews, all participants gave informed consent after receiving information about the study. Patients participated in individual telephone interviews after their final PST session. An overview of the qualitative data collection is provided in . All interviews were completed by AM and/or AS and were audio-recorded. Interviews were based on semi-structured interview guides with open-ended questions aiming to explore the implementation of the intervention by assessing implementation outcomes, as described in . The participants were asked about their experiences with delivering (healthcare providers) or receiving (patients) the intervention, how well the intervention fitted the general practice setting and the patient group, and their general perception of PST. Overall, the interviewers encouraged the participants to freely express their thoughts and opinions. The interview guides were developed during multiple discussion rounds in the multidisciplinary research group and are available on request.
Data were coded deductively based on operationalisations of the implementation outcomes: appropriateness, acceptability and fidelity . As suggested by Nevedal et al. , the analyses were conducted directly from the audio-recordings, thereby deriving detailed notes and captured quotes. To validate the coding, this was done independently by two authors (AS and SER), and any disagreements were negotiated until agreement was reached. A third author (AM) validated the analysis, and it was condensed into one joint narrative describing both healthcare providers’ and patients’ perspectives on appropriateness, acceptability and fidelity. Inspired by the concept of information power, we continuously considered the study aim, sample specificity, use of theory, quality of dialogue, and analysis strategy to further validate the analysis .
The study complied with the Declaration of Helsinki, which outlines some general ethical principles for good medical research practice . Patients were informed, orally and in writing, about the study. They were informed that participation was voluntary and that they could withdraw from the study at any point in time. Written consent to participate was obtained from the patients. General practices were remunerated in accordance with their time spent on study-related activities. Data storage and access complied with the General Data Protection Regulation (GDPR) of the European Union, and the project was listed in the record of processing activities at the Research Unit for General Practice, Aarhus . According to the Danish National Committee on Health Research Ethics, no ethical approval was required for this study, since it did not include any human material .
Participants provides an overview of patient characteristics and healthcare provider/patient relationships. All healthcare providers completed the training programme. However, two PNs (PN4 and PN5), from two different general practices, left the workplace shortly after completing the PST course without having delivered PST to any patients. Of the 54 patients screened during the study, nine patients (17%) were found to have poor mental well-being (WHO-5 score < 50 points). All patients agreed to participate in PST sessions. Two patients were excluded from the study based on the healthcare providers’ assessments, and three patients declined to participate in the interviews after the PST sessions. Three main themes were derived from the analysis: the fit of PST to setting and patient group, content and structure of PST, and delivery of PST. Theme 1: Fit of PST to setting and patient group Increasing the focus on mental well-being Broadening the focus of the annual chronic care consultation to include a systematic focus on both mental and somatic aspects was positively viewed by healthcare providers and patients. A PN explained that she considered somatic and mental health to be interconnected, but she often found herself more preoccupied with the somatic aspects of the disease during the chronic care consultations. The patient-centered approach during the study helped her balance her attention to both somatic and mental aspects. [It is useful] to shift the focus away from all the biochemical aspects and values, and rather talk about how the patients are actually doing. Because their diabetes may be well managed… But [it is also useful] to be more interested in their mental well-being. Because sometimes we care so much about the former, right? That’s also important, but there is something else that is important, too. And it is so interconnected. (PN2) All patients expressed positive attitudes towards the increased focus on their mental well-being in their chronic care. One patient described how the increased focus on mental health contributed positively to his chronic care: In the past, I would get a blood test and my medication was adjusted. But this time, we also delved into the personal aspects, and it was a relief to me that I could talk to her about these things. (PT7) All patients were prepared and willing to discuss mental and emotional issues with their healthcare provider. One patient thought that the mental issues being addressed had to be directly linked to aspects of the somatic disease, such as how to cope with having to follow a strict diet or taking medication on a daily basis, which was not the case. One patient described that the close professional relationship with his practice nurse made him participate, even though he was generally reluctant to talk about and cope with emotional issues. The healthcare providers often had a pre-existing professional relationship with the patients due to the continuity in chronic care. One PN explained how her pre-existing confidentiality and acquaintance with the patients made it easy for her to increase the focus on mental aspects and engage her patients in the PST sessions. I know these patients […] very well. I already know their challenges, and they are used to talking with me. (PN1) Based on their pre-existing knowledge of the patients and their professional relationships with them, the healthcare providers considered the intervention appropriate for most patients. However, participation was considered inappropriate for two patients, who were excluded from the study due to a fragile professional alliance between healthcare provider and patient. A PN elaborated: I have spent three years building up a trusting relationship with one patient, who has an extremely dysregulated diabetes, and she is such a person where I would think that this [the intervention] is not appropriate. We are already working with the mental aspects – there are all sorts of things in her life. [When] you finally convince her to come for a [chronic care] consultation and – POW – she drops out again. (PN2) Navigating time constraints Time was described as the biggest challenge for delivering the intervention. Both GPs and PNs reflected that if the intervention was to be implemented in real life, PNs would have to be the main intervention providers. Compared to the GPs, the PNs’ schedules were generally better suited for long consultations, which made it easier for them to deliver the intervention. In two of the general practices, a PN left the workplace during the study period, which caused the general workload to increase for the remaining healthcare providers, and time became an important barrier for offering the intervention. A GP reflected: The biggest challenge is time […]. I think it has been exciting [to participate in the study], but I was overwhelmed by busy days and then it becomes a burden. (GP3) The patients noticed that the healthcare providers seemed to have a busy schedule. One patient described that, even though he felt the need for additional psychological support during the days after the first PST sessions, he was reluctant to ask for more consultations when he saw how busy the GP seemed to be: I am sure that he [the GP] would have taken me in – I do not doubt that – but I also know they have a busy schedule, and he probably does not need to spend his time on long psychological consultations. (PT1) Theme 2: Content and structure of PST The tangible and action-oriented nature of PST was positively viewed by both healthcare providers and patients, and PST was regarded as an easily accessible and comprehensive treatment and a valuable clinical tool for managing mental health issues in general practice. A GP elaborates: It [the intervention] feeds into everyday life instead of dealing with a personality structure or something more complicated. (GP3) The predetermined stepwise structure of PST was perceived as particularly helpful by healthcare providers with limited previous experience in treating mental health issues, and treatment progress was found to be facilitated by the structure of PST. One PN stated: It is nice to have a tool that is a little stringent. You stick to what you discussed last time instead of moving on. It transforms into more action. It becomes more committing – both to the provider and to the patient. (PN1) One healthcare provider, who was experienced in managing mental health issues, reflected that combining PST with other psychological treatment methods, such as cognitive behavioural therapy, was compelling to her. Still, she also reflected that using PST alone may be more time efficient compared to other treatments. All patients expressed an overall positive view on the content and structure of PST. The straightforward and tangible nature of PST was comprehensible to the patients. A patient expressed that the use of worksheets facilitated the identification and in-depth analysis of her problems. Another patient reflected that the method allowed him to see things from a new perspective, which helped him find new ways to manage his problems. Theme 3: Delivery of PST All healthcare providers reported that they followed the stepwise methodology during PST sessions, with the worksheets as a central part of the delivery. The duration of all PST sessions was 30 min. All GPs expressed that it was difficult to stay in the role as a guide and facilitator rather than taking an active part in the problem-solving process. A GP explained: The method is not like when we were on the course. The patients do not behave like they are supposed to. You need to be more of an idea generator and come up with solutions to a larger extent than what the method implies. (GP2) Another GP reflected that the patients expect the GPs to be problem fixers: I have come up with more ideas than my patients have. But maybe that is what is expected. So, now you come to the doctor, now you need some advice and then you are not supposed to sit and…. It is probably on our shoulders as well, since we have a hard time holding our tongues and giving time. (GP3) One patient felt that the healthcare provider dictated which problem to address during the PST sessions: We wrote down 3-4-5 topics on the list, and he chose the one with physical activity. I had expected him to ask me what I thought was the most important – and it [physical activity] was not what I would have said. (PT4) Due to the patient’s lack of influence on choosing which problem to work with, he perceived the problem as trivial and did not feel committed to the treatment. All healthcare providers reported that they encouraged the patients to take home the worksheets and to continue the problem-solving process at home after each consultation. Nevertheless, none of the healthcare providers expected their patients to be able to independently complete the more analytical problem-solving steps of the process. All patients brought home the worksheets and reported that they were encouraged to use them between the PST sessions as a guide during the problem-solving process. However, they reported to have sporadically reflected on the problem-solving process in an unstructured manner rather than using the worksheets systematically. All patients described that the treatment helped them take action and implement the solutions that they had come up with during PST sessions. For example, one patient changed jobs, and another reorganised his everyday chores to make time for more social activities.
provides an overview of patient characteristics and healthcare provider/patient relationships. All healthcare providers completed the training programme. However, two PNs (PN4 and PN5), from two different general practices, left the workplace shortly after completing the PST course without having delivered PST to any patients. Of the 54 patients screened during the study, nine patients (17%) were found to have poor mental well-being (WHO-5 score < 50 points). All patients agreed to participate in PST sessions. Two patients were excluded from the study based on the healthcare providers’ assessments, and three patients declined to participate in the interviews after the PST sessions. Three main themes were derived from the analysis: the fit of PST to setting and patient group, content and structure of PST, and delivery of PST.
Increasing the focus on mental well-being Broadening the focus of the annual chronic care consultation to include a systematic focus on both mental and somatic aspects was positively viewed by healthcare providers and patients. A PN explained that she considered somatic and mental health to be interconnected, but she often found herself more preoccupied with the somatic aspects of the disease during the chronic care consultations. The patient-centered approach during the study helped her balance her attention to both somatic and mental aspects. [It is useful] to shift the focus away from all the biochemical aspects and values, and rather talk about how the patients are actually doing. Because their diabetes may be well managed… But [it is also useful] to be more interested in their mental well-being. Because sometimes we care so much about the former, right? That’s also important, but there is something else that is important, too. And it is so interconnected. (PN2) All patients expressed positive attitudes towards the increased focus on their mental well-being in their chronic care. One patient described how the increased focus on mental health contributed positively to his chronic care: In the past, I would get a blood test and my medication was adjusted. But this time, we also delved into the personal aspects, and it was a relief to me that I could talk to her about these things. (PT7) All patients were prepared and willing to discuss mental and emotional issues with their healthcare provider. One patient thought that the mental issues being addressed had to be directly linked to aspects of the somatic disease, such as how to cope with having to follow a strict diet or taking medication on a daily basis, which was not the case. One patient described that the close professional relationship with his practice nurse made him participate, even though he was generally reluctant to talk about and cope with emotional issues. The healthcare providers often had a pre-existing professional relationship with the patients due to the continuity in chronic care. One PN explained how her pre-existing confidentiality and acquaintance with the patients made it easy for her to increase the focus on mental aspects and engage her patients in the PST sessions. I know these patients […] very well. I already know their challenges, and they are used to talking with me. (PN1) Based on their pre-existing knowledge of the patients and their professional relationships with them, the healthcare providers considered the intervention appropriate for most patients. However, participation was considered inappropriate for two patients, who were excluded from the study due to a fragile professional alliance between healthcare provider and patient. A PN elaborated: I have spent three years building up a trusting relationship with one patient, who has an extremely dysregulated diabetes, and she is such a person where I would think that this [the intervention] is not appropriate. We are already working with the mental aspects – there are all sorts of things in her life. [When] you finally convince her to come for a [chronic care] consultation and – POW – she drops out again. (PN2) Navigating time constraints Time was described as the biggest challenge for delivering the intervention. Both GPs and PNs reflected that if the intervention was to be implemented in real life, PNs would have to be the main intervention providers. Compared to the GPs, the PNs’ schedules were generally better suited for long consultations, which made it easier for them to deliver the intervention. In two of the general practices, a PN left the workplace during the study period, which caused the general workload to increase for the remaining healthcare providers, and time became an important barrier for offering the intervention. A GP reflected: The biggest challenge is time […]. I think it has been exciting [to participate in the study], but I was overwhelmed by busy days and then it becomes a burden. (GP3) The patients noticed that the healthcare providers seemed to have a busy schedule. One patient described that, even though he felt the need for additional psychological support during the days after the first PST sessions, he was reluctant to ask for more consultations when he saw how busy the GP seemed to be: I am sure that he [the GP] would have taken me in – I do not doubt that – but I also know they have a busy schedule, and he probably does not need to spend his time on long psychological consultations. (PT1)
Broadening the focus of the annual chronic care consultation to include a systematic focus on both mental and somatic aspects was positively viewed by healthcare providers and patients. A PN explained that she considered somatic and mental health to be interconnected, but she often found herself more preoccupied with the somatic aspects of the disease during the chronic care consultations. The patient-centered approach during the study helped her balance her attention to both somatic and mental aspects. [It is useful] to shift the focus away from all the biochemical aspects and values, and rather talk about how the patients are actually doing. Because their diabetes may be well managed… But [it is also useful] to be more interested in their mental well-being. Because sometimes we care so much about the former, right? That’s also important, but there is something else that is important, too. And it is so interconnected. (PN2) All patients expressed positive attitudes towards the increased focus on their mental well-being in their chronic care. One patient described how the increased focus on mental health contributed positively to his chronic care: In the past, I would get a blood test and my medication was adjusted. But this time, we also delved into the personal aspects, and it was a relief to me that I could talk to her about these things. (PT7) All patients were prepared and willing to discuss mental and emotional issues with their healthcare provider. One patient thought that the mental issues being addressed had to be directly linked to aspects of the somatic disease, such as how to cope with having to follow a strict diet or taking medication on a daily basis, which was not the case. One patient described that the close professional relationship with his practice nurse made him participate, even though he was generally reluctant to talk about and cope with emotional issues. The healthcare providers often had a pre-existing professional relationship with the patients due to the continuity in chronic care. One PN explained how her pre-existing confidentiality and acquaintance with the patients made it easy for her to increase the focus on mental aspects and engage her patients in the PST sessions. I know these patients […] very well. I already know their challenges, and they are used to talking with me. (PN1) Based on their pre-existing knowledge of the patients and their professional relationships with them, the healthcare providers considered the intervention appropriate for most patients. However, participation was considered inappropriate for two patients, who were excluded from the study due to a fragile professional alliance between healthcare provider and patient. A PN elaborated: I have spent three years building up a trusting relationship with one patient, who has an extremely dysregulated diabetes, and she is such a person where I would think that this [the intervention] is not appropriate. We are already working with the mental aspects – there are all sorts of things in her life. [When] you finally convince her to come for a [chronic care] consultation and – POW – she drops out again. (PN2)
Time was described as the biggest challenge for delivering the intervention. Both GPs and PNs reflected that if the intervention was to be implemented in real life, PNs would have to be the main intervention providers. Compared to the GPs, the PNs’ schedules were generally better suited for long consultations, which made it easier for them to deliver the intervention. In two of the general practices, a PN left the workplace during the study period, which caused the general workload to increase for the remaining healthcare providers, and time became an important barrier for offering the intervention. A GP reflected: The biggest challenge is time […]. I think it has been exciting [to participate in the study], but I was overwhelmed by busy days and then it becomes a burden. (GP3) The patients noticed that the healthcare providers seemed to have a busy schedule. One patient described that, even though he felt the need for additional psychological support during the days after the first PST sessions, he was reluctant to ask for more consultations when he saw how busy the GP seemed to be: I am sure that he [the GP] would have taken me in – I do not doubt that – but I also know they have a busy schedule, and he probably does not need to spend his time on long psychological consultations. (PT1)
The tangible and action-oriented nature of PST was positively viewed by both healthcare providers and patients, and PST was regarded as an easily accessible and comprehensive treatment and a valuable clinical tool for managing mental health issues in general practice. A GP elaborates: It [the intervention] feeds into everyday life instead of dealing with a personality structure or something more complicated. (GP3) The predetermined stepwise structure of PST was perceived as particularly helpful by healthcare providers with limited previous experience in treating mental health issues, and treatment progress was found to be facilitated by the structure of PST. One PN stated: It is nice to have a tool that is a little stringent. You stick to what you discussed last time instead of moving on. It transforms into more action. It becomes more committing – both to the provider and to the patient. (PN1) One healthcare provider, who was experienced in managing mental health issues, reflected that combining PST with other psychological treatment methods, such as cognitive behavioural therapy, was compelling to her. Still, she also reflected that using PST alone may be more time efficient compared to other treatments. All patients expressed an overall positive view on the content and structure of PST. The straightforward and tangible nature of PST was comprehensible to the patients. A patient expressed that the use of worksheets facilitated the identification and in-depth analysis of her problems. Another patient reflected that the method allowed him to see things from a new perspective, which helped him find new ways to manage his problems.
All healthcare providers reported that they followed the stepwise methodology during PST sessions, with the worksheets as a central part of the delivery. The duration of all PST sessions was 30 min. All GPs expressed that it was difficult to stay in the role as a guide and facilitator rather than taking an active part in the problem-solving process. A GP explained: The method is not like when we were on the course. The patients do not behave like they are supposed to. You need to be more of an idea generator and come up with solutions to a larger extent than what the method implies. (GP2) Another GP reflected that the patients expect the GPs to be problem fixers: I have come up with more ideas than my patients have. But maybe that is what is expected. So, now you come to the doctor, now you need some advice and then you are not supposed to sit and…. It is probably on our shoulders as well, since we have a hard time holding our tongues and giving time. (GP3) One patient felt that the healthcare provider dictated which problem to address during the PST sessions: We wrote down 3-4-5 topics on the list, and he chose the one with physical activity. I had expected him to ask me what I thought was the most important – and it [physical activity] was not what I would have said. (PT4) Due to the patient’s lack of influence on choosing which problem to work with, he perceived the problem as trivial and did not feel committed to the treatment. All healthcare providers reported that they encouraged the patients to take home the worksheets and to continue the problem-solving process at home after each consultation. Nevertheless, none of the healthcare providers expected their patients to be able to independently complete the more analytical problem-solving steps of the process. All patients brought home the worksheets and reported that they were encouraged to use them between the PST sessions as a guide during the problem-solving process. However, they reported to have sporadically reflected on the problem-solving process in an unstructured manner rather than using the worksheets systematically. All patients described that the treatment helped them take action and implement the solutions that they had come up with during PST sessions. For example, one patient changed jobs, and another reorganised his everyday chores to make time for more social activities.
Main findings This study aimed to evaluate the feasibility of a PST intervention, the Healthy Mind intervention, offered to patients in general practice by assessing appropriateness, acceptability and fidelity. The intervention was found to be appropriate. Increasing focus on psychological aspects during chronic care visits was viewed positively by both patients and healthcare providers. Patients were prepared and willing to engage in PST sessions. The pre-existing professional relationship between patient and healthcare provider facilitated delivery of PST and influenced the patients’ decision to participate in the treatment. Concerns about time constraints were considerable. GPs and PNs reflected that PNs should be the main deliverers of the intervention since their schedules were generally more accommodating for longer consultations. The intervention was found to be acceptable. Healthcare providers perceived PST as a valuable intervention for handling psychological issues in patients, and PST was considered suitable for both GPs and PNs to deliver. The patients viewed PST as a relevant and comprehensible treatment method. The intervention was delivered with an acceptable level of fidelity. The stepwise methodology was followed in all cases. However, the GPs sometimes struggled with remaining in their roles as facilitators and guides, which was facilitated by a preconceived anticipation among GPs that the patients expected them to take a more directive approach. Strengths and limitations An important strength of this study was the inclusion of perspectives from both patients and healthcare providers on important aspects of implementation. This allowed the research team to obtain a broad and nuanced understanding of the perceptions of both deliverers and receivers of the intervention. Further, we interviewed all healthcare providers and most patients who tested the intervention. Moreover, the intervention was tested in three general practices that differed in size, geographical location and workflows, thereby capturing insights from various types of organisational structures. However, including more general practices, participating healthcare providers and patients could have contributed with more insights on the feasibility of the intervention. Two healthcare providers were unable to attend the focus group interview after termination of the study and subsequently gave individual interviews. Thus, the dynamic nature of focus group interviews where participants can interact in a dialogue and discuss different views and opinions could not be applied. However, the same interview guides applied for both the focus group interview and individual interviews and the perspectives that emerged during the focus group interview were introduced in the individual interviews. Our study explored the feasibility of only the intervention, not the evaluation design. Yet, the analysis of recruitment and refusal rates, number of sessions per patient etc. is not described further, although they are relevant in the design of the subsequent larger trial. Also, we focused mainly on the early implementation of PST. Insights into sustained use of the intervention may have been obtained if the study had been conducted over a longer period, thereby allowing for other aspects of intervention feasibility to be evaluated, e.g. follow-up response rates. All included general practices had previously expressed that working with mental health issues was an area of special interest to them, which may have affected their perception of the intervention. Thus, the findings may not be generalisable to general practices with no special interest in mental health. Findings in relation to other literature Patient-centered care is a core value in general practice . However, biomedical aspects remain the primary focus in the chronic care consultations . In the present study, all participating patients were ready and willing to discuss psychological issues with their healthcare provider. Although it is not a prerequisite for the intervention, a pre-existing relationship with the healthcare provider seemed to facilitate the patients’ readiness for participation, which is also found in other studies . One of the reasons why general practice is a relevant setting for psychological interventions is that patients here often have longitudinal and continuous relationships with healthcare providers and, consequently, a trusting relationship is established prior to the treatment. However, two patients reported uncertainty about the framework of the PST consultations; one patient expected that the issues addressed during PST should be related to the somatic disease, and another patient was reluctant to schedule more PST sessions when considering how busy the GP seemed to be. This may reflect preconceptions that physical health is the main concern in chronic care in general practice and that psychological treatment is not the GP’s main focus. Such patient expectations have also been found in other studies and need to be addressed to facilitate successful implementation. One of the most important barriers identified in our study was time constraints. The PNs’ schedules were generally more accommodating, and it was easier for PNs to make time for longer consultations. However, the unexpected attrition of two of the healthcare providers led to an increase in workload that burdened the remaining healthcare providers and compromised the implementation of the intervention. In healthcare interventions, it is important to acknowledge that time is a scarce resource, and interventions need to be designed to fit clinical realities and accommodate potential challenges, such as unexpected staff attrition, which could be addressed by increasing the number of intervention providers. However, if the providers’ increasing experience with delivering the intervention is expected to increase the quality of the delivery, it is necessary to balance the vulnerability of attrition (increasing the number of intervention providers) with the need for the provider to gain sufficient skills in providing the intervention (ensuring that the provider gains sufficient experience). In this study, we found that delivery of PST was appropriate for both GPs and PNs. However, since the PNs’ schedules were more flexible, the healthcare providers reflected that delivery by PNs was most appropriate. In recent years, workforce shortage among GPs combined with an increasingly ageing population and expansion of general practice core tasks have led to a transfer of tasks to an increasingly multidisciplinary workforce in general practice. This task delegation to trained practice staff has been shown to be both safe and effective, and it is an important component in overcoming the challenges faced in general practice . Trained practice staff is increasingly involved in chronic care services, which should be considered when designing interventions targeting patients with chronic diseases in general practice. Preferably, such interventions should be flexible in terms of delivery to ensure that both nurses and GPs can deliver the intervention according to the preferences of each individual general practice. Some GPs anticipated that their patients would expect them to take the lead by generating possible solutions and giving advice on how to pursue the goals, which is not the intended approach in PST . At times, it was challenging for the GPs to balance this expectation and remain in a facilitating and guiding role during PST sessions, which ultimately challenged the implementation of the intervention. Thus, it is important to consider the preconceptions of both patients and healthcare providers when introducing an intervention in general practice. To ensure that participants are aware of their own preconceptions and to facilitate consensus between healthcare providers and patients, they should be encouraged to articulate their expectations prior to commencing the intervention. Furthermore, it is crucial to teach healthcare providers how to negotiate the agenda of the consultation when the roles in and the structure of the consultation differ from an ordinary consultation. Prior to a definitive trial, the PST course is therefore recommended to increase focus on preparing healthcare providers for delivery of the intervention, including awareness of possible patient expectations and the importance of delivering the intervention with high fidelity – including remaining in a facilitating and guiding role.
This study aimed to evaluate the feasibility of a PST intervention, the Healthy Mind intervention, offered to patients in general practice by assessing appropriateness, acceptability and fidelity. The intervention was found to be appropriate. Increasing focus on psychological aspects during chronic care visits was viewed positively by both patients and healthcare providers. Patients were prepared and willing to engage in PST sessions. The pre-existing professional relationship between patient and healthcare provider facilitated delivery of PST and influenced the patients’ decision to participate in the treatment. Concerns about time constraints were considerable. GPs and PNs reflected that PNs should be the main deliverers of the intervention since their schedules were generally more accommodating for longer consultations. The intervention was found to be acceptable. Healthcare providers perceived PST as a valuable intervention for handling psychological issues in patients, and PST was considered suitable for both GPs and PNs to deliver. The patients viewed PST as a relevant and comprehensible treatment method. The intervention was delivered with an acceptable level of fidelity. The stepwise methodology was followed in all cases. However, the GPs sometimes struggled with remaining in their roles as facilitators and guides, which was facilitated by a preconceived anticipation among GPs that the patients expected them to take a more directive approach.
An important strength of this study was the inclusion of perspectives from both patients and healthcare providers on important aspects of implementation. This allowed the research team to obtain a broad and nuanced understanding of the perceptions of both deliverers and receivers of the intervention. Further, we interviewed all healthcare providers and most patients who tested the intervention. Moreover, the intervention was tested in three general practices that differed in size, geographical location and workflows, thereby capturing insights from various types of organisational structures. However, including more general practices, participating healthcare providers and patients could have contributed with more insights on the feasibility of the intervention. Two healthcare providers were unable to attend the focus group interview after termination of the study and subsequently gave individual interviews. Thus, the dynamic nature of focus group interviews where participants can interact in a dialogue and discuss different views and opinions could not be applied. However, the same interview guides applied for both the focus group interview and individual interviews and the perspectives that emerged during the focus group interview were introduced in the individual interviews. Our study explored the feasibility of only the intervention, not the evaluation design. Yet, the analysis of recruitment and refusal rates, number of sessions per patient etc. is not described further, although they are relevant in the design of the subsequent larger trial. Also, we focused mainly on the early implementation of PST. Insights into sustained use of the intervention may have been obtained if the study had been conducted over a longer period, thereby allowing for other aspects of intervention feasibility to be evaluated, e.g. follow-up response rates. All included general practices had previously expressed that working with mental health issues was an area of special interest to them, which may have affected their perception of the intervention. Thus, the findings may not be generalisable to general practices with no special interest in mental health.
Patient-centered care is a core value in general practice . However, biomedical aspects remain the primary focus in the chronic care consultations . In the present study, all participating patients were ready and willing to discuss psychological issues with their healthcare provider. Although it is not a prerequisite for the intervention, a pre-existing relationship with the healthcare provider seemed to facilitate the patients’ readiness for participation, which is also found in other studies . One of the reasons why general practice is a relevant setting for psychological interventions is that patients here often have longitudinal and continuous relationships with healthcare providers and, consequently, a trusting relationship is established prior to the treatment. However, two patients reported uncertainty about the framework of the PST consultations; one patient expected that the issues addressed during PST should be related to the somatic disease, and another patient was reluctant to schedule more PST sessions when considering how busy the GP seemed to be. This may reflect preconceptions that physical health is the main concern in chronic care in general practice and that psychological treatment is not the GP’s main focus. Such patient expectations have also been found in other studies and need to be addressed to facilitate successful implementation. One of the most important barriers identified in our study was time constraints. The PNs’ schedules were generally more accommodating, and it was easier for PNs to make time for longer consultations. However, the unexpected attrition of two of the healthcare providers led to an increase in workload that burdened the remaining healthcare providers and compromised the implementation of the intervention. In healthcare interventions, it is important to acknowledge that time is a scarce resource, and interventions need to be designed to fit clinical realities and accommodate potential challenges, such as unexpected staff attrition, which could be addressed by increasing the number of intervention providers. However, if the providers’ increasing experience with delivering the intervention is expected to increase the quality of the delivery, it is necessary to balance the vulnerability of attrition (increasing the number of intervention providers) with the need for the provider to gain sufficient skills in providing the intervention (ensuring that the provider gains sufficient experience). In this study, we found that delivery of PST was appropriate for both GPs and PNs. However, since the PNs’ schedules were more flexible, the healthcare providers reflected that delivery by PNs was most appropriate. In recent years, workforce shortage among GPs combined with an increasingly ageing population and expansion of general practice core tasks have led to a transfer of tasks to an increasingly multidisciplinary workforce in general practice. This task delegation to trained practice staff has been shown to be both safe and effective, and it is an important component in overcoming the challenges faced in general practice . Trained practice staff is increasingly involved in chronic care services, which should be considered when designing interventions targeting patients with chronic diseases in general practice. Preferably, such interventions should be flexible in terms of delivery to ensure that both nurses and GPs can deliver the intervention according to the preferences of each individual general practice. Some GPs anticipated that their patients would expect them to take the lead by generating possible solutions and giving advice on how to pursue the goals, which is not the intended approach in PST . At times, it was challenging for the GPs to balance this expectation and remain in a facilitating and guiding role during PST sessions, which ultimately challenged the implementation of the intervention. Thus, it is important to consider the preconceptions of both patients and healthcare providers when introducing an intervention in general practice. To ensure that participants are aware of their own preconceptions and to facilitate consensus between healthcare providers and patients, they should be encouraged to articulate their expectations prior to commencing the intervention. Furthermore, it is crucial to teach healthcare providers how to negotiate the agenda of the consultation when the roles in and the structure of the consultation differ from an ordinary consultation. Prior to a definitive trial, the PST course is therefore recommended to increase focus on preparing healthcare providers for delivery of the intervention, including awareness of possible patient expectations and the importance of delivering the intervention with high fidelity – including remaining in a facilitating and guiding role.
The Healthy Mind intervention was found to fit the setting in general practice and the patient group. The content and structure of PST was positively viewed by the participants, and PST was delivered with an acceptable level of fidelity. Healthcare providers expressed concerns about time constraints and GPs sometimes struggled to remain in a guiding role. Delivery of PST was considered suitable for both GPs and PNs, however healthcare providers reflected that PNs should be the main deliverers of the intervention. In consideration of these findings, we perceive the Healthy Mind intervention to be feasible, and it is considered appropriate to proceed with a full-scale evaluation study.
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Nickel-induced labial angioedema in a pediatric patient with orthodontic braces: a case report | 58f04dda-4484-4c7c-893a-1a07e9703004 | 11702119 | Dentistry[mh] | Although both are reactions to external substances, Angioedema (AE) and contact dermatitis (CT) have distinct immunological mechanisms. This distinction is crucial in the clinical arena, especially in the complex interaction between these conditions and external allergens, such as nickel (Ni), commonly encountered in everyday materials, including orthodontic devices. AE is primarily associated with Type I hypersensitivity reactions, involving the release of histamine and other chemicals from mast cells and basophils, or with deficiencies or dysfunctions of the C1 Inhibitor (C1INH), leading to uncontrolled release of bradykinin . These mechanisms result in deep tissue swelling, often without itchiness, manifesting rapidly after exposure to the allergen . The complexity increases with angioedema resulting from drug reactions, such as those caused by ACE inhibitors, which decrease the breakdown of bradykinin or hereditary conditions involving specific mutations . In contrast, CT falls under the Type IV hypersensitivity category, which is characterized by a delayed immune response . This process begins when allergens such as nickel make contact with the skin, are processed by antigen-presenting cells (APCs), and are subsequently recognized by T cells, leading to inflammation, rash, and itching that develops over days. The primary mediators in CT are T lymphocytes and cytokines, affecting the epidermal layers of the skin, as opposed to the deeper, subcutaneous and submucosal tissues affected in angioedema . The main differences between these two entities are reported in Table . This case report presents a scenario in which prolonged exposure to nickel from an orthodontic device triggers angioedema, a condition typically associated with more acute allergic reactions or genetic factors rather than delayed-type hypersensitivity reactions like contact dermatitis.
The patient is a 12-year-old girl with a relevant medical history of episodes of labial and lingual angioedema (Fig. ), mainly triggered by the ingestion of specific foods, including chocolate, lentils, nuts, cooked ham, clams, mussels, and certain types of tomato sauce. Additionally, she experienced similar episodes while playing a metal flute, characterized by swelling of the lips and tongue accompanied by limb itching. A switch to a wooden flute resulted in symptom improvement. Complete resolution of symptoms was achieved within 2–3 days with oral betamethasone. Her family history is notable for nickel allergy in her mother and maternal and paternal aunts. Despite the family history of nickel allergy, no one in the family has ever had recurrent episodes of angioedema. She does not have a history of atopic dermatitis, ocular or rhinitis symptoms, or asthma. During episodes of labial angioedema, she had a single-time mild respiratory failure resolved after taking betamethasone; she never had abdominal pain or other associated symptoms. Patch tests confirmed a significant allergy to nickel sulfate , along with positive reactions to paraben mix and methyl(chloro)-isothiazolinone . Routine blood tests, including complete blood count, liver function tests, iron panel, thyroid function tests, and screening for celiac disease, were within normal limits. Her total IgE level was 184 kU/L, with specific IgE tests for cypress, house dust mites (Dermatophagoides pteronyssinus and farinae), dog dander, timothy grass, olive, and wall pellitory being negative. ALEX (Allergy Xplorer) test results were negative, and skin prick tests for inhalants, cocoa, tomato, pork, mussels, peach, LTP, and profilin were negative. Quantitative and qualitative measurements of C1INH were within the normal range. Despite adherence to a nickel avoidance strategy, she continued to experience angioedema episodes, which appear not related to direct nickel contact or substances identified in patch testing. Then, she required further evaluation at our Pediatric Allergology and Immunology Unit for additional insights. This prompted further investigation into less obvious nickel exposure sources, leading to her discovering her orthodontic device. Consultation with her orthodontist confirmed that the orthodontic archwires and brackets contained the nickel-titanium (NiTi) alloy. After the removal of the orthodontic device, she presented a last episode of labial angioedema, which resolved in a few hours without taking any drugs. Complete improvement without treatment was observed in 2 weeks, and no recurrence was observed at 3 and 6 months of patient follow-up.
This case underscores the importance of considering all potential sources of allergen exposure, even those that may not be immediately obvious, such as orthodontic devices, in patients with persistent symptoms despite adherence to known allergen avoidance strategies. The resolution of angioedema symptoms following the removal of the orthodontic device underscores the role of nickel as a trigger, blurring the lines between direct immunological mechanisms and non-immunological mast cell activation by nickel. Nickel sensitization caused by orthodontic treatments has been studied . Although we did not assess our patient’s serum and saliva nickel levels, it’s critical to acknowledge that these concentrations may rise following the application of orthodontic devices . The release of nickel into the intraoral cavity may be caused by saliva corroding the dental devices (brackets, bands, mesh, pads, and arches). Some studies discussed the phenomena of corrosion and wear of nickel in orthodontic devices. Studies on the corrosion of Ni-containing orthodontic alloys elucidate how in vivo exposure affects these archwires’ electrochemical properties and wear rates . The protective nature of oral deposits on in vivo exposed NiTi and stainless steel archwires signifies a lesser corrosive impact than new devices. Yet, an increased wear and friction coefficient was noted, particularly in NiTi wires . In orthodontic treatment, key processes such as galvanic corrosion, metal ion release, and surface degradation of nickel-containing appliances significantly influence their interaction with the oral environment and patient health, as highlighted by studies on fluoride mouthwashes, salivary concentrations of metals over 12 weeks, and galvanic interactions between dental alloys . Furthermore, as discussed in the comprehensive review on Ni leaching and its biological implications, the metallurgical aspects and intraorally reactions underpin our understanding of the potential for increased nickel levels post orthodontic appliance application . This phenomenon, compounded by the invasive nature of appliance removal, may enhance nickel absorption by the oral mucosa, thereby justifying the observed clinical manifestations even after the device’s removal. This underlines that removing the dental device should also be done in total safety in such patients. The oral mucosa’s unique attributes, such as its rich vascularization and non-keratinized epithelium, make it a particular area for allergen absorption, possibly explaining systemic symptoms such as respiratory wheezing observed in some cases. The predominant symptom will result in labial angioedema due, however, to a mechanism of contact, as demonstrated in other case reports . The differentiation between Type I and Type IV hypersensitivity reactions to nickel ( Fig. ) and the potential for nickel to directly activate mast cells on a non-immunological basis highlights the complexity of diagnosing and managing angioedema and contact dermatitis. However, we do not execute prick tests for nickel; this kind of angioedema could be oriented towards type I hypersensitivity, as reported in a case series of patients with contact urticaria to nickel . Although it is possible to measure specific IgE antibodies against nickel, as demonstrated by the detection of specific IgE to nickel-conjugated human serum albumin (Ni-HSA) and nickel-conjugated exchange resin (Ni-resin) in patients with hard metal asthma , we were unfortunately unable to perform these tests in our case. In this case clinical history and symptom correlation remain key. However, other studies have shown the co-presence of Type I and IV hypersensitivity to nickel . It has been postulated that nickel can act as an activator of mast cells on a non-immunological basis : the release of histamine is triggered directly by the nickel ions without the involvement of IgE. These reactions mimic true IgE-mediated responses, they occur via different pathways. This could explain the occurrence of symptoms like angioedema in patients where IgE to nickel is not detectable, but mast cell degranulation still occurs. In Type IV hypersensitivity nickel ions come into contact with the skin, they act as haptens, forming a new antigenic structure: this model is recognized as foreign by the immune system. Langerhans cells in the skin, capture this hapten-protein complex; then they process and present the complex to CD4 + T cells in the lymph nodes, initiating a cascade of immune activation. During the sensitization, these T cells become specific to the nickel antigen. When the individual is re-exposed to nickel, these memory T cells are rapidly recruited to the site of contact and after activation, they release various cytokines, including IFN-γ and TNF-α, which are central to the Th1 response and mediate the inflammatory reaction. The Th17, also plays a role, producing IL-17, which helps recruit neutrophils to the site, sustaining inflammation and contributing to the tissue damage associated with allergic contact dermatitis. The hallmark of Type IV hypersensitivity is the chronic inflammation driven by these immune cells and cytokines, which manifests clinically as redness, itching, swelling, and vesicle formation at the site of contact. The clinical presentation of angioedema that we observed could potentially be interpreted as a form of mucositis. While angioedema typically involves the swelling of deeper layers of skin and mucosa, in this case, the swelling might represent a contact mucositis induced by direct contact with the metal apparatus: the device was exclusively touching the mucosal surfaces, leading to a localized inflammatory reaction. Although clinically similar to angioedema in its appearance, the underlying process may be more in line with mucosal irritation or inflammation, hence introducing the term “contact mucositis” to better describe this phenomenon. This inflammatory response the complexity of nickel-induced hypersensitivity. While Type IV hypersensitivity is the most recognized mechanism of nickel allergy, it’s essential to consider that nickel can also induce Type I hypersensitivity reactions, although this is rare. In such cases, the immunological mechanism shifts from T-cell mediated to IgE-mediated, resulting in mast cell degranulation and immediate hypersensitivity reactions, such as urticaria and angioedema. In expanding this understanding, it becomes crucial to recognize the genetic predispositions that may make certain individuals more susceptible to nickel allergy. For example, certain HLA alleles have been associated with increased sensitivity to nickel, particularly in individuals with repeated exposure, whether through jewelry, implants, or occupational contact . In our case, there was a strong family history of nickel allergy: unfortunately, one of the limitations in this study was the inability to perform HLA typing in both the patient and her family. Other reactions related to nickel allergy include a range of both localized and systemic manifestations. Beyond the classic presentation of allergic contact dermatitis, individuals sensitized to nickel may experience respiratory symptoms, such as allergic rhinitis or asthma-like symptoms, particularly in occupational settings where nickel exposure occurs through inhalation . Additionally, oral allergy symptoms, such as swelling of the lips or oral mucosa (mucositis), can occur after contact with or ingestion of nickel-containing foods or materials . Ocular symptoms, such as itching or swelling around the eyes, are often observed, particularly with the use of certain cosmetics containing nickel: these reactions can occur due to the direct contact of nickel-containing products triggering a localized allergic response . Systemic Nickel Allergy Syndrome (SNAS) is a broader and more complex manifestation of nickel allergy . This condition involves systemic symptoms following the ingestion of nickel through food, water, or environmental exposure. SNAS is characterized by gastrointestinal discomfort (such as nausea, bloating, and abdominal pain), generalized eczema, fatigue, and headaches. The mechanism of SNAS likely involves a combination of Type I and Type IV hypersensitivity reactions, along with systemic absorption of nickel that triggers widespread immune responses, contributing to the diverse range of symptoms. This concept challenges the idea that localized symptoms are purely driven by a single type of hypersensitivity. The braces release small amounts of nickel, which could be absorbed into the systemic circulation. This provides a plausible explanation for the complex reaction observed, going beyond the simple contact of nickel with the oral mucosa. Thus, the complexity of nickel-induced reactions, particularly in individuals with a high degree of sensitivity, requires us to consider that what presents as a local reaction may, in fact, involve systemic absorption and a combination of immune responses, extending the implications of nickel exposure beyond the site of contact. SNAS, along with other systemic and localized reactions, underscores the complexity of nickel allergy and the broad spectrum of clinical manifestations that can result from exposure to this metal allergen. The implications of this case extend beyond clinical diagnosis to include considerations in dental and orthodontic material selection, underscoring the importance of screening for metal allergies before device placement and the potential benefits of alternative materials free from common allergens like nickel . This case emphasizes the necessity of considering all potential sources of allergen exposure, particularly in environments as intricate as the oral cavity, where the combination of saliva, microbial presence, and dietary acids can accelerate metal corrosion and increase allergen release. Moreover, the differentiation between Type I and Type IV hypersensitivity reactions to nickel and the potential for nickel to directly activate mast cells on a non-immunological basis highlights the complexity of diagnosing and managing angioedema and contact dermatitis cases. The implications of this case extend beyond clinical diagnosis to include considerations in dental and orthodontic material selection, underscoring the importance of screening for metal allergies before device placement and the potential benefits of alternative materials free from common allergens like nickel.
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Potential Safety Issues with Use of Sodium-Glucose Cotransporter 2 Inhibitors, Particularly in People with Type 2 Diabetes and Chronic Kidney Disease | e43bd5ee-ba47-4e88-917b-0586ea75c535 | 7582030 | Patient Education as Topic[mh] |
Diabetes is the leading cause of chronic kidney disease (CKD) , and the coexistence of both conditions places an individual at high risk of cardiovascular disease and death . Sodium-glucose cotransporter 2 (SGLT2) inhibitors inhibit proximal tubular glucose reabsorption, leading to glycosuria . These drugs have important cardiovascular and renal benefits [ – ], leading one expert to refer to SGLT2 inhibitors as “cardiorenal risk reducing agents that have glucose lowering as a side effect” . Furthermore, the cardiovascular and renal benefits of SGLT2 inhibitors appear to be largely independent of their glycaemic effects [ , – ]. This point is pertinent to the use of these drugs in people with type 2 diabetes and CKD where glycosuria secondary to SGLT2 inhibition is reduced, resulting in potentially limited anti-hyperglycaemic efficacy [ – ]. Indeed, in two heart failure trials (DAPA-HF and EMPEROR-Reduced) and a CKD trial (DAPA-CKD) where the primary endpoint was met, the effect of the SGLT2 inhibitor on the primary outcome was consistent in participants irrespective of the presence or absence of diabetes [ , , ]. There are a multitude of proposed mechanisms for the cardioprotective properties of SGLT2 inhibitors, including natriuresis and osmotic diuresis, inhibition of the sodium-hydrogen exchanger in the myocardium, potential use of ketone bodies for cardiac metabolism, and reduced cardiac fibrosis and inflammation . These results suggest that the benefits may be independent of effects on glycaemia. Given the use of these drugs by not only endocrinologists and primary care physicians, but also nephrologists and cardiologists, clinicians need to become familiar with the physiology, efficacy, and safety of SGLT2 inhibitors [ , , ]. Indeed, SGLT2 inhibitors are associated with a number of adverse effects, including diabetic ketoacidosis (DKA), which is potentially life-threatening. Furthermore, adverse effects can occur very quickly. There have been many reviews exploring the efficacy of these agents in different population groups. Hence, this article examines the potential safety issues associated with the use of SGLT2 inhibitors, with a particular focus on the safety of these drugs in people with type 2 diabetes and CKD. We highlight measures that clinicians can implement to minimise the risk of adverse effects, including DKA, which is of particular relevance during the current coronavirus disease 2019 (COVID-19) pandemic.
The cardiovascular, renal and heart failure outcome trials to date have differed with respect to inclusion and exclusion criteria, including estimated glomerular filtration rate (eGFR) cut-offs [ , – , , , ]. Importantly, there is limited available data specifically about SGLT2 inhibitor use in patients with severe CKD. Subgroup analyses from the EMPA-REG OUTCOME, CANVAS, and DECLARE-TIMI 58 trials found similar adverse event profiles with respect to specific SGLT2 inhibitors among participants with different baseline eGFR levels (Table ) [ – ]. In a subgroup analysis of the CREDENCE trial, severe adverse events were consistent among screening eGFR categories. There was a significant interaction test for volume depletion, with a higher risk with canagliflozin apparent in participants with screening eGFR 30 to < 45, but not eGFRs 45 to < 60 or 60 to < 90 mL/min/1.73 m 2 (Table ) . Of note, empagliflozin, dapagliflozin, canagliflozin, and ertugliflozin exposure increases with advancing renal impairment; however, the area under the concentration–time curve does not exceed by twofold that reported in subjects with normal renal function [ – ]. Canagliflozin is the only one of these four SGLT2 inhibitors for which use of the lower dose (100 mg once daily) is recommended for patients with renal impairment (specifically eGFR 30 to < 60 mL/min/1.73 m 2 ) . However, in Australia, empagliflozin, dapagliflozin, and ertugliflozin are contraindicated in patients with eGFR persistently < 45 mL/min/1.73 m 2 , largely due to limited anti-hyperglycaemic efficacy [ – ].
Diabetes, particularly with poor control and glycosuria, is a known risk factor for genital infection . Diabetes may suppress the immune response to infection . Glucose present on the genitalia due to SGLT2 inhibition is believed to aid growth and adherence of yeast and impair the local immune response . A meta-analysis of randomised controlled trials (RCTs) and two large population-based studies have demonstrated an approximate threefold increase in risk of genital infection with SGLT2 inhibitor use compared with placebo or other diabetes drug classes [ – ]. In the CREDENCE trial, the event rate for genital mycotic infection in the canagliflozin versus placebo groups for females was 12.6 versus 6.1 per 1000 person-years, and for men, 8.4 versus 0.9 per 1000 person-years . A study of two large cohorts of commercially insured patients in the United States found that the elevated risk of genital infections was apparent within the first month of SGLT2 inhibitor treatment and remained elevated during the course of treatment . Furthermore, the risk of genital infections with SGLT2 inhibitor use was greater in the subgroup of patients aged 60 years and over. History of prior genital infection (especially recent history) is a clear risk factor for the development of genital infection during SGLT2 inhibitor treatment . Toyama et al. conducted a meta-analysis of RCTs of SGLT2 inhibitors in patients with type 2 diabetes and CKD (defined as eGFR < 60 mL/min/1.73 m 2 ) and found an approximate threefold increase in the risk of genital infections . This suggests that the increase in the risk of genital infections in patients with CKD is similar to that in the non-CKD population. Most genital infections are mild to moderate in severity and are responsive to topical antifungals or a single dose of fluconazole . These infections do not necessitate cessation of the agent . Clinicians should recommend good perineal hygiene to patients . In 2018, the Food and Drug Administration (FDA) issued a warning about the risk of necrotising fasciitis of the perineum (Fournier’s gangrene) with SGLT2 inhibitor treatment . There were 55 cases of Fournier’s gangrene requiring surgical debridement associated with SGLT2 inhibitor use reported to the FDA Adverse Event Reporting System (FAERS) between March 2013 and January 2019 . All patients were severely ill, at least 25 patients needed more than one surgery, and three patients died. A number of patients had complicating DKA, sepsis, and/or acute kidney injury (AKI). The time between initiation of SGLT2 inhibitor therapy and infection was variable—5 days to 49 months. Large RCTs have not demonstrated an increased risk of Fournier’s gangrene with SGLT2 inhibitor treatment. In the DECLARE-TIMI 58 trial, one patient in the dapagliflozin group compared with five patients in the placebo group experienced Fournier’s gangrene . However, RCTs are not designed or powered to demonstrate or refute an increased risk of extremely rare events such as Fournier’s gangrene. The documented case reports submitted to regulatory and surveillance agencies need to be carefully considered.
In 2015, the FDA issued a warning about the risk of serious urinary tract infections (UTIs) with SGLT2 inhibitor use due to 19 cases of urosepsis and pyelonephritis reported over an 18-month period . In contrast, SGLT2 inhibitors have generally not been associated with an elevated risk of UTIs in large meta-analyses and population-based studies [ , , , ]. One of the four cardiovascular outcome trials to date, however, has demonstrated a significant increase in risk of UTIs with SGLT2 inhibitor therapy; in the VERTIS CV trial approximately 12% versus 10% of participants randomised to ertugliflozin and placebo, respectively, experienced a UTI . In the CREDENCE trial, there was no significant difference in the rate of UTIs between the canagliflozin and placebo groups . The exact rate of pyelonephritis and urosepsis was not reported. Whether there are differences in the risk of UTI based on the type of SGLT2 inhibitor is yet to be established. With respect to why SGLT2 inhibition may not increase the risk of UTIs despite causing glycosuria, Fralick and MacFadden have hypothesised that diuresis and polyuria secondary to SGLT2 inhibition counters potential bacterial growth due to glycosuria and/or prevents bacterial ascension of the urinary tract . Severe CKD may lead to reduced urine output, and glycosuria secondary to SGLT2 inhibition is reduced [ – ]. The influence of these factors on the risk of UTIs is uncertain as data regarding SGLT2 inhibitor treatment in patients with stage 4 and 5 CKD (eGFR 15–29 mL/min/1.73 m 2 and < 15 mL/min/1.73 m 2 or requiring dialysis, respectively) are limited. Also unclear is the risk of UTIs in higher-risk populations such as people with urinary tract structural or functional abnormalities or people who are immunosuppressed. With regard to post-transplant diabetes mellitus in renal transplant recipients, in one RCT ( n = 49 patients), which compared empagliflozin or placebo treatment for 24 weeks, three patients in both the empagliflozin and placebo groups experienced a UTI . However, two patients in the empagliflozin group had to discontinue treatment—one because of urosepsis and one because of repeated UTIs. The patient who experienced urosepsis had a history of recurrent UTIs, and clinicians should be cautious when considering prescribing SGLT2 inhibitors to patients with a history of recurrent UTIs.
Given the insulin-independent mechanism of action of SGLT2 inhibitors, these agents are not associated with an increased risk of hypoglycaemia [ , , , ]. These drugs lower plasma glucose by inducing glycosuria, resulting in a reduction in plasma insulin concentration and an increase in plasma glucagon concentration, leading to an increase in endogenous glucose production . The absence of hypoglycaemia risk was clearly seen in the DAPA-HF trial, where 55% of patients did not have diabetes . However, if a patient is prescribed insulin and/or a sulfonylurea, the doses of these medications may need to be reduced, as SGLT2 inhibitors reduce glycated haemoglobin (HbA1c) by 0.6–0.9% (Table ) . With regard to adjusting concomitant diabetes medications in patients with moderate to severe CKD, clinicians should note that SGLT2 inhibitors have limited anti-hyperglycaemic efficacy, due to reduced glycosuria .
In 2015/2016, the FDA issued a warning about the risk of AKI with canagliflozin and dapagliflozin based on 101 cases over an approximate 2.5-year period, some requiring hospitalisation and dialysis . In approximately half of the cases, AKI occurred within 1 month of SGLT2 inhibitor initiation. CREDENCE and the cardiovascular outcome trials have clearly demonstrated the important renoprotective effects of SGLT2 inhibitors [ , , ]. In CREDENCE and the cardiovascular outcome trials (EMPA-REG OUTCOME, CANVAS Program, and DECLARE-TIMI 58), there was a 25% lower risk of AKI with SGLT2 inhibitor treatment compared with placebo . There is often a mild acute decrease in eGFR with SGLT2 inhibitor initiation that is reversible on treatment cessation—the reduction in the CREDENCE trial at 3 weeks was −3.7 mL/min/1.73 m 2 . In the trial, the mean change in eGFR slope was lower in the canagliflozin group compared with the placebo group (−3.19 vs −4.71 mL/min/1.73 m 2 per year) . The mild reduction in eGFR with SGLT2 inhibitor initiation does not represent AKI. This effect is thought to be due to increased tubular sodium delivery to the macula densa activating tubuloglomerular feedback, resulting in afferent arteriolar vasoconstriction, which is protective in the long-term because of the reduction in intraglomerular pressure . This theory is partly based on data in young adults with type 1 diabetes and hyperfiltration . However, the recent Renoprotective Effects of Dapagliflozin in Type 2 Diabetes trial questioned this theory . In patients with type 2 diabetes without overt nephropathy, 12 weeks of dapagliflozin reduced GFR, filtration fraction, and intraglomerular pressure without increasing renal vascular resistance—suggesting that the acute eGFR decline is due to efferent arteriolar vasodilation rather than afferent arteriolar vasoconstriction . In the EMPA-REG OUTCOME trial, the acute dip in eGFR with empagliflozin treatment was greater in users of an angiotensin converting enzyme (ACE) inhibitor, an angiotensin receptor blocker (ARB), or any diuretic compared with non-users . However, in users of these medications, adding empagliflozin did not increase the risk of AKI compared with adding placebo . SGLT2 inhibitors should not be initiated in patients who are hypovolaemic and/or hypotensive, because this could contribute to AKI. Further, patients prescribed loop and/or thiazide diuretics may need dose reduction of these medications to prevent volume depletion (Table ) . Patients should be instructed when acutely unwell (for example, vomiting, diarrhoea, and reduced oral intake) to withhold their SGLT2 inhibitor (part of a sick day management plan) . There has been a recent reported case of AKI secondary to osmotic nephrosis attributed to recent prescription of canagliflozin, postulated to be due to increased tubular osmotic pressure secondary to glucose reabsorption inhibition . The authors of this report recommend consideration of a kidney biopsy in cases of prolonged AKI despite SGLT2 inhibitor discontinuation. Furthermore, the issue of possible hypoxic medullary injury secondary to SGLT2 inhibition, due to increased distal natriuresis augmenting transport workload in the medulla and oxygen consumption, has been raised by Heyman et al. . These authors caution against concomitant administration of agents that could worsen medullary hypoxia, and recommend cessation of SGLT2 inhibitors prior to radiocontrast studies.
DKA is a rare but potentially life-threatening adverse effect of SGLT2 inhibitor therapy , estimated to occur in approximately one in 1000 SGLT2 users who have type 2 diabetes (although the precise incidence is unknown) . The event rate of SGLT2 inhibitor-associated DKA in the CREDENCE trial was higher compared with the cardiovascular outcome trials (2.2 vs < 1 event per 1000 patient-years) . This may be, at least in part, related to the higher use of insulin at baseline in CREDENCE; all except one of the 12 patients in CREDENCE who developed DKA had concomitant insulin treatment [ , – ]. In contrast, there were no reported DKA events in patients randomised to dapagliflozin in the DAPA-CKD trial; however, this trial included patients with and without diabetes . The higher risk of DKA in insulin-treated patients is pertinent to nephrologists as patients with advanced CKD have relatively limited therapeutic options for the management of type 2 diabetes. SGLT2 inhibitor-associated DKA is commonly referred to as “euglycaemic” DKA as the degree of hyperglycaemia is often lower than expected due to glycosuria . However, in a review of 105 cases of SGLT2 inhibitor-associated DKA, 35% of cases had an admission plasma glucose concentration < 200 mg/dL (11.1 mmol/L) . A more precise term for this adverse effect is “DKA with lower-than-anticipated glucose levels”, as recommended by the American Association of Clinical Endocrinologists and American College of Endocrinology . The duration of SGLT2 inhibitor treatment prior to the onset of DKA is highly variable (0.3–420 days) . With regard to the pathophysiology of DKA, SGLT2 inhibitor use leads to a reduction in plasma insulin concentration and an increase in plasma glucagon concentration . Additionally, free fatty acid suppression post-meal is impaired . This decrease in the insulin-to-glucagon ratio and increase in free fatty acids promotes ketogenesis . SGLT2 inhibitor treatment increases plasma ketone levels, and an elevated ketone level does not necessarily indicate DKA [ – ]. SGLT2 inhibitor-associated DKA most frequently occurs in patients with one or more additional risk factor(s) for insulin deficiency and/or ketogenesis (Table ) [ – ]. In a recent Australian retrospective cohort study of SGLT2 inhibitor-associated DKA cases, 22% of patients with presumed type 2 diabetes were subsequently diagnosed as having type 1 diabetes . Fourteen of 37 cases of DKA related to SGLT2 inhibition occurred during hospital admission. Eleven of the 14 patients were fasting due to surgery, and SGLT2 inhibitor therapy was continued during admission in six of these cases. Eleven of the 14 inpatients were on insulin treatment prior to hospitalisation, and insulin was generally ceased prior to the onset of DKA. These findings highlight the need to employ specific strategies to reduce the risk of DKA, including educating patients to temporarily withhold their SGLT2 inhibitor when acutely unwell with reduced oral intake (Table ) [ , , ]. Treatment of SGLT2 inhibitor-associated DKA involves rehydration and an insulin-dextrose infusion . A higher rate of intravenous dextrose (10–20%) is often needed to enable sufficient dosage of insulin for resolution of ketoacidosis . An endocrinologist should be involved in the management of DKA and decisions regarding subsequent diabetes pharmacotherapy.
The CANVAS Program is the only cardiovascular outcome trial that has shown an increased risk of lower limb amputation with SGLT2 inhibitor therapy compared with placebo [ , , ]. Approximately six versus three participants per 1000 person-years in the canagliflozin versus placebo groups experienced lower limb amputation; 71% of amputations occurred at the level of the toe or metatarsal . Multivariate modelling revealed a number of baseline characteristics that were significantly associated with amputation during follow-up, including male sex, prior amputation, peripheral vascular disease, neuropathy, albuminuria, and higher HbA1c . However, the effect of canagliflozin on amputation risk did not vary according to any baseline characteristic or dose of canagliflozin (100 or 300 mg daily) . Interestingly, there was no difference in the risk of amputation between the canagliflozin and placebo groups in the CREDENCE trial . During the CREDENCE trial, there was a protocol amendment asking investigators to examine patients’ feet and temporarily withhold the study drug if there was any active condition present that might lead to amputation . A cohort study using nationwide health and administrative registers in Sweden and Denmark found that compared with new users of glucagon-like peptide 1 (GLP-1) receptor agonists, new users of SGLT2 inhibitors had an increased risk of lower limb amputation (incidence rate 2.7 vs 1.1 events per 1000 person-years, hazard ratio 2.3) . Ninety-nine per cent of SGLT2 inhibitor users were taking dapagliflozin (61%) or empagliflozin (38%). These results contrast with safety results of the EMPA-REG OUTCOME, DECLARE-TIMI 58, and DAPA-HF trials [ , , ]. In summary, whether there is a definite increase in risk of lower limb amputation with canagliflozin treatment is unclear. Furthermore, the mechanisms underlying such a potential adverse effect are unknown. Postulated mechanisms include volume depletion secondary to diuresis, and an effect on calcium, magnesium, and vitamin D metabolism that may impair foot ulcer healing . Based on available evidence to date, we recommend that clinicians provide education to patients about preventive foot care and perform regular foot screening, as well as avoiding canagliflozin in patients with an acute heightened risk of amputation (as per the CREDENCE protocol—history of amputation within past 12 months, active ulcer, osteomyelitis, gangrene, or critical leg ischaemia within 6 months) .
Blau et al. examined the acute effect of canagliflozin on mineral metabolism in healthy adults . Subjects received canagliflozin 300 mg daily or placebo for 5 days, and later crossed over to the other treatment. Canagliflozin administration rapidly increased serum phosphorus, corresponding to an increase in urinary phosphorus reabsorption. Additionally, canagliflozin treatment increased plasma fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) and reduced 1,25-dihydroxyvitamin D. The differences in mean serum phosphorus and plasma FGF23 between the canagliflozin and placebo groups were no longer significant by day 5. In contrast, differences in 1,25-dihydroxyvitamin D and PTH were still significant at this time point. There was no significant difference in serum calcium, but there was a significant decrease in urinary calcium excretion on day 4. de Jong et al. performed a post hoc analysis of the IMPROVE trial, a randomised, placebo-controlled, crossover trial involving dapagliflozin in patients with type 2 diabetes and albuminuric CKD (eGFR ≥ 45 mL/min/1.73 m 2 ) . Compared with the start of treatment, 6 weeks of dapagliflozin increased serum phosphorus (+ 11%), PTH (+ 15%), and FGF23 (+ 20%) and decreased 1,25-dihydroxyvitamin D (− 19%). Importantly, these changes did not correlate with change in eGFR. The increase in serum phosphorus with SGLT2 inhibition is believed to be due to increased sodium in the proximal tubule driving sodium-dependent phosphate reabsorption . This is postulated to increase FGF23, which decreases 1,25-dihydroxyvitamin D, leading to an increase in PTH . In the study by Blau et al., the increase in serum phosphorus correlated with urinary sodium excretion, but not urinary glucose excretion . In severe CKD, the effects of SGLT2 inhibition on urinary glucose and presumably also urinary sodium excretion are attenuated, perhaps resulting in a less marked effect on serum phosphorus. However, more data are needed with regard to patients with stage 4 CKD, where control of hyperphosphataemia can be difficult. Changes to mineral metabolism secondary to SGLT2 inhibition may be relevant to the heightened risk of fracture evident in the CANVAS Program (15.4 vs 11.9 participants with fracture randomised to canagliflozin vs placebo per 1000 patient-years) , although this is the only very large RCT to date with a fracture safety signal. Meta-analyses of RCTs of SGLT2 inhibitors have not demonstrated an increased risk of fractures compared with placebo . The increased risk of fracture with canagliflozin was only seen in one of the two trials that compose the CANVAS Program (CANVAS, not CANVAS-R). Furthermore, there was no difference in risk of fracture between the canagliflozin and placebo groups in CREDENCE . The mean follow-up was longer in CANVAS compared with CANVAS-R (5.7 vs 2.1 years). The median follow-up of CREDENCE was 2.6 years. Interestingly, in a fracture analysis of CANVAS, there was no difference between canagliflozin-treated patients with or without fractures with respect to post-randomisation per cent changes from baseline in serum phosphate . There was a similar fracture incidence in the canagliflozin 100 mg and 300 mg groups. A possible relationship between falls (potentially caused by volume depletion) and fractures cannot be excluded. An RCT of dapagliflozin in patients with stage 3 CKD demonstrated a higher risk of fracture with the SGLT2 inhibitor compared with placebo; seven of the 13 participants randomised to dapagliflozin who sustained a fracture exhibited orthostatic hypotension or had a history of diabetic neuropathy . In summary, meta-analyses and population-based studies of SGLT2 inhibitor therapy have largely not demonstrated an increased risk of fracture [ , , , , ]. However, given the changes in mineral metabolism and the results of the CANVAS Program described, longer-term data are needed with respect to risk of fracture. This issue is of relevance to the population of patients with CKD, who have or are at risk of CKD–mineral and bone disorder (CKD-MBD).
Patients with comorbidities including diabetes, older age and hypertension are at risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection . Therefore, it is important that clinicians inform patients taking SGLT2 inhibitors that they should temporarily stop their SGLT2 inhibitor when acutely unwell with reduced oral intake (as part of a sick day management plan), and that patients understand this advice, to try to reduce the risk of DKA and dehydration potentially leading to AKI . Patients admitted to hospital with SARS-CoV-2 infection should have their SGLT2 inhibitor discontinued . In a systematic literature review of cases involving patients with COVID-19 and DKA or combined DKA and hyperglycaemic hyperosmolar syndrome, at least seven of the 110 patients were taking SGLT2 inhibitors. Self-monitoring of plasma ketone levels when DKA is suspected has been recommended , although there are cost and possibly test kit availability issues. Additionally, patients must understand how to correctly use a meter that measures plasma ketone levels and be able to seek advice, preferably from a diabetes educator or endocrinologist, as to what actions to take based on their symptoms, plasma glucose, and ketone level.
SGLT2 inhibitors have clinically important cardio-renal benefits, especially for people with type 2 diabetes and CKD, who are at high risk of cardiovascular disease and end-stage kidney disease. Clinicians need to be aware of the potential safety issues with SGLT2 inhibitor therapy in order to try to minimise the occurrence of adverse events, as well as to detect and intervene early if these events occur. Our understanding regarding the safety of these agents is evolving, and longer-term data will provide greater knowledge. Additionally, there is a need for greater specific data with respect to people with severe CKD.
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Maternal mental health: a key area for future research among women with congenital heart disease | 58395ae9-9863-47b6-8a26-860ae0094781 | 10582993 | Internal Medicine[mh] | The recently published national priorities for research in congenital heart disease (CHD) among adults, established through the James Lind Alliance (JLA) Priority Setting Partnership, include both mental health and maternal health among the top 10 priorities : #3—What is the impact of living with CHD on mental health in adults and how can this be improved through access to psychological support and other therapies? #5—What are the risks and limitations associated with pregnancy, childbirth, and motherhood for women with CHD, and what information and support is available? Having recently completed a policy impact project focusing on the mental health of women with CHD during pregnancy and the postnatal period, we welcome the inclusion of both areas as priorities, and suggest that future research focus on the considerable overlap between them. In particular, we argue that mental health should be considered a core component of the ‘risks and limitations associated with pregnancy, childbirth and motherhood’ in priority 5. Research attention to the experiences of pregnant women and new mothers with CHD is crucial. One per cent of all children have CHD, with 13 born in the UK every day. In recent decades, with advances in medical and surgical knowledge and technology, 97% of children with CHD have reached adulthood, albeit with increased risk for cardiac morbidity. However, even years after successful surgical repair and treatment, many adults with CHD face lifelong psychosocial difficulties, with high rates of depression and anxiety, including anxieties about being ‘normal’. The haemodynamic changes of pregnancy and the immediate postnatal period pose physiological challenges to women with CHD. As such, pregnant women with CHD are classified as high risk, and often experience medicalised pregnancies and managed births. For women with CHD, particularly those who live otherwise healthy lives, pregnancies may lead to emotional distress concerning one’s identity and future, the immediate risk to cardiac function and the potential for life-threatening complications for both mother and baby. Prolonged exposure to distress, in turn, may have cardiovascular consequences: a recent scientific statement from the American Heart Association suggested that, even within the general population, accumulated psychological stress places women at risk for cardiovascular morbidity in pregnancy, such that prepregnancy interventions should include a stress-reduction component. As seen in a previous investigation of the healthcare priorities of pregnant women with heart disease, both congenital and acquired, the mental health impact of pregnancy is a key concern for women. However, although women with heart disease prioritise mental health outcomes, the research literature emphasises the clinical outcomes of pregnancies complicated by heart disease, suggesting a gap between extant research approaches and women’s experiences. Moreover, while both the research literature and adult congenital heart disease (ACHD) clinical services tend to focus on the risks inherent in pregnancy, a successful pregnancy and transition to motherhood can be psychosocially normalising events which can boost the mental health of women with CHD. Between January and March 2022, we undertook a policy impact project which brought together researchers, clinicians and women with lived experience of CHD and pregnancy to inform service improvement in two National Health Service (NHS) Trusts (University Hospitals Birmingham and Birmingham Women’s and Children’s) and develop recommendations that could be applied more broadly. This project was the first to turn urgent attention to the mental health needs of women with CHD during pregnancy and the postnatal period. Group discussions with clinicians and women with lived experience identified (1) mental health needs and priorities specific to women with CHD; (2) barriers to accessing mental healthcare among women with CHD during and following pregnancy; and (3) recommendations for service improvement. We highlight two key findings from these discussions, showing that both clinical and research attention to the overlaps between mental and maternal health in ACHD are crucial. Anxiety: a common experience for women with CHD in pregnancy and the postnatal period Anxiety was the mental health challenge mentioned most frequently in our multidisciplinary project discussions, by both clinicians and women with lived experience. Importantly, the discussions established that anxiety developed during pregnancy and the postnatal period may lead to further anxiety over time, with women feeling anxious about their future health, particularly in relation to parenting. Anxiety also extends to women’s immediate families, especially their partners. Our group identified anxiety as related to several factors, many of which arise from patient experiences of maternity care in both community and hospital settings. Emphasis on risks (to both mother and baby) in ACHD discussions ACHD and obstetric teams emphasise risks to the baby’s heart as well as risks to the mother’s cardiovascular function. Women with lived experience identified this discourse of immediate and potentially life-altering risk as a key trigger of anxiety, which also places an emotional strain on the mother’s partner and wider family. Additionally, both women with lived experience and clinicians said that, given the emphasis on risks within ACHD maternity services, infrequent contact with obstetric care before 20 weeks of pregnancy can be anxiety inducing. Our group also discussed the ACHD emphasis on risk beyond the context of pregnancy. ACHD teams tend to emphasise possible complications and limitations on what patients can do physically. This direct, honest and realistic communication style can be beneficial. However, without providing reassurance or a positive counter-focus on what the body can do, ACHD discussions can be anxiety-provoking, and some women live in fear of the future, even avoiding exercise for fear of straining their hearts. Lack of CHD understanding across hospital departments When women with CHD encounter hospital departments beyond specialist ACHD services—such as the emergency department, delivery suite or postnatal ward—they may find that non-specialist clinicians minimise or dismiss their concerns. Lack of CHD understanding in primary care Women with CHD may find it difficult to rely on antenatal and postnatal support in the community, because many midwives and health visitors are uninformed about CHD and sometimes give advice that conflicts with that of the ACHD team. The social isolation of living with CHD The experience of growing up with CHD can be socially isolating, and this sense of isolation often persists into adulthood. Women with CHD may feel their experiences of pregnancy cannot be understood by friends, relatives, or other pregnant women (eg, National Childbirth Trust (NCT) group members). While support may be found through ACHD groups on social media, some of the experiences related by social media group members may be distressing. In addition, a small number of women with CHD experience serious complications, requiring surgical or medical interventions in pregnancy. These experiences add a layer of trauma to an already anxious time, and can lead to a greater sense of social isolation, given how rare they are. While clinical teams are focused on delivering the best cardiac care, the deep psychological impact on the woman and her partner is left unaddressed, both in pregnancy and in the postnatal period. Barriers to help-seeking: timing and availability of resources Both the clinicians and the women with lived experience emphasised that it is important to provide mental health support to women with CHD during pregnancy and the postnatal period. However, several barriers prevent patients with CHD from seeking and receiving appropriate support. Specialist mental healthcare for the CHD population is difficult to obtain Women with lived experience and clinicians felt that psychological support would be most effective if delivered by a therapist familiar with CHD-related issues. People with CHD have unique lifelong experiences that necessitate a psychologist or therapist with specialist knowledge. However, this is difficult to obtain without the help of the ACHD service, and patients must resort either to seeking treatment privately, facing long waits in the community, or forgoing mental healthcare entirely. Mental health support is made available when women are unable to use it Mental health support may be offered during the immediate postnatal period, but not later. This is often a time of great upheaval for women with CHD, who are overwhelmed by recovering from childbirth and caring for a newborn baby, and who may be burdened by frequent hospital visits related to their heart condition in the first few weeks following the birth.
Anxiety was the mental health challenge mentioned most frequently in our multidisciplinary project discussions, by both clinicians and women with lived experience. Importantly, the discussions established that anxiety developed during pregnancy and the postnatal period may lead to further anxiety over time, with women feeling anxious about their future health, particularly in relation to parenting. Anxiety also extends to women’s immediate families, especially their partners. Our group identified anxiety as related to several factors, many of which arise from patient experiences of maternity care in both community and hospital settings. Emphasis on risks (to both mother and baby) in ACHD discussions ACHD and obstetric teams emphasise risks to the baby’s heart as well as risks to the mother’s cardiovascular function. Women with lived experience identified this discourse of immediate and potentially life-altering risk as a key trigger of anxiety, which also places an emotional strain on the mother’s partner and wider family. Additionally, both women with lived experience and clinicians said that, given the emphasis on risks within ACHD maternity services, infrequent contact with obstetric care before 20 weeks of pregnancy can be anxiety inducing. Our group also discussed the ACHD emphasis on risk beyond the context of pregnancy. ACHD teams tend to emphasise possible complications and limitations on what patients can do physically. This direct, honest and realistic communication style can be beneficial. However, without providing reassurance or a positive counter-focus on what the body can do, ACHD discussions can be anxiety-provoking, and some women live in fear of the future, even avoiding exercise for fear of straining their hearts. Lack of CHD understanding across hospital departments When women with CHD encounter hospital departments beyond specialist ACHD services—such as the emergency department, delivery suite or postnatal ward—they may find that non-specialist clinicians minimise or dismiss their concerns. Lack of CHD understanding in primary care Women with CHD may find it difficult to rely on antenatal and postnatal support in the community, because many midwives and health visitors are uninformed about CHD and sometimes give advice that conflicts with that of the ACHD team. The social isolation of living with CHD The experience of growing up with CHD can be socially isolating, and this sense of isolation often persists into adulthood. Women with CHD may feel their experiences of pregnancy cannot be understood by friends, relatives, or other pregnant women (eg, National Childbirth Trust (NCT) group members). While support may be found through ACHD groups on social media, some of the experiences related by social media group members may be distressing. In addition, a small number of women with CHD experience serious complications, requiring surgical or medical interventions in pregnancy. These experiences add a layer of trauma to an already anxious time, and can lead to a greater sense of social isolation, given how rare they are. While clinical teams are focused on delivering the best cardiac care, the deep psychological impact on the woman and her partner is left unaddressed, both in pregnancy and in the postnatal period.
ACHD and obstetric teams emphasise risks to the baby’s heart as well as risks to the mother’s cardiovascular function. Women with lived experience identified this discourse of immediate and potentially life-altering risk as a key trigger of anxiety, which also places an emotional strain on the mother’s partner and wider family. Additionally, both women with lived experience and clinicians said that, given the emphasis on risks within ACHD maternity services, infrequent contact with obstetric care before 20 weeks of pregnancy can be anxiety inducing. Our group also discussed the ACHD emphasis on risk beyond the context of pregnancy. ACHD teams tend to emphasise possible complications and limitations on what patients can do physically. This direct, honest and realistic communication style can be beneficial. However, without providing reassurance or a positive counter-focus on what the body can do, ACHD discussions can be anxiety-provoking, and some women live in fear of the future, even avoiding exercise for fear of straining their hearts.
When women with CHD encounter hospital departments beyond specialist ACHD services—such as the emergency department, delivery suite or postnatal ward—they may find that non-specialist clinicians minimise or dismiss their concerns.
Women with CHD may find it difficult to rely on antenatal and postnatal support in the community, because many midwives and health visitors are uninformed about CHD and sometimes give advice that conflicts with that of the ACHD team.
The experience of growing up with CHD can be socially isolating, and this sense of isolation often persists into adulthood. Women with CHD may feel their experiences of pregnancy cannot be understood by friends, relatives, or other pregnant women (eg, National Childbirth Trust (NCT) group members). While support may be found through ACHD groups on social media, some of the experiences related by social media group members may be distressing. In addition, a small number of women with CHD experience serious complications, requiring surgical or medical interventions in pregnancy. These experiences add a layer of trauma to an already anxious time, and can lead to a greater sense of social isolation, given how rare they are. While clinical teams are focused on delivering the best cardiac care, the deep psychological impact on the woman and her partner is left unaddressed, both in pregnancy and in the postnatal period.
Both the clinicians and the women with lived experience emphasised that it is important to provide mental health support to women with CHD during pregnancy and the postnatal period. However, several barriers prevent patients with CHD from seeking and receiving appropriate support. Specialist mental healthcare for the CHD population is difficult to obtain Women with lived experience and clinicians felt that psychological support would be most effective if delivered by a therapist familiar with CHD-related issues. People with CHD have unique lifelong experiences that necessitate a psychologist or therapist with specialist knowledge. However, this is difficult to obtain without the help of the ACHD service, and patients must resort either to seeking treatment privately, facing long waits in the community, or forgoing mental healthcare entirely. Mental health support is made available when women are unable to use it Mental health support may be offered during the immediate postnatal period, but not later. This is often a time of great upheaval for women with CHD, who are overwhelmed by recovering from childbirth and caring for a newborn baby, and who may be burdened by frequent hospital visits related to their heart condition in the first few weeks following the birth.
Women with lived experience and clinicians felt that psychological support would be most effective if delivered by a therapist familiar with CHD-related issues. People with CHD have unique lifelong experiences that necessitate a psychologist or therapist with specialist knowledge. However, this is difficult to obtain without the help of the ACHD service, and patients must resort either to seeking treatment privately, facing long waits in the community, or forgoing mental healthcare entirely.
Mental health support may be offered during the immediate postnatal period, but not later. This is often a time of great upheaval for women with CHD, who are overwhelmed by recovering from childbirth and caring for a newborn baby, and who may be burdened by frequent hospital visits related to their heart condition in the first few weeks following the birth.
While our policy impact project was designed to inform service improvement in two NHS Trusts, the outcomes of our multidisciplinary discussions suggest that the overlap between maternal and mental health in ACHD is an important area for future research. As the JLA Priority Setting Partnership has highlighted, adults with CHD face lifelong mental health challenges. Against this broader background, we suggest that, for women with CHD, these mental health challenges are particularly acute during pregnancy and the postnatal period, with current services not yet equipped to address them. With research on maternal mental health in ACHD still scarce, there is an urgent need for both cross-sectional and longitudinal studies that investigate the lived experiences of women with CHD in the preconception period, pregnancy, and the immediate and extended postnatal periods. Also needed is research on health service innovations to reduce barriers to accessing mental healthcare among pregnant women and new mothers with CHD, including trials focused on different modalities of prevention, intervention and joined-up care specific to this patient population.
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Implementation and outcomes in benign gynecological surgery with HUGO™ RAS system 12 months initial experience | 872c5bed-2b7b-49df-9609-ee9bac09eed3 | 11427502 | Robotic Surgical Procedures[mh] | Minimally invasive gynecological surgery has emerged as the main approach in the realm of benign gynecological procedures, with well-documented benefits. These advantages include a reduction in postoperative pain, complication rates, and hospitalization durations while concurrently facilitating an accelerated recovery period when compared with traditional laparotomy. . Over the past 20 years, robotic-assisted laparoscopic surgery has shifted significantly from traditional laparoscopic surgeries. In just two years, the use of robotic-assisted laparoscopic surgery has increased by 36% for common gynecological procedures including hysterectomy, myomectomy, sacrocolpopexy and management of severe endometriosis in Europe and the United States [ – ]. Robotic surgeries are designed with the potential to offer advantages, and have been more established and researched, particularly on the da Vinci platform (Intuitive, United states) . These include the use of three-dimensional vision as opposed to the traditional two-dimensional view, higher magnification, tremor filtration, improved articulation with a wider range of motion, and enhanced ergonomic design . These innovative features have substantially elevated precision, accuracy, and efficiency, especially in complex surgical procedures, potentially leading to reduced complications and shorter recovery times [ – ]. However, it is important to acknowledge that despite its numerous advantages, robotic surgery is not without limitations, such as higher costs and longer operative times . The Hugo™ RAS system (Medtronic, United states) introduced in 2022 is distinguished by its concept of independent robotic arms, akin to the Versius™ (Cambridge Medical, UK) robot , Hugo™ offers an enhanced configuration flexibility and an open console with diverse gripping techniques. However, our understanding of the optimal utilization of Hugo™ is still in its nascent stages, and we are in the process of gaining insights into harnessing its full potential . The majority of data related to the system, from a gynecological perspective, have been presented through case reports or case series, with a particular focus on key parameters, such as docking time, operative time, and major safety events . One of the most extensive study conducted on this system involved 192 cases and aimed to address previously unexplored aspects of the learning curve associated with docking and operation time. Their principal finding indicated that these procedures can be performed efficiently in terms of time, and the specific robotic learning curve appears to be consistent with the existing data for other platforms . The Hugo™ RAS, while promising, still requires testing across essential parameters to provide guidance for newcomers. In this manuscript, we share our experiences and insights through the utilization of the Hugo™ RAS system. Our focus was on the critical aspects of port placement, arm positioning, and docking time. The study was conducted by the same team, which has undergone training for the Hugo™ RAS. The primary robotic surgeon has experience with over a thousand cases using the Da Vinci robotic system prior to study period. We aim to describe the broader surgical experience, with the goal of establishing Hugo™ RAS as a versatile and efficacious platform applicable to various benign gynecological procedures.
Patients This retrospective observational study included all patients who underwent elective robotic surgeries using the Hugo™ RAS system for benign gynecological conditions between February 2023 and February 2024. Patients aged between 18 and 65 years were included. The procedures were performed by a single robotic surgeon and consistent robotic team, all of whom had undergone robotic training as described later. The procedures included elective robotic cases of endometriosis resection (stages I-IV, including rectal shaving), hysterectomy (with or without salpingectomy or oophorectomy), myomectomy, cervical cerclage, and cesarean section scar defect repair. These procedures were chosen based on clinical assessments, physical examinations, and sonographic evaluations in accordance with the established and accepted medical criteria. All surgeries that fell under the previously described categories were included without exclusion. Outcomes The primary outcomes were perioperative port placement, docking time, arm configuration, and console time. Secondary outcomes were defined as team satisfaction, system troubleshooting, arm repositioning, and complications at Clavien–Dindo Scale grade 3–4. Data Data were collected for this study from electronic medical records, including patient demographics, medical history, indication for surgery, surgical findings and reports, and a two-month postoperative follow-up. Specific perioperative data were recorded with respect to time from patient entrance to first skin incision, port placement, arm configurations, time from the first incision to port insertion, duration of docking up to console time, and whole surgery time (console time). In addition, information was documented regarding procedural outcome data, including troubleshooting, arm repositioning, instances of arm clashes, instrument breakages, and overall satisfaction of the surgical team. Ethical considerations This study was conducted in accordance with the principles of the Declaration of Helsinki. All methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Institutional Review Board #2023/ETH02370. Given the observational nature of this study, informed consent was not required. Robotic surgical team Elective gynecological surgeries for benign conditions were conducted using the Hugo™ RAS system, led by a minimally invasive gynecology specialist with more than 1200 procedures using the Da Vinci robotic system prior to the study period and a team of anesthetists, surgical fellows, and nurses who have been trained together in robotic surgery. Robotic training program All team members as described above completed the Hugo™ RAS Ascend training program which included three stages: 1. introduction phase: Hugo™ demonstration, e-learning modules and practice on a virtual simulator. 2. Hands-on training: technical training, dry lab exercises, and collaborative training with the entire theater team. 3. Proctored Case: live case observation, video case review and continuous support to ensure competence in utilizing the system. Robotic surgery Various time-related events have also been documented. These events included the time from entering the operating theater to making the first incision, initiation of port placement, docking time (including instrument insertion), console time, skin closure, dressing time, and the moment when the patient left the operating theater. The patient was placed in a supine low lithotomy position. Following general anesthesia, a Verres needle was inserted at the umbilicus to create pneumoperitoneum, followed by the introduction of an 11 mm robotic port through the umbilicus for the endoscope. A 3D 30-° scope was used. After achieving the Trendelenburg position, two to three additional 8 mm robotic ports were inserted, along with usually a 5 mm AirSeal port for an assistant. The choice between standard and low-port placement was determined based on the specific surgical procedure being performed (Fig. ). Arm positioning was performed by the surgeon and first assistant on each side of the patient. The arm that is furthest away is docked first. Tilt angles were preset on each of the robotic arms by the scrub nurse before being positioned next to the patient. The docking angle was set during the docking. The tilt and dock angles were determined based on arm placement. “ Symmetric ” arm positioning included two arm positions on each side of the patient. The tilt of the top arms from both sides was configured to + 15°, and that of the lower arms on both sides was set to −30°. The clockwise angles from the left upper part to the right upper part were 100°, 140°, 220°, and 260°. The assistant port was inserted on the left side between Arms 1 and 2. (Fig. ). “ Asymmetric ” arm positioning included one arm positioned on the left side of the patient and three arms on the right side of the patient. Arm 1 had a tilt of −30° and a dock angle of 140°, arm 2 also had a −30° tilt and an angle of 220°, arm 3 had a tilt of + 15° with a dock angle of 280°, and the fourth arm was tilted to 0 / −15° with a dock angle of 340°. The assistant port was placed on the left side between arms 1 and 2 (Fig. ). Typically, our standard port configuration consists of four robotic arms with Symmetrical arm positioning, which we find works best for more complex cases. For more minor cases, particularly in young patients, we aimed for low-port placement with a reduced port configuration with only two robotic accessory ports and no assistant port. In such cases, we found that Asymmetrical arm positioning worked well, giving the assistant more room. Statistical analysis Descriptive statistics were used to describe the parameters. Categorical variables are summarized as numbers and percentages. For ease of reading, all continuous variables are reported as means and standard deviations, unless mentioned otherwise.
This retrospective observational study included all patients who underwent elective robotic surgeries using the Hugo™ RAS system for benign gynecological conditions between February 2023 and February 2024. Patients aged between 18 and 65 years were included. The procedures were performed by a single robotic surgeon and consistent robotic team, all of whom had undergone robotic training as described later. The procedures included elective robotic cases of endometriosis resection (stages I-IV, including rectal shaving), hysterectomy (with or without salpingectomy or oophorectomy), myomectomy, cervical cerclage, and cesarean section scar defect repair. These procedures were chosen based on clinical assessments, physical examinations, and sonographic evaluations in accordance with the established and accepted medical criteria. All surgeries that fell under the previously described categories were included without exclusion.
The primary outcomes were perioperative port placement, docking time, arm configuration, and console time. Secondary outcomes were defined as team satisfaction, system troubleshooting, arm repositioning, and complications at Clavien–Dindo Scale grade 3–4.
Data were collected for this study from electronic medical records, including patient demographics, medical history, indication for surgery, surgical findings and reports, and a two-month postoperative follow-up. Specific perioperative data were recorded with respect to time from patient entrance to first skin incision, port placement, arm configurations, time from the first incision to port insertion, duration of docking up to console time, and whole surgery time (console time). In addition, information was documented regarding procedural outcome data, including troubleshooting, arm repositioning, instances of arm clashes, instrument breakages, and overall satisfaction of the surgical team.
This study was conducted in accordance with the principles of the Declaration of Helsinki. All methods were performed in accordance with relevant guidelines and regulations. All experimental protocols were approved by the Ethics Institutional Review Board #2023/ETH02370. Given the observational nature of this study, informed consent was not required.
Elective gynecological surgeries for benign conditions were conducted using the Hugo™ RAS system, led by a minimally invasive gynecology specialist with more than 1200 procedures using the Da Vinci robotic system prior to the study period and a team of anesthetists, surgical fellows, and nurses who have been trained together in robotic surgery.
All team members as described above completed the Hugo™ RAS Ascend training program which included three stages: 1. introduction phase: Hugo™ demonstration, e-learning modules and practice on a virtual simulator. 2. Hands-on training: technical training, dry lab exercises, and collaborative training with the entire theater team. 3. Proctored Case: live case observation, video case review and continuous support to ensure competence in utilizing the system.
Various time-related events have also been documented. These events included the time from entering the operating theater to making the first incision, initiation of port placement, docking time (including instrument insertion), console time, skin closure, dressing time, and the moment when the patient left the operating theater. The patient was placed in a supine low lithotomy position. Following general anesthesia, a Verres needle was inserted at the umbilicus to create pneumoperitoneum, followed by the introduction of an 11 mm robotic port through the umbilicus for the endoscope. A 3D 30-° scope was used. After achieving the Trendelenburg position, two to three additional 8 mm robotic ports were inserted, along with usually a 5 mm AirSeal port for an assistant. The choice between standard and low-port placement was determined based on the specific surgical procedure being performed (Fig. ). Arm positioning was performed by the surgeon and first assistant on each side of the patient. The arm that is furthest away is docked first. Tilt angles were preset on each of the robotic arms by the scrub nurse before being positioned next to the patient. The docking angle was set during the docking. The tilt and dock angles were determined based on arm placement. “ Symmetric ” arm positioning included two arm positions on each side of the patient. The tilt of the top arms from both sides was configured to + 15°, and that of the lower arms on both sides was set to −30°. The clockwise angles from the left upper part to the right upper part were 100°, 140°, 220°, and 260°. The assistant port was inserted on the left side between Arms 1 and 2. (Fig. ). “ Asymmetric ” arm positioning included one arm positioned on the left side of the patient and three arms on the right side of the patient. Arm 1 had a tilt of −30° and a dock angle of 140°, arm 2 also had a −30° tilt and an angle of 220°, arm 3 had a tilt of + 15° with a dock angle of 280°, and the fourth arm was tilted to 0 / −15° with a dock angle of 340°. The assistant port was placed on the left side between arms 1 and 2 (Fig. ). Typically, our standard port configuration consists of four robotic arms with Symmetrical arm positioning, which we find works best for more complex cases. For more minor cases, particularly in young patients, we aimed for low-port placement with a reduced port configuration with only two robotic accessory ports and no assistant port. In such cases, we found that Asymmetrical arm positioning worked well, giving the assistant more room.
Descriptive statistics were used to describe the parameters. Categorical variables are summarized as numbers and percentages. For ease of reading, all continuous variables are reported as means and standard deviations, unless mentioned otherwise.
During the study period, 60 (17.2%) patients were operated by Hugo™ RAS system. Table provides an overview of the demographic data of the entire cohort. The surgeries performed using Hugo™ RAS included 32 (53%) endometriosis cases with stages ranging from 1 to 4, including two rectal shaving and one bowel resection, 16 (27%) hysterectomies, 6 (10%) adnexal surgeries, 3 (5%) myomectomy, one (2%) each of sacrocolpopexy, cervical cerclage and cesarean section scar defect repair. Table presents the operative data, configuration data and satisfaction levels for the study cohort, including various surgical timelines. The mean port-placement time, defined from the first skin incision to the completion of port placement, was recorded at 13 min and 41 s (range 5–23). The mean docking time, measured from the end of port placement to the end of docking, averaged 5 min and 51 s (range 2–11). Mean console time, covering the entire operative period from the start of the main operation in the console to the end of surgery, was recorded as a mean of 1 h, 5 min, and 20 s (range 18–176). 37% of cases used the “Symmetric” configuration, while 63% opted for the “Asymmetric” configuration. A total of ten cases required port changes due to troubleshooting, with an average of 0.47 changes per case (± 0.6). Arm repositioning occurred nineteen times, with an average of 0.75 adjustments (± 0.8). There were a total of 35 reported clashes, averaging 2.2 clashes per case (± 2.8). Surgeon satisfaction was rated at 8.4 (± 1.9) on a scale of 1 to 10, where 1 was least satisfied and 10 completely satisfied, while assistant satisfaction was reported at 6.9 (± 3.1), primarily due to space limitations and assistant arm collision. Figure illustrates time of port placement and docking time throughout the study period. For patients in the Hugo™ RAS cohort, postoperative complications were monitored up until the of data collection. To date, no cases of intra-, peri- and postoperative complications at Clavien–Dindo Scale grade 3–5 were reported.
With experience in performing robotic surgery using both the da Vinci RAS and Hugo™ RAS systems, this study aims to share our early leaning in its application in advanced gynecological robotic surgery. In this article, we discuss the crucial aspects of port placement, arm configuration, docking time and essential insights for individuals new to the Hugo™ RAS system. We have detailed our experiences, learning curves, levels of satisfaction, and address specific challenges encountered. Despite the available data on the Hugo™ RAS, undisclosed aspects remain while the technology is still in the learning curve period. Our primary outcome was to investigate the perioperative learning curves for port placement, arm positioning, docking and console time. Pertaining to port placement, young gynecological patients and gynecologist alike, typically favor a low-port configuration for cosmesis, but it can also be relevant in some surgeries such as endometriosis wherein high port placement can be uncomfortable and is one of the inherent disadvantages of robotic surgery . Studies comparing robotic-assisted surgery and laparoscopic surgery for endometriosis have primarily focused on factors such as surgery duration, postoperative outcomes, and precision . Here, we provide a different perspective on port placement. The da Vinci robotic system comprises four arms attached to a single boom, with one arm for the scope and three arms serving as accessory arms. In its default configuration, there are limits to how far these arms can be positioned from the scope and from each other . In contrast, the Hugo™ RAS system’s modular arm design provides us with the flexibility to place the accessory ports in low port for needed cases such as endometriosis, without any constrain to the proximity to umbilical optical port . Docking time has been an interesting topic of research for robotic surgery, with initial earlier publications reporting docking times ranging from 6 to 9 min [ , , ]. As larger datasets were examined, they reinforced this trend . In our study, the “Asymmetric” configuration was mainly utilized. We observed a docking time of 5 min and 51 s (with a standard deviation) range (2–11 min) for the Hugo™ RAS system, whereas for our team, the docking time with the da Vinci system typically was 2 min (range 1–8 min), this difference could be due to our learning curve with the docking of the Hugo™ RAS. Studies showed longer docking time with da Vinci then our report and almost similar to Hugo™ RAS system . Our experience presented in this study indicates that a well-trained team with experience in robotic-assisted surgery can rapidly adapt to the docking process, as illustrated in Fig. and supported in other manuscripts . More insight regarding this issue, the modular nature of Hugo™ RAS arms, could give the impression that bringing in the four arms carts to the patient would prolong the docking process compared to the da Vinci system, where there is a single patient’s cart with four arms on it, but in practice that is not the case. With the da Vinci system, after placing the ports, surgeons need to wait as the da Vinci’s patient cart is driven over to the patient and have the laser guide aligned to the umbilical port. The endoscope arm is docked, and the endoscope is inserted and targeted before the remaining arms are docked. With the Hugo™ RAS there is no wait required as four individual carts are brought in while the ports are placed and immediately available for docking. Our results shows that console time remained largely consistent between the two robotic systems. This observation further substantiates the notion of a rapid learning curve and highlights the absence of significant differences between the systems, particularly when utilized by well-trained surgeons. The secondary outcomes of this study were focused on team satisfaction, system troubleshooting, arm repositioning, and complications at Clavien–Dindo Scale grade 3–4. External clashes were encountered in earlier cases, which is one of the most common challenges in the system. Resolution of these events requires a clear understanding of how the arm cart operates. This situation can often be resolved by placing the arm carts apart, by changing the docking angle and / or tilt in one or both arms. Occasionally surgeons may be able to overcome an arm collision situation or limitation of the range of instrument reach without bedside assistance by modifying surgical techniques. This can be achieved by bringing mobile tissue more centrally, use of an alternate robotic arm or by pushing a tissue instead of pulling while still achieving the same desired tissue effect. Despite the encouraging data, there are a few issues with the Hugo™ RAS system that deserve attention, as they can influence the satisfaction of surgeons. In cases such as endometriosis surgeries the use of bipolar and monopolar scissors, as well as forceps, mirrors the experience with the da Vinci system. However, in myomectomy procedures, the absence of a tenaculum, and the lack of a vessel sealer in cases of hysterectomy, may impact surgical satisfaction. While the needle holder remains the same, the absence of a suture cut feature is noticeable. Our understanding that these relatively minor issues will be improved upon in the near future. Several other minor issues can contribute to discomfort during console time when using the Hugo™ RAS system. Notably, there is no indicator on tracking instruments that are out of sight. In addition, some inconveniences were noted, such as requiring a long press on the left side for switching between arms 3 and 4, and the double press needed to activate diathermy. Another issue we encountered was arm tremoroccurring when the robotic arm reaches its maximum range of movement, which can potentially impact surgical precision. In addition, we were unable to utilize our port-hopping technique due to the 10 mm scope, which cannot be easily transferred to other 8 mm ports. Consequently, when morcellation was required and access through one of the 8 mm ports became necessary, we had to employ a different scope for this purpose. These details highlight areas where refinement in the system can enhance the user experience. In our comprehensive evaluation of our surgical team satisfaction while using Hugo™ RAS system, we considered all of the previously mentioned issues, including the occasional necessity for port changes due to troubleshooting and considered factors such as the low number of arm repositioning instances, as well as a relatively higher number of arm clashes. Despite these challenges, overall satisfaction of the lead surgeon remained high, with a rating of 8.4. This reflects a positive assessment of the system performance, even in the presence of certain operational issues. Assistant satisfaction, while still favorable, was slightly lower. This can be attributed to the limited space available for the assistant in our arm configuration, which increases the risk of exposure to injuries resulting from arm movements . In addition, there were instances where the assistant faced challenges when operating in the pelvic area, primarily due to arm occlusions and clashes. These factors, while not diminishing overall satisfaction, underscore areas for potential improvement in terms of safety and workspace optimization. Another parameter for surgeon satisfaction is the ergonomic advantage that emerged through our experience with the Hugo™ RAS, which has been substantiated in other research papers, related to open console design . Prior studies conducted with the da Vinci system highlighted certain shortcomings and potential eye discomfort , the open console design can lessen these issues, subsequently reducing eye fatigue and allowing for an upright sitting position, which, in turn, can alleviate muscle stiffness and discomfort and indeed allows the operator to sit or stand at the console as desired. Finally, no complications at Clavien–Dindo Scale grade 3–4 were noted during the study period with all different surgical procedures, port placements and arm positioning. This study has several strengths. First, it is one of the few investigations delving into the initial experiences with the Hugo™ RAS system, comprehensively addressing its advantages, limitations, and serving as a practical guide for newcomers to the field. Furthermore, the strength of the study lies in its singular centre-based approach, where all cases were managed consistently by a single surgeon, complemented by a proficient team of surgical assistants, nurses, and anesthetists, all of whom were well-versed in the realm of robotic surgery. This study has some limitations. Most notably, the sample size was relatively small, potentially affecting the generalizability of the findings. Being drawn from a single institution, there may also be constraints on the diversity of surgeons that can learn from it.
Our study offers insights into the realm of advanced gynecological robotic surgery, drawn from our diverse experience with both the da Vinci and Hugo™ RAS systems. We have documented our experiences, learning curves, levels of satisfaction, and addressed specific challenges faced during our journey highlighting key considerations that contribute to this learning process. Our study contributes to the growing body of knowledge on advanced gynecological robotic surgery, emphasizing the need for ongoing exploration and refinement of the Hugo™ RAS system to maximize its potential and benefit surgeons and patients.
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The wisdom of claiming ownership of human genomic data: A cautionary tale for research institutions | 2e317faf-b7ae-48c6-acb3-48c7f2842cbb | 11289161 | Psychiatry[mh] | INTRODUCTION We are witnessing a rapid increase in the volume of human genomic data. However, given the relative novelty of this new type of legal object, there are debates in many countries over the exact legal nature of human genomic data—in particular, whether such data can be owned, and if so, by whom. Some have argued that data are not susceptible of ownership since they are not capable of exclusive control by a putative owner. For example, university X may be in possession of one instance of person Y's human genomic data, but Y can provide a blood sample to a genomics research study of university Z, which will then also be in possession of an instance of Y's human genomic data. Y does not even need to donate blood again, as the data instance in X's possession can simply be copied. The data instance in X's possession can be copied an infinite number of times (subject to practical limitations) and even published online in the style of the Harvard Personal Genome Project. Are any claims of ownership of Y's human genomic data legally tenable in light of these realities? Surely, Y has a legitimate interest in controlling what happens with his or her genomic data. If such an interest is recognised by the law, does it not imply that Y has ownership in his or her genomic data? And where does this leave X that invested effort and resources in sequencing Y's genome? In this article, I consider the practical question of how research institutions should best structure their legal relationship with human genomic data. My analysis is presented in two parts: The first part is a positivist legal analysis of whether human genomic data can be owned, and if so, by whom. Once it has been established what the current law is, I propose my answer to the main research question (of how research institutions should best structure their legal relationship with human genomic data), substantiate my answer, and defend it against potential counterarguments. My analysis is based in South African law, but I suggest that many of the principles are applicable more broadly in other countries.
THE LEGAL LANDSCAPE 2.1 Understanding ownership Ownership is the most comprehensive set of rights that a person can have in an object, and it is in principle enforceable against anyone else—regardless of who they are. Ownership can be understood as a bundle of rights entailing (in very general terms) the right to use an object, the right to enjoy the fruits of the object, and the right to dispose of an object. However, ownership is not absolute. It is always qualified depending on the nature of the owned object and the context. For example, I may be the owner of my car, but I cannot drive it on a public road without a driver's licence. And while driving my car on a public road (assuming I have a driver's licence), I must adhere to the many rules of the road. I can decide to destroy my car, but I may not do so by setting fire to it in my backyard, as this would contravene municipal waste disposal and fire hazard ordinances. But the legal fact that I am the owner of my car means that nobody else may, without my consent, take my car keys and drive my car around or destroy it. If someone does so in contravention of the law, there will be civil and criminal consequences. So, despite the numerous rules that qualify ownership of an object in certain contexts, ownership provides a broad range of default legal rules that govern who may do what with a particular object. In this sense, ownership can be perceived as a vital legal safety net. 2.2 Can data be owned? An object's susceptibility of being controlled is often viewed as the preeminent condition for its susceptibility of ownership. How does this apply to data? Data often seem ubiquitous, like the air around us, and the vast oceans—beyond human control, and thus not susceptible of ownership. But, if one fills a gas cylinder with air, or fills a bottle with seawater, then the air or water becomes part of an object that is indeed susceptible of ownership. Can the same be done with data? Yes, when data are recorded in a computer file—a single data instance—the data are brought within human control. However, one might argue that this does not settle the question about the control of data, because—different from the gas cylinder filled with air, or the bottle of seawater—data are intangible and can be reproduced indefinitely and can thus be published online and downloaded by any number of people. Consider again the example used in the introduction above. University X is in possession of an instance of person Y's human genomic data. Does this amount to control of such data? It can be argued that the answer is 'no', since Y can provide a blood sample to a genomics research study of university Z, which will then be able to generate its own instance of Y's human genomic data; and perhaps Y is a data altruist who publishes his or her genomic data online for anyone in the world to access. I suggest that, despite the tangible/intangible difference, this argument commits the same category error as with the ocean versus bottle of seawater example above. Having control over a bottle of seawater does not mean that one is claiming Poseidon‐like control of the ocean. Similarly, control over Y's human genomic data that are recorded in a computer file is not a claim to control Y's human genomic data generally —only the specific instance of it on the computer file. This is entirely compatible with the fact that other persons also control their respective instances of the same data, as data is nonrivalrous—one person using data in no way depletes the data, nor prevents other persons from using it. Accordingly, once the object that is the candidate of the ownership inquiry is conceptually pinned down as a data instance —not to be confused with data generally —control (and thus ownership) becomes realistic. In light of this conceptual clarity, I next explore how data ownership has been dealt with in South African caselaw and academic literature. 2.3 Data ownership in South African caselaw and academic literature It is well‐established in South African law that confidential information can be owned. For information to be confidential it must be (a) useful in the sense that it must be capable of application in trade or industry, (b) of economic value to the person seeking to protect it, and (c) known only to a restricted number of people, and not be public knowledge or public property. For example, a medical scheme brokerage firm can claim ownership of all the information of clients that it collects. Does this mean that such clients cannot provide the same information to anybody else, as their information is now owned by the firm? Of course not. The firm can only claim ownership of the instance of the information of a client that it has collected, not the information of a client in general . Accordingly, the clients are at liberty to provide the same information about themselves to anybody else. But can information be owned if it is shared with outside parties, for example, if it is sold to other firms? In a case that dealt with electronic point‐of‐sale data, which have obvious usefulness and economic value, the South African Competition Tribunal treated such data as private property that can be bought and sold. It is interesting that the Competition Tribunal did not even find it necessary to consider whether electronic point‐of‐sale data constitute confidential information. However, provided that the sale agreements prohibit the purchasers from further sharing the electronic point‐of‐sale data or making such data public, the electronic point of sale data remain known only to a restricted number of people, and thus clearly constitute confidential information. The essence of the caselaw discussed above is that information can be owned, provided that it is useful, valuable, and remain known only to a restricted number of people. Importantly, there is no caselaw that explicitly excludes non‐confidential information from being owned. Academic literature in South African property law lends support to the position that not just confidential information, but information more generally can be owned. Njotini examines the historical development of the concept of an object of property rights in South African law, and suggests that property rights should expand to include information qua object. Erlank suggests that ‘digital objects’, such as websites email addresses, bank accounts, e‐books, smartphone apps, and digital music, are indeed the objects of ownership in extant law. In a recent article, Thaldar et al. focus specifically on human genomic data. The authors suggest that a human genomic data instance fulfils all the general criteria for an object to be susceptible of private ownership in South African law. These criteria are that an object must be (a) useful and valuable; (b) not merely part of something else; (c) not part of a human body; and (d) capable of human control. Accordingly, there is a solid theoretical basis for recognising private ownership in instances of human genomic data. It follows that such data instances can be sold or out‐licenced like any other digital asset—subject, of course, to other rights that may apply to such data, such as data protection rights. 2.4 Excursus: Ownership and intellectual property rights It is important to differentiate ownership of data from intellectual property rights related to data, such as copyright and patents. Most pertinently, the objects of rights entailed by each of these legal concepts in the context of human genomic data are different: The object of ownership is genomic sequence data as contained in a computer file—i.e., a data instance. The object of copyright is a genomic dataset, which is a compilation of the genomic sequence data of multiple individuals. The object of patent rights is an invention, which in certain jurisdictions, such as the European Union and likely South Africa, can be a gene sequence that has a proven functional application. Note however, that in other jurisdictions, such as the United States and Australia, naturally occurring gene sequences are not patentable. Ownership of data and the various intellectual property rights related to data are best conceived of as separate layers of rights related to data, interacting with each other, rather than replacing or subsuming one another. Accordingly, patenting a gene sequence (assuming it is permissible in one's jurisdiction) does not mean that one becomes the owner of the gene sequence—these are different legal concepts, each with its own rules. Patenting a gene sequence provides one with patent rights, but it does not change who owns the gene sequence qua data instance. Similarly, ownership of an object to which an invention pertains is not a prerequisite for obtaining a patent in such an invention. Accordingly, the need to give attention to the ownership of genomic data remains, irrespective of whether such genomic data are incorporated into a dataset or have a proven functional application that makes such data patentable. 2.5 Who owns human genomic data? Since it has been established that human genomic data instances are susceptible of ownership, the next question is: who owns such data instances? The question of who owns an object must be answered with reference to the rules of property law. If a new object N is made from or produced by another object M, the owner of M would typically also acquire ownership in N. Is a newly generated human genomic data instance made from or produced by DNA? If so, the default legal position in South African law would be that the newly generated human genomic data instance would be owned by a research institution, as the statutory position in South Africa is that the research institution to whom human biological material is donated acquires exclusive rights in such material. However, this does not assist much, as it would be inaccurate to state that a human genomic data instance is made from or produced by DNA. More accurately, a human genomic data instance is a description of DNA, analogous to an entomologist studying a newly discovered insect and making extensive notes and sketches that describe the insect. However, the analogy is of limited validity, as the entomologist will automatically have intellectual property rights (in the form of copyright) in his or her descriptive notes, while no intellectual property rights vest in respect of a human genomic data instance, as it is machine‐generated. My point is that there is no existing rule in property law that determines who owns a newly generated human genomic data instance. It is a new object that was neither made from nor produced by any antecedent object. Accordingly, as concluded by Thaldar et al., a newly generated human genomic data instance is res nullius , meaning that it does not belong to anyone—at least not at its genesis. This conclusion has an important consequence: The first person to take effective control of res nullius with the intention of being its owner immediately becomes its owner. Since the research institution that conducts the sequencing is presumably already in control of the human genomic data instance, such a research institution is best placed to legally claim ownership of such newly generated data instance. Building on this conclusion, Swales et al. suggest that South African research institutions should claim ownership of the data that they generate to ensure that they have the legal means to effectively protect such data. In the next section of this article, I elaborate on this argument and consider possible counterarguments.
Understanding ownership Ownership is the most comprehensive set of rights that a person can have in an object, and it is in principle enforceable against anyone else—regardless of who they are. Ownership can be understood as a bundle of rights entailing (in very general terms) the right to use an object, the right to enjoy the fruits of the object, and the right to dispose of an object. However, ownership is not absolute. It is always qualified depending on the nature of the owned object and the context. For example, I may be the owner of my car, but I cannot drive it on a public road without a driver's licence. And while driving my car on a public road (assuming I have a driver's licence), I must adhere to the many rules of the road. I can decide to destroy my car, but I may not do so by setting fire to it in my backyard, as this would contravene municipal waste disposal and fire hazard ordinances. But the legal fact that I am the owner of my car means that nobody else may, without my consent, take my car keys and drive my car around or destroy it. If someone does so in contravention of the law, there will be civil and criminal consequences. So, despite the numerous rules that qualify ownership of an object in certain contexts, ownership provides a broad range of default legal rules that govern who may do what with a particular object. In this sense, ownership can be perceived as a vital legal safety net.
Can data be owned? An object's susceptibility of being controlled is often viewed as the preeminent condition for its susceptibility of ownership. How does this apply to data? Data often seem ubiquitous, like the air around us, and the vast oceans—beyond human control, and thus not susceptible of ownership. But, if one fills a gas cylinder with air, or fills a bottle with seawater, then the air or water becomes part of an object that is indeed susceptible of ownership. Can the same be done with data? Yes, when data are recorded in a computer file—a single data instance—the data are brought within human control. However, one might argue that this does not settle the question about the control of data, because—different from the gas cylinder filled with air, or the bottle of seawater—data are intangible and can be reproduced indefinitely and can thus be published online and downloaded by any number of people. Consider again the example used in the introduction above. University X is in possession of an instance of person Y's human genomic data. Does this amount to control of such data? It can be argued that the answer is 'no', since Y can provide a blood sample to a genomics research study of university Z, which will then be able to generate its own instance of Y's human genomic data; and perhaps Y is a data altruist who publishes his or her genomic data online for anyone in the world to access. I suggest that, despite the tangible/intangible difference, this argument commits the same category error as with the ocean versus bottle of seawater example above. Having control over a bottle of seawater does not mean that one is claiming Poseidon‐like control of the ocean. Similarly, control over Y's human genomic data that are recorded in a computer file is not a claim to control Y's human genomic data generally —only the specific instance of it on the computer file. This is entirely compatible with the fact that other persons also control their respective instances of the same data, as data is nonrivalrous—one person using data in no way depletes the data, nor prevents other persons from using it. Accordingly, once the object that is the candidate of the ownership inquiry is conceptually pinned down as a data instance —not to be confused with data generally —control (and thus ownership) becomes realistic. In light of this conceptual clarity, I next explore how data ownership has been dealt with in South African caselaw and academic literature.
Data ownership in South African caselaw and academic literature It is well‐established in South African law that confidential information can be owned. For information to be confidential it must be (a) useful in the sense that it must be capable of application in trade or industry, (b) of economic value to the person seeking to protect it, and (c) known only to a restricted number of people, and not be public knowledge or public property. For example, a medical scheme brokerage firm can claim ownership of all the information of clients that it collects. Does this mean that such clients cannot provide the same information to anybody else, as their information is now owned by the firm? Of course not. The firm can only claim ownership of the instance of the information of a client that it has collected, not the information of a client in general . Accordingly, the clients are at liberty to provide the same information about themselves to anybody else. But can information be owned if it is shared with outside parties, for example, if it is sold to other firms? In a case that dealt with electronic point‐of‐sale data, which have obvious usefulness and economic value, the South African Competition Tribunal treated such data as private property that can be bought and sold. It is interesting that the Competition Tribunal did not even find it necessary to consider whether electronic point‐of‐sale data constitute confidential information. However, provided that the sale agreements prohibit the purchasers from further sharing the electronic point‐of‐sale data or making such data public, the electronic point of sale data remain known only to a restricted number of people, and thus clearly constitute confidential information. The essence of the caselaw discussed above is that information can be owned, provided that it is useful, valuable, and remain known only to a restricted number of people. Importantly, there is no caselaw that explicitly excludes non‐confidential information from being owned. Academic literature in South African property law lends support to the position that not just confidential information, but information more generally can be owned. Njotini examines the historical development of the concept of an object of property rights in South African law, and suggests that property rights should expand to include information qua object. Erlank suggests that ‘digital objects’, such as websites email addresses, bank accounts, e‐books, smartphone apps, and digital music, are indeed the objects of ownership in extant law. In a recent article, Thaldar et al. focus specifically on human genomic data. The authors suggest that a human genomic data instance fulfils all the general criteria for an object to be susceptible of private ownership in South African law. These criteria are that an object must be (a) useful and valuable; (b) not merely part of something else; (c) not part of a human body; and (d) capable of human control. Accordingly, there is a solid theoretical basis for recognising private ownership in instances of human genomic data. It follows that such data instances can be sold or out‐licenced like any other digital asset—subject, of course, to other rights that may apply to such data, such as data protection rights.
Excursus: Ownership and intellectual property rights It is important to differentiate ownership of data from intellectual property rights related to data, such as copyright and patents. Most pertinently, the objects of rights entailed by each of these legal concepts in the context of human genomic data are different: The object of ownership is genomic sequence data as contained in a computer file—i.e., a data instance. The object of copyright is a genomic dataset, which is a compilation of the genomic sequence data of multiple individuals. The object of patent rights is an invention, which in certain jurisdictions, such as the European Union and likely South Africa, can be a gene sequence that has a proven functional application. Note however, that in other jurisdictions, such as the United States and Australia, naturally occurring gene sequences are not patentable. Ownership of data and the various intellectual property rights related to data are best conceived of as separate layers of rights related to data, interacting with each other, rather than replacing or subsuming one another. Accordingly, patenting a gene sequence (assuming it is permissible in one's jurisdiction) does not mean that one becomes the owner of the gene sequence—these are different legal concepts, each with its own rules. Patenting a gene sequence provides one with patent rights, but it does not change who owns the gene sequence qua data instance. Similarly, ownership of an object to which an invention pertains is not a prerequisite for obtaining a patent in such an invention. Accordingly, the need to give attention to the ownership of genomic data remains, irrespective of whether such genomic data are incorporated into a dataset or have a proven functional application that makes such data patentable.
Who owns human genomic data? Since it has been established that human genomic data instances are susceptible of ownership, the next question is: who owns such data instances? The question of who owns an object must be answered with reference to the rules of property law. If a new object N is made from or produced by another object M, the owner of M would typically also acquire ownership in N. Is a newly generated human genomic data instance made from or produced by DNA? If so, the default legal position in South African law would be that the newly generated human genomic data instance would be owned by a research institution, as the statutory position in South Africa is that the research institution to whom human biological material is donated acquires exclusive rights in such material. However, this does not assist much, as it would be inaccurate to state that a human genomic data instance is made from or produced by DNA. More accurately, a human genomic data instance is a description of DNA, analogous to an entomologist studying a newly discovered insect and making extensive notes and sketches that describe the insect. However, the analogy is of limited validity, as the entomologist will automatically have intellectual property rights (in the form of copyright) in his or her descriptive notes, while no intellectual property rights vest in respect of a human genomic data instance, as it is machine‐generated. My point is that there is no existing rule in property law that determines who owns a newly generated human genomic data instance. It is a new object that was neither made from nor produced by any antecedent object. Accordingly, as concluded by Thaldar et al., a newly generated human genomic data instance is res nullius , meaning that it does not belong to anyone—at least not at its genesis. This conclusion has an important consequence: The first person to take effective control of res nullius with the intention of being its owner immediately becomes its owner. Since the research institution that conducts the sequencing is presumably already in control of the human genomic data instance, such a research institution is best placed to legally claim ownership of such newly generated data instance. Building on this conclusion, Swales et al. suggest that South African research institutions should claim ownership of the data that they generate to ensure that they have the legal means to effectively protect such data. In the next section of this article, I elaborate on this argument and consider possible counterarguments.
THE WISDOM OF CLAIMING OWNERSHIP OF HUMAN GENOMIC DATA 3.1 The sweat of one's brow As the generator of the human genomic data, a research institution is the entity that invested effort and resources in generating the data. As such, based on classic Lockean property theory, the research institution has a moral right to be the owner of the human genomic data instance that it generated. Furthermore, research institutions generally have an interest in being able to conduct research and continue to do so with as little unnecessary cost and interruption as possible. Thus, it makes practical sense for research institutions to place themselves in a position that would afford them with the most comprehensive set of rights in the human genomic data instance that they have generated—while, at the same time, being cognisant and respectful of their legal and ethical duties towards research participants. Based on a combination of moral entitlement and self‐interest, it appears that the prudent course of action for research institutions would be to proactively claim ownership of the human genomic data that they generate. How should this be accomplished in practice? I suggest that the research institution should make its ownership claim clear to all interested parties. This would include, first, a clear policy that is available online to its employees and any outside party; second, by explicitly stating in informed consent forms that the research institution will be the owner of the resulting human genomic data instances; and third, by maintaining the integrity of its ownership of its human genomic data by including relevant ownership clauses in data transfer agreements. An example of such a data ownership clause can be found in the freely available data transfer agreement template developed by Swales et al. But, what about the research participants—do they not have a stronger legal and moral claim to ownership of the human genomic data that relate to them? Moreover, should the concept of ownership of human genomic data not rather be abandoned in favour of custodianship ? In the following subsection, I analyse these opposing views to thesis that research institutions should claim ownership of the human genomic data instances that they generate. Through this analysis, I suggest that the thesis gains clarity and proves its robustness. 3.2 The first opposing position: Research participants as data owners Given that a human genomic data instance relates to an individual research participant, should he or she not be deemed the owner by virtue of this personal connection with the data? The answer must be ‘no’. The acquisition of ownership is determined by the rules of property law . Property law provides for a closed number of ways in which ownership can be acquired, and the existence of a personal connection between a person and an object is not one of them. Note, however, that having a personal connection with an object is not necessarily legally irrelevant—it may indeed be legally relevant, but in another branch of the law, namely data protection law . South African data protection law is codified in the Protection of Personal Information Act (POPIA). POPIA applies to personal information , which is information that relates to and that identifies or can identify a person, and therefore includes a person's genomic data. POPIA provides for a wide array of data protection rights that persons— data subjects in data protection law terminology—can exercise with respect of their personal information, in the same mould as the European Union's General Data Protection Regulation (GDPR). These rights include the rights (i) to have one's personal information processed in accordance with POPIA's eight conditions for processing of personal information; (ii) to be notified if one's personal information is being collected, or has been accessed by an unauthorised person; (iii) to establish whether a person holds one's personal information; (iv) to request where necessary, the correction, destruction or deletion of one's personal information; (v) to object, on reasonable grounds, to the processing of one's personal information; (vi) to object to the processing of one's personal information for purposes of direct marketing; (vii) to not be subjected to automated decision‐making based on one's personal information (under certain circumstances); (viii) to submit a complaint to the Information Regulator regarding the alleged interference with the protection of the personal information; and (ix) to institute civil proceedings regarding the alleged interference with the protection of their personal information. Some of these data protection rights have characteristics that resemble ownership, in the sense that they correspond to a lesser or greater degree with rights that are part of the ownership bundle of rights. Examples are (a) the data protection right to have one's personal information deleted, which corresponds with the right of an owner to destroy the owned object; (b) the data protection right to have one's personal information processed only if a legal ground for processing it is present, such as consent by the data subject, which can be perceived to correspond with the scenario where an owner agrees to transfer the right to use the owned object to another person, such as in the case of loan for use. However, these few instances of resemblance should not lull one into confusing data protection rights with ownership, or into thinking that data protection rights are somehow indicative of ownership. Although some data protection rights resemble ownership, they stem from a different branch of the law and have different natures and functions. For example, while ownership can in principle be transferred from one person to another, it is not possible to transfer one's data protection rights, as they are bound to one's personality. The proper legal relationship between data protection rights and ownership is that if any data protection right comes into conflict with ownership, the data protection right will trump ownership. The existence of data protection rights in no way influence the acquisition of ownership. However, the idea that research participants own the data that relate to them is found in the controversial material transfer agreement that was published by the South African Minister of Health in 2018 (SA MTA). In clause 3.3 it states that the ‘donor remains the owner of the material until such materials are destroyed’. ‘Materials’ in turn is defined as including both biological samples and data. The choice of words in clause 3.3 is strikingly contradictory, as a donor per definition transfers ownership and does not remain the owner. If ownership remains with persons who provide biological samples from their bodies, they cannot be donors and the transaction cannot be a donation . The bad drafting aside, it should be noted that the SA MTA provides that it is only a ‘framework’, meaning that its substantive provisions need not be followed. Moreover, there are good reasons why clause 3.3 of the SA MTA should not be followed: (1) The idea that research participants can acquire ownership in the data generated from their biological samples is in conflict with primary legislation and thus legally impermissible; and (2) even if it were legally permissible for research participants to acquire ownership in the data generated from their biological samples, there would be very little if any benefit to the research participants, while there would be significant downsides to the research institution. I analyse these reasons seriatim. I commence with the idea that research participants can acquire ownership in the data generated from their biological samples. To the extent that the word ‘materials’ in the SA MTA pertains to data, it can be divided into (a) data directly collected from research participants, such as their medical history, and (b) data generated using biological samples collected from research participants, such as genomic data. In the case of (a), clause 3.3 of the SA MTA can be applied, as there is nothing in South African law that would in principle militate against research participants being the owners of the data collected from them. However, the same does not apply in the case of (b), as (b) entails the collection of biological samples from research participants, which triggers section 60(4) of the National Health Act. This section prohibits persons who donate biological samples from their bodies from receiving any kind of reward for such a donation. This would likely render unlawful receiving ownership of one's newly generated genomic data instance. It can potentially be argued that the value of such ownership is so trivial that the law should ignore it. This may depend on the type of research. For example, the cost of sequencing a single gene is significantly less than the cost of sequencing a whole genome, which costs several hundred US dollars depending on the platform. But, as highlighted by Thaldar and Shozi, South African courts view the theft of goods in the amount of less than ten US dollars as non‐trivial and sufficient for a guilty verdict. Accordingly, the cost of generating the data should be truly insignificant to be legally deemed trivial and of no legal consequence. It follows that the legal tenability of clause 3.3 of the SA MTA is probably limited to (a) data directly collected from research participants, and (b) data that are generated using biological samples collected from research participants and that truly have insignificant value. However, with respect to data that are generated using biological samples collected from research participants and that have non‐trivial value—which would for practical purposes be all human genomic data instances—agreeing with research participants that they will acquire ownership in the data instances that are generated from the samples that they donated, would likely be unlawful and void. Even if it were legally permissible for research participants to be the owners of data instances that are generated from their donated samples and that have non‐trivial value, I suggest that a policy of bestowing ownership of their genomic data instances on research participants would offer little or no benefit to research participants over and above the comprehensive data protection rights that they already enjoy in terms of POPIA, while presenting considerable practical obstacles for research institutes. To understand these obstacles, one should be cognisant of the different scopes of application between data protection law and ownership. While data protection rights exist exclusively in respect of research data that qualify as personal information (research data from which research participants can be identified), ownership rights are not similarly limited, and extend to non‐personal information. In this light, consider the following scenario: A genetics research group is doing research on a specific human gene and have sequenced the alleles of tens of thousands of South Africans. Before sharing their research data, they intend to deidentify the data—i.e., ensure that none of their research participants can be identified from the allelic and associated data. Deidentification is often implemented by researchers so as to share the data more easily—especially across national borders. While the research participants qua data subjects in data protection law need not consent to such deidentification and have no data protection rights in respect of deidentified data, the research participants qua owners in property law retain their rights in their data, irrespective of whether it is identifiable or deidentified. In fact, the very act by a researcher of deidentifying data without a research participant's consent would infringe such a research participant's ownership rights. Now consider the following quotidian facts: I borrow a friend's car to drive to the supermarket. Upon returning from the supermarket, I must return the car to my friend. There is of course also the possibility of extending the borrowing of the car, by asking my friend's permission to use the car for a further period or for a further specified task. From a legal perspective, I never become the owner of the car. I just have a right to use it. But, if I wilfully continue to use my friend's car beyond the agreed borrowing period, I would be unlawfully appropriating the property of another—i.e., I would be committing theft. Would this change if I remove the car's number plates and scratch out the engine number? Can I, by removing the identifying elements of the car, argue that the car has ceased to be owned by my friend, that the car is now free‐for‐all and that I am not committing theft? Clearly, not. The same applies to research data. Hence, from the perspective of the genetics research group in the scenario sketched above, there are none of the typical benefits of deidentifying research data if the data are owned by the research participants. This is clearly a serious limitation on the researchers. Can the genetics research group approach their institutional research ethics committee to provide permission to create and share a deidentified version of their allelic dataset—given the enormous effort that obtaining consent from tens of thousands of research participants would entail? The answer is ‘no’. The institutional research ethics committee will be powerless to assist researchers in this regard, for the simple reason that once research participants are endowed with ownership , it falls beyond the remit of research ethics committees to limit such ownership in any way, as that would be an ultra vires encroachment in the field of property law. For example, if a research ethics committee allows the sharing of the deidentified dataset of alleles with a research collaborator without the consent of each one of the tens of thousands of research participants, such sharing remains an unlawful violation of the research participants’ ownership rights. Furthermore, the members of the research ethics committee would be opening themselves up to criminal prosecution (as accessories to theft) in their personal capacities. (Given that the dataset contains the allelic data of tens of thousands of research participants, it is unlikely that it can be argued that the value of the dataset is insignificant. Therefore, the risk of criminal prosecution would be real.) The solution from the perspective of the research institution would be to require research participants ( qua owners) from the outset to consent to deidentified versions of their (owned) data to be made and used freely by the research institution. However, then research participants will be owners in name only—but to what end? Clearly, the idea of research participants as data owners offers little benefit, if any, at significant cost. 3.3 The second opposing position: Abandoning the concept of ownership Should ownership of genomic data not be abandoned altogether? This is indeed what was proposed by the Academy of Science of South Africa (ASSAf) in a 2018 report on the ethical, legal and social implications of genetics and genomics in South Africa. The ASSAf report suggested that ownership of human biological samples and genomic data should be ‘avoided’ and replaced with ‘custodianship’. The reason for the proposed move away from ownership seems to be an ideological commitment to perceiving human biological samples and genomic data as a kind of natural resource, akin to the country's water resources and gold deposits, which should be managed by the state in the public interest. This natural resource view of human biological samples and genomic data has been criticised as insufficiently respectful of individual rights. It is also worth pointing out that the natural resource view provides a basis to argue against private ownership of human genomic data, but not necessarily for the non‐existence of ownership altogether. Instead, it provides a basis for arguing in favour of the public ownership of human genomic data. However, given that human genomic data may already be privately owned, for instance if a research institution claimed ownership of the genomic data sequences that it generated, public ownership of such data would require the expropriation of such data by the state. This may require an excessive amount of state resources to accomplish, which raises the question of whether the public interest in fair access to research data cannot be served in less drastic ways, such as through policy initiatives to promote data sharing. Instead of confronting the issue of expropriation, the ASSAf report proposes an ostrich policy of ‘avoiding’ ownership. However, in our law, human genomic data are susceptible of private ownership, and whether a specific human genomic data instance is owned and by whom are legal facts that can be proven in a court of law. Thus, avoiding reference to ownership is a denial of reality and hence not useful policy. Consider a scenario where a research institution generates human genomic sequence data, but decides to shun ownership and instead declares that it is the ‘custodian’ of the data that it generated. Say someone, person X, who has lawful access to the data, such as a research collaborator or a student, makes a copy of the file containing the relevant data on her own memory stick and deletes the original file from the research institution's system. X declares herself the owner of the data contained in the file on the memory stick. What are the legal consequences of these actions? First, had the research institution been the owner of the file, X's actions would have been theft. Note that section 12 of the Cybercrimes Act provides that the common law crime of theft must be interpreted so as to not exclude the theft of incorporeal property. Moreover, had the research institution been the owner of the file, the research institution would also have had civil remedies. The research institution would be able to claim the file back from X—or anyone to whom X might have transferred it—using the ancient but efficient actio rei vindicatio. However, in the scenario under consideration, the research institution failed to claim ownership. Consequently, no theft was committed (something that is not owned cannot be stolen) and the research institution cannot use the actio rei vindicatio to reclaim the file. The research institution would need to rely on contractual remedies against X—if it has any available. Contractual remedies are, however, limited in scope. For example, if X transferred the file to a third person, such a person would not be bound by the contract between the research institution and X. This is not the end of the research institution's woes. Given its express failure to claim ownership, the data was owned by no one. The fact that X has taken effective control of the data instance and has the intention to own it, means that X acquires ownership in the data instance through appropriation. This is the same as catching a wild animal like a dove that belongs to no one. The first person to take effective control of the dove with the intention of owning it, acquires ownership in it. The fact that someone declared himself or herself the ‘custodian’ of all the doves that are flying around in a market square is of no legal consequence. The research institution can attempt to insert clauses in its contracts with, among others, collaborators and students, in an attempt to stop them from claiming ownership of data that is within the research institution's deemed ‘custodianship’. However, this is weaker than if the research institution had acquired ownership. A contract will only provide civil remedies against the counterparty, not against any third parties who may come into possession of the data.
The sweat of one's brow As the generator of the human genomic data, a research institution is the entity that invested effort and resources in generating the data. As such, based on classic Lockean property theory, the research institution has a moral right to be the owner of the human genomic data instance that it generated. Furthermore, research institutions generally have an interest in being able to conduct research and continue to do so with as little unnecessary cost and interruption as possible. Thus, it makes practical sense for research institutions to place themselves in a position that would afford them with the most comprehensive set of rights in the human genomic data instance that they have generated—while, at the same time, being cognisant and respectful of their legal and ethical duties towards research participants. Based on a combination of moral entitlement and self‐interest, it appears that the prudent course of action for research institutions would be to proactively claim ownership of the human genomic data that they generate. How should this be accomplished in practice? I suggest that the research institution should make its ownership claim clear to all interested parties. This would include, first, a clear policy that is available online to its employees and any outside party; second, by explicitly stating in informed consent forms that the research institution will be the owner of the resulting human genomic data instances; and third, by maintaining the integrity of its ownership of its human genomic data by including relevant ownership clauses in data transfer agreements. An example of such a data ownership clause can be found in the freely available data transfer agreement template developed by Swales et al. But, what about the research participants—do they not have a stronger legal and moral claim to ownership of the human genomic data that relate to them? Moreover, should the concept of ownership of human genomic data not rather be abandoned in favour of custodianship ? In the following subsection, I analyse these opposing views to thesis that research institutions should claim ownership of the human genomic data instances that they generate. Through this analysis, I suggest that the thesis gains clarity and proves its robustness.
The first opposing position: Research participants as data owners Given that a human genomic data instance relates to an individual research participant, should he or she not be deemed the owner by virtue of this personal connection with the data? The answer must be ‘no’. The acquisition of ownership is determined by the rules of property law . Property law provides for a closed number of ways in which ownership can be acquired, and the existence of a personal connection between a person and an object is not one of them. Note, however, that having a personal connection with an object is not necessarily legally irrelevant—it may indeed be legally relevant, but in another branch of the law, namely data protection law . South African data protection law is codified in the Protection of Personal Information Act (POPIA). POPIA applies to personal information , which is information that relates to and that identifies or can identify a person, and therefore includes a person's genomic data. POPIA provides for a wide array of data protection rights that persons— data subjects in data protection law terminology—can exercise with respect of their personal information, in the same mould as the European Union's General Data Protection Regulation (GDPR). These rights include the rights (i) to have one's personal information processed in accordance with POPIA's eight conditions for processing of personal information; (ii) to be notified if one's personal information is being collected, or has been accessed by an unauthorised person; (iii) to establish whether a person holds one's personal information; (iv) to request where necessary, the correction, destruction or deletion of one's personal information; (v) to object, on reasonable grounds, to the processing of one's personal information; (vi) to object to the processing of one's personal information for purposes of direct marketing; (vii) to not be subjected to automated decision‐making based on one's personal information (under certain circumstances); (viii) to submit a complaint to the Information Regulator regarding the alleged interference with the protection of the personal information; and (ix) to institute civil proceedings regarding the alleged interference with the protection of their personal information. Some of these data protection rights have characteristics that resemble ownership, in the sense that they correspond to a lesser or greater degree with rights that are part of the ownership bundle of rights. Examples are (a) the data protection right to have one's personal information deleted, which corresponds with the right of an owner to destroy the owned object; (b) the data protection right to have one's personal information processed only if a legal ground for processing it is present, such as consent by the data subject, which can be perceived to correspond with the scenario where an owner agrees to transfer the right to use the owned object to another person, such as in the case of loan for use. However, these few instances of resemblance should not lull one into confusing data protection rights with ownership, or into thinking that data protection rights are somehow indicative of ownership. Although some data protection rights resemble ownership, they stem from a different branch of the law and have different natures and functions. For example, while ownership can in principle be transferred from one person to another, it is not possible to transfer one's data protection rights, as they are bound to one's personality. The proper legal relationship between data protection rights and ownership is that if any data protection right comes into conflict with ownership, the data protection right will trump ownership. The existence of data protection rights in no way influence the acquisition of ownership. However, the idea that research participants own the data that relate to them is found in the controversial material transfer agreement that was published by the South African Minister of Health in 2018 (SA MTA). In clause 3.3 it states that the ‘donor remains the owner of the material until such materials are destroyed’. ‘Materials’ in turn is defined as including both biological samples and data. The choice of words in clause 3.3 is strikingly contradictory, as a donor per definition transfers ownership and does not remain the owner. If ownership remains with persons who provide biological samples from their bodies, they cannot be donors and the transaction cannot be a donation . The bad drafting aside, it should be noted that the SA MTA provides that it is only a ‘framework’, meaning that its substantive provisions need not be followed. Moreover, there are good reasons why clause 3.3 of the SA MTA should not be followed: (1) The idea that research participants can acquire ownership in the data generated from their biological samples is in conflict with primary legislation and thus legally impermissible; and (2) even if it were legally permissible for research participants to acquire ownership in the data generated from their biological samples, there would be very little if any benefit to the research participants, while there would be significant downsides to the research institution. I analyse these reasons seriatim. I commence with the idea that research participants can acquire ownership in the data generated from their biological samples. To the extent that the word ‘materials’ in the SA MTA pertains to data, it can be divided into (a) data directly collected from research participants, such as their medical history, and (b) data generated using biological samples collected from research participants, such as genomic data. In the case of (a), clause 3.3 of the SA MTA can be applied, as there is nothing in South African law that would in principle militate against research participants being the owners of the data collected from them. However, the same does not apply in the case of (b), as (b) entails the collection of biological samples from research participants, which triggers section 60(4) of the National Health Act. This section prohibits persons who donate biological samples from their bodies from receiving any kind of reward for such a donation. This would likely render unlawful receiving ownership of one's newly generated genomic data instance. It can potentially be argued that the value of such ownership is so trivial that the law should ignore it. This may depend on the type of research. For example, the cost of sequencing a single gene is significantly less than the cost of sequencing a whole genome, which costs several hundred US dollars depending on the platform. But, as highlighted by Thaldar and Shozi, South African courts view the theft of goods in the amount of less than ten US dollars as non‐trivial and sufficient for a guilty verdict. Accordingly, the cost of generating the data should be truly insignificant to be legally deemed trivial and of no legal consequence. It follows that the legal tenability of clause 3.3 of the SA MTA is probably limited to (a) data directly collected from research participants, and (b) data that are generated using biological samples collected from research participants and that truly have insignificant value. However, with respect to data that are generated using biological samples collected from research participants and that have non‐trivial value—which would for practical purposes be all human genomic data instances—agreeing with research participants that they will acquire ownership in the data instances that are generated from the samples that they donated, would likely be unlawful and void. Even if it were legally permissible for research participants to be the owners of data instances that are generated from their donated samples and that have non‐trivial value, I suggest that a policy of bestowing ownership of their genomic data instances on research participants would offer little or no benefit to research participants over and above the comprehensive data protection rights that they already enjoy in terms of POPIA, while presenting considerable practical obstacles for research institutes. To understand these obstacles, one should be cognisant of the different scopes of application between data protection law and ownership. While data protection rights exist exclusively in respect of research data that qualify as personal information (research data from which research participants can be identified), ownership rights are not similarly limited, and extend to non‐personal information. In this light, consider the following scenario: A genetics research group is doing research on a specific human gene and have sequenced the alleles of tens of thousands of South Africans. Before sharing their research data, they intend to deidentify the data—i.e., ensure that none of their research participants can be identified from the allelic and associated data. Deidentification is often implemented by researchers so as to share the data more easily—especially across national borders. While the research participants qua data subjects in data protection law need not consent to such deidentification and have no data protection rights in respect of deidentified data, the research participants qua owners in property law retain their rights in their data, irrespective of whether it is identifiable or deidentified. In fact, the very act by a researcher of deidentifying data without a research participant's consent would infringe such a research participant's ownership rights. Now consider the following quotidian facts: I borrow a friend's car to drive to the supermarket. Upon returning from the supermarket, I must return the car to my friend. There is of course also the possibility of extending the borrowing of the car, by asking my friend's permission to use the car for a further period or for a further specified task. From a legal perspective, I never become the owner of the car. I just have a right to use it. But, if I wilfully continue to use my friend's car beyond the agreed borrowing period, I would be unlawfully appropriating the property of another—i.e., I would be committing theft. Would this change if I remove the car's number plates and scratch out the engine number? Can I, by removing the identifying elements of the car, argue that the car has ceased to be owned by my friend, that the car is now free‐for‐all and that I am not committing theft? Clearly, not. The same applies to research data. Hence, from the perspective of the genetics research group in the scenario sketched above, there are none of the typical benefits of deidentifying research data if the data are owned by the research participants. This is clearly a serious limitation on the researchers. Can the genetics research group approach their institutional research ethics committee to provide permission to create and share a deidentified version of their allelic dataset—given the enormous effort that obtaining consent from tens of thousands of research participants would entail? The answer is ‘no’. The institutional research ethics committee will be powerless to assist researchers in this regard, for the simple reason that once research participants are endowed with ownership , it falls beyond the remit of research ethics committees to limit such ownership in any way, as that would be an ultra vires encroachment in the field of property law. For example, if a research ethics committee allows the sharing of the deidentified dataset of alleles with a research collaborator without the consent of each one of the tens of thousands of research participants, such sharing remains an unlawful violation of the research participants’ ownership rights. Furthermore, the members of the research ethics committee would be opening themselves up to criminal prosecution (as accessories to theft) in their personal capacities. (Given that the dataset contains the allelic data of tens of thousands of research participants, it is unlikely that it can be argued that the value of the dataset is insignificant. Therefore, the risk of criminal prosecution would be real.) The solution from the perspective of the research institution would be to require research participants ( qua owners) from the outset to consent to deidentified versions of their (owned) data to be made and used freely by the research institution. However, then research participants will be owners in name only—but to what end? Clearly, the idea of research participants as data owners offers little benefit, if any, at significant cost.
The second opposing position: Abandoning the concept of ownership Should ownership of genomic data not be abandoned altogether? This is indeed what was proposed by the Academy of Science of South Africa (ASSAf) in a 2018 report on the ethical, legal and social implications of genetics and genomics in South Africa. The ASSAf report suggested that ownership of human biological samples and genomic data should be ‘avoided’ and replaced with ‘custodianship’. The reason for the proposed move away from ownership seems to be an ideological commitment to perceiving human biological samples and genomic data as a kind of natural resource, akin to the country's water resources and gold deposits, which should be managed by the state in the public interest. This natural resource view of human biological samples and genomic data has been criticised as insufficiently respectful of individual rights. It is also worth pointing out that the natural resource view provides a basis to argue against private ownership of human genomic data, but not necessarily for the non‐existence of ownership altogether. Instead, it provides a basis for arguing in favour of the public ownership of human genomic data. However, given that human genomic data may already be privately owned, for instance if a research institution claimed ownership of the genomic data sequences that it generated, public ownership of such data would require the expropriation of such data by the state. This may require an excessive amount of state resources to accomplish, which raises the question of whether the public interest in fair access to research data cannot be served in less drastic ways, such as through policy initiatives to promote data sharing. Instead of confronting the issue of expropriation, the ASSAf report proposes an ostrich policy of ‘avoiding’ ownership. However, in our law, human genomic data are susceptible of private ownership, and whether a specific human genomic data instance is owned and by whom are legal facts that can be proven in a court of law. Thus, avoiding reference to ownership is a denial of reality and hence not useful policy. Consider a scenario where a research institution generates human genomic sequence data, but decides to shun ownership and instead declares that it is the ‘custodian’ of the data that it generated. Say someone, person X, who has lawful access to the data, such as a research collaborator or a student, makes a copy of the file containing the relevant data on her own memory stick and deletes the original file from the research institution's system. X declares herself the owner of the data contained in the file on the memory stick. What are the legal consequences of these actions? First, had the research institution been the owner of the file, X's actions would have been theft. Note that section 12 of the Cybercrimes Act provides that the common law crime of theft must be interpreted so as to not exclude the theft of incorporeal property. Moreover, had the research institution been the owner of the file, the research institution would also have had civil remedies. The research institution would be able to claim the file back from X—or anyone to whom X might have transferred it—using the ancient but efficient actio rei vindicatio. However, in the scenario under consideration, the research institution failed to claim ownership. Consequently, no theft was committed (something that is not owned cannot be stolen) and the research institution cannot use the actio rei vindicatio to reclaim the file. The research institution would need to rely on contractual remedies against X—if it has any available. Contractual remedies are, however, limited in scope. For example, if X transferred the file to a third person, such a person would not be bound by the contract between the research institution and X. This is not the end of the research institution's woes. Given its express failure to claim ownership, the data was owned by no one. The fact that X has taken effective control of the data instance and has the intention to own it, means that X acquires ownership in the data instance through appropriation. This is the same as catching a wild animal like a dove that belongs to no one. The first person to take effective control of the dove with the intention of owning it, acquires ownership in it. The fact that someone declared himself or herself the ‘custodian’ of all the doves that are flying around in a market square is of no legal consequence. The research institution can attempt to insert clauses in its contracts with, among others, collaborators and students, in an attempt to stop them from claiming ownership of data that is within the research institution's deemed ‘custodianship’. However, this is weaker than if the research institution had acquired ownership. A contract will only provide civil remedies against the counterparty, not against any third parties who may come into possession of the data.
CONCLUSION The only way in which a research institution can ensure that it has the full force of the law behind it—especially in a situation where someone surreptitiously takes human genomic data from the research institution's systems—is to ensure that its legal status as owner of its research data is unassailable. This does not mean that the research institution will have carte blanche to do with the data as it pleases. In our contemporary society, absolute ownership does not exist—ownership is always qualified. A research institution's ownership of human genomic data will be qualified in two pertinent ways: First, the research institution is subject to ethics oversight. All the health research that the research institution intends to conduct with the human genomic data that it generated must be approved by its health research ethics committee, which is mandated to protect the interests of research participants. Secondly, the research institution must also comply with POPIA. POPIA applies to all data that can—through the use of a reasonably foreseeable method by the research institution—identify the research participants (the data subjects). Whenever this is the case, the research institution and the relevant principal investigator are responsible parties in terms of POPIA, with all the legal duties of a responsible party in relation to data subjects. In these ways, the research institution's ownership of its research data is clearly a qualified ownership. Yet, such qualified ownership is necessary to overcome the practical problems associated with the alternatives to research institution ownership of human genomic data, as highlighted in this article. The concept of data custodianship might still prove useful. Beyond its (rather rudimentary) ordinary meaning of someone who looks after data, it could be defined in the public policy context as referring to this rich collection of legal rights and duties that a research institution qua data owner has in respect of its data. But what is clear is that a research institution can only be an effective data custodian if it is also empowered as the data owner. Accordingly, it would be wise for South African research institutions to explicitly claim ownership of all human genomic data instances that they generate.
Work on this article was supported by the US National Institute of Mental Health and the US National Institutes of Health (award number U01MH127690) under the Harnessing Data Science for Health Discovery and Innovation in Africa (DS‐I Africa) program. The content of this article is solely the author's responsibility and does not necessarily represent the official views of the US National Institute of Mental Health or the US National Institutes of Health.
The author has no financial, personal, academic or any other interest that could be construed as a potential conflict of interest in the subject matter of this manuscript.
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Do clinical and communication skills scores on credentialing exams predict potentially inappropriate antibiotic prescribing? | da9b9a5f-da39-49b8-b3da-c76547ba17c3 | 10621187 | Family Medicine[mh] | Design and population A retrospective cohort study of international medical graduates was conducted. Physicians were eligible for inclusion in the cohort if they successfully completed the Educational Commission for Foreign Medical Graduates (ECFMG) clinical assessment OSCE examination, entered practice in the United States in primary care (family medicine, general practice, internal medicine), and conducted an evaluation and management visit for a Medicare patient whom they diagnosed as having an upper respiratory infection or sinusitis. All physicians who completed the required ECFMG clinical assessment examination were first identified. Physicians were then linked by first and last name, sex, and birthdate to the American Medical Association (AMA) Masterfile to identify those who had acquired a license to practice in the United States. The AMA national provider identifier of each physician was used to link to the Center for Medicare and Medicaid Services (CMS) administrative files to identify physicians in the cohort who had billed for Medicare patients. All patients seen by these physicians in 2014 and 2015 were identified in the Medicare Carrier RIF file, inpatient files, outpatient file, and Part D files and then all health care services and medications received by these patients by any health professional were retrieved. As earlier studies have shown associations between clinical competence, communication ability and quality of care indicators after 3 to 7 years in practice , we aimed to determine if these associations would persist, even after physicians had been in practice 8 to 15 years. Patients were eligible for consideration if they were enrolled in Medicare, had an evaluation and management visit with a study physician in an outpatient setting between July 2014 and November 2015 with a diagnosis of upper respiratory infection or acute sinusitis (Appendix for ICD codes), had continuous Part D drug coverage, had not been diagnosed with chronic sinusitis in the 6 months prior to the evaluation visit, and did not have an active supply of an antibiotic prescription on the date of the visit. The start date of follow-up allowed us to obtain at least 6 months of baseline information about patient characteristics before the evaluation visit for sinusitis or URI with the study physician. The end date allowed us a 30 day follow-up period to evaluate whether an antibiotic was prescribed/dispensed after the evaluation visit. If patients had visits for both upper respiratory infection and sinusitis, the first visit for either condition was selected. Outcome: antibiotic prescription The dispensation of an antibiotic was measured in the 30 days following the evaluation and management visit to the study physician using the Part D drug insurance file. Antibiotic drugs included the penicillins, cephalosporins, tetracyclines, macrolides, clindamycin, sulfonamides, quinolones, and metronidazole. The prescribing physician had to be the study physician who conducted the evaluation visit. Predictors Clinical skills and communication ability Scores from the Clinical Skills Assessment (CSA) OSCE Examination between 1998 and 2004 were used to measure clinical skills and communication ability. The CSA consisted of 10 or 11 modeled encounters between the candidate and a standardized patient. An overall clinical skills score was given based on history taking and physical examination conducted in these encounters and each candidate’s diagnosis and management plan as written in a post-encounter clinical note. An overall communication score was given based on the candidate’s interpersonal skills, assessed in each encounter by the standardized patient, as well as their spoken English proficiency. An acceptable clinical skills and communication score was required to pass the examination. The reliability for the various test components ranges from 0.71 to 0.82 . The CSA was put in place to ensure that all IMGs could demonstrate an acceptable level of clinical skills necessary for entry into US graduate medical education programs. The CSA was subsequently replaced by USMLE Step 2 Clinical Skills, which, as of 2004, was required for graduates of all US and foreign medical schools, up until the start of the pandemic . Other physician characteristics Physician age, sex, specialty, and practice region have been associated with a variety of quality of care indicators . These data were retrieved from the ECFMG database and the AMA Masterfile. As the type and rate of infections varies between different countries, we hypothesized that the physician’s country of origin may influence the likelihood of antibiotic prescribing. Physician citizenship at the time of medical school graduation was obtained from the ECFMG database and grouped into twelve geographic regions. Patient characteristics Patient characteristics that could influence the likelihood of antibiotic prescribing may differ between physician practices. For this reason, we measured patient sex, and race (White, Black, Asian, Hispanic, NA Native, other) using data from the CMS Master Beneficiary Summary File. Multimorbidity has been found to increase the likelihood antibiotic drug prescribing. We used the Elixhauser index to measure co-morbidities using diagnostic data from the outpatient, inpatient and carrier files in the six months prior to the evaluation and management visit. A count of the number of active medications at the time of the evaluation and management visit was also measured using the Part D files. Analysis Descriptive statistics were used to summarize physician and patient characteristics. To estimate the association between clinical skills, communication ability and the odds of antibiotic prescribing for primary and secondary prevention, we used GEE logistic regression. Patient was the unit of analysis and physician was the clustering factor, accounted for using an exchangeable correlation coefficient. The component sub-scores for clinical skills (history and physical examination, diagnosis and management) and communication (interpersonal skills and English proficiency) were fit in separate models as the adequacy of history and physical examination, diagnostic ability, and interpersonal skill proficiency have all been shown to be associated with antibiotic prescribing in prior studies . Each score was treated as a continuous variable, and models were adjusted for other physician and patient characteristics. There may be a non-linear association between clinical and communication skills scores and antibiotic prescribing, possibly a minimum threshold of skills needed to manage these encounters. To assess for non-linear effects, we included a score squared term in the model and used the Wald Chi-square to assess significance. All analyses were done using SAS version 9.4.
A retrospective cohort study of international medical graduates was conducted. Physicians were eligible for inclusion in the cohort if they successfully completed the Educational Commission for Foreign Medical Graduates (ECFMG) clinical assessment OSCE examination, entered practice in the United States in primary care (family medicine, general practice, internal medicine), and conducted an evaluation and management visit for a Medicare patient whom they diagnosed as having an upper respiratory infection or sinusitis. All physicians who completed the required ECFMG clinical assessment examination were first identified. Physicians were then linked by first and last name, sex, and birthdate to the American Medical Association (AMA) Masterfile to identify those who had acquired a license to practice in the United States. The AMA national provider identifier of each physician was used to link to the Center for Medicare and Medicaid Services (CMS) administrative files to identify physicians in the cohort who had billed for Medicare patients. All patients seen by these physicians in 2014 and 2015 were identified in the Medicare Carrier RIF file, inpatient files, outpatient file, and Part D files and then all health care services and medications received by these patients by any health professional were retrieved. As earlier studies have shown associations between clinical competence, communication ability and quality of care indicators after 3 to 7 years in practice , we aimed to determine if these associations would persist, even after physicians had been in practice 8 to 15 years. Patients were eligible for consideration if they were enrolled in Medicare, had an evaluation and management visit with a study physician in an outpatient setting between July 2014 and November 2015 with a diagnosis of upper respiratory infection or acute sinusitis (Appendix for ICD codes), had continuous Part D drug coverage, had not been diagnosed with chronic sinusitis in the 6 months prior to the evaluation visit, and did not have an active supply of an antibiotic prescription on the date of the visit. The start date of follow-up allowed us to obtain at least 6 months of baseline information about patient characteristics before the evaluation visit for sinusitis or URI with the study physician. The end date allowed us a 30 day follow-up period to evaluate whether an antibiotic was prescribed/dispensed after the evaluation visit. If patients had visits for both upper respiratory infection and sinusitis, the first visit for either condition was selected.
The dispensation of an antibiotic was measured in the 30 days following the evaluation and management visit to the study physician using the Part D drug insurance file. Antibiotic drugs included the penicillins, cephalosporins, tetracyclines, macrolides, clindamycin, sulfonamides, quinolones, and metronidazole. The prescribing physician had to be the study physician who conducted the evaluation visit.
Clinical skills and communication ability Scores from the Clinical Skills Assessment (CSA) OSCE Examination between 1998 and 2004 were used to measure clinical skills and communication ability. The CSA consisted of 10 or 11 modeled encounters between the candidate and a standardized patient. An overall clinical skills score was given based on history taking and physical examination conducted in these encounters and each candidate’s diagnosis and management plan as written in a post-encounter clinical note. An overall communication score was given based on the candidate’s interpersonal skills, assessed in each encounter by the standardized patient, as well as their spoken English proficiency. An acceptable clinical skills and communication score was required to pass the examination. The reliability for the various test components ranges from 0.71 to 0.82 . The CSA was put in place to ensure that all IMGs could demonstrate an acceptable level of clinical skills necessary for entry into US graduate medical education programs. The CSA was subsequently replaced by USMLE Step 2 Clinical Skills, which, as of 2004, was required for graduates of all US and foreign medical schools, up until the start of the pandemic . Other physician characteristics Physician age, sex, specialty, and practice region have been associated with a variety of quality of care indicators . These data were retrieved from the ECFMG database and the AMA Masterfile. As the type and rate of infections varies between different countries, we hypothesized that the physician’s country of origin may influence the likelihood of antibiotic prescribing. Physician citizenship at the time of medical school graduation was obtained from the ECFMG database and grouped into twelve geographic regions. Patient characteristics Patient characteristics that could influence the likelihood of antibiotic prescribing may differ between physician practices. For this reason, we measured patient sex, and race (White, Black, Asian, Hispanic, NA Native, other) using data from the CMS Master Beneficiary Summary File. Multimorbidity has been found to increase the likelihood antibiotic drug prescribing. We used the Elixhauser index to measure co-morbidities using diagnostic data from the outpatient, inpatient and carrier files in the six months prior to the evaluation and management visit. A count of the number of active medications at the time of the evaluation and management visit was also measured using the Part D files.
Scores from the Clinical Skills Assessment (CSA) OSCE Examination between 1998 and 2004 were used to measure clinical skills and communication ability. The CSA consisted of 10 or 11 modeled encounters between the candidate and a standardized patient. An overall clinical skills score was given based on history taking and physical examination conducted in these encounters and each candidate’s diagnosis and management plan as written in a post-encounter clinical note. An overall communication score was given based on the candidate’s interpersonal skills, assessed in each encounter by the standardized patient, as well as their spoken English proficiency. An acceptable clinical skills and communication score was required to pass the examination. The reliability for the various test components ranges from 0.71 to 0.82 . The CSA was put in place to ensure that all IMGs could demonstrate an acceptable level of clinical skills necessary for entry into US graduate medical education programs. The CSA was subsequently replaced by USMLE Step 2 Clinical Skills, which, as of 2004, was required for graduates of all US and foreign medical schools, up until the start of the pandemic .
Physician age, sex, specialty, and practice region have been associated with a variety of quality of care indicators . These data were retrieved from the ECFMG database and the AMA Masterfile. As the type and rate of infections varies between different countries, we hypothesized that the physician’s country of origin may influence the likelihood of antibiotic prescribing. Physician citizenship at the time of medical school graduation was obtained from the ECFMG database and grouped into twelve geographic regions.
Patient characteristics that could influence the likelihood of antibiotic prescribing may differ between physician practices. For this reason, we measured patient sex, and race (White, Black, Asian, Hispanic, NA Native, other) using data from the CMS Master Beneficiary Summary File. Multimorbidity has been found to increase the likelihood antibiotic drug prescribing. We used the Elixhauser index to measure co-morbidities using diagnostic data from the outpatient, inpatient and carrier files in the six months prior to the evaluation and management visit. A count of the number of active medications at the time of the evaluation and management visit was also measured using the Part D files.
Descriptive statistics were used to summarize physician and patient characteristics. To estimate the association between clinical skills, communication ability and the odds of antibiotic prescribing for primary and secondary prevention, we used GEE logistic regression. Patient was the unit of analysis and physician was the clustering factor, accounted for using an exchangeable correlation coefficient. The component sub-scores for clinical skills (history and physical examination, diagnosis and management) and communication (interpersonal skills and English proficiency) were fit in separate models as the adequacy of history and physical examination, diagnostic ability, and interpersonal skill proficiency have all been shown to be associated with antibiotic prescribing in prior studies . Each score was treated as a continuous variable, and models were adjusted for other physician and patient characteristics. There may be a non-linear association between clinical and communication skills scores and antibiotic prescribing, possibly a minimum threshold of skills needed to manage these encounters. To assess for non-linear effects, we included a score squared term in the model and used the Wald Chi-square to assess significance. All analyses were done using SAS version 9.4.
A total of 2,526 physicians were included in the cohort, 54.8% of whom were male with a mean age of 43.8 years (Table ). The majority were citizens of India (25.7%), United States (21.1%), or Asia/Oceania (13.8%), 58.3% specialized in family medicine, and 37.7% practiced in the south of the United States. With respect to clinical skills, physicians scored higher on their skill in conducting a history and physical examination (mean 67.8%) compared to diagnosis and management (mean: 59.3%). For communication, English proficiency scores (mean 86.0%) were higher than interpersonal skills (mean 76.6%). Overall, 48,394 patients were eligible for inclusion, 66.5% were female, 81.2% were white, with a mean age of 68.9 years (Table ). The presenting problem was diagnosed as upper respiratory infection (URI) in 56.5% of encounters and acute sinusitis in 43.5%. Most patients had co-morbid conditions, with 34.8% having five or more conditions, and 33.6% were taking six or more prescribed medications. In 59.5% of encounters, an antibiotic was prescribed, in 71.1% of encounters for sinusitis and in 50.5% for upper respiratory infections. The most common antibiotics prescribed were azithromycin (URI 53.8%; sinusitis 33.3%) and amoxicillin (URI 21.2%; sinusitis 38.5%). Greater proficiency in history and physical examination (OR 0.97) and diagnosis and management (OR 0.99) was associated with a non-significant reduction in the odds of antibiotic prescribing (Table ). While there was no association between English proficiency and antibiotic prescribing, interpersonal skills scores were associated with a significant reduction in the odds of antibiotic prescribing. For every decile increase in score, the odds of antibiotic prescribing were reduced by 7% (OR 0.93, 95% CI 0.87–0.99). This translates into a probability of receiving an antibiotic prescription of 57% for a physician who scores 62% in interpersonal skills and 49% for a physician scoring 92%; respectively two standard deviations below and above the mean interpersonal skills score. There was no evidence of a non-linear relationship. After adjusting for interpersonal skills and other physician and patient characteristics, female physicians were 14% (OR 1.14, 95% CI 1.03–1.26) more likely to prescribe antibiotics compared to males (Table ). Compared to physicians who were citizens of the United States at the time they completed medical school, physicians from all other countries were less likely to prescribe antibiotics, differences that were significant for Africa (OR 0.71, 95% CI 0.57–0.88), Central America (OR 0.49, 95% CI 0.38–0.63), and South America (OR 0.67, 95% CI 0.51–0.87). Physicians specializing in family medicine were 19% (OR 0.81, 95% CI 0.74–0.90) less likely to prescribe antibiotics compared to internal medicine specialists, and those practicing in the south and west regions of the United States were more and less likely to prescribe compared to the northeast, respectively. Patients were more likely to receive an antibiotic if they presented with acute sinusitis (OR 2.76, 95% CI 2.52–3.02) compared to upper respiratory infection, and were using a greater number of medications. They were less likely to receive an antibiotic if they were black compared to white and had higher levels of co-morbidity.
The ECFMG was the first agency in the world to introduce a standardized assessment of clinical and communication skills as part of the credentialing process. We found that physicians with higher scores for interpersonal skills on this examination were less likely to prescribe antibiotics for upper respiratory infection and acute sinusitis, even after 8 to 15 years in practice. Greater proficiency in history and physical examination, as well as diagnosis and management on the examination, were not significantly associated with antibiotic prescribing. Female physicians, internal medicine specialists, and those practicing in the south regions of the United States were also more likely to prescribe antibiotics. Physicians who were citizens of South or Central America were also less likely to prescribe compared to US citizens. Although better interpersonal skills were associated with lower odds of antibiotic prescribing, better clinical skills were not. There may be several reasons for these differences in findings. First, this examination is taken before entering U.S residency programs that are designed to improve proficiency in data collection as well as diagnosis and management skills . Thus, measurement of these clinical skills at the time of entry into residency may not reflect abilities 8 to 15 years after starting clinical practice. Second, while communications training has been shown to improve skills for some individuals, it may represent a more stable skill set that is less amenable to sustained improvement with training. Indeed, interpersonal skills assessment has been introduced as part of the admissions process by some medical schools in order to select applicants who excel in this area. While the ECFMG was the first agency to introduce the OSCE as part of the credentialing process, it has since been implemented as a requirement for licensure in both Canada and the United States . Communication skills measured in the Canadian OSCE credentialing examination have been shown to predict the frequency of complaints to medical licensing authorities in the first 5 to 7 years in practice . Other studies have found that the quality of a physician’s interpersonal skills increases patient satisfaction and adherence to treatment as well as health outcomes . Combined, these findings suggest that there may be benefits in measuring proficiency in interpersonal skills at medical school admission and on credentialing examinations because of their importance in optimizing the quality of clinical practice. There are several mechanisms by which better communication skills may reduce the likelihood of antibiotic prescribing. First, physicians may be more likely to engage in patient education about the differences between viral and bacterial infections and their treatment . Second, they may be more likely to address patient’s concerns about the severity of their symptoms and effective treatment . Failure to address patient concerns has been shown to increase the likelihood of antibiotic prescribing . Third, physicians with better communication skills may be more effective at addressing patient demand for antibiotics, using such strategies as online commentary during the physical examination about normal findings, the provision of contingency plans, and patient empowerment . Indeed intervention studies to improve physician communication in managing patient expectations has been shown to reduce antibiotic prescribing . The clinical and communication skills assessment component of both the United States Medical Licensure Examination (USMLE) and the Canadian Medical Council Examination was interrupted during the pandemic. The USLME Step 2 CS was subsequently permanently cancelled in 2021. Opponents to the examination pointed to the high cost for students who needed to travel to test centres for the examination, the existence of performance-based assessment of clinical and communication skills using OSCE -like examinations in most North American medical schools , and the need for the examination given an overall pass rate of over 90% . Others have pointed to the adverse effects of eliminating the examination for international medical graduates and physicians from allopathic and osteopathic medical schools . Regardless of the outcome, there is consensus that performance based assessment of clinical and communication skills is important and new methods of assessment are needed whether that be imbedded in accreditation processes of medical schools or as a component of licensing examinations. Our findings are consistent with other studies that have shown that internal medicine specialists are more likely to prescribe antibiotics than family physicians , as are physicians practicing in the south . Once we adjusted for interpersonal skills, we did not find that older physicians or males were more likely to prescribe antibiotics, associations that have been reported in a number of other studies . Female physicians receive higher scores on OSCE examinations, primarily related to better communication skills . However, even after adjustment for interpersonal skills, female physicians were more likely to prescribe antibiotics. In exploratory analysis, we found that female physicians were more likely to prescribe antibiotics for patients presenting with sinusitis (OR: 1.46, 95% CI 1.28–1.67), not URI (OR: 1.10, 95% CI 0.99–1.24). Female physicians are more likely than male physicians to prescribe medication for pain . Compared to URIs, patients with sinusitis are more likely to have considerable pain in relationship to their infection , which may be why, on the off chance that it is a bacterial not a viral infection, female physicians are more likely to prescribe antibiotics. Another possibility is that patients are more likely to ask or demand antibiotics from female physicians, as there are differences in male and female patient behavior by physician gender . The citizenship of the international medical graduate at the time of their medical school education was significantly associatied with antibiotic prescribing, with the odds of prescribing being lower among citizens of all other countries compared to U.S. citizens, significantly so for Africa, South and Central America. Substantial differences in antibiotic prescribing by dentists in the U.S, Canada, Australia and England has also been reported with US dentists being the highest . Future research should focus on the role of culture and medical education in promulgation of unnecessary antibiotic prescribing. Similar to prior studies, we found patients with sinusitis are more likely to receive antibiotics than patients with upper respiratory infections , as were white patients compared to black patients and patients with higher levels of co-morbidity when measured by the number of concurrent medications. Our research has limitations that need to be considered in interpreting the results. First, we had no information on the clinical signs and symptoms that patients presented with at the time of the visit for sinusitis or upper respiratory infection with the study physicians. Both positive and worsening symptoms increase the risk of antibiotic prescribing . While we do not expect there to be differences in the severity of a patient’s presentation in relationship to the physician’s clinical or communication skills, internal medicine specialists may see more symptomatic, co-morbid patients, which may explain the greater risk of antibiotic prescribing, although these differences, if any, appear to be modest . We also had no measure of visit length or daily practice volume. Shorter visits and higher daily volume increase the risk of antibiotic prescribing . Physicians with better interpersonal skills may spend more time with patients, and as a result see fewer patients per day. This may be one plausible mechanism for why shorter visits and high volume increase the risk of antibiotic prescribing. In conclusion, physicians who are international medical school graduates with better interpersonal skills are less likely to prescribe antibiotics to patients presenting with upper respiratory infection and acute sinusitis. Future research should examine whether tailored interpersonal skills training to help physicians manage patient expectations for antibiotics during medical school or continuing professional education would reduce the risk of unnecessary antibiotic prescribing.
Below is the link to the electronic supplementary material. Supplementary Material 1
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Comparison of PD-L1 tumor cell expression with 22C3, 28-8, and SP142 IHC assays across multiple tumor types | 793b5329-2533-4ded-8e5a-1d357129ebdb | 9621188 | Anatomy[mh] | Prior studies have shown high concordance between 22C3 and 28-8 programmed death ligand 1 (PD-L1) immunohistochemistry (IHC) assays, with lower tumor cell (TC) expression with the SP142 assay in various controlled and clinical settings, which focused on either a limited number (<100) of tumor specimens, or a larger number (>100) of tumor specimens but of a single tumor type, or were unable to assess all three (22C3, 28-8, and SP142 assays) on each tumor specimen.
We present the PD-L1 TC expression scores of 22C3, 28-8, and SP142 IHC assays performed on 418 specimens of multiple tumor types encountered in routine clinical practice. We demonstrate that 22C3 is the most sensitive PD-L1 IHC assay, followed in turn by 28-8 and SP-142.
The findings represent an additional factor for clinicians to consider when selecting which PD-L1 IHC assay is most appropriate for each individual patient.
Immunotherapy has revolutionized cancer care over the past decade, and new treatment strategies continue to evolve. Programmed death-1/programmed death ligand 1 (PD-1/PD-L1) inhibitors have been approved in the USA and around the world to treat and improve outcomes for patients with a range of tumor types, including non-small cell lung cancer (NSCLC), melanoma, triple-negative breast cancer, and small cell lung cancer. PD-L1 is a transmembrane protein that binds to receptors, PD-1 or B7.1, to downregulate the immune response. PD-1 is an inhibitory receptor predominantly expressed on T cells following T cell activation in response to chronic inflammation, as may occur during infection or within the tumor microenvironment. Binding of PD-L1 to PD-1 inhibits T cell proliferation, cytokine production, and cytolytic activity, leading to functional inactivation of T cells. PD-L1 expression can occur on the surface of both tumor cells (TCs) and tumor-associated immune cells (ICs). Upregulated PD-L1 expression on TCs can therefore enable the tumor to evade the immune response. Blockade of PD-L1/PD-1 ligation has become a strategy to restore tumor-specific T cell immunity, and positive expression of PD-L1 on the surface of TCs or ICs has been correlated with clinical benefit and successful treatment with PD-L1/PD-1 inhibitors across a range of cancer types. The PD-1/PD-L1 treatment landscape is continually adapting to new clinical trial and outcomes data and new Food and Drug Administration (FDA) approvals. Alongside the generation of new PD-1/PD-L1 inhibitors, various immunohistochemistry (IHC) assays have been developed to measure PD-L1 expression and have received approval from the FDA as either companion or complementary diagnostic assays. A companion diagnostic assay (CDx) is defined by the FDA as a diagnostic test that provides required information that is ‘essential for the safe and effective use of a corresponding drug or biological product’. In the European Union, a CDx is defined as ‘a device which is essential for the safe and effective use of a corresponding medicinal product to (a) identify, before and/or during treatment, patients who are most likely to benefit from the corresponding medicinal product; or (b) identify, before and/or during treatment, patients likely to be at increased risk of serious adverse reactions as a result of treatment with the corresponding medicinal product’. To date, the FDA has approved four CDx PD-L1 IHC assays: Dako 22C3 (hereon referred to as 22C3; as a CDx for treatment with pembrolizumab in patients with a range of solid tumors, see ); Ventana SP142 (hereon referred to as SP142; as a CDx for treatment with atezolizumab in patients with urothelial carcinoma or NSCLC); Dako 28-8 (hereon referred to as 28-8; as a CDx for treatment with combination of ipilimumab and nivolumab in patients with NSCLC); and Ventana SP263 (hereon referred to as SP263; as a CDx for treatment with atezolizumab in patients with NSCLC). It should be noted, however, that pembrolizumab is also FDA approved for the treatment of all solid tumors regardless of PD-L1 status if microsatellite instability - high (MSI-H) or tumor mutational burden ≥10 mutations/megabase. A complementary diagnostic test is broadly defined as ‘a test that identifies a biomarker-defined subset of patients that respond particularly well to a drug and aid risk/benefit assessments for individual patients, but that are not prerequisites for receiving the drug’. In the USA, the currently approved complementary diagnostic PD-L1 assays include the 28-8 assay for identifying patients with non-squamous NSCLC, head and neck squamous cell carcinoma (HNSCC) and urothelial carcinoma for treatment with nivolumab and the SP263 assay for identifying patients with urothelial carcinoma for treatment with durvalumab. Treatment with PD-1/PD-L1 inhibitors is indicated when the FDA-approved companion or complementary diagnostic assay demonstrates PD-L1 expression in the patient’s tumor above a specified cut-off. lists the cut-offs and various other features of the four main PD-L1 assays available in the USA. Prior studies have investigated the concordance between two or more PD-L1 IHC assays in controlled and clinical settings. The consensus of these reports is that there is high concordance in TC expression scores between 22C3 and 28-8 IHC assays and much lower TC expression with the SP142 assay. However, several of these studies focused on a limited number (<100) of tumor specimens, while others that evaluated a larger number (>100) of specimens were unable to assess all three IHC assays (22C3, 28-8, and SP142) or analyze more than a single tumor type. In the current study, we analyzed 418 specimens from multiple tumor types (including 199 NSCLC specimens) with three PD-L1 IHC assays (22C3, 28-8, and SP142) during routine clinical practice. These specimens included a range of resection, core needle biopsy, and cytology patient samples. We present the PD-L1 TC expression scores for each specimen, as well as the histology and IHC staining patterns for select clinical cases to showcase important implications for patient treatment and clinical decision making.
Patient cohort The Foundation Medicine database (Morrisville, North Carolina, USA) was searched to collect advanced solid tumor specimens that were tested with all three PD-L1 IHC assays (DAKO 22C3, DAKO 28-8, and Ventana SP142) during the year starting in December 2020 and ending in December 2021. A total of 418 patient specimens (including 199 patient NSCLC specimens) were identified that fulfilled these criteria. Of these 418 specimens, 55.3% (231/418) were biopsies, 41.6% (174/418) were resection specimens, and 3.1% (13/418) were cytology specimens. DAKO PD-L1 IHC 22C3 pharmDx assay The DAKO PD-L1 IHC 22C3 pharmDx assay was performed per manufacturer’s instructions in a Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited reference laboratory (Foundation Medicine). The DAKO PD-L1 22C3 pharmDx assay is an immunohistochemical assay using mouse monoclonal anti-PD-L1 (22C3 clone) for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) tissue using the EnVision FLEX visualization system on Autostainer Link 48 and associated staining protocol provided by the package insert and interpreted with the guidelines of the DAKO interpretation guide. All cases had accompanying controls, H&E-stained patient slide, negative reagent control stained patient slide, and a DAKO PD-L1 22C3-stained patient slide. PD-L1 stained slides were evaluated using a tumor proportion score (TPS) method, where TPS=(number of PD-L1-stained tumor cells/total number of viable tumor cells) × 100. A cut-off of TPS ≥1 was used to determine positivity for the 22C3 assay. It should be noted here that the terms TPS and TC expression (see below for 28-8) are often used interchangeably and refer to essentially the same scoring system. Of note, positivity in NSCLC was determined using a cut-off of TPS ≥1 per CDx for pembrolizumab. DAKO PD-L1 28-8 pharmDx assays The DAKO PD-L1 28-8 pharmDx assay was performed per manufacturer’s instructions in a CLIA-certified and CAP-accredited reference laboratory (Foundation Medicine). In brief, the DAKO PD-L1 28-8 pharmDx assay is an immunohistochemical assay using rabbit monoclonal anti-PD-L1, for use in the detection of PD-L1 protein in FFPE tissue with the EnVision FLEX visualization system on Autostainer Link 48, and associated staining protocol provided by the package insert and interpreted with the guidelines of the DAKO interpretation guide. All cases had accompanying controls, H&E-stained patient slide, negative reagent control-stained patient slide, and a DAKO PD-L1 28-8-stained patient slide. PD-L1 stained slides were evaluated using a tumor cell expression (TC) scoring method, where TC=(number of PD-L1-stained tumor cells/total number of viable tumor cells) × 100. A cut-off of TC ≥1% was used to determine positivity for the DAKO PD-L1 28-8 pharmDx assay. Of note, positivity in NSCLC was determined using a cut-off per CDx for nivolumab in combination with ipilimumab using a TC cut-off score of 1. Ventana PD-L1 SP142 CDx assay PD-L1 SP142 testing was performed using the Ventana SP142 CDx assay per manufacturer’s instructions in a CLIA-certified and CAP-accredited reference laboratory (Foundation Medicine). In brief, the Ventana SP142 CDx assay consists of the rabbit monoclonal anti-PD-L1 SP142 clone, the Opti-View DAB IHC detection kit, and the Opti-View Amplification Kit stained on the Ventana BenchMark ULTRA instrument using the staining protocol provided by the package insert and interpreted with the guidelines of the Ventana interpretation guide. All cases had an accompanying H&E-stained patient slide, negative regent control stained patient slide with an on-slide tonsil control and a Ventana PD-L1 SP142 stained patient slide with an on-slide tonsil control. TC levels were determined similarly to the TPS method. A cut-off level of ≥1% staining TCs was used to determine tumor cell expression. (As part of routine clinical practice, the tumor infiltrating ICs scoring method was also used to generate an IC score, which is defined as proportion of tumor area that is occupied by PD-L1 staining IC of any intensity and is not reported in this study). Data analysis Comparison of all continuous variables were performed using Kruskal-Wallis test using the R software V.4.0.3.
The Foundation Medicine database (Morrisville, North Carolina, USA) was searched to collect advanced solid tumor specimens that were tested with all three PD-L1 IHC assays (DAKO 22C3, DAKO 28-8, and Ventana SP142) during the year starting in December 2020 and ending in December 2021. A total of 418 patient specimens (including 199 patient NSCLC specimens) were identified that fulfilled these criteria. Of these 418 specimens, 55.3% (231/418) were biopsies, 41.6% (174/418) were resection specimens, and 3.1% (13/418) were cytology specimens.
The DAKO PD-L1 IHC 22C3 pharmDx assay was performed per manufacturer’s instructions in a Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited reference laboratory (Foundation Medicine). The DAKO PD-L1 22C3 pharmDx assay is an immunohistochemical assay using mouse monoclonal anti-PD-L1 (22C3 clone) for use in the detection of PD-L1 protein in formalin-fixed, paraffin-embedded (FFPE) tissue using the EnVision FLEX visualization system on Autostainer Link 48 and associated staining protocol provided by the package insert and interpreted with the guidelines of the DAKO interpretation guide. All cases had accompanying controls, H&E-stained patient slide, negative reagent control stained patient slide, and a DAKO PD-L1 22C3-stained patient slide. PD-L1 stained slides were evaluated using a tumor proportion score (TPS) method, where TPS=(number of PD-L1-stained tumor cells/total number of viable tumor cells) × 100. A cut-off of TPS ≥1 was used to determine positivity for the 22C3 assay. It should be noted here that the terms TPS and TC expression (see below for 28-8) are often used interchangeably and refer to essentially the same scoring system. Of note, positivity in NSCLC was determined using a cut-off of TPS ≥1 per CDx for pembrolizumab.
The DAKO PD-L1 28-8 pharmDx assay was performed per manufacturer’s instructions in a CLIA-certified and CAP-accredited reference laboratory (Foundation Medicine). In brief, the DAKO PD-L1 28-8 pharmDx assay is an immunohistochemical assay using rabbit monoclonal anti-PD-L1, for use in the detection of PD-L1 protein in FFPE tissue with the EnVision FLEX visualization system on Autostainer Link 48, and associated staining protocol provided by the package insert and interpreted with the guidelines of the DAKO interpretation guide. All cases had accompanying controls, H&E-stained patient slide, negative reagent control-stained patient slide, and a DAKO PD-L1 28-8-stained patient slide. PD-L1 stained slides were evaluated using a tumor cell expression (TC) scoring method, where TC=(number of PD-L1-stained tumor cells/total number of viable tumor cells) × 100. A cut-off of TC ≥1% was used to determine positivity for the DAKO PD-L1 28-8 pharmDx assay. Of note, positivity in NSCLC was determined using a cut-off per CDx for nivolumab in combination with ipilimumab using a TC cut-off score of 1.
PD-L1 SP142 testing was performed using the Ventana SP142 CDx assay per manufacturer’s instructions in a CLIA-certified and CAP-accredited reference laboratory (Foundation Medicine). In brief, the Ventana SP142 CDx assay consists of the rabbit monoclonal anti-PD-L1 SP142 clone, the Opti-View DAB IHC detection kit, and the Opti-View Amplification Kit stained on the Ventana BenchMark ULTRA instrument using the staining protocol provided by the package insert and interpreted with the guidelines of the Ventana interpretation guide. All cases had an accompanying H&E-stained patient slide, negative regent control stained patient slide with an on-slide tonsil control and a Ventana PD-L1 SP142 stained patient slide with an on-slide tonsil control. TC levels were determined similarly to the TPS method. A cut-off level of ≥1% staining TCs was used to determine tumor cell expression. (As part of routine clinical practice, the tumor infiltrating ICs scoring method was also used to generate an IC score, which is defined as proportion of tumor area that is occupied by PD-L1 staining IC of any intensity and is not reported in this study).
Comparison of all continuous variables were performed using Kruskal-Wallis test using the R software V.4.0.3.
Patient cohort demographics A detailed summary of patient cohort demographics is provided in . The mean and median ages of the entire patient cohort were 67.3 and 68.5 years old, respectively. A total of 48.1% (201/418 cases) of the patients were female. A total of 39.0% (163/418) specimens received were from a primary tumor site, 32.8% (137/418) specimens from metastatic site, and in the remaining 28.2% (118/418) of specimens the tumor site was ambiguous or unknown. The mean and median ages of the NSCLC patient cohort were 69.7 and 69.0 years old, respectively. A total of 41.7% (83/199 cases) of the patients were female. A total of 52.8% (105/199) NSCLC specimens were from the primary tumor site (lung), 30.7% (61/199) specimens from a metastatic site, and 16.6% (33/199) from a specimen site that was either ambiguous or unknown. In addition, NSCLC specimens were received from patients with stage IV (61.3%, 122/199), stage III (16.6%, 33/199), stage II (5.0%, 10/199), and stage I disease (8.0%, 16/199); the remaining specimens were received from patients with unknown stage of disease (9.0%, 18/199). The disease diagnoses for NSCLC specimens included lung adenocarcinoma (61.3%, 122/199 cases), lung squamous cell carcinoma (26.6%, 53/199 cases), NSCLC (not otherwise specified) (11.6%, 23/199 cases), and adenosquamous carcinoma (0.5%, 1/199 cases). In addition, the total tumor cohort included eight small cell lung cancer (SCLC) specimens (8/418 total tumor cases, or 1.9% of all tumor cases). Overall PD-L1 tumor cell expression patterns shows the PD-L1 status with all three antibody clones in resection, biopsy and cytology specimens. No appreciable difference in tumor cell expression was observed between specimen types. A higher percentage of triple negative (22C3-/28-8−/SP142−) cases are observed in cytology specimens, but this corresponds to only 7 out of 13 total cytology specimens. The proportion of resection, biopsy and cytology specimens with an IC score of ≥1 were 62.6% (65/174), 46.3% (107/231), and 30.8% (4/13), respectively, and the proportion of resection, biopsy and cytology specimens with an IC score <1 were 37.4% (65/174), 53.2% (123/231), and 69.2% (9/13), respectively. Tumor cell PD-L1 expression as detected with 22C3, 28-8 and SP142 IHC assays across all tumor types are shown in . Higher positive tumor cell staining was observed with 22C3 compared with 28-8 and SP142 in NSCLC, neuroendocrine tumor, melanoma, soft tissue, and overall tumor specimens. There were no clear or significant differences in PD-L1 tumor cell expression status between the three different antibody clones for GYN, genitourinary, GI, or CNS tumor specimens. provides an analytical comparison of tumor cell expression staining, by case, for 22C3 (dark blue triangles), 28-8 (light green circles), and SP142 (yellow squares) PD-L1 IHC assays across all tumor cases (n=418; ) and all NSCLC cases (n=199; ). The ‘best fit’ curves show that for all tumor cases (and NSCLC cases) that were positive with all three IHC assays, the average 22C3 TC score was typically 10–20 percentage points higher than 28-8 scores, which were in turn approximately 40–60 percentage points higher than SP142 scores. The analytical concordance between the three antibody clones across all tumor specimens (n=418) is shown in . The same PDL-1 status with all three antibody clones (either TC ≥1% with all three assays or TC <1% with all three assays) was observed in 60.0% (251/418) of all tumor specimens: 45.9% (192/418) of all tumor cases were triple negative and 14.1% (59/418) of all tumor cases were triple positive. A different PD-L1 status was observed with the three antibody clones in 40.0% (167/418) of all tumor specimens; and among these cases, 62.3% (104/167) were 22C3+/28-8+/SP142−, and 28.7% (48/167) were 22C3+/28-8−/SP142−. A total of 54.1% (226/418) of all tumor cases were positive with at least 1 IHC assay (22C3, 28-8 or SP142) and among these cases, 94.2% (213/226) were positive for 22C3, 77.0% (174/226) were positive for 28-8, and 28.8% (65/226) were positive for SP142. shows the positive and negative PD-L1 status with all three antibody clones for NSCLC specimens (n=199). The same PD-L1 status with all three antibody clones (either all assays demonstrating TC ≥1% or all demonstrating TC<1%) was observed in 48.7% (97/199) of all NSCLC specimens. Of these NSCLC specimens showing the same PD-L1 status with all three antibody clones, 54.6% (53/97) were triple negative, and 45.4% (44/97) were triple positive. A different PD-L1 status was observed among all three antibody clones in 51.3% (102/199) of all NSCLC specimens. Among these NSCLC cases showing a different status, 67.6% (69/102) of cases were 22C3+/28-8+/SP142−, and 23.5% (24/102) were 22C3+/28-8−/SP142−. A total of 73.4% (146/199) of all NSCLC cases were positive with at least one IHC assay (22C3, 28-8, or SP142), compared with 54.1% (226/418) of tumor specimens overall. Among these cases that were positive with at least one IHC assay, 22C3 was positive in 95.2% (139/146) of cases, 82.2% (120/146) were positive for 28-8, and 32.2% (47/146) were positive for SP142. shows the positive and negative PD-L1 status with all three antibody clones for different types of lung cancer specimens. The PD-L1 status was the same (≥ 1% TC expression with all three clones, or <1% TC expression with all three clones) with all three antibody clones in 50.8% (62/122) of all lung adenocarcinoma specimens, 32.1% (17/53) of all lung squamous cell carcinoma specimens, 69.6% (16/23) of all NSCLC (NOS) specimens, and 62.5% (5/8) of all small cell lung carcinoma specimens. A total of 72.1% (88/122) of adenocarcinoma cases were positive with at least one antibody clone, compared with 81.1% (43/53) of all squamous cell carcinoma specimens, 69.6% (16/23) of all NSCLC (NOS) specimens, and 50.0% (4/8) of all small cell lung carcinoma specimens. Among lung adenocarcinoma specimens that were positive with at least one antibody clone: 95.5% (84/88) were positive for 22C3, 83.0% (73/88) were positive for 28-8, and 23.8% (29/122) were positive for SP142. Among squamous cell carcinoma specimens that were positive with at least one antibody clone: 93.0% (40/43) were positive for 22C3, 79.1% (34/43) were positive for 28-8, and 16.3% (7/43) were positive for SP142. Among NSCLC (NOS) specimens that were positive with at least one antibody clone: 100.0% (16/16) were positive for 22C3, 75.0% (12/16) were positive for 28-8, and 62.5% (10/23) were positive for SP142. Finally, among small cell lung carcinoma specimens that were positive with at least one antibody clone: 100.0% (4/4) were positive for 22C3, 75.0% (3/4) were positive for 28-8, and 25.0% (1/4) were positive for SP142. shows the positive and negative PD-L1 status with all three antibody clones for primary versus metastatic NSCLC specimens. The PD-L1 status was the same (either ≥1% TC expression with all three clones, or <1% TC expression with all three clones) with all three antibody clones in 42.5% (45/106) of all primary NSCLC specimens, 50.7% (35/69) of all metastatic NSCLC specimens, and 50.0% (12/24) of all NSCLC specimens with undetermined specimen location. It was observed that 78.3% (83/106) primary NSCLC specimens were positive with at least one antibody clone, compared with 65.2% (45/69) of metastatic NSCLC specimens, and 83.3% (20/24) of all NSCLC specimens with undetermined specimen location. Among primary NSCLC specimens that were positive with at least one antibody clone: 97.6% (81/83) were positive for 22C3, 78.3% (65/83) were positive for 28-8, and 27.7% (23/83) were positive for SP142. Among metastatic NSCLC specimens that were positive with at least one antibody clone: 93.3% (42/45) were positive for 22C3, 80.0% (36/45) were positive for 28-8, and 31.1% (14/45) were positive for SP142. Among NSCLC cases with undetermined specimen location that were positive with at least one antibody clone: 91.7% (22/24) were positive for 22C3, 100.0% (24/24) were positive for 28-8, and 50.0% (12/24) were positive for SP142. Clinical implication with clinical vignettes The histology and PD-L1 tumor cell expression pattern with each of the three antibody clones (22C3, 28-8 and SP142) for three separate NSCLC patient specimens are shown in . These three cases are representative of most tumor specimens observed with a positive PD-L1 status, in that TC score is greatest with 22C3, followed by 28-8, followed by SP142 having the lowest TC score. A score of TC ≥1 was reported with 22C3 for all three specimens, qualifying all three patients for treatment with pembrolizumab or cemiplimab-rwlc. Furthermore, a score of TC ≥1 with 28-8 was reported for all three specimens, qualifying all three patients for treatment with nivolumub or nivolumab in combination with ipilimumab. While specimens from patient 1 and patient 3 both exhibited SP142 scores of TC ≥1 (and are therefore considered having a positive PD-L1 status for purposes of this study), the scores of TC 5 for each of these specimens did not reach the NSCLC SP142 CDx threshold of TC ≥50 to qualify for treatment with atezolizumab. However, in should be noted that patient 3 also had an IC ≥10 (not reported here), which reached the NSCLC SP142 CDx threshold of IC ≥10 for treatment with atezolizumab.
A detailed summary of patient cohort demographics is provided in . The mean and median ages of the entire patient cohort were 67.3 and 68.5 years old, respectively. A total of 48.1% (201/418 cases) of the patients were female. A total of 39.0% (163/418) specimens received were from a primary tumor site, 32.8% (137/418) specimens from metastatic site, and in the remaining 28.2% (118/418) of specimens the tumor site was ambiguous or unknown. The mean and median ages of the NSCLC patient cohort were 69.7 and 69.0 years old, respectively. A total of 41.7% (83/199 cases) of the patients were female. A total of 52.8% (105/199) NSCLC specimens were from the primary tumor site (lung), 30.7% (61/199) specimens from a metastatic site, and 16.6% (33/199) from a specimen site that was either ambiguous or unknown. In addition, NSCLC specimens were received from patients with stage IV (61.3%, 122/199), stage III (16.6%, 33/199), stage II (5.0%, 10/199), and stage I disease (8.0%, 16/199); the remaining specimens were received from patients with unknown stage of disease (9.0%, 18/199). The disease diagnoses for NSCLC specimens included lung adenocarcinoma (61.3%, 122/199 cases), lung squamous cell carcinoma (26.6%, 53/199 cases), NSCLC (not otherwise specified) (11.6%, 23/199 cases), and adenosquamous carcinoma (0.5%, 1/199 cases). In addition, the total tumor cohort included eight small cell lung cancer (SCLC) specimens (8/418 total tumor cases, or 1.9% of all tumor cases).
shows the PD-L1 status with all three antibody clones in resection, biopsy and cytology specimens. No appreciable difference in tumor cell expression was observed between specimen types. A higher percentage of triple negative (22C3-/28-8−/SP142−) cases are observed in cytology specimens, but this corresponds to only 7 out of 13 total cytology specimens. The proportion of resection, biopsy and cytology specimens with an IC score of ≥1 were 62.6% (65/174), 46.3% (107/231), and 30.8% (4/13), respectively, and the proportion of resection, biopsy and cytology specimens with an IC score <1 were 37.4% (65/174), 53.2% (123/231), and 69.2% (9/13), respectively. Tumor cell PD-L1 expression as detected with 22C3, 28-8 and SP142 IHC assays across all tumor types are shown in . Higher positive tumor cell staining was observed with 22C3 compared with 28-8 and SP142 in NSCLC, neuroendocrine tumor, melanoma, soft tissue, and overall tumor specimens. There were no clear or significant differences in PD-L1 tumor cell expression status between the three different antibody clones for GYN, genitourinary, GI, or CNS tumor specimens. provides an analytical comparison of tumor cell expression staining, by case, for 22C3 (dark blue triangles), 28-8 (light green circles), and SP142 (yellow squares) PD-L1 IHC assays across all tumor cases (n=418; ) and all NSCLC cases (n=199; ). The ‘best fit’ curves show that for all tumor cases (and NSCLC cases) that were positive with all three IHC assays, the average 22C3 TC score was typically 10–20 percentage points higher than 28-8 scores, which were in turn approximately 40–60 percentage points higher than SP142 scores. The analytical concordance between the three antibody clones across all tumor specimens (n=418) is shown in . The same PDL-1 status with all three antibody clones (either TC ≥1% with all three assays or TC <1% with all three assays) was observed in 60.0% (251/418) of all tumor specimens: 45.9% (192/418) of all tumor cases were triple negative and 14.1% (59/418) of all tumor cases were triple positive. A different PD-L1 status was observed with the three antibody clones in 40.0% (167/418) of all tumor specimens; and among these cases, 62.3% (104/167) were 22C3+/28-8+/SP142−, and 28.7% (48/167) were 22C3+/28-8−/SP142−. A total of 54.1% (226/418) of all tumor cases were positive with at least 1 IHC assay (22C3, 28-8 or SP142) and among these cases, 94.2% (213/226) were positive for 22C3, 77.0% (174/226) were positive for 28-8, and 28.8% (65/226) were positive for SP142. shows the positive and negative PD-L1 status with all three antibody clones for NSCLC specimens (n=199). The same PD-L1 status with all three antibody clones (either all assays demonstrating TC ≥1% or all demonstrating TC<1%) was observed in 48.7% (97/199) of all NSCLC specimens. Of these NSCLC specimens showing the same PD-L1 status with all three antibody clones, 54.6% (53/97) were triple negative, and 45.4% (44/97) were triple positive. A different PD-L1 status was observed among all three antibody clones in 51.3% (102/199) of all NSCLC specimens. Among these NSCLC cases showing a different status, 67.6% (69/102) of cases were 22C3+/28-8+/SP142−, and 23.5% (24/102) were 22C3+/28-8−/SP142−. A total of 73.4% (146/199) of all NSCLC cases were positive with at least one IHC assay (22C3, 28-8, or SP142), compared with 54.1% (226/418) of tumor specimens overall. Among these cases that were positive with at least one IHC assay, 22C3 was positive in 95.2% (139/146) of cases, 82.2% (120/146) were positive for 28-8, and 32.2% (47/146) were positive for SP142. shows the positive and negative PD-L1 status with all three antibody clones for different types of lung cancer specimens. The PD-L1 status was the same (≥ 1% TC expression with all three clones, or <1% TC expression with all three clones) with all three antibody clones in 50.8% (62/122) of all lung adenocarcinoma specimens, 32.1% (17/53) of all lung squamous cell carcinoma specimens, 69.6% (16/23) of all NSCLC (NOS) specimens, and 62.5% (5/8) of all small cell lung carcinoma specimens. A total of 72.1% (88/122) of adenocarcinoma cases were positive with at least one antibody clone, compared with 81.1% (43/53) of all squamous cell carcinoma specimens, 69.6% (16/23) of all NSCLC (NOS) specimens, and 50.0% (4/8) of all small cell lung carcinoma specimens. Among lung adenocarcinoma specimens that were positive with at least one antibody clone: 95.5% (84/88) were positive for 22C3, 83.0% (73/88) were positive for 28-8, and 23.8% (29/122) were positive for SP142. Among squamous cell carcinoma specimens that were positive with at least one antibody clone: 93.0% (40/43) were positive for 22C3, 79.1% (34/43) were positive for 28-8, and 16.3% (7/43) were positive for SP142. Among NSCLC (NOS) specimens that were positive with at least one antibody clone: 100.0% (16/16) were positive for 22C3, 75.0% (12/16) were positive for 28-8, and 62.5% (10/23) were positive for SP142. Finally, among small cell lung carcinoma specimens that were positive with at least one antibody clone: 100.0% (4/4) were positive for 22C3, 75.0% (3/4) were positive for 28-8, and 25.0% (1/4) were positive for SP142. shows the positive and negative PD-L1 status with all three antibody clones for primary versus metastatic NSCLC specimens. The PD-L1 status was the same (either ≥1% TC expression with all three clones, or <1% TC expression with all three clones) with all three antibody clones in 42.5% (45/106) of all primary NSCLC specimens, 50.7% (35/69) of all metastatic NSCLC specimens, and 50.0% (12/24) of all NSCLC specimens with undetermined specimen location. It was observed that 78.3% (83/106) primary NSCLC specimens were positive with at least one antibody clone, compared with 65.2% (45/69) of metastatic NSCLC specimens, and 83.3% (20/24) of all NSCLC specimens with undetermined specimen location. Among primary NSCLC specimens that were positive with at least one antibody clone: 97.6% (81/83) were positive for 22C3, 78.3% (65/83) were positive for 28-8, and 27.7% (23/83) were positive for SP142. Among metastatic NSCLC specimens that were positive with at least one antibody clone: 93.3% (42/45) were positive for 22C3, 80.0% (36/45) were positive for 28-8, and 31.1% (14/45) were positive for SP142. Among NSCLC cases with undetermined specimen location that were positive with at least one antibody clone: 91.7% (22/24) were positive for 22C3, 100.0% (24/24) were positive for 28-8, and 50.0% (12/24) were positive for SP142.
The histology and PD-L1 tumor cell expression pattern with each of the three antibody clones (22C3, 28-8 and SP142) for three separate NSCLC patient specimens are shown in . These three cases are representative of most tumor specimens observed with a positive PD-L1 status, in that TC score is greatest with 22C3, followed by 28-8, followed by SP142 having the lowest TC score. A score of TC ≥1 was reported with 22C3 for all three specimens, qualifying all three patients for treatment with pembrolizumab or cemiplimab-rwlc. Furthermore, a score of TC ≥1 with 28-8 was reported for all three specimens, qualifying all three patients for treatment with nivolumub or nivolumab in combination with ipilimumab. While specimens from patient 1 and patient 3 both exhibited SP142 scores of TC ≥1 (and are therefore considered having a positive PD-L1 status for purposes of this study), the scores of TC 5 for each of these specimens did not reach the NSCLC SP142 CDx threshold of TC ≥50 to qualify for treatment with atezolizumab. However, in should be noted that patient 3 also had an IC ≥10 (not reported here), which reached the NSCLC SP142 CDx threshold of IC ≥10 for treatment with atezolizumab.
This report represents one of the largest studies to date of tumor specimens analyzed with multiple PD-L1 IHC assays in routine clinical practice. The result of each FDA-approved PD-L1 IHC CDx assay is an indication for a specific cancer treatment . Some tumor types, such as NSCLC, have multiple associated PD-L1 IHC CDx assays, each specifically linked to a different cancer treatment. For example, there are four PD-L1 IHC CDx assays for NSCLC, including 22C3 (linked to pembrolizumab or cemiplimab-rwlc treatment), 28-8 (linked to nivolumab in combination with ipilimumab), SP142 and SP263 (both linked to atezolizumab treatment). Several factors might be considered when selecting a PD-L1 IHC assay for each individual patient, including which IHC assay has a CDx indication for the patient’s specific tumor type, which CDx-associated cancer treatment is preferred by the clinician, which immunotherapy has the least side effects, as well as the current clinical status of the patient. The findings presented here, including the sensitivity of various PD-L1 IHC assays across a range of tumor types, represent an additional factor to consider when selecting which PD-L1 IHC assay is most appropriate for each individual patient. It should also be noted that, while PD-L1 assays can help to establish the eligibility of patients for immune checkpoint inhibitors, additional biomarkers such as tumor mutational burden (TMB) can be useful to anticipate the efficacy and durability of benefit of these drugs, in an additive and independent manner across many tumor types. Selecting the most appropriate PD-L1 assay to run for a particular patient, especially in cases of tissue specimen scarcity, is important. For example, an NSCLC case with a positive 22C3 result (tumor cell expression, or TC ≥1) and negative 28-8 result (TC <1), would lead to an FDA-approved indication for treatment with pembrolizumab or cemiplimab-rwlc, but not with nivolumab in combination with ipilimumab (the CDx-associated treatment for 28-8). In this scenario, if the clinician had ordered a 28-8 assay only, without 22C3 (eg, due to limited tissue), a single negative 28-8 PD-L1 score would likely be reported, and the patient would not be eligible for any specific PD-L1 immunotherapy. In this study of 418 tumor specimens tested with 22C3, 28-8 and SP142 assays, the same PD-L1 status (either all three assays scored with TC <1, or all scored with TC ≥1) was observed in 60.0% (251/418) of all tumor specimens, and 48.7% (97/199) of all NSCLC specimens. In these cases that received the same PD-L1 status with all three IHC assays, if one IHC assay was ordered instead of all three, the final ‘positive’ PD-L1 status reported for that patient would not have been altered (although the patient would only be eligible for the CDx indicated treatment associated with the single IHC assay that was ordered). However, in a significant proportion of cases (ie, 40.0% (167/418) of all tumor specimens, and 51.3% (102/199) of all NSCLC specimens), the PD-L1 status was different among the three assays. In these cases that received a different PD-L1 status, if one IHC assay was ordered instead of all three, the final PD-L1 status reported for that patient may have been altered if only one IHC assay was ordered instead of all three assays. This raises the question, which PD-L1 IHC assay should the patient’s clinical team choose, especially if they have no CDx treatment preference, or if the sample size is not sufficient for multiple PD-L1 IHC assays? In this study, our data suggest that 22C3 is the most sensitive IHC CDx assay for tumor cell PD-L1 expression and identifies a positive PD-L1 status more frequently than 28-8, which in turn is positive more often than SP142. These findings are consistent with previous reports, such as Blueprint phase 1 and phase 2 studies, as well as analyses of PD-L1 IHC assays in cell lines and lung cancer specimens, all of which reported greater sensitivity with 22C3 and 28-8 assays and consistently lower TC staining with the Ventana SP142 assay. In this study, the 22C3, 28-8 and SP142 assays were positive (TC ≥1) in 94.2% (213/226), 77.0% (174/226), and 28.8% (65/226), respectively, of all tumor cases that were positive with at least one IHC assay (ie, 54.1% (226/418) of all tumor cases). Similarly, among the 73.4% (146/199) NSCLC specimens that were positive with at least one IHC assay, 22C3 was positive in 95.2% (139/146), 28-8 was positive in 82.2% (120/146), and SP142 was positive in 32.2% (47/146) of cases. While the Blueprint phase 2 study (which evaluated 39 NSCLC tumor cases), concluded that 22C3 and 28-8 assays were highly comparable, the current study suggests that the 22C3 assay returns a positive PD-L1 status to a slightly greater extent than 28-8 in routine clinical practice. Furthermore, as shown in , in tumor cases that are positive with all three assays, the average 22C3 score is consistently 10–20 percentage points higher than 28-8. This conclusion is further supported by our findings that among tumor specimens that showed a different PD-L1 status between the three IHC assays, 22C3 was positive in most cases: among all tumor specimens with a different PD-L1 status, 62.3% (104/167) were 22C3+/28-8+/SP142−, and 28.7% (48/167) were 22C3+/28-8−/SP142−, with only 7.8% (13/167) reported as negative for 22C3. Similarly, among all NSCLC cases with a different PD-L1 status, 67.6% (69/102 cases) were 22C3+/28-8+/SP142−, 23.5% (24/102 cases) were 22C3+/28-8−/SP142−, with only 6.9% (7/102) reported as negative for 22C3. While 22C3 may not be the optimal PD-L1 IHC choice for every tumor type or every clinical scenario (eg, some clinicians may prefer to treat with atezolizumab in certain situations, for which 22C3 is not a CDx assay), our data indicate that a positive tumor cell PD-L1 status is more often reported with the 22C3 assay compared with other IHC assays. In reference to the current study, it is important to point out that the Ventana SP142 assay scoring system also includes an IC score, which is not included in the 22C3 and 28-8 assays (see ). For example, an SP142 IC score of ≥5 and ≥10 are indications for treatment of urothelial carcinoma and NSCLC, respectively (both with atezolizumab), regardless of SP142 TC score. Therefore, while an SP142 case would be regarded as negative for the purposes of our study if TC <1, there remains a possibility that the IC score may have been at or above the CDx threshold to be regarded as having a ‘positive PD-L1 status’. Many explanations have been postulated to explain different staining characteristics among the various PD-L1 IHC assays available today. Some reports suggest that differences in the number, size, and accessibility of specific epitopes recognized by the various PD-L1 antibodies (22C3, 28-8 and SP142) could be a factor. PD-L1 is a 290-amino acid transmembrane glycoprotein, with two extracellular immunoglobulin (Ig) domains and a 31-amino acid cytoplasmic domain. The greater sensitivity of 22C3 and 28-8 IHC assays (compared with SP142) might be explained by the findings that DAKO 22C3 and DAKO 28-8 antibodies each bind multiple epitopes in the extracellular domain of PD-L1 (although at different sites), while the Ventana SP142 antibody binds to a single 7-amino acid stretch ( 284 DTHLEET 290 ) epitope in the cytoplasmic domain and that therefore the 22C3 and 28-8 antibodies have more binding ‘targets’ per PD-L1 molecule with which to bind. Furthermore, the 22C3 and 28-8 antibodies recognize larger epitopes that are possibly more accessible to antibodies during the IHC assay. The epitope recognized by 22C3 spans 31 amino acids and lies predominantly in extracellular residues 166–190, while the main epitopes recognized by 28-8 lie within extracellular residues 86–93, 125–145, and 205–223. However, it is also important to point out that other factors could contribute to the different sensitivities and staining characteristics among the three assays. Some studies indicate that variation in TC PD-L1 staining between 22C3, 28-8 and SP142 assays is independent of antibody used (and epitope recognized), and more likely due to differences in tumor heterogeneity, as well as platform and assay variables. Other reports indicate that variation in patient treatment (eg, with chemotherapy regimens) at the time of biopsy may alter tumor cell expression of PD-L1. Finally, others have proposed that initial negative PD-L1 test results could be followed by repeat PD-L1 testing at a later point with alternative specimens, or with a different clone if necessary. Our data show that 81.1% (43/53) of lung squamous cell carcinoma cases were positive with at least one IHC assay, compared with 72.1% (88/122) cases of lung adenocarcinoma. This finding is consistent with several recent studies that have shown greater tumor cell PD-L1 expression in squamous cell carcinoma compared with lung adenocarcinoma. While it is a limited number of samples, our data also show that an even lower proportion (50.0%, 4/8 cases) of SCLC cases demonstrate positive PD-L1 status with at least one IHC assay, a finding consistent with previous reports of lower, although variable, PD-L1 tumor cell expression in SCLC ranging from 0–3% to 71%. Finally, we show that a greater proportion (34.8%, 24/69 cases) of metastatic NSCLC specimens were triple negative, compared with only 21.7% (23/106) of primary NSCLC cases. While some reports have demonstrated similarly lower PD-L1 tumor cell expression in metastatic versus primary sites, others have reported higher PD-L1 tumor cell expression in metastatic specimens. This variation in reported TC expression in metastatic versus primary sites might be due to differences in the specific tumor type studied, differences in the local tumor microenvironment such as the local cytokine milieu, or the specific metastatic site in question. For example, in metastatic triple negative breast cancer, TC expression of PD-L1 is reported to be substantially lower in liver, skin and bone sites, compared with those in lung, soft tissue, and lymph nodes. One limitation of this study is that all the specimens were stained using the manufacturer’s staining protocol for their assay, and our results are therefore not generalizable if a lab uses a different staining protocol. A further limitation is that each of the IHC assays were performed on different tissue sections from the same block for each specimen, rather than an IHC multiplex assay on the same tissue section. As such, not all tissue sections through a tumor specimen are identical, due to variations in tissue size and shape, as well as tumor heterogeneity, a factor that likely caused approximately 5–6 ‘outlier’ specimens to show variant 28-8 TC expression, compared with the best fit 28-8 curve. The 28-8 IHC assay was disproportionately affected by these factors in our study, because tissue sections for 22C3 and SP142 IHC assays were often cut first, followed by tissue sections cut for next-generation sequencing testing, and only finally followed by tissue sections cut for 28-8 IHC. Therefore, the tissue profile and amount of tumor present on the 28-8 tissue section could on occasion vary significantly from that seen in the 22C3 and SP142 tissue sections. Conclusion In conclusion, we show that 22C3 is the most sensitive PD-L1 IHC assay for tumor cell expression and returns a positive PD-L1 status more frequently than 28-8, which in turn is positive more often than SP-142. Multiple factors should be considered in the selection of a PD-L1 IHC assay for a particular patient, including which IHC assay is a CDx for the patient’s specific tumor type, which CDx-associated therapy is most clinically appropriate, the side effect profile of that therapy, as well as the current clinical status of the patient. The findings presented here represent an additional resource to help guide clinicians select which PD-L1 IHC CDx assay is most suitable for their patient.
In conclusion, we show that 22C3 is the most sensitive PD-L1 IHC assay for tumor cell expression and returns a positive PD-L1 status more frequently than 28-8, which in turn is positive more often than SP-142. Multiple factors should be considered in the selection of a PD-L1 IHC assay for a particular patient, including which IHC assay is a CDx for the patient’s specific tumor type, which CDx-associated therapy is most clinically appropriate, the side effect profile of that therapy, as well as the current clinical status of the patient. The findings presented here represent an additional resource to help guide clinicians select which PD-L1 IHC CDx assay is most suitable for their patient.
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Measurement of serum adropin levels in chronic renal failure patients receiving routine hemodialysis treatment | f80a194a-3ae6-45c1-bb7f-d1f38e30c129 | 11936645 | Pathologic Processes[mh] | Chronic renal failure (CRF) is a serious health problem that is becoming increasingly common worldwide, and renal replacement therapies such as hemodialysis are widely used to prolong the life span of these patients. Patients with CRF are at high risk for cardiovascular diseases and problems such as inflammation, malnutrition and lipid imbalances are common in these patients. Adropin is a peptide involved in the regulation of energy balance and metabolism, and studies on the importance of this biomarker in renal failure and hemodialysis patients are ongoing. Studies in the literature show that serum adropin levels are generally low in CRF and hemodialysis patients and this may be associated with malnutrition, inflammation, and cardiovascular complications. The association of adropin with complications such as malnutrition and inflammation, especially in patients undergoing hemodialysis treatment, has not been adequately investigated. Current research suggests that adropin levels are associated with renal dysfunction, but the specific role of this biomolecule has not been fully elucidated. Furthermore, it has been suggested that low levels of adropin may be a potential biomarker for predicting impaired renal function in patients with diabetes and chronic heart failure. In this study, we aimed to compare serum adropin levels in CRF patients receiving regular hemodialysis treatment with normal healthy subjects and to evaluate the relationship with clinical parameters.
2.1. Study subjects, materials, and participants In this study, 49 patients with chronic renal failure receiving routine hemodialysis treatment in the hemodialysis unit of the Internal Medicine Clinic of Erzurum City Hospital and 36 healthy control patients who did not receive treatment during the same period were evaluated. Detailed demographic and clinical data such as age, gender, creatinine levels were obtained retrospectively from the files. Inclusion criteria were defined as being over 18 years of age, having a diagnosis of chronic renal failure and receiving hemodialysis treatment for at least 6 months. The control group consisted of individuals with normal renal function and no diagnosis of diabetes mellitus. Exclusion criteria included kidney transplantation, diagnosis of acute renal failure, presence of systemic inflammatory diseases, and acute infection. 2.2. Participant preparation and selection In the participant selection process, care was taken to ensure that the patient and control groups were comparable, especially patients with renal function and hemodialysis history were taken into consideration. The control group was selected among individuals who were similar to the patient group in terms of age and gender, but who did not have renal failure. In this way, a meaningful comparison between the groups was aimed. 2.3. Measurement and calculation methods Serum adropin levels were measured by enzyme-linked immunosorbent assay method using sera obtained from blood samples taken from the participants. The human adropin enzyme-linked immunosorbent assay kit (catalog number: E-EL-H5307, Elabscience Biotechnology Inc., USA) was used for measurements. The assay range was 31.25 to 2000 ng/L with a minimum detectable concentration of 18.75 ng/L. The intra-assay coefficient of variation was <8% and inter-assay coefficient of variation was <10%. For quality control, each sample was measured in duplicate, and the mean value was used for analysis. Blood samples were collected in the morning after overnight fasting, centrifuged within 30 minutes of collection at 3000 rpm for 10 minutes, and serum was stored at −80 °C until analysis. Other biochemical parameters such as creatinine, uric acid, sodium, and potassium were measured using standard biochemical analyzers. No additional blood samples were taken from the patients for the study and adropin level was measured with the blood samples taken during the examination. 2.4. Statistical analysis Statistical analyses were performed using SPSS 22.0 software. The sample size was calculated using G*Power 3.1.9.4 software, with an α error of 0.05 and a power of 0.80. The conformity of the data to normal distribution was evaluated by Shapiro–Wilk test. In the analysis of differences between groups, Student’s t test was used for normally distributed data and Mann–Whitney U test was used for non-normally distributed data. For the correlation analysis, Pearson correlation coefficient was used after confirming normal distribution of variables. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of serum adropin levels. The area under the curve (AUC), sensitivity, and specificity were calculated. Results with P -values below .05 were considered statistically significant. 2.5. Ethical approval and compliance This study was approved by the Scientific Research Ethics Committee of Erzurum Faculty of Medicine, University of Health Sciences (ethics committee approval number: 2023/03-29, approval date: 12.07.2023). The study complied with the ethical principles of the 1964 Declaration of Helsinki. All patients included in the study were informed about the study and verbal and written consent was obtained.
In this study, 49 patients with chronic renal failure receiving routine hemodialysis treatment in the hemodialysis unit of the Internal Medicine Clinic of Erzurum City Hospital and 36 healthy control patients who did not receive treatment during the same period were evaluated. Detailed demographic and clinical data such as age, gender, creatinine levels were obtained retrospectively from the files. Inclusion criteria were defined as being over 18 years of age, having a diagnosis of chronic renal failure and receiving hemodialysis treatment for at least 6 months. The control group consisted of individuals with normal renal function and no diagnosis of diabetes mellitus. Exclusion criteria included kidney transplantation, diagnosis of acute renal failure, presence of systemic inflammatory diseases, and acute infection.
In the participant selection process, care was taken to ensure that the patient and control groups were comparable, especially patients with renal function and hemodialysis history were taken into consideration. The control group was selected among individuals who were similar to the patient group in terms of age and gender, but who did not have renal failure. In this way, a meaningful comparison between the groups was aimed.
Serum adropin levels were measured by enzyme-linked immunosorbent assay method using sera obtained from blood samples taken from the participants. The human adropin enzyme-linked immunosorbent assay kit (catalog number: E-EL-H5307, Elabscience Biotechnology Inc., USA) was used for measurements. The assay range was 31.25 to 2000 ng/L with a minimum detectable concentration of 18.75 ng/L. The intra-assay coefficient of variation was <8% and inter-assay coefficient of variation was <10%. For quality control, each sample was measured in duplicate, and the mean value was used for analysis. Blood samples were collected in the morning after overnight fasting, centrifuged within 30 minutes of collection at 3000 rpm for 10 minutes, and serum was stored at −80 °C until analysis. Other biochemical parameters such as creatinine, uric acid, sodium, and potassium were measured using standard biochemical analyzers. No additional blood samples were taken from the patients for the study and adropin level was measured with the blood samples taken during the examination.
Statistical analyses were performed using SPSS 22.0 software. The sample size was calculated using G*Power 3.1.9.4 software, with an α error of 0.05 and a power of 0.80. The conformity of the data to normal distribution was evaluated by Shapiro–Wilk test. In the analysis of differences between groups, Student’s t test was used for normally distributed data and Mann–Whitney U test was used for non-normally distributed data. For the correlation analysis, Pearson correlation coefficient was used after confirming normal distribution of variables. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value of serum adropin levels. The area under the curve (AUC), sensitivity, and specificity were calculated. Results with P -values below .05 were considered statistically significant.
This study was approved by the Scientific Research Ethics Committee of Erzurum Faculty of Medicine, University of Health Sciences (ethics committee approval number: 2023/03-29, approval date: 12.07.2023). The study complied with the ethical principles of the 1964 Declaration of Helsinki. All patients included in the study were informed about the study and verbal and written consent was obtained.
The study revealed important differences regarding serum adropin levels in patients suffering from chronic renal failure. The patient group’s adropin levels were considerably less than control group’s adropin levels (522.7 ± 169.4 vs 789.6 ± 259.3 ng/L, t = ‐5.842, P = .000). There was no statistically significant difference in the mean ages of 2 groups such as 54.7 ± 13.4 years in the first group and 51.4 ± 7.6 years in second group ( t = 1.359, P = .178). Furthermore, in other factors that are the markers of renal activity and metabolism status, the patient sample differed from the control sample in creatinine, uric acid and phosphate levels as well. Creatinine levels were reported to be significantly higher in the patient group which has chronic renal failure (9.7 ± 2.9 vs 0.7 ± 0.2 mg/dL, t = 18.456, P = .000). Statistically significant differences were also proven in case of sodium ( t = ‐4.532, P = .000) and potassium levels ( t = 2.657, P = .010). C-reactive protein (CRP) level which indicates the presence of inflammation was recorded to be higher in the patient group (21.8 ± 28.9 vs 1.4 ± 2.6 mg/L, t = 4.234, P = .000) thus suggesting that inflammation is increased among CRF patients. Other important biochemical parameters included calcium ( t = ‐5.321, P = .000), albumin ( t = ‐10.234, P = .000), hemoglobin ( t = ‐9.654, P = .000) and parathyroid hormone (PTH) (t = 7.234, P = .000) levels, and also, for these set of parameters, statistically significant differences were proved for both groups (Table ) (Figs. and ). According to the correlation analysis, serum adropin levels showed significant negative correlations with creatinine ( r = −0.584, P < .01), PTH ( r = −0.431, P < .01), and CRP ( r = −0.392, P < .01). There was no significant correlation between adropin and age ( r = −0.213, P > .05). Additionally, a moderate positive correlation was found between creatinine and age ( r = 0.613, P < .01) and between creatinine and parathyroid hormone ( r = 0.621, P < .01). The correlation between age and parathyroid hormone was weaker but still showed a positive association ( r = 0.413, P < .01). These results suggest that decreased adropin levels in CRF patients are significantly associated with markers of renal dysfunction (creatinine), mineral bone disorder (PTH), and inflammation (CRP). When all these variables are considered together, these relationships may differ in regression analysis and the results may become more complex (Table ). The correlation analysis revealed significant associations between serum adropin levels and various clinical parameters in hemodialysis patients. Strong negative correlations were observed between adropin levels and both creatinine ( r = −0.613, P < .001) and parathyroid hormone ( r = −0.621, P < .001). A moderate negative correlation was found between adropin and C-reactive protein ( r = −0.487, P < .001), suggesting a potential link between adropin and inflammatory processes. Notably, there was a moderate positive correlation between adropin and albumin levels ( r = 0.534, P < .001), which might indicate a relationship between adropin and nutritional status. Additional significant correlations were observed with sodium ( r = 0.413, P < .001), calcium ( r = 0.398, P < .001), and hemoglobin ( r = 0.445, P < .001), demonstrating moderate positive associations. Weaker but still significant negative correlations were noted with red cell distribution width ( r = −0.378, P = .001) and hemoglobin A1c ( r = −0.321, P = .044). All correlations remained significant within their respective 95% confidence intervals, supporting the robustness of these associations. These findings suggest that adropin levels in hemodialysis patients are closely intertwined with markers of renal function, inflammation, and nutritional status (Table ). The chronicle depression of serum adropin levels in chronic renal failure was evaluated using an ROC analysis which determined sensitivity and specificity of said AA protein. However, this analysis showed a barely low relationship with the average of 0.22 showing an area of the curve. Such a low AUC would prompt the conclusion that indeed adropin levels are not quite efficient as a diagnostic criterion for CRF. These findings stress that the application of adropin as a biomolecule in CRF should be interpreted with caution (see Fig. ).
The results showed that levels of serum adropin in patients with CRF were significantly lower than in relatives. Moreover, adropin levels and serum creatinine and PTH levels were significantly associated, while no significant age-related influence on adropin was noted. This would suggest that adropin may potentially be involved in renal dysfunction and the pathophysiology of CRF patients. However, it is stressed that that this interconnectedness is probably more complicated than it appears and that adropin’s role in kidney disease needs more research. It was found out that out of the studied cohorts, only the chronic renal failure patients on hemodialysis treatment showed significantly lower serum adropin levels when compared to healthy subjects, and such findings are also recorded elsewhere in literature. For example, Es-haghi et al (2021) reported that adropin levels were significantly lower in CRF patients with type 2 diabetes mellitus compared to healthy controls and that this decrease was associated with impaired renal function. In a similar study, Kałużna et al (2019) reported that adropin levels were lower in hemodialysis patients compared to the control group. Several hypotheses have been proposed about the potential role of this decrease in adropin levels in CRF. Kaur et al (2023) suggested that adropin may be involved in the deterioration of renal function and the pathogenesis of complications associated with CRF. Considering the regulatory effects of adropin on energy metabolism and insulin sensitivity, low adropin levels in CRF may contribute to the metabolic complications of the disease. Berezina et al (2023) emphasized the metabolic modulation of adropin and stated that low adropin levels may be a potential biomarker for CRF. This study suggests that adropin may be a biomarker for early detection of CRF and monitoring of disease progression. Our findings and other studies in the literature consistently demonstrate that serum adropin levels are low in CRF patients. However, no significant correlation was found between adropin levels and clinical parameters such as age, creatinine and PTH in the regression analysis ( P > .05). This result suggests that further research is needed to fully understand the precise role and clinical significance of adropin in CRF. The relationship between adropin and inflammation is an area of interest in patients with chronic renal failure. In our study, we found that CRF patients had significantly lower adropin levels compared to the healthy control group (522.7 ± 169.4 vs 789.6 ± 259.3 ng/L, P < .01) and higher CRP levels, an indicator of inflammation (21.8 ± 28.9 vs 1.4 ± 2.6 mg/L, P < .01). These findings suggest that there may be a relationship between adropin and inflammation. The study by Memi and Yazgan (2021) in an adenine-induced CRF model also supports this relationship. In their study, they showed that adropin treatment decreased IL-17A and IL-33 gene expression levels by regulating the inflammatory response and thus provided an anti-inflammatory effect. In addition, Li et al (2020) found a negative correlation between adropin levels and high-sensitivity C-reactive protein in type 2 diabetes patients. This supports the effects of adropin on inflammation. Regarding the molecular mechanisms of adropin’s effects on inflammation, it has been suggested that it reduces oxidative stress and inflammation by activating the Nrf2/ARE pathway. Nrf2 has been shown to reduce inflammation and strengthen cell defense by suppressing pro-inflammatory cytokine production. Kutlu et al (2019) suggested that adropin may be associated with metabolic irregularities and inflammation. All these findings suggest that adropin may play a potential role in the regulation of inflammation processes in CRF patients. Future research should further investigate the anti-inflammatory effects of adropin and its therapeutic potential in CRF. In our correlation analysis, significant relationships were found between creatinine, age and parathyroid hormone levels. There was a positive correlation between creatinine and age ( r = 0.613) and a significant positive correlation between creatinine and PTH ( r = 0.621). These results suggest that deterioration of renal function (high creatinine) may be associated with advancing age and increased PTH. Furthermore, a weak but significant positive correlation ( r = 0.413) was also found between age and PTH, which may indicate the complex role of adropin in the pathophysiology of kidney disease. Regression analysis showed that age, creatinine and PTH together had a significant effect on creatinine levels ( P < .05). In particular, age and PTH stood out as independent variables affecting creatinine levels. This suggests that deterioration in renal function is associated with age and PTH. Such relationships are also supported in the literature. For example, Kamel et al reported that adropin levels were associated with malnutrition and lipid profile in hemodialysis patients, but found limited association with other clinical parameters. Similarly, Hu and Chen found that adropin levels were associated with renal function in type 2 diabetic nephropathy patients, but no significant correlation with age and other parameters. Akkaya et al reported that adropin levels in coronary artery disease patients showed a weak correlation with age and creatinine, but did not reach clinical significance. Depboylu et al also found that adropin levels were more closely associated with nutritional status and inflammation in hemodialysis patients, but showed limited correlation with parameters such as creatinine and age. These findings suggest that adropin plays an important role in energy homeostasis and metabolic regulation, but may have less impact on some clinical parameters. these studies suggest that further research is needed to fully understand the relationship of adropin with clinical parameters. Our results suggest that the connection between adropin and renal function is still inadequately elucidated and is consistent with other similar studies in the literature. Lin et al reported that adropin levels are low in patients with CRF and this is associated with renal dysfunction, but the mechanism of this relationship is not yet fully understood. Similarly, Chen et al emphasized that adropin acts through multiple pathways in different diseases, but its role on renal function is complex and more research is needed. Brnić et al also addressed the relationship of adropin with inflammatory processes and drew attention to the difficulty of clarifying its effect on renal dysfunction. The ROC analysis in our study also revealed that adropin has a low accuracy in the diagnosis of chronic renal failure (AUC = 0.22). This finding implies that adropin has low promise as a potential biomarker in CRF and the role of this protein in the development of renal impairment needs further exploration. Berezin et al noted the possible relevance of adropin as a biomarker in chronic kidney disease and vascular diseases. On the other hand, they all believed that this biomarker should not be used in clinical practice until many more studies are undertaken. Recent studies have highlighted the potential role of adropin in CRF, though larger-scale investigations are needed to fully validate its clinical utility. According to a study conducted in 2024 by Elfedawy et al, adropin can be associated with a biomarker for cardiovascular disease for patients that suffer from CRF. The study indicated that adropin is an important factor of cardiovascular disease risk estimation in patients suffering from CRF as it also linked metabolic homeostasis and cardiovascular system. Berezina et al (2023) undertook a study concerning the predictive utility of adropin as a biomarker of chronic kidney diseases in patients suffering from type 2 diabetes mellitus and chronic heart failure. The findings of the study highlighted that serum adropin levels of <2.30 ng/mL were independently associated with and accurately predicted a CRF ‐1 to 3 stage in patients suffering from type 2 diabetes mellitus (T2DM) and chronic heart failure. Notably, the adropin levels as well as the serum levels of T2DM patients suffering from heart failure was comparably lower than that of healthy volunteers and of T2DM patients not experiencing heart failure. Definitely, adropin has a high potential of being an advanced marker based system for clinicians during clinical management of stratification of heart failure patients with chronic kidney disease. Kaur et al (2023) conducted a cross-sectional analysis of adropin and afamin and their possible role as biomarkers for chronic kidney disease (CKD) and accompanying cardiovascular complications. Their analysis revealed that as CKD increased in severity, serum adropin concentration exhibited a substantial decline whilst afamin concentration had a marked increase. In a related study, Boric-Skaro et al (2021) identified a significant negative correlation between adropin levels and malnutrition-inflammation scores, dialysis malnutrition scores, and high-sensitivity C-reactive protein suggesting that adropin plays a role in the pathophysiological mechanisms of CRF and hemodialysis-related complications. Rooban et al (2024) conducted a comprehensive review of adropin’s role in cardiovascular health and metabolic regulation, highlighting its potential as a biomarker for cardiovascular disease risk and its protective effects on endothelial function, lipid metabolism, and glucose homeostasis. Combined with our results, these recent findings suggest that although adropin seems a promising biomarker in CKD large scale multicenter prospective studies will be necessary to determine its clinical relevance and to set standardized reference values. In conclusion, it should not be forgotten that the measures taken to analyze the relationship between adropin and kidneys function give credence to the effects of this protein but much more data and research are required for firm conclusions. A better understanding of the function of adropin in the mechanism of renal failure progression will help to argue more firmly why this biomolecule can be used in clinical practice in the future. Our study has some limitations. First, due to the use of a retrospective design, the data collection process could not be intervened and data control was limited. All of these, however, might contribute to possible biases as well as certain constraints in reading the pages. Apart from that, relative smallness of the sample consists also the factor which weakens the contextuality of the results. Although adropin levels have been found to be associated with CRF, the exact role of this biomarker in CRF has not been fully clarified. Although significant associations have been found between creatinine, age and PTH, no significant association between adropin levels and these clinical parameters has been observed. The limited sample size and the exclusion of other potential influencing factors may have made it difficult to fully assess the effects of adropin on kidney disease. In this regard, it has been suggested that the findings be replicated with a larger and more ethnic diversity. We also studied in details the level of serum adropin in patients with chronic renal failure and controlled such patients with healthy subjects. Furthermore, the impact of hemodialysis procedures on adropin level production has been analyzed in a historical manner. It is possible that further larger, and more prolonged investigations will be able to reveal the role of adropin as a marker in depth in conjunction with complications like inflammation, and malnutrition.
Our study demonstrates that patients with chronic renal failure undergoing routine hemodialysis treatment have significantly lower serum adropin levels compared to healthy individuals (522.7 ± 169.4 vs 789.6 ± 259.3 ng/L, P < .01). Strong negative correlations between adropin levels and both creatinine ( r = −0.613, P < .001) and PTH ( r = −0.621, P < .001); along with positive correlations with albumin ( r = 0.534, P < .001) and hemoglobin ( r = 0.445, P < .001); suggest adropin’s significant role in renal dysfunction, inflammation and nutritional status. These findings provide evidence for adropin’s potential involvement in the pathophysiology of CRF and its associated complications. However, the clinical utility of adropin as a biomarker requires validation through larger, multicenter prospective studies with diverse patient populations and longer follow-up periods to better elucidate its role in disease progression and potential therapeutic applications.
Methodology: Lale Duysak. Visualization: Lale Duysak. Writing – original draft: Adil Furkan Kiliç. Writing – review & editing: Edip Erkuş.
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Are the anatomical, clinical, and ultrasound characteristics of thyroid nodules with Bethesda III or IV cytology and ACR TI-RADS 3, 4, or 5 able to refine the indications for molecular diagnostic tests? | 824f8c1d-af2c-40fe-99fd-dd68d5bd23b1 | 10528568 | Pathology[mh] | The detection of thyroid nodules has increased considerably in recent decades, and most present with benign histology. The expansion of the use of ultrasound (US) for diagnosis and screening has led to the greater detection of nodules, causing a “diagnostic epidemic” . US can be used to estimate the probability of malignant nodule status and select whether to perform a fine-needle aspiration biopsy (FNAB) . FNABs of thyroid nodules accurately classify most nodules; however, the results remain undetermined in approximately 20% (10%-38%) when specific cytological characteristics necessary for definitive diagnosis are lacking . In this scenario, the growing number of biopsies, with the indeterminate cytological results that accompany the increased detection of thyroid nodules and cancers, is relevant . Indeterminate cytology involves three Bethesda categories : Bethesda category III (III-B), which includes atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS); Bethesda category IV (IV-B), which includes follicular neoplasm/suspicious for follicular neoplasm or Hurthle cells (FN/SFN); and Bethesda category V (V-B), which is suggestive of malignancy. In a recent meta-analysis , thyroid malignancy corresponded to one-third of the resected nodules, with half of all resected nodules corresponding to indeterminate cytologies, and two-thirds of these cytologies representing benign nodules . In addition, approximately 25% of thyroid nodule FNABs are classified in the indeterminate Bethesda categories (III-B, IV-B, V-B), and more than 75% of III-B or IV-B cytology nodules have benign histology . Therefore, many of these nodules are referred for surgery due to the 10%-40% risk (category III-B or IV-B) of malignancy. This excess treatment exposes patients to short- and long-term surgical complications, in addition to permanent hormonal replacement with levothyroxine following total thyroidectomy and sometimes following lobectomy. Currently, the most used US classifications for the evaluation of thyroid nodules are from the “American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer” (ATA 2015) and “American College Radiology (ACR) Thyroid Imaging, Reporting and Data System (TI-RADS)” (ACR TI-RADS) , which are concordant in most thyroid nodules . The classification of ATA 2015 involves five categories: benign, very low risk, low risk, intermediate risk, and high risk, with the risk of malignancy being <1%, <3%, 5%-10%, 10%-20%, and >70%, respectively. The ACR TI-RADS classification is a system that uses scoring based on whether the nodule presents or does not present with malignant characteristics, and is classified from ACR TI-RADS 1 to ACR TI-RADS 5. The score ranges from 0 to 14 points, with higher scores representing a greater risk of malignancy for a given nodule. The characteristics involved in this classification include composition (solid, cystic, mixed), echogenicity (anechoic, hyperechoic, isoechoic, hypoechoic), shape (taller than wide or wider than tall), margins (well-defined, ill-defined, lobed, extrathyroid extension), and presence of echogenic foci (macrocalcifications, peripheral calcifications, punctiform echogenic foci). US characteristics influence the pretest value of the risk of malignancy when examining the cytology of a nodule, as shown by Rosario in a prospective study; in the AUS/FLUS category, US analysis showed a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 79.4%, 90.5%, 71%, and 93.75%, respectively, in predicting the malignancy of the nodules. Solid nodules that are markedly hypoechogenic or with microcalcifications, or that are hypoechogenic associated with another suspicious characteristic (irregular/microlobulated margins, anteroposterior diameter greater than transverse diameter, or central vascularization) are considered “suspect” . According to the second edition of the Bethesda categorization published in 2017, the risk of malignancy is 10%-30% for III-B, 25%-40% for IV-B, and 50%-75% for V-B, disregarding non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), and 6%-18%, 10%-40%, and 45%-60%, respectively, when considering NIFTP histologies . There is heterogeneity within III-B, in which groups with nuclear atypia (NA) have a 2.6-fold risk (odds ratio [OR] = 3.63 in III-B; OR = 4.38 in IV-B) compared to groups with only architectural changes (AA) . Finally, V-B, generally considered indeterminate, is found in 2%-3% of all FNABs and may present with a malignancy rate of 50%-75% or 53%-97% according to the study . The focus of this study is III-B and IV-B, with each found in approximately 10% of all FNABs, since V-B already presents with a high risk of malignancy, which is sufficient surgical indication in most cases (high pre-test probability). In this scenario, the use of molecular tests has gained importance , since it is necessary to use tools to assist in the preoperative diagnostic definition of thyroid nodules that are not highly suspected of being malignant or benign because of the characteristics available at this stage of the investigation (US, cytological, risk factors for thyroid carcinoma). Molecular diagnostic tests can be useful to avoid unnecessary surgeries in patients with thyroid nodules with III-B or IV-B cytology; however, their use is limited by their high cost. Zanocco and cols. recently suggested that molecular tests are cost-effective in nodules with III-B or IV-B cytology with ACR TI-RADS 3 or 4 classification on US and cases with a pretest probability of benignity above 31% (some cases of ACR TI-RADS 2 with other risk factors and ACR TI-RADS 3 and 4), since in nodules with highly suspicious US (ACR TI-RADS 5), the tests showed benignity in only 8.3% of nodules, which means the tests are not cost-effective in these cases . ACR TI-RADS 2 and ACR TI-RADS 5 nodules already have a high pre-test probability for benignity and malignancy, respectively. Typically, the molecular test does not modify the treatment strategy, except in ACR TI-RADS 2 nodules with several other risk factors for malignancy (age, sex, size, previous irradiation history, positive family history, growth in follow-up period) or in ACR TI-RADS 5 nodules (<1.5 cm) without the presence of any of the other risk factors mentioned before. Molecular tests, initially classified as “rule-in” and “rule-out”, were developed in recent years to improve the diagnostic accuracy of FNAB cytology . They consisted of small panels of genetic mutations that offered high PPV for cancer detection; however, they did not achieve sufficient NPV to reliably exclude malignancy in negative samples . Later, more advanced molecular tests were developed using gene expression technology, wider panels of mutational markers or combinations of different markers , and algorithms built with the aid of artificial intelligence, which offered greater sensitivity and improved NPV . Specifically, when the diagnostic test is designed to predict benign nodules and exclude malignancy, a high NPV is required. However, when predicting malignancy, a high PPV is required. According to Vargas-Salas and cols. , to consider a test to have a good ability to rule out malignancy, the test must have an NPV of at least 95%, which means that the residual risk of malignancy would be less than 5% for a negative result. This is close to the risk of malignancy for II-B cytology , and a minimum sensitivity above 86% is required to keep the NPV above 95% for a wide range of disease prevalences. There is no consensus on the minimum PPV necessary to consider a rule test adequate; however, a specificity rate above 87% would result in a PPV above 70% for a disease with a prevalence rate above 25% . The two tests available in Brazil have an NPV and sensitivity greater than 94% . Therefore, the use of diagnostic molecular tests has gained feasibility to assist in the preoperative diagnostic definition, mainly of ACR TI-RADS 3 and 4 nodules with III-B and IV-B cytology. However, there are still doubts as to whether the clinical, cytological, and US characteristics associated with the risk of malignancy, whether alone or considered together, would be sufficient to dispense with the use of molecular diagnostic tests. To answer these questions, this study evaluated whether anatomical and clinical data and the risk of malignancy based on US available in the preoperative period could contribute to a more selective indication for these tests in III-B or IV-B thyroid nodules. Risk factors for thyroid cancer were prospectively analyzed , including a positive family history of thyroid cancer, age, sex, and nodule size, in addition to Bethesda cytological and US ACR TI-RADS classifications, to determine which anatomical, clinical, and US characteristics, alone or considered together, would be able to predict the malignancy or benignity of III-B or IV-B nodules, in the preoperative period, to dispense with the use of molecular panels. In a cohort of 62 participants with III-B and IV-B cytology, who underwent thyroidectomy (total or partial) at the recommendation of the attending physician, with ACR TI-RADS 3 and 4 or ACR TI-RADS 5 on US, the association of malignancy with the following variables was analyzed: sex (female vs. male), age (<55 years; ≥55 years), family history of thyroid cancer, presence of thyroid cancer in first-degree relatives (yes vs. no), size of thyroid nodule (<2 cm vs. 2-4 cm vs. >4 cm), Bethesda classification (III-B only with AA, III-B with NA without cracks or pseudoinclusions, III-B with NA with cracks or pseudoinclusions, and IV-B), and ACR TI-RADS classification (ACR TI-RADS 3, 4, and 5). The variables are described in terms of absolute and relative frequencies. Logistic regression was used to assess factors associated with malignancy. This prospective cohort study was approved by the Research Ethics Committee of the Federal University of Minas Gerais (CAAE number 17794719.9.0000.5149). All patients were informed about the study objectives and freely signed an informed consent form. Sixty-two nodules were analyzed in 62 participants (one nodule in each participant), of whom 87.1% (54) were women, 74.2% were < 55 years old, and 95.2% (59) had no family history of thyroid cancer. Among these, 56.5% had nodules < 2 cm in size, 9.7% had nodules > 4 cm in size, 62.9% were IV-B, and 69.4% were ACR TI-RADS 4. All nodules were resected; 32 were determined to be thyroid carcinoma and 30 were benign tumors . The average age of patients was 45.27 years, and the majority of patients were female; however, there was no statistical significance regarding carcinomas (p = 0.1006, ). There was also no statistical significance regarding family history of thyroid cancer in first-degree relatives or size of the nodules divided into < 2 cm, 2-4 cm, and > 4 cm in size. However, it is important to highlight a reduction in the p-value (0.06172) when simplifying the analysis to nodules ≤ 4 cm and > 4 cm, which indicates a tendency towards greater risk of malignancy in larger nodules, as suggested by some studies . The III-B classification was divided into three subtypes: III-B with AA, III-B with NA other than cracks or pseudoinclusions, and III-B with NA with cracks or pseudoinclusions. These subtypes were described in the latest edition of the Bethesda classification published in 2017 as nuclear atypia due to the presence of enlarged and prominent nuclei with pale chromatin, in addition to rare pseudoinclusions in the AUS/FLUS classification and that in these groups with nuclear atypia there was a 2.6-fold risk in relation to the group that presented with architectural changes only . The results obtained were 100% benignity in nodules where only architectural changes were found. In nodules with nuclear alterations, other than cracks or pseudoinclusions, there was a predominance of malignant cases (77% malignant vs. 23% benign) and in nodules that were III-B with cracks and pseudoinclusions, the presence of malignancy was even greater (86% malignant vs. 14% benign), as shown in . Again, there was a reduction in the p-value (0.0040 to 0.0014), suggesting a greater statistical association with malignancy when simplifying the analysis to III-B with nuclear atypia vs. IV-B . The high rate of malignancy found (32/62) in the III-B and IV-B categories (51.6% vs. 20%-25%, which would be the expected malignancy rate for these two groups together), is justified by the careful surgical indication for all patients (ACR TI-RADS 5 and III-B and IV-B with some risk factors for malignancy, such as suggestive US characteristics). Regarding the high rate of malignancy in category III-B with NA (around 80%), the result was surprising and can be justified by the absence of well-defined criteria for this category. According to the latest Bethesda classification , the diagnosis of atypia of undetermined significance is reserved for samples containing cells (follicular, lymphoid, or others) with architectural atypia and/or atypia that are not sufficient to be classified as a suspected follicular neoplasia, suspected to be malignant, or malignant, reinforcing that atypia that are more accentuated than can be convincingly attributed to benign cytologies. The criteria for defining III-B are variable: cytological atypia, architectural atypia or both, presence of Hurthle cells, atypia not specified in another category, atypia with lymphoid cells, and lymphoma being ruled out. This broad definition allows the subjectivity of the examiner (depending on their experience and experience of the service in which they find themself) to be relevant in determining this classification. Although all cases in this study were examined by pathologists who perform a high volume of thyroid exams in a service where doubtful cases are reviewed by peers before making the diagnosis, we propose that a portion of the nodules classified as category III-B with NA are actually category V-B, and this is a risk that exists and has already been described in the literature . In the second step, it is possible to review these category III-B cases blindly using another reference service to assess the agreement and discuss the possible pitfalls of III-B classification. The rate of malignancy in IV-B patients (41% malignant) was slightly higher than that reported in the literature (15%-30%). The selective indication for surgery in IV-B cases at higher risk (when there are clinical, cytological, and US risk factors) justifies this finding. Finally, in the US analysis, nodules classified as ACR TI-RADS 5 and high risk according to the ATA 2015 classification, presented with a 100% malignancy rate on histology. There was a small difference in the classification of low-risk and intermediate-risk ATA 2015 nodules compared to ACR TI-RADS 3 and ACR TI-RADS 4 . However, when combining “low risk/intermediate risk” and “ACR TI-RADS 3/ACR TI-RADS 4” in one category, the difference in the classification of the nodules was minimal, in addition to supporting the association between malignancy and ACR TI-RADS 5 and high-risk ATA 2015 (p = 0.003475 and 0.008038, respectively), as predicted in the literature . To assess risk factors for thyroid cancer and its prognosis, nomograms have already been described to validate their clinical use . However, the predictive power of these risk factors has not been compared with the predictive power of molecular tests to the point of dispensing with the indication of molecular tests. The agreement between the ACR TI-RADS and ATA 2015 classification was assessed using the kappa index, which had a value of 0.848 , showing substantial agreement. Similar to the analysis of this study, researchers at the Clinical Hospital of the Federal University of Paraná (UFPR) in Brazil noticed the less aggressive behavior of some nodules classified as ACR TI-RADS 4 (TR4). Furthermore, the authors divided this category into TR4a (4 points), TR4b (5 points), and TR4c (6 points), where TR4a was equivalent to intermediate risk in ATA 2015 (with intermediate risk equivalent to low risk in the study). This study demonstrated a high NPV for III-B associated with a “favorable” US (TR2, TR3, TR4a/ATA 2015, very low risk to intermediate risk), similar to those presented in the molecular tests. The characteristics that demonstrated statistical significance were nodule size, markedly hypoechoic, higher than wide, irregular margins, and presence of microcalcifications, with no significant difference between age and sex, as shown in this study. In 2013, two studies evaluated factors indicative of worse prognosis related to thyroid cancer and death . In the first study, the probability of death at 5 and 10 years increased with advancing age, male sex, lesion size, and radiotherapy . The second study corroborated that the predictors of worse prognosis are age at diagnosis, male sex, TNM status, histology, presence of distant metastasis, post-treatment macroscopic residue, and lymph node involvement . In 2015, Lang and cols. developed a simpler nomogram to contribute to the individual assessment of patients with thyroid cancer instead of staging-based approaches that work better in population analyses than individual ones . The nomogram proposed the use of age, size, presence or absence of multifocality, lymph node involvement, and distant metastasis, and developed a numerical score, with values < 28 having high NPV, indicating a 99% chance of not dying in the next 10 years due to thyroid cancer. The study also indicated that the performance of molecular tests may contribute to better accuracy of the nomogram . The variables age and size were also included in our study, but did not contribute significantly to distinguishing between benign and malignant lesions. A limitation of this study is the small number of patients, which may explain the lack of association of known risk factors for thyroid cancer related to the malignancy of the lesions studied (such as age, sex, family history, and previous exposure to radiation). Nevertheless, a significant statistical association between III-B cytology was found, with an emphasis on nuclear changes (which were more evident when these nuclear changes were cracks and/or pseudoinclusions), IV-B, and US with a high risk of malignancy, similar to analyses found in the literature . We concluded that, despite the small number of participants, 85% (53/62) of thyroid nodules were classified as III-B (in this study, only those with NA) or IV-B, and in those that are also ACR TI-RADS 3 or ACR TI-RADS 4, molecular tests may provide benefits when determining the indication for surgery, given the inability of most analyzed anatomical and clinical characteristics, whether considered alone or together, to predict malignancy or benignity with statistical significance. The exception would be already high-risk nodules on US, ACR TI-RADS 5 (in this study, they were all malignant histologically) and, if confirmed in new studies, ACR TI-RADS 3 and ACR TI-RADS 4 III-B nodules with AA (in this study, there were three and all were benign). Therefore, regardless of the importance of using molecular tests, when suitably indicated (i.e., nodules that are category III-B or category IV-B and ACR TI-RADS 3 or ACR TI-RADS 4), we must always remember to individualize each approach, considering the high cost of these tests. We should also continually analyze categories III-B and IV-B before deciding on whether a molecular test is indicated and consider factors such as US characteristics (ACR TI-RADS 2 and ACR TI-RADS 5, usually presenting with a high pretest probability for benignity and malignancy, respectively, dispensing with the need for molecular testing), positive family history (three or more first-degree relatives affected by thyroid cancer), sex (increased risk of malignancy in men), nodules showing significant growth on serial US (if performed and available), history of previous head and neck or whole body irradiation, and cytology (III-B with NA or IV-B). Before using the molecular panel, it is necessary to evaluate the possibility of revising the slide and/or the new FNAB in category III-B. Only after analyzing all clinical, cytological, and US characteristics should whether molecular tests can assist in therapeutic decisions be considered. Finally, all the factors analyzed in this study allowed us to reach the conclusions already mentioned regarding the benefits of molecular tests in ACR TI-RADS 3 or ACR TI-RADS 4 and III-B or IV-B. |
The Impact of Coronavirus Information-Seeking Behavior on Dental Care Access: A Cross-Sectional Questionnaire-Based Study | 5a7ae0c3-8942-4ae5-99aa-d29701543d19 | 8622317 | Dental[mh] | The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has posed a challenge to healthcare systems across the world, and its rapidly evolving spread has become a global health crisis. In this scenario, Italy has been severely affected by COVID-19, registering more than 4.5 million cases since the beginning of the pandemic and reaching more than 130,000 deaths. Soon after the declaration of the pandemic by the WHO, national governments started issuing advisories and regulations to their people with the aim of restricting the spread of SARS-CoV-2. These policies included several limitations, ranging from social isolation to travel and movement restrictions. Governments used print media, mass media, and the web to inform the community. The communities themselves used a variety of tools and media sources, predominantly web based, to learn more about COVID-19: people started looking for symptoms and precautionary measures to avoid contagion . During a disaster or a public health emergency, information sources help people be more conscious and aware of the situation, learn precautionary measures, and reduce anxiety caused by the uncertainty of a newly emerged situation . Health information-seeking behavior, meant as the “purposive acquisition of health information from selected information carriers” , can provide a variety of benefits, above all the potential to reduce knowledge gaps across social groups and educate individuals outside the doctors’ office . At the same time, information sources can create new problems, especially if not properly accessed by the people. Scholars believe that new media, especially social media, have great potentials to support information searching and decision making on self-care and health-related issues . The proliferation of new media active in healthcare poses several problems and challenges . First of all, the quality of health-related information present on social media is far from perfect, with contents being misleading, inconsistent, or not trustworthy . Secondly, the excessive internet use recorded may have led to both cyberchondria and information overload . An overexposure to information can increase the initial rates of post-traumatic stress disorder symptoms and can positively be related to forming risk perceptions. Nevertheless, Farooq et al. stated that, during the SARS-CoV-2 pandemic, cyberchondria and information overload contributed to the adoption of recommended health behavior, suggesting that strategies aimed at motivating citizens should focus both on stressing the gravity of the situation and on enhancing the quality of information through the reduction of overloading information . Previous studies investigated how information sources have different impacts on psychological well-being and coping behavior during COVID-19, highlighting different results. Chao et al. have proved that information seeking on social media is generally associated with adverse psychological outcomes, while this negative relation does not apply to traditional mass media, such as television and the press . Additionally, health information-seeking behavior has varied during the pandemic. COVID-19 has reached great public attention as a result of information-seeking behavior, and a considerable amount of information about the virus, its outbreak, and spread has been published and searched . Although in Italy, dental offices practice was never formally stopped by specific policies regulating the spread of coronavirus, doctors suspended routine dental treatments, limiting their practice to just urgent and emergency dental care. In addition, the anxiety and fear generated by the spread of SARS-CoV-2 affected the willingness of the population to undergo dental treatment, further restricting access to dental care . Especially during the very first phases of the pandemic, dental practice and dental professionals were considered to be subjected to a high risk of becoming infected with SARS-CoV-2. However, recent studies showed that the risk of SARS-CoV-2 transmission in dental settings is low, as the adherence to national infection control guidelines is high among clinicians [ , , ]. Several studies have investigated the levels of anxiety and risk perceptions around the world during the COVID-19 pandemic so far, but, to the best of our knowledge, little attention has been focused on how different channels of communication could affect the availability to access dental care in the future . With this background in mind, the aim of this study was to assess whether access to different communication tools (online, offline, social media, etc.) by the Italian population, with the purpose of seeking coronavirus-related information, as well as the level of trust shown by the population toward these sources of information, could somehow influence the availability to undergo dental treatment.
Questionnaires are useful tools for investigating the perspectives and attitudes of a population. For this study, an online survey was conducted using the free-access platform powered by Google ( https://forms.gle/sAP8E5D3pWgs9pn46 , accessed on 19 May 2020). Data collection occurred between 11 May and 18 May 2020, and a total of 1003 responses were obtained. The questionnaire, in the Italian language, was composed of 43 questions, divided into 3 sections ( ). The first segment was designed to obtain socio-demographic information (age, gender, level of education, employment) and to survey the main financial consequences (i.e., family income) caused by the SARS-CoV-2 pandemic and the consequent modifications to the lifestyle it brought along. The following sections were aimed at understanding the relationship between information sources and future availability to access oral care after the pandemic. Respondents were asked about a variety of channels of information used to obtain information on COVID-19 and, for each channel, they were asked to assign a value of reliability ranging from 1 (not reliable) to 5 (very reliable). The questions were aimed at comprehending how trust in different communication means could influence the willingness of Italian individuals to undergo dental treatment. Initially, the questionnaire was revised by experts (expert colleagues in statistics, economics, and medicine), and a pilot study was conducted with a group of 20 people, heterogeneous for demographic traits, education, and employment in order to assess the understandability of the questionnaire. On the basis of the respondents’ suggestions and reactions, some questions were adjusted to make the questionnaire more understandable and clearer. The study was based on the questionnaire previously published and analyzed elsewhere . In particular, the purpose of the analysis was to assess how much means of communication used to receive information and news on the SARS-CoV-2 pandemic were perceived as reliable by the population and whether this perceived reliability would affect the willingness of the Italian people to access upcoming dental care appointments. For this study, it was deemed appropriate to use a non-probability sampling technique called snowball sampling that has the advantage of expanding the sample size, reducing at the same time cost and time of research . Moreover, snowball sampling allowed the recruitment of participants that were not easily accessible to the researchers because of the lockdown imposed by the pandemic [ , , ]. The self-administrated standard anonymous questionnaire, composed of 43 questions, was posted on social media belonging to the Facebook Group (Facebook, Instagram, and WhatsApp), on the personal accounts of two members of the research group. Using the snowball approach, all the respondents were asked to share the link with other people . Several research studies have demonstrated that Facebook is an effective tool for data collection: snowball sampling adopted on Facebook manages to provide higher response rates than traditional snowball sampling, because of the strong personal links that exist on the social network between the researcher and the respondents . Social media platforms belonging to the Facebook group were chosen as preferred social media for the purposes of the research because of their increasing relevance as social networks, as communication tools for the users, and also for the significant amount of time that users spend on these platforms . This study was conducted in full accordance with national and international regulations and the Declaration of Helsinki. All the respondents were fully informed about the aims of the study and were required to accept informed consent about the data sharing and privacy policy before participating in the survey, in compliance with Italian Legislative Decree 196/2003 and UE GDPR 679/2016. The anonymous nature of the questionnaire did not allow the identification of any personal data. Statistical Analysis Descriptive analysis was initially performed to evaluate the main characteristics of the participants in the study. Absolute and percentage frequencies were calculated to summarize the characteristics collected. Descriptive statistics were performed, and the dependent variable influence of COVID-19 information-seeking behavior on accessing future dental appointments (from now on, just “influence” for brevity) was introduced in a multiple logistic regression to estimate the effect of different sources of information on the probability of being influenced by COVID-19 in accessing future dental appointments. This binary variable was defined as “no” or “yes”, on the basis of the influence of COVID-19 on future dental appointments. In particular, the variable was defined as “no” if the corresponding value of the question n. 38 ( ) was minor, or equal to 3, while it was defined as “yes” if it was major, i.e., more than 3. Statistical analyses were performed with Stata statistical software, and the outcomes were considered both at the level of the whole sample and at the level of two subgroups divided by gender (male and female), in order to investigate possible gender differences in the willingness to undergo future dental treatment, also considering the impact of the communication channels used by the respondents to gather information about COVID-19.
Descriptive analysis was initially performed to evaluate the main characteristics of the participants in the study. Absolute and percentage frequencies were calculated to summarize the characteristics collected. Descriptive statistics were performed, and the dependent variable influence of COVID-19 information-seeking behavior on accessing future dental appointments (from now on, just “influence” for brevity) was introduced in a multiple logistic regression to estimate the effect of different sources of information on the probability of being influenced by COVID-19 in accessing future dental appointments. This binary variable was defined as “no” or “yes”, on the basis of the influence of COVID-19 on future dental appointments. In particular, the variable was defined as “no” if the corresponding value of the question n. 38 ( ) was minor, or equal to 3, while it was defined as “yes” if it was major, i.e., more than 3. Statistical analyses were performed with Stata statistical software, and the outcomes were considered both at the level of the whole sample and at the level of two subgroups divided by gender (male and female), in order to investigate possible gender differences in the willingness to undergo future dental treatment, also considering the impact of the communication channels used by the respondents to gather information about COVID-19.
Overall, out of the total 1003 respondents, 60.7% were female and 39.3% male. The largest share of respondents belonged to the age group 25–34 (32.4%), followed by the age groups 45–54 (18.1%), 18–24 (17.6%), 33–44 (15.7%), 55–64 (12.2%), 65–74 (2.6%) and over 75 (1.4%). describes the characteristics of participants. After dropping missing values of the dependent variable influence, statistics were carried out on 854 individuals, with the purpose of analyzing how different communication means were used to gather information concerning COVID-19. shows results for the seven information channels considered: television, newspapers, journals or websites of health and medicine, blog/forum, social media (e.g., Facebook, YouTube, and Instagram), friends and relatives, chemists, and family doctors. It was revealed that respondents collect information from several means of communication, but they clearly distinguish reliable channels (assigned value of 4 and/or 5) from unreliable ones (assigned value of 1, 2, and 3), in terms of the quality of the information provided. The most used source of information was newspapers/online newspapers, followed by TV/radio, while the least used sources were blogs and forums. Focusing on the number of individuals who believe the information to be true, journals or websites of medicine, health, wellness, and family doctor/other doctors/chemists were considered the most reliable channels, followed by TV and newspapers or online newspapers. Few individuals seek information from blogs and forums, while the information provided by social media and friends, despite being accessed by many, was generally not reliable for most of the respondents. The interaction terms used as predictors in the logistic regression model took into account the following information channels: TV/radio, newspapers/online newspapers, journals or websites of medicine, health, and wellness, blog/forum, social media (e.g., Facebook, YouTube, and Instagram), friends/relatives and family doctor/other doctors/chemist. The interaction terms were dichotomous variables, assigned a value of 0 if the respondent did not gather information from that specific channel, 1 if otherwise. The associated variable reliability represents the trust of respondents with reference to the corresponding information channel. In question n.16 ( ), respondents were asked to assign a level of perceived reliability to each channel of communication investigated: the values were ranging from 1 (not at all) to 5 (extremely). During the analysis, the reliability variable was converted into a dichotomous one: if the respondents showed trust toward the investigated information channel (assigning values of 4 and/or 5), the value of the dichotomous variable reliability reached 1; otherwise, in case the respondents did not trust the channel of communication, the value reached 0. shows the association among information means and the influence of COVID-19 on upcoming dental appointments for the whole sample and for the two population subgroups of females and males. Odds ratio estimates have been computed also for combinations of variables (e.g., social media and reliability). The communication tools that were used by the respondents and, at the same time, were deemed to be reliable were considered (reliability = 1). The purpose was to understand to what extent using and trusting a communication channel could influence the willingness to undergo dental appointments. Multiple logistic regression showed that the risk of being influenced by COVID-19 information as regards upcoming dental care appointments, relative to the risk of belonging to the “no influence” category, was 2.05 times higher (more than double) for people who spontaneously gathered further information to feel safer with concern to access to oral care, regardless of the sample dimension considered. In other words, the more an individual becomes informed about COVID-19, the higher is the risk that the upcoming dental care appointments will be affected. On the contrary, if the information gathered included all the necessary details on the sanitization procedures adopted in the dental office, then the risk of being influenced by COVID-19 information in relation to upcoming dentist appointments decreased, but this was only valid for males. Moreover, it was also found that individuals feeling informed and having a higher degree of education (bachelor, master or PhD) showed a risk of being influenced by COVID-19 information as regards upcoming dentist appointments that was 1.66 times higher for the whole sample and 2.42 for the male group ( p < 0.05). Focusing on gender differences, results showed that women tended to perceive less secure channels such as social media and friends as reliable sources, differently from males, who preferred to gather information from the family doctor, hence preferring more institutional sources. For both subgroups, collecting information from journals and newspapers increased the risk of being influenced by COVID-19 information (compared with not being influenced) by 1.68 times ( p < 0.01).
The current study aimed at investigating the factors affecting consumers’ availability to undergo future dental appointments after the SARS-CoV-2 pandemic, focusing on users’ information-seeking behavior and attempting to assess differences over risk perception depending on the channel of information used. The presented results showed that communication tools affect the risk of being influenced by fear of contagion by COVID-19 as regards accessing dental care, but also that there are demographic and especially gender differences that influence the risk perception over future visits at the dentist. In this study, a significant positive relationship was found between information about COVID-19 and the perceived risk of infection: this finding is consistent with previous studies on the Middle East respiratory syndrome (MERS) , demonstrating that MERS-related knowledge was significantly correlated with preventive behaviors. This may imply that individuals who are more informed about COVID-19 and its modalities of contagion are also more aware of the high infectious characteristics of the disease and hence less willing to be exposed to the risk of contagion through dental care. From the analysis, it was also revealed that communication is a key point in determining patients’ availability to undergo dental care: individuals who seek health-related information today have access to many more sources than they had in the past. While knowledge can be acquired from several different means of communication, patients clearly understood that reliability was not equal for all. This study highlighted that health professionals such as family doctors and chemists were entrusted as the most reliable source of information about COVID-19, on par with journals or websites of medicine, health, wellness. The finding is in accordance with previous studies on COVID-19 and on H1N1 influenza . Confirming our results, studies also show that, already in 2004, the internet was one of the main information sources, and it was already expected to play a key role in healthcare communication in the future . In 2008, the internet was the second-most popular source for seeking health-related information, although its believability was found to be relatively low . The aspect of believability is confirmed by this study, especially for what concerns social media, since only 5.34% of users looking for information on these channels consider the information gathered reliable. The evident discrepancy between the most credible and most accessible source of information (family doctor/other doctors are most credible with 61.83% but second-least accessible with 32.79%) should be covered to enhance COVID-19-related communication and minimize risks of negative influence over the population. In this sense, health professionals and family doctors, in particular, should enhance their presence on social media platforms, and play a more focal role in delivering information about COVID-19, and contact more widely and more often with their patients (i.e., using broadcast lists, newsletters, mailing lists, etc.) to convey secure and reliable information. The presence of dentists on social media should also be enhanced in light of the fact that the COVID-19 outbreak increased the use of social media communication because patients feel anxiety about infection through close contact . The study also highlighted severe discrepancies between health information-seeking behavior and risk perceptions of men and women: in fact, not all groups sought health information equally. Females were more likely to engage in information seeking than males. Gender is a factor that discriminates e-health information quality perception, with males perceiving e-health information quality lower than females . Our results also confirmed previous studies according to which women rather than men tended to gather more health-related information . This may be due to the fact that women are more interested in health-related issues because of the care-taking role they generally have in families, and this makes them more aggressive in searching for this kind of information . Women tended to search for information on a wider variety of information channels compared with men, becoming more involved in quality evaluation of e-health information than males but also trusting less secure channels such as personal networks (family, friends) and social media. Our results also confirmed those of the study by Bidmon and Terlutter (2015) according to which, in comparison with men, women reported a higher frequency of using health forums and blogs, and the internet was a more important source of information for them . Nevertheless, social media appeared to be a prominent and emerging information channel. Parmar et al. highlighted that social media may offer critical opportunities for dentists to facilitate patient-dentist communication and that 44% of the patients make contact with their physicians through social media . Especially for women, it would be appropriate to create official social media channels to convey information and raise operational efficiency to create patient-friendly services. Since women may be more easily convinced by health awareness information found online, they should be a primary target group for this kind of communication, and this should be interesting for government institutions (i.e., for health consciousness campaigns) but also for doctors wanting to promote their activity or reaching out to their patients. In this sense, social media use has the potential to improve health by spreading health information and may allow dentists to deliver the service and support outside the dental practice environment, increasing patients’ trust and the appreciation of the services received . For men, who, in this study, demonstrated to rely on secure and official channels such as family doctors, teledentistry, started by the US military in 1994 to serve the US troops all around the world, can provide an innovative solution to continue dental practice during pandemics and beyond . Today, teledentistry needs to be implemented into routine dental practice: if not fully replaced, it can at least implement the compromised dental system during the pandemic. This study has certain limitations that provide opportunities for future work. As the snowball sampling technique is a non-probabilistic method of sampling, it may limit the representativeness of the sample because of self-selection bias, despite having been proven to be an efficient tool for data collection. Snowball sampling through social media may introduce sampling bias toward characteristics of those who have an active online presence . This study, despite not being population based, still managed to reach almost all Italian regions with a wide range of demographic characteristics of the respondents. Future research extending the current study should recruit a more diverse sample to overcome these limitations and broaden the interpretation of results. Another limitation of this study may be that gender differences are likely to be dependent on the cultural background, so we cannot consider the results generalizable beyond the Italian population, even if comparable studies in other countries could confirm the generalizability of our findings. The data analyzed in this study refer to the early phases of the pandemic, in May 2020, when the levels of fear and anxiety were at very high levels ; the quality of data collected is very much time related, but it has also great importance because the research allowed the perception of patients to be analyzed in the early moments of the emergency and to draw implications in the physician–patient relationship that will be applicable in daily communication strategies and in case of future emergencies. The purpose of this study was to understand the influence of seeking knowledge about COVID-19 on the Italian population with a specific reference to the willingness to undergo upcoming dental care appointments. Several factors, such as the variety of information channels used and some demographic variables such as gender were the focal points of the analysis. It was clearly evident that respondents collect information from a wide variety of means of communication, but they distinguish reliable channels from unreliable ones. In general, it was revealed that gathering information about COVID-19 increases the risk of being influenced over future dental appointments: this may be due to the fact that an informed individual is more conscious about the modalities of contagion by SARS-CoV-2 and perceives a higher level of risk, in line with the results by Motta Zanin et al. on overestimation of risk perception during COVID-19 . From the current study, it also emerged that there are clear differences in the information sources used by men and women: men tend to rely on official sources such as health care professionals and family doctors, while women, who, on average, search for more information probably also because of their care-taking role in families , tend to rely on less secure sources such as personal links (family, friends) and social media. This study revealed important implications for dental service providers and for other healthcare services professionals in general. In particular, the opportunity to develop an adequate presence on social channels was highlighted in order to educate and provide useful information to users [ , , ], especially if the target audience is female, given that women are more likely to rely on online sources. The fact that the source of information is institutional or represented by a healthcare professional can provide greater credibility and reassure users to a greater extent; in fact, there is a certain awareness that misleading information and news mostly circulate on the web. In the case of male users, trust can be gained through direct doctor–user communication, and therefore, it becomes essential to develop the relationship between them.
In the event of a crisis such as that generated by the COVID-19 pandemic, social media can play an important role in serving as a source of information and in addressing people’s opinions: social media should hence have a greater presence on the side of medical service providers in order to avoid distortions of information and fake news that ultimately cause fear among citizens and compromise their health by avoiding, for example, preventive checks or unnecessarily postponing necessary interventions. Taking into consideration these results, healthcare professionals and institutions should be able to adapt their communication channels based on the audience they want to address, with the purpose of conveying official and secure information on health-related issues such as COVID-19.
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Exploring culturally-preferred communication approaches for increased uptake of voluntary medical male circumcision (VMMC) services in rural Malawi | f187bd24-cb76-4964-94e2-8495c120d2ee | 10061708 | Health Communication[mh] | In 2010, approximately 54, 000 new HIV infections were registered in Malawi. In response to this alarming figure, Malawi committed to reducing new infections by 75% by 2020 . By that year, Malawi had registered 19,000 new infections . In light of Malawi’s prevailing HIV burden, voluntary medical male circumcision (VMMC) became a key HIV prevention strategy as per joint World Health Organization (WHO) and The Joint United Nations Programme on HIV/AIDS (UNAIDS) recommendation of 2007 . Supported by evidence from three randomized studies conducted in South Africa, Uganda and Kenya which revealed that circumcision was effective in reducing HIV transmission among heterosexual men by 60–70%, in 2007, both UNAIDS and WHO recommended VMMC as a new HIV prevention measure . The recommendation specifically targeted 14 countries with high HIV burden in the sub-Saharan Africa region, including Malawi . Globally, by 2019 nearly 26.8 million cumulative male circumcisions for HIV prevention were performed between 2008 and 2019 in the 15 priority countries of East and Southern Africa . This number of circumcisions had averted about 340,000 new cases of HIV by 2019. As a result of the registered success, it was projected that by 2030 some 1.8 million new infections would be prevented . In the same period, Malawi performed cumulatively 887,205 medical circumcisions representing approximately 31% of the target for 2020 . As in the case of Uganda, Malawi’s VMMC implementation faced some hesitancy due to among others, lack of local evidence on benefits of the intervention . Malawi developed its first national policy on VMMC in 2012, which was incorporated into a mix of HIV prevention strategies in the country . However, performance towards the set targets has generally been slacking . With VMMC prevalence currently at 31% of men aged between 15 and 49, Malawi trailed the 60% target by almost 50% . Communication has been integral to VMMC implementation and specifically in VMMC demand creation in Malawi . The first VMMC communication strategy in Malawi was implemented from 2012 to 2016 . The 2010 Situation Analysis Report recommended a VMMC communication strategy to create demand for services . At regional level, findings of a study in Kenya adds weight behind communication as a tool for increasing VMMC uptake . Accordingly, a communication strategy was embedded into the implementation package for VMMC for purposes of demand creation . Malawi Government acknowledges that VMMC demand creation through communication interventions has proved challenging . Though there is a high awareness of VMMC’s health benefits among the target audience, there is low uptake among both circumcising and non-circumcising communities of Malawi . Several studies have shown that most tribes and religious groups regard circumcision as ‘amoral’ and ‘intrusive’ to their social-cultural values, beliefs, and traditions; thus, also viewed as ‘threatening’ their identity of tribes and religious groups . In addition, fear of pain, the long recovery period, perceived fertility loss, and medical complications are some other factors fueling the resistance to medical circumcision among adults . The impact of these factors on uptake among adult men in Malawi suggests the need for efforts to increase uptake . Communication is integral to countering these factors by boosting knowledge, understanding and positive attitudes toward the service and in turn stimulating demand and uptake among Malawian men, boys, parents/guardians and partners . Hence, the Malawi government developed the first VMMC Communication Strategy in 2012 to guide design and implementation of strategic communications within the framework of the national VMMC policy towards achieving the 80% uptake of VMMC among eligible males aged 10–49 by 2025 . By 2007, the Sub-Sahara African Region had the highest HIV prevalence globally and had very low male circumcision prevalence allegedly due to the widespread presence of Christianity which did not encourage male circumcision and the historical legacy of British colonialism . Following three randomized clinical trials in Uganda, Kenya and South Africa in 2005, in 2007 WHO and UNAIDS recommended to 14 East and Southern Africa countries, to take VMMC as an extra arsenal in the fight against HIV transmission . These countries included Botswana, Ethiopia, Kenya, Lesotho, Malawi, Mozambique, Namibia, Rwanda, South Africa, Swaziland, Uganda, United Republic of Tanzania, Zambia and Zimbabwe . By 2017 nearly 15,269,720 million boys and men had been circumcised in a decade with support from PEPFAR alone and figures have continued to rise . In 2013, UNAIDS estimated that the world had 35 Million people living with HIV; in the same year, 2.1 million more people got infected and 1.5 people died of AIDS-related illnesses . In Malawi, HIV prevalence among persons aged between 15 and 49 has been reducing from 16.4% in 1999 to 8.5 in 2020 . Overall, new infections among the 15–49 brackets were reducing. While in 2011 the country registered 55,000 new infections, it only registered 19, 000 in 2020 . This, however, remains an unacceptably very high number compared to the population size of approximately 19 million people and calls for concerted effort in a drive to cut the number of new infections to 0 by 2030 . Although national average adult HIV prevalence hovered around 8.5% the country had witnessed about 10, 000 HIV-related deaths at the end of 2020 . In Malawi, Male Circumcision is most common in Southern Region with prevalence rate at 47% among men aged 15–49 seconded by Central Region at 15% and lastly at 6% in the Northern Region of the country . By 2018, overall VMMC performance in the country hovered around 30% of the total target population of males in the age bracket of 15 to 49 . According to available data, low performance of VMMC in Malawi could be attributed to social-cultural factors and perceived adverse effects following the procedure at social-ecological level . Specifically, VMMC was perceived by both traditionally circumcising and non-circumcising ethnic groups as a threat to cultural identity, religious beliefs and values . At personal level, males avoided VMMC for fear of being tested for HIV at the health facility; concerns of meeting travel costs to the health facility; concerns over losing income during the recuperation period; fear of infidelity on the other partner among married couples either during the recuperation period (on the part of males) and after recuperation (on the part of females); unwillingness to abstain from sexual activity six weeks after undergoing the VMMC procedure and perception that VMMC diminishes sexual pleasure . Related to VMMC communication, Mhagama et al had concluded that although VMMC was purportedly voluntary, most up-takers did not do so voluntarily . According to the study that was conducted in Lilongwe, men were influenced to uptake VMMC mostly by peer pressure and the need for conformity; partner/girlfriend demand and considerations; and advice from health personnel . That men opt for VMMC under duress impedes on VMMC uptake . It is therefore the task of communication to bring to the fore the salience of voluntariness in VMMC in order to increase uptake among men. The word “communication” finds its roots in the Latin word, “communis” which translates “common”. The key purpose of communication interventions is to create a common understanding between the communicator and the audience . Linking “communication” to behavior change, Carl Hovland defines communication as: “…the process by which an individual (the communicator) transmits stimuli (usually verbal symbols) to modify the behavior of the other individuals (communicates)” . In order to effectively identify the preferred communication approaches for the promotion of VMMC services among the Yao of Mangochi and the Chewa of Dowa in Southern and Central regions respectively, these researchers used Laswell’s Transmission Theory which is anchored by the dictum “Who says What, in Which Channel, to Whom and for What Effect?” (as indicated in Fig. : A typical conception of Laswell’s construct as a graphic model of communication ) . This theory was named after Harold Dwight Laswell an American political scientist and a communication theorist who in 1948, while he was a professor at Yale Law School, wrote in his article entitled: “The Structure and Function of Communication in Society” , : …the most convenient way to describe an act of communication is to answer the following questions: a. Who b. Says What c. In Which Channel d. To Whom e. With What Effect? We call that the “5Ws” model . This framework effectively divides up the field of communication into five key areas of study and research. These researchers framed questions from these five fields to unearth the preferred communication approaches to be used by health communicators in both circumcising and non-circumcising communities. Informants answered these five most important questions: (a) What is your preferred source of information for VMMC information? (b) What is your preferred message for VMMC communication interventions? (c) What is your preferred audience for VMMC messaging? (d) What is your preferred channel for VMMC communication interventions? (e) What are your expected effects (or impacts) of VMMC communication interventions? When data were collected and analyzed these researchers noted, just like other scholars have, that feedback is the key missing element in Laswell’s 5Ws Model since communication is a two-way and not a one-way road . They noted through this study too that current communication interventions meant to promote VMMC services in Malawi did not pay adequate attention to these five elements. These researchers also discovered that, in Malawi, the most popular and preferred communication approaches were community engagement and interpersonal communication since they allowed the audience to give real-time feedback to communicators on VMMC messages. This study was informed by the works and commentaries on the Social-Ecological Model, Laswell’s Transmission Theory, and numerous communication and behaviour change theories. Secondly, investigators collected data among the predominantly circumcising Yao of Mangochi and the predominantly non-circumcising Chewa of Dowa. Researchers used FGDs, KIIs and IDIs to gain a comprehensive and deep understanding of the values, beliefs and traditions that underlie the resistance to VMMC. Data were thematically analyzed to decipher the people’s communication needs and preferred communication approaches by VMMC campaigners. Sample population Total number of participants in this study was 276 (160 males and 116 females. We conducted 24 focus group discussions (FGDs); 13 in Mangochi involving 118 participants (66 males and 52 females), 11 in Dowa involving 97 participants (54 males and 43 females). This makes a total of 215 participants for both Mangochi and Dowa. There were 9 key informant interviews involving 14 participants 4 interviews of Mangochi (7 males 0 females) and 5 interviews in Dowa involving 7 participants (4 males and 3 females). There were 16 in-depth interviews, involving 19 participants (14 males and 5 females). In Dowa there were 7 in-depth interviews involving 10 people (6 males and 4 females). In Mangochi there were 9 in-depth interviews involving 10 participants (9 males and 1 female). There were 5 life histories involving 5 participants (2 in Dowa and 3 in Mangochi). In Dowa 1 was male and 2 were females. In Mangochi there was 1 male and 1 female. There were two PRAs involving 23 people. In Dowa there were 11 participants (6 males and 5 females). In Mangochi there were 12 participants (6 males and 6 females) The interview guides during this study were based on the five key questions as indicated in Fig. below. By collecting data from 160 males and 116 females including youths and minors, we explored the social and cultural values that influence people’s decisions to seek health care. Specifically, the study explored the reasons why uptake of VMMC services was low. We also explored the preferred communication approaches that VMMC campaigners could use in order to motivate men and boys to access services. Figure below shows the details of data collection channels disintegrated according to districts, research tools and participant categories. Total number of participants in this study was 276 (160 males and 116 females. We conducted 24 focus group discussions (FGDs); 13 in Mangochi involving 118 participants (66 males and 52 females), 11 in Dowa involving 97 participants (54 males and 43 females). This makes a total of 215 participants for both Mangochi and Dowa. There were 9 key informant interviews involving 14 participants 4 interviews of Mangochi (7 males 0 females) and 5 interviews in Dowa involving 7 participants (4 males and 3 females). There were 16 in-depth interviews, involving 19 participants (14 males and 5 females). In Dowa there were 7 in-depth interviews involving 10 people (6 males and 4 females). In Mangochi there were 9 in-depth interviews involving 10 participants (9 males and 1 female). There were 5 life histories involving 5 participants (2 in Dowa and 3 in Mangochi). In Dowa 1 was male and 2 were females. In Mangochi there was 1 male and 1 female. There were two PRAs involving 23 people. In Dowa there were 11 participants (6 males and 5 females). In Mangochi there were 12 participants (6 males and 6 females) The interview guides during this study were based on the five key questions as indicated in Fig. below. By collecting data from 160 males and 116 females including youths and minors, we explored the social and cultural values that influence people’s decisions to seek health care. Specifically, the study explored the reasons why uptake of VMMC services was low. We also explored the preferred communication approaches that VMMC campaigners could use in order to motivate men and boys to access services. Figure below shows the details of data collection channels disintegrated according to districts, research tools and participant categories. Findings of this study revolved around the five critical questions of transmission inquiry as suggested by Harold Laswell namely ‘who says what, to whom, through what channel, and with what effect? ’ Five questions framed the presentation of results namely: (a) What is the preferred source of information for VMMC information? (b) What is the preferred message for VMMC communication interventions? (c) What is the preferred channel for VMMC communication interventions? (d) What is the preferred audience for VMMC messaging? (e) What are the expected effects (or impacts) of VMMC communication interventions? Communication ‘noise’ behind the low attention to information on VMMC services in the two study sites We first report on the cultural and religious factors that impeded VMMC communication in the two data collection sites. Overall, this study found that royalty to culture and religion prevented people from the two study sites from acting on messages. These messages were being disseminated by various VMMC implementing agencies, predominantly NGOs, with support from various development partners, under the leadership of the Ministry of Health. However, the traditionally circumcising Yao people avoided VMMC services on the fear that it undermined jando, a cultural initiation ritual where boys were circumcised to symbolize transition from childhood to adulthood. VMMC messages and the VMMC program as a whole, were perceived to be in conflict with their cultural identities and, therefore, a threat to the survival of their initiation practices which, for the circumcising Yaos, went beyond the mere cutting of the foreskin but also inculcated character in the initiates: “Ours was the circumcision of the brain not of the penis as they are portraying it now… As Yaos we were circumcised for cleanliness, moral discipline and transition to manhood,” [KII, Yao Culture Expert ]. Jando also had a political significance since boys were prepared for fiduciary duties towards their local chief and enabled them to practice a ritual that was sanctioned by Islamic Scriptures. “…This time around you are telling the chiefs that circumcision should be done at the hospital. The chief cannot promote this because he is benefiting nothing ….,” [ KII, Yao Cultural Expert & Sheikh]. On the other hand, being a predominantly traditionally non-circumcising and Christian ethnic group, the Chewa people did not pay much attention to VMMC messages. Their understanding was twofold: first, male circumcision was not part of their culture. Secondly, male circumcision was not part of their religion but an attempt by government to popularize Islam in their area. Informants, therefore, equated getting circumcised with becoming a Yao or a Muslim. One man said: “ I am a Chewa man, why should I go for circumcision? Do you want me to become a Yao?” Another one was more conscious of his faith: “ As Christians, we do not make [put] emphasis on circumcision,” [IDI, Religious leader - Dowa]. These researchers posit, therefore, that cultural and religious perception, accounted for low uptake of VMMC services in the two data collection communities. Culturally-preferred communication approaches based on Laswell’s theory Based on Harold Laswell’s fivefold question we now present results of the study as follows: What is the preferred source of information for VMMC information? Both cultures under study perceived the chief as a primary source of information and a mouth-piece of the community. Hence any information to be delivered to the community needed to pass through and vetted by him or her. Despite that among the Yao allegiance was determined by one’s economic standing, among the Chewa it was more of the cultural royalty. From that end, the study informants first noted that promoters of VMMC had overlooked this important community entry protocol thereby creating a communication barrier. “They must tell the chief, [and] the chief will announce to his people that the hospital personnel have something to say, so everyone will go to the meeting…The chief will open the floor and hand it over to the hospital staff to address the people.” [FGD, Unmarried Youth – Dowa]. From the foregoing, the chief was traditionally, the primary source of information. However, for lack of technical knowledge and skills on the subject of VMMC services study participants in both communities identified health workers as a preferred source of information. Study participants held that health workers were well trained hence knowledgeable, experienced and holding reliable information. “…medical personnel should be assigned to spread the message of circumcision because they are well trained. If they were to convey the message it will be better understood,” [IDI, Traditional Leader – Dowa]. What is the preferred message for VMMC communication interventions? The most preferred message that communities want to hear from those implementing VMMC interventions is benefits that VMMC offers to communities, households, and men and boys in particular. Regarding pre-adolescents, parents, clan leaders, community and religious leaders from Mangochi district indicated that they needed to be schooled on how VMMC provided long-term health benefits to males. For example, they needed to learn about such long-term benefits as the reduction of the risk of contracting HIV during traditional circumcision rituals during ndagala . Parents and guardians also wanted assurance that VMMC offered their wards protection against HIV infection during heterosexual relationships before and after they began to raise their future families. An unmarried youth in Dowa narrated as follows: “ I think the most vital information is that people should know the benefits… of circumcision and to my understanding circumcision primarily protects you from cervical cancer so if they are told the women will encourage the men to go,” [ FP1, Dowa Unmarried Youth]. A female respondent in Dowa was of the view that men were not provided with sufficient details regarding VMMC procedure. From her perspective, VMMC messages needed to clearly explain what exactly made a circumcised person different from a non-circumcised man. She observed that VMMC messages had scanty details on what the circumcision procedure involved and what really happened to the man. She argued that men, who were the target of opportunity for VMMC interventions, were blank on details of the procedure: “…We just said they circumcise each other but we did not really know what they really cut. People just say the ‘foreskin’ of the penis others just say the ‘[head of the] penis’ so when you think about it, eeh! It’s scaring.” [FGD, Gate Keepers - Dowa]. The study established that the role of VMMC in the prevention of cervical cancer was more attractive than the role that it played in the reduction of HIV infection since everybody in the area knew at least one woman who had died of cervical cancer. They, therefore, wanted messages to focus on how VMMC would protect females from cervical cancer. “The protection from cervical cancer in women is most important in this area…we don’t have the desire for promiscuity and our women don’t have the desire of seeking sexual satisfaction in bed from circumcised men… Let us put HIV aside, currently the lives of people are at risk. You can manage HIV when infected, but there is no cure for cancer,” [FGD, Traditional Leaders - Dowa]. Female participants wanted VMMC messages to focus more on how their sons would be circumcised by trained medical personnel, which would result in less pain, loss of blood and reduce the risk of complications. “…in the past we had STIs like chindoko mabomu but they were treatable but these days there is AIDS which is very dangerous. There is [cervical] cancer and chisonono too. VMMC is helpful since we hear that it protects our youth and their [future] families from these. These are messages that we need hear about,” [FGD, Adult Women – Mangochi]. VMMC messages also needed to fight against stigma and must assure both Christians and Muslims that VMMC did not contradict the Bible and the Qur’an respectively. One Sheikh, for example, was pleased with VMMC, as a religious authority: “…when I look at the current situation as an Islamic leader, I am very pleased because what is happening now [in clinics] is real jando as prescribed by the Islamic faith.… VMMC fully agrees with Islam.” [KII, Religious Leaders - Mangochi] What is the preferred channel for VMMC communication interventions? Regarding the channel of communication for VMMC intervention, many informants reported that the media, particularly radio was prominently used in both study communities. “…to avoid the huge cost [that would come with village meetings] then I can only agree with the media approaches that are being used currently… just think about how MBC Radio One is boasting that 87% of the Malawi population are able to hear them… Now if 87% are listening to MBC [alone] how about those listening to Zodiak?” [IDI, Religious Leader – Mangochi]. However, not many informants were excited about the use of such open channels to promote male circumcision both in Dowa and Mangochi district. In the two districts the use of open media channels was frowned upon for infringing on the secrecy that surrounds rites of passage rituals. First, male circumcision among the Yao was not a subject for public discussion. Second, although both the Quran and the Bible legitimized it, the praxis in the Muslim Yao community was to veil it in secrecy since those not circumcised and females, in particular, were not supposed to know what happened out in the bush: “In the past, circumcision was a total secret such that even the boy’s mother did not know why her child was going to ndagala . The majority of the mothers celebrated jando blindly. And when we were there they strictly told us not to tell anybody what had happened to us. They told us that when we went back home we must bath with pants on lest the mother notices the difference in the appearance of the member….” [KII, Religious Leaders - Mangochi]. Another key concern with the use of radio, television and other forms of mass media was the sexual explicitness that such messages were associated with. This concern was particularly common among adult men and women who feared that such messages would have negative consequences since they promoted sexual immorality among the youth. This, they argued, was contrary to the purpose of male circumcision in the Yao culture: “The problem comes in because … [through the media] you are telling people that once you have been circumcised, you are free from HIV. These are wrong messages…You will [now] hear a man telling a woman that ‘I was circumcised you can come for sex I can’t contract diseases,’” [KII, Yao Culture Expert - Mangochi]. Informants’ most preferred communication channel both among the Yaos of Mangochi and the Chewas of Dowa district was face-to-face engagement meetings attended by health promotion experts on one hand and community leaders, cultural gatekeepers and community members on the other. Among these methods were: meetings where traditional leaders would bring their subjects to one place and engage them in a dialogue; cultural performances such as gule wankulu; religious gatherings such as regular weekly services of worship; interactive drama and door-to-door visits just to mention some. The key reasons for preference were twofold: first, unlike the mass media, they provided room to control the type of audience to hear the message or not. Secondly, they provided an opportunity for the audience to ask questions and seek clarifications as messages are delivered. Engagement meetings organized by traditional leaders were important since chiefs and their councils of advisors acted as a bridge between citizens and government and its stakeholders: “… gulewankulu characters can dance then the drama can come next to teach things like these or you can introduce the topic at first so that the people can have an idea so when the drama on circumcision comes it will entertain them,” [FGD, Gatekeepers – Dowa]. Although informants from all Protestant Christian churches including the Catholic leadership in Dowa sounded very negative about using the church to promote VMMC messages, in Mangochi the Catholic Church was already disseminating VMMC messages. I was also carrying out surgeries. One gulupa said: “ … [Children] are also assisted to get circumcised. When they get to that stage, they are kept at the parish, which is like a simba ….” In Chowe area, Mangochi, FGD participants proposed that VMMC messages should be disseminated through their mosques and churches. This is how one traditional leader put it: “In this community, Churches and Mosques are more ideal. Religious gatherings are easy places to disseminate VMMC information,” [FGD, Traditional Leaders, Mangochi]. In Mangochi, a representative of an NGO added that they used males from within the community to deliver VMMC messages to fellow men. The health worker posited that men tended to listen better to health messages when delivered by their peers. “ We have community mobilizers who are recruited from within communities. They reach out to men in the communities,” [KII Participant, NGO Worker – Mangochi]. Another NGO worker said: “Our main approach with demand creation is interpersonal communication.” [KII Participant, NGO Worker - Mangochi] . What is the preferred audience for VMMC messaging? The study established that VMMC messages should target all community members including men, women and boys to avoid misunderstandings that may arise from misinterpretation of the messages particularly among married couples. This is what one IDI participant said: “These days I think that everyone [should be targeted] because, in a family, the man may hear the message alone but when he brings the message into his household the wife may misinterpret it… If the message was received by all - the man, the woman and the boy … people would easily embrace it,” [IDI, Traditional Leader – Dowa]. This view was supported by another community leader who argued that the message must go to both males and females to avoid disagreements between married couples: “The message has to go to both because the women are the ones who stay with the children. They can get them circumcised,” [FGD, Gatekeepers – Dowa]. The study also established that another key entry point into VMMC among the Yao men of Mangochi was the pre-adolescence age bracket of 5 to 10 years. They were minors such that they needed consent from their parents or guardians or those in authority of a village of institution. Two KII participants in Mangochi suggested that VMMC messaging must also target teachers and schools: “…I think that teachers also are very important in their own right because they interact with the community [and learners] in particular. I have a view that teachers can help us clear a lot of misconceptions …,” [KII, Service Providers – Mangochi]. This view point agrees with the observation that one participant in an FGD with Service Providers made on the same matter in Dowa. He lamented that although schools had a lot of HIV/AIDS-related clubs authorities had failed to utilize them to popularized VMMC services among adolescents and youth. “At school we have HIV&AIDS youth clubs but because they were not oriented on [VMMC] services … it is hard for you [as a teacher] to explain it in clear details [to learners] particularly the benefits of VMMC… The main topic in youth clubs is HIV&AIDS so I think if HSAs and teachers were oriented we can pass on the knowledge when we are with the youth clubs ,” [FGD, Service Providers – Dowa]. What are the expected effects (or impact) of messages on VMMC services on the audience and policy? Regarding the expected effects (or impact) of VMMC communication interventions and the VMMC program in general, the study established that the anticipated effects were at four different levels: at personal level, at household level, at community level and at policy level. At personal level, interventions were expected to create awareness of VMMC and how it contributes to HIV prevention in heterosexual men, cervical cancer (among women) and other sexually transmitted infections. There was also an expectation of increased preventive post-circumcision behaviour among VMMC clients: “…one man did not observe the six weeks -sexual-abstinence window like he was instructed by the service provider. They resumed sex earlier and he developed complications,” [KII, Service Providers, Chowe – Mangochi]. In Dowa, the overall anticipated impact of VMMC communication interventions was the reduction of cervical cancer cases that were considered to be on the rise. One sex workers’ expectation had this to say: “The main issue is reduction of diseases; it is the same when the hospital is giving counseling on condoms, they say ‘every man should abstain from sex. However, if they fail then using a condom is Plan B’. It is the same with circumcision. It is Plan B,” [FGD, Commercial Sex Worker - Dowa]. In Dowa, there was also fear that VMMC communication interventions could also lead to rising culture of promiscuity that could lead to increased HIV prevalence: “It [VMMC] is encouraging promiscuity among the youth here because if one boy is told that he is very sweet by a girl he wants to sleep with every girl…,” [FGD, Service Provider – Dowa]. Community members and health personnel working in local health facilities wanted to see household leaders, particularly mothers and clan leaders, to mobilize adult men and boys under their influence for services since they were primary producers of health. Sexual partners, mothers, aunties and grandmothers were considered to have the duty of motivating male members of their households to access services. They were also expected to support circumcised males to adopt post-circumcision prevention measures: “…females are the ones who go to the hospital… when the mother and other females are taught and understand [advantages of VMMC] it will be easy for the child to get circumcised,” [FGD, Community Leaders, Dowa]. Some traditional circumcisers were still reported using one razor blade or knife to circumcise more than one initiate. But, change had already started happening at community level since VMMC communication intervention rolled out in Mangochi: “Two weeks ago I was on a tour … Angaliba themselves were asking us: ‘Please, government should send us circumcision knives for use in initiation camps ,” [IDI, Senior Chief – Mangochi]. In Mangochi, informants felt that interventions enabled Yao Muslims practice jando as prescribed in the Holy Quran. One Yao Islamic cleric posited that VMMC already contributed to the demystification of male circumcision which, in the Yao culture, had been shrouded in secrecy from women, children and uncircumcised males for centuries: “…I am very pleased because what is happening now [in clinics] is real jando as prescribed by the Islamic faith.… VMMC fully agrees with Islam,” [KII, Religious Leaders - Mangochi] . In Dowa district, gatekeepers also expected non-formal institutions such as the gulewankulu supporting the VMMC campaign through information dissemination. They also expected it to lead in the reduction of cancer-related illness and deaths and to harness VMMC clients as advocates for services in their respective communities. In Dowa interventions were also expected to lobby for a more stable availability of funds to support community outreach programs: “In the past we used to go on outreach programs and we would discuss (VMMC) with community members… We have no money for outreach programs,” [FGD, Service Providers– Mponela, Dowa]. Although they completely rejected the proposals to adopt male circumcision as a standard cultural practice in gulewankulu bases called dambwe , the greatest change was that the Chewas were, nonetheless, ready to use gule wankulu characters as crowd puller to community meetings to ensure that VMMC messages quickly diffused into communities. “…They can spread the message via songs since gule wankulu characters have a special talent when it comes to composing songs,” [FGD, Community Leaders – Dowa]. Informants both in Mangochi and Dowa also wanted to see secondary facility (i.e. district-level-hospital) circumcisers and community-level-hospital (i.e. local health facilities) personnel working together when carrying out circumcisions. To achieve this, informants were of the view that the district health system needed to ensure that there was adequate coordination and collaboration by all key stakeholders in the VMMC service delivery value chain in each district. “If, on average, they had at least enrolled one nurse or clinician from each rural facility in the district …These could have been assigned to promote VMMC services at community level,” [KII, Service Provider, Mangochi]. Another service provider in Dowa : “The job is done by the top dogs so… we can’t follow-up because we don’t know how they counsel [clients]. Maybe there isn’t any follow-up [in their plans],” [FGD, Service Providers -Dowa]. Informants also complained that VMMC circumcisions that were offered at health facilities lacked privacy for clients. They expected this to change if the program would be successful. In Mangochi, due to lack of a proper transport arrangements, older men were sometimes being ferried from rural areas to the secondary facility on the same open lorry with boys as young as 5 years. At one facility in Dowa, informants reported that circumcisions were taking place in a room whose entry was inside the maternity ward such that adult men shunned services. Many parents who were willing to get their sons circumcised shunned services. “ … they had no choice due to lack of proper space … [VMMC clients] had to go past a group of pregnant women [in the maternity wing]. Boys were shy. The room should have [had] two doors so that after getting circumcised they should use the other door… ,” [FGD, Service Providers – Dowa]. At two facilities in Mangochi, informants told these researchers that VMMC had not been integrated into other services offered by facility and no special day or time had been allocated to these services since services were just being provided when circumcisers came all the way from Mangochi District Hospital. The other challenge both in Mangochi and Dowa was the long distances to service points. In the FGD with VMMC clients in Dowa said: “Hospitals are very far from our area so it costs a lot of money to go there and sometimes we don’t have the money,” [FGD, VMMC Clients – Dowa] . We first report on the cultural and religious factors that impeded VMMC communication in the two data collection sites. Overall, this study found that royalty to culture and religion prevented people from the two study sites from acting on messages. These messages were being disseminated by various VMMC implementing agencies, predominantly NGOs, with support from various development partners, under the leadership of the Ministry of Health. However, the traditionally circumcising Yao people avoided VMMC services on the fear that it undermined jando, a cultural initiation ritual where boys were circumcised to symbolize transition from childhood to adulthood. VMMC messages and the VMMC program as a whole, were perceived to be in conflict with their cultural identities and, therefore, a threat to the survival of their initiation practices which, for the circumcising Yaos, went beyond the mere cutting of the foreskin but also inculcated character in the initiates: “Ours was the circumcision of the brain not of the penis as they are portraying it now… As Yaos we were circumcised for cleanliness, moral discipline and transition to manhood,” [KII, Yao Culture Expert ]. Jando also had a political significance since boys were prepared for fiduciary duties towards their local chief and enabled them to practice a ritual that was sanctioned by Islamic Scriptures. “…This time around you are telling the chiefs that circumcision should be done at the hospital. The chief cannot promote this because he is benefiting nothing ….,” [ KII, Yao Cultural Expert & Sheikh]. On the other hand, being a predominantly traditionally non-circumcising and Christian ethnic group, the Chewa people did not pay much attention to VMMC messages. Their understanding was twofold: first, male circumcision was not part of their culture. Secondly, male circumcision was not part of their religion but an attempt by government to popularize Islam in their area. Informants, therefore, equated getting circumcised with becoming a Yao or a Muslim. One man said: “ I am a Chewa man, why should I go for circumcision? Do you want me to become a Yao?” Another one was more conscious of his faith: “ As Christians, we do not make [put] emphasis on circumcision,” [IDI, Religious leader - Dowa]. These researchers posit, therefore, that cultural and religious perception, accounted for low uptake of VMMC services in the two data collection communities. Based on Harold Laswell’s fivefold question we now present results of the study as follows: What is the preferred source of information for VMMC information? Both cultures under study perceived the chief as a primary source of information and a mouth-piece of the community. Hence any information to be delivered to the community needed to pass through and vetted by him or her. Despite that among the Yao allegiance was determined by one’s economic standing, among the Chewa it was more of the cultural royalty. From that end, the study informants first noted that promoters of VMMC had overlooked this important community entry protocol thereby creating a communication barrier. “They must tell the chief, [and] the chief will announce to his people that the hospital personnel have something to say, so everyone will go to the meeting…The chief will open the floor and hand it over to the hospital staff to address the people.” [FGD, Unmarried Youth – Dowa]. From the foregoing, the chief was traditionally, the primary source of information. However, for lack of technical knowledge and skills on the subject of VMMC services study participants in both communities identified health workers as a preferred source of information. Study participants held that health workers were well trained hence knowledgeable, experienced and holding reliable information. “…medical personnel should be assigned to spread the message of circumcision because they are well trained. If they were to convey the message it will be better understood,” [IDI, Traditional Leader – Dowa]. What is the preferred message for VMMC communication interventions? The most preferred message that communities want to hear from those implementing VMMC interventions is benefits that VMMC offers to communities, households, and men and boys in particular. Regarding pre-adolescents, parents, clan leaders, community and religious leaders from Mangochi district indicated that they needed to be schooled on how VMMC provided long-term health benefits to males. For example, they needed to learn about such long-term benefits as the reduction of the risk of contracting HIV during traditional circumcision rituals during ndagala . Parents and guardians also wanted assurance that VMMC offered their wards protection against HIV infection during heterosexual relationships before and after they began to raise their future families. An unmarried youth in Dowa narrated as follows: “ I think the most vital information is that people should know the benefits… of circumcision and to my understanding circumcision primarily protects you from cervical cancer so if they are told the women will encourage the men to go,” [ FP1, Dowa Unmarried Youth]. A female respondent in Dowa was of the view that men were not provided with sufficient details regarding VMMC procedure. From her perspective, VMMC messages needed to clearly explain what exactly made a circumcised person different from a non-circumcised man. She observed that VMMC messages had scanty details on what the circumcision procedure involved and what really happened to the man. She argued that men, who were the target of opportunity for VMMC interventions, were blank on details of the procedure: “…We just said they circumcise each other but we did not really know what they really cut. People just say the ‘foreskin’ of the penis others just say the ‘[head of the] penis’ so when you think about it, eeh! It’s scaring.” [FGD, Gate Keepers - Dowa]. The study established that the role of VMMC in the prevention of cervical cancer was more attractive than the role that it played in the reduction of HIV infection since everybody in the area knew at least one woman who had died of cervical cancer. They, therefore, wanted messages to focus on how VMMC would protect females from cervical cancer. “The protection from cervical cancer in women is most important in this area…we don’t have the desire for promiscuity and our women don’t have the desire of seeking sexual satisfaction in bed from circumcised men… Let us put HIV aside, currently the lives of people are at risk. You can manage HIV when infected, but there is no cure for cancer,” [FGD, Traditional Leaders - Dowa]. Female participants wanted VMMC messages to focus more on how their sons would be circumcised by trained medical personnel, which would result in less pain, loss of blood and reduce the risk of complications. “…in the past we had STIs like chindoko mabomu but they were treatable but these days there is AIDS which is very dangerous. There is [cervical] cancer and chisonono too. VMMC is helpful since we hear that it protects our youth and their [future] families from these. These are messages that we need hear about,” [FGD, Adult Women – Mangochi]. VMMC messages also needed to fight against stigma and must assure both Christians and Muslims that VMMC did not contradict the Bible and the Qur’an respectively. One Sheikh, for example, was pleased with VMMC, as a religious authority: “…when I look at the current situation as an Islamic leader, I am very pleased because what is happening now [in clinics] is real jando as prescribed by the Islamic faith.… VMMC fully agrees with Islam.” [KII, Religious Leaders - Mangochi] What is the preferred channel for VMMC communication interventions? Regarding the channel of communication for VMMC intervention, many informants reported that the media, particularly radio was prominently used in both study communities. “…to avoid the huge cost [that would come with village meetings] then I can only agree with the media approaches that are being used currently… just think about how MBC Radio One is boasting that 87% of the Malawi population are able to hear them… Now if 87% are listening to MBC [alone] how about those listening to Zodiak?” [IDI, Religious Leader – Mangochi]. However, not many informants were excited about the use of such open channels to promote male circumcision both in Dowa and Mangochi district. In the two districts the use of open media channels was frowned upon for infringing on the secrecy that surrounds rites of passage rituals. First, male circumcision among the Yao was not a subject for public discussion. Second, although both the Quran and the Bible legitimized it, the praxis in the Muslim Yao community was to veil it in secrecy since those not circumcised and females, in particular, were not supposed to know what happened out in the bush: “In the past, circumcision was a total secret such that even the boy’s mother did not know why her child was going to ndagala . The majority of the mothers celebrated jando blindly. And when we were there they strictly told us not to tell anybody what had happened to us. They told us that when we went back home we must bath with pants on lest the mother notices the difference in the appearance of the member….” [KII, Religious Leaders - Mangochi]. Another key concern with the use of radio, television and other forms of mass media was the sexual explicitness that such messages were associated with. This concern was particularly common among adult men and women who feared that such messages would have negative consequences since they promoted sexual immorality among the youth. This, they argued, was contrary to the purpose of male circumcision in the Yao culture: “The problem comes in because … [through the media] you are telling people that once you have been circumcised, you are free from HIV. These are wrong messages…You will [now] hear a man telling a woman that ‘I was circumcised you can come for sex I can’t contract diseases,’” [KII, Yao Culture Expert - Mangochi]. Informants’ most preferred communication channel both among the Yaos of Mangochi and the Chewas of Dowa district was face-to-face engagement meetings attended by health promotion experts on one hand and community leaders, cultural gatekeepers and community members on the other. Among these methods were: meetings where traditional leaders would bring their subjects to one place and engage them in a dialogue; cultural performances such as gule wankulu; religious gatherings such as regular weekly services of worship; interactive drama and door-to-door visits just to mention some. The key reasons for preference were twofold: first, unlike the mass media, they provided room to control the type of audience to hear the message or not. Secondly, they provided an opportunity for the audience to ask questions and seek clarifications as messages are delivered. Engagement meetings organized by traditional leaders were important since chiefs and their councils of advisors acted as a bridge between citizens and government and its stakeholders: “… gulewankulu characters can dance then the drama can come next to teach things like these or you can introduce the topic at first so that the people can have an idea so when the drama on circumcision comes it will entertain them,” [FGD, Gatekeepers – Dowa]. Although informants from all Protestant Christian churches including the Catholic leadership in Dowa sounded very negative about using the church to promote VMMC messages, in Mangochi the Catholic Church was already disseminating VMMC messages. I was also carrying out surgeries. One gulupa said: “ … [Children] are also assisted to get circumcised. When they get to that stage, they are kept at the parish, which is like a simba ….” In Chowe area, Mangochi, FGD participants proposed that VMMC messages should be disseminated through their mosques and churches. This is how one traditional leader put it: “In this community, Churches and Mosques are more ideal. Religious gatherings are easy places to disseminate VMMC information,” [FGD, Traditional Leaders, Mangochi]. In Mangochi, a representative of an NGO added that they used males from within the community to deliver VMMC messages to fellow men. The health worker posited that men tended to listen better to health messages when delivered by their peers. “ We have community mobilizers who are recruited from within communities. They reach out to men in the communities,” [KII Participant, NGO Worker – Mangochi]. Another NGO worker said: “Our main approach with demand creation is interpersonal communication.” [KII Participant, NGO Worker - Mangochi] . What is the preferred audience for VMMC messaging? The study established that VMMC messages should target all community members including men, women and boys to avoid misunderstandings that may arise from misinterpretation of the messages particularly among married couples. This is what one IDI participant said: “These days I think that everyone [should be targeted] because, in a family, the man may hear the message alone but when he brings the message into his household the wife may misinterpret it… If the message was received by all - the man, the woman and the boy … people would easily embrace it,” [IDI, Traditional Leader – Dowa]. This view was supported by another community leader who argued that the message must go to both males and females to avoid disagreements between married couples: “The message has to go to both because the women are the ones who stay with the children. They can get them circumcised,” [FGD, Gatekeepers – Dowa]. The study also established that another key entry point into VMMC among the Yao men of Mangochi was the pre-adolescence age bracket of 5 to 10 years. They were minors such that they needed consent from their parents or guardians or those in authority of a village of institution. Two KII participants in Mangochi suggested that VMMC messaging must also target teachers and schools: “…I think that teachers also are very important in their own right because they interact with the community [and learners] in particular. I have a view that teachers can help us clear a lot of misconceptions …,” [KII, Service Providers – Mangochi]. This view point agrees with the observation that one participant in an FGD with Service Providers made on the same matter in Dowa. He lamented that although schools had a lot of HIV/AIDS-related clubs authorities had failed to utilize them to popularized VMMC services among adolescents and youth. “At school we have HIV&AIDS youth clubs but because they were not oriented on [VMMC] services … it is hard for you [as a teacher] to explain it in clear details [to learners] particularly the benefits of VMMC… The main topic in youth clubs is HIV&AIDS so I think if HSAs and teachers were oriented we can pass on the knowledge when we are with the youth clubs ,” [FGD, Service Providers – Dowa]. What are the expected effects (or impact) of messages on VMMC services on the audience and policy? Regarding the expected effects (or impact) of VMMC communication interventions and the VMMC program in general, the study established that the anticipated effects were at four different levels: at personal level, at household level, at community level and at policy level. At personal level, interventions were expected to create awareness of VMMC and how it contributes to HIV prevention in heterosexual men, cervical cancer (among women) and other sexually transmitted infections. There was also an expectation of increased preventive post-circumcision behaviour among VMMC clients: “…one man did not observe the six weeks -sexual-abstinence window like he was instructed by the service provider. They resumed sex earlier and he developed complications,” [KII, Service Providers, Chowe – Mangochi]. In Dowa, the overall anticipated impact of VMMC communication interventions was the reduction of cervical cancer cases that were considered to be on the rise. One sex workers’ expectation had this to say: “The main issue is reduction of diseases; it is the same when the hospital is giving counseling on condoms, they say ‘every man should abstain from sex. However, if they fail then using a condom is Plan B’. It is the same with circumcision. It is Plan B,” [FGD, Commercial Sex Worker - Dowa]. In Dowa, there was also fear that VMMC communication interventions could also lead to rising culture of promiscuity that could lead to increased HIV prevalence: “It [VMMC] is encouraging promiscuity among the youth here because if one boy is told that he is very sweet by a girl he wants to sleep with every girl…,” [FGD, Service Provider – Dowa]. Community members and health personnel working in local health facilities wanted to see household leaders, particularly mothers and clan leaders, to mobilize adult men and boys under their influence for services since they were primary producers of health. Sexual partners, mothers, aunties and grandmothers were considered to have the duty of motivating male members of their households to access services. They were also expected to support circumcised males to adopt post-circumcision prevention measures: “…females are the ones who go to the hospital… when the mother and other females are taught and understand [advantages of VMMC] it will be easy for the child to get circumcised,” [FGD, Community Leaders, Dowa]. Some traditional circumcisers were still reported using one razor blade or knife to circumcise more than one initiate. But, change had already started happening at community level since VMMC communication intervention rolled out in Mangochi: “Two weeks ago I was on a tour … Angaliba themselves were asking us: ‘Please, government should send us circumcision knives for use in initiation camps ,” [IDI, Senior Chief – Mangochi]. In Mangochi, informants felt that interventions enabled Yao Muslims practice jando as prescribed in the Holy Quran. One Yao Islamic cleric posited that VMMC already contributed to the demystification of male circumcision which, in the Yao culture, had been shrouded in secrecy from women, children and uncircumcised males for centuries: “…I am very pleased because what is happening now [in clinics] is real jando as prescribed by the Islamic faith.… VMMC fully agrees with Islam,” [KII, Religious Leaders - Mangochi] . In Dowa district, gatekeepers also expected non-formal institutions such as the gulewankulu supporting the VMMC campaign through information dissemination. They also expected it to lead in the reduction of cancer-related illness and deaths and to harness VMMC clients as advocates for services in their respective communities. In Dowa interventions were also expected to lobby for a more stable availability of funds to support community outreach programs: “In the past we used to go on outreach programs and we would discuss (VMMC) with community members… We have no money for outreach programs,” [FGD, Service Providers– Mponela, Dowa]. Although they completely rejected the proposals to adopt male circumcision as a standard cultural practice in gulewankulu bases called dambwe , the greatest change was that the Chewas were, nonetheless, ready to use gule wankulu characters as crowd puller to community meetings to ensure that VMMC messages quickly diffused into communities. “…They can spread the message via songs since gule wankulu characters have a special talent when it comes to composing songs,” [FGD, Community Leaders – Dowa]. Informants both in Mangochi and Dowa also wanted to see secondary facility (i.e. district-level-hospital) circumcisers and community-level-hospital (i.e. local health facilities) personnel working together when carrying out circumcisions. To achieve this, informants were of the view that the district health system needed to ensure that there was adequate coordination and collaboration by all key stakeholders in the VMMC service delivery value chain in each district. “If, on average, they had at least enrolled one nurse or clinician from each rural facility in the district …These could have been assigned to promote VMMC services at community level,” [KII, Service Provider, Mangochi]. Another service provider in Dowa : “The job is done by the top dogs so… we can’t follow-up because we don’t know how they counsel [clients]. Maybe there isn’t any follow-up [in their plans],” [FGD, Service Providers -Dowa]. Informants also complained that VMMC circumcisions that were offered at health facilities lacked privacy for clients. They expected this to change if the program would be successful. In Mangochi, due to lack of a proper transport arrangements, older men were sometimes being ferried from rural areas to the secondary facility on the same open lorry with boys as young as 5 years. At one facility in Dowa, informants reported that circumcisions were taking place in a room whose entry was inside the maternity ward such that adult men shunned services. Many parents who were willing to get their sons circumcised shunned services. “ … they had no choice due to lack of proper space … [VMMC clients] had to go past a group of pregnant women [in the maternity wing]. Boys were shy. The room should have [had] two doors so that after getting circumcised they should use the other door… ,” [FGD, Service Providers – Dowa]. At two facilities in Mangochi, informants told these researchers that VMMC had not been integrated into other services offered by facility and no special day or time had been allocated to these services since services were just being provided when circumcisers came all the way from Mangochi District Hospital. The other challenge both in Mangochi and Dowa was the long distances to service points. In the FGD with VMMC clients in Dowa said: “Hospitals are very far from our area so it costs a lot of money to go there and sometimes we don’t have the money,” [FGD, VMMC Clients – Dowa] . Both cultures under study perceived the chief as a primary source of information and a mouth-piece of the community. Hence any information to be delivered to the community needed to pass through and vetted by him or her. Despite that among the Yao allegiance was determined by one’s economic standing, among the Chewa it was more of the cultural royalty. From that end, the study informants first noted that promoters of VMMC had overlooked this important community entry protocol thereby creating a communication barrier. “They must tell the chief, [and] the chief will announce to his people that the hospital personnel have something to say, so everyone will go to the meeting…The chief will open the floor and hand it over to the hospital staff to address the people.” [FGD, Unmarried Youth – Dowa]. From the foregoing, the chief was traditionally, the primary source of information. However, for lack of technical knowledge and skills on the subject of VMMC services study participants in both communities identified health workers as a preferred source of information. Study participants held that health workers were well trained hence knowledgeable, experienced and holding reliable information. “…medical personnel should be assigned to spread the message of circumcision because they are well trained. If they were to convey the message it will be better understood,” [IDI, Traditional Leader – Dowa]. The most preferred message that communities want to hear from those implementing VMMC interventions is benefits that VMMC offers to communities, households, and men and boys in particular. Regarding pre-adolescents, parents, clan leaders, community and religious leaders from Mangochi district indicated that they needed to be schooled on how VMMC provided long-term health benefits to males. For example, they needed to learn about such long-term benefits as the reduction of the risk of contracting HIV during traditional circumcision rituals during ndagala . Parents and guardians also wanted assurance that VMMC offered their wards protection against HIV infection during heterosexual relationships before and after they began to raise their future families. An unmarried youth in Dowa narrated as follows: “ I think the most vital information is that people should know the benefits… of circumcision and to my understanding circumcision primarily protects you from cervical cancer so if they are told the women will encourage the men to go,” [ FP1, Dowa Unmarried Youth]. A female respondent in Dowa was of the view that men were not provided with sufficient details regarding VMMC procedure. From her perspective, VMMC messages needed to clearly explain what exactly made a circumcised person different from a non-circumcised man. She observed that VMMC messages had scanty details on what the circumcision procedure involved and what really happened to the man. She argued that men, who were the target of opportunity for VMMC interventions, were blank on details of the procedure: “…We just said they circumcise each other but we did not really know what they really cut. People just say the ‘foreskin’ of the penis others just say the ‘[head of the] penis’ so when you think about it, eeh! It’s scaring.” [FGD, Gate Keepers - Dowa]. The study established that the role of VMMC in the prevention of cervical cancer was more attractive than the role that it played in the reduction of HIV infection since everybody in the area knew at least one woman who had died of cervical cancer. They, therefore, wanted messages to focus on how VMMC would protect females from cervical cancer. “The protection from cervical cancer in women is most important in this area…we don’t have the desire for promiscuity and our women don’t have the desire of seeking sexual satisfaction in bed from circumcised men… Let us put HIV aside, currently the lives of people are at risk. You can manage HIV when infected, but there is no cure for cancer,” [FGD, Traditional Leaders - Dowa]. Female participants wanted VMMC messages to focus more on how their sons would be circumcised by trained medical personnel, which would result in less pain, loss of blood and reduce the risk of complications. “…in the past we had STIs like chindoko mabomu but they were treatable but these days there is AIDS which is very dangerous. There is [cervical] cancer and chisonono too. VMMC is helpful since we hear that it protects our youth and their [future] families from these. These are messages that we need hear about,” [FGD, Adult Women – Mangochi]. VMMC messages also needed to fight against stigma and must assure both Christians and Muslims that VMMC did not contradict the Bible and the Qur’an respectively. One Sheikh, for example, was pleased with VMMC, as a religious authority: “…when I look at the current situation as an Islamic leader, I am very pleased because what is happening now [in clinics] is real jando as prescribed by the Islamic faith.… VMMC fully agrees with Islam.” [KII, Religious Leaders - Mangochi] Regarding the channel of communication for VMMC intervention, many informants reported that the media, particularly radio was prominently used in both study communities. “…to avoid the huge cost [that would come with village meetings] then I can only agree with the media approaches that are being used currently… just think about how MBC Radio One is boasting that 87% of the Malawi population are able to hear them… Now if 87% are listening to MBC [alone] how about those listening to Zodiak?” [IDI, Religious Leader – Mangochi]. However, not many informants were excited about the use of such open channels to promote male circumcision both in Dowa and Mangochi district. In the two districts the use of open media channels was frowned upon for infringing on the secrecy that surrounds rites of passage rituals. First, male circumcision among the Yao was not a subject for public discussion. Second, although both the Quran and the Bible legitimized it, the praxis in the Muslim Yao community was to veil it in secrecy since those not circumcised and females, in particular, were not supposed to know what happened out in the bush: “In the past, circumcision was a total secret such that even the boy’s mother did not know why her child was going to ndagala . The majority of the mothers celebrated jando blindly. And when we were there they strictly told us not to tell anybody what had happened to us. They told us that when we went back home we must bath with pants on lest the mother notices the difference in the appearance of the member….” [KII, Religious Leaders - Mangochi]. Another key concern with the use of radio, television and other forms of mass media was the sexual explicitness that such messages were associated with. This concern was particularly common among adult men and women who feared that such messages would have negative consequences since they promoted sexual immorality among the youth. This, they argued, was contrary to the purpose of male circumcision in the Yao culture: “The problem comes in because … [through the media] you are telling people that once you have been circumcised, you are free from HIV. These are wrong messages…You will [now] hear a man telling a woman that ‘I was circumcised you can come for sex I can’t contract diseases,’” [KII, Yao Culture Expert - Mangochi]. Informants’ most preferred communication channel both among the Yaos of Mangochi and the Chewas of Dowa district was face-to-face engagement meetings attended by health promotion experts on one hand and community leaders, cultural gatekeepers and community members on the other. Among these methods were: meetings where traditional leaders would bring their subjects to one place and engage them in a dialogue; cultural performances such as gule wankulu; religious gatherings such as regular weekly services of worship; interactive drama and door-to-door visits just to mention some. The key reasons for preference were twofold: first, unlike the mass media, they provided room to control the type of audience to hear the message or not. Secondly, they provided an opportunity for the audience to ask questions and seek clarifications as messages are delivered. Engagement meetings organized by traditional leaders were important since chiefs and their councils of advisors acted as a bridge between citizens and government and its stakeholders: “… gulewankulu characters can dance then the drama can come next to teach things like these or you can introduce the topic at first so that the people can have an idea so when the drama on circumcision comes it will entertain them,” [FGD, Gatekeepers – Dowa]. Although informants from all Protestant Christian churches including the Catholic leadership in Dowa sounded very negative about using the church to promote VMMC messages, in Mangochi the Catholic Church was already disseminating VMMC messages. I was also carrying out surgeries. One gulupa said: “ … [Children] are also assisted to get circumcised. When they get to that stage, they are kept at the parish, which is like a simba ….” In Chowe area, Mangochi, FGD participants proposed that VMMC messages should be disseminated through their mosques and churches. This is how one traditional leader put it: “In this community, Churches and Mosques are more ideal. Religious gatherings are easy places to disseminate VMMC information,” [FGD, Traditional Leaders, Mangochi]. In Mangochi, a representative of an NGO added that they used males from within the community to deliver VMMC messages to fellow men. The health worker posited that men tended to listen better to health messages when delivered by their peers. “ We have community mobilizers who are recruited from within communities. They reach out to men in the communities,” [KII Participant, NGO Worker – Mangochi]. Another NGO worker said: “Our main approach with demand creation is interpersonal communication.” [KII Participant, NGO Worker - Mangochi] . The study established that VMMC messages should target all community members including men, women and boys to avoid misunderstandings that may arise from misinterpretation of the messages particularly among married couples. This is what one IDI participant said: “These days I think that everyone [should be targeted] because, in a family, the man may hear the message alone but when he brings the message into his household the wife may misinterpret it… If the message was received by all - the man, the woman and the boy … people would easily embrace it,” [IDI, Traditional Leader – Dowa]. This view was supported by another community leader who argued that the message must go to both males and females to avoid disagreements between married couples: “The message has to go to both because the women are the ones who stay with the children. They can get them circumcised,” [FGD, Gatekeepers – Dowa]. The study also established that another key entry point into VMMC among the Yao men of Mangochi was the pre-adolescence age bracket of 5 to 10 years. They were minors such that they needed consent from their parents or guardians or those in authority of a village of institution. Two KII participants in Mangochi suggested that VMMC messaging must also target teachers and schools: “…I think that teachers also are very important in their own right because they interact with the community [and learners] in particular. I have a view that teachers can help us clear a lot of misconceptions …,” [KII, Service Providers – Mangochi]. This view point agrees with the observation that one participant in an FGD with Service Providers made on the same matter in Dowa. He lamented that although schools had a lot of HIV/AIDS-related clubs authorities had failed to utilize them to popularized VMMC services among adolescents and youth. “At school we have HIV&AIDS youth clubs but because they were not oriented on [VMMC] services … it is hard for you [as a teacher] to explain it in clear details [to learners] particularly the benefits of VMMC… The main topic in youth clubs is HIV&AIDS so I think if HSAs and teachers were oriented we can pass on the knowledge when we are with the youth clubs ,” [FGD, Service Providers – Dowa]. Regarding the expected effects (or impact) of VMMC communication interventions and the VMMC program in general, the study established that the anticipated effects were at four different levels: at personal level, at household level, at community level and at policy level. At personal level, interventions were expected to create awareness of VMMC and how it contributes to HIV prevention in heterosexual men, cervical cancer (among women) and other sexually transmitted infections. There was also an expectation of increased preventive post-circumcision behaviour among VMMC clients: “…one man did not observe the six weeks -sexual-abstinence window like he was instructed by the service provider. They resumed sex earlier and he developed complications,” [KII, Service Providers, Chowe – Mangochi]. In Dowa, the overall anticipated impact of VMMC communication interventions was the reduction of cervical cancer cases that were considered to be on the rise. One sex workers’ expectation had this to say: “The main issue is reduction of diseases; it is the same when the hospital is giving counseling on condoms, they say ‘every man should abstain from sex. However, if they fail then using a condom is Plan B’. It is the same with circumcision. It is Plan B,” [FGD, Commercial Sex Worker - Dowa]. In Dowa, there was also fear that VMMC communication interventions could also lead to rising culture of promiscuity that could lead to increased HIV prevalence: “It [VMMC] is encouraging promiscuity among the youth here because if one boy is told that he is very sweet by a girl he wants to sleep with every girl…,” [FGD, Service Provider – Dowa]. Community members and health personnel working in local health facilities wanted to see household leaders, particularly mothers and clan leaders, to mobilize adult men and boys under their influence for services since they were primary producers of health. Sexual partners, mothers, aunties and grandmothers were considered to have the duty of motivating male members of their households to access services. They were also expected to support circumcised males to adopt post-circumcision prevention measures: “…females are the ones who go to the hospital… when the mother and other females are taught and understand [advantages of VMMC] it will be easy for the child to get circumcised,” [FGD, Community Leaders, Dowa]. Some traditional circumcisers were still reported using one razor blade or knife to circumcise more than one initiate. But, change had already started happening at community level since VMMC communication intervention rolled out in Mangochi: “Two weeks ago I was on a tour … Angaliba themselves were asking us: ‘Please, government should send us circumcision knives for use in initiation camps ,” [IDI, Senior Chief – Mangochi]. In Mangochi, informants felt that interventions enabled Yao Muslims practice jando as prescribed in the Holy Quran. One Yao Islamic cleric posited that VMMC already contributed to the demystification of male circumcision which, in the Yao culture, had been shrouded in secrecy from women, children and uncircumcised males for centuries: “…I am very pleased because what is happening now [in clinics] is real jando as prescribed by the Islamic faith.… VMMC fully agrees with Islam,” [KII, Religious Leaders - Mangochi] . In Dowa district, gatekeepers also expected non-formal institutions such as the gulewankulu supporting the VMMC campaign through information dissemination. They also expected it to lead in the reduction of cancer-related illness and deaths and to harness VMMC clients as advocates for services in their respective communities. In Dowa interventions were also expected to lobby for a more stable availability of funds to support community outreach programs: “In the past we used to go on outreach programs and we would discuss (VMMC) with community members… We have no money for outreach programs,” [FGD, Service Providers– Mponela, Dowa]. Although they completely rejected the proposals to adopt male circumcision as a standard cultural practice in gulewankulu bases called dambwe , the greatest change was that the Chewas were, nonetheless, ready to use gule wankulu characters as crowd puller to community meetings to ensure that VMMC messages quickly diffused into communities. “…They can spread the message via songs since gule wankulu characters have a special talent when it comes to composing songs,” [FGD, Community Leaders – Dowa]. Informants both in Mangochi and Dowa also wanted to see secondary facility (i.e. district-level-hospital) circumcisers and community-level-hospital (i.e. local health facilities) personnel working together when carrying out circumcisions. To achieve this, informants were of the view that the district health system needed to ensure that there was adequate coordination and collaboration by all key stakeholders in the VMMC service delivery value chain in each district. “If, on average, they had at least enrolled one nurse or clinician from each rural facility in the district …These could have been assigned to promote VMMC services at community level,” [KII, Service Provider, Mangochi]. Another service provider in Dowa : “The job is done by the top dogs so… we can’t follow-up because we don’t know how they counsel [clients]. Maybe there isn’t any follow-up [in their plans],” [FGD, Service Providers -Dowa]. Informants also complained that VMMC circumcisions that were offered at health facilities lacked privacy for clients. They expected this to change if the program would be successful. In Mangochi, due to lack of a proper transport arrangements, older men were sometimes being ferried from rural areas to the secondary facility on the same open lorry with boys as young as 5 years. At one facility in Dowa, informants reported that circumcisions were taking place in a room whose entry was inside the maternity ward such that adult men shunned services. Many parents who were willing to get their sons circumcised shunned services. “ … they had no choice due to lack of proper space … [VMMC clients] had to go past a group of pregnant women [in the maternity wing]. Boys were shy. The room should have [had] two doors so that after getting circumcised they should use the other door… ,” [FGD, Service Providers – Dowa]. At two facilities in Mangochi, informants told these researchers that VMMC had not been integrated into other services offered by facility and no special day or time had been allocated to these services since services were just being provided when circumcisers came all the way from Mangochi District Hospital. The other challenge both in Mangochi and Dowa was the long distances to service points. In the FGD with VMMC clients in Dowa said: “Hospitals are very far from our area so it costs a lot of money to go there and sometimes we don’t have the money,” [FGD, VMMC Clients – Dowa] . Both the Chewa and Yao people performed rites of passage with the same purpose of shaping the character of boys and transitioning them from childhood to adulthood. Nonetheless, s they differed in the rituals they performed for both boys’ initiation rites. While the Chewa initiated boys throughgulewamkulu the Yao people initiated them through jando where male circumcision was administered both for cultural and religious beliefs. The negative attitude to VMMC observable among the two ethnic groups is the major reason why there is low uptake of VMMC services both among the Chewa people of Dowa and the Yao people of Mangochi. They feared that the procedure was in conflict with their cultural identities, traditions and value systems. In particular, both data collection communities perceived VMMC as lacking in character formation which was the main drive behind rites of passage rituals in their areas. However, the two cultures also differed on motivators for VMMC services. A fraction of the Yao people accepted VMMC because they did not have to pay anything to access services, except meeting transport costs, reduced pain, quicker healing of wounds, reduced risk of contracting HIV due to the use of sterilized razor blades and receiving services in a hygienic environment. Some Chewa people, on the other hand, were motivated to access VMMC services mainly because of its perceived ability to protect women from cervical cancer. On culturally-preferred communication approaches, generally, both study communities opted for interpersonal face-to-face communication approaches since they gave them the opportunity to interface with health promoters and provide real-time feedback on the messages that they disseminated. The study also established that since the two ethnic groups were different from each other culturally and religiously such that, although they had similar communication needs in many respects, these two groups had different communication needs. This phenomenon suggested that communication programs should not be generic but must be designed to address the specific needs of each treatment group. Regarding the source of VMMC messages , informants in both study groups posited that trained health workers were preferred. Health workers were preferred to disseminate such information because they were considered trained and well experience in disseminating health information hence expected to possess the required competence to carry out the task of reaching out to various audiences with VMMC messages. This finding is also consistent with other studies that have been conducted in Malawi and elsewhere on VMMC communication and have shown that health personnel are among the most trusted and preferred sources of information among males . The Ministry of Health in Kenya, for example, prioritized capacity building of health personnel including those involved in health communication . It was intriguing, however, to learn that much as informants preferred health workers as sources of VMMC messages, study participants both in Mangochi and Dowa were of the view that community entry must be done in consultation with leaders of their communities. Thus, iformation delivery must take place in the presence of chiefs or anyone delegated to stand in. The study demonstrated that health workers needed to work with chiefs in order to create a platform on which the message can be disseminated to the maximum number of people in the area. The contact between the health experts and traditional leaders provides the first opportunity for establishing a shared vision between health experts and the target audience. This suggest that the starting point for designing a good health communication campaign is the establishment of a shared vision between the health promoters and the target audience . This finding agrees with Lozare et al. who positulated that since the primary producers of health are the people themselves there is need to engage them in the most meaningful way . In the Malawi setting, when the chief and the rest of the cultural structures reject a message, the whole community follow suit. Generally, informants approved the current practice where health workers were at the center of message dissemination. However, they expressed worry that, in the current set up VMMC workers from the district hospital or the NGO partners, invaded their villages and communities with loud hailing before adequately engaging traditional leaders, cultural gatekeepers and without building partnerships with cultural structures that carried out similar work in the local community. They further deplored the failure by health experts to hold community meetings through which community members would have a chance to ask questions and seek clarification on aspects of VMMC that were not clear. Regarding messaging , informants wanted messages that were contextual and unambiguous and promoted HIV prevention. They expressed worry that although VMMC had the potential of positively contributing to the reduction of HIV prevalence sexually explicit messages, which were frequently heard in the media, were perceived to have the potential of increasing promiscuity among the youth, a phenomenon that could potentially worsen the HIV situation in their areas. This notion reflects the general Malawian worldview that the function of sex surpasses the procreation and sensual function as it connotes survival of the extended family, clan and tribe . In his writings Augustine Musopole argues that, in Africa sexuality is one of those vital forces that make life secure, meaningful and worthwhile . He, therefore, advises that it is largely a taboo to openly discuss sex and sexuality across many African cultures where adolescents were separated from the community and taken to the bush to be taught sexual rituals, moral values, and cultural norms . In this study informants generally agreed that messages that put emphasis on the sexual pleasure as a benefit of VMMC services were not welcome to the majority of Malawians. Both in Dowa and Mangochi, older informants expressed concern that sexual pleasure was dominating the VMMC messaging instead of focusing on HIV prevention outcomes. These findings add value to the conclusions of the study in Malawi by Patrick Makono et al. who observed that lack of a proper research prior to the design of Malawi’s Ndife Otsogola VMMC brand, resulted in messages that failed to effectively promote the uptake of VMMC services among men and boys in Malawi . Both study sites preferred messages that focused on health benefits of VMMC such as the prevention of HIV and cervical cancer. The Chewa people, on one hand, were almost complacent about their vulnerability to HIV infection because they did not practice polygamy and their instruction, both in the cultural and religious initiation rituals, was perceived to be strong on its emphasis on sexual abstinence. They, therefore, felt that if VMMC messages were to appeal to the majority of them, they needed to create an awareness of how male circumcision contributed to the reduction of deaths due to cervical cancer among women. Yao informants, on the other hand, posited that both HIV and cervical cancer prevention was a motivating factor. They, therefore, preferred messages that created the awareness, that facility circumcision service reduced pain, ensured early healing of the wound and the risk of post-op complications. They also preferred messages that portrayed VMMC services as being cheaper and more hygienic than traditional circumcision. Findings on the need for benefit-centered messages and their indispensability in promoting the uptake of VMMC services are similar to outcomes of other studies conducted in Malawi. For example, Mhagama et al. and Makono, et al. established that men and boys will opt for medical circumcision after obtaining benefit-centered information from medical personnel and personalized information from their peers . In a study conducted in KwaZulu-Natal in South Africa by George Gavin et al. established that knowledge-rich personalized-information was a vital facilitator of service uptake in South Africa . Another study carried out in Rakai, Uganda, by Ssekubugu, et al., demonstrated that males acted on VMMC messages that emphasize on health benefits . Regarding channels of communication , radio came out prominently as a common channel that government agencies and NGOs were using to disseminate messages on VMMC services. However, as Tilson et al. have noted, one key obstacle to effective communication in the past pertained to conceptualizing communication as a simple one-way transmission from the source to a receiver with the intention of producing some effect . Many study participants pointed out that radio and loud hailing was not interactive enough because it did not provide them with an opportunity to ask questions and seek clarification on the messages that it delivered to listeners. They, instead, preferred community-based interpersonal communication to any other form of communication. For example, the Chewa people of Mwancheka area, in Dowa district, preferred approaches that brought families and communities together. Messages could therefore be delivered during funeral receptions, religious functions, drama performances and gule wankulu functions, just to mention a few. One community leader said: “Even when there is a funeral the chief can stand [up] and deliver information on VMMC.” Study participants in Dowa were completely opposed to mainstreaming male circumcision into the male-dominated gulewankulu. However, they were open to the use of gulewamkulu to accelerate the dissemination of messages on VMMC services in their communities. It is for this reason that communication channels that enabled families and communities to collectively have direct access to VMMC messages in an interactive manner were preferred. This view point was consistent with Benjamin Lozare’s assertion that since the households and communities were the primary producers of health, communication approaches that sought to empower such units needed to be prioritized . About the audience , the key problem with VMMC campaigns in Malawi, according to informants from both study ethnic groups, was the focus on the individual male rather than every ‘individual citizen’ of the community in the VMMC messaging. It was recommended by study communities that VMMC messages must target the man, the boy, the woman, and all social structures in the social-ecology. They argued that the message must be directed to close family members, peers of eligible men and boys, sexual partners and caregivers who happen to be females generally. Decisions either to access VMMC or not are not entirely personal for men or boys. Like it was discovered in Uganda, this study, both in Dowa and Mangochi, found that for married men, female sexual partners had a strong say on the decision a man makes . This finding also agrees with studies conducted in Papua New Guinea, Zimbabwe and Botswana. In these studies, it was observed that women played a significant role to improve uptake of voluntary medical male circumcision (VMMC) and in the post-op behaviors of the client . These studies established that women could motivate men, particularly their sexual partners, to access VMMC services. Women were willing to engage their husbands on the benefits of VMMC such as improved penile hygiene, reduced risk of HIV transmission and other sexually transmitted infections, as well as increased sexual satisfaction . In the matrilineal marriage system which is generally practiced in Malawi and in the study areas i.e. Mangochi and Dowa, extended family members particularly uncles, aunts, and grandmothers, as well as clan heads (eni mbumba) , have a great deal of influence in decision making processes at household level . Among the Chewa people of Dowa for example, the woman generally moves to live in the man’s village under a practice called chitengwa while among the Yao of Mangochi, the man is expected to move and live in the woman’s village under a practice called chikamwini . This means that apart from aiming messages at eligible men and boys, the campaign must also aim at reaching the entire community, particularly women, if access to services is to improve. This study has unearthed mixed preferred effects of VMMC messages. These preferred effects , according to the Social-Ecological Model that was used in identifying informants to this study, manifested at four different levels of the social ecology namely: at individual level of a circumcised male; at household level; at community or society level and, finally, at policy level. The preferred effect of VMMC messages on the primary audience, adult men and boys, is sixfold. First, is an awareness and an understanding of VMMC with a view to demystify the concept of voluntary medical male circumcision - a phenomenon that would lead to increased uptake of services, reduced transmission of HIV, cervical cancer among women and girls and sexually transmitted infections. Secondly, an assurance both among Christians and Muslims that VMMC had not been introduced neither to contradict the Bible or the Qur’an respectively nor for political reasons since its purpose is purely biomedical. Thirdly, informants mentioned increased post-circumcision preventive behaviour among clients. Fourthly, they expected increased sexual pleasure and a reduced risk of complications due to early sexual resumption and a reduced fear of pain particularly among adults. At household level informants expected two key impacts. First, mothers and female household members, as primary producers of health , were expected to use their ‘authority of matrilineality’ to encourage men to access services and get their sons, particularly infants and pre-adolescents and adolescents, circumcised at the facility. Secondly, clan leaders were expected to play their rightful role as mwini mtundu or mwini mbumba , by encouraging men, pre-adolescents and adolescents to get circumcised at the facility and supporting them adequately after accessing services. At community level in Mangochi, informants expected to see the reduction of three risky practices: adult sexual instruction that initiates as young as 5 or 6 years were exposed to in rituals called kusasa fumbi for girls and kutaya mafuta for boys; sexually explicit language and songs and the use one razor blade or knife to circumcise more than one initiate by traditional circumcisers. In Dowa, community leaders and gatekeepers expected to see the gulewankulu community support the cause of the VMMC campaign by disseminating messages in their own communities; the increased adoption of VMMC services by adult men who were said to be reticent on access to health services generally. Lastly, the key effect that informants expected from VMMC communication programming was community development as a result of good health at household level. These authors have noted, however, that feedback is the key missing link in Laswell’s Transmission Theory since the theory discusses communication purely as a linear process on the assumption that all there is in a communicative process are sources of information, messages, the audience, medium or channel through which sources speak, and effects. This observation was also made by Peng Wenxiu . This was not complete because, for real effect to occur, like Lozare puts it, the communicator (source of the message) and the audience (the consumer of the message) need to have a common vision or understanding for true behaviour change to occur . For this to happen there is need for those involved in VMMC communication to appreciate that communication is not a one-way lane but a two-way road with interaction between the communicator and the audience. It is for this reason that this study concluded that community engagement and interpersonal communication which provide room for real-time feedback in any communicative event are the preferred communication interventions for VMMC services among Yaos and Chewas of Southern and Central Malawi respectively. Participants both in Mangochi and Dowa raised a serious concern that health practitioners involved in the promotion of VMMC services in their districts did not adequately engage them in their villages. This, they observed, robbed them of the chance to ask questions and to seek clarification on matters that concerned them or were not clear regarding facility circumcision. Study strength The study has clarified five key elements in the communication continuum of VMMC interventions including source, audience, message, channel, effect and feedback and has demonstrated the place of communication in health promotion. The study has demonstrated the indispensability of interpersonal communication health communication with a special emphasis on the need for real-time feedback. It has also provided a platform for further inquiry into health promotion programming particularly in the use of Laswell’s Transmission Theory in health communication in the African context. Limitations Being a by-product of a purely qualitative study, this report lacks statistical basis upon which conclusions could effectively be grounded. Although the principal investigator sought the services of two Yao insiders (a male and a female) and one Chewa insider (a female in-training health worker) to collect and support data analysis, he is aware that his own personal biases may have been introduced in the data analysis and presentation of findings. To counter the bias the data analysis approach was guided by the thematic framework approach whereby rigorous reading and re-reading of each transcript was undertaken with the view to getting familiar with the content of the transcripts. The repeated review of transcripts informed the identification of codes which were then classified into sub-themes which were further synthesized into bigger themes. This effectively minimized the possibility of introducing biases into the content. The codes were developed from the emerging issues from the responses as was required by the study questions which fully underwent a review and approach of the National Committee on Research Ethics in the Social Sciences and Humanities (NCRSH). In this regard, the approach to data collection and analysis was guided by the stipulated ethical obligations by a competent review IRB board implying that the researchers were guided and made sure to adhere to the stipulated ethical obligations and principles of thematic framework approach which is the analysis methodology followed. Further to this, this is a product of team work involving the corresponding author and a team of co-authors who participated in the drafting of manuscript and review of the same suffice to say that co-authors were from different cultural backgrounds. The study has clarified five key elements in the communication continuum of VMMC interventions including source, audience, message, channel, effect and feedback and has demonstrated the place of communication in health promotion. The study has demonstrated the indispensability of interpersonal communication health communication with a special emphasis on the need for real-time feedback. It has also provided a platform for further inquiry into health promotion programming particularly in the use of Laswell’s Transmission Theory in health communication in the African context. Being a by-product of a purely qualitative study, this report lacks statistical basis upon which conclusions could effectively be grounded. Although the principal investigator sought the services of two Yao insiders (a male and a female) and one Chewa insider (a female in-training health worker) to collect and support data analysis, he is aware that his own personal biases may have been introduced in the data analysis and presentation of findings. To counter the bias the data analysis approach was guided by the thematic framework approach whereby rigorous reading and re-reading of each transcript was undertaken with the view to getting familiar with the content of the transcripts. The repeated review of transcripts informed the identification of codes which were then classified into sub-themes which were further synthesized into bigger themes. This effectively minimized the possibility of introducing biases into the content. The codes were developed from the emerging issues from the responses as was required by the study questions which fully underwent a review and approach of the National Committee on Research Ethics in the Social Sciences and Humanities (NCRSH). In this regard, the approach to data collection and analysis was guided by the stipulated ethical obligations by a competent review IRB board implying that the researchers were guided and made sure to adhere to the stipulated ethical obligations and principles of thematic framework approach which is the analysis methodology followed. Further to this, this is a product of team work involving the corresponding author and a team of co-authors who participated in the drafting of manuscript and review of the same suffice to say that co-authors were from different cultural backgrounds. The key finding of the study is that community stakeholder engagement, complemented by various interpersonal communication and small-group approaches such as village town-hall meetings, drama, religious and cultural gatherings (including gulewankulu festivities for the Chewa people), is the preferred communication approach both in Yao and Chewa communities. The main reason is that apart from its strong emphasis on exchanging knowledge between the source and the audience in a human-to-human fashion, community stakeholder engagement and interpersonal communication allow the communicator and the audience to foster a common vision which, according to Ben Lozare, is a prerequisite for behavioral change. This study also established that one critical element that must be created in every health communication situation is feedback . It is this element that makes the development of a common vision between the health communicator and the target audience. It is in this context that true adoption of a desired behavior can be achieved. |
TaiNi: Maximizing research output whilst improving animals’ welfare in neurophysiology experiments | 582209b7-ee44-44ba-961c-480e6f00eb4c | 5556067 | Physiology[mh] | Recent advances in the development of transgenic mice have provided unprecedented insight into the mechanisms of mammalian brain function and human disease processes, and have led to a dramatic shift from rats to mice as the preferred preclinical model used in drug discovery. However, their small size makes neuronal recording in freely-moving mice challenging. The ability to make direct electrophysiological recordings from populations of neurons requires multiple parallel recording channels and high sampling rates (>10 KHz) in order to properly characterize action potentials , . The circuitry required is consequently energy-intensive and traditionally requires a multi-wire tether to provide power and to carry the analogue signal to the recording equipment. While this is practical in larger rodents, it presents a serious burden for a mouse . Recently developed wireless recording systems allow both greater freedom of movement and the possibility of entirely new experimental designs, such as recording from complex enclosed environments – . However, there has always been a trade-off between the weight of the device and recording density or duration. Current off-the-shelf solutions are limited to less than 4 hours recording unless a harness is employed to support the additional battery weight, or provide longer recording only at reduced sample-rates which are suitable for recording local field potentials (LFP) or electroencephalograms (EEG), but not action potentials (APs). In either case, the devices are also still cumbersome and relatively heavy (4 g or more, Table ). This represents >10% of the weight of an adult mouse - similar to a human subject carrying a 6 Kg weight on their head. To improve on existing wireless technology, the circuitry needed to be redesigned from the bottom-up, with an emphasis on improving the energy efficiency. Here we test the performance of TaiNi - a novel wireless system based on a customized Application-Specific Integrated Circuit (ASIC) created using innovative low power design techniques. The circuit blocks, as shown in Fig. , incorporated in the ASIC include filters, amplifiers, analogue-to-digital converters, multiplexing and wireless transmission. This maximized efficiency and reduced total weight to 1.5 g. This system represents a significant advance in scalable recording systems for small rodents, and provides a robust foundation for additional designs.
Bench tests Packet loss Bench tests to assess the packet loss of the TaiNi devices were performed with the receiving antenna oriented horizontally (25 cm above the bench) and directly connected to a receiver (USRP X300, Ettus Research, USA) equipped with a pair of wideband transceiver daughter-boards (SBX-120 USRP). The TaiNi device was positioned on the bench, with the loop of the transmitting antenna directed towards the receiving antenna. The horizontal distance between the two antennas was increased in 10 cm increments between 0.1 and 3.0 m. With the transmitter operating at 2.31 GHz, the packet loss was quantified over a 2 minute period in each location. Frequency response The frequency response of the system was calculated using two series of chirp signals (arbitrary waveform generator 33522A, Agilent Technologies, USA), one low, and one high. For the low-frequency, a 2 mV peak-to-peak sinusoid with a logarithmic frequency sweep from 0.1 Hz–1 Hz was used. The sweep time was 100 s. For the high-frequency response we used a 10 s 2 mV 1–100,000 Hz logarithmic sweep. Animals All experiments were performed in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986 with approval from the United Kingdom Home Office. For the current experiment 16 female rTg4510 mice , were bred by Envigo (Loughborough, GB), before being delivered to Eli Lilly and Company for testing (Windlesham, GB). Here, mice were singly housed on a 12 hour light/dark cycle with water provided ad libitum. Food was restricted as required prior to behavioural testing, with mice maintained at no less than 85% of their free-feeding weight. Because the mice used had previously participated in another experiment, 9 of them were treated from 2–3 months of age with doxycycline (DOX), initially via a daily bolus of 10 mg/kg p.o. doxycycline hyclate (Sigma Aldrich, GB) for the first two days. They were then maintained on doxycycline-mixed chow diet (Harlan Teklad Rodent Diet, 200 mg DOX per kg of dietary chow) until the end of the study. All tests involving a comparison of different systems required all animals to run in all conditions, so the DOX treatment could not affect the results. Surgery Mice were implanted with either a single shank 16 channel probe attached to a microdrive (Cambridge Neurotech, GB) targeting the CA1 region of the hippocampus (n = 14 mice; AP: +1.7 mm, ML: −0.3 mm relative to Bregma), or a static dual shank silicon probe (n = 2 mice; 8 sites per shank (Neuronexus, USA)) targeting the medial prefrontal cortex (AP: +1.7 mm, ML: −0.3 mm relative to Bregma & DV: −2.0 mm from brain surface) & dorsal hippocampus (AP: −1.9 mm, ML: −2.0 mm relative to Bregma & DV: −2.0 mm from brain surface). Mice were initially anaesthetised with 3% isoflurane, before being transferred to a stereotaxic frame and maintained for the remainder of the procedure at 1.5–2%. Five stainless steel screws (00–96 × 1/16, PlasticsOne, USA) were attached to the skull and used as an anchor point for implant. One of the screws was placed over the cerebellum and acted as the ground electrode. Craniotomies were made at the specified stereotaxic coordinates, and the probe lowered into position before the hole was sealed with silicone sealant (3–4680 silicone gel kit, Dow Corning, USA). Light curable composite (Revolution Formula 2, Kerr Dental, USA) was used to bind the implant/microdrive to the skull. Mice were allowed to recover for a minimum of 2 weeks before entering into any experiments, however probes attached to microdrives were slowly lowered to span the CA1 pyramidal layer over multiple days between 1 & 2 weeks post-surgery (initially implanted 500 µm below the brain surface). Electrophysiological recording Room and system configuration All in vivo tests were conducted in a 3.1 × 4.0 m windowless room with black walls and ceiling, lit by two uplighters and separated by a door from an anteroom containing the recording equipment. The room was on a 12-12 dark-light cycle with lights-on at 7am, and all experiments except the 72-hour recording were conducted in the light portion of the cycle. The room contained four bespoke infrared tables (100 × 100 cm), two on either side of the room and separated by 90 cm. A near- infrared emitter array (4 x IR emitters, ~650 nmm wavelength) and a miniature infrared-sensitive video camera (model M700LD, Henrys, UK) were centrally positioned above each table. The tables and emitter provided illumination of the subject from above and below to facilitate high-quality video tracking under a variety of lighting conditions. To receive the signals from the TaiNi transmitter, each table was equipped with a pair of omnidirectional rod-antennas (CSL 12dBi, 2.4 GHz) elevated 30 cm above the table, oriented horizontally and at 90-degrees to one another, forming an open bracket around the recording environments described below. The two antennas from each of the two tables on either side of the room were connected to a 4-way splitter (model DBD-PD-8426-4, dBD Communications UK) by 1.5 m lengths of flexible antenna cable (LBC195 ExtraFlex, MS Distribution UK). Each splitter was in turn connected to one of two inputs on the receiver in the anteroom via 12 m lengths of heavy-gauge coaxial cable (LBC 400, MS Distribution UK). This heavy cable has very low 0.2 dB/m signal-attenuation. In the anteroom, video signals from all four cameras were fed to a quad video combiner (VQM801P, Sanyo, JPN) and the merged video stream processed by EthoVision XT 8.5 (Noldus, NL), enabling the position of each mouse (body-centre and nose) to be tracked. To allow synchronization of the electrophysiology and behavioural data, an I/O interface (PTIO-0020, Noldus, NL) was used to generate a signal at the beginning and end of each video tracking recording, which was converted to a TTL pulse by a hardware interface (MED-PC model SG-233-48) and was recorded by the Taini acquisition PC. The synchronization was performed on the receiving unit during the data post-processing using the local timings of the TTL pulse recorded in both the electrophysiology and behavioural data. The antenna signals were fed to the same model and configuration receiver used in the bench tests (see above). Acquired signals were passed via a 10 gigabit ethernet cable to a Linux workstation running custom software for channelization, demultiplexing, visualization and storage. Channelization was parallelized using the graphics processing unit (Quadro K420, Nvidia) to handle the large data bandwidth. The software also registered TTL sync-pulses sent by the EthoVision PC. All 16 channels from each transmitter were recorded “single-ended” (non-differential), using only the cerebellar skull screw as the signal ground. Training and testing During the 72 hour recordings, mice (n = 2) with dual shank probes were placed in a novel home-cage (15 × 30 cm) with free access to food and water for the duration of the recording. Open field pellet-chasing experiments were performed using 4 food restricted, five month old female rTg4510 mice (2 nonDOX & 2 DOX treated) implanted with single shank silicon probes. These mice were trained twice daily on a pellet chasing task (10 minutes per session) for a total of 10 days in the month proceeding testing. Training consisted of mice being placed in a transparent, circular arena (diameter: 30 cm; height: 50 cm) with pellets (12 mg chocolate flavour AIN-76A Rodent Tablet, TestDiet, USA) dispensed from above at 20 s intervals. On the day of recording, a 60 min sleep recording was performed in a familiar home-cage environment before the mice were transferred to the familiar open field arena for 20 mins of pellet chasing (20 s inter-pellet interval). Throughout both the 72 hour and open field recordings, the cameras above each mouse continually captured data. In the open field recordings the animal’s body was tracked as a silhouette against the infrared emitting table under the arena, while during the home-cage recordings the overhead infrared emitters provided the necessary contrast against the bedding material, which obscured the light from the table below. Split signal tests Concurrent home-cage recordings (n = 3 mice implanted with single shank probes in CA1) were performed using the TaiNi and tethered Digital Lynx SX (Software: Cheetah v5.6.0, Neuralynx, USA) recording systems by utilising a signal splitter placed inline between the silicon probe and headstages. For the Neuralynx recordings, data was band-pass filtered at 0.5–9000 Hz, and sampled at 20 KHz. Weight tests on the automated T-maze 11 food restricted, five month old female rTg4510 mice (6 nonDOX & 5 DOX treated) were tested on a non-match to place T-maze task. The mice had previously been tested on the maze at 2 months of age and also in the week preceding this weight comparison test. On each of the 3 testing days, mice were randomly allocated a treatment condition (no transmitter, Triangle Biosystems International (TBSI) W16 transmitter or the TaiNi transmitter) in a fully counterbalanced manner. The T-maze apparatus was custom-made (Apogee Engineering Analysis solutions, GB) and consisted of a matt black Perspex track (width: 6 cm) with clear Perspex walls (height: 20 cm). The centre-arm of the maze was 100 cm in length and each of the reward arms 36 cm long. The maze was raised 100 cm from the ground and sited in a rectangular room containing multiple visual cues. 3 pellet dispensers were situated around the maze (one per reward arm & one in the delay area at the beginning of the centre arm) to allow rewards (12 mg unflavoured AIN-76A Rodent Tablet, TestDiet, USA) to be delivered for successful performance. The maze protocol was automatically controlled by a microcontroller board (Arduino Mega 2560) connected via a USB serial connection to a host-PC running Matlab (Mathworks, Natick, MA, USA). The animals’ position was detected by infrared beam breaks, which enabled the maze controller to adjust the position of 5 pneumatically driven, black Perspex doors. These could be to be raised from below to block entry into areas of the maze as appropriate. The T-maze task comprised of a sample and choice component. In the initial forced phase, mice were guided along the centre arm by the doors and forced to turn left/right in a pseudo-random manner (maximum of 3 consecutive sample runs to the same arm) in order to receive a reward. Mice then returned to the holding area in return for a second reward, at which time the choice phase of the task began. Mice were again guided along the central arm of the maze, before being offered a free choice to enter either reward arm. Mice were given a reward only if they chose to enter the opposite arm visited during the sample phase. A further reward was provided at the waiting area for mice making a correct choice. Mice were held in this zone for 5 s prior to be start of the next trial. On each day mice were given 60 minutes to complete as many trials as possible. Statistics and Analyses Running-speed estimates were calculated from the animals’ body centre location (x/y coordinates) integrated over 400 ms non-overlapping epochs . Nose-position was used to provide the high-precision data required for analysing neuronal spatial firing properties. Immobility was defined as a period when the mouse moved at a rate of less than 0.5 cm/s for at least 300 s. Running was defined as any period where the mouse moved at more than 5 cm/s for at least 100 ms. For the automated T-maze weight tests, a repeated-measures ANOVA was used, with orthogonal planned contrasts between the NONE and TaiNi condition, and between TaiNi and TBSI. Local field potentials (LFPs) were analysed using an anti-aliased version of the raw data, downsampled to 1 KHz. A short-time FFT using 2-second sliding windows with 50% overlap was used to estimate spectral power. Ripple-detection was performed according to the methods described by Sullivan et al . . Phase-amplitude-coupling in the hippocampus was analysed according to the “cross-frequency-coupling” methods described in Onslow et al . A simple Pearson’s correlation was used to analyse the relationship between 0.5–4 HZ delta power and ripple density. Spike detection and cluster analysis was performed using the KlustaKiwk suite of software , with the maximum number of clusters set to 100 and using the Akaike Information Critierion to determine whether a given cluster should be divided. For spike-detection, the data was filtered (500 Hz high-pass 3rd-order Butterworth filter) and z-scored, with a 4.5 standard-deviation detection threshold. Clusters were discarded if their autocorrelograms exhibited excessive spiking in the 2 ms refractory zone, and were combined where they shared clear refractoriness with other clusters. Place fields for each cluster (putative neuron) were generated according to the methods described previously , but using a linear colour scale for representing firing rates. Briefly, mean dwell-time (seconds) and spike-counts in 1 × 1 cm bins were calculated to generate the firing rate in each bin, before applying a 5 cm Gaussian smoother to the map. Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request.
Packet loss Bench tests to assess the packet loss of the TaiNi devices were performed with the receiving antenna oriented horizontally (25 cm above the bench) and directly connected to a receiver (USRP X300, Ettus Research, USA) equipped with a pair of wideband transceiver daughter-boards (SBX-120 USRP). The TaiNi device was positioned on the bench, with the loop of the transmitting antenna directed towards the receiving antenna. The horizontal distance between the two antennas was increased in 10 cm increments between 0.1 and 3.0 m. With the transmitter operating at 2.31 GHz, the packet loss was quantified over a 2 minute period in each location. Frequency response The frequency response of the system was calculated using two series of chirp signals (arbitrary waveform generator 33522A, Agilent Technologies, USA), one low, and one high. For the low-frequency, a 2 mV peak-to-peak sinusoid with a logarithmic frequency sweep from 0.1 Hz–1 Hz was used. The sweep time was 100 s. For the high-frequency response we used a 10 s 2 mV 1–100,000 Hz logarithmic sweep.
Bench tests to assess the packet loss of the TaiNi devices were performed with the receiving antenna oriented horizontally (25 cm above the bench) and directly connected to a receiver (USRP X300, Ettus Research, USA) equipped with a pair of wideband transceiver daughter-boards (SBX-120 USRP). The TaiNi device was positioned on the bench, with the loop of the transmitting antenna directed towards the receiving antenna. The horizontal distance between the two antennas was increased in 10 cm increments between 0.1 and 3.0 m. With the transmitter operating at 2.31 GHz, the packet loss was quantified over a 2 minute period in each location.
The frequency response of the system was calculated using two series of chirp signals (arbitrary waveform generator 33522A, Agilent Technologies, USA), one low, and one high. For the low-frequency, a 2 mV peak-to-peak sinusoid with a logarithmic frequency sweep from 0.1 Hz–1 Hz was used. The sweep time was 100 s. For the high-frequency response we used a 10 s 2 mV 1–100,000 Hz logarithmic sweep.
All experiments were performed in accordance with the United Kingdom Animal (Scientific Procedures) Act 1986 with approval from the United Kingdom Home Office. For the current experiment 16 female rTg4510 mice , were bred by Envigo (Loughborough, GB), before being delivered to Eli Lilly and Company for testing (Windlesham, GB). Here, mice were singly housed on a 12 hour light/dark cycle with water provided ad libitum. Food was restricted as required prior to behavioural testing, with mice maintained at no less than 85% of their free-feeding weight. Because the mice used had previously participated in another experiment, 9 of them were treated from 2–3 months of age with doxycycline (DOX), initially via a daily bolus of 10 mg/kg p.o. doxycycline hyclate (Sigma Aldrich, GB) for the first two days. They were then maintained on doxycycline-mixed chow diet (Harlan Teklad Rodent Diet, 200 mg DOX per kg of dietary chow) until the end of the study. All tests involving a comparison of different systems required all animals to run in all conditions, so the DOX treatment could not affect the results.
Mice were implanted with either a single shank 16 channel probe attached to a microdrive (Cambridge Neurotech, GB) targeting the CA1 region of the hippocampus (n = 14 mice; AP: +1.7 mm, ML: −0.3 mm relative to Bregma), or a static dual shank silicon probe (n = 2 mice; 8 sites per shank (Neuronexus, USA)) targeting the medial prefrontal cortex (AP: +1.7 mm, ML: −0.3 mm relative to Bregma & DV: −2.0 mm from brain surface) & dorsal hippocampus (AP: −1.9 mm, ML: −2.0 mm relative to Bregma & DV: −2.0 mm from brain surface). Mice were initially anaesthetised with 3% isoflurane, before being transferred to a stereotaxic frame and maintained for the remainder of the procedure at 1.5–2%. Five stainless steel screws (00–96 × 1/16, PlasticsOne, USA) were attached to the skull and used as an anchor point for implant. One of the screws was placed over the cerebellum and acted as the ground electrode. Craniotomies were made at the specified stereotaxic coordinates, and the probe lowered into position before the hole was sealed with silicone sealant (3–4680 silicone gel kit, Dow Corning, USA). Light curable composite (Revolution Formula 2, Kerr Dental, USA) was used to bind the implant/microdrive to the skull. Mice were allowed to recover for a minimum of 2 weeks before entering into any experiments, however probes attached to microdrives were slowly lowered to span the CA1 pyramidal layer over multiple days between 1 & 2 weeks post-surgery (initially implanted 500 µm below the brain surface).
Room and system configuration All in vivo tests were conducted in a 3.1 × 4.0 m windowless room with black walls and ceiling, lit by two uplighters and separated by a door from an anteroom containing the recording equipment. The room was on a 12-12 dark-light cycle with lights-on at 7am, and all experiments except the 72-hour recording were conducted in the light portion of the cycle. The room contained four bespoke infrared tables (100 × 100 cm), two on either side of the room and separated by 90 cm. A near- infrared emitter array (4 x IR emitters, ~650 nmm wavelength) and a miniature infrared-sensitive video camera (model M700LD, Henrys, UK) were centrally positioned above each table. The tables and emitter provided illumination of the subject from above and below to facilitate high-quality video tracking under a variety of lighting conditions. To receive the signals from the TaiNi transmitter, each table was equipped with a pair of omnidirectional rod-antennas (CSL 12dBi, 2.4 GHz) elevated 30 cm above the table, oriented horizontally and at 90-degrees to one another, forming an open bracket around the recording environments described below. The two antennas from each of the two tables on either side of the room were connected to a 4-way splitter (model DBD-PD-8426-4, dBD Communications UK) by 1.5 m lengths of flexible antenna cable (LBC195 ExtraFlex, MS Distribution UK). Each splitter was in turn connected to one of two inputs on the receiver in the anteroom via 12 m lengths of heavy-gauge coaxial cable (LBC 400, MS Distribution UK). This heavy cable has very low 0.2 dB/m signal-attenuation. In the anteroom, video signals from all four cameras were fed to a quad video combiner (VQM801P, Sanyo, JPN) and the merged video stream processed by EthoVision XT 8.5 (Noldus, NL), enabling the position of each mouse (body-centre and nose) to be tracked. To allow synchronization of the electrophysiology and behavioural data, an I/O interface (PTIO-0020, Noldus, NL) was used to generate a signal at the beginning and end of each video tracking recording, which was converted to a TTL pulse by a hardware interface (MED-PC model SG-233-48) and was recorded by the Taini acquisition PC. The synchronization was performed on the receiving unit during the data post-processing using the local timings of the TTL pulse recorded in both the electrophysiology and behavioural data. The antenna signals were fed to the same model and configuration receiver used in the bench tests (see above). Acquired signals were passed via a 10 gigabit ethernet cable to a Linux workstation running custom software for channelization, demultiplexing, visualization and storage. Channelization was parallelized using the graphics processing unit (Quadro K420, Nvidia) to handle the large data bandwidth. The software also registered TTL sync-pulses sent by the EthoVision PC. All 16 channels from each transmitter were recorded “single-ended” (non-differential), using only the cerebellar skull screw as the signal ground. Training and testing During the 72 hour recordings, mice (n = 2) with dual shank probes were placed in a novel home-cage (15 × 30 cm) with free access to food and water for the duration of the recording. Open field pellet-chasing experiments were performed using 4 food restricted, five month old female rTg4510 mice (2 nonDOX & 2 DOX treated) implanted with single shank silicon probes. These mice were trained twice daily on a pellet chasing task (10 minutes per session) for a total of 10 days in the month proceeding testing. Training consisted of mice being placed in a transparent, circular arena (diameter: 30 cm; height: 50 cm) with pellets (12 mg chocolate flavour AIN-76A Rodent Tablet, TestDiet, USA) dispensed from above at 20 s intervals. On the day of recording, a 60 min sleep recording was performed in a familiar home-cage environment before the mice were transferred to the familiar open field arena for 20 mins of pellet chasing (20 s inter-pellet interval). Throughout both the 72 hour and open field recordings, the cameras above each mouse continually captured data. In the open field recordings the animal’s body was tracked as a silhouette against the infrared emitting table under the arena, while during the home-cage recordings the overhead infrared emitters provided the necessary contrast against the bedding material, which obscured the light from the table below. Split signal tests Concurrent home-cage recordings (n = 3 mice implanted with single shank probes in CA1) were performed using the TaiNi and tethered Digital Lynx SX (Software: Cheetah v5.6.0, Neuralynx, USA) recording systems by utilising a signal splitter placed inline between the silicon probe and headstages. For the Neuralynx recordings, data was band-pass filtered at 0.5–9000 Hz, and sampled at 20 KHz.
All in vivo tests were conducted in a 3.1 × 4.0 m windowless room with black walls and ceiling, lit by two uplighters and separated by a door from an anteroom containing the recording equipment. The room was on a 12-12 dark-light cycle with lights-on at 7am, and all experiments except the 72-hour recording were conducted in the light portion of the cycle. The room contained four bespoke infrared tables (100 × 100 cm), two on either side of the room and separated by 90 cm. A near- infrared emitter array (4 x IR emitters, ~650 nmm wavelength) and a miniature infrared-sensitive video camera (model M700LD, Henrys, UK) were centrally positioned above each table. The tables and emitter provided illumination of the subject from above and below to facilitate high-quality video tracking under a variety of lighting conditions. To receive the signals from the TaiNi transmitter, each table was equipped with a pair of omnidirectional rod-antennas (CSL 12dBi, 2.4 GHz) elevated 30 cm above the table, oriented horizontally and at 90-degrees to one another, forming an open bracket around the recording environments described below. The two antennas from each of the two tables on either side of the room were connected to a 4-way splitter (model DBD-PD-8426-4, dBD Communications UK) by 1.5 m lengths of flexible antenna cable (LBC195 ExtraFlex, MS Distribution UK). Each splitter was in turn connected to one of two inputs on the receiver in the anteroom via 12 m lengths of heavy-gauge coaxial cable (LBC 400, MS Distribution UK). This heavy cable has very low 0.2 dB/m signal-attenuation. In the anteroom, video signals from all four cameras were fed to a quad video combiner (VQM801P, Sanyo, JPN) and the merged video stream processed by EthoVision XT 8.5 (Noldus, NL), enabling the position of each mouse (body-centre and nose) to be tracked. To allow synchronization of the electrophysiology and behavioural data, an I/O interface (PTIO-0020, Noldus, NL) was used to generate a signal at the beginning and end of each video tracking recording, which was converted to a TTL pulse by a hardware interface (MED-PC model SG-233-48) and was recorded by the Taini acquisition PC. The synchronization was performed on the receiving unit during the data post-processing using the local timings of the TTL pulse recorded in both the electrophysiology and behavioural data. The antenna signals were fed to the same model and configuration receiver used in the bench tests (see above). Acquired signals were passed via a 10 gigabit ethernet cable to a Linux workstation running custom software for channelization, demultiplexing, visualization and storage. Channelization was parallelized using the graphics processing unit (Quadro K420, Nvidia) to handle the large data bandwidth. The software also registered TTL sync-pulses sent by the EthoVision PC. All 16 channels from each transmitter were recorded “single-ended” (non-differential), using only the cerebellar skull screw as the signal ground.
During the 72 hour recordings, mice (n = 2) with dual shank probes were placed in a novel home-cage (15 × 30 cm) with free access to food and water for the duration of the recording. Open field pellet-chasing experiments were performed using 4 food restricted, five month old female rTg4510 mice (2 nonDOX & 2 DOX treated) implanted with single shank silicon probes. These mice were trained twice daily on a pellet chasing task (10 minutes per session) for a total of 10 days in the month proceeding testing. Training consisted of mice being placed in a transparent, circular arena (diameter: 30 cm; height: 50 cm) with pellets (12 mg chocolate flavour AIN-76A Rodent Tablet, TestDiet, USA) dispensed from above at 20 s intervals. On the day of recording, a 60 min sleep recording was performed in a familiar home-cage environment before the mice were transferred to the familiar open field arena for 20 mins of pellet chasing (20 s inter-pellet interval). Throughout both the 72 hour and open field recordings, the cameras above each mouse continually captured data. In the open field recordings the animal’s body was tracked as a silhouette against the infrared emitting table under the arena, while during the home-cage recordings the overhead infrared emitters provided the necessary contrast against the bedding material, which obscured the light from the table below.
Concurrent home-cage recordings (n = 3 mice implanted with single shank probes in CA1) were performed using the TaiNi and tethered Digital Lynx SX (Software: Cheetah v5.6.0, Neuralynx, USA) recording systems by utilising a signal splitter placed inline between the silicon probe and headstages. For the Neuralynx recordings, data was band-pass filtered at 0.5–9000 Hz, and sampled at 20 KHz.
11 food restricted, five month old female rTg4510 mice (6 nonDOX & 5 DOX treated) were tested on a non-match to place T-maze task. The mice had previously been tested on the maze at 2 months of age and also in the week preceding this weight comparison test. On each of the 3 testing days, mice were randomly allocated a treatment condition (no transmitter, Triangle Biosystems International (TBSI) W16 transmitter or the TaiNi transmitter) in a fully counterbalanced manner. The T-maze apparatus was custom-made (Apogee Engineering Analysis solutions, GB) and consisted of a matt black Perspex track (width: 6 cm) with clear Perspex walls (height: 20 cm). The centre-arm of the maze was 100 cm in length and each of the reward arms 36 cm long. The maze was raised 100 cm from the ground and sited in a rectangular room containing multiple visual cues. 3 pellet dispensers were situated around the maze (one per reward arm & one in the delay area at the beginning of the centre arm) to allow rewards (12 mg unflavoured AIN-76A Rodent Tablet, TestDiet, USA) to be delivered for successful performance. The maze protocol was automatically controlled by a microcontroller board (Arduino Mega 2560) connected via a USB serial connection to a host-PC running Matlab (Mathworks, Natick, MA, USA). The animals’ position was detected by infrared beam breaks, which enabled the maze controller to adjust the position of 5 pneumatically driven, black Perspex doors. These could be to be raised from below to block entry into areas of the maze as appropriate. The T-maze task comprised of a sample and choice component. In the initial forced phase, mice were guided along the centre arm by the doors and forced to turn left/right in a pseudo-random manner (maximum of 3 consecutive sample runs to the same arm) in order to receive a reward. Mice then returned to the holding area in return for a second reward, at which time the choice phase of the task began. Mice were again guided along the central arm of the maze, before being offered a free choice to enter either reward arm. Mice were given a reward only if they chose to enter the opposite arm visited during the sample phase. A further reward was provided at the waiting area for mice making a correct choice. Mice were held in this zone for 5 s prior to be start of the next trial. On each day mice were given 60 minutes to complete as many trials as possible.
Running-speed estimates were calculated from the animals’ body centre location (x/y coordinates) integrated over 400 ms non-overlapping epochs . Nose-position was used to provide the high-precision data required for analysing neuronal spatial firing properties. Immobility was defined as a period when the mouse moved at a rate of less than 0.5 cm/s for at least 300 s. Running was defined as any period where the mouse moved at more than 5 cm/s for at least 100 ms. For the automated T-maze weight tests, a repeated-measures ANOVA was used, with orthogonal planned contrasts between the NONE and TaiNi condition, and between TaiNi and TBSI. Local field potentials (LFPs) were analysed using an anti-aliased version of the raw data, downsampled to 1 KHz. A short-time FFT using 2-second sliding windows with 50% overlap was used to estimate spectral power. Ripple-detection was performed according to the methods described by Sullivan et al . . Phase-amplitude-coupling in the hippocampus was analysed according to the “cross-frequency-coupling” methods described in Onslow et al . A simple Pearson’s correlation was used to analyse the relationship between 0.5–4 HZ delta power and ripple density. Spike detection and cluster analysis was performed using the KlustaKiwk suite of software , with the maximum number of clusters set to 100 and using the Akaike Information Critierion to determine whether a given cluster should be divided. For spike-detection, the data was filtered (500 Hz high-pass 3rd-order Butterworth filter) and z-scored, with a 4.5 standard-deviation detection threshold. Clusters were discarded if their autocorrelograms exhibited excessive spiking in the 2 ms refractory zone, and were combined where they shared clear refractoriness with other clusters. Place fields for each cluster (putative neuron) were generated according to the methods described previously , but using a linear colour scale for representing firing rates. Briefly, mean dwell-time (seconds) and spike-counts in 1 × 1 cm bins were calculated to generate the firing rate in each bin, before applying a 5 cm Gaussian smoother to the map.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
System overview and bench-tests The TaiNi system (Fig. ) weighs 1.5 g (including battery) and transmits 16 channels sampled at 19.531 KHz. The system employs a high-pass hardware filter (−3 dB at 0.35 Hz) to reduce high-amplitude, low-frequency signal components, yielding a total bandwidth (after anti-aliasing) of 9.7 KHz. The device measures 20 × 12 × 14 mm and includes a plastic shield for the loop antenna. Omnetics connectors were used for the current version of the system, although they could easily be changed without altering performance. A summary of the system’s electrical specification is given in Table . Figure shows the device in use, with the transmitter comprising about 50% of the volume of materials on the mouse’s head, the rest being the electrode implant assembly as described below. For bench tests, data from a single transmitter was detected by an off-the shelf 2.4 GHz WiFi antenna elevated 25 cm above the bench. Signals were fed to a receiver (Ettus, model USRP X300) and passed via a gigabit ethernet link to a Linux (Ubuntu) workstation running custom acquisition software. For all recordings, we used a 180 mAh zinc-air battery weighing 0.58 g. Packet loss was averaged over two minutes at each distance from 10 cm to 300 cm in 10 cm steps (Fig. ). Average loss was 0.015% (1.5 packets per million) for distances under 260 cm, rising to 0.13% at 300 cm. The frequency response of the device was estimated using a 100 s chirp signal ranging from 0.1 Hz to 1 Hz, and a 10 s chirp ranging from 1 Hz to 100 KHz (Fig. ). There was no signal attenuation between 1 Hz and 2000 Hz (Fig. ). The 0.5 Hz signal was only attenuated by 0.95 dB at 0.5 Hz, below the generally-held lower-bound for delta oscillations , and by 7.5 dB at 0.1 Hz (Fig. ). In vivo tests Female Tg4510 mice (fourteen 5 month olds and two 9.5 months old) which had been used in previous experiments were used to test the impact of the TaiNi system on behaviour and its performance in experimental settings. Each mouse was implanted with a 16-channel silicon probe (Cambridge NeuroTech, UK) directed at the dorsal CA1 region of the hippocampus. For the younger mice, the electrode was fixed to a miniature microdrive to allow positioning of the recording sites in the pyramidal cell layer. Behavioural impact: TaiNi versus existing wireless technology Supplementary video shows a mouse wearing the TaiNi system and exhibiting typical head-bobbing movements, as well as very normal looking exploratory behaviour, rearing and grooming. The same mouse was recorded 10 minutes later in Supplementary video , wearing a lightweight 16-channel head-stage and recording tether attached to a load-bearing cable-management system. To quantify the behavioural benefits of using an ultra-light system, we tested spatial working memory in eleven mice on an automated delayed non-match-to-place T-maze task (Fig. ). All animals had been previously trained to perform the task with no transmitter. On testing days animals were run in the maze wearing either no transmitter (NONE), the 1.5 g TaiNi, or a comparable 4.0 g commercially available transmitter (TBSI model W16). Over three days the mice were exposed to all three conditions in counterbalanced order. Figure shows there was no difference in the percentage of correct choices from each animal under each condition. In contrast, mice completed significantly more trials with TaiNi compared with TBSI (repeated-measures ANOVA, n = 11, t = 4.29, p < 0.01), and there was no significant difference between TaiNi and the NONE condition (n = 11, t = 1.27, p = 0.23) (Fig. ). The magnitude of the impact of wearing the heavier TBSI transmitter is clear from Fig. : 35.9% fewer trials than when tested wearing no transmitter at all, as compared with a 6.5% reduction in the TaiNi condition. Anecdotally, by the end of each session mice wearing the TaiNi transmitter were behaving normally, while animals wearing the heavier 4 g transmitter were clearly struggling to maintain an upright head posture. Simultaneous high-density recording from 4 animals We recorded LFP and action potentials from four animals on a backlit infrared table, monitored by overhead cameras connected to a PC running whole-body tracking software. Output of the TaiNi transmitter was picked up by two antennas per table, and fed to a single receiver. A schematic of the recording configuration is shown in Fig. . A 30 cm diameter striped arena and a modified home-cage were used to record during alternating pellet-foraging and sleep/rest trials, respectively (Fig. ). The TaiNi transmitter and recording system comfortably handled 64 channels of data sampled at 19.5 KHz (16 channels per mouse). As illustrated in Fig. , traces from each animal exhibited normal local field potential oscillations, evidence of action potentials, and in one of the examples shown, the muscular artefacts normally associated with chewing in a pellet-foraging task. A small number of defective channels were identified, but these were specific to each mouse and therefore represent a problem with the electrodes, not the TaiNi system. It is also worth noting that we observed some instances where a drop of water could disrupt performance, which was resolved by using an air-duster and absorbent cotton to remove the moisture. This will be addressed in the production version of the device by the addition of an extended housing to shield the battery enclosure and circuit board in a manner that does not impact the total weight. Using the KlustaKwik and KlustaViewa offline spike sorting tools , we detected action potentials and assigned them to individual hippocampal neurons. We combined the action potential data with the time-stamped whole-body tracking coordinates corresponding with the concurrent position of the animal’s nose. The spatial firing probability of each neuron was calculated as described previously . Figure shows the firing fields from one of the four mice. From this recording it is clear that we obtained a collection of pyramidal cells with well-defined spatial firing fields (place cells ), interneurons with more uniform patterns of activity , and sparsely firing neurons which were essentially silent for most of the recording. Figure compares the average waveforms of the putative pyramidal cell and interneuron highlighted in the previous panel. The spike-timing autocorrelograms for these two neurons (Fig. ), reveal the interneuron’s characteristic tonic firing probability, while the pyramidal cell tends to fire bursts of action potentials within 10 ms of one another , . Figure shows a spectral analysis of the data from a single channel in the hippocampal pyramidal cell layer of the same mouse, comparing a foraging and a rest trial. The running-speed of the mouse is overlaid to highlight the high degree of temporal dependence of 0.5–4 Hz delta and 4–12 Hz theta oscillations on momentary running speed. During the foraging trial (top panel) the animal frequently pauses to wait for the next pellet to drop to the floor of the arena before resuming foraging. During these pauses we observed brief pronounced reductions in theta power, as expected. Conversely, during the rest trials, power in the 0.5–4 Hz delta band predominated, but was seen to be disrupted by momentary periods of arousal when the mouse began moving again. The neuronal and LFP results described here would be impossible without confidence in the sub-second synchronization of the electrophysiology and behavioural data, indicating that the time-stamping for both the TaiNi and the EthoVision systems is very robust. Battery life test: 72-hour recording To test the battery-life of the device, we recorded behaviour and LFPs from two 9.5 month old Tg4510 mice during 72-hours of natural behaviour in a modified home-cage with ad libitum access to food and water. A small drop of hot-glue applied to either side of the connector ensured that the device remained attached for the duration of the recording. A small amount of electrical tape was used to shield the circuit board and battery enclosure from water. The home cages were placed centrally on an infra-red table and the room was placed on a 12-12 hour light cycle with lights-on at 7AM. During these 72 hours the animals were monitored via the overhead camera but nobody entered the room. Recording duration exceeded our expectations, with the devices continuing to function beyond the end of the 72-hour trial. The spectral time-course of hippocampal LFP oscillations over 72 hours revealed slow cyclic variations in the balance between low- and high-frequency oscillations which followed the light cycles in one of the mice but not in the other (Fig. ). This difference is recapitulated in the time-course of running speed for the animals, with the second animal exhibiting long periods of activity in what is normally considered the rodent sleep-phase. This highlights the traditional difficulty in quantifying neuronal activity from a given sleep/wake state when experimenters are limited to shorter recordings. At this timescale it is evident that running and 4–12 Hz theta are associated with each other as well as with increases in the 30–100 Hz gamma power band and reductions in 13–30 Hz beta. The reverse holds during immobile periods, which exhibit low theta and stronger 0.5–4 Hz delta. Analysis of the density of 140–220 Hz ripple oscillations (black trace, Fig. ), which are associated with slow-wave sleep and memory consolidation – , confirmed that their numbers increase during periods when delta power is highest. Figure shows an unfiltered ripple from mouse #31, and the same ripple band-pass filtered at 140–220 Hz. Figure shows the average filtered ripple for this mouse, aligned to the highest amplitude peak in each oscillatory event. We used 10-minute bins to compare the mean power in the 0.5–4.0 Hz delta band and the mean amplitude of detected ripple oscillations over 72 hours (Fig. ). For the 4320 10-minute epochs analysed, this revealed a robust Pearson’s correlation (r = 0.49, p < 0.001), and a clear bimodal distribution in ripple amplitude. This bimodality emerged only when the data was averaged on behavioural time-scales, capturing amplitude changes as the mouse transitions between low and high delta power states during running and immobility, respectively. However, using a z-score of zero as the arbitrary threshold for categorization, ripple amplitude was also strongly correlated with delta power for both low (r = 0.48, p < 0.001) and high (r = 0.38, p < 0.001) amplitude ripples. Spike and LFP signal quality compared with 16-bit tethered system Simultaneous recordings from TaiNi and the TBSI devices were impossible due to the size and shape of the respective systems, and the limited space on the mouse’s head. However we were able to create a signal splitter which allowed comparison of data acquired with 12-bit TaiNi to the same data processed via a tethered 16-bit system (Digital Lynx SX, Neuralynx). This is not an entirely fair comparison, given that tethered systems always offer greater bit-depth and/or voltage range to represent their input, compared with wireless devices. Nevertheless, we collected 10 minutes worth of recording from each of three mice, splitting the signal at the implant connector. We were particularly interested in whether the traces, power spectra, and measures of frequency coupling based on the LFP would be comparable. We also wanted to investigate how spike-sorting algorithms would handle the two datasets. Figure compares results from the two data streams, with TaiNi on the left and Neuralynx on the right. Sample traces from the CA1 pyramidal cell layer of the hippocampus in one of the mice are shown in Fig. . The LFP appears virtually identical in both systems (top panel) while very subtle differences became apparent when looking at the data on a much shorter 5 ms timescale (bottom panel). However, the action potential in this example is extremely similar. Next we analysed the power spectrum using a short-time Fourier transform with a 1-second sliding window (Fig. ). The resulting spectra were also nearly identical. Next we looked at phase-amplitude coupling - a measure of the degree to which the amplitude of fast oscillations is modulated by the phase of slower ones. Both systems produced a robust peak in modulation of 35 Hz gamma by 9 Hz theta oscillations, as expected, with only minor differences in the pattern of background modulation (Fig. ). Finally, we tested whether the subtle differences in the signals would affect the classification of action potentials based on their waveforms across the 16 recording sites of the electrode. The depth-profile of the mean action potential waveform is shown alongside the 100 ms activity histogram for each detected cell (Fig. ) . A total of ten neurons were detected, 8 by the TaiNi device, and 9 by the Neuralynx system. Seven cells were detected by both systems, there was 1 cell detected by TaiNi only, and another two which were detected only by Neuralynx. For the seven cells detected by both systems, TaiNi detected on average 17.3 ± 4.1% fewer action potentials than Neuralynx. However, the cells identified by TaiNi had 55.9 ± 22.3% better refractoriness scores, suggesting that the action potentials excluded by TaiNi were lower amplitude and therefore more likely to be of ambiguous classification. It is worth noting that the sample size (7 common cells) is small, and running the same data through KlustaKwik does not yield identical results every time. Nevertheless we believe these findings, with this dataset, are representative.
The TaiNi system (Fig. ) weighs 1.5 g (including battery) and transmits 16 channels sampled at 19.531 KHz. The system employs a high-pass hardware filter (−3 dB at 0.35 Hz) to reduce high-amplitude, low-frequency signal components, yielding a total bandwidth (after anti-aliasing) of 9.7 KHz. The device measures 20 × 12 × 14 mm and includes a plastic shield for the loop antenna. Omnetics connectors were used for the current version of the system, although they could easily be changed without altering performance. A summary of the system’s electrical specification is given in Table . Figure shows the device in use, with the transmitter comprising about 50% of the volume of materials on the mouse’s head, the rest being the electrode implant assembly as described below. For bench tests, data from a single transmitter was detected by an off-the shelf 2.4 GHz WiFi antenna elevated 25 cm above the bench. Signals were fed to a receiver (Ettus, model USRP X300) and passed via a gigabit ethernet link to a Linux (Ubuntu) workstation running custom acquisition software. For all recordings, we used a 180 mAh zinc-air battery weighing 0.58 g. Packet loss was averaged over two minutes at each distance from 10 cm to 300 cm in 10 cm steps (Fig. ). Average loss was 0.015% (1.5 packets per million) for distances under 260 cm, rising to 0.13% at 300 cm. The frequency response of the device was estimated using a 100 s chirp signal ranging from 0.1 Hz to 1 Hz, and a 10 s chirp ranging from 1 Hz to 100 KHz (Fig. ). There was no signal attenuation between 1 Hz and 2000 Hz (Fig. ). The 0.5 Hz signal was only attenuated by 0.95 dB at 0.5 Hz, below the generally-held lower-bound for delta oscillations , and by 7.5 dB at 0.1 Hz (Fig. ).
tests Female Tg4510 mice (fourteen 5 month olds and two 9.5 months old) which had been used in previous experiments were used to test the impact of the TaiNi system on behaviour and its performance in experimental settings. Each mouse was implanted with a 16-channel silicon probe (Cambridge NeuroTech, UK) directed at the dorsal CA1 region of the hippocampus. For the younger mice, the electrode was fixed to a miniature microdrive to allow positioning of the recording sites in the pyramidal cell layer. Behavioural impact: TaiNi versus existing wireless technology Supplementary video shows a mouse wearing the TaiNi system and exhibiting typical head-bobbing movements, as well as very normal looking exploratory behaviour, rearing and grooming. The same mouse was recorded 10 minutes later in Supplementary video , wearing a lightweight 16-channel head-stage and recording tether attached to a load-bearing cable-management system. To quantify the behavioural benefits of using an ultra-light system, we tested spatial working memory in eleven mice on an automated delayed non-match-to-place T-maze task (Fig. ). All animals had been previously trained to perform the task with no transmitter. On testing days animals were run in the maze wearing either no transmitter (NONE), the 1.5 g TaiNi, or a comparable 4.0 g commercially available transmitter (TBSI model W16). Over three days the mice were exposed to all three conditions in counterbalanced order. Figure shows there was no difference in the percentage of correct choices from each animal under each condition. In contrast, mice completed significantly more trials with TaiNi compared with TBSI (repeated-measures ANOVA, n = 11, t = 4.29, p < 0.01), and there was no significant difference between TaiNi and the NONE condition (n = 11, t = 1.27, p = 0.23) (Fig. ). The magnitude of the impact of wearing the heavier TBSI transmitter is clear from Fig. : 35.9% fewer trials than when tested wearing no transmitter at all, as compared with a 6.5% reduction in the TaiNi condition. Anecdotally, by the end of each session mice wearing the TaiNi transmitter were behaving normally, while animals wearing the heavier 4 g transmitter were clearly struggling to maintain an upright head posture. Simultaneous high-density recording from 4 animals We recorded LFP and action potentials from four animals on a backlit infrared table, monitored by overhead cameras connected to a PC running whole-body tracking software. Output of the TaiNi transmitter was picked up by two antennas per table, and fed to a single receiver. A schematic of the recording configuration is shown in Fig. . A 30 cm diameter striped arena and a modified home-cage were used to record during alternating pellet-foraging and sleep/rest trials, respectively (Fig. ). The TaiNi transmitter and recording system comfortably handled 64 channels of data sampled at 19.5 KHz (16 channels per mouse). As illustrated in Fig. , traces from each animal exhibited normal local field potential oscillations, evidence of action potentials, and in one of the examples shown, the muscular artefacts normally associated with chewing in a pellet-foraging task. A small number of defective channels were identified, but these were specific to each mouse and therefore represent a problem with the electrodes, not the TaiNi system. It is also worth noting that we observed some instances where a drop of water could disrupt performance, which was resolved by using an air-duster and absorbent cotton to remove the moisture. This will be addressed in the production version of the device by the addition of an extended housing to shield the battery enclosure and circuit board in a manner that does not impact the total weight. Using the KlustaKwik and KlustaViewa offline spike sorting tools , we detected action potentials and assigned them to individual hippocampal neurons. We combined the action potential data with the time-stamped whole-body tracking coordinates corresponding with the concurrent position of the animal’s nose. The spatial firing probability of each neuron was calculated as described previously . Figure shows the firing fields from one of the four mice. From this recording it is clear that we obtained a collection of pyramidal cells with well-defined spatial firing fields (place cells ), interneurons with more uniform patterns of activity , and sparsely firing neurons which were essentially silent for most of the recording. Figure compares the average waveforms of the putative pyramidal cell and interneuron highlighted in the previous panel. The spike-timing autocorrelograms for these two neurons (Fig. ), reveal the interneuron’s characteristic tonic firing probability, while the pyramidal cell tends to fire bursts of action potentials within 10 ms of one another , . Figure shows a spectral analysis of the data from a single channel in the hippocampal pyramidal cell layer of the same mouse, comparing a foraging and a rest trial. The running-speed of the mouse is overlaid to highlight the high degree of temporal dependence of 0.5–4 Hz delta and 4–12 Hz theta oscillations on momentary running speed. During the foraging trial (top panel) the animal frequently pauses to wait for the next pellet to drop to the floor of the arena before resuming foraging. During these pauses we observed brief pronounced reductions in theta power, as expected. Conversely, during the rest trials, power in the 0.5–4 Hz delta band predominated, but was seen to be disrupted by momentary periods of arousal when the mouse began moving again. The neuronal and LFP results described here would be impossible without confidence in the sub-second synchronization of the electrophysiology and behavioural data, indicating that the time-stamping for both the TaiNi and the EthoVision systems is very robust. Battery life test: 72-hour recording To test the battery-life of the device, we recorded behaviour and LFPs from two 9.5 month old Tg4510 mice during 72-hours of natural behaviour in a modified home-cage with ad libitum access to food and water. A small drop of hot-glue applied to either side of the connector ensured that the device remained attached for the duration of the recording. A small amount of electrical tape was used to shield the circuit board and battery enclosure from water. The home cages were placed centrally on an infra-red table and the room was placed on a 12-12 hour light cycle with lights-on at 7AM. During these 72 hours the animals were monitored via the overhead camera but nobody entered the room. Recording duration exceeded our expectations, with the devices continuing to function beyond the end of the 72-hour trial. The spectral time-course of hippocampal LFP oscillations over 72 hours revealed slow cyclic variations in the balance between low- and high-frequency oscillations which followed the light cycles in one of the mice but not in the other (Fig. ). This difference is recapitulated in the time-course of running speed for the animals, with the second animal exhibiting long periods of activity in what is normally considered the rodent sleep-phase. This highlights the traditional difficulty in quantifying neuronal activity from a given sleep/wake state when experimenters are limited to shorter recordings. At this timescale it is evident that running and 4–12 Hz theta are associated with each other as well as with increases in the 30–100 Hz gamma power band and reductions in 13–30 Hz beta. The reverse holds during immobile periods, which exhibit low theta and stronger 0.5–4 Hz delta. Analysis of the density of 140–220 Hz ripple oscillations (black trace, Fig. ), which are associated with slow-wave sleep and memory consolidation – , confirmed that their numbers increase during periods when delta power is highest. Figure shows an unfiltered ripple from mouse #31, and the same ripple band-pass filtered at 140–220 Hz. Figure shows the average filtered ripple for this mouse, aligned to the highest amplitude peak in each oscillatory event. We used 10-minute bins to compare the mean power in the 0.5–4.0 Hz delta band and the mean amplitude of detected ripple oscillations over 72 hours (Fig. ). For the 4320 10-minute epochs analysed, this revealed a robust Pearson’s correlation (r = 0.49, p < 0.001), and a clear bimodal distribution in ripple amplitude. This bimodality emerged only when the data was averaged on behavioural time-scales, capturing amplitude changes as the mouse transitions between low and high delta power states during running and immobility, respectively. However, using a z-score of zero as the arbitrary threshold for categorization, ripple amplitude was also strongly correlated with delta power for both low (r = 0.48, p < 0.001) and high (r = 0.38, p < 0.001) amplitude ripples. Spike and LFP signal quality compared with 16-bit tethered system Simultaneous recordings from TaiNi and the TBSI devices were impossible due to the size and shape of the respective systems, and the limited space on the mouse’s head. However we were able to create a signal splitter which allowed comparison of data acquired with 12-bit TaiNi to the same data processed via a tethered 16-bit system (Digital Lynx SX, Neuralynx). This is not an entirely fair comparison, given that tethered systems always offer greater bit-depth and/or voltage range to represent their input, compared with wireless devices. Nevertheless, we collected 10 minutes worth of recording from each of three mice, splitting the signal at the implant connector. We were particularly interested in whether the traces, power spectra, and measures of frequency coupling based on the LFP would be comparable. We also wanted to investigate how spike-sorting algorithms would handle the two datasets. Figure compares results from the two data streams, with TaiNi on the left and Neuralynx on the right. Sample traces from the CA1 pyramidal cell layer of the hippocampus in one of the mice are shown in Fig. . The LFP appears virtually identical in both systems (top panel) while very subtle differences became apparent when looking at the data on a much shorter 5 ms timescale (bottom panel). However, the action potential in this example is extremely similar. Next we analysed the power spectrum using a short-time Fourier transform with a 1-second sliding window (Fig. ). The resulting spectra were also nearly identical. Next we looked at phase-amplitude coupling - a measure of the degree to which the amplitude of fast oscillations is modulated by the phase of slower ones. Both systems produced a robust peak in modulation of 35 Hz gamma by 9 Hz theta oscillations, as expected, with only minor differences in the pattern of background modulation (Fig. ). Finally, we tested whether the subtle differences in the signals would affect the classification of action potentials based on their waveforms across the 16 recording sites of the electrode. The depth-profile of the mean action potential waveform is shown alongside the 100 ms activity histogram for each detected cell (Fig. ) . A total of ten neurons were detected, 8 by the TaiNi device, and 9 by the Neuralynx system. Seven cells were detected by both systems, there was 1 cell detected by TaiNi only, and another two which were detected only by Neuralynx. For the seven cells detected by both systems, TaiNi detected on average 17.3 ± 4.1% fewer action potentials than Neuralynx. However, the cells identified by TaiNi had 55.9 ± 22.3% better refractoriness scores, suggesting that the action potentials excluded by TaiNi were lower amplitude and therefore more likely to be of ambiguous classification. It is worth noting that the sample size (7 common cells) is small, and running the same data through KlustaKwik does not yield identical results every time. Nevertheless we believe these findings, with this dataset, are representative.
Supplementary video shows a mouse wearing the TaiNi system and exhibiting typical head-bobbing movements, as well as very normal looking exploratory behaviour, rearing and grooming. The same mouse was recorded 10 minutes later in Supplementary video , wearing a lightweight 16-channel head-stage and recording tether attached to a load-bearing cable-management system. To quantify the behavioural benefits of using an ultra-light system, we tested spatial working memory in eleven mice on an automated delayed non-match-to-place T-maze task (Fig. ). All animals had been previously trained to perform the task with no transmitter. On testing days animals were run in the maze wearing either no transmitter (NONE), the 1.5 g TaiNi, or a comparable 4.0 g commercially available transmitter (TBSI model W16). Over three days the mice were exposed to all three conditions in counterbalanced order. Figure shows there was no difference in the percentage of correct choices from each animal under each condition. In contrast, mice completed significantly more trials with TaiNi compared with TBSI (repeated-measures ANOVA, n = 11, t = 4.29, p < 0.01), and there was no significant difference between TaiNi and the NONE condition (n = 11, t = 1.27, p = 0.23) (Fig. ). The magnitude of the impact of wearing the heavier TBSI transmitter is clear from Fig. : 35.9% fewer trials than when tested wearing no transmitter at all, as compared with a 6.5% reduction in the TaiNi condition. Anecdotally, by the end of each session mice wearing the TaiNi transmitter were behaving normally, while animals wearing the heavier 4 g transmitter were clearly struggling to maintain an upright head posture.
We recorded LFP and action potentials from four animals on a backlit infrared table, monitored by overhead cameras connected to a PC running whole-body tracking software. Output of the TaiNi transmitter was picked up by two antennas per table, and fed to a single receiver. A schematic of the recording configuration is shown in Fig. . A 30 cm diameter striped arena and a modified home-cage were used to record during alternating pellet-foraging and sleep/rest trials, respectively (Fig. ). The TaiNi transmitter and recording system comfortably handled 64 channels of data sampled at 19.5 KHz (16 channels per mouse). As illustrated in Fig. , traces from each animal exhibited normal local field potential oscillations, evidence of action potentials, and in one of the examples shown, the muscular artefacts normally associated with chewing in a pellet-foraging task. A small number of defective channels were identified, but these were specific to each mouse and therefore represent a problem with the electrodes, not the TaiNi system. It is also worth noting that we observed some instances where a drop of water could disrupt performance, which was resolved by using an air-duster and absorbent cotton to remove the moisture. This will be addressed in the production version of the device by the addition of an extended housing to shield the battery enclosure and circuit board in a manner that does not impact the total weight. Using the KlustaKwik and KlustaViewa offline spike sorting tools , we detected action potentials and assigned them to individual hippocampal neurons. We combined the action potential data with the time-stamped whole-body tracking coordinates corresponding with the concurrent position of the animal’s nose. The spatial firing probability of each neuron was calculated as described previously . Figure shows the firing fields from one of the four mice. From this recording it is clear that we obtained a collection of pyramidal cells with well-defined spatial firing fields (place cells ), interneurons with more uniform patterns of activity , and sparsely firing neurons which were essentially silent for most of the recording. Figure compares the average waveforms of the putative pyramidal cell and interneuron highlighted in the previous panel. The spike-timing autocorrelograms for these two neurons (Fig. ), reveal the interneuron’s characteristic tonic firing probability, while the pyramidal cell tends to fire bursts of action potentials within 10 ms of one another , . Figure shows a spectral analysis of the data from a single channel in the hippocampal pyramidal cell layer of the same mouse, comparing a foraging and a rest trial. The running-speed of the mouse is overlaid to highlight the high degree of temporal dependence of 0.5–4 Hz delta and 4–12 Hz theta oscillations on momentary running speed. During the foraging trial (top panel) the animal frequently pauses to wait for the next pellet to drop to the floor of the arena before resuming foraging. During these pauses we observed brief pronounced reductions in theta power, as expected. Conversely, during the rest trials, power in the 0.5–4 Hz delta band predominated, but was seen to be disrupted by momentary periods of arousal when the mouse began moving again. The neuronal and LFP results described here would be impossible without confidence in the sub-second synchronization of the electrophysiology and behavioural data, indicating that the time-stamping for both the TaiNi and the EthoVision systems is very robust.
To test the battery-life of the device, we recorded behaviour and LFPs from two 9.5 month old Tg4510 mice during 72-hours of natural behaviour in a modified home-cage with ad libitum access to food and water. A small drop of hot-glue applied to either side of the connector ensured that the device remained attached for the duration of the recording. A small amount of electrical tape was used to shield the circuit board and battery enclosure from water. The home cages were placed centrally on an infra-red table and the room was placed on a 12-12 hour light cycle with lights-on at 7AM. During these 72 hours the animals were monitored via the overhead camera but nobody entered the room. Recording duration exceeded our expectations, with the devices continuing to function beyond the end of the 72-hour trial. The spectral time-course of hippocampal LFP oscillations over 72 hours revealed slow cyclic variations in the balance between low- and high-frequency oscillations which followed the light cycles in one of the mice but not in the other (Fig. ). This difference is recapitulated in the time-course of running speed for the animals, with the second animal exhibiting long periods of activity in what is normally considered the rodent sleep-phase. This highlights the traditional difficulty in quantifying neuronal activity from a given sleep/wake state when experimenters are limited to shorter recordings. At this timescale it is evident that running and 4–12 Hz theta are associated with each other as well as with increases in the 30–100 Hz gamma power band and reductions in 13–30 Hz beta. The reverse holds during immobile periods, which exhibit low theta and stronger 0.5–4 Hz delta. Analysis of the density of 140–220 Hz ripple oscillations (black trace, Fig. ), which are associated with slow-wave sleep and memory consolidation – , confirmed that their numbers increase during periods when delta power is highest. Figure shows an unfiltered ripple from mouse #31, and the same ripple band-pass filtered at 140–220 Hz. Figure shows the average filtered ripple for this mouse, aligned to the highest amplitude peak in each oscillatory event. We used 10-minute bins to compare the mean power in the 0.5–4.0 Hz delta band and the mean amplitude of detected ripple oscillations over 72 hours (Fig. ). For the 4320 10-minute epochs analysed, this revealed a robust Pearson’s correlation (r = 0.49, p < 0.001), and a clear bimodal distribution in ripple amplitude. This bimodality emerged only when the data was averaged on behavioural time-scales, capturing amplitude changes as the mouse transitions between low and high delta power states during running and immobility, respectively. However, using a z-score of zero as the arbitrary threshold for categorization, ripple amplitude was also strongly correlated with delta power for both low (r = 0.48, p < 0.001) and high (r = 0.38, p < 0.001) amplitude ripples.
Simultaneous recordings from TaiNi and the TBSI devices were impossible due to the size and shape of the respective systems, and the limited space on the mouse’s head. However we were able to create a signal splitter which allowed comparison of data acquired with 12-bit TaiNi to the same data processed via a tethered 16-bit system (Digital Lynx SX, Neuralynx). This is not an entirely fair comparison, given that tethered systems always offer greater bit-depth and/or voltage range to represent their input, compared with wireless devices. Nevertheless, we collected 10 minutes worth of recording from each of three mice, splitting the signal at the implant connector. We were particularly interested in whether the traces, power spectra, and measures of frequency coupling based on the LFP would be comparable. We also wanted to investigate how spike-sorting algorithms would handle the two datasets. Figure compares results from the two data streams, with TaiNi on the left and Neuralynx on the right. Sample traces from the CA1 pyramidal cell layer of the hippocampus in one of the mice are shown in Fig. . The LFP appears virtually identical in both systems (top panel) while very subtle differences became apparent when looking at the data on a much shorter 5 ms timescale (bottom panel). However, the action potential in this example is extremely similar. Next we analysed the power spectrum using a short-time Fourier transform with a 1-second sliding window (Fig. ). The resulting spectra were also nearly identical. Next we looked at phase-amplitude coupling - a measure of the degree to which the amplitude of fast oscillations is modulated by the phase of slower ones. Both systems produced a robust peak in modulation of 35 Hz gamma by 9 Hz theta oscillations, as expected, with only minor differences in the pattern of background modulation (Fig. ). Finally, we tested whether the subtle differences in the signals would affect the classification of action potentials based on their waveforms across the 16 recording sites of the electrode. The depth-profile of the mean action potential waveform is shown alongside the 100 ms activity histogram for each detected cell (Fig. ) . A total of ten neurons were detected, 8 by the TaiNi device, and 9 by the Neuralynx system. Seven cells were detected by both systems, there was 1 cell detected by TaiNi only, and another two which were detected only by Neuralynx. For the seven cells detected by both systems, TaiNi detected on average 17.3 ± 4.1% fewer action potentials than Neuralynx. However, the cells identified by TaiNi had 55.9 ± 22.3% better refractoriness scores, suggesting that the action potentials excluded by TaiNi were lower amplitude and therefore more likely to be of ambiguous classification. It is worth noting that the sample size (7 common cells) is small, and running the same data through KlustaKwik does not yield identical results every time. Nevertheless we believe these findings, with this dataset, are representative.
Our tests confirm that the TaiNi wireless system is lighter than any commercially available wireless device able to record neuronal action potentials, and indeed lighter even than multi-channel devices capable only of recording LFPs. The light weight resulted in significant improvements in the ability to complete trials on the T-maze task compared to the most similar commercially available alternative, the TBSI transmitter. While our relatively unimpaired animals showed no concomitant reduction in the percentage of correct choices on the T-maze task, the ability to complete additional trials increases statistical power and reduces the number of animals required for a given experiment. This provides an animal welfare benefit beyond the obvious ones for the individual animal wearing the transmitter. TaiNi was capable of undisturbed recordings spanning multiple diurnal cycles. This is particularly important for studies using mice, for which human contact has a prolonged effect on behaviour, and which often have (as we observed) erratic sleep patterns. Consequently the TaiNi transmitter both removes the stress associated with repeated handling to change batteries or untwist tethers, and also enables state-dependent analyses of neuronal activity which are traditionally very challenging in mice. Long-duration recording also enables the discovery of novel phenomena such as the discrete categories of 140–220 Hz ripple-oscillations revealed by the current experiments. This might not have been possible without many hours of recordings integrated across fluctuating behavioural states. While we were unable to process the enormous volume of 72-hour action potential data at the time of writing, the potential to do so in the near future opens up many novel avenues of scientific investigation. It is also worth noting that while our tests ran to 72 hours using a 180 mAh battery, using a 310 mAh better would extend recording to 120 hours (5 days) while adding only 0.25 g to the total weight. We also confirm that the acquisition system is capable of simultaneously acquiring 16 channels of 19.5 KHz data streams from four devices using a single receiver, and accurately synchronising the data with movement and position information recorded by the Ethovision whole-body tracking software. This demonstrates the scalability of the system given additional receivers, and the time-stamp integrity required for analyses such as the spatial selectivity of hippocampal neurons and the instantaneous behavioural correlates of local field potential oscillations. Both benefits are important in industry-scale experimental programs where recording from many animals simultaneously is a benefit, and integration of multiple data streams is essential. Finally, split-signal recordings confirm that, for LFP recordings, the results from TaiNi are qualitatively and quantitatively similar to those obtained using a higher-power, higher bit-rate tethered system. As expected, there is some compromise in terms of detection of very low amplitude events, which manifests as slightly different spike clustering results and somewhat lower action-potential counts in each cluster. However, we believe this is typical of any 12-bit wireless recording system, and does not represent an obstacle to collecting high-quality data. In summary, we believe the TaiNi wireless transmitter is a transformative technology which opens up entirely new areas of investigation using long-duration, high-density recordings in mice. The experiments described here illustrate, for the first time, the capabilities of this device in a real-world experimental setting, and reveal a feature of high-frequency neuronal oscillations which might otherwise have been impossible to observe.
Supplementary Video 1 - TaiNi Supplementary Video 2 - Tether
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Analyzing 2,589 child neurology telehealth encounters necessitated by the COVID-19 pandemic | a7ccd9b5-accf-40e3-b92f-80160e77ebef | 7538222 | Pediatrics[mh] | Standard protocol approvals, registrations, and patient consents This was a quality improvement initiative that did not require review by an Internal Review Board. We applied Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting standards. Setting This study was performed by the Division of Neurology at Children's Hospital of Philadelphia, a pediatric specialty care network composed of an urban quaternary care hospital, an ambulatory center, and an additional 8 satellite locations offering child neurology care. The practice includes 55 child neurologists, 7 advanced practice providers, and 21 graduate medical trainees. The program provides 32,000 outpatient encounters per year across general child neurology and specialty programs in epilepsy, neuromuscular, neuroimmune disorders, brain protection, stroke, neurocardiac care, fetal and neonatal neurology, headache, cognitive/behavioral neurology, leukodystrophy, and Friedreich ataxia. All clinical care is documented with the Epic (Verona, WI) electronic medical record (EMR) system. Before the pandemic, our practice offered no outpatient telehealth services. All providers in the practice were licensed in the state of Pennsylvania. During the pandemic, all providers received emergency licenses for the care of children in New Jersey through telehealth services. Implementation of telehealth, including audio-video telemedicine and scheduled telephone encounters On March 9, 2020, our institution suspended all nonurgent in-person neurology office visits due to regional adoption of social distancing measures at the onset of the COVID-19 pandemic. New patient referrals were screened by a physician, and a small number were seen in person in a dedicated urgent clinic. Most new and all established patients were scheduled for audio-video telemedicine encounters. Established patients who lacked access to a smartphone or computer application required to enable telemedicine encounters were scheduled for structured (audio-only) telephone encounters. Unscheduled telephone calls by a provider in response to a patient message were not considered telephone encounters, and they were not included in our analyses. Providers used the telemedicine software embedded in the Epic EMR to conduct telemedicine encounters. Telephone encounters were conducted with a phone. Coordinators instructed patients or caregivers regarding the download of the patient portal smartphone application required for telemedicine encounters when appointments were scheduled. One day before the encounter, a nursing assistant called the family or caregivers to provide additional instructions about the encounter procedure to increase the likelihood of successfully accessing the patient portal. Families were instructed to initiate the encounter 15 minutes before the scheduled time to sign consents and to verify allergies, medications, and the active problem list. If patients did not successfully connect to the encounter within 5 minutes of the scheduled encounter time, then staff reached out to patients to troubleshoot. Neurology providers designated as EMR superusers trained all providers to perform telemedicine and telephone encounters through a narrated slide presentation or a one-on-one tutorial that included a simulated encounter. Providers reviewed resources on conducting video telemedicine neurologic examinations from the American Academy of Neurology. At the time of the encounter, providers accessed the system through a smartphone (Haiku) or tablet (Canto) application and completed documentation by accessing Epic on a computer with a remote desktop connection (Citrix Systems). Providers documented telemedicine and telephone encounters within the EMR using structured documentation templates that contained key medical data fields from typical in-person note templates. In addition, new fields were added, including the need for the encounter, identification of participants within the encounter, and appointment duration. Audio-video telemedicine encounters contained a template for a physical examination, which was not included in templates for telephone encounters. Design of provider questionnaires embedded in telemedicine notes To assess the effectiveness of telemedicine encounters, we embedded 5 multiple choice questions in the note templates. Because these questions were aimed at evaluating the quality of the newly established telemedicine encounters, these questions were not included in the telephone encounter template. The questions assessed (question 1) provider satisfaction, (question 2) follow-up plans using telemedicine, (question 3) presence of technical issues, (question 4) presence of concerns requiring sooner in-patient assessment, and (question 5) caregiver evaluation of the telemedicine encounter assessed by the provider. The question regarding caregiver evaluation was added to the survey ≈4 weeks after the initial survey deployment. Providers were asked to query families about their satisfaction with telemedicine at the conclusion of the encounter. Data abstraction Data from the Epic EMR were accessed via the Clarity database (Epic). We developed a dedicated EMR data extraction protocol that identified all outpatient encounters including the 3 categories of telehealth encounters (telemedicine new, telemedicine follow-up, and telephone follow-up). We extracted demographic variables, including age, sex, race, and ethnicity, for all encounters. We mapped patient zip codes to median household income (MHI) using 2018 US Census data. We retrieved the primary ICD-10 code for each encounter and used higher-level grouping of primary ICD-10 codes for analysis (e.g., G40 instead of G40.10). For the small minority of encounters with primary diagnoses mapping on 2 ICD-10 codes, we used the more common ICD-10 code for the analyses to allow grouping of common codes. We performed data extraction and analysis within an institutional Health Insurance Portability and Accountability Act–compliant framework. We developed a Natural Language Processing pipeline within Oracle SQL that detected the quality improvement question text within the full text and parsed the semistructured answers, including free-text options. We used the R analysis framework (R Foundation for Statistical Computing, Vienna, Austria) for data analysis and visualization. For our study, the unit of analysis was a patient encounter (not patient). We present data as descriptive statistics or comparisons using Fisher exact tests and Wilcoxon rank sum tests. Analyses We assessed all in-person encounters conducted from October 1, 2019 until March 15, 2020 and all telehealth encounters, including telemedicine encounters and telephone encounters, from March 16, 2020 until April 24, 2020. We used the term telemedicine to refer exclusively to patient encounters performed through the audio and video software embedded in the Epic EMR, while telehealth includes both telemedicine encounters and telephone encounters. The rationale for including both encounter types was that the combination of both encounter types reflects the overall care provided by our care network after cessation of in-person encounters and that telephone encounters included all components of a telemedicine encounter except visualization of the patient and remote physical examination. However, telemedicine encounters and telephone encounters are often considered conceptually different. , Therefore, we also compared in-person encounters with telemedicine encounters, excluding telephone encounters. Because telephone encounters included only follow-up encounters for established patients, we performed a separate analysis of follow-up encounters. First, we compared in-person encounters with telehealth encounters to determine whether practice had changed. Second, within the telehealth cohort for return encounters, we compared patient and diagnosis variables between telemedicine and telephone encounters. Third, we compared telemedicine encounters for which providers did or did not complete the quality improvement questionnaires to determine whether the assessed cohort was representative of the overall population. Fourth, we assessed responses to the quality improvement questionnaire. For the analysis of provider satisfaction (question 1), the options “very satisfied” and “somewhat satisfied” were collapsed. For the analysis of follow-up plans (question 2), the options “Yes, I only need to see them in person if there is a new symptom or major change” and “Yes, but as a mix of telemedicine and in-person encounters” were collapsed. For the analysis of technical issues (question 3), answers to any option were analyzed jointly and separately. A binary choice (yes/no) was given to providers for questions regarding concerning features requiring in-person evaluation sooner than if the encounter had occurred in person (visits of concern, question 4) and provider-assessed caregiver reception of telemedicine encounters (question 5). Finally, a board-certified child neurologist manually assessed all encounters flagged as concerning by providers in the embedded questionnaire, including the reason for concern, primary diagnosis, documented follow-up plan, placement of orders, and disposition. The primary encounter diagnosis was used to compare frequency of diagnostic subgroups in the cohort with concerns to the full telemedicine cohort. Data availability Data in a deidentified format will be made available by request to the corresponding author.
This was a quality improvement initiative that did not require review by an Internal Review Board. We applied Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting standards.
This study was performed by the Division of Neurology at Children's Hospital of Philadelphia, a pediatric specialty care network composed of an urban quaternary care hospital, an ambulatory center, and an additional 8 satellite locations offering child neurology care. The practice includes 55 child neurologists, 7 advanced practice providers, and 21 graduate medical trainees. The program provides 32,000 outpatient encounters per year across general child neurology and specialty programs in epilepsy, neuromuscular, neuroimmune disorders, brain protection, stroke, neurocardiac care, fetal and neonatal neurology, headache, cognitive/behavioral neurology, leukodystrophy, and Friedreich ataxia. All clinical care is documented with the Epic (Verona, WI) electronic medical record (EMR) system. Before the pandemic, our practice offered no outpatient telehealth services. All providers in the practice were licensed in the state of Pennsylvania. During the pandemic, all providers received emergency licenses for the care of children in New Jersey through telehealth services.
On March 9, 2020, our institution suspended all nonurgent in-person neurology office visits due to regional adoption of social distancing measures at the onset of the COVID-19 pandemic. New patient referrals were screened by a physician, and a small number were seen in person in a dedicated urgent clinic. Most new and all established patients were scheduled for audio-video telemedicine encounters. Established patients who lacked access to a smartphone or computer application required to enable telemedicine encounters were scheduled for structured (audio-only) telephone encounters. Unscheduled telephone calls by a provider in response to a patient message were not considered telephone encounters, and they were not included in our analyses. Providers used the telemedicine software embedded in the Epic EMR to conduct telemedicine encounters. Telephone encounters were conducted with a phone. Coordinators instructed patients or caregivers regarding the download of the patient portal smartphone application required for telemedicine encounters when appointments were scheduled. One day before the encounter, a nursing assistant called the family or caregivers to provide additional instructions about the encounter procedure to increase the likelihood of successfully accessing the patient portal. Families were instructed to initiate the encounter 15 minutes before the scheduled time to sign consents and to verify allergies, medications, and the active problem list. If patients did not successfully connect to the encounter within 5 minutes of the scheduled encounter time, then staff reached out to patients to troubleshoot. Neurology providers designated as EMR superusers trained all providers to perform telemedicine and telephone encounters through a narrated slide presentation or a one-on-one tutorial that included a simulated encounter. Providers reviewed resources on conducting video telemedicine neurologic examinations from the American Academy of Neurology. At the time of the encounter, providers accessed the system through a smartphone (Haiku) or tablet (Canto) application and completed documentation by accessing Epic on a computer with a remote desktop connection (Citrix Systems). Providers documented telemedicine and telephone encounters within the EMR using structured documentation templates that contained key medical data fields from typical in-person note templates. In addition, new fields were added, including the need for the encounter, identification of participants within the encounter, and appointment duration. Audio-video telemedicine encounters contained a template for a physical examination, which was not included in templates for telephone encounters.
To assess the effectiveness of telemedicine encounters, we embedded 5 multiple choice questions in the note templates. Because these questions were aimed at evaluating the quality of the newly established telemedicine encounters, these questions were not included in the telephone encounter template. The questions assessed (question 1) provider satisfaction, (question 2) follow-up plans using telemedicine, (question 3) presence of technical issues, (question 4) presence of concerns requiring sooner in-patient assessment, and (question 5) caregiver evaluation of the telemedicine encounter assessed by the provider. The question regarding caregiver evaluation was added to the survey ≈4 weeks after the initial survey deployment. Providers were asked to query families about their satisfaction with telemedicine at the conclusion of the encounter.
Data from the Epic EMR were accessed via the Clarity database (Epic). We developed a dedicated EMR data extraction protocol that identified all outpatient encounters including the 3 categories of telehealth encounters (telemedicine new, telemedicine follow-up, and telephone follow-up). We extracted demographic variables, including age, sex, race, and ethnicity, for all encounters. We mapped patient zip codes to median household income (MHI) using 2018 US Census data. We retrieved the primary ICD-10 code for each encounter and used higher-level grouping of primary ICD-10 codes for analysis (e.g., G40 instead of G40.10). For the small minority of encounters with primary diagnoses mapping on 2 ICD-10 codes, we used the more common ICD-10 code for the analyses to allow grouping of common codes. We performed data extraction and analysis within an institutional Health Insurance Portability and Accountability Act–compliant framework. We developed a Natural Language Processing pipeline within Oracle SQL that detected the quality improvement question text within the full text and parsed the semistructured answers, including free-text options. We used the R analysis framework (R Foundation for Statistical Computing, Vienna, Austria) for data analysis and visualization. For our study, the unit of analysis was a patient encounter (not patient). We present data as descriptive statistics or comparisons using Fisher exact tests and Wilcoxon rank sum tests.
We assessed all in-person encounters conducted from October 1, 2019 until March 15, 2020 and all telehealth encounters, including telemedicine encounters and telephone encounters, from March 16, 2020 until April 24, 2020. We used the term telemedicine to refer exclusively to patient encounters performed through the audio and video software embedded in the Epic EMR, while telehealth includes both telemedicine encounters and telephone encounters. The rationale for including both encounter types was that the combination of both encounter types reflects the overall care provided by our care network after cessation of in-person encounters and that telephone encounters included all components of a telemedicine encounter except visualization of the patient and remote physical examination. However, telemedicine encounters and telephone encounters are often considered conceptually different. , Therefore, we also compared in-person encounters with telemedicine encounters, excluding telephone encounters. Because telephone encounters included only follow-up encounters for established patients, we performed a separate analysis of follow-up encounters. First, we compared in-person encounters with telehealth encounters to determine whether practice had changed. Second, within the telehealth cohort for return encounters, we compared patient and diagnosis variables between telemedicine and telephone encounters. Third, we compared telemedicine encounters for which providers did or did not complete the quality improvement questionnaires to determine whether the assessed cohort was representative of the overall population. Fourth, we assessed responses to the quality improvement questionnaire. For the analysis of provider satisfaction (question 1), the options “very satisfied” and “somewhat satisfied” were collapsed. For the analysis of follow-up plans (question 2), the options “Yes, I only need to see them in person if there is a new symptom or major change” and “Yes, but as a mix of telemedicine and in-person encounters” were collapsed. For the analysis of technical issues (question 3), answers to any option were analyzed jointly and separately. A binary choice (yes/no) was given to providers for questions regarding concerning features requiring in-person evaluation sooner than if the encounter had occurred in person (visits of concern, question 4) and provider-assessed caregiver reception of telemedicine encounters (question 5). Finally, a board-certified child neurologist manually assessed all encounters flagged as concerning by providers in the embedded questionnaire, including the reason for concern, primary diagnosis, documented follow-up plan, placement of orders, and disposition. The primary encounter diagnosis was used to compare frequency of diagnostic subgroups in the cohort with concerns to the full telemedicine cohort.
Data in a deidentified format will be made available by request to the corresponding author.
Recovery of patient volume by telehealth during the COVID-19 pandemic Patient volume initially decreased but then recovered. Compared to baseline outpatient in-person clinical volume (average 610.75 encounters per week), patient volume during the initial 2 weeks of telehealth care decreased by 41% (average 247.5 encounters per week). However, for the next 2 weeks, the mean volume increased to 3% over baseline (average 626.5 encounters per week). Telemedicine encounters accounted for 80% of all telehealth encounters. Only 21 of 2,459 patient encounters (1%) were performed in person after transition to telehealth. Patient demographics before and after telehealth transition We compared patient demographics and spectrum of diagnoses between 14,780 in-person encounters and 2,589 telehealth encounters (including 2,093 telemedicine and 496 telephone encounters) between October 1, 2019 and April 24, 2020 ( and ). The median age was 11.6 and 11.4 years in the in-person and telehealth cohorts, respectively. The age distribution in both cohorts was virtually identical. Self-reported ethnicity and race were not different between the cohorts except for a small increase in the number of individuals self-reporting as multiple races in the telehealth cohort. MHI was identical between the cohorts. There were no differences in age, self-reported ethnicity, race, and MHI when the in-person cohort was compared to only the telemedicine component of the telehealth cohort (excluding telephone encounters). The ratio of new to established patients was higher in the telemedicine cohort compared to the broader telehealth cohort but still lower compared to the in-person cohort (645 of 2,093 [31%] vs 5,103 of 14,780 [35%], odds ratio [OR] 1.2, 95% confidence interval [CI] 1.1–1.3). Diagnostic spectrum before and after telehealth The most common primary diagnoses in the in-person and telehealth cohorts were epilepsy (G40) in 30% and migraine (G43) in 20% . Epilepsy diagnoses were slightly more prevalent in the telehealth cohort than in the in-person cohort (telehealth 30% vs in-person 27%, OR 1.2, 95% CI 1.1–1.3). The proportion of patients with migraine was 20% in both cohorts. In an assessment of all 376 primary diagnoses seen in the overall cohort, 16 diagnoses were significantly different between the in-person and telehealth cohort. Diagnoses that were overrepresented in the telehealth cohort included metabolic disorders (E75, OR 3.3, 95% CI 1.4–7.2), while underrepresented diagnoses in the telehealth cohort included back pain (M54, OR 0, 95% CI 0–0.7), malaise and fatigue (R53, OR 0.15, 95% CI 0–0.9), and syncope (R55, OR 0.45, 95% CI 0.2–1.0). When the in-person cohort was compared to the telemedicine cohort (excluding telephone encounters), there were no differences in the frequency of epilepsy or migraine diagnoses. Metabolic disorders remained overrepresented in the telemedicine vs in-person cohort (E75, OR 4.1, 95% CI 1.8–8.9), while back pain was underrepresented (M54, OR 0, 95% CI 0–0.9). Provider and parent/caregiver assessments of telemedicine encounters The provider replied to at least 1 question in the provider questionnaire in 63% (1,314 of 2,093) of telemedicine encounters. Questions were not included for telephone encounters. The patient encounters with questionnaire answers were representative of the overall telemedicine cohort . Providers indicated overall satisfaction with the telemedicine encounters in 93% (1,200 of 1,286) of encounters, including a subset of 60% (767 of 1,286) of encounters for which the providers were very satisfied with the encounters. Providers indicated that they would use telemedicine for at least a component of the follow-up plan for the patient in 89% (1,144 of 1,286) of encounters, including 38% (484 of 1,286) of patient encounters for which telemedicine could be used exclusively unless the patient had new symptoms or a clinical change. In 40% (519 of 1,314) of encounters, the technical quality was impaired, and the most frequent single causes affecting quality were poor audio (19%), poor video (13%), and interruption of the encounter (9%). In 5% of encounters, providers documented additional technical quality problems in free-text notes. After a patient/caregiver questionnaire was implemented, responses were provided for 217 of 559 encounters. Caregivers indicated an interest in telemedicine as part of future care for 86% (187 of 217) of encounters. Evaluation of visits of concern In 5% (65 of 1,285) of telemedicine encounters, the provider flagged the clinical scenario as concerning enough to necessitate in-person evaluation. These 65 encounters were evaluated further by chart review . Patient age in visits of concerns was not significantly different from that in visits without concern. Epilepsy (G40) was the most common primary diagnosis in visits of concern, but the frequency was not significantly different from the overall telemedicine or telehealth cohort. Migraine (G43) was significantly underrepresented in the visits of concern, while metabolic disorders (E75), facial nerve disorders (G51), sensory disturbance (R20), neuromuscular disorders (G71), and abnormal movements (R25) were significantly overrepresented. In all visits of concern, manual review revealed an adequate plan documented in the provider notes. Comparison of telemedicine and telephone follow-up encounters As a result of parent/caregiver preference or feasibility, 496 follow-up encounters were conducted as telephone encounters instead of telemedicine encounters. Given that telephone encounters were used only for follow-up of established patients, we compared demographics and patient diagnoses of the 496 telephone and 1,448 telemedicine follow-up encounters. Patients with telephone encounters were more likely to be male compared to follow-up telemedicine encounters (56% male telephone encounter, 48% follow-up telemedicine encounter, OR 1.4, 95% CI 1.1–1.7). Age did not differ between the cohorts. Patients in racial or ethnic minority groups were evaluated by telephone encounters instead of telemedicine encounters more often than patients self-identified as white. Patients self-identified as black made up 21% of the group opting for telephone encounters compared to 11% of the group using telemedicine encounters (OR 2.2, 95% CI 1.7–3.0). Hispanic/Latino patients made up of 14% of telephone encounters compared to 9% of telemedicine encounters (OR 1.7, 95% CI 1.2–2.3). MHI was lower in patients evaluated by telephone encounters compared to telemedicine encounters ($72,373 MHI telephone vs $79,997 MHI telemedicine encounters, difference $10,656, 95% CI 7,509–13,723). Epilepsy (G40) was overrepresented in telephone encounters compared to telemedicine encounters (42% of telephone vs 35% of telemedicine encounters, OR 1.3, 95% 1.1–1.7). The frequency of patients with a primary diagnosis of migraine (G43) was 19% in both cohorts. Given the differences in both cohorts, we also compared telephone encounters with the 9,677 in-person follow-up encounters, which showed similar results. Again, we did not observe age differences, but male (OR 1.3, 95% CI 1.1–1.6), black (OR 1.63, 95% CI 1.3–2.1), and Hispanic/Latino (OR 1.4, 95% CI 1.1–1.8) patients were overrepresented in telephone encounters compared to in-person follow-up encounters. In addition, we saw similar differences in MHI ($72,373 MHI telephone vs $78,540 MHI in-person follow-up encounters, difference $7,915, 95% CI 5,162–10,695) and epilepsy diagnoses (42% of telephone vs 33% of follow-up in-person encounters, OR 1.4, 95% CI 1.2–1.7). These results suggest that the differences between telephone encounters and telemedicine follow-up encounters can be attributed to changes in the patient population evaluated by telephone encounters rather than changes in the patient group evaluated by telemedicine.
Patient volume initially decreased but then recovered. Compared to baseline outpatient in-person clinical volume (average 610.75 encounters per week), patient volume during the initial 2 weeks of telehealth care decreased by 41% (average 247.5 encounters per week). However, for the next 2 weeks, the mean volume increased to 3% over baseline (average 626.5 encounters per week). Telemedicine encounters accounted for 80% of all telehealth encounters. Only 21 of 2,459 patient encounters (1%) were performed in person after transition to telehealth.
We compared patient demographics and spectrum of diagnoses between 14,780 in-person encounters and 2,589 telehealth encounters (including 2,093 telemedicine and 496 telephone encounters) between October 1, 2019 and April 24, 2020 ( and ). The median age was 11.6 and 11.4 years in the in-person and telehealth cohorts, respectively. The age distribution in both cohorts was virtually identical. Self-reported ethnicity and race were not different between the cohorts except for a small increase in the number of individuals self-reporting as multiple races in the telehealth cohort. MHI was identical between the cohorts. There were no differences in age, self-reported ethnicity, race, and MHI when the in-person cohort was compared to only the telemedicine component of the telehealth cohort (excluding telephone encounters). The ratio of new to established patients was higher in the telemedicine cohort compared to the broader telehealth cohort but still lower compared to the in-person cohort (645 of 2,093 [31%] vs 5,103 of 14,780 [35%], odds ratio [OR] 1.2, 95% confidence interval [CI] 1.1–1.3).
The most common primary diagnoses in the in-person and telehealth cohorts were epilepsy (G40) in 30% and migraine (G43) in 20% . Epilepsy diagnoses were slightly more prevalent in the telehealth cohort than in the in-person cohort (telehealth 30% vs in-person 27%, OR 1.2, 95% CI 1.1–1.3). The proportion of patients with migraine was 20% in both cohorts. In an assessment of all 376 primary diagnoses seen in the overall cohort, 16 diagnoses were significantly different between the in-person and telehealth cohort. Diagnoses that were overrepresented in the telehealth cohort included metabolic disorders (E75, OR 3.3, 95% CI 1.4–7.2), while underrepresented diagnoses in the telehealth cohort included back pain (M54, OR 0, 95% CI 0–0.7), malaise and fatigue (R53, OR 0.15, 95% CI 0–0.9), and syncope (R55, OR 0.45, 95% CI 0.2–1.0). When the in-person cohort was compared to the telemedicine cohort (excluding telephone encounters), there were no differences in the frequency of epilepsy or migraine diagnoses. Metabolic disorders remained overrepresented in the telemedicine vs in-person cohort (E75, OR 4.1, 95% CI 1.8–8.9), while back pain was underrepresented (M54, OR 0, 95% CI 0–0.9).
The provider replied to at least 1 question in the provider questionnaire in 63% (1,314 of 2,093) of telemedicine encounters. Questions were not included for telephone encounters. The patient encounters with questionnaire answers were representative of the overall telemedicine cohort . Providers indicated overall satisfaction with the telemedicine encounters in 93% (1,200 of 1,286) of encounters, including a subset of 60% (767 of 1,286) of encounters for which the providers were very satisfied with the encounters. Providers indicated that they would use telemedicine for at least a component of the follow-up plan for the patient in 89% (1,144 of 1,286) of encounters, including 38% (484 of 1,286) of patient encounters for which telemedicine could be used exclusively unless the patient had new symptoms or a clinical change. In 40% (519 of 1,314) of encounters, the technical quality was impaired, and the most frequent single causes affecting quality were poor audio (19%), poor video (13%), and interruption of the encounter (9%). In 5% of encounters, providers documented additional technical quality problems in free-text notes. After a patient/caregiver questionnaire was implemented, responses were provided for 217 of 559 encounters. Caregivers indicated an interest in telemedicine as part of future care for 86% (187 of 217) of encounters.
In 5% (65 of 1,285) of telemedicine encounters, the provider flagged the clinical scenario as concerning enough to necessitate in-person evaluation. These 65 encounters were evaluated further by chart review . Patient age in visits of concerns was not significantly different from that in visits without concern. Epilepsy (G40) was the most common primary diagnosis in visits of concern, but the frequency was not significantly different from the overall telemedicine or telehealth cohort. Migraine (G43) was significantly underrepresented in the visits of concern, while metabolic disorders (E75), facial nerve disorders (G51), sensory disturbance (R20), neuromuscular disorders (G71), and abnormal movements (R25) were significantly overrepresented. In all visits of concern, manual review revealed an adequate plan documented in the provider notes.
As a result of parent/caregiver preference or feasibility, 496 follow-up encounters were conducted as telephone encounters instead of telemedicine encounters. Given that telephone encounters were used only for follow-up of established patients, we compared demographics and patient diagnoses of the 496 telephone and 1,448 telemedicine follow-up encounters. Patients with telephone encounters were more likely to be male compared to follow-up telemedicine encounters (56% male telephone encounter, 48% follow-up telemedicine encounter, OR 1.4, 95% CI 1.1–1.7). Age did not differ between the cohorts. Patients in racial or ethnic minority groups were evaluated by telephone encounters instead of telemedicine encounters more often than patients self-identified as white. Patients self-identified as black made up 21% of the group opting for telephone encounters compared to 11% of the group using telemedicine encounters (OR 2.2, 95% CI 1.7–3.0). Hispanic/Latino patients made up of 14% of telephone encounters compared to 9% of telemedicine encounters (OR 1.7, 95% CI 1.2–2.3). MHI was lower in patients evaluated by telephone encounters compared to telemedicine encounters ($72,373 MHI telephone vs $79,997 MHI telemedicine encounters, difference $10,656, 95% CI 7,509–13,723). Epilepsy (G40) was overrepresented in telephone encounters compared to telemedicine encounters (42% of telephone vs 35% of telemedicine encounters, OR 1.3, 95% 1.1–1.7). The frequency of patients with a primary diagnosis of migraine (G43) was 19% in both cohorts. Given the differences in both cohorts, we also compared telephone encounters with the 9,677 in-person follow-up encounters, which showed similar results. Again, we did not observe age differences, but male (OR 1.3, 95% CI 1.1–1.6), black (OR 1.63, 95% CI 1.3–2.1), and Hispanic/Latino (OR 1.4, 95% CI 1.1–1.8) patients were overrepresented in telephone encounters compared to in-person follow-up encounters. In addition, we saw similar differences in MHI ($72,373 MHI telephone vs $78,540 MHI in-person follow-up encounters, difference $7,915, 95% CI 5,162–10,695) and epilepsy diagnoses (42% of telephone vs 33% of follow-up in-person encounters, OR 1.4, 95% CI 1.2–1.7). These results suggest that the differences between telephone encounters and telemedicine follow-up encounters can be attributed to changes in the patient population evaluated by telephone encounters rather than changes in the patient group evaluated by telemedicine.
In this quality improvement study after rapid implementation of telehealth services for outpatient child neurology care, we made 5 key observations. First, conversion of outpatient care to telehealth encounters occurred across our patients with a similar distribution of demographic and clinical characteristics compared to prepandemic in-person encounters. Second, when using dedicated telemedicine services, providers reported that telemedicine was satisfactory for almost all encounters and that they would opt for ongoing use of telemedicine for most patients. Providers reported a high level of satisfaction despite technical issues in about one-third of encounters, suggesting that these issues did not substantially interfere with care delivery. Third, in a single basic measure, most parents/caregivers reported satisfaction with the telemedicine encounter. Fourth, urgent in-person evaluation was needed in a small percentage of patients. Fifth, access to telemedicine encounters compared to telephone encounters was lower in racial and ethnic minority groups, highlighting an inequity that must be addressed. In the context of the COVID-19 pandemic, telemedicine as a subset of remote health care services that include audio and video equipment aligns with the 6 health care quality domains described by the Institute of Medicine. By providing a way to receive care without increasing the risk of pathogen exposure, telemedicine facilitates safe care . By allowing information exchange with patients and caregivers to inform medical decision-making, providers can offer effective care. Accessibility and convenience while maintaining high patient and family satisfaction allow patient-centered care. Telemedicine provides timely care by avoiding suspension or delays in care during a pandemic that requires social distancing, efficient care by reducing the burden on providers or families related to travel and time required for an in-person encounter, and equitable care by ensuring that we continue to meet the needs of our diverse patient population. High levels of satisfaction with the telemedicine process in a practice where few providers had prior telemedicine experience suggest that this method of health care delivery is sustainable during and after the current pandemic. High levels of satisfaction despite frequent technical issues may have been influenced by lack of alternative methods in the setting of the COVID-19 pandemic, and infrastructural improvements to rapidly address technical issues are needed. These improvements may include software updates and bandwidth expansion given the massive increase in data traffic across the hospital's networks. The vast majority of our providers indicated that they would continue to perform telemedicine encounters beyond the current pandemic if given the opportunity. This finding demonstrates that remote history taking and virtual examinations are effective for providing most child neurology care. In some instances, telemedicine may be able to remove barriers to care that result from in-person encounters. This benefit may be especially true for underserved patients whose caregivers cannot afford to miss work or travel to the clinic in person, who live far from our facilities, or who have complex transportation needs. However, this study uncovered disparities in the delivery of telemedicine care to patients in racial and ethnic minority groups, who received care in the same proportion as in-person encounters but were less likely to have access to the potentially more robust care that telemedicine encounters can provide compared to telephone encounters. The need for rapid implementation of quality assurance measures alongside rapid implementation of telemedicine led to some notable limitations in our study. First, the provider questionnaire was completed in only 63% of telemedicine encounters. While the cohort with completed questionnaires was representative of the overall population of encounters, it is possible that providers who did not routinely complete the surveys in their telemedicine encounters were less technically savvy and therefore may have had differing opinions on the utility of telemedicine. Second, the survey questions used in the telemedicine encounters were not validated assessments of care effectiveness but rather offered targeted insights that will be used to implement future systems changes. Third, while our data suggest that most providers perceive telemedicine to be at least equivalent to in-person care for a variety of neurologic conditions, we cannot conclude that outcomes from telemedicine encounters are comparable to those of in-person encounters. Prospective studies of process measures and patient-centered outcome measures are needed to evaluate the effectiveness of child neurology telemedicine more robustly. Fourth, a measurement of patient satisfaction was carried out with only a single question asked by the clinician providing care, and this approach may influence answers toward a positive reply. Using electronic survey technology to gather anonymous satisfaction assessments may provide a less biased view in future studies. While telemedicine encounters are often strictly defined as encounters with both audio and video components, we chose to include both audio-video telemedicine encounters and audio-only telephone encounters in our analyses. We opted for the inclusion of telephone encounters for 2 reasons. First, the combination of both telemedicine encounters and telephone encounters represented the total scheduled patient encounters provided by our care network. Therefore, the combination of both encounter types reflected the outpatient services provided more adequately than telemedicine encounters alone. Second, scheduled telephone encounters were structured and mirrored audio-video telemedicine encounters in all ways except remote physical examination and nonverbal communication. The rapid implementation of both encounter types occurred simultaneously, and we deemed both types of care as important within our care delivery model. Given the disparities among racial or ethnic groups uncovered by our analyses, further work is ongoing to address these differences. Priority questions for future studies include determining whether previously documented benefits of telemedicine such as reduction in no-show burden, , in costly emergency department visits, or in miles traveled for patients , occur when telemedicine is implemented more broadly in child neurology practice. While many of our patients travel far for subspecialty care, those who live in our primary catchment region may also benefit from telemedicine child neurology services. In addition, evaluation of the impact of remote monitoring technologies, including seizure detection devices, long-term remote EEG monitoring, electronic pill boxes, and actigraphy might further bolster the future use of telehealth services beyond provider consultation. We describe the successful implementation of telehealth services across all the subspecialties of a pediatric neurology program with a detailed evaluation of audio-video telemedicine encounters. We expect that further research into optimizing these technologies will show telemedicine to be even more valuable than demonstrated in our study. We recognize that implementation during the COVID-19 crisis was possible because legislators and payers quickly accommodated this approach, and we hope evidence of effectiveness and benefit across all 6 Institute of Medicine health care quality domains will help ensure that children with neurologic conditions have continued access to telemedicine care.
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Cancer Survivors with Sub-Optimal Patient-Centered Communication Before and During the Early COVID-19 Pandemic. | 392ceca9-1664-4852-be8f-7dd552ec4b12 | 10299944 | Patient-Centered Care[mh] | Introduction Patient Centered Communication (PCC) is defined as interactions and communications between patients and providers to meet patients’ needs and respond to their preferences . The National Cancer Institute (NCI) outlined six core domains of PCC that could influence patients’ essential health outcomes: exchanging information, responding to emotions, making decisions, enabling self-management, fostering healing relationships, and managing uncertainty . PCC allows patients to have time with providers to ask questions and receive the relevant information to care for themselves, acquire support from the providers for health decision-making, and help to express emotions and deal with uncertainty and anxiety , , , . The Institute of Medicine (IOM) identified PCC as an essential element of patient-centered care in 2013 . People who experienced PCC reported benefits from mental distress management , . They also showed higher cancer care quality, treatment adherence, emotional well-being, and health-related quality of life , , . During the early COVID-19 pandemic in 2020, cancer survivors faced disrupted cancer care (e.g., delayed cancer care, changed treatment plans) and fear of disease progression , , , . After the unprecedented Stay At Home Order in March 2020 in United States (U.S.), in-person clinic visits were extremely limited. In addition, cancer survivors experienced additional fear of COVID-19 infection because those with chronic medical conditions, including cancer, showed worse COVID-19 infection outcomes , . The restricted in-person patient-provider interactions due to Stay At Home Order might have hindered optimal PCC during this time. In addition to this actual limitation of providers' communicational capacity (e.g., closed health care facility, lack of health providers), patients' perceived distance from providers due to the interaction-discouraged atmosphere during this unique time might have been also at play in preventing optimal PCC performance , , . A study reported that physicians’ responsiveness to patients during conversations to help address uncertain and difficult emotions was associated with better health and coping and less psychological distress during COVID-19 , highlighting the importance of PCC. Prior studies have found PCC disparities by sociodemographic and health status factors among cancer survivors in the U.S. Cancer survivors who were racial/ethnic minorities, were more educated, had low income, had no usual source of care, or had poor physical or mental health reported lower perceived PCC , , , , while age showed inconsistent associations. For example, older cancer survivors had higher perceived PCC in HINTS 4 (2011-2013) , yet age was not related among newly diagnosed colon or rectal cancer patients . Previously, Blanch-Hartigan et al. assessed the trends in cancer survivors' PCC experience using HINTS 2007-2013 . However, a systematic evaluation of all six PCC domains among cancer survivors during the early pandemic has not been conducted, limiting our ability to examine the impact of COVID-19 pandemic overall PCC performance and across the subgroups. Therefore, this study used the nationally representative HINTS data (2017 to 2020) to assess the prevalence of optimal PCC, defined as always having perceived PCC , among cancer survivors during COVID-19 compared to those before COVID-19. This study also investigated sociodemographic and health status characteristics associated with optimal PCC during COVID-19 to identify subgroups of cancer survivors who would need support to have optimal PCC. We hypothesized that the prevalence of optimal PCC would decrease during the pandemic and the subgroups of cancer survivors with sub-optimal PCC would differ during COVID-19 than before COVID-19. Findings from this study can inform targeted interventions to support those in need. Furthermore, the knowledge could also contribute to improving PCC during telehealth visits that became rapidly and widely implemented during COVID-19 .
Methods 2.1 Data source We used nationally representative survey data from Health Information National Trends Survey (HINTS) for this study . HINTS is a self-administered, publicly available, cross-sectional survey data distributed and collected by National Cancer Institute (NCI) . This study used the HINTS 5 data, Cycles 1-3 (2017-2019) for before COVID-19 and Cycle 4 (2020) for during COVID-19. Of note, the COVID-19 sample was collected from February to June 2020. The survey questionnaires were administered to non-institutionalized civilians 18 years and older in the United States. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to report for an observational study (Supplemental ). The total number of survey responses in HINTS 5 Cycles 1-4 was 16,092 and the average response rate was 33% . Among the total responses, those with a history of cancer diagnosis were designated as cancer survivors’ responses (n=2,579) in this study as we followed the NCI definition of cancer survivor, a person with cancer from the time of diagnosis until the end of life . The HINTS reconciled the data from the different survey modes (mailed, push-to-web with a paper return, push-to-web with web return). We examined our variables of interest before combining 4 survey cycles to make sure the variable names and codes were consistent across the cycles . We used the HINTS Data Merging Code Tool that the HINTS provides to merge the data of HINTS 5 Cycle 1 to Cycle 4 . We obtained 200 replicate weights and used those to calculate standard errors. The full-sample weights were applied for the data to be nationally representative, intending to account for household-level base weight, non-response, and person-level initial weight . 2.2 Outcomes PCC was defined by the NCI framework and measured using the following questions: "In your communication with all doctors, nurses, or other health professionals in the past 12 months, how often did they 1) give you a chance to ask health questions? (Exchanging information), 2) had the attention you needed to your feelings and emotions? (Responding to emotions), 3) involve you in decisions about your health care as much as you wanted? (Making decisions), 4) make sure you understood the things you needed to do to take care of your health? (Enabling self-management), 5) explain things in a way you could understand? (Enabling self-management), 6) spend enough time with you? (Fostering healing relationships), 7) help you deal with uncertain feelings about your health or health care? (Managing uncertainty)." Responses for each question were measured on a Likert scale (1=always, 2=usually, 3=sometimes, 4=never). As done previously with HINTS data , , overall PCC was analyzed as a dichotomous outcome when all 6 domains were “always” for optimal PCC. Given this stringent cut-off, responses were combined and recoded using the Likert scale numbers to generate a new continuous PCC outcome variable, ranging from score 0 (the least optimal, when all 6 domains were scored “ never ”) to score 100 (the most optimal, when all 6 domains were scored “ always ”) to allow for comparisons to prior studies , . Furthermore, we dichotomized response options of each of the 6 domains as optimal (always) vs. sub-optimal (usually, sometimes, never) for our analysis and a sensitivity analysis was done to assess if the different cut-points [optimal (always/usually) vs. sub-optimal (sometimes/never)] would affect the associations, consistent with prior work . 2.3 Covariates 2.3.1 Sociodemographic characteristics We chose sociodemographic factors as independent variables of this study based on the social determinants of health conceptual framework from the Healthy People 2030 : age, birth gender, race/ethnicity, household income, educational attainment, marital status (married or living with a romantic partner as a married vs. not married including divorced, widowed, separated, single/never been married), employment status (employed vs. unemployed including homemaker, student, retired, disabled), health insurance type, usual source of care, and rurality of residence (metropolitan, micropolitan, small town, rural). HINTS used Urban Rural Commuting Area (RUCA) to designate the rurality of residence of the survey respondents, which categorized census tracts using population density, urbanization, and commuting patterns developed by the United States Department of Agriculture . 2.3.2 Health status characteristics Health status factors included general health status (excellent, very good, good, fair, poor), chronic medical conditions (diabetes, high blood pressure, heart disease, lung disease, depression), time since cancer diagnosis (less than a year, 2-5 years, 6-10 years, more than 11 years), cancer type (breast, cervical, prostate, colon, lung, skin cancer, melanoma, other cancer, or multiple cancer), and measures of psychological distress (little interest, hopelessness, nervousness, worrying). The psychological distress measurements were converted to depression or anxiety symptoms (past 2 weeks) using Patient-Health Questionnaire-4 (PHQ-4), and following its clinical cut-off (score ≥ 3, then symptom presents) . 2.4 Statistical analysis Weighted descriptive analyses [percentage with standard error (SE)] was conducted to describe cancer survivors’ sociodemographic and health status characteristics. To assess the prevalence of optimal PCC for each of the 6 domains and overall [dichotomized response (optimal = the response was ‘always’)] by time period (before and during COVID-19), we calculated the weighted percentage (%) with SE. Additionally, to examine the overall continuous PCC by sociodemographic and health status factors over the entire study period and in before and during COVID-19 time periods, we calculated the overall mean PCC and SE. To investigate the factors associated with optimal PCC (optimal=the responses of each domain was ‘always’), multivariable-adjusted weighted logistic regression models were developed to estimate the odds ratio (OR) and 95% confidence intervals (95% CI) of optimal PCC using dichotomized response for each domain. The same model was applied for a dichotomous overall PCC (optimal=the responses of all 6 domains were ‘always’). To explore the factors associated with a continuous overall PCC score, a multivariable-adjusted weighted linear regression model was developed to obtain coefficients (β) with SE. Sociodemographic and health status variables for the logistic and linear regression models, included age, gender, race/ethnicity, education, income, usual source of care, general health status, depression or anxiety symptoms, time since diagnosis, and cancer type. These variables were retained in the final model because they were considered as confounders (e.g., the covariate effect estimate changed by more than 10%), significantly associated with the outcome in univariable models ( P <0.05) or were associated with PCC in prior studies , , . To investigate whether the PCC differed during COVID-19 compared with before COVID-19, we assessed the associations between selected sociodemographic and health status factors (age, income, gender, usual source of care, race/ethnicity, and depression/anxiety symptoms) and time period (before vs. during COVID-19) in each model. Sensitivity analysis was conducted with [optimal (always/usually) vs. sub-optimal (sometimes/never)] to investigate the associated factors further by domain, as done previously . We assessed the interactions of selected sociodemographic and health status factors (age, income, gender, usual source of care, race/ethnicity, and depression/anxiety symptoms) with time period (before vs. during COVID-19) with overall PCC score. For these interaction assessments, we included interaction terms in multivariable weighted linear regression models. We performed hot deck imputation, which the HINTS used to account for the non-response , to account for the missing data in the covariates, which ranged from 1.0% to 13.3% (see footnotes of ). For all descriptive and regression analyses, the imputed data were used in SAS 9.4 (SAS studio 3.8, Cary, NC, USA). The statistical significance was determined at a P < 0.05.
Data source We used nationally representative survey data from Health Information National Trends Survey (HINTS) for this study . HINTS is a self-administered, publicly available, cross-sectional survey data distributed and collected by National Cancer Institute (NCI) . This study used the HINTS 5 data, Cycles 1-3 (2017-2019) for before COVID-19 and Cycle 4 (2020) for during COVID-19. Of note, the COVID-19 sample was collected from February to June 2020. The survey questionnaires were administered to non-institutionalized civilians 18 years and older in the United States. We followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to report for an observational study (Supplemental ). The total number of survey responses in HINTS 5 Cycles 1-4 was 16,092 and the average response rate was 33% . Among the total responses, those with a history of cancer diagnosis were designated as cancer survivors’ responses (n=2,579) in this study as we followed the NCI definition of cancer survivor, a person with cancer from the time of diagnosis until the end of life . The HINTS reconciled the data from the different survey modes (mailed, push-to-web with a paper return, push-to-web with web return). We examined our variables of interest before combining 4 survey cycles to make sure the variable names and codes were consistent across the cycles . We used the HINTS Data Merging Code Tool that the HINTS provides to merge the data of HINTS 5 Cycle 1 to Cycle 4 . We obtained 200 replicate weights and used those to calculate standard errors. The full-sample weights were applied for the data to be nationally representative, intending to account for household-level base weight, non-response, and person-level initial weight .
Outcomes PCC was defined by the NCI framework and measured using the following questions: "In your communication with all doctors, nurses, or other health professionals in the past 12 months, how often did they 1) give you a chance to ask health questions? (Exchanging information), 2) had the attention you needed to your feelings and emotions? (Responding to emotions), 3) involve you in decisions about your health care as much as you wanted? (Making decisions), 4) make sure you understood the things you needed to do to take care of your health? (Enabling self-management), 5) explain things in a way you could understand? (Enabling self-management), 6) spend enough time with you? (Fostering healing relationships), 7) help you deal with uncertain feelings about your health or health care? (Managing uncertainty)." Responses for each question were measured on a Likert scale (1=always, 2=usually, 3=sometimes, 4=never). As done previously with HINTS data , , overall PCC was analyzed as a dichotomous outcome when all 6 domains were “always” for optimal PCC. Given this stringent cut-off, responses were combined and recoded using the Likert scale numbers to generate a new continuous PCC outcome variable, ranging from score 0 (the least optimal, when all 6 domains were scored “ never ”) to score 100 (the most optimal, when all 6 domains were scored “ always ”) to allow for comparisons to prior studies , . Furthermore, we dichotomized response options of each of the 6 domains as optimal (always) vs. sub-optimal (usually, sometimes, never) for our analysis and a sensitivity analysis was done to assess if the different cut-points [optimal (always/usually) vs. sub-optimal (sometimes/never)] would affect the associations, consistent with prior work .
Covariates 2.3.1 Sociodemographic characteristics We chose sociodemographic factors as independent variables of this study based on the social determinants of health conceptual framework from the Healthy People 2030 : age, birth gender, race/ethnicity, household income, educational attainment, marital status (married or living with a romantic partner as a married vs. not married including divorced, widowed, separated, single/never been married), employment status (employed vs. unemployed including homemaker, student, retired, disabled), health insurance type, usual source of care, and rurality of residence (metropolitan, micropolitan, small town, rural). HINTS used Urban Rural Commuting Area (RUCA) to designate the rurality of residence of the survey respondents, which categorized census tracts using population density, urbanization, and commuting patterns developed by the United States Department of Agriculture . 2.3.2 Health status characteristics Health status factors included general health status (excellent, very good, good, fair, poor), chronic medical conditions (diabetes, high blood pressure, heart disease, lung disease, depression), time since cancer diagnosis (less than a year, 2-5 years, 6-10 years, more than 11 years), cancer type (breast, cervical, prostate, colon, lung, skin cancer, melanoma, other cancer, or multiple cancer), and measures of psychological distress (little interest, hopelessness, nervousness, worrying). The psychological distress measurements were converted to depression or anxiety symptoms (past 2 weeks) using Patient-Health Questionnaire-4 (PHQ-4), and following its clinical cut-off (score ≥ 3, then symptom presents) .
Sociodemographic characteristics We chose sociodemographic factors as independent variables of this study based on the social determinants of health conceptual framework from the Healthy People 2030 : age, birth gender, race/ethnicity, household income, educational attainment, marital status (married or living with a romantic partner as a married vs. not married including divorced, widowed, separated, single/never been married), employment status (employed vs. unemployed including homemaker, student, retired, disabled), health insurance type, usual source of care, and rurality of residence (metropolitan, micropolitan, small town, rural). HINTS used Urban Rural Commuting Area (RUCA) to designate the rurality of residence of the survey respondents, which categorized census tracts using population density, urbanization, and commuting patterns developed by the United States Department of Agriculture .
Health status characteristics Health status factors included general health status (excellent, very good, good, fair, poor), chronic medical conditions (diabetes, high blood pressure, heart disease, lung disease, depression), time since cancer diagnosis (less than a year, 2-5 years, 6-10 years, more than 11 years), cancer type (breast, cervical, prostate, colon, lung, skin cancer, melanoma, other cancer, or multiple cancer), and measures of psychological distress (little interest, hopelessness, nervousness, worrying). The psychological distress measurements were converted to depression or anxiety symptoms (past 2 weeks) using Patient-Health Questionnaire-4 (PHQ-4), and following its clinical cut-off (score ≥ 3, then symptom presents) .
Statistical analysis Weighted descriptive analyses [percentage with standard error (SE)] was conducted to describe cancer survivors’ sociodemographic and health status characteristics. To assess the prevalence of optimal PCC for each of the 6 domains and overall [dichotomized response (optimal = the response was ‘always’)] by time period (before and during COVID-19), we calculated the weighted percentage (%) with SE. Additionally, to examine the overall continuous PCC by sociodemographic and health status factors over the entire study period and in before and during COVID-19 time periods, we calculated the overall mean PCC and SE. To investigate the factors associated with optimal PCC (optimal=the responses of each domain was ‘always’), multivariable-adjusted weighted logistic regression models were developed to estimate the odds ratio (OR) and 95% confidence intervals (95% CI) of optimal PCC using dichotomized response for each domain. The same model was applied for a dichotomous overall PCC (optimal=the responses of all 6 domains were ‘always’). To explore the factors associated with a continuous overall PCC score, a multivariable-adjusted weighted linear regression model was developed to obtain coefficients (β) with SE. Sociodemographic and health status variables for the logistic and linear regression models, included age, gender, race/ethnicity, education, income, usual source of care, general health status, depression or anxiety symptoms, time since diagnosis, and cancer type. These variables were retained in the final model because they were considered as confounders (e.g., the covariate effect estimate changed by more than 10%), significantly associated with the outcome in univariable models ( P <0.05) or were associated with PCC in prior studies , , . To investigate whether the PCC differed during COVID-19 compared with before COVID-19, we assessed the associations between selected sociodemographic and health status factors (age, income, gender, usual source of care, race/ethnicity, and depression/anxiety symptoms) and time period (before vs. during COVID-19) in each model. Sensitivity analysis was conducted with [optimal (always/usually) vs. sub-optimal (sometimes/never)] to investigate the associated factors further by domain, as done previously . We assessed the interactions of selected sociodemographic and health status factors (age, income, gender, usual source of care, race/ethnicity, and depression/anxiety symptoms) with time period (before vs. during COVID-19) with overall PCC score. For these interaction assessments, we included interaction terms in multivariable weighted linear regression models. We performed hot deck imputation, which the HINTS used to account for the non-response , to account for the missing data in the covariates, which ranged from 1.0% to 13.3% (see footnotes of ). For all descriptive and regression analyses, the imputed data were used in SAS 9.4 (SAS studio 3.8, Cary, NC, USA). The statistical significance was determined at a P < 0.05.
Results 3.1 Cancer survivor characteristics In HINTS 5 2017-2020, there were 2,579 cancer survivors, 75% before (n=1,953) and 25% during COVID-19 (n=626) time periods ( ). About half (51%) were older adults ( ≥ 65 years), non-Hispanic Whites were the majority (80%), 66% had some college education or more, more than half (53%) reported $50,000 or more income, 57% had public/government-supported insurance, 84% had a regular provider, and 75% rated their health status as excellent/good. High blood pressure (54%) was the most common co-morbid chronic condition, followed by diabetes (24%) and depression (23%). Nearly one in three cancer survivors reported depression or anxiety symptoms in the past 2 weeks (33%). Almost half have been cancer survivors for more than 11 years (47%). There were no significant differences in population characteristics of cancer survivors between before and during COVID-19 (Supplemental ). 3.2 Prevalence of optimal PCC: before vs. during COVID-19 describes the prevalence of optimal PCC before and during COVID-19 by 6 PCC domains and overall. The prevalence of optimal PCC decreased during COVID-19 in most domains, except for exchanging information. The largest decrease of 7.3% was observed for managing uncertainty. In both periods, exchanging information was the domain with the highest prevalence of optimal PCC (64.5% before and 70% during COVID-19) while managing uncertainty was the domain with the lowest prevalence of optimal PCC (47.4% before and 40.1% during COVID-19). However, none of these differences were statistically significant. shows the mean PCC by sociodemographic and health status factors over the entire study period and before and during COVID-19 ( higher score refers to better PCC). The PCC mean score significantly differed in some sociodemographic subgroups by time. From before to during COVID-19, the PCC mean score increased in non-Hispanic Black/African Americans and decreased in those in the middle-income bracket ($50,000 to < $75,000). 3.3 Impact of COVID-19 We did not observe interactions between COVID-19 time period and sociodemographic or health status factors with overall PCC score. Thus, associations of sociodemographic and health status factors with optimal PCC before and during COVID-19 were combined in and Supplemental . 3.4 Factors associated with optimal PCC in each domain and overall Compared with the time before COVID-19, cancer survivors during COVID-19 were less likely to have optimal PCC overall (OR=0.73, 95% CI 0.54-0.98) and in the domain of managing uncertainty (OR=0.74, 0.55-0.99) ( ). However, other PCC functions did not differ between the two time periods . Cancer survivors who had a usual source of care were 1.5-2 times as likely to have optimal PCC than those without it overall (OR=1.53, 1.04-2.25) and in all domains (ORs=1.64-2.29), except for managing uncertainty . Similarly, cancer survivors who had no depression or anxiety symptoms had 1.62-2.17 times the odds of having optimal PCC overall and each domain, compared with those with anxiety or depression symptoms. Cancer survivors with excellent/good general health had 1.40-1.66 times the odds of having optimal PCC overall and in the domains of responding to emotion, making decisions, enabling self-management, and fostering healing relationships. The second oldest age group (ORs=1.37-1.61, 65-74 years) was more likely to have optimal PCC than the oldest ( ≥ 75 years) in making decisions and enabling self-management domains ( ). Females were more likely to have optimal PCC in exchanging information, enabling self-management, and fostering healing relationship compared to males. Hispanic cancer survivors were approximately 2 times as likely to have optimal PCC compared with Whites in exchanging information and enabling self-management (ORs=1.71-1.89). Compared to those with the lowest income (<$20,000), cancer survivors in the middle -income group ($50,000 to <$75,000) were less likely to have optimal PCC in the responding to emotions, fostering healing relationship, and managing uncertainty domains (ORs=0.51-0.61). Individuals diagnosed with cancer more recently (2-5 years ago) had a higher odds of having optimal PCC (ORs=1.51-1.53) in exchanging information and enabling self-management than those diagnosed 11 years ago. In the linear regression models considering overall PCC score, most associations were similar to optimal PCC, with the exception that the COVID-19 time period was not significantly related to the overall PCC score (Supplemental ). Sensitivity analysis revealed that the associations remained the same for most sociodemographic and health status factors, except for gender. Gender was not associated with PCC outcomes when ‘always/usually’ were treated as optimal PCC.
Cancer survivor characteristics In HINTS 5 2017-2020, there were 2,579 cancer survivors, 75% before (n=1,953) and 25% during COVID-19 (n=626) time periods ( ). About half (51%) were older adults ( ≥ 65 years), non-Hispanic Whites were the majority (80%), 66% had some college education or more, more than half (53%) reported $50,000 or more income, 57% had public/government-supported insurance, 84% had a regular provider, and 75% rated their health status as excellent/good. High blood pressure (54%) was the most common co-morbid chronic condition, followed by diabetes (24%) and depression (23%). Nearly one in three cancer survivors reported depression or anxiety symptoms in the past 2 weeks (33%). Almost half have been cancer survivors for more than 11 years (47%). There were no significant differences in population characteristics of cancer survivors between before and during COVID-19 (Supplemental ).
Prevalence of optimal PCC: before vs. during COVID-19 describes the prevalence of optimal PCC before and during COVID-19 by 6 PCC domains and overall. The prevalence of optimal PCC decreased during COVID-19 in most domains, except for exchanging information. The largest decrease of 7.3% was observed for managing uncertainty. In both periods, exchanging information was the domain with the highest prevalence of optimal PCC (64.5% before and 70% during COVID-19) while managing uncertainty was the domain with the lowest prevalence of optimal PCC (47.4% before and 40.1% during COVID-19). However, none of these differences were statistically significant. shows the mean PCC by sociodemographic and health status factors over the entire study period and before and during COVID-19 ( higher score refers to better PCC). The PCC mean score significantly differed in some sociodemographic subgroups by time. From before to during COVID-19, the PCC mean score increased in non-Hispanic Black/African Americans and decreased in those in the middle-income bracket ($50,000 to < $75,000).
Impact of COVID-19 We did not observe interactions between COVID-19 time period and sociodemographic or health status factors with overall PCC score. Thus, associations of sociodemographic and health status factors with optimal PCC before and during COVID-19 were combined in and Supplemental .
Factors associated with optimal PCC in each domain and overall Compared with the time before COVID-19, cancer survivors during COVID-19 were less likely to have optimal PCC overall (OR=0.73, 95% CI 0.54-0.98) and in the domain of managing uncertainty (OR=0.74, 0.55-0.99) ( ). However, other PCC functions did not differ between the two time periods . Cancer survivors who had a usual source of care were 1.5-2 times as likely to have optimal PCC than those without it overall (OR=1.53, 1.04-2.25) and in all domains (ORs=1.64-2.29), except for managing uncertainty . Similarly, cancer survivors who had no depression or anxiety symptoms had 1.62-2.17 times the odds of having optimal PCC overall and each domain, compared with those with anxiety or depression symptoms. Cancer survivors with excellent/good general health had 1.40-1.66 times the odds of having optimal PCC overall and in the domains of responding to emotion, making decisions, enabling self-management, and fostering healing relationships. The second oldest age group (ORs=1.37-1.61, 65-74 years) was more likely to have optimal PCC than the oldest ( ≥ 75 years) in making decisions and enabling self-management domains ( ). Females were more likely to have optimal PCC in exchanging information, enabling self-management, and fostering healing relationship compared to males. Hispanic cancer survivors were approximately 2 times as likely to have optimal PCC compared with Whites in exchanging information and enabling self-management (ORs=1.71-1.89). Compared to those with the lowest income (<$20,000), cancer survivors in the middle -income group ($50,000 to <$75,000) were less likely to have optimal PCC in the responding to emotions, fostering healing relationship, and managing uncertainty domains (ORs=0.51-0.61). Individuals diagnosed with cancer more recently (2-5 years ago) had a higher odds of having optimal PCC (ORs=1.51-1.53) in exchanging information and enabling self-management than those diagnosed 11 years ago. In the linear regression models considering overall PCC score, most associations were similar to optimal PCC, with the exception that the COVID-19 time period was not significantly related to the overall PCC score (Supplemental ). Sensitivity analysis revealed that the associations remained the same for most sociodemographic and health status factors, except for gender. Gender was not associated with PCC outcomes when ‘always/usually’ were treated as optimal PCC.
Discussion and conclusions 4.1 Discussion We found that cancer survivors were less likely to have optimal PCC overall and in the managing uncertainty domain during the early COVID-19 period compared to before COVID-19, using nationally representative survey data. We identified sociodemographic and health status factors associated with optimal PCC among cancer survivors in recent years, including during the initial COVID-19 pandemic. Cancer survivors least likely to have optimal PCC in most domains were those without a usual source of care, with depression or anxiety symptoms or poor general health status. Additionally, older, male, non-Hispanic White and middle-income cancer survivors were less likely to have optimal PCC in some PCC domains. More efforts need to focus on improving PCC among cancer survivors, particularly those identified in this study. Multifaceted approaches may be required to enhance the perception of PCC through patient education and clinician training. We observed that the overall optimal PCC prevalence was lower (6.3% lower) during COVID-19 compared to before the pandemic among cancer survivors, particularly for responding to emotions (3.6% lower) and managing uncertainty (7.3% lower) domains , yet this unadjusted prevalence did not significantly differ. However, in the fully adjusted model , cancer survivors during COVID-19 were less likely to have optimal PCC overall and in managing uncertainty than cancer survivors before pandemic. Substantially limited interactions with providers (actual restriction) and pervasive social distancing policies (perceived distance from providers) might have prevented them from having quality communications for managing uncertainty despite their elevated fear and uncertainties. Before COVID-19, several efforts to enhance the quality of PCC have been put into the practice , including educational PCC training for healthcare providers (e.g., family physician residents, nursing students) , and attempts to improve PCC assessment tools (e.g., standardization and validation of PCC check list, engagement of patient advocate s to improve PCC design and content) , . However, systematic PCC practice guidelines and evaluations for clinicians , and consistent and broadly available education for patients are still lacking. Despite those previous efforts, the prevalence of optimal PCC and some domains have decreased over time, even lower than estimated from a study during 2008-2013 , highlighting the need to focus more attention and resources on promoting PCC. We observed that having depression or anxiety symptoms or poor general health status were consistently associated with sub-optimal PCC in most PCC domains among cancer survivors, which aligns with previous reports , , . While PCC is ideal at all times, under the situations like COVID-19 pandemic, when individuals with compromised health conditions, including cancer patients, experienced additional fear due to COVID-19 , the PCC’s role is crucial as a channel to address those uncertainties and receive necessary care and support . It is possible that those with poor health status were less likely to be engaged in the communications with providers (e.g., disinterested or unable to), and the providers were also less likely to be patient-centered for those less attentive during the communication , . Our findings highlight the importance of preparing targeted approaches for those with poor physical or mental health to improve PCC, which has been found to be positively related to better health-related outcomes, including disease outcomes, quality of life, and mental health , , . In our study, those without a usual source of care were less likely to have optimal PCC in most domains, as found previously . This finding may relate to consistent medical encounters enabling quality patient-physician relationships and positively impacting optimal PCC , . Previously, cancer survivors with low-income were less likely to have optimal PCC, and had a higher rate of discontinuation of treatment or disease care , , , which may relate to inconsistent or less frequent office visits due to financial barriers. However, in our study, those in the middle-income bracket ($50,000 to <$75,000) had a lower likelihood of having optimal PCC, compared with those with the lowest income in the emotion, relationship, and uncertainty domains. We also observed that the overall PCC score significantly worsened in this middle-income bracket during COVID-19 compared to before the pandemic. Further investigations, including qualitative approaches, are warranted to understand the underlying dynamics in this observation. It is notable that 16% of cancer survivors did not have a usual source of care and 19% were middle income in our study. Cancer survivors 65-74 years-old had a higher likelihood of optimal PCC than those 75 years of age and older in the enabling self-management and making decisions domains. The 75+ group may prefer to have strong control in care, asking direct questions, refusing some treatment options, or valuing 'being understood' during the communication , . Demanding more quality in care among the oldest could potentially contribute to less satisfactory PCC in these patient involvement related domains . More than half of cancer survivors (53%) were age 70 or older in the U.S. in 2022, and it is projected to be growing . Thus, our findings indicate that more resources will need to be put into the oldest group to support them to achieve optimal PCC. Perhaps, national efforts for healthy aging could potentially incorporate opportunities to inform and educate older adults to improve PCC , . Male cancer survivors were consistently less likely to have optimal PCC than females in most domains. It aligns with the previous literature, which reported that male cancer survivors experienced sub-optimal PCC in managing uncertainty . This may reflect gender differences in communicational styles, as women are more likely to share their issues or concerns with providers than men , . Typically, care providers can be more informative and supportive when they better understand patients’ issues . Given the gender gap in optimal PCC widened in recent years, further investigations to understand the underlying reasons for PCC differences are warranted. 4.2 Practice implications To improve PCC among the vulnerable cancer survivors identified in this study, educational programs and guidelines/ policies for both healthcare providers and patients are suggested to raise awareness of PCC roles for both groups and guide them to practice PCC in clinical settings , , . For example, early-stage trainings could be offered to health professionals on performing PCC and identifying vulnerable subgroups, particularly those with poor general health or mental health symptoms , , . Moreover, patient advocate groups for the older or male cancer survivors could play roles in tailored patient education. Also, healthcare providers are usually more responsive to the patients who ask questions and share concerns, like other social interactions . Additionally, there is evidence that racial/ethnic provider-patient concordance could facilitate positive interactions and relationships . Last, exploring opportunities to enhance optimal PCC through online platforms (e.g., communications using Electronic Health Record to increase patients engagement) are timely with the widespread use of digital devices , . Online platforms could reach broad populations, including those without a usual source of care. Furthermore, given the rapid adoption and wide dissemination of telehealth during the pandemic, efforts may need to focus on engaging clinicians with PCC in telehealth services , , . 4.3 Limitations Some limitations need to be acknowledged. First, the present study used self-report ed survey data. Although the HINTS is a nationally representative, high-quality dataset, there is the possibility of reporting bias (e.g., some PCC responses could be reported subjectively, including the ‘spending enough time with you’ question because the same amount of time could be enough for some and not for others). Second, the possibility of selection bias needs to be acknowledged due to low overall response rate (33%, 2017-2020). Third, because the data are cross-sectional, we were not able to determine the prospective and longitudinal associations with optimal PCC . Fourth, the COVID-19 data were collected from February to June 2020, during the early COVID-19 pandemic. Hence, the findings should be interpreted in the early COVID-19 pandemic context, and the findings may differ in later or post COVID-19 periods. Despite the limitations, this study has strengths, including the comprehensive investigations of the prevalence and associations by sociodemographic and health status factors with the optimal PCC by domains as well as the overall PCC with recent data, including in the context of the COVID-19 pandemic on a population level. This information contributes to our knowledge base of the PCC performance of vulnerable populations with chronic conditions, like cancer, during COVID-19. 4.4 Conclusions Our findings highlight that cancer survivors without a usual source of care, with depression or anxiety symptoms or with poor general health status, or those who were older, males, non-Hispanic Whites, or had middle-income require additional support to achieve optimal PCC during the extended COVID-19 pandemic. Raising awareness of PCC roles among both providers and cancer survivors and guiding them to practice it are suggested strategies to improve PCC. The knowledge generated by this study informs related stakeholders, including healthcare professionals, public health professionals and policymakers, of the subgroups of cancer survivors to target with approaches to improve PCC performance and potentially prevent further disparities in health outcomes in these vulnerable populations.
Discussion We found that cancer survivors were less likely to have optimal PCC overall and in the managing uncertainty domain during the early COVID-19 period compared to before COVID-19, using nationally representative survey data. We identified sociodemographic and health status factors associated with optimal PCC among cancer survivors in recent years, including during the initial COVID-19 pandemic. Cancer survivors least likely to have optimal PCC in most domains were those without a usual source of care, with depression or anxiety symptoms or poor general health status. Additionally, older, male, non-Hispanic White and middle-income cancer survivors were less likely to have optimal PCC in some PCC domains. More efforts need to focus on improving PCC among cancer survivors, particularly those identified in this study. Multifaceted approaches may be required to enhance the perception of PCC through patient education and clinician training. We observed that the overall optimal PCC prevalence was lower (6.3% lower) during COVID-19 compared to before the pandemic among cancer survivors, particularly for responding to emotions (3.6% lower) and managing uncertainty (7.3% lower) domains , yet this unadjusted prevalence did not significantly differ. However, in the fully adjusted model , cancer survivors during COVID-19 were less likely to have optimal PCC overall and in managing uncertainty than cancer survivors before pandemic. Substantially limited interactions with providers (actual restriction) and pervasive social distancing policies (perceived distance from providers) might have prevented them from having quality communications for managing uncertainty despite their elevated fear and uncertainties. Before COVID-19, several efforts to enhance the quality of PCC have been put into the practice , including educational PCC training for healthcare providers (e.g., family physician residents, nursing students) , and attempts to improve PCC assessment tools (e.g., standardization and validation of PCC check list, engagement of patient advocate s to improve PCC design and content) , . However, systematic PCC practice guidelines and evaluations for clinicians , and consistent and broadly available education for patients are still lacking. Despite those previous efforts, the prevalence of optimal PCC and some domains have decreased over time, even lower than estimated from a study during 2008-2013 , highlighting the need to focus more attention and resources on promoting PCC. We observed that having depression or anxiety symptoms or poor general health status were consistently associated with sub-optimal PCC in most PCC domains among cancer survivors, which aligns with previous reports , , . While PCC is ideal at all times, under the situations like COVID-19 pandemic, when individuals with compromised health conditions, including cancer patients, experienced additional fear due to COVID-19 , the PCC’s role is crucial as a channel to address those uncertainties and receive necessary care and support . It is possible that those with poor health status were less likely to be engaged in the communications with providers (e.g., disinterested or unable to), and the providers were also less likely to be patient-centered for those less attentive during the communication , . Our findings highlight the importance of preparing targeted approaches for those with poor physical or mental health to improve PCC, which has been found to be positively related to better health-related outcomes, including disease outcomes, quality of life, and mental health , , . In our study, those without a usual source of care were less likely to have optimal PCC in most domains, as found previously . This finding may relate to consistent medical encounters enabling quality patient-physician relationships and positively impacting optimal PCC , . Previously, cancer survivors with low-income were less likely to have optimal PCC, and had a higher rate of discontinuation of treatment or disease care , , , which may relate to inconsistent or less frequent office visits due to financial barriers. However, in our study, those in the middle-income bracket ($50,000 to <$75,000) had a lower likelihood of having optimal PCC, compared with those with the lowest income in the emotion, relationship, and uncertainty domains. We also observed that the overall PCC score significantly worsened in this middle-income bracket during COVID-19 compared to before the pandemic. Further investigations, including qualitative approaches, are warranted to understand the underlying dynamics in this observation. It is notable that 16% of cancer survivors did not have a usual source of care and 19% were middle income in our study. Cancer survivors 65-74 years-old had a higher likelihood of optimal PCC than those 75 years of age and older in the enabling self-management and making decisions domains. The 75+ group may prefer to have strong control in care, asking direct questions, refusing some treatment options, or valuing 'being understood' during the communication , . Demanding more quality in care among the oldest could potentially contribute to less satisfactory PCC in these patient involvement related domains . More than half of cancer survivors (53%) were age 70 or older in the U.S. in 2022, and it is projected to be growing . Thus, our findings indicate that more resources will need to be put into the oldest group to support them to achieve optimal PCC. Perhaps, national efforts for healthy aging could potentially incorporate opportunities to inform and educate older adults to improve PCC , . Male cancer survivors were consistently less likely to have optimal PCC than females in most domains. It aligns with the previous literature, which reported that male cancer survivors experienced sub-optimal PCC in managing uncertainty . This may reflect gender differences in communicational styles, as women are more likely to share their issues or concerns with providers than men , . Typically, care providers can be more informative and supportive when they better understand patients’ issues . Given the gender gap in optimal PCC widened in recent years, further investigations to understand the underlying reasons for PCC differences are warranted.
Practice implications To improve PCC among the vulnerable cancer survivors identified in this study, educational programs and guidelines/ policies for both healthcare providers and patients are suggested to raise awareness of PCC roles for both groups and guide them to practice PCC in clinical settings , , . For example, early-stage trainings could be offered to health professionals on performing PCC and identifying vulnerable subgroups, particularly those with poor general health or mental health symptoms , , . Moreover, patient advocate groups for the older or male cancer survivors could play roles in tailored patient education. Also, healthcare providers are usually more responsive to the patients who ask questions and share concerns, like other social interactions . Additionally, there is evidence that racial/ethnic provider-patient concordance could facilitate positive interactions and relationships . Last, exploring opportunities to enhance optimal PCC through online platforms (e.g., communications using Electronic Health Record to increase patients engagement) are timely with the widespread use of digital devices , . Online platforms could reach broad populations, including those without a usual source of care. Furthermore, given the rapid adoption and wide dissemination of telehealth during the pandemic, efforts may need to focus on engaging clinicians with PCC in telehealth services , , .
Limitations Some limitations need to be acknowledged. First, the present study used self-report ed survey data. Although the HINTS is a nationally representative, high-quality dataset, there is the possibility of reporting bias (e.g., some PCC responses could be reported subjectively, including the ‘spending enough time with you’ question because the same amount of time could be enough for some and not for others). Second, the possibility of selection bias needs to be acknowledged due to low overall response rate (33%, 2017-2020). Third, because the data are cross-sectional, we were not able to determine the prospective and longitudinal associations with optimal PCC . Fourth, the COVID-19 data were collected from February to June 2020, during the early COVID-19 pandemic. Hence, the findings should be interpreted in the early COVID-19 pandemic context, and the findings may differ in later or post COVID-19 periods. Despite the limitations, this study has strengths, including the comprehensive investigations of the prevalence and associations by sociodemographic and health status factors with the optimal PCC by domains as well as the overall PCC with recent data, including in the context of the COVID-19 pandemic on a population level. This information contributes to our knowledge base of the PCC performance of vulnerable populations with chronic conditions, like cancer, during COVID-19.
Conclusions Our findings highlight that cancer survivors without a usual source of care, with depression or anxiety symptoms or with poor general health status, or those who were older, males, non-Hispanic Whites, or had middle-income require additional support to achieve optimal PCC during the extended COVID-19 pandemic. Raising awareness of PCC roles among both providers and cancer survivors and guiding them to practice it are suggested strategies to improve PCC. The knowledge generated by this study informs related stakeholders, including healthcare professionals, public health professionals and policymakers, of the subgroups of cancer survivors to target with approaches to improve PCC performance and potentially prevent further disparities in health outcomes in these vulnerable populations.
This study was a secondary data analysis. Human subject was not involved, and identifiable information was not included. Thus, this was deemed exempt for review by the Institutional Review Board at University of California, Davis
Theresa H. Keegan’s time was supported, in part, by the UC Davis Comprehensive Cancer Center and National Cancer Institute of the National Institutes of Health under award number P30CA093373. Melanie S. Dove was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number UL1 TR001860 and linked award KL2 TR001859. However, these funding sources did not have any roles in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication.
Jiyeong Kim: Conceptualization, Methodology, Formal analysis, Investigation, Writing-original draft, Editing, Reviewing final submission. Nathan P. Fairman: Writing, Editing, Reviewing final submission. Melanie S. Dove: Methodology, Investigation, Writing, Editing, Reviewing final submission. Jeffrey S. Hoch: Writing, Editing, Reviewing final submission. Theresa H. Keegan: Conceptualization, Methodology, Investigation, Writing, Editing, Reviewing final submission.
Jiyeong Kim: Conceptualization of study, Writing, Data analysis, Editing, Reviewing final submission. Nathan P. Fairman: Writing, Editing, Reviewing final submission. Melanie S. Dove: Writing, Data analysis, Editing, Reviewing final submission. Jeffrey S. Hoch: Writing, Editing, Reviewing final submission. Theresa H. Keegan: Conceptualization of study, Writing, Data analysis, Editing, Reviewing final submission.
Keegan Theresa H.: Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Conceptualization. Fairman Nathan P.: Writing – review & editing, Validation. Kim Jiyeong: Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. Hoch Jeffrey S.: Writing – review & editing, Resources. Dove Melanie S.: Writing – review & editing, Methodology, Investigation.
The authors declared no potential conflicts of interest.
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Policy and care in tandem: structuring youth volunteerism for psychological benefits in pediatric palliative care | e2242c88-3310-4c11-8dd5-a3006aa28ec3 | 11907901 | Pediatrics[mh] | Terminally ill children (TIC), particularly those abandoned by their parents and residing in a pediatric palliative care center in southern China, are adversely impacted by delays in self-discovery. This issue is primarily due to enforced geographical isolation in remote urban areas, compounded by social isolation characterized by limited social interaction beyond their caregivers. The government recommends isolation to protect TIC’s health, especially during the COVID-19 and post-pandemic period. A policy recommendation suggests establishing a registration system for all personnel entering and existing, and avoiding unnecessary visits . This measure aims to safeguard their fragile health, as they are more vulnerable to secondary infections. For instance, one such risk is pulmonary tuberculosis, which can be transmitted through airborne droplets from visitors carrying Mycobacterium tuberculosis, posing serious harm to immunosuppressed TICs . However, an unintended consequence of protective isolation is psychological developmental delays, characterized by a limited understanding of themselves and the external world . In palliative care centers, managers and caregivers serve as the legal guardians and often assume parental roles for these children, primarily because many are orphans. They strive to meet the needs of TICs and avoid conflicts, given the children’s limited life expectancy. This situation leads to prevalent hyper-parenting behaviors among the caregiving staff, who, through overprotection and restricted autonomy, unintentionally limit the TIC’s ability to navigate and internalize norms of interpersonal relationships . Consequently, there is a tendency towards self-centeredness, as the focus is predominantly on safeguarding their physical health rather than fostering psychological development . The lack of opportunities for socialization in external environments further exacerbates delays in the self-discovery process, inhibiting the TIC from engaging in enriching interactions like playing with peers and exploring diverse settings . These constrained childhood experiences adversely affect children’s understanding of their identity, including talents, interests, and vision by limiting their exploration of the world . The impact of peer interaction in the self-discovery process Self-discovery, which includes identifying personal interests, character traits, and social roles, plays a crucial role in mental health, particularly during adolescence. This process aids in understanding social interactions and daily functioning, mainly including living habits and recreational activities . It is important for identity formation, a process of understanding one’s self within a social context, where group interactions play a facilitating role . Adolescents typically experience a transition period where their life focus shifts from family members to peers , moving from parent-dependent self-definition to a self-identity integrated within society . Peer interactions are critical in developing social-emotional competence, enabling adolescents to self-regulate and maintain relationships . Moreover, the quality of peer relationships significantly influences this process. Positive peer interactions can enhance mental health and psychological well-being, reducing issues like social anxiety and depression . However, interactions with at-risk peers can negatively affect the TIC’s identity formation . This emphasizes the need for careful selection and training in volunteerism. Volunteerism’s impact on pediatric palliative care Acknowledging the delays in the self-discovery process of TIC compared to their peers highlights their unique developmental challenges. Children raised in institutions often experience developmental delays and psychological deprivation, and positive peer interactions are a viable solution . In this context, China’s Young Pioneer Voluntary Teams (YPVTs) in pediatric palliative care centers emerge as a promising strategy to address these issues. The YPVTs are groups of adolescents who engage in voluntary activities as a form of social practice, which are under the control of the Ministry of Education . YPVTs typically volunteer during weekends and summer and winter holidays, ensuring consistent operation of these programs. Traditional voluntary activities in the health sector include outpatient medical guidance and assistance with self-service registration, with the goal of equipping adolescents with healthcare knowledge. However, these programs often fall short in imparting practical health knowledge, underscoring the additional purpose of volunteer intervention in pediatric palliative care. Moving to the impact on TIC, research by Haski-Leventhal et al. emphasized the essential role of youth volunteerism in addressing social exclusion and discord among TIC, highlighting that, in contrast to the service-oriented approach of adults, youth volunteering tends to be relationship-oriented . Unlike their adult counterparts, teenage volunteers within the YPVTs are less inclined towards excessive coddling, thereby enabling TIC to cultivate interpersonal skills and emotional intelligence through more naturalistic social interactions. Moreover, the proximity in age and interests between YPVTs and TIC facilitates a more authentic understanding and engagement . These volunteers bring diverse experiences, from understanding popular culture to sharing travel stories, enriching TIC’s learning and broadening their worldviews. This facilitates personal identity development and a deeper self-discovery process in TIC. The viewpoints of YPVTs bring to light new challenges, significantly influencing the development of interventions within pediatric care. Further exploring the volunteers’ perspective, YPVTs also experience benefits in terms of psychological well-being. Engaging in voluntary programs allows YPVTs to interact with people from diverse socio-economic backgrounds, which gives them a sense of community and being needed. Moreover, lower possibility of depression and higher life satisfaction and self-esteem is linked with youth volunteering . However, challenges exist in the implementation of YPVTs. Inconsistencies in volunteer selection and training can lead to unpreparedness and behaviors that are not conducive to the sensitive environment required for TIC. Inadequate psychological readiness may result in inappropriate interactions, impacting the TIC’s psychological well-being and hindering their growth. Additionally, volunteers may experience psychological distress, including anxiety, depression, and even post-traumatic stress disorder, when confronted with the suffering or death of the children they assist . This issue is exacerbated by the lack of comprehensive death education in China, emphasizing the need for rigorous selection and training processes . Another concern is the high turnover rate among volunteers, which can disrupt the continuity of care and reduce service quality . Increased investment in the selection and training processes is necessary to enhance the overall performance of organizations, as it directly impacts the quality of care provided . Research has investigated adult volunteer selection and training in palliative care and the effect of youth volunteering, but most of them are fragmented, indicating an absence of research focus on the impact of youth volunteering on TIC and a lack of systematic frameworks for adolescent volunteers working in pediatric palliative care. Studies like Niinomi demonstrate the effectiveness of training programs in enhancing volunteers’ confidence in providing palliative care through a series of five progressive lectures, but these methods are not directly transferable to adolescent volunteers due to differences in age and maturity. Training content for adolescent volunteers should be simpler and more relatable, emphasizing easy-to-grasp concepts in pediatric palliative care. Furthermore, the incorporation of adolescent volunteers’ perspectives, crucial for feedback collection, has been largely overlooked in policy-making . A formal feedback mechanism for regular volunteerism is essential for efficient improvement. Zeanah also highlighted the issue of attachment disruption in orphanage volunteering, where short-term volunteer relationships can negatively impact children’s psychological well-being, leading to poor social behavior and even serious psychiatric disorders. To address this gap, this paper advocates for youth volunteerism as a transformative element of pediatric palliative care. A comprehensive framework aims to achieve mutual psychological development in both YPVTs and TIC. The effectiveness of this framework will be assessed through two distinct psychological questionnaires, tailored respectively to the YPVTs and TIC, to understand the benefits each group derived from the volunteer program and guide future enhancement.
Self-discovery, which includes identifying personal interests, character traits, and social roles, plays a crucial role in mental health, particularly during adolescence. This process aids in understanding social interactions and daily functioning, mainly including living habits and recreational activities . It is important for identity formation, a process of understanding one’s self within a social context, where group interactions play a facilitating role . Adolescents typically experience a transition period where their life focus shifts from family members to peers , moving from parent-dependent self-definition to a self-identity integrated within society . Peer interactions are critical in developing social-emotional competence, enabling adolescents to self-regulate and maintain relationships . Moreover, the quality of peer relationships significantly influences this process. Positive peer interactions can enhance mental health and psychological well-being, reducing issues like social anxiety and depression . However, interactions with at-risk peers can negatively affect the TIC’s identity formation . This emphasizes the need for careful selection and training in volunteerism.
Acknowledging the delays in the self-discovery process of TIC compared to their peers highlights their unique developmental challenges. Children raised in institutions often experience developmental delays and psychological deprivation, and positive peer interactions are a viable solution . In this context, China’s Young Pioneer Voluntary Teams (YPVTs) in pediatric palliative care centers emerge as a promising strategy to address these issues. The YPVTs are groups of adolescents who engage in voluntary activities as a form of social practice, which are under the control of the Ministry of Education . YPVTs typically volunteer during weekends and summer and winter holidays, ensuring consistent operation of these programs. Traditional voluntary activities in the health sector include outpatient medical guidance and assistance with self-service registration, with the goal of equipping adolescents with healthcare knowledge. However, these programs often fall short in imparting practical health knowledge, underscoring the additional purpose of volunteer intervention in pediatric palliative care. Moving to the impact on TIC, research by Haski-Leventhal et al. emphasized the essential role of youth volunteerism in addressing social exclusion and discord among TIC, highlighting that, in contrast to the service-oriented approach of adults, youth volunteering tends to be relationship-oriented . Unlike their adult counterparts, teenage volunteers within the YPVTs are less inclined towards excessive coddling, thereby enabling TIC to cultivate interpersonal skills and emotional intelligence through more naturalistic social interactions. Moreover, the proximity in age and interests between YPVTs and TIC facilitates a more authentic understanding and engagement . These volunteers bring diverse experiences, from understanding popular culture to sharing travel stories, enriching TIC’s learning and broadening their worldviews. This facilitates personal identity development and a deeper self-discovery process in TIC. The viewpoints of YPVTs bring to light new challenges, significantly influencing the development of interventions within pediatric care. Further exploring the volunteers’ perspective, YPVTs also experience benefits in terms of psychological well-being. Engaging in voluntary programs allows YPVTs to interact with people from diverse socio-economic backgrounds, which gives them a sense of community and being needed. Moreover, lower possibility of depression and higher life satisfaction and self-esteem is linked with youth volunteering . However, challenges exist in the implementation of YPVTs. Inconsistencies in volunteer selection and training can lead to unpreparedness and behaviors that are not conducive to the sensitive environment required for TIC. Inadequate psychological readiness may result in inappropriate interactions, impacting the TIC’s psychological well-being and hindering their growth. Additionally, volunteers may experience psychological distress, including anxiety, depression, and even post-traumatic stress disorder, when confronted with the suffering or death of the children they assist . This issue is exacerbated by the lack of comprehensive death education in China, emphasizing the need for rigorous selection and training processes . Another concern is the high turnover rate among volunteers, which can disrupt the continuity of care and reduce service quality . Increased investment in the selection and training processes is necessary to enhance the overall performance of organizations, as it directly impacts the quality of care provided . Research has investigated adult volunteer selection and training in palliative care and the effect of youth volunteering, but most of them are fragmented, indicating an absence of research focus on the impact of youth volunteering on TIC and a lack of systematic frameworks for adolescent volunteers working in pediatric palliative care. Studies like Niinomi demonstrate the effectiveness of training programs in enhancing volunteers’ confidence in providing palliative care through a series of five progressive lectures, but these methods are not directly transferable to adolescent volunteers due to differences in age and maturity. Training content for adolescent volunteers should be simpler and more relatable, emphasizing easy-to-grasp concepts in pediatric palliative care. Furthermore, the incorporation of adolescent volunteers’ perspectives, crucial for feedback collection, has been largely overlooked in policy-making . A formal feedback mechanism for regular volunteerism is essential for efficient improvement. Zeanah also highlighted the issue of attachment disruption in orphanage volunteering, where short-term volunteer relationships can negatively impact children’s psychological well-being, leading to poor social behavior and even serious psychiatric disorders. To address this gap, this paper advocates for youth volunteerism as a transformative element of pediatric palliative care. A comprehensive framework aims to achieve mutual psychological development in both YPVTs and TIC. The effectiveness of this framework will be assessed through two distinct psychological questionnaires, tailored respectively to the YPVTs and TIC, to understand the benefits each group derived from the volunteer program and guide future enhancement.
A four-step framework to improve volunteerism in pediatric palliative care To address identified deficiencies and optimize the contributions of YPVTs, a four-step framework is proposed. It is grounded in three important virtues: psychological resilience, respectful demeanour, and a proclivity for enthusiastic sharing. Firstly, psychological resilience is crucial as it promotes mutual psychological well-being among TIC and YPVTs, allowing volunteers to navigate the complexities of rare diseases without undue distress . This resilience is vital in maintaining a supportive presence and safeguarding TIC from potential negative reactions. Secondly, a respectful attitude is imperative for building a foundation of mutual trust. Physical demeanor, such as maintaining eye contact, is an essential expression of this respect, which contributes to creating an environment conducive to equitable communication . Lastly, the embodiment of sharing enthusiasm is also important to facilitate interaction between TIC and YPVTs and further encourage TIC’s self-discovery process. The volunteers are required to show a willingness to openly share their interests, talents, and experiences, cultivating a rich exchange of perspectives. This enthusiastic sharing fosters a reciprocal interaction, encouraging TIC in their journey of self-discovery while also allowing volunteers to glean deeper insights into the lived experiences of the TIC. This framework (Fig. ) includes meticulous selection processes, comprehensive training program, and dedicated follow-up procedures. Each stage is designed to reinforce these core qualities, ensuring that volunteers are well-prepared. The goal is to minimize the risks of adverse impacts and establish a platform that facilitates the recognition and constructive utilization of the YPVT’s insights.
To address identified deficiencies and optimize the contributions of YPVTs, a four-step framework is proposed. It is grounded in three important virtues: psychological resilience, respectful demeanour, and a proclivity for enthusiastic sharing. Firstly, psychological resilience is crucial as it promotes mutual psychological well-being among TIC and YPVTs, allowing volunteers to navigate the complexities of rare diseases without undue distress . This resilience is vital in maintaining a supportive presence and safeguarding TIC from potential negative reactions. Secondly, a respectful attitude is imperative for building a foundation of mutual trust. Physical demeanor, such as maintaining eye contact, is an essential expression of this respect, which contributes to creating an environment conducive to equitable communication . Lastly, the embodiment of sharing enthusiasm is also important to facilitate interaction between TIC and YPVTs and further encourage TIC’s self-discovery process. The volunteers are required to show a willingness to openly share their interests, talents, and experiences, cultivating a rich exchange of perspectives. This enthusiastic sharing fosters a reciprocal interaction, encouraging TIC in their journey of self-discovery while also allowing volunteers to glean deeper insights into the lived experiences of the TIC. This framework (Fig. ) includes meticulous selection processes, comprehensive training program, and dedicated follow-up procedures. Each stage is designed to reinforce these core qualities, ensuring that volunteers are well-prepared. The goal is to minimize the risks of adverse impacts and establish a platform that facilitates the recognition and constructive utilization of the YPVT’s insights.
Selection process The selection process for YPVTs consists of two stages: a questionnaire and an interview. Initially, candidates complete a series of closed-ended questions covering personal background, interests, and talents, providing program managers with preliminary insights. Further, the process mainly evaluates volunteers’ qualities of psychological resilience and a proclivity for enthusiastic sharing by incorporating the General Self-Efficacy Scale (GSES) (Fig. ) and the Motivation to Volunteer Scale (MTVS) (Fig. ), both pivotal in assessing crucial traits for volunteering. The GSES is a 10-item questionnaire using a four-point Likert scale to measure self-efficacy, reflecting a volunteer’s commitment to caring for TIC and their ability to effectively navigate challenges while volunteering . Each item is rated as “Not at all true,” “Hardly true,” “Moderately true,” and “Exactly true.” “Not at all true” indicates no confidence in managing the described situation or task. “Hardly true” suggests minimal confidence, with occasional capability under limited conditions. “Moderately true” reflects a moderate belief in handling challenges, indicating capability in many situations, though not consistently. “Exactly true” signifies strong confidence, showing the ability to effectively dealing with challenges and tasks in most situations. Higher GSES scores indicate commendable psychological resilience among YPVTs. Considering the nature of volunteerism, special attention should be paid to voluntary motivation. The MTVS uses the same scoring method to assess the candidates’ voluntary commitment to the program . It categorizes motivation into voluntary and involuntary, reflecting their enthusiasm when meeting TIC. An evaluative approach will be applied to the MTVS by computing the average scores for both voluntary (e.g., “Volunteering makes me feel good about myself”) and involuntary motivation (e.g., “They told me to volunteer in the school”), which is to assess YPVTs' underlying motivation. Those who are self-motivated will be preferred. To achieve the accurate assessment, the GSES and MTVS must be precisely translated and culturally adapted for Chinese youth volunteers. This process involves multiple steps to guarantee accuracy and cultural appropriateness. The translation team is composed of at least two independent subgroups, including native speakers of both English and Chinese with bicultural backgrounds. This diversity allows culturally specific terms, such as “self-efficacy,” to be appropriately translated and adapted to the Chinese cultural context . Team members experienced with the target population help align terminology with the group’s understanding and needs. Professional child psychologists play a key role in refining the translations for clarity and comprehensibility. The independent teams then compare their translations and reach a consensus, followed by a separate team’s back-translation into the original language. Finally, pilot testing with a small sample identifies and resolves any ambiguities or misunderstandings, ensuring the final versions of the scales are accurate and contextually appropriate for the intended population . With the translation process confirming cultural relevance and clarity, the refined scales are now poised for effective deployment in evaluating candidates. When considering the two scales comprehensively, criteria for identifying “relatively lower scores” on the GSES will be established through using normative data and statistical benchmarks derived from the pilot tests. Previous research with a sample size of 9,578 students in China indicates a mean of 28.75, which is used as a benchmark to measure the self-efficacy level for the YPVTs . Scores below 28.74 may be classified as having a relatively lower self-efficacy level. However, candidates with strong motivation on the MTVS will still be eligible for admission, provided they undergo additional training to build their self-efficacy and better equip them to handle challenges effectively during their volunteer service. The scores from scales will not be the sole determinants for the eligibility of YPVTs to participate in volunteering, unless combining the results from further interviews. The interview phase builds upon the insights gained from these scales. During this stage, evaluators pay attention to how candidates respond and their level of enthusiasm, which are key indicators of their genuineness and suitability. Given that the candidates are adolescents, a trait-based selection method is preferred to minimize stress and potential subjectivity . The use of the GSES and MTVS in this phase ensures a standardized and objective approach to scoring the candidates’ performance. In addition to standardized scales, the selection process incorporates scenario-based interviews. These interviews utilize real-life cases from pediatric palliative care centers, featuring virtual avatars and narrative explanations to create realistic scenarios. The goal is to elicit natural responses from candidates, allowing them to demonstrate their problem-solving abilities in practical situations . Candidates who feel uncomfortable during the interview have the right to withdraw at any time. The materials used in these interviews will be reviewed by volunteer managers and child psychologists to mitigate potential psychological risks, underscoring commitment to safeguarding the mental well-being of all participants. Candidates who exhibit psychological resilience, intrinsic self-motivation, and adeptness in addressing scenario-based interviews will be prioritized for entry into the training process. Conversely, those with transmittable diseases, a history of misconduct, or insufficient time commitment will be excluded from the program. These exclusion criteria are designed to protect the psychological and physical health of TIC and to enhance the overall quality of the volunteer program. This rigorous selection acts as an effective way to protect both TIC’s and YPVTs’ physical and psychological health during the program. Training Upon the conscientious selection, a comprehensive training session is imperative to aptly equip YPVTs with the requisite competencies, especially the three qualities mentioned above, to navigate the complexities of their volunteering roles effectively. This training initiates with an orientation, aiming to imbue the YPVTs with a deeper understanding of the program’s objectives . It’s essential that YPVTs are furnished with foundational knowledge of palliative care paradigms, coupled with an insight into the specificities governing the experiences of the TIC, such as their daily routines and prevalent symptomatic manifestations. This pedagogical approach seeks to promote an atmosphere of familiarity and contextual sensitivity, enabling the YPVTs to better resonate with the challenges faced by the TIC as well as a psychological preparation for themselves . Elaboration on the responsibilities of YPVTs also constitutes a pivotal aspect of the training, ensuring alignment with the program’s ethos and objectives. The training incorporates lectures, workshops and exercises to foster competency and preparedness among the YPVTs. Key topics including “Cultivating Empathetic Perspectives” and “Nurturing Care in Interpersonal Engagements” will be emphasized. These topics are designed to reinforce the three main qualities mentioned in the selection section, and help YPVT to empathize effectively with TIOs . Feedback collection To further refine the program, a sophisticated feedback collection and evaluation framework is proposed, envisioning a synergistic collaboration between various pediatric palliative care centers. Throughout their volunteer engagements, YPVTs acquire invaluable insights into the obscured challenges and needs represented within the palliative environments through observation and communication . Moreover, the personal feelings of YPVTs should be collected to ensure they are not stressful after the interaction with TIC, protecting their psychological well-being . Feedback, reflective of these insights, will be systematically harvested through a variety of methods, including questionnaires, post-engagement interviews and discussions. Such feedback endeavours to drive internal enhancements, tailored specifically to improve the program and daily care provision. To uphold the integrity and authenticity of the feedback, the preliminary collection process will be led by professional’s adept in child psychology. This approach is strategic in circumventing potential biases or power imbalances inherent in adult-centric interpretations, as it acknowledges the propensity for adults to unconsciously position teenagers as their subordinates . Naturalistic settings characterized by peer companionship, where teenagers feel emboldened to express their perspectives, is also important, aiming to foster an environment conducive to open expression and reflection. Utilizing a holistic evaluative lens, results collated from group discussions and interviews will be synergistically analyzed alongside field observations, ensuring a consideration of all pertinent insights . Ultimately, this feedback and evaluation framework is instrumental in driving continuous refinement within this program. Effectiveness evaluation To assess the impact of the four-step framework on the psychological well-being of YPVTs and TIC, three different scales will be administered post-volunteering. For TIC, evaluations will be conducted by volunteer team managers in pediatric palliative care centers with caregiver assistance. The Identity Scale for Adolescents, suitable for individuals aged 13–18, will be employed. This 39-items self-report questionnaire uses a 4-point Likert rating scale 0 (never), 1 (rarely), 2 (sometimes) and 3 (often) to categorize adolescents into three personality types: positive, negative, and arrogant self-identity . It measures identity formation, with an expectation of a shift towards a positive self-identity, characterized by sociability and optimism, after interactions with YPVTs. Negative identity shows a lack of confidence and social skills and arrogant identity indicates egoistic and feeling superior which potentially results from hyper-parenting behaviors. In evaluating YPVTs, the GSES and MTVS will be reapplied to develop deeper into their motivations and the changes they experience through the program. This approach aims to assess personal traits such as dedication and professionalism among the youth volunteers, ensuring alignment with the framework’s objectives. By comparing the results obtained during the selection process with those gathered post-program, any increase in scores will indicate an improvement in self-efficacy, a desirable outcome of the volunteer experience. In addition, it is anticipated that responses on the MTVS will shift towards affirmations like “Volunteering makes me feel good about myself” and “It will help me in the future,” reflecting a positive transformation in their perception of volunteer work. Opposite responses would indicate the potential reasons for dropping out of the voluntary program, where more attention should be paid to retaining the youth volunteers. These two scales can be used to regularly measure and track the volunteers' self-efficacy and motivation. This ensures they are engaged and find meaning in their work, while also helping to identify suitable roles and responsibilities for future adaptation. For instance, volunteers who resonate with the statement “I can handle whatever comes my way” and score high in feeling good about themselves through volunteering could be encouraged to take on leadership roles within the volunteer group. They can also provide immediate feedback on the impact of their work. Moreover, this approach helps identify when volunteers might need additional support or a change in role to stay motivated. Such a strategy not only aids in their self-discovery process but also contributes to fostering a culture of continuous improvement within the volunteer program. In practical terms, these self-report scales will be utilized for self-evaluation by the young volunteers. They will rate their agreement on a Likert scale, ranging from 1 (“Not at all true”) to 4 (“Exactly true”), under condition of confidentiality to encourage honest responses. Post-volunteering, these scales will be re-administered to assess any changes in self-efficacy and motivations. This approach provides valuable insights into the program’s impact and the volunteers’ development. The longitudinal data obtained will help in understanding the volunteers’ personal traits, their dedication, and professionalism. This information is significant for informing the refinement of future volunteer training and the overall development of the program. Limitations One of the challenges in youth volunteerism is the high turnover rate. The first step to controlling the turnover rate is to improve the recruiting process to gain more qualified candidates since recruitment and retention are two interrelated processes . Moreover, peer groups positively strengthen the relationships within the volunteering groups, improve their commitment to the task, and help control the turnover rate . Additionally, the volunteers in palliative care centers, who bear the sadness of witnessing their clients suffering, tend to be overstressed psychologically, being a major reason for withdrawing from the volunteering program . Timely counselling and taking time off are common ways to release stress, indicating that professional psychological support and a proper volunteering schedule may help volunteers maintain a good status and retain them . In the feedback collection section, YPVTs should be interviewed about their motivation to join and reasons for leaving the program, which can comprehensively improve the framework from both TIC’s and YPVTs’ perspectives, striving for a sustainable program . There might be some potential flaws or weaknesses in the framework worth noting. In the selection section, the threshold for determining YPVTs’ eligibility to enroll in the program is unclear due to the limited data available. A significant issue in the framework evaluation section is the self-assessment bias inherent in these tools. Since all motivation items and statements are self-reported, responses could be influenced by the individual’s current mood or self-perception. This might lead to an inaccurate portrayal of one’s true capabilities or motivations. Furthermore, complex personal traits such as dedication and professionalism may not be fully captured through self-reporting, as such qualities are often better demonstrated through actions rather than introspection. Moreover, governmental regulations pose challenges in data collection from local palliative care centers in collaboration with welfare institutions, primarily due to concerns over data and privacy protection. To address these limitations, the questionnaire scores should be collected in the pilot experiments to determine a more reliable threshold by calculating the range of scores of youth volunteers who withdraw from the program. The incorporation of performance-based assessment with multiple raters is planned. This approach will include assessments from peers, supervisors, or community members, providing a more objective measure of the volunteer's traits and performance. Additionally, the process of synergistically analyzing feedback from various sources, including group discussions, interviews, and observations, though complex, is essential. Ensuring accurate reflection of all viewpoints in this data synthesis is significant for a comprehensive evaluation. Combining both qualitative feedback collection and quantitative methods, such as graded scales, offers a more balanced approach to evaluation.
The selection process for YPVTs consists of two stages: a questionnaire and an interview. Initially, candidates complete a series of closed-ended questions covering personal background, interests, and talents, providing program managers with preliminary insights. Further, the process mainly evaluates volunteers’ qualities of psychological resilience and a proclivity for enthusiastic sharing by incorporating the General Self-Efficacy Scale (GSES) (Fig. ) and the Motivation to Volunteer Scale (MTVS) (Fig. ), both pivotal in assessing crucial traits for volunteering. The GSES is a 10-item questionnaire using a four-point Likert scale to measure self-efficacy, reflecting a volunteer’s commitment to caring for TIC and their ability to effectively navigate challenges while volunteering . Each item is rated as “Not at all true,” “Hardly true,” “Moderately true,” and “Exactly true.” “Not at all true” indicates no confidence in managing the described situation or task. “Hardly true” suggests minimal confidence, with occasional capability under limited conditions. “Moderately true” reflects a moderate belief in handling challenges, indicating capability in many situations, though not consistently. “Exactly true” signifies strong confidence, showing the ability to effectively dealing with challenges and tasks in most situations. Higher GSES scores indicate commendable psychological resilience among YPVTs. Considering the nature of volunteerism, special attention should be paid to voluntary motivation. The MTVS uses the same scoring method to assess the candidates’ voluntary commitment to the program . It categorizes motivation into voluntary and involuntary, reflecting their enthusiasm when meeting TIC. An evaluative approach will be applied to the MTVS by computing the average scores for both voluntary (e.g., “Volunteering makes me feel good about myself”) and involuntary motivation (e.g., “They told me to volunteer in the school”), which is to assess YPVTs' underlying motivation. Those who are self-motivated will be preferred. To achieve the accurate assessment, the GSES and MTVS must be precisely translated and culturally adapted for Chinese youth volunteers. This process involves multiple steps to guarantee accuracy and cultural appropriateness. The translation team is composed of at least two independent subgroups, including native speakers of both English and Chinese with bicultural backgrounds. This diversity allows culturally specific terms, such as “self-efficacy,” to be appropriately translated and adapted to the Chinese cultural context . Team members experienced with the target population help align terminology with the group’s understanding and needs. Professional child psychologists play a key role in refining the translations for clarity and comprehensibility. The independent teams then compare their translations and reach a consensus, followed by a separate team’s back-translation into the original language. Finally, pilot testing with a small sample identifies and resolves any ambiguities or misunderstandings, ensuring the final versions of the scales are accurate and contextually appropriate for the intended population . With the translation process confirming cultural relevance and clarity, the refined scales are now poised for effective deployment in evaluating candidates. When considering the two scales comprehensively, criteria for identifying “relatively lower scores” on the GSES will be established through using normative data and statistical benchmarks derived from the pilot tests. Previous research with a sample size of 9,578 students in China indicates a mean of 28.75, which is used as a benchmark to measure the self-efficacy level for the YPVTs . Scores below 28.74 may be classified as having a relatively lower self-efficacy level. However, candidates with strong motivation on the MTVS will still be eligible for admission, provided they undergo additional training to build their self-efficacy and better equip them to handle challenges effectively during their volunteer service. The scores from scales will not be the sole determinants for the eligibility of YPVTs to participate in volunteering, unless combining the results from further interviews. The interview phase builds upon the insights gained from these scales. During this stage, evaluators pay attention to how candidates respond and their level of enthusiasm, which are key indicators of their genuineness and suitability. Given that the candidates are adolescents, a trait-based selection method is preferred to minimize stress and potential subjectivity . The use of the GSES and MTVS in this phase ensures a standardized and objective approach to scoring the candidates’ performance. In addition to standardized scales, the selection process incorporates scenario-based interviews. These interviews utilize real-life cases from pediatric palliative care centers, featuring virtual avatars and narrative explanations to create realistic scenarios. The goal is to elicit natural responses from candidates, allowing them to demonstrate their problem-solving abilities in practical situations . Candidates who feel uncomfortable during the interview have the right to withdraw at any time. The materials used in these interviews will be reviewed by volunteer managers and child psychologists to mitigate potential psychological risks, underscoring commitment to safeguarding the mental well-being of all participants. Candidates who exhibit psychological resilience, intrinsic self-motivation, and adeptness in addressing scenario-based interviews will be prioritized for entry into the training process. Conversely, those with transmittable diseases, a history of misconduct, or insufficient time commitment will be excluded from the program. These exclusion criteria are designed to protect the psychological and physical health of TIC and to enhance the overall quality of the volunteer program. This rigorous selection acts as an effective way to protect both TIC’s and YPVTs’ physical and psychological health during the program.
Upon the conscientious selection, a comprehensive training session is imperative to aptly equip YPVTs with the requisite competencies, especially the three qualities mentioned above, to navigate the complexities of their volunteering roles effectively. This training initiates with an orientation, aiming to imbue the YPVTs with a deeper understanding of the program’s objectives . It’s essential that YPVTs are furnished with foundational knowledge of palliative care paradigms, coupled with an insight into the specificities governing the experiences of the TIC, such as their daily routines and prevalent symptomatic manifestations. This pedagogical approach seeks to promote an atmosphere of familiarity and contextual sensitivity, enabling the YPVTs to better resonate with the challenges faced by the TIC as well as a psychological preparation for themselves . Elaboration on the responsibilities of YPVTs also constitutes a pivotal aspect of the training, ensuring alignment with the program’s ethos and objectives. The training incorporates lectures, workshops and exercises to foster competency and preparedness among the YPVTs. Key topics including “Cultivating Empathetic Perspectives” and “Nurturing Care in Interpersonal Engagements” will be emphasized. These topics are designed to reinforce the three main qualities mentioned in the selection section, and help YPVT to empathize effectively with TIOs .
To further refine the program, a sophisticated feedback collection and evaluation framework is proposed, envisioning a synergistic collaboration between various pediatric palliative care centers. Throughout their volunteer engagements, YPVTs acquire invaluable insights into the obscured challenges and needs represented within the palliative environments through observation and communication . Moreover, the personal feelings of YPVTs should be collected to ensure they are not stressful after the interaction with TIC, protecting their psychological well-being . Feedback, reflective of these insights, will be systematically harvested through a variety of methods, including questionnaires, post-engagement interviews and discussions. Such feedback endeavours to drive internal enhancements, tailored specifically to improve the program and daily care provision. To uphold the integrity and authenticity of the feedback, the preliminary collection process will be led by professional’s adept in child psychology. This approach is strategic in circumventing potential biases or power imbalances inherent in adult-centric interpretations, as it acknowledges the propensity for adults to unconsciously position teenagers as their subordinates . Naturalistic settings characterized by peer companionship, where teenagers feel emboldened to express their perspectives, is also important, aiming to foster an environment conducive to open expression and reflection. Utilizing a holistic evaluative lens, results collated from group discussions and interviews will be synergistically analyzed alongside field observations, ensuring a consideration of all pertinent insights . Ultimately, this feedback and evaluation framework is instrumental in driving continuous refinement within this program.
To assess the impact of the four-step framework on the psychological well-being of YPVTs and TIC, three different scales will be administered post-volunteering. For TIC, evaluations will be conducted by volunteer team managers in pediatric palliative care centers with caregiver assistance. The Identity Scale for Adolescents, suitable for individuals aged 13–18, will be employed. This 39-items self-report questionnaire uses a 4-point Likert rating scale 0 (never), 1 (rarely), 2 (sometimes) and 3 (often) to categorize adolescents into three personality types: positive, negative, and arrogant self-identity . It measures identity formation, with an expectation of a shift towards a positive self-identity, characterized by sociability and optimism, after interactions with YPVTs. Negative identity shows a lack of confidence and social skills and arrogant identity indicates egoistic and feeling superior which potentially results from hyper-parenting behaviors. In evaluating YPVTs, the GSES and MTVS will be reapplied to develop deeper into their motivations and the changes they experience through the program. This approach aims to assess personal traits such as dedication and professionalism among the youth volunteers, ensuring alignment with the framework’s objectives. By comparing the results obtained during the selection process with those gathered post-program, any increase in scores will indicate an improvement in self-efficacy, a desirable outcome of the volunteer experience. In addition, it is anticipated that responses on the MTVS will shift towards affirmations like “Volunteering makes me feel good about myself” and “It will help me in the future,” reflecting a positive transformation in their perception of volunteer work. Opposite responses would indicate the potential reasons for dropping out of the voluntary program, where more attention should be paid to retaining the youth volunteers. These two scales can be used to regularly measure and track the volunteers' self-efficacy and motivation. This ensures they are engaged and find meaning in their work, while also helping to identify suitable roles and responsibilities for future adaptation. For instance, volunteers who resonate with the statement “I can handle whatever comes my way” and score high in feeling good about themselves through volunteering could be encouraged to take on leadership roles within the volunteer group. They can also provide immediate feedback on the impact of their work. Moreover, this approach helps identify when volunteers might need additional support or a change in role to stay motivated. Such a strategy not only aids in their self-discovery process but also contributes to fostering a culture of continuous improvement within the volunteer program. In practical terms, these self-report scales will be utilized for self-evaluation by the young volunteers. They will rate their agreement on a Likert scale, ranging from 1 (“Not at all true”) to 4 (“Exactly true”), under condition of confidentiality to encourage honest responses. Post-volunteering, these scales will be re-administered to assess any changes in self-efficacy and motivations. This approach provides valuable insights into the program’s impact and the volunteers’ development. The longitudinal data obtained will help in understanding the volunteers’ personal traits, their dedication, and professionalism. This information is significant for informing the refinement of future volunteer training and the overall development of the program.
One of the challenges in youth volunteerism is the high turnover rate. The first step to controlling the turnover rate is to improve the recruiting process to gain more qualified candidates since recruitment and retention are two interrelated processes . Moreover, peer groups positively strengthen the relationships within the volunteering groups, improve their commitment to the task, and help control the turnover rate . Additionally, the volunteers in palliative care centers, who bear the sadness of witnessing their clients suffering, tend to be overstressed psychologically, being a major reason for withdrawing from the volunteering program . Timely counselling and taking time off are common ways to release stress, indicating that professional psychological support and a proper volunteering schedule may help volunteers maintain a good status and retain them . In the feedback collection section, YPVTs should be interviewed about their motivation to join and reasons for leaving the program, which can comprehensively improve the framework from both TIC’s and YPVTs’ perspectives, striving for a sustainable program . There might be some potential flaws or weaknesses in the framework worth noting. In the selection section, the threshold for determining YPVTs’ eligibility to enroll in the program is unclear due to the limited data available. A significant issue in the framework evaluation section is the self-assessment bias inherent in these tools. Since all motivation items and statements are self-reported, responses could be influenced by the individual’s current mood or self-perception. This might lead to an inaccurate portrayal of one’s true capabilities or motivations. Furthermore, complex personal traits such as dedication and professionalism may not be fully captured through self-reporting, as such qualities are often better demonstrated through actions rather than introspection. Moreover, governmental regulations pose challenges in data collection from local palliative care centers in collaboration with welfare institutions, primarily due to concerns over data and privacy protection. To address these limitations, the questionnaire scores should be collected in the pilot experiments to determine a more reliable threshold by calculating the range of scores of youth volunteers who withdraw from the program. The incorporation of performance-based assessment with multiple raters is planned. This approach will include assessments from peers, supervisors, or community members, providing a more objective measure of the volunteer's traits and performance. Additionally, the process of synergistically analyzing feedback from various sources, including group discussions, interviews, and observations, though complex, is essential. Ensuring accurate reflection of all viewpoints in this data synthesis is significant for a comprehensive evaluation. Combining both qualitative feedback collection and quantitative methods, such as graded scales, offers a more balanced approach to evaluation.
Due to the relatively recent introduction of pediatric palliative care in China, the application of youth volunteering as an effective intervention to promote the self-discovery process among TIC remains an area not yet extensively explored by researchers and policymakers. A further research direction involves conducting pilot experiments to assess the effectiveness of the proposed framework. This paper presents an innovative approach, advocating for the empowerment of private palliative care centers to address the psychological challenges among disadvantaged children.
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Transient bradycardia during local anaesthesia to the oral cavity under intravenous sedation and its associate factors: a prospective observational study | c048e441-bffd-46d0-b49a-a41b5e6e30ca | 11443804 | Dentistry[mh] | The incidence of hypotension during local dental anaesthesia is less than 0.1%. Reports have stated that discomfort or transient ischaemic-like symptoms observed during dental procedures or the administration of local anaesthesia are often attributed to a decline in heart rate (HR) and blood pressure (BP) resulting from an emotional syncope, known as the vasovagal reflex (VVR) . Another mechanism implicated in hypotension induced by stimulation in the maxillofacial-oral region is the trigeminal cardiac reflex (TCR) . This distinctive brainstem reflex is characterised by abrupt reductions in HR, BP, cardiac arrhythmias, asystole, and other autonomic responses, such as apnoea and gastric hypermotility . In dental procedures or oral surgery, TCR is triggered by stimulation of the maxillary and mandibular branches and their corresponding innervated area . Consequently, TCR can be elicited through invasive procedures during dental treatments. TCR frequently occurs during ophthalmic surgeries (25–90%) and skull base surgeries (8–18%) under general anaesthesia, although the incidence of TCR during oral surgery ranges from 1–2%. The reasons for this low incidence remain unclear . Previous studies on TCR generally employed a threshold of a 20% decrease in HR from baseline. This threshold could reduce the risk of misinterpreting minor HR changes influenced by anaesthetics or patient movements, which are frequently observed in surgical settings. Nevertheless, this criterion may potentially underestimate the incidence of TCR . We conducted a retrospective analysis to confirm the incidence and potential precipitating factors contributing to a ≥ 10% decrease in HR during local anaesthesia . Our findings indicated that a decrease in HR during local anaesthesia is comparable to that reported for TCR occurrences in skull base surgery and that a history of gag reflex during dental treatment is associated with transient bradycardia (TB). However, nausea may also be associated with anxiety, stress, and depression, and anxiety about dental treatment might influence nausea and TB occurrence . Additionally, unresolved questions remain because of the retrospective nature of the study. Firstly, the study could not verify that only cases in which actual HR reduction occurred during local anaesthesia were included. Furthermore, it did not ensure the accuracy of the classification of discomfort symptoms during past dental treatment, which was the reason for using intravenous sedation. Therefore, the previous study did not completely exclude the possibility that anxiety about dental treatment may trigger TB . To accurately assess the factors associated with TB occurrence, we required an objective quantitative assessment of anxiety during dental treatment, as well as an accurate classification of past discomfort, including the gag reflex. In this study, we conducted a prospective analysis to assess HR fluctuations during local anaesthesia administration in patients receiving intravenous sedation for dental treatment. A questionnaire was used to obtain comprehensive data on the discomfort experienced during dental treatment (Additional File). We utilised the Dental Fear Survey (DFS) and Modified Dental Anxiety Scale (MDAS) to assess specific anxiety in detail during dental treatment. Additionally, the State-Trait Anxiety Inventory (STAI) was used to assess anxiety, which can evaluate both the intensity of situational temporary anxiety and relatively stable anxiety, independent of the environment and based on individual characteristics . The primary outcome is the incidence of TB during local anaesthesia under sedation, with or without the gag reflex. The secondary outcome focuses on identifying the associations between TB occurrence and accurately categorising dental treatment-associated discomfort and various anxiety scales.
This study included patients scheduled for oral surgery under intravenous sedation between March 2022 and March 2023 at the dental anaesthesia outpatient clinic of the hospital. Eligible participants were adults classified under the American Society of Anesthesiologists Physical Status (ASA PS) 1–2. Exclusion criteria included individuals aged < 17 years, those with artificial pacemakers, recipients of catecholamine agonists/blockers or circulatory medications, users of antipsychotic/antidepressant medications, and those with a history of cardiovascular disease. Informed consent was obtained from all participants after thoroughly explaining the study’s purpose. Upon providing consent to participate in the study, a comprehensive questionnaire was administered to document the occurrence and characteristics of the discomfort experienced during dental treatment and their detailed symptoms. Demographic data of participants were also collected. Subsequently, a certified dental anaesthesiologist categorised each discomfort based on the questionnaire responses. The classification included dental phobia, hyperventilation, gag reflex, VVR, intraoperative hypertension, drug allergy, and allergic reactions to local anaesthesia. Symptoms that could not be classified into these categories were classified as ‘other’ (invasive). Anxiety related to dental phobia was assessed using DFS (60 or more points positive) and MDAS (19 or more points positive). We used the Japanese translations of the DFS and MDAS, and each threshold was defined based on previous publications . Additionally, the anxiety scale was assessed using STAI and was considered positive if it met or exceeded certain thresholds: for State Anxiety (Y-1), men who scored 41 or higher and women who scored 42 or higher, and for Trait Anxiety (Y-2), men who scored 44 or higher and women who scored 45 or higher were considered positive. We used the Japanese version of the STAI, and the thresholds were based on its manual . For intravenous sedation, a combination of midazolam and propofol constituted the anaesthetic regimen, and the attending anaesthesiologist determined the dosage. The target sedation depth for the participants was maintained within the range of 4–5 on the Ramsay Sedation Scale (RSS), indicating brisk to sluggish responses to stimuli. Vital signs, including electrocardiogram, HR, non-invasive blood pressure, and peripheral capillary oxygen saturation, were monitored and recorded by the patient monitors (BSM3562, Nihon-kohden, Tokyo, Japan) and documented through the anaesthesia record system (paperChart; https://paperchart.net/ech/ ) at 2-s intervals. Following the stabilisation of BP and HR, the baseline HR before local anaesthesia (HRLA) was recorded alongside the sedation level assessment. HRLA was defined as the mean value over 2 min before local anaesthesia administration. Subsequently, a local anaesthetic injection was administered by the treating dentist, and the largest decrease in HR (HRmin) within 2 min immediately after the start of the local anaesthetic injection was recorded. Sedation management and the recording of HR variability (HRR) were performed by a dental anaesthesiologist who did not participate in this study. HRR after local anaesthesia was determined according to a previous publication per the following equation : [12pt]{minimal}
$$=([]-[])/[]100\;$$ HRR = ( [ HRMin ] - [ HRLA ] ) / [ HRLA ] × 100 A ≥ 10% decrease in HRR was defined as TB. The patients were divided into two groups: those with observed TB (TB group) and those without observed TB (normal group). Sample size estimation and statistical analysis To prove the hypothesis that a history of nausea is not associated with TB, we established a significance level of 0.05, with a power of 0.8 and an effect size of 0.27. The effect size was determined based on the results of a previous retrospective study. Assuming an expected dropout rate of 25%, a total of 235 cases were required. Comparative analyses of the experience of nausea and TB occurrence or other variables were conducted using the chi-square test or Fisher’s two-tailed exact probability test for categorical variables and an independent-sample t-test for continuous variables. Furthermore, a binary logistic regression model was used to identify risk factors associated with TB occurrence. The dependent variable was TB, and the independent variables were selected using a likelihood-based variable reduction method. Initially, all candidate factors were included in the model. A two-tailed P -value of < 0.05 indicated statistical significance.
To prove the hypothesis that a history of nausea is not associated with TB, we established a significance level of 0.05, with a power of 0.8 and an effect size of 0.27. The effect size was determined based on the results of a previous retrospective study. Assuming an expected dropout rate of 25%, a total of 235 cases were required. Comparative analyses of the experience of nausea and TB occurrence or other variables were conducted using the chi-square test or Fisher’s two-tailed exact probability test for categorical variables and an independent-sample t-test for continuous variables. Furthermore, a binary logistic regression model was used to identify risk factors associated with TB occurrence. The dependent variable was TB, and the independent variables were selected using a likelihood-based variable reduction method. Initially, all candidate factors were included in the model. A two-tailed P -value of < 0.05 indicated statistical significance.
This study was conducted between March 2022 and March 2023. Figure illustrates the patient flow. Of the initial 234 patients attending our outpatient dental anaesthesia clinic during this period, 46 were excluded due to incomplete questionnaire responses, insufficient anaesthesia records, or the absence of local anaesthetic administration, leaving a final cohort of 188 patients for data analysis. Table presents the patients’ demographic, cardiac, and pharmacological characteristics in both cohorts according to the presence or absence of gag reflex. Among the 188 patients, 21 (11.2%) experienced vomiting reflexes during dental treatment. No significant differences were observed in height, weight, HRR, or sedative doses between the two groups. Table presents the relationship between the presence or absence of the gag reflex and age distribution, categories of discomfort during dental treatment, and positive findings of DFS, MDAS, and STAI. TB occurrence was significantly higher in the group of patients with the gag reflex. Furthermore, the prevalence of dental phobia was lower in this group. No significant differences were found between the presence or absence of gag reflex and age group. No apparent differences were observed in other discomfort symptoms experienced during dental treatment between the groups with and without the gag reflex. Positive findings on DFS and MDAS, in addition to positive findings on STAI, were not significantly different between the two groups. Among the 188 participants, 77 (41.0%) exhibited TB symptoms, as illustrated in Fig. . The largest decrease in HR recorded was 30 beats per minute (bpm), whereas the lowest HRmin observed was 41 bpm. Additionally, 32 (17.0%) patients had an HR below 60 bpm. Notably, no patients experienced severe hypotension accompanied by loss of or decreased consciousness, nor did they require pharmacological intervention or therapeutic measures. Furthermore, none of the patients had postoperative memories of the administration of local anaesthesia. Table presents the results of the univariate and binary logistic regression analyses performed to identify the independent factors associated with the occurrence of TB. A significant association was found between the prevalence of the gag reflex (odds ratio [OR], 5.578; 95% confidence interval [CI], 1.726–18.025; P = 0.004) and positive findings for trait anxiety on the STAI (Y-2) (OR, 2.529; 95% CI, 1.160–5.514; P = 0.020) with TB occurrence. Additionally, HRLA expression was slightly higher in the TB group (OR, 1.027; 95% CI, 1.000–1.055; P = 0.047). Age and sex exhibited no association with TB occurrence.
Our results revealed a higher occurrence of TB in patients with a history of nausea. Furthermore, a history of gag reflex was identified as an independent factor associated with the occurrence of TB. These results are consistent with those of a previous retrospective analysis; however, the present study was conducted using a prospective approach, ensuring the results were more robust. One possible explanation for our results is that afferent stimuli through the glossopharyngeal nerve overstimulate the emetic centre in individuals with an enhanced gag reflex, efferently activating the parasympathetic nervous system . As neurally modulated syncope also induces bradycardia via afferent vagal branches in response to various stimuli, symptoms such as the swallowing reflex and nausea could precede the bradycardia reflex . Excitation of the upper gastrointestinal tract by vomiting stimuli can also increase vagal activity, leading to a vagal reflex (known as vomiting syncope) . As stimulation of the gastrointestinal vagal afferent nerve can cause nausea, bradycardia may occur more frequently in patients with frequent episodes of the gag reflex. Moreover, animal studies suggest that the activation of gastrointestinal vagal afferent fibres may be a pathway that could initiate the prodromal symptoms of vomiting and activate the efferent vagus nerve . However, vagus nerve stimulators reportedly cause a sustained vomiting reflex . Afferent stimulation via the trigeminal nerve may induce vomiting or nausea by stimulating the efferent gastrointestinal vagus nerve, in addition to generating bradycardia . In this study, trait anxiety tendency emerges as an independent predictor of TB occurrence. Unlike state anxiety, which fluctuates rapidly in response to situational factors, such as dental treatment or local anaesthesia, trait anxiety remains relatively stable regardless of the situation . Patients with trait anxiety experience persistent anxiety and continuously significant psychological stress. In such situations, the sympathetic nervous system is activated, and the parasympathetic nervous system is hyperactive, leading to vagal reflexes in case of disruption of this delicate balance by minor intraoral stimuli during dental treatment . In our investigation, we observed a slight elevation in HR among the TB group before local anaesthesia administration. This finding may indicate trait anxiety, characterised by chronic hyperactivity of the sympathetic nervous system. Moreover, the gag reflex increases when the parasympathetic activity is preponderant . This may provide a physiological basis for the development of vomiting and nausea. In a study of haematopoietic stem cell transplant recipients, participants with frequent nausea had higher anxiety levels, and the severity and frequency of nausea were predictors of trait anxiety . In addition, persistent stress caused by anxiety affects the autonomic nervous system and increases the secretion of stress hormones. This may cause nausea by decreasing blood flow to the gastrointestinal tract to prepare for the ‘fight or flight’ response . Conversely, we investigated the association between dental phobia and TB, and no correlation with TB was established using quantitative measures, such as DFS or MDAS, or qualitative assessments of dental phobia classification by dental anaesthetists using structured questionnaires . This finding is consistent with our previous study . Dental phobia is considered a risk factor for bradycardia and associated syncope during local anaesthesia administration; however, it may not directly affect bradycardia caused by TCR. Our study revealed a 41% incidence of TCR during local anaesthesia under sedation, which exceeds that of previous reports and our previous retrospective study. In this study, we evaluated the depth of intravenous sedation and confirmed that the patients had no memory of local anaesthesia after arousal. Thus, the present study indicates that TCR, rather than VVR, is the primary inducing factor . Variations in TCR incidence among surgical fields might be due to factors, including the intensity of the stimulation and whether the stimulation originates from direct stimulation of the nerve branches . Dental treatment can provide only relatively low-intensity stimulation, resulting in a minor decrease in HR compared with otolaryngological or ophthalmological procedures. Another reason for the low incidence of TCR is that local anaesthesia operations do not directly stimulate nerve branches but rather nerve endings distributed over the innervated area, which may result in minor fluctuations in HR . This study had some limitations. First, the study did not distinguish between bradycardia occurrence induced by the gag reflex and bradycardia-induced vomiting or nausea. Therefore, the actual or potential likelihood of bradycardia in patients with a gag reflex remains unexplored. Second, because autonomic nerve activity has not been directly assessed during local anaesthesia, an increase in the parasympathetic tone of activity has not been proven. Finally, the study cohort comprised patients who required intravenous sedation during dental treatment for various reasons. Therefore, they might have encountered more discomfort during previous dental treatments than the cohort who visited a general dental clinic. This may have influenced the analysis of this study.
The results of this study indicate a high frequency of occurrence of local anaesthesia-induced TB, likely caused by TCR. Furthermore, TB was associated with gag reflex and the presence of trait anxiety, as identified by the STAI. Whether the occurrence of TB during local anaesthesia is solely the result of TCR remains controversial. Therefore, future studies are required, including the direct measurement of neural activity.
Supplementary Material 1.
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Interpretation of bacterial composition patterns and community assembly processes in the rhizosphere soil of tea trees in karst areas | 7609c397-74f1-4b8b-985a-60a30f63e361 | 11585092 | Microbiology[mh] | Prevention and control of plant diseases are major concerns in modern agriculture, and effective measures are consistent with modern farm management strategies . Recent studies have shown that one of the most important factors affecting plant health is the presence of soil microorganisms closely associated with plants . Soil microorganisms are vital in maintaining soil health and long-term production because they execute major soil processes such as decomposition, nutrient cycling, and soil formation , and crop growth . Plant pathogens can emerge and proliferate to invade plants and cause diseases . However, soil microorganisms can prevent the occurrence and development of plant diseases by effectively inhibiting the activity of pathogenic bacteria and thus maintaining the healthy life activities of plants . Research on plant diseases is currently quite extensive, and positive results have been achieved, particularly in relation to fungal infestations and diseases caused by insect-plant interactions . Nevertheless, given the intricacies involved in the relationship between the soil environment and microorganisms, gaining a clearer understanding of the community status of such microorganisms under stress exerted by plant diseases is required. Studying soil microbial communities is important for the prevention and control of plant diseases. Advancements in high-throughput sequencing technology have shifted the focus of scientists to soil microbial communities because of the potential and practicality of their role in increasing and improving plant disease prevention and control . Plant diseases can cause changes in the rhizosphere soil microbial community, including changes in the abundance, composition, and structure of microorganisms and core microbiota, which may be attributed to plant disease mediation . Research has shown that a high degree of microbial community diversity significantly increases plant resistance to pathogen invasion, which may be related to the complexity of the soil-microbial interaction network . Changes in the environment of soil microorganisms are crucial for stabilising the community structure and protecting plants from pathogen infestation . Hence, exploring the effect of plant diseases on soil microbial communities is crucial for designing novel preventive measures against diseases and advancing modern agriculture. Microbial communities commonly display imbalanced distribution characteristics, featuring a considerable number of low-abundance taxa and a limited number of high-abundance taxonomic groups. The objective evaluation of community composition is essential for advancing our understanding of the ecological dynamics of microbial systems . Research has shown that the high abundance of taxonomic groups in soil microbial communities plays an important role in the carbon cycling process . However, growing emphasis is now on the importance of rare taxonomic groups because rare bacterial species have been shown to play an important role in maintaining ecosystem stability, and a study has pointed out that soil rare taxa have a stronger response to desertification restoration than abundant taxa . Currently, insufficient knowledge regarding the influence of plant diseases on both the richness and rarity of soil microbes is observed, and the representation of particular bacterial strains in community modules remains unclear. Diseases in the tea tree ( Camellia sinensis ) are currently one of the major factors contributing to declining tea yield and quality . Guizhou Province is located at the heart of the South China Karst, an area famous for its karst landscape and rich history of tea cultivation, which plays a key role in the local economy . The prevalence of rainfall and heat during the summer increases the occurrence of tree diseases, including tea leaf blight ( Colletotrichum cameliae Masse ) and tea ring rot ( Pseudopestalotiopsis camelliae-sinensis ) in tea crops . Tea leaf blight, also known as tea-tree leaf blight, mostly affects leaves but can also affect young shoots, branches, and fruits. Infected tea trees lose their leaves early, and fresh shoots may wither, resulting in diminished tree vigour . Effectively preventing the outbreak of tea leaf blight (pathogen: Colletotrichum camelliae Masse ) is crucial for advancing modern agriculture in this area. At the same time, karst areas urgently require green and effective modern agricultural development methods to boost the area’s environmental carrying capacity because of its distinctive geological landforms , high degree of habitat fragmentation , and delicate ecological environment . Therefore, studying the microbial communities in the rhizosphere soil of tea trees in this area can provide a theoretical basis for the prevention and control of tea tree diseases. At the beginning of this study, we hypothesized that: (i) tea leaf blight would significantly alter the composition and diversity of abundant and rare bacterial communities in the rhizosphere soil of tea trees; (ii) tea leaf blight will alter the general pattern of rhizosphere bacteria in tea trees; (iii)tea leaf blight will significantly alter the assembly processes of rare and abundant taxa; (iv) tea leaf blight will significantly alter the co-occurrence network relationships and key species information of rhizosphere bacteria. Sample collection In mid-June 2023, tea gardens affected by tea leaf blight were selected for research in Guizhou Province, southwestern China (Fig. A). The collection site is located in Qingzhen City (26º51’75"N, 106º38’38"E) and Weng’an County (27º08’47"N, 107º35’16"E), Guizhou Province, the geological background of the areas where the tested tea gardens are located is dominated by covered karst in the Guizhou Karst Plateau. The tea garden management mode is consistent, and the climate background and average meteorological data in the past three years (2020–2022) are similar (Table ). In order to avoid unnecessary interference from human and animal activities on the sample site, areas with high vegetation diversity were selected for sampling. All plots are located in the subtropical monsoon climate zone, with widely distributed karst landforms and a long history of tea tree cultivation. The tea tree types and cultivation history in the tested tea gardens are the same, and there was no physical or chemical intervention in the quarter before sampling. When collecting samples from a single tea garden, healthy and diseased areas were determined based on the actual condition of tea trees affected by cloud leaf blight, with a total of 5 small areas set up as biological replicates for each area. Set up 5 mini sampling points (1 m × 1 m) in each small area, and mix the samples from the 5 mini sampling points to form 1 biological replicate of the area. The numbering rule for collecting samples: (1) Based on the impact of tea tree diseases, samples collected from healthy areas are numbered H and disease areas are numbered M; (2) According to different collection locations, the samples collected in Qingzhen City will be numbered QS, and the samples collected in Weng’an County will be numbered WS. Please refer to Table for specific sample numbers. The following are the specific steps in the soil sample collection process. Soil samples were collected from the rhizosphere of tea trees as follows: Standardised procedures for collecting soil samples from tea tree rhizospheres Humus was cleared from the surface at the bottom of the tea tree crown, and a sterilised shovel was used to extract soil from the ground until the tea tree roots were visible. Tea tree roots were collected (using sterile scissors to cut representative root systems) and shaken, and a 1 mm layer of soil was left on them. The collected soil samples were temporarily placed in a cooler and stored in a -80 ℃ refrigerator upon returning to the laboratory. The roots were placed in a 50 ml centrifuge tube and washed with 3 ml of phosphate-buffered saline at 180 rpm for 20 min to remove the soil from the root surface. The solution was centrifuged at 4000 rpm for 20 min, the supernatant was drained, and the sediment was collected for extraction (Fig. B) . During the process of collecting rhizosphere soil, a soil layer of 0–20 cm from the sampling point was also collected for physical and chemical property testing. The test results are detailed in Table . The average pH value of the soil sampling points in the tested tea garden is 4.48, which is acidic soil suitable for tea tree growth. The average organic matter content is 30.83 g/kg, and the organic matter content of all sampling points exceeds 20 g/kg, all of which meet the third level soil standard. Molecular biology analysis Sub-area sequencing DNA was extracted from the target soil samples using HiPure Soil DNA Kits (Magen, Guangzhou, China) according to the manufacturer’s instructions. The 341 F/806R primer pair was used to amplify the V3-V4 hypervariable area of the 16 S rRNA gene (341 F: 5’-CCTACGGGNGGCWGCAG-3′ and 806R: 5’-GGACTACHVGGGTATCTAAT-3′). Polymerase chain reaction products were recovered and validated using a Qubit3.0 (New England Biolabs, USA). Using Illumina DNA Prep Kit (Illumina, CA, USA) Construct a sequencing library. Use the ABI StepOnePlus Real Time PCR System (Life Technologies, Foster City, USA) for library quality testing. Qualified libraries were sequenced using Novaseq 6000 PE250 mode pooling machine sequencing. The specific details of PCR amplification are as follows: The 16 S rDNA target area of the ribosomal RNA gene was amplified by PCR (95 °C for 5 min, followed by 30 cycles at 95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 7 min) with primers specified in the above. A 50 µL combination includes 10 µL of 5 × Q5@ Reaction Buffer, 10 µL of 5 × Q5@ High GC Enhancer, 1.5 µL of 2.5 mM dNTPs, 1.5 µL of each primer (10 µM), 0.2 µL of Q5@ High-Fidelity DNA Polymerase, and 50 ng template DNA. Related PCR reagents were obtained from New England Biolabs in the US. Quality control and clusterin FASTP (version 0.18.0) was used to filter low-quality raw paired-end sequences . FLASH (version 1.2.11) was used to combine the acquired sequences . The raw reads were filtered to obtain high-quality clean reads. At a confidence level of 80%, the sample readings from each operational taxonomic unit (OTU) were classified into taxonomic groups using the ribosomal database project classifier against the SILVA database (version 138.1). The detailed quality control statistics are as follows (Illumina + Usearch method): (1) Reads filtering: remove reads containing unknown nucleotides (N) ≥ 10%; remove reads with phred quality score ≤ 20 and base ≥ 50%; delete reads with connectors. (2) Reads merging: use FLASH to merge clean reads with a minimum overlap of 10 bp and a maximum mismatch rate of 2% threshold into reads. (3) Raw reads filtering: truncate the Raw reads from the first low-quality (default quality threshold < = 3) base site of the set length (default length value of 3) of consecutive low-quality base numbers; after intercepting the Reads dataset, further filter out Reads with consecutive high-quality base lengths less than 75% of the Reads length. (4) Clustering and chimera removal: the clean reads were clustered into operational taxonomic units (OTUs) of ≥ 97% similarity using UPARSE; all chimeric reads were removed using UCHIME algorithm and finally obtained effective reads for further analysis; the reads sequence with highest abundance was selected as representative sequence within each cluster. Data processing OTUs with fewer than 20 reads were excluded from the analysis. Based on the lowest number of samples, randomly select the sequences of all samples to a uniform data size (Figure ). The criteria for defining abundant and rare OTUs were based on the published literature . The definition is as follows: OTUs with relative abundance (i) higher than 0.1% are referred to as “abundant”, (ii) below 0.01% are referred to as “rare”, and (iii) the remaining OTUs are referred to as “intermediate”. The relative abundance values were based on the relative abundance of each OTU in the total sample. Most data analyses in this study were performed using R (version 4.3.1). Phylogenetic tree is constructed using MEGA (version 11) and phylogenetic tree beautification treatment was assessed using the Chiplot online platform ( https://www.chiplot.online/ ). Difference analysis was performed based on the systems-theoretic accident model and processes (STAMP: version 2.1.3), it is a graphical software for analyzing differences between microbial communities ( https://beikolab.cs.dal.ca/software/STAMP ). The “vegan” package was used to compute the α-diversity index. Venn plots were used to count the number of common and unique OTUs in each sample. The Bray–Curtis dissimilarity index was used to calculate the β-diversity using the “vegan” package’s non-metric multidimensional scaling (NMDS) analysis. Analysis of similarities was used to quantify variations in abundant and rare bacterial communities in the rhizosphere soil of different groups . Additionally, community differences across the soil strata were calculated using permutation multivariate analysis of variance. Niche width for the overall sample and abundant and rare bacterial taxa were calculated using the “spaa” package . Neutral community model (NCM) analysis was performed using the “Hmisc” package and fitted with non-linear least squares. The “normalised stochasticity ratio (NST)”, “picante,” and “ape” packages were used to calculate phylogenetic NST (pNST) and β-nearest taxon index (βNTI) values. Null model analysis based on phylogenetic bins was used to quantify the contributions of different ecological processes to the community assembly. The “iCAMP” and “ape” packages in R are used for community assembly analysis and parameter settings: rand.time = 500, nworker = 4, memory = 50, and bin-size limit = 18. Co-occurrence network analysis used the function rcorr in the “Hmisc” package to calculate the Spearman correlation coefficient between OTUs. Multiple test corrections were performed using the Benjamini (“FDR-BH”) method. Threshold settings: r = 0.65 and P = 0.05. Furthermore, network diagrams were constructed with the “igraph” software, visualised with Gephi (V 0. (10)) and their topological properties were computed. In within- and among-module connectivity analysis of bacterial networks, the “igraph” package was used to calculate the network modules, and “ggplot2” was used to classify and plot the results . Bacterial network vulnerability analysis based on Yuan et al.‘s research . The calculation results are based on the implementation of info.centrality.R function in Rstudio. The vulnerability of each node measures its relative contribution to network efficiency. The vulnerability of a network is represented by the maximum vulnerability of its nodes, as follows: [12pt]{minimal} $$ $$ In the formula, E is the global efficiency of the network; E i is the global efficiency after removing node i and all its connections. The global efficiency of a network graph is calculated as the average efficiency between all pairs of nodes: [12pt]{minimal} $$E = {1 {n(n - 1)}}_{i j} {{1 {d(i,j)}}}$$ In the formula, d ( i , j ) represents the number of edges on the shortest path between node i and node j . In mid-June 2023, tea gardens affected by tea leaf blight were selected for research in Guizhou Province, southwestern China (Fig. A). The collection site is located in Qingzhen City (26º51’75"N, 106º38’38"E) and Weng’an County (27º08’47"N, 107º35’16"E), Guizhou Province, the geological background of the areas where the tested tea gardens are located is dominated by covered karst in the Guizhou Karst Plateau. The tea garden management mode is consistent, and the climate background and average meteorological data in the past three years (2020–2022) are similar (Table ). In order to avoid unnecessary interference from human and animal activities on the sample site, areas with high vegetation diversity were selected for sampling. All plots are located in the subtropical monsoon climate zone, with widely distributed karst landforms and a long history of tea tree cultivation. The tea tree types and cultivation history in the tested tea gardens are the same, and there was no physical or chemical intervention in the quarter before sampling. When collecting samples from a single tea garden, healthy and diseased areas were determined based on the actual condition of tea trees affected by cloud leaf blight, with a total of 5 small areas set up as biological replicates for each area. Set up 5 mini sampling points (1 m × 1 m) in each small area, and mix the samples from the 5 mini sampling points to form 1 biological replicate of the area. The numbering rule for collecting samples: (1) Based on the impact of tea tree diseases, samples collected from healthy areas are numbered H and disease areas are numbered M; (2) According to different collection locations, the samples collected in Qingzhen City will be numbered QS, and the samples collected in Weng’an County will be numbered WS. Please refer to Table for specific sample numbers. The following are the specific steps in the soil sample collection process. Soil samples were collected from the rhizosphere of tea trees as follows: Standardised procedures for collecting soil samples from tea tree rhizospheres Humus was cleared from the surface at the bottom of the tea tree crown, and a sterilised shovel was used to extract soil from the ground until the tea tree roots were visible. Tea tree roots were collected (using sterile scissors to cut representative root systems) and shaken, and a 1 mm layer of soil was left on them. The collected soil samples were temporarily placed in a cooler and stored in a -80 ℃ refrigerator upon returning to the laboratory. The roots were placed in a 50 ml centrifuge tube and washed with 3 ml of phosphate-buffered saline at 180 rpm for 20 min to remove the soil from the root surface. The solution was centrifuged at 4000 rpm for 20 min, the supernatant was drained, and the sediment was collected for extraction (Fig. B) . During the process of collecting rhizosphere soil, a soil layer of 0–20 cm from the sampling point was also collected for physical and chemical property testing. The test results are detailed in Table . The average pH value of the soil sampling points in the tested tea garden is 4.48, which is acidic soil suitable for tea tree growth. The average organic matter content is 30.83 g/kg, and the organic matter content of all sampling points exceeds 20 g/kg, all of which meet the third level soil standard. Sub-area sequencing DNA was extracted from the target soil samples using HiPure Soil DNA Kits (Magen, Guangzhou, China) according to the manufacturer’s instructions. The 341 F/806R primer pair was used to amplify the V3-V4 hypervariable area of the 16 S rRNA gene (341 F: 5’-CCTACGGGNGGCWGCAG-3′ and 806R: 5’-GGACTACHVGGGTATCTAAT-3′). Polymerase chain reaction products were recovered and validated using a Qubit3.0 (New England Biolabs, USA). Using Illumina DNA Prep Kit (Illumina, CA, USA) Construct a sequencing library. Use the ABI StepOnePlus Real Time PCR System (Life Technologies, Foster City, USA) for library quality testing. Qualified libraries were sequenced using Novaseq 6000 PE250 mode pooling machine sequencing. The specific details of PCR amplification are as follows: The 16 S rDNA target area of the ribosomal RNA gene was amplified by PCR (95 °C for 5 min, followed by 30 cycles at 95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 7 min) with primers specified in the above. A 50 µL combination includes 10 µL of 5 × Q5@ Reaction Buffer, 10 µL of 5 × Q5@ High GC Enhancer, 1.5 µL of 2.5 mM dNTPs, 1.5 µL of each primer (10 µM), 0.2 µL of Q5@ High-Fidelity DNA Polymerase, and 50 ng template DNA. Related PCR reagents were obtained from New England Biolabs in the US. Quality control and clusterin FASTP (version 0.18.0) was used to filter low-quality raw paired-end sequences . FLASH (version 1.2.11) was used to combine the acquired sequences . The raw reads were filtered to obtain high-quality clean reads. At a confidence level of 80%, the sample readings from each operational taxonomic unit (OTU) were classified into taxonomic groups using the ribosomal database project classifier against the SILVA database (version 138.1). The detailed quality control statistics are as follows (Illumina + Usearch method): (1) Reads filtering: remove reads containing unknown nucleotides (N) ≥ 10%; remove reads with phred quality score ≤ 20 and base ≥ 50%; delete reads with connectors. (2) Reads merging: use FLASH to merge clean reads with a minimum overlap of 10 bp and a maximum mismatch rate of 2% threshold into reads. (3) Raw reads filtering: truncate the Raw reads from the first low-quality (default quality threshold < = 3) base site of the set length (default length value of 3) of consecutive low-quality base numbers; after intercepting the Reads dataset, further filter out Reads with consecutive high-quality base lengths less than 75% of the Reads length. (4) Clustering and chimera removal: the clean reads were clustered into operational taxonomic units (OTUs) of ≥ 97% similarity using UPARSE; all chimeric reads were removed using UCHIME algorithm and finally obtained effective reads for further analysis; the reads sequence with highest abundance was selected as representative sequence within each cluster. DNA was extracted from the target soil samples using HiPure Soil DNA Kits (Magen, Guangzhou, China) according to the manufacturer’s instructions. The 341 F/806R primer pair was used to amplify the V3-V4 hypervariable area of the 16 S rRNA gene (341 F: 5’-CCTACGGGNGGCWGCAG-3′ and 806R: 5’-GGACTACHVGGGTATCTAAT-3′). Polymerase chain reaction products were recovered and validated using a Qubit3.0 (New England Biolabs, USA). Using Illumina DNA Prep Kit (Illumina, CA, USA) Construct a sequencing library. Use the ABI StepOnePlus Real Time PCR System (Life Technologies, Foster City, USA) for library quality testing. Qualified libraries were sequenced using Novaseq 6000 PE250 mode pooling machine sequencing. The specific details of PCR amplification are as follows: The 16 S rDNA target area of the ribosomal RNA gene was amplified by PCR (95 °C for 5 min, followed by 30 cycles at 95 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 7 min) with primers specified in the above. A 50 µL combination includes 10 µL of 5 × Q5@ Reaction Buffer, 10 µL of 5 × Q5@ High GC Enhancer, 1.5 µL of 2.5 mM dNTPs, 1.5 µL of each primer (10 µM), 0.2 µL of Q5@ High-Fidelity DNA Polymerase, and 50 ng template DNA. Related PCR reagents were obtained from New England Biolabs in the US. FASTP (version 0.18.0) was used to filter low-quality raw paired-end sequences . FLASH (version 1.2.11) was used to combine the acquired sequences . The raw reads were filtered to obtain high-quality clean reads. At a confidence level of 80%, the sample readings from each operational taxonomic unit (OTU) were classified into taxonomic groups using the ribosomal database project classifier against the SILVA database (version 138.1). The detailed quality control statistics are as follows (Illumina + Usearch method): (1) Reads filtering: remove reads containing unknown nucleotides (N) ≥ 10%; remove reads with phred quality score ≤ 20 and base ≥ 50%; delete reads with connectors. (2) Reads merging: use FLASH to merge clean reads with a minimum overlap of 10 bp and a maximum mismatch rate of 2% threshold into reads. (3) Raw reads filtering: truncate the Raw reads from the first low-quality (default quality threshold < = 3) base site of the set length (default length value of 3) of consecutive low-quality base numbers; after intercepting the Reads dataset, further filter out Reads with consecutive high-quality base lengths less than 75% of the Reads length. (4) Clustering and chimera removal: the clean reads were clustered into operational taxonomic units (OTUs) of ≥ 97% similarity using UPARSE; all chimeric reads were removed using UCHIME algorithm and finally obtained effective reads for further analysis; the reads sequence with highest abundance was selected as representative sequence within each cluster. OTUs with fewer than 20 reads were excluded from the analysis. Based on the lowest number of samples, randomly select the sequences of all samples to a uniform data size (Figure ). The criteria for defining abundant and rare OTUs were based on the published literature . The definition is as follows: OTUs with relative abundance (i) higher than 0.1% are referred to as “abundant”, (ii) below 0.01% are referred to as “rare”, and (iii) the remaining OTUs are referred to as “intermediate”. The relative abundance values were based on the relative abundance of each OTU in the total sample. Most data analyses in this study were performed using R (version 4.3.1). Phylogenetic tree is constructed using MEGA (version 11) and phylogenetic tree beautification treatment was assessed using the Chiplot online platform ( https://www.chiplot.online/ ). Difference analysis was performed based on the systems-theoretic accident model and processes (STAMP: version 2.1.3), it is a graphical software for analyzing differences between microbial communities ( https://beikolab.cs.dal.ca/software/STAMP ). The “vegan” package was used to compute the α-diversity index. Venn plots were used to count the number of common and unique OTUs in each sample. The Bray–Curtis dissimilarity index was used to calculate the β-diversity using the “vegan” package’s non-metric multidimensional scaling (NMDS) analysis. Analysis of similarities was used to quantify variations in abundant and rare bacterial communities in the rhizosphere soil of different groups . Additionally, community differences across the soil strata were calculated using permutation multivariate analysis of variance. Niche width for the overall sample and abundant and rare bacterial taxa were calculated using the “spaa” package . Neutral community model (NCM) analysis was performed using the “Hmisc” package and fitted with non-linear least squares. The “normalised stochasticity ratio (NST)”, “picante,” and “ape” packages were used to calculate phylogenetic NST (pNST) and β-nearest taxon index (βNTI) values. Null model analysis based on phylogenetic bins was used to quantify the contributions of different ecological processes to the community assembly. The “iCAMP” and “ape” packages in R are used for community assembly analysis and parameter settings: rand.time = 500, nworker = 4, memory = 50, and bin-size limit = 18. Co-occurrence network analysis used the function rcorr in the “Hmisc” package to calculate the Spearman correlation coefficient between OTUs. Multiple test corrections were performed using the Benjamini (“FDR-BH”) method. Threshold settings: r = 0.65 and P = 0.05. Furthermore, network diagrams were constructed with the “igraph” software, visualised with Gephi (V 0. (10)) and their topological properties were computed. In within- and among-module connectivity analysis of bacterial networks, the “igraph” package was used to calculate the network modules, and “ggplot2” was used to classify and plot the results . Bacterial network vulnerability analysis based on Yuan et al.‘s research . The calculation results are based on the implementation of info.centrality.R function in Rstudio. The vulnerability of each node measures its relative contribution to network efficiency. The vulnerability of a network is represented by the maximum vulnerability of its nodes, as follows: [12pt]{minimal} $$ $$ In the formula, E is the global efficiency of the network; E i is the global efficiency after removing node i and all its connections. The global efficiency of a network graph is calculated as the average efficiency between all pairs of nodes: [12pt]{minimal} $$E = {1 {n(n - 1)}}_{i j} {{1 {d(i,j)}}}$$ In the formula, d ( i , j ) represents the number of edges on the shortest path between node i and node j . General distribution of abundant and rare bacteria in the rhizosphere soil of tea trees Following rigorous data processing, 787,876 high-quality sequences were assigned to 18,741 OTUs (Domain: Bacteria). According to the grouping scheme based on the criterion of the minimum number of sample sequences, the sequences of all samples were randomly extracted into a uniform data volume, and 16,960 OTUs were obtained. A total of 1536 OTUs (reads ≥ 20) were used for subsequent analysis (Table ). Overall, abundant taxa were annotated with 178 OTUs (11.59%), and their abundance was as high as 68.85%. In contrast, rare taxa were annotated in up to 560 OTUs; however, their abundance accounted for only 3.9% of the entire bacterial community. Additionally, the intermediate included 798 OTUs, with an abundance of approximately 27.25%. Similar distributions (QS-H, QS-M, WS-H, and WS-M) were observed in the soil layers of four groups in the experimental tea gardens. Figure shows the composition of soil bacterial abundant and rare taxa based on phylum level for different groups. All annotated phyla were visualised. Additionally to unclassified bacteria, 12 and 19 phyla were annotated at the phylum level for the abundant and rare taxa, respectively, which were significantly higher for the rare taxa compared to the abundant taxa. Regarding the abundant taxa, Acidobacterota (31.87%) and Proteobacteria (26.97%) were the dominant bacterial phyla in tea garden soil, and Chloroflexi tended to aggregate in QS-H (5.78%) and QS-M (2.65%). Regarding rare taxa, Chloroflexi (15.93%), Patescibacteria (13.60%), and Proteobacteria (12.48%) were the dominant phyla, and the bacterial abundances in QS-M (23.84%) and WS-M (35.78%) were higher than QS-H (22.03%) and WS-H (18.34%). A phylogenetic tree was constructed based on the richness of the 50 most abundant taxa at the class level (Fig. C). The results showed that Actinobacteria occupied the majority of the target species (top 50) and were far from other important species (Alphaproteobacteria, Gammaproteobacteria, and Ktedonobacteria). Systems-theoretic accident model and process analysis showed that the genus Granulicella had a clear distribution in the healthy areas among the abundant taxa ( P < 0.01), followed by the family Acidobacteriaceae_subgroup_1 ( P < 0.01, not identified at the genus level), and the morbidity area was mainly clustered at the family level KF-JG30-B3 (Table ). In rare taxa, the genera Aquisphaera and Bacillus favoured healthy areas ( P < 0.01), whereas the genera Pedosphaera and Candidatus Koribacter were more frequently found in the disease areas. Diversity analysis of abundant and rare bacterial taxa The rarefaction-curve trends supported the reliability of the sequencing data volume and species information (Figure ). Venn diagrams showed that all abundant taxa coexisted in different single groups, whereas over half of the rare taxa (60.78%) coexisted or became unique species in different groups (Fig. C). Higher α-diversity in abundant and rare taxa was more prone to morbidity groups. (Table ). Generally, the community richness of rare taxa was significantly higher than that of abundant taxa, demonstrating that rare taxa have a more complicated and diversified taxonomy (Fig. A and B). Both the ACE and Chao 1 indices of QS-M and WS-M were higher than those of QS-H and WS-H (Table ), indicating that tea leaf blight may be an important factor affecting the formation of bacterial communities in rhizosphere soil. NMD analysis showed a moderate difference (analysis of similarities, R > 0.5, P = 0.001) between abundant (stress < 0.2) and rare bacterial taxa (stress < 0.2) in all four sets of samples (Fig. B and C). A similar significance (stress < 0.2) was observed for all the samples (Fig. A). Additionally, permutation multivariate analysis of variance showed that the total sample bacterial communities, abundant bacterial taxa, and rare bacterial taxa significantly differed ( P < 0.05) between sample groups. Niche width was assessed based on the relative abundance of species at the OTU level. Species with a niche width > 3 were defined as generalists, whereas species with a niche width < 1.5 were defined as specialists. The results showed that the abundant taxa had a larger niche width than the rare taxa (Fig. D), with an average niche width of approximately 7.33, and generalists accounted for approximately 90.45% of the total abundant taxa. The average niche width of the species in the rare taxa was approximately 3.98, and specialists accounted for approximately 13.2% of the total rare taxa. Assembly process of bacterial community in the tea rhizosphere soil Ecological processes of microbial community assembly can be used to understand the mechanisms of microbial community formation. The NCM analysis results indicated that the model describes the relationship between the frequency of sample species occurrence and their abundance (Fig. C, D, E, and F). The relative contribution of neutral processes between the healthy and disease areas was higher in the morbidity group than in the healthy group (Fig. ), explaining 10.8%, 41.3%, 30.8%, 31.3%, and 52.8% of the QS-H, QS-M, WS-H, WS-M, and community variance, respectively. The same pattern was observed in the NCM analysis of the overall sample ( R 2 = 0.528) (Fig. A), reflecting higher stochasticity contribution to the rhizosphere soil bacterial community of tea trees in morbid areas. Simultaneously, pNST calculations showed that the distributions of pNST values were all above the 0.5 threshold line (Fig. B), suggesting that stochastic processes were more important in the overall community and among different subgroups (pNST < 0.5, deterministic processes dominated; pNST > 0.5, stochastic processes dominated). βNTI value is calculated to evaluate the relative importance of bacterial community assembly in the tea-tree rhizosphere soil in abundant and rare taxa. βNTI > 2, < -2, and between − 2 and 2 are interpreted as a variable selection of deterministic processes, a homogeneous selection of deterministic processes, and the result of a stochastic process, respectively. The difference analysis of βNTI values between the two taxa groups was conducted through a T-test ( P = 0.15, NS), indicating no significant differences between the two taxa groups (Fig. A). Of the abundant taxa, 91.58% of βNTI values were between − 2 and 2, and the rare taxa’s 95.26% of the βNTI values fell within the range of [-2, 2] (Fig. A), indicating that stochastic is the main factor affecting bacterial community assembly in the total sample, and certainty is more involved in the community assembly process of abundant taxa. The assembly of different taxonomic groups was explored using the iCAMP method. The results showed that dispersal limitation was the most important ecological process for abundant taxa (Fig. B and C), accounting for 92.4% of all community variations, followed by homogeneous selection (3.8%). Drift and dispersal limitations were the most important ecological processes in rare taxa (Fig. B and C), accounting for approximately 44.9% and 35.1%, respectively. Additionally, homogeneous selection (8.6%), homogenising dispersal (5.9%), and heterogeneous selection (5.5%) participated in the community assembly process to varying degrees, and the balance between the different ecological processes in the sample was relatively conservative. Co-occurrence network analysis of the abundant and rare taxa of bacteria in the rhizosphere soil of tea trees To clarify the interactions between abundant and rare bacterial taxa in the rhizosphere soil of tea trees, a bacterial co-occurrence network was constructed based on the relevant results (Fig. A and C). The relevant topological parameters are listed in Table . The results showed that the network constructed by the enriched taxa contained 178 nodes (OTU) associated with 1644 edges (Fig. A). Most of the edges between the OTUs were positively correlated (73.05%). The rare taxa comprised a network of 560 nodes (OTU) and 7248 edges (Fig. C). Almost all edges between the OTUs exhibited a negative correlation (97.46%). Based on the four phyla with the highest levels in the network, the topological characteristics of their specific nodes were quantified (Fig. B and D). The results showed that in the network of rare taxa, the degree, betweenness, eigenvector, and closeness centrality of Planctomycetota were generally higher than those of other phyla (Fig. D). To better elucidate the interaction patterns between species, we quantified the co-occurring network relationships among the species (abundant, intermediate, and rare taxa). The corresponding topological parameters are listed in Table . The results indicated that the network had 1510 nodes (OTUs), and species were associated with 42,686 edges. Most species exhibited a positive correlation with each other (Fig. A). Positive correlations were significantly more prevalent in the rare-intermediate taxa (11,344 edges, 91.02%) and intermediate-intermediate taxa (12,805 edges, 87.22%) than in the abundant-intermediate taxa (5632 edges, 72.50%), abundant-rare taxa (1405 edges, 70.15%), and abundant-abundant taxa (981 edges, 69.82%); rare taxa also had higher positive correlations (Table ). More connections were observed between species in the rare and intermediate taxa, indicating closer interactions between the intermediate and rare taxa. Although abundant taxa were associated with intermediate and rare taxa to some extent, they were not sufficient to support their strong relationships with intermediate and rare taxa. Specific node topology features were then compared (Table ). Generally, the degree, betweenness, eigenvector, and closeness centrality values of the abundant taxa were higher than those of intermediate and rare taxa (Fig. B), reflecting the central nature of abundant taxa in the network. Keystone species were found to have a high degree (> 70) and low betweenness centrality (< 5000), which are crucial for sustaining the structure and function of bacterial communities. A total of 365 OTUs were identified as key taxa, including abundant (59 OTUs, 33.15%), intermediate (215 OTUs, 26.94%), and rare (91 OTUs, 16.25%). Among them, the family Acidothermaceae had the highest proportion of abundant taxa (seven OTUs, 11.86%). The stability assessment of bacterial networks shows that in all groups (Fig. C), the vulnerability of bacterial networks in disease areas is generally lower than that in healthy areas, while in different bacterial taxa, the vulnerability of rare taxa is higher than that of abundant taxa, which is negatively correlated (Fig. A and C) with their network connectivity attributes (positive and negative). Following rigorous data processing, 787,876 high-quality sequences were assigned to 18,741 OTUs (Domain: Bacteria). According to the grouping scheme based on the criterion of the minimum number of sample sequences, the sequences of all samples were randomly extracted into a uniform data volume, and 16,960 OTUs were obtained. A total of 1536 OTUs (reads ≥ 20) were used for subsequent analysis (Table ). Overall, abundant taxa were annotated with 178 OTUs (11.59%), and their abundance was as high as 68.85%. In contrast, rare taxa were annotated in up to 560 OTUs; however, their abundance accounted for only 3.9% of the entire bacterial community. Additionally, the intermediate included 798 OTUs, with an abundance of approximately 27.25%. Similar distributions (QS-H, QS-M, WS-H, and WS-M) were observed in the soil layers of four groups in the experimental tea gardens. Figure shows the composition of soil bacterial abundant and rare taxa based on phylum level for different groups. All annotated phyla were visualised. Additionally to unclassified bacteria, 12 and 19 phyla were annotated at the phylum level for the abundant and rare taxa, respectively, which were significantly higher for the rare taxa compared to the abundant taxa. Regarding the abundant taxa, Acidobacterota (31.87%) and Proteobacteria (26.97%) were the dominant bacterial phyla in tea garden soil, and Chloroflexi tended to aggregate in QS-H (5.78%) and QS-M (2.65%). Regarding rare taxa, Chloroflexi (15.93%), Patescibacteria (13.60%), and Proteobacteria (12.48%) were the dominant phyla, and the bacterial abundances in QS-M (23.84%) and WS-M (35.78%) were higher than QS-H (22.03%) and WS-H (18.34%). A phylogenetic tree was constructed based on the richness of the 50 most abundant taxa at the class level (Fig. C). The results showed that Actinobacteria occupied the majority of the target species (top 50) and were far from other important species (Alphaproteobacteria, Gammaproteobacteria, and Ktedonobacteria). Systems-theoretic accident model and process analysis showed that the genus Granulicella had a clear distribution in the healthy areas among the abundant taxa ( P < 0.01), followed by the family Acidobacteriaceae_subgroup_1 ( P < 0.01, not identified at the genus level), and the morbidity area was mainly clustered at the family level KF-JG30-B3 (Table ). In rare taxa, the genera Aquisphaera and Bacillus favoured healthy areas ( P < 0.01), whereas the genera Pedosphaera and Candidatus Koribacter were more frequently found in the disease areas. The rarefaction-curve trends supported the reliability of the sequencing data volume and species information (Figure ). Venn diagrams showed that all abundant taxa coexisted in different single groups, whereas over half of the rare taxa (60.78%) coexisted or became unique species in different groups (Fig. C). Higher α-diversity in abundant and rare taxa was more prone to morbidity groups. (Table ). Generally, the community richness of rare taxa was significantly higher than that of abundant taxa, demonstrating that rare taxa have a more complicated and diversified taxonomy (Fig. A and B). Both the ACE and Chao 1 indices of QS-M and WS-M were higher than those of QS-H and WS-H (Table ), indicating that tea leaf blight may be an important factor affecting the formation of bacterial communities in rhizosphere soil. NMD analysis showed a moderate difference (analysis of similarities, R > 0.5, P = 0.001) between abundant (stress < 0.2) and rare bacterial taxa (stress < 0.2) in all four sets of samples (Fig. B and C). A similar significance (stress < 0.2) was observed for all the samples (Fig. A). Additionally, permutation multivariate analysis of variance showed that the total sample bacterial communities, abundant bacterial taxa, and rare bacterial taxa significantly differed ( P < 0.05) between sample groups. Niche width was assessed based on the relative abundance of species at the OTU level. Species with a niche width > 3 were defined as generalists, whereas species with a niche width < 1.5 were defined as specialists. The results showed that the abundant taxa had a larger niche width than the rare taxa (Fig. D), with an average niche width of approximately 7.33, and generalists accounted for approximately 90.45% of the total abundant taxa. The average niche width of the species in the rare taxa was approximately 3.98, and specialists accounted for approximately 13.2% of the total rare taxa. Ecological processes of microbial community assembly can be used to understand the mechanisms of microbial community formation. The NCM analysis results indicated that the model describes the relationship between the frequency of sample species occurrence and their abundance (Fig. C, D, E, and F). The relative contribution of neutral processes between the healthy and disease areas was higher in the morbidity group than in the healthy group (Fig. ), explaining 10.8%, 41.3%, 30.8%, 31.3%, and 52.8% of the QS-H, QS-M, WS-H, WS-M, and community variance, respectively. The same pattern was observed in the NCM analysis of the overall sample ( R 2 = 0.528) (Fig. A), reflecting higher stochasticity contribution to the rhizosphere soil bacterial community of tea trees in morbid areas. Simultaneously, pNST calculations showed that the distributions of pNST values were all above the 0.5 threshold line (Fig. B), suggesting that stochastic processes were more important in the overall community and among different subgroups (pNST < 0.5, deterministic processes dominated; pNST > 0.5, stochastic processes dominated). βNTI value is calculated to evaluate the relative importance of bacterial community assembly in the tea-tree rhizosphere soil in abundant and rare taxa. βNTI > 2, < -2, and between − 2 and 2 are interpreted as a variable selection of deterministic processes, a homogeneous selection of deterministic processes, and the result of a stochastic process, respectively. The difference analysis of βNTI values between the two taxa groups was conducted through a T-test ( P = 0.15, NS), indicating no significant differences between the two taxa groups (Fig. A). Of the abundant taxa, 91.58% of βNTI values were between − 2 and 2, and the rare taxa’s 95.26% of the βNTI values fell within the range of [-2, 2] (Fig. A), indicating that stochastic is the main factor affecting bacterial community assembly in the total sample, and certainty is more involved in the community assembly process of abundant taxa. The assembly of different taxonomic groups was explored using the iCAMP method. The results showed that dispersal limitation was the most important ecological process for abundant taxa (Fig. B and C), accounting for 92.4% of all community variations, followed by homogeneous selection (3.8%). Drift and dispersal limitations were the most important ecological processes in rare taxa (Fig. B and C), accounting for approximately 44.9% and 35.1%, respectively. Additionally, homogeneous selection (8.6%), homogenising dispersal (5.9%), and heterogeneous selection (5.5%) participated in the community assembly process to varying degrees, and the balance between the different ecological processes in the sample was relatively conservative. To clarify the interactions between abundant and rare bacterial taxa in the rhizosphere soil of tea trees, a bacterial co-occurrence network was constructed based on the relevant results (Fig. A and C). The relevant topological parameters are listed in Table . The results showed that the network constructed by the enriched taxa contained 178 nodes (OTU) associated with 1644 edges (Fig. A). Most of the edges between the OTUs were positively correlated (73.05%). The rare taxa comprised a network of 560 nodes (OTU) and 7248 edges (Fig. C). Almost all edges between the OTUs exhibited a negative correlation (97.46%). Based on the four phyla with the highest levels in the network, the topological characteristics of their specific nodes were quantified (Fig. B and D). The results showed that in the network of rare taxa, the degree, betweenness, eigenvector, and closeness centrality of Planctomycetota were generally higher than those of other phyla (Fig. D). To better elucidate the interaction patterns between species, we quantified the co-occurring network relationships among the species (abundant, intermediate, and rare taxa). The corresponding topological parameters are listed in Table . The results indicated that the network had 1510 nodes (OTUs), and species were associated with 42,686 edges. Most species exhibited a positive correlation with each other (Fig. A). Positive correlations were significantly more prevalent in the rare-intermediate taxa (11,344 edges, 91.02%) and intermediate-intermediate taxa (12,805 edges, 87.22%) than in the abundant-intermediate taxa (5632 edges, 72.50%), abundant-rare taxa (1405 edges, 70.15%), and abundant-abundant taxa (981 edges, 69.82%); rare taxa also had higher positive correlations (Table ). More connections were observed between species in the rare and intermediate taxa, indicating closer interactions between the intermediate and rare taxa. Although abundant taxa were associated with intermediate and rare taxa to some extent, they were not sufficient to support their strong relationships with intermediate and rare taxa. Specific node topology features were then compared (Table ). Generally, the degree, betweenness, eigenvector, and closeness centrality values of the abundant taxa were higher than those of intermediate and rare taxa (Fig. B), reflecting the central nature of abundant taxa in the network. Keystone species were found to have a high degree (> 70) and low betweenness centrality (< 5000), which are crucial for sustaining the structure and function of bacterial communities. A total of 365 OTUs were identified as key taxa, including abundant (59 OTUs, 33.15%), intermediate (215 OTUs, 26.94%), and rare (91 OTUs, 16.25%). Among them, the family Acidothermaceae had the highest proportion of abundant taxa (seven OTUs, 11.86%). The stability assessment of bacterial networks shows that in all groups (Fig. C), the vulnerability of bacterial networks in disease areas is generally lower than that in healthy areas, while in different bacterial taxa, the vulnerability of rare taxa is higher than that of abundant taxa, which is negatively correlated (Fig. A and C) with their network connectivity attributes (positive and negative). Exploring complex rhizosphere soil microbial communities and their diversity is crucial for plant protection and biodiversity, particularly in karst areas with fragile and sensitive ecological environments . Additionally, focusing on the microbial composition of the rhizosphere soil affected by diseases, especially the composition of abundant and rare microbial taxa, is of great significance for protecting plants from disease invasion and for developing modern agricultural development models . In this study, we revealed the composition and diversity of abundant and rare bacterial taxa, general assembly processes, and endemic species in the tea-tree rhizosphere soil in two tea gardens affected by diseases in Guizhou Province, China. Tea leaf blight affects the composition of bacterial communities in the rhizosphere soil The compositional status of a given bacterial species (abundant and rare taxa) has been correlated with plant disease, with some phylum-level classifications of bacteria showing a propensity to aggregate, which is reflected in the stacking of bacterial species from different taxa (Fig. A and B). This study indicates that the distribution of bacterial communities affected by tea leaf blight in the rhizosphere soil is more complex than that in healthy rhizosphere soil bacteria of tea trees because of the higher relative abundance of bacteria in the rhizosphere soil of tea trees in the disease areas α-diversity. This phenomenon also applies to a given species (abundant and rare) taxa (Fig. ). Microbial communities are complex groups of organisms that play important roles in ecological restoration and biogeochemical processes in environmentally fragile areas . Rare bacterial taxa had a more diverse taxon level than abundant bacterial taxa, and significant differences were observed in the species stacks of abundant and rare taxa (Fig. A and B). Furthermore, at the genus level, Acidothermus , Bryobater , and Acidibacter are the most abundant taxa and have strong adaptability to rhizospheres soil that are not affected by tea leaf blight. Among them, the genus Bryobater has been proven beneficial for decomposing lignin and cellulose . In contrast, Burkholderia-Caballeronia-Paraburkholderia and Candidatus solibacter were commonly observed in the overall samples of the abundant taxa but were more abundant in the morbidity group. Burkholderia-Caballeronia-Paraburkholderia strongly antagonises the root-rot pathogen Ilyonectria destructans and is involved in stabilising the rhizosphere soil microbiological environment . Excluding unclassified OTUs, the genera Aquicella , Bryobacter , Chthoniobacter , Gemmatimonas , Haliangium , and Pajaroellobacter were the dominant genera among the rare taxa. Notably, Chthonioacter members can degrade complex organic compounds . Gemmatimonas is a beneficial bacterium that promotes plant growth and participates in photosynthesis . These results indicate that in disease infestation, abundant bacterial taxa can resist pathogen invasions and participate in the construction of a stable microecological environment. Moreover, rare taxa contribute to more diverse ecological functions than abundant taxa and are key factors in the microbial ecological environment, enhancing the functional redundancy of microbial communities . Abundant and rare taxa work together and show different environmental adaptations Clarifying the changes in the diversity of a given species (abundant and rare taxa) in the rhizosphere soil mediated by plant diseases is crucial for a better understanding of the characteristics and related ecological functions and processes of a given species taxa . In our study, rare taxa had a higher α-diversity than abundant taxa (Table ), which is consistent with similar studies conducted under conditions of disturbance by other biotic and abiotic factors, such as controlled organic-contaminated sites and rice agroecosystems . Furthermore, in all given species, the α-diversity of the morbidity group is generally higher than that of the healthy group (Table ), suggesting that the increased diversity of rare taxa is associated with tea leaf blight and has a sharper perception of environmental change than the abundant taxa . This phenomenon can be explained by the narrow niche width of rare taxa (Fig. D) . We assessed the α-diversity of the bacterial community in the rhizosphere soil from three dimensions: the overall bacterial community, abundant taxa, and rare taxa, where a more pronounced difference was observed between the overall bacterial community and abundant taxa. Although the tea leaf blight had an impact on the α-diversity of abundant taxa, the importance of the rare taxa could not be ignored . Some studies have pointed out that rare microbial communities are the main driving force for the multifunctionality of soil ecosystems under long-term fertilization , while indirectly improving soil quality and rhizosphere microecology, which has a positive effect on the prevention and control of tea tree diseases . Similarly, NMDS analysis based on Bray–Curtis distance quantifies that rare classifications have a higher β-diversity characterised by a clear distinction in the distance between samples, while the distinction between overall bacterial and abundant taxa communities is not significant. This may be attributed to the influence of external microorganisms on the bacterial community structure through diffused input . Additionally, compared with rare taxa, abundant taxa had wider ecological niche widths (Fig. D), which is sufficient to demonstrate that abundant taxa have better potential adaptability to the living environment. Abundant taxa have larger niche widths than rare taxa, indicating that abundant taxa play a key role in maintaining community stability. The presence of more generalists (niche width > 3) in the abundant taxa suggests that they play an important role in maintaining the ecosystem functions of tea gardens . Assembly of bacterial communities in the tea tree rhizosphere soil, driven by stochastic processes Studying the relative importance of deterministic and stochastic processes in community assembly is crucial for understanding factors affecting community composition . In our study, we combined the NCM with the null model and found that the stochastic process dominated the assembly of abundant and rare taxa (Fig. B) and that the abundant taxa were, to some extent, influenced by deterministic processes (Fig. B). Dispersal limitation and homogeneous selection were jointly involved in the community assembly of abundant taxa. Rare taxa were more conserved in each ecological process (Fig. C), and the difference between the two could be determined by the width of the niche (Fig. D). Rare taxa with lower phylogenetic clustering (Fig. B) and narrower niche widths may be dispersed through environmental filtering and interspecific interactions . Notably, in the assembly of rare taxa, non-dominant processes accounted for a relatively high proportion, indicating that complex mechanisms of community assembly may form rare taxa (Fig. C). Neutral processes are equally important for shaping bacterial communities. Our neutral model explained a significant portion of the frequency variation in bacterial occurrence in the rhizosphere soil of tea trees ( R 2 > 0.5) (Fig. A). The explanation of neutral processes in the rhizosphere soil of diseased tea trees was higher than that in healthy groups (Fig. C–F). This indicated that neutral processes affect the formation of bacterial communities in the roots of diseased tea trees. Previous studies showed that rare taxa are more affected by dispersal limitations in soils from oil-contaminated areas and in subtropical bays, and these differences may be attributed to the differences in geographic location and habitat. Other studies have shown that diffusion has a significant impact on abundant taxa but has no effect on rare taxa . A conceptual review of the effects of disturbances on bacterial assembly processes in terrestrial ecosystems has been presented , showing that disturbances can change the relative importance of certainty and stochastic assembly processes. Based on previous studies, we identified that various anthropogenic environmental disturbances (such as fertilisation, mowing, and irrigation) increase stochastic processes in soil bacterial community assembly . Because of the lack of a unified threshold for dividing abundant and rare taxa, different assembly results may occur . Furthermore, the rare taxa of the WS tea gardens were dominated by stochastic aggregation processes (whether in morbid or healthy areas) corresponding to their higher OTU abundances (Fig. C). This phenomenon is supported by a previous study, which suggests that communities with higher α-diversity indices are more dominated by stochastic assembly processes . It is worth noting that in evaluating the community assembly process between disease areas and healthy areas, the pNST index of disease areas was significantly higher than that of healthy areas (Fig. B), indicating that the significant bacterial community diversity in disease areas has expanded the ecological niche width of their rhizosphere community habitats, thereby weakening the importance of environmental filtering . The deterministic process emphasizes environmental filtering , and different habitat environments reflect the role of environmental filtering by changing community composition (including biological species) . In this study, we emphasize the dominant role of randomness in the assembly process of bacterial communities. The climate and management patterns in the study area are almost the same, and the assembly process of bacterial communities is mainly reflected in the diversity changes within them, such as the increase in niche width, rather than species characteristics or environmental filtering emphasized by habitat or environmental heterogeneity. Co-occurrence network reveals that abundant taxa occupy more central positions Complex networks are formed when microbial species with distinct ecological niches interact with one another . In this study, a co-occurrence network was constructed based on the correlations between species to clarify species action patterns in abundant and rare taxa. The connections between species in rare taxa were mostly positive (Fig. C). These positive interactions (cooperative relationships) between species are driven by external plant disease pressures, which largely support the ecological function of the rhizosphere soil and the stability of bacterial communities under the influence of tea leaf blight . Of the top four phyla with the highest abundance of rare taxa, Planctomycetota had higher topological value indicators than the other three phyla, indicating that Planctomycetota occupied a central position among the rare taxa. Notably, Planctomycetota is a type of aquatic bacteria, of which the genera Planctomyces and Pirellula are specialised aerobic bacteria, and their presence may provide a favourable environment for constructing rare bacterial communities . To explore network interactions among species, we found that the correlation between abundant and rare taxa was substantially weaker than that between intermediate and rare taxa (Fig. A; Table ). This may be due to ecological niche differentiation of both abundant and rare bacterial communities . The intermediate and rare taxa were observed to maintain a positive correlation while exhibiting a close correlation (Fig. A), but in the evaluation of network stability, it showed opposite characteristics (Figs. A and C and C), which may be due to the increased diversity of bacterial communities mediated by tea tree diseases (Fig. A and B), which strengthened the resistance of tea tree rhizosphere to disease stress . By observing the topological values of a given species network, we found that the topological feature values of abundant taxa were slightly higher than those of intermediate and rare taxa. From another perspective, abundant taxa occupied a more central position in co-occurrence networks, a pattern supported by previous studies . Abundant taxa did not aggregate in the network, whereas rare taxa were intertwined with intermediate taxa (Fig. A). There are relatively few connections observed between abundant and rare taxa, and we inferred that rare taxa were more specific than abundant taxa, resulting in a statistically lower frequency of coexistence with abundant taxa. The emergence of the key bacterial family Acidothermaceae in the abundant taxa indicates that the pH value of tea garden soil was maintained at a reasonable level, which is of great significance for promoting the stability of tea garden soil properties. In this work, we selected typical tea garden cases in karst areas. Due to the wide cultivation area of tea gardens in Guizhou Province, in future work, we will expand our research scope, introduce relevant concepts of biogeography, and deeply explore the biological process of tea tree rhizosphere soil. The compositional status of a given bacterial species (abundant and rare taxa) has been correlated with plant disease, with some phylum-level classifications of bacteria showing a propensity to aggregate, which is reflected in the stacking of bacterial species from different taxa (Fig. A and B). This study indicates that the distribution of bacterial communities affected by tea leaf blight in the rhizosphere soil is more complex than that in healthy rhizosphere soil bacteria of tea trees because of the higher relative abundance of bacteria in the rhizosphere soil of tea trees in the disease areas α-diversity. This phenomenon also applies to a given species (abundant and rare) taxa (Fig. ). Microbial communities are complex groups of organisms that play important roles in ecological restoration and biogeochemical processes in environmentally fragile areas . Rare bacterial taxa had a more diverse taxon level than abundant bacterial taxa, and significant differences were observed in the species stacks of abundant and rare taxa (Fig. A and B). Furthermore, at the genus level, Acidothermus , Bryobater , and Acidibacter are the most abundant taxa and have strong adaptability to rhizospheres soil that are not affected by tea leaf blight. Among them, the genus Bryobater has been proven beneficial for decomposing lignin and cellulose . In contrast, Burkholderia-Caballeronia-Paraburkholderia and Candidatus solibacter were commonly observed in the overall samples of the abundant taxa but were more abundant in the morbidity group. Burkholderia-Caballeronia-Paraburkholderia strongly antagonises the root-rot pathogen Ilyonectria destructans and is involved in stabilising the rhizosphere soil microbiological environment . Excluding unclassified OTUs, the genera Aquicella , Bryobacter , Chthoniobacter , Gemmatimonas , Haliangium , and Pajaroellobacter were the dominant genera among the rare taxa. Notably, Chthonioacter members can degrade complex organic compounds . Gemmatimonas is a beneficial bacterium that promotes plant growth and participates in photosynthesis . These results indicate that in disease infestation, abundant bacterial taxa can resist pathogen invasions and participate in the construction of a stable microecological environment. Moreover, rare taxa contribute to more diverse ecological functions than abundant taxa and are key factors in the microbial ecological environment, enhancing the functional redundancy of microbial communities . Clarifying the changes in the diversity of a given species (abundant and rare taxa) in the rhizosphere soil mediated by plant diseases is crucial for a better understanding of the characteristics and related ecological functions and processes of a given species taxa . In our study, rare taxa had a higher α-diversity than abundant taxa (Table ), which is consistent with similar studies conducted under conditions of disturbance by other biotic and abiotic factors, such as controlled organic-contaminated sites and rice agroecosystems . Furthermore, in all given species, the α-diversity of the morbidity group is generally higher than that of the healthy group (Table ), suggesting that the increased diversity of rare taxa is associated with tea leaf blight and has a sharper perception of environmental change than the abundant taxa . This phenomenon can be explained by the narrow niche width of rare taxa (Fig. D) . We assessed the α-diversity of the bacterial community in the rhizosphere soil from three dimensions: the overall bacterial community, abundant taxa, and rare taxa, where a more pronounced difference was observed between the overall bacterial community and abundant taxa. Although the tea leaf blight had an impact on the α-diversity of abundant taxa, the importance of the rare taxa could not be ignored . Some studies have pointed out that rare microbial communities are the main driving force for the multifunctionality of soil ecosystems under long-term fertilization , while indirectly improving soil quality and rhizosphere microecology, which has a positive effect on the prevention and control of tea tree diseases . Similarly, NMDS analysis based on Bray–Curtis distance quantifies that rare classifications have a higher β-diversity characterised by a clear distinction in the distance between samples, while the distinction between overall bacterial and abundant taxa communities is not significant. This may be attributed to the influence of external microorganisms on the bacterial community structure through diffused input . Additionally, compared with rare taxa, abundant taxa had wider ecological niche widths (Fig. D), which is sufficient to demonstrate that abundant taxa have better potential adaptability to the living environment. Abundant taxa have larger niche widths than rare taxa, indicating that abundant taxa play a key role in maintaining community stability. The presence of more generalists (niche width > 3) in the abundant taxa suggests that they play an important role in maintaining the ecosystem functions of tea gardens . Studying the relative importance of deterministic and stochastic processes in community assembly is crucial for understanding factors affecting community composition . In our study, we combined the NCM with the null model and found that the stochastic process dominated the assembly of abundant and rare taxa (Fig. B) and that the abundant taxa were, to some extent, influenced by deterministic processes (Fig. B). Dispersal limitation and homogeneous selection were jointly involved in the community assembly of abundant taxa. Rare taxa were more conserved in each ecological process (Fig. C), and the difference between the two could be determined by the width of the niche (Fig. D). Rare taxa with lower phylogenetic clustering (Fig. B) and narrower niche widths may be dispersed through environmental filtering and interspecific interactions . Notably, in the assembly of rare taxa, non-dominant processes accounted for a relatively high proportion, indicating that complex mechanisms of community assembly may form rare taxa (Fig. C). Neutral processes are equally important for shaping bacterial communities. Our neutral model explained a significant portion of the frequency variation in bacterial occurrence in the rhizosphere soil of tea trees ( R 2 > 0.5) (Fig. A). The explanation of neutral processes in the rhizosphere soil of diseased tea trees was higher than that in healthy groups (Fig. C–F). This indicated that neutral processes affect the formation of bacterial communities in the roots of diseased tea trees. Previous studies showed that rare taxa are more affected by dispersal limitations in soils from oil-contaminated areas and in subtropical bays, and these differences may be attributed to the differences in geographic location and habitat. Other studies have shown that diffusion has a significant impact on abundant taxa but has no effect on rare taxa . A conceptual review of the effects of disturbances on bacterial assembly processes in terrestrial ecosystems has been presented , showing that disturbances can change the relative importance of certainty and stochastic assembly processes. Based on previous studies, we identified that various anthropogenic environmental disturbances (such as fertilisation, mowing, and irrigation) increase stochastic processes in soil bacterial community assembly . Because of the lack of a unified threshold for dividing abundant and rare taxa, different assembly results may occur . Furthermore, the rare taxa of the WS tea gardens were dominated by stochastic aggregation processes (whether in morbid or healthy areas) corresponding to their higher OTU abundances (Fig. C). This phenomenon is supported by a previous study, which suggests that communities with higher α-diversity indices are more dominated by stochastic assembly processes . It is worth noting that in evaluating the community assembly process between disease areas and healthy areas, the pNST index of disease areas was significantly higher than that of healthy areas (Fig. B), indicating that the significant bacterial community diversity in disease areas has expanded the ecological niche width of their rhizosphere community habitats, thereby weakening the importance of environmental filtering . The deterministic process emphasizes environmental filtering , and different habitat environments reflect the role of environmental filtering by changing community composition (including biological species) . In this study, we emphasize the dominant role of randomness in the assembly process of bacterial communities. The climate and management patterns in the study area are almost the same, and the assembly process of bacterial communities is mainly reflected in the diversity changes within them, such as the increase in niche width, rather than species characteristics or environmental filtering emphasized by habitat or environmental heterogeneity. Complex networks are formed when microbial species with distinct ecological niches interact with one another . In this study, a co-occurrence network was constructed based on the correlations between species to clarify species action patterns in abundant and rare taxa. The connections between species in rare taxa were mostly positive (Fig. C). These positive interactions (cooperative relationships) between species are driven by external plant disease pressures, which largely support the ecological function of the rhizosphere soil and the stability of bacterial communities under the influence of tea leaf blight . Of the top four phyla with the highest abundance of rare taxa, Planctomycetota had higher topological value indicators than the other three phyla, indicating that Planctomycetota occupied a central position among the rare taxa. Notably, Planctomycetota is a type of aquatic bacteria, of which the genera Planctomyces and Pirellula are specialised aerobic bacteria, and their presence may provide a favourable environment for constructing rare bacterial communities . To explore network interactions among species, we found that the correlation between abundant and rare taxa was substantially weaker than that between intermediate and rare taxa (Fig. A; Table ). This may be due to ecological niche differentiation of both abundant and rare bacterial communities . The intermediate and rare taxa were observed to maintain a positive correlation while exhibiting a close correlation (Fig. A), but in the evaluation of network stability, it showed opposite characteristics (Figs. A and C and C), which may be due to the increased diversity of bacterial communities mediated by tea tree diseases (Fig. A and B), which strengthened the resistance of tea tree rhizosphere to disease stress . By observing the topological values of a given species network, we found that the topological feature values of abundant taxa were slightly higher than those of intermediate and rare taxa. From another perspective, abundant taxa occupied a more central position in co-occurrence networks, a pattern supported by previous studies . Abundant taxa did not aggregate in the network, whereas rare taxa were intertwined with intermediate taxa (Fig. A). There are relatively few connections observed between abundant and rare taxa, and we inferred that rare taxa were more specific than abundant taxa, resulting in a statistically lower frequency of coexistence with abundant taxa. The emergence of the key bacterial family Acidothermaceae in the abundant taxa indicates that the pH value of tea garden soil was maintained at a reasonable level, which is of great significance for promoting the stability of tea garden soil properties. In this work, we selected typical tea garden cases in karst areas. Due to the wide cultivation area of tea gardens in Guizhou Province, in future work, we will expand our research scope, introduce relevant concepts of biogeography, and deeply explore the biological process of tea tree rhizosphere soil. Summarily, this study proposed a paradigm that describes the diversity and assembly mechanisms of abundant and rare bacterial communities in the rhizosphere soil systems of tea trees under the influence of tea leaf blight in karst areas. Rare taxa have a more diverse species composition, and tea leaf blight significantly affects the bacterial community diversity and neutral processes. Stochastic processes dominated the community assembly of abundant and rare taxa. The contribution of deterministic processes to the abundant taxa was higher than that of the rare taxa. The co-occurrence network revealed that abundant taxa occupied more central nodes; however, their role as key species still needs to be explored. The increase in bacterial community diversity has a significant effect on stabilizing ecological networks. In the future, we will further explore the dynamic changes and interaction patterns of the species in the rhizosphere soil affected by tea tree diseases, and their ecological functions and importance in areas of habitat fragmentation. Especially in karst areas with a high degree of fragmentation, paying attention to the functional genes of microorganisms in plant rhizosphere microecology and their contribution to soil ecological processes is of great significance for enhancing the environmental carrying capacity of karst areas. Understanding the interactions between plant health and soil microbial communities is crucial for designing novel disease-preventive measures and sustainable agricultural practices. Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2 |
Mechanisms of Ion Transport Across the Mouse Retinal Pigment Epithelium Measured In Vitro | 6f2640f8-ac44-4cff-874c-e349759ed6cf | 7416899 | Physiology[mh] | Experimental Animals and Tissue Preparation Healthy adult mice (C57Bl/6J), purchased from Taconic Denmark (Silkeborg, Denmark) were kept on a regular 12:12 light/dark cycle. All animal experiments were approved by the Icelandic Food and Veterinary Authority (MAST license numbers 0112-0101 and 0312-0102), and all experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animals were sacrificed at about the same time in the morning, as it is known that circadian rhythms and light/dark entrainment can affect the RPE short-circuit current. One animal at a time was anesthetized with carbon dioxide (CO 2 ) in a closed box for 15 to 20 seconds; thereafter, a cervical dislocation was performed, and the carotid arteries were severed. Whiskers were removed, and the eyes were enucleated and placed in a Petri dish with a cold Krebs solution. Using a 0.45 × 12-mm Sterican needle (B. Braun, Melsungen, Germany), a small puncture was then made in the cornea of one eye under a microscope, and the cornea and the iris were removed with incisions along the border of the pars plana. The lens was gently removed by pressure to avoid retinal detachment. Details of the Ussing chamber recording system are shown in . The RPE, together with the retina, choroid and sclera, was mounted in miniature epithelial chambers with an aperture of 0.031 cm 2 (EasyMount; Physiologic Instruments Inc., San Diego, CA, USA), depicted in B and C, with a normal Krebs solution on both sides kept at 38°C by a thermocirculator (Heto Lab Equipment, Allerod, Denmark) and aerated with an air mixture of 5% CO 2 and 95% O 2 . Drugs and Solutions Adenosine triphosphate (ATP) was purchased from Tocris Bioscience (Oxford, UK), and epinephrine, bumetanide, and ouabain were purchased from Sigma-Aldrich (St. Louis, MO, USA). The normal Krebs solution had the following final bath ion concentration (mmol/L): Na + , 138.6; K + , 4.6; Ca 2+ , 2.5; Mg 2+ , 1.2; Cl – , 125.1; HCO 3 – , 21.9; PO4 2 – , 1.2; SO4 2 – , 1.2; and glucose, 11.1. A K + -free Krebs solution was made by replacing K + ions with Na + , with the following composition (mmol/L): Na + , 143.2; K + , 0.0; Ca 2+ , 2.5; Mg 2+ , 1.2; Cl – , 125.1; HCO 3 – , 21.9; PO 4 2 – , 1.2; SO 4 2 – , 1.2; and glucose, 11.1. The osmolarity of these two types of Krebs solutions was the same and measured to be 295 ± 5 mOsm. All solutions were aerated with an air mixture of 5% CO 2 and 95% O 2 , with pH maintained at 7.4. Electrophysiological Recordings Two pairs of silver chloride (AgCl) electrodes placed in agar bridges, made using Krebs without glucose, were used for the Ussing chamber recordings ( A, B). One pair of electrodes was used to measure the TEP across the tissue. The second pair of electrodes was used to pass a current to clamp the TEP at zero millivolts ( B). A DVC-1000 Voltage/Current Clamp unit (World Precision Instruments, Sarasota, FL, USA) ( A) was used for voltage clamping and measurements of the resulting I SC . The DVC-1000 unit was programmed to pass a current through the tissue, causing a 1-mV deflection in the TEP every 5th minute, and the resulting deflections in TEP and I SC were used to calculate TER, according to Ohm’s law. The TEP was measured in millivolts (mV), the I SC in µA/cm 2 , and TER in ohm·cm 2 . The output from the DVC-1000 unit was digitized by an analog/digital converter unit (PowerLab 16/30; ADInstruments, Dunedin, New Zealand) ( A), and the data were collected by LabChart 7 data acquisition software (ADInstruments). Experimental Protocols Both eyes of each animal were used, but only one eye from each animal was used for each experimental series. The number of mice tested in each experimental series was six to eight. Each control period or treatment period lasted 30 minutes. The recording of the I SC was continuous, but for data analysis we took an average of a 10-second period just before each current pulse (and an average of the I sc during the pulse itself), which provided six data points for each 30-minute period to use for statistical analysis and to plot as means in figures. In all experiments, the tissue was allowed to stabilize in the Ussing chamber for at least 30 minutes, after which a control period (Control 1) was recorded for 30 minutes, except in the first experiment depicted in , where the initial control period lasted 2 hours. In the second set of experiments, there was only one initial control period followed by 30-minute drug periods . In the later ion exchange and barium experiments, a second control period was added at the end, where the depleted ions were replenished again and the drugs were washed out . In the first experiment depicted in , which was to test the viability of the retina/RPE/choroid/sclera preparation under the normal conditions of the Ussing chamber environment, the preparation was left without any experimental manipulation for 2 hours, after which the Krebs solution was replaced with a fresh solution; finally, 1-mM ouabain was added to the apical solution. In the second set of experiments, after the initial control period, the following substances were added in the order listed: ATP (100 µM), epinephrine (100 µM), bumetanide (100) µM), and ouabain (1 mM). All of the experimental substances were added to both the apical and basolateral side of the tissue. In the ion exchange and BaCl 2 experiments, after the initial control period (Control 1), the normal Krebs solution was washed off three times with K + -free Krebs. After 30 minutes of recording, these solutions were replaced three times with fresh normal Krebs, and the I SC and TER were recorded for another 30 minutes (Control 2). After Control 2, we examined the effect of adding the potassium channel blocker BaCl 2 to both sides with the tissue bathed in the normal Krebs, after which we changed to K + -free Krebs, followed by a Control 3 period with normal Krebs only. In the data analysis, we treated these treatments separately for the sake of clarity, such that the first ion exchange experiments without BaCl 2 were treated as one experimental series, and the second ion exchange experiments with BaCl 2 were treated as another experimental series. Thus, the Control 2 period of the ion exchange experiments without BaCl 2 is the same period as the Control 1 period of the ion exchange experiments with BaCl 2 . It was found that the absolute values of the I SC were variable, resulting in large deviations of the means; therefore, all of our tests of significance are based on paired t -tests. We compared the measured values of I SC and TER at each time point of the experimental period to regressed values at the same time point calculated from regression lines derived from the previous period, or between two control periods (see Data Analysis), rather than, for example, comparing the last time point of the previous period with the last time point of the experimental period, which would have led to erroneous conclusions due to the time factor involved. The regression method coupled with the paired t -test is, in our opinion, a simple but powerful method to eliminate both the time factor and the variability of the absolute I SC and TER values, particularly when using two control periods, one before and one after the experimental periods, as done in the later experiments presented here. Thus, all statistical P values hereafter refer only to comparisons between the measured I sc and TER values and the corresponding extrapolated control values along the control regression line at the same time points. Data Analysis In preliminary experiments, it was observed that the I SC and TER of the RPE preparation generally changed slowly with time, which is a common problem encountered with measurements of epithelial tissue in Ussing chambers, possibly due to degeneration of the cells in the tissue. To control for the time factor, the effects of each treatment (drug or ion exchange) were evaluated by comparing the measured I SC and TER values with calculated I SC and TER values at the same time points according to a linear regression line extrapolated either from the previous control or experimental period (first and second experiments) or between the Control 1 and Control 2 periods (all other experiments). The values used for the regression line in each case were either all of the points of the previous period or the last three points of the Control 1 period plus the last three points of the Control 2 period. These methods are depicted graphically in A. The difference at each time point between the measured I SC and TER and the values calculated from the slope and intercept of the control regression line were tested by the paired option of the Student's t -test in Excel 2010 (Microsoft, Inc., Redmond, WA, USA). A value of P below 0.05 was considered significant for a treatment. In some cases, the mean ± SEM slopes of the I SC are reported with paired t -test comparisons between control and experimental periods. Also, the percentage change in either I SC or TER was calculated in some cases, based on the deviation of the measured values at each time point from the extrapolated values derived from the control regression lines. The figures were made using SigmaPlot Version 13 (Systat Software, Inc., San Jose, CA, USA). All results are reported or depicted as means ± SEM.
Healthy adult mice (C57Bl/6J), purchased from Taconic Denmark (Silkeborg, Denmark) were kept on a regular 12:12 light/dark cycle. All animal experiments were approved by the Icelandic Food and Veterinary Authority (MAST license numbers 0112-0101 and 0312-0102), and all experimental procedures adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animals were sacrificed at about the same time in the morning, as it is known that circadian rhythms and light/dark entrainment can affect the RPE short-circuit current. One animal at a time was anesthetized with carbon dioxide (CO 2 ) in a closed box for 15 to 20 seconds; thereafter, a cervical dislocation was performed, and the carotid arteries were severed. Whiskers were removed, and the eyes were enucleated and placed in a Petri dish with a cold Krebs solution. Using a 0.45 × 12-mm Sterican needle (B. Braun, Melsungen, Germany), a small puncture was then made in the cornea of one eye under a microscope, and the cornea and the iris were removed with incisions along the border of the pars plana. The lens was gently removed by pressure to avoid retinal detachment. Details of the Ussing chamber recording system are shown in . The RPE, together with the retina, choroid and sclera, was mounted in miniature epithelial chambers with an aperture of 0.031 cm 2 (EasyMount; Physiologic Instruments Inc., San Diego, CA, USA), depicted in B and C, with a normal Krebs solution on both sides kept at 38°C by a thermocirculator (Heto Lab Equipment, Allerod, Denmark) and aerated with an air mixture of 5% CO 2 and 95% O 2 .
Adenosine triphosphate (ATP) was purchased from Tocris Bioscience (Oxford, UK), and epinephrine, bumetanide, and ouabain were purchased from Sigma-Aldrich (St. Louis, MO, USA). The normal Krebs solution had the following final bath ion concentration (mmol/L): Na + , 138.6; K + , 4.6; Ca 2+ , 2.5; Mg 2+ , 1.2; Cl – , 125.1; HCO 3 – , 21.9; PO4 2 – , 1.2; SO4 2 – , 1.2; and glucose, 11.1. A K + -free Krebs solution was made by replacing K + ions with Na + , with the following composition (mmol/L): Na + , 143.2; K + , 0.0; Ca 2+ , 2.5; Mg 2+ , 1.2; Cl – , 125.1; HCO 3 – , 21.9; PO 4 2 – , 1.2; SO 4 2 – , 1.2; and glucose, 11.1. The osmolarity of these two types of Krebs solutions was the same and measured to be 295 ± 5 mOsm. All solutions were aerated with an air mixture of 5% CO 2 and 95% O 2 , with pH maintained at 7.4.
Two pairs of silver chloride (AgCl) electrodes placed in agar bridges, made using Krebs without glucose, were used for the Ussing chamber recordings ( A, B). One pair of electrodes was used to measure the TEP across the tissue. The second pair of electrodes was used to pass a current to clamp the TEP at zero millivolts ( B). A DVC-1000 Voltage/Current Clamp unit (World Precision Instruments, Sarasota, FL, USA) ( A) was used for voltage clamping and measurements of the resulting I SC . The DVC-1000 unit was programmed to pass a current through the tissue, causing a 1-mV deflection in the TEP every 5th minute, and the resulting deflections in TEP and I SC were used to calculate TER, according to Ohm’s law. The TEP was measured in millivolts (mV), the I SC in µA/cm 2 , and TER in ohm·cm 2 . The output from the DVC-1000 unit was digitized by an analog/digital converter unit (PowerLab 16/30; ADInstruments, Dunedin, New Zealand) ( A), and the data were collected by LabChart 7 data acquisition software (ADInstruments).
Both eyes of each animal were used, but only one eye from each animal was used for each experimental series. The number of mice tested in each experimental series was six to eight. Each control period or treatment period lasted 30 minutes. The recording of the I SC was continuous, but for data analysis we took an average of a 10-second period just before each current pulse (and an average of the I sc during the pulse itself), which provided six data points for each 30-minute period to use for statistical analysis and to plot as means in figures. In all experiments, the tissue was allowed to stabilize in the Ussing chamber for at least 30 minutes, after which a control period (Control 1) was recorded for 30 minutes, except in the first experiment depicted in , where the initial control period lasted 2 hours. In the second set of experiments, there was only one initial control period followed by 30-minute drug periods . In the later ion exchange and barium experiments, a second control period was added at the end, where the depleted ions were replenished again and the drugs were washed out . In the first experiment depicted in , which was to test the viability of the retina/RPE/choroid/sclera preparation under the normal conditions of the Ussing chamber environment, the preparation was left without any experimental manipulation for 2 hours, after which the Krebs solution was replaced with a fresh solution; finally, 1-mM ouabain was added to the apical solution. In the second set of experiments, after the initial control period, the following substances were added in the order listed: ATP (100 µM), epinephrine (100 µM), bumetanide (100) µM), and ouabain (1 mM). All of the experimental substances were added to both the apical and basolateral side of the tissue. In the ion exchange and BaCl 2 experiments, after the initial control period (Control 1), the normal Krebs solution was washed off three times with K + -free Krebs. After 30 minutes of recording, these solutions were replaced three times with fresh normal Krebs, and the I SC and TER were recorded for another 30 minutes (Control 2). After Control 2, we examined the effect of adding the potassium channel blocker BaCl 2 to both sides with the tissue bathed in the normal Krebs, after which we changed to K + -free Krebs, followed by a Control 3 period with normal Krebs only. In the data analysis, we treated these treatments separately for the sake of clarity, such that the first ion exchange experiments without BaCl 2 were treated as one experimental series, and the second ion exchange experiments with BaCl 2 were treated as another experimental series. Thus, the Control 2 period of the ion exchange experiments without BaCl 2 is the same period as the Control 1 period of the ion exchange experiments with BaCl 2 . It was found that the absolute values of the I SC were variable, resulting in large deviations of the means; therefore, all of our tests of significance are based on paired t -tests. We compared the measured values of I SC and TER at each time point of the experimental period to regressed values at the same time point calculated from regression lines derived from the previous period, or between two control periods (see Data Analysis), rather than, for example, comparing the last time point of the previous period with the last time point of the experimental period, which would have led to erroneous conclusions due to the time factor involved. The regression method coupled with the paired t -test is, in our opinion, a simple but powerful method to eliminate both the time factor and the variability of the absolute I SC and TER values, particularly when using two control periods, one before and one after the experimental periods, as done in the later experiments presented here. Thus, all statistical P values hereafter refer only to comparisons between the measured I sc and TER values and the corresponding extrapolated control values along the control regression line at the same time points.
In preliminary experiments, it was observed that the I SC and TER of the RPE preparation generally changed slowly with time, which is a common problem encountered with measurements of epithelial tissue in Ussing chambers, possibly due to degeneration of the cells in the tissue. To control for the time factor, the effects of each treatment (drug or ion exchange) were evaluated by comparing the measured I SC and TER values with calculated I SC and TER values at the same time points according to a linear regression line extrapolated either from the previous control or experimental period (first and second experiments) or between the Control 1 and Control 2 periods (all other experiments). The values used for the regression line in each case were either all of the points of the previous period or the last three points of the Control 1 period plus the last three points of the Control 2 period. These methods are depicted graphically in A. The difference at each time point between the measured I SC and TER and the values calculated from the slope and intercept of the control regression line were tested by the paired option of the Student's t -test in Excel 2010 (Microsoft, Inc., Redmond, WA, USA). A value of P below 0.05 was considered significant for a treatment. In some cases, the mean ± SEM slopes of the I SC are reported with paired t -test comparisons between control and experimental periods. Also, the percentage change in either I SC or TER was calculated in some cases, based on the deviation of the measured values at each time point from the extrapolated values derived from the control regression lines. The figures were made using SigmaPlot Version 13 (Systat Software, Inc., San Jose, CA, USA). All results are reported or depicted as means ± SEM.
Baseline Electrophysiological Values In the series of experiments with only normal Krebs present for 2 hours, n = 6 , there was no significant change in either the I SC or TER over this period of time. The mean I SC of the last 50 minutes was −7.7 ± 2.7 µA/cm 2 , with the basolateral side being negative, and the mean TER was 41.2 ± 4.8 ohm·cm 2 . These values indicate a low TEP, 0.32 mV (apical side positive), based on Ohm's law. After a 2-hour bath, the normal Krebs solution was replaced with a fresh normal Krebs solution, which caused no change in the I SC and only a small decrease in TER (about 3 ohm·cm 2 ). In most, if not all, of the species studied an active Na/K-ATPase-dependent mechanism has been demonstrated on the apical side of the retinal pigment epithelium, which can be effectively blocked by ouabain. We therefore tested the viability of the RPE preparation by adding ouabain to the apical side. This resulted in a rapid and large increase in the negative I SC , which nearly tripled from −8.14 ± 2.96 to −22.66 ± 6.34 µA/cm 2 in about 5 minutes, after which the I SC decreased and reached −4.16 ± 2.21 µA/cm 2 after 30 minutes, which was significantly below the control regression line ( P = 0.011). Ouabain caused TER to increase steadily, and after 30 minutes it had increased significantly to 52.46 ± 7.70 ohms·cm 2 ( P = 0.007). These results indicate that the RPE preparation was in good condition for 165 minutes and responsive during that time to drugs, as demonstrated by the ouabain response . We also calculated the means of I SC and TER of the Control 1 periods of all of the mouse preparations that were tested with drugs or ion exchange, and these values are shown in . The mean I SC ranged between 4 and 18 µA/cm 2 , and the mean TER ranged between 44 and 86 ohm·cm 2 , which resulted in a low calculated TEP in almost all of the experiments, around 1 mV . We found that the stable I SC measured in the control series shown in was the exception rather than the rule, as it was more common that the I SC decreased slowly with time, as evident from the experimental series shown in to . This has previously been observed in bovine RPE tissue. In our case, the decrease in I SC was 0.078 ± 0.033 µA/cm 2 ·hr −1 with an intercept at −14.0 ± 4.1 µA/cm 2 (mean slopes of all Control 1 periods). This phenomenon was taken into account in our data analysis (see Methods). Although TER was found to be generally steady during the control periods of most experiments, we analyzed the TER data in a similar way. Effects of Purinergic and Adrenergic Agonists, and Transporter Blockers In the second series of experiments (n = 7), we tested four drugs in an additive manner, with only one control period at the beginning. Each drug tested was applied simultaneously on both the apical and basolateral sides. The results are here depicted for each substance in to , where the control values for each substance were obtained during the 30-minute period immediately before the addition of that substance. As shows, there was a steady but slow decrease in the negative I SC during the control period, and this decrease seemed to continue during the entire course of these experiments, independent of the effects of drug applications. The first drug tested was ATP (100 µM). As can be seen from B, the slope of the negative I SC decreased significantly from 0.097 ± 0.046 to 0.041 ± 0.028 µA/cm 2 /min ( P = 0.040), indicating that the ATP induced an increase in the I SC , which counteracted the underlying decrease in the I SC . When the measured I SC values were compared by paired t -test to the regressed values at the same time points, the difference between these values was highly significant within 6 minutes ( P = 0.018), and this difference increased as time progressed, reaching 2.22 ± 0.41 µA/cm 2 after 30 minutes ( P = 0.003). This amounts to a 24% increase. ATP had no effect on TER ( C). We then added 100-µM epinephrine to both sides of the tissue with ATP still present, as shown in A, and the tissue was left in the bath for an additional 30 minutes. Epinephrine caused a small but rapid increase in the I SC of 1.14 ± 0.19 µA/cm 2 ( P = 0.011), which lasted for 30 minutes. No further change occurred, as evidenced by no difference in the slope of the I SC over time, which remained at 0.046 ± 0.031 µA/cm 2 /min. There was no significant change in TER during epinephrine application ( B). Bumetanide (100 µM) was then added with both ATP and epinephrine present in the Ussing chambers to block the Na-K-2Cl transporter. , It induced a reduction in the I SC , which reached significance after 12 minutes ( P = 0.009), with a decrease of 1.72 ± 0.73 µA/cm 2 after 30 minutes ( P = 0.027). The slope increased from 0.052 ± 0.015 to 0.130 ± 0.024 µA/cm −2 /min ( P = 0.022). The blocker did not induce any significant changes in TER . Finally, ouabain (1 mM) was added to the Ussing chambers and to both sides, as with the previous drugs. As B shows, there was a biphasic response as observed in a previous experiment , with I SC increasing rapidly in 6 minutes from −7.9 ± 2.4 to −15.49 ± 2.12 µA/cm 2 , which is an increase of 6.92 ± 2.76 µA/cm 2 ( P = 0.023). A gradual decrease followed thereafter and reached −3.7 ± 2.2 ( P = 0.031) after 25 minutes. Ouabain also induced an increase in mean TER, from 83.8 ± 8.2 to 99.8 ± 6.0 ohm·cm 2 in 30 minutes ( P = 0.017) ( C). Role of Potassium Channels To mimic the light-induced changes in the subretinal space, which has been found to induce a decrease in subretinal [K + ] o from 5 to 2 mM, we reduced the apical [K + ] o to 0 mM. The results are shown in B (n = 8). This caused a transient significant increase in the negative I SC during the first 5 minutes by 3.36 ± 1.06 µA/cm 2 (i.e., from −14.15 ± 4.37 to −17.43 ± 3.65 µA/cm 2 ; P = 0.008). This increase remained steady during a significant portion of the 30-minute period when the zero-K + Krebs solution was present in the bath. Returning the apical [K + ] o to normal levels evoked a change in the opposite direction, as the I SC decreased by 3.45 ± 1.60 µA/cm 2 at peak ( P = 0.034), which then returned slowly to baseline in 20 minutes. TER, which was 70.6 ± 8.9 ohm·cm 2 immediately before the change, first increased and then decreased significantly compared to the regression line ( P = 0.02), but the changes were relatively small, approximately 2 ohm·cm 2 in either direction ( C). We next examined the role of K + channels in the net transepithelial ion transport across the RPE preparation. After the RPE tissues had adjusted to the normal Krebs solution again for 30 minutes, we blocked barium-sensitive K + channels on both sides of the preparation by adding 1-mM BaCl 2 to the extracellular fluid. The I SC decreased from −10.4 ± 3.1 to −6.6 ± 1.8 µA/cm 2 ( P = 0.01), and TER increased from 74.8 ± 9.6 to 75.4 ± 8.7 in 30 minutes, a significant change compared to the regression line ( P = 0.03) . After a 30-minute period when barium was present in the bath, the normal Krebs solution was replaced with a 0-mM K + Krebs solution, keeping the barium concentration at 1 mM, to test for the role of barium-sensitive potassium channels in the observed response to apical K + -free solution. When analyzing this part of the experiment, the barium period was used as Control 1. The I SC decreased significantly from −5.65 ± 1.24 to −3.37 ± 0.79 µA/cm 2 ( P = 0.029) 10 minutes after switching to apical K + -free solution with barium, and it remained significantly different from the control regression line for the next 20 minutes ( A). TER decreased significantly and returned to the pre-barium level, as indicated by the regression line between the two control periods in B.
In the series of experiments with only normal Krebs present for 2 hours, n = 6 , there was no significant change in either the I SC or TER over this period of time. The mean I SC of the last 50 minutes was −7.7 ± 2.7 µA/cm 2 , with the basolateral side being negative, and the mean TER was 41.2 ± 4.8 ohm·cm 2 . These values indicate a low TEP, 0.32 mV (apical side positive), based on Ohm's law. After a 2-hour bath, the normal Krebs solution was replaced with a fresh normal Krebs solution, which caused no change in the I SC and only a small decrease in TER (about 3 ohm·cm 2 ). In most, if not all, of the species studied an active Na/K-ATPase-dependent mechanism has been demonstrated on the apical side of the retinal pigment epithelium, which can be effectively blocked by ouabain. We therefore tested the viability of the RPE preparation by adding ouabain to the apical side. This resulted in a rapid and large increase in the negative I SC , which nearly tripled from −8.14 ± 2.96 to −22.66 ± 6.34 µA/cm 2 in about 5 minutes, after which the I SC decreased and reached −4.16 ± 2.21 µA/cm 2 after 30 minutes, which was significantly below the control regression line ( P = 0.011). Ouabain caused TER to increase steadily, and after 30 minutes it had increased significantly to 52.46 ± 7.70 ohms·cm 2 ( P = 0.007). These results indicate that the RPE preparation was in good condition for 165 minutes and responsive during that time to drugs, as demonstrated by the ouabain response . We also calculated the means of I SC and TER of the Control 1 periods of all of the mouse preparations that were tested with drugs or ion exchange, and these values are shown in . The mean I SC ranged between 4 and 18 µA/cm 2 , and the mean TER ranged between 44 and 86 ohm·cm 2 , which resulted in a low calculated TEP in almost all of the experiments, around 1 mV . We found that the stable I SC measured in the control series shown in was the exception rather than the rule, as it was more common that the I SC decreased slowly with time, as evident from the experimental series shown in to . This has previously been observed in bovine RPE tissue. In our case, the decrease in I SC was 0.078 ± 0.033 µA/cm 2 ·hr −1 with an intercept at −14.0 ± 4.1 µA/cm 2 (mean slopes of all Control 1 periods). This phenomenon was taken into account in our data analysis (see Methods). Although TER was found to be generally steady during the control periods of most experiments, we analyzed the TER data in a similar way.
In the second series of experiments (n = 7), we tested four drugs in an additive manner, with only one control period at the beginning. Each drug tested was applied simultaneously on both the apical and basolateral sides. The results are here depicted for each substance in to , where the control values for each substance were obtained during the 30-minute period immediately before the addition of that substance. As shows, there was a steady but slow decrease in the negative I SC during the control period, and this decrease seemed to continue during the entire course of these experiments, independent of the effects of drug applications. The first drug tested was ATP (100 µM). As can be seen from B, the slope of the negative I SC decreased significantly from 0.097 ± 0.046 to 0.041 ± 0.028 µA/cm 2 /min ( P = 0.040), indicating that the ATP induced an increase in the I SC , which counteracted the underlying decrease in the I SC . When the measured I SC values were compared by paired t -test to the regressed values at the same time points, the difference between these values was highly significant within 6 minutes ( P = 0.018), and this difference increased as time progressed, reaching 2.22 ± 0.41 µA/cm 2 after 30 minutes ( P = 0.003). This amounts to a 24% increase. ATP had no effect on TER ( C). We then added 100-µM epinephrine to both sides of the tissue with ATP still present, as shown in A, and the tissue was left in the bath for an additional 30 minutes. Epinephrine caused a small but rapid increase in the I SC of 1.14 ± 0.19 µA/cm 2 ( P = 0.011), which lasted for 30 minutes. No further change occurred, as evidenced by no difference in the slope of the I SC over time, which remained at 0.046 ± 0.031 µA/cm 2 /min. There was no significant change in TER during epinephrine application ( B). Bumetanide (100 µM) was then added with both ATP and epinephrine present in the Ussing chambers to block the Na-K-2Cl transporter. , It induced a reduction in the I SC , which reached significance after 12 minutes ( P = 0.009), with a decrease of 1.72 ± 0.73 µA/cm 2 after 30 minutes ( P = 0.027). The slope increased from 0.052 ± 0.015 to 0.130 ± 0.024 µA/cm −2 /min ( P = 0.022). The blocker did not induce any significant changes in TER . Finally, ouabain (1 mM) was added to the Ussing chambers and to both sides, as with the previous drugs. As B shows, there was a biphasic response as observed in a previous experiment , with I SC increasing rapidly in 6 minutes from −7.9 ± 2.4 to −15.49 ± 2.12 µA/cm 2 , which is an increase of 6.92 ± 2.76 µA/cm 2 ( P = 0.023). A gradual decrease followed thereafter and reached −3.7 ± 2.2 ( P = 0.031) after 25 minutes. Ouabain also induced an increase in mean TER, from 83.8 ± 8.2 to 99.8 ± 6.0 ohm·cm 2 in 30 minutes ( P = 0.017) ( C).
To mimic the light-induced changes in the subretinal space, which has been found to induce a decrease in subretinal [K + ] o from 5 to 2 mM, we reduced the apical [K + ] o to 0 mM. The results are shown in B (n = 8). This caused a transient significant increase in the negative I SC during the first 5 minutes by 3.36 ± 1.06 µA/cm 2 (i.e., from −14.15 ± 4.37 to −17.43 ± 3.65 µA/cm 2 ; P = 0.008). This increase remained steady during a significant portion of the 30-minute period when the zero-K + Krebs solution was present in the bath. Returning the apical [K + ] o to normal levels evoked a change in the opposite direction, as the I SC decreased by 3.45 ± 1.60 µA/cm 2 at peak ( P = 0.034), which then returned slowly to baseline in 20 minutes. TER, which was 70.6 ± 8.9 ohm·cm 2 immediately before the change, first increased and then decreased significantly compared to the regression line ( P = 0.02), but the changes were relatively small, approximately 2 ohm·cm 2 in either direction ( C). We next examined the role of K + channels in the net transepithelial ion transport across the RPE preparation. After the RPE tissues had adjusted to the normal Krebs solution again for 30 minutes, we blocked barium-sensitive K + channels on both sides of the preparation by adding 1-mM BaCl 2 to the extracellular fluid. The I SC decreased from −10.4 ± 3.1 to −6.6 ± 1.8 µA/cm 2 ( P = 0.01), and TER increased from 74.8 ± 9.6 to 75.4 ± 8.7 in 30 minutes, a significant change compared to the regression line ( P = 0.03) . After a 30-minute period when barium was present in the bath, the normal Krebs solution was replaced with a 0-mM K + Krebs solution, keeping the barium concentration at 1 mM, to test for the role of barium-sensitive potassium channels in the observed response to apical K + -free solution. When analyzing this part of the experiment, the barium period was used as Control 1. The I SC decreased significantly from −5.65 ± 1.24 to −3.37 ± 0.79 µA/cm 2 ( P = 0.029) 10 minutes after switching to apical K + -free solution with barium, and it remained significantly different from the control regression line for the next 20 minutes ( A). TER decreased significantly and returned to the pre-barium level, as indicated by the regression line between the two control periods in B.
The electrical correlates of ion transport across the RPE have been recorded from a number of vertebrate species, although the mouse RPE has been a notable exception so far. Here, we establish that, despite the small size of the mouse eye, the I SC and TER of the RPE can be reliably recorded from a mouse retina/RPE/choroid/sclera preparation in the modified Ussing chamber system with an aperture of 0.031 cm 2 as used in this study. Both the I SC and TER recorded from the preparation were stable over more than 2 hours. The effects of a Na-K-ATPase blocker, ouabain, demonstrated that the RPE preparation was live and responsive after this long time. A slow, gradual reduction of the I SC occurred over time, comparable to what has been observed in long-term recordings from similar preparations. , , We took this natural decrease of the current into account in all our analyses. The present study, which, to our knowledge is the first to examine ion transport across the mouse RPE, has several limitations. The mean values of the I SC and TER are presented in . These values are rather low compared to preparations from other species and resulted in a low calculated TEP, around 1 mV, with the apical side being positive, indicative of a rather leaky preparation. Nevertheless, we found the preparation to be robust and responsive to various treatments that affect ion transport, lasting for 2 to 3 hours. One difference between the mouse preparation used in the present study and larger ones from other species used in previous work is that both the neuroretina on the apical side and choroid and sclera on the basolateral side were kept in situ. This may constitute a diffusion barrier to drugs reaching the RPE, and thus represents a limitation of our study, but the present results suggest that is not a serious hindrance to the use of this preparation, as most responses were rapid and within seconds. Because the preparation leaves the retina in situ, Müller cells are still present; spatial buffering of K + from the RPE to the vitreous side mediated by these glial cells is likely to occur and may have affected measurements of ion transport, which can be considered a limitation of the study. TER values presented in this study are calculated as ohms·cm 2 and are based on passing a current very briefly through the tissue every 5th minute during the experiments, causing a 1-mV deflection of the TEP from the clamped value of 0 mV. The corresponding shift in the I sc required to keep the TEP at 1 mV was then measured, and these deflections in the TEP and I sc were then used to calculate TER based on Ohm's law. The TEP, TER, and I sc values thus obtained can only be regarded as an indication of the non-clamped or open circuit values, as the ion transport capabilities and ion gradients across the tissue under voltage-clamped conditions are not the exact same as under an open circuit. The above calculations are also based on the assumption that there was no unnatural leakage through the preparation, which is probably not true but difficult to ascertain. We suspect that some leakage is the reason for the relatively low TEP values found and was perhaps caused by edge damage that may have occurred during mounting of the tissue in the Ussing chambers or was due to discreet damage caused by preparation of the tissue that was not visible in the dissecting microscope. The TER values calculated are based on the assumption that the surface area of the tissue was nearly the same as that of the opening area of the Ussing chamber, which was 0.0013 cm 2 . However, it is likely that the area of the RPE is actually larger than the opening, given that there are basal deep infoldings and apical microvilli that increase the surface area of the RPE and probably not to the same extent on the apical and basolateral surfaces. The TER values should thus be regarded as a close approximation of the actual transepithelial resistance in our preparation. summarizes the effect of the various treatments known to affect RPE ion transport that were examined in this study, in comparison with similar findings from other mammalian species. It should be noted that some of the studies referred to in combined recordings from the RPE in a classic Ussing chamber preparation similar to the one used here with intracellular recordings from individual RPE cells. , , , – ATP has been shown to increase transepithelial ion and fluid transport across the RPE measured in Ussing chambers – and to induce ionic currents in isolated rat RPE cells studied with the patch clamp technique. ATP is converted into adenosine monophosphate (AMP) by the eNPP family of enzymes, and AMP then dephosphorylates to adenosine by ecto-5′-nucleotidase, and this process has been shown to be present on the apical side of the RPE. This conversion of ATP is rapid; therefore, it cannot be ruled out that the effects of ATP were in part mediated by adenosine receptors, as well as P2 purine receptors. Both P2X and P2Y receptors, as well as adenosine receptors, have been found on RPE tissues in various animals. But, up to this point, it is not known which subtypes of P2Y receptors are expressed in the mouse RPE, and an extensive literature search suggests that this has not been examined. It has been shown that in both the bovine and human retinal pigment epithelia, epinephrine stimulates transport of fluid and ions such as Cl – and K + across the RPE via alpha-1 receptors located on the apical side. , , , It is believed to act in vivo as a paracrine signal from the retina to the RPE. , It has been known for some time that adrenergic receptors are present on RPE cells, and recently it has been shown that there are adrenergic nerve endings in close proximity to the basolateral membrane of the mouse RPE and β1 and β2 adrenergic receptors are present on mouse RPE cells. Unlike what has been found in rabbit, bovine, and human RPE, the effect of epinephrine on the mouse RPE short-circuit current was small, but significant, whereas there was no effect on TER. This is similar to what has been found in the rabbit RPE . Epinephrine also does not affect TER measured from the bovine RPE/choroid, but it lowers TER measured from a human fetal RPE/choroid preparation. , Based on these findings, our results suggest that epinephrine has a limited function as a paracrine signal affecting ion transport and fluid absorption in the mouse RPE. The effect of epinephrine on the RPE transport has been found in some species to be dependent on the apical Na-K-2Cl cotransporter, , as bumetanide blocks the adrenergic effect on the apical side of the RPE . This includes blockage of the effect of epinephrine on fluid transport across the bovine RPE. The effects of blocking the Na-K-2Cl cotransporter of the mouse RPE with bumetanide are comparable to its effects on the short-circuit current found in tissue from several other species, , , As found in the mouse, bumetanide induces little or no change in pigment epithelial total resistance in any of the species tested. The apical Na-K-2Cl cotransporter on the RPE serves a vital role in the transport of ions and fluid across the tissue. Under normal conditions, the bovine RPE has been found to absorb chloride across the apical membrane, and this absorption is blocked by apical bumetanide. The cotransporter also has been found to absorb K + across the apical membrane of the bovine RPE. Thus, the role of the Na-K-2Cl cotransporter in the RPE seems to vary between species , and the present results suggest it plays a lesser role in the function of the mouse RPE than in most of the other species examined, except the rabbit. It should be noted, however, that the Na-K-2Cl cotransporter, as well as other RPE ion transport mechanisms, may function differently under an open-circuit condition than the short-circuit condition that was applied in the present work. This may to some extent account for the variability in the results reported with bumetanide, as some of the previous studies were done under open-circuit conditions while measuring the TEP. Na/K-ATPase is very important for transepithelial ion transport across the RPE tissue, as it maintains the ionic homeostasis of the RPE cells. , – It is electrogenic; for example, it has been shown to create a membrane voltage of about 5 to 10 mV in the bovine RPE. This electrogenic effect is quickly removed by apically applied ouabain. In retinal pigment epithelia in general, the Na/K-ATPase is situated on the apical surface. , Blocking the Na/K-ATPase transporter with ouabain applied to either the apical side only or both sides of the mouse retina/RPE/choroid preparation evoked a two-phase response in the I SC . These results are comparable to what has been found in most other species when ouabain has been applied to the apical side of the RPE , , , , , – . It has been suggested that the first phase is due to blocking of the electrogenic current generated by the Na/K-ATPase activity, and the second phase is a slow run down of ionic gradients across the apical membrane leading to a gradual depolarization for hours. Thus, our results with ouabain are consistent with an apical Na/K-ATPase transporter being present on the mouse RPE that shows electrogenic activity. When ouabain is applied to the basolateral side of the RPE, there is no effect on the I SC or the TEP, which is consistent with a distribution of RPE Na/K-ATPase subunits being restricted to the apical membrane surface. , , As the experiment shown in demonstrates, these effects of ouabain are due to its blockage of apically located Na/K-ATPase. It should be emphasized that ATP, epinephrine, bumetanide, and ouabain were added to the bath sequentially, which may have to some extent altered the response of the tissue, but the effects of all of these (except for ouabain) on ion transport were reversible, so this approach is not likely to have affected the main conclusions of the study. Light stimulation induces a decrease in subretinal [K + ] o , caused by the hyperpolarization of the photoreceptors in response to light, which remains lowered to a great extent during maintained light. , It has been suggested that RPE chloride transport can be activated by alterations in K + concentration that occur in the subretinal space, particularly during transitions between light and dark. The effect of subretinal potassium on RPE transport is dependent on K + channels present on the apical membrane. We found that lowering the apical [K + ] o produced a pattern in the I SC similar to that observed in the TEP of a bovine RPE/choroid preparation. , Only minor effects on TER were found in these experiments. Although our results generally show a pattern similar to that of the bovine results, , they deviate somewhat, as the I SC did not decrease below previous levels but remained higher during most of the remainder of the experiment ( B). It seems that the transepithelial ion transport of the RPE is dependent on barium-sensitive K + channels, as indicated by the findings summarized in . We found that 1-mM barium on both sides of the mouse RPE preparation caused a significant decrease in I SC and a small increase in TER. These results are also comparable to changes found in TER measures from several other mammalian preparations . , Previous results from the bovine RPE/choroid preparation suggest that a Ba ++ -sensitive apical K + conductance is critical in mediating the RPE response to [K + ] o changes in the subretinal space. , , This was also the case in the present mouse RPE preparation, as barium not only blocked the response to lowering the apical K + concentration but also caused a further decrease in the I SC by approximately 35% ( A). Three main types of K + channels have been found on RPE cells of most species examined in patch-clamp experiments, although the heterogeneity of these channels is considerable: inward rectifier channels blocked by extracellular Ba ++ , outward rectifier voltage-gated K + channels (KCNQ/K channels), and Ca ++ -activated K + channels. , , – However, whole-cell patch-clamp recordings from cultured mouse RPE cells show that they primarily express a delayed rectifier outward K + current. Although the effects of Ba ++ on these K + currents in mouse RPE cells has not been examined, this may to some extent account for the differences in the effect of extracellular Ba ++ and lowering the apical [K + ] o on the I SC and TER recorded from the mouse retina/RPE/choroid/sclera preparation as compared to other species tested. , , , Isolated bovine and human RPE cells show a prominent inward rectifying current, with a conductance that is inversely dependent on the extracellular [K + ] o and is almost completely blocked by Ba ++ . , , Thus, there appears to be a correspondence between the types and relative expression of K + channels on RPE cells of the different species and the effect of extracellular Ba ++ on the I SC and TER. Taken together, the present work is, to our knowledge, the first study of ionic transport mechanisms across the mouse retinal pigment epithelium. It allows comparison with similar transport mechanisms in other species, despite differences in size. In addition, a successful and reliable mouse retina/RPE/choroid/sclera preparation can be utilized to examine RPE transport mechanisms in mice with known defects in RPE function. ATP seems to increase transepithelial ion transport across the mouse RPE, and lowering the apical potassium concentration produced results in the I sc similar to those seen previously in TEP. The most salient differences between the present results for mouse RPE and those for other species are that epinephrine appears to have a limited function as a paracrine signal, affecting ion transport and fluid absorption in the mouse RPE. Also, the apical Na-K-2Cl cotransporter may play a lesser role in its function than in most of the other species examined, apart from the rabbit. As found in RPE of other larger mammals, part of the transepithelial ion transport is dependent on potassium channels, which also are part of the response to low subretinal K + postulated to cause the light-induced effects on RPE ion transport. With the use of a modified Ussing chamber, with an aperture as small as 0.031 cm 2 in diameter, the mechanisms of ionic transport across the normal mouse RPE can be examined.
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A case of malignant melanoma, a possible primary site in the digit, with
systemic metastasis in a mini-Rex | c6896541-4d47-485d-821a-0d98f8154943 | 9791239 | Anatomy[mh] | The authors declare no potential conflicts of interest with respect to the publication of
this manuscript.
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Predictors of institutional delivery service utilization in Ethiopia: an umbrella review | 69c12959-e681-4451-b7e0-7251690a3311 | 11927173 | Surgical Procedures, Operative[mh] | Though maternal health improvement is one of the key priorities, approximately 287,000 women die following pregnancy and childbirth in 2020 worldwide, where 95% of these avoidable maternal deaths occurred in low- and middle-income countries (LMICs) . By 2030, the global MMR should be below 70 per 100,000 live births, with no country exceeding 140 per 100,000 live births . Even though there has been a 38% reduction in maternal mortality ratio (MMR) worldwide and a 40% reduction in sub-Saharan Africa (SSA) from 2000 to 2017, reaching these targets needs substantial effort. The reports of MMR in Ethiopia are inconsistent. In 2020, for example, the World Bank Groups reported an MMR of 267 per 100,000 live births ; however, the national study by Geleto et al. showed 149 per 100,000 live births in Ethiopia during the same year . In 2019, Mekonnen et al. reported that the maternal mortality ratio in rural Ethiopia was 326 deaths for every 100,000 live births . Further, the goal set in 2017 during the launching of the quality network to lower MMR from 412 to 199 per 100,000 live births by 2020 has not been achieved and achieving the sustainable development goal (SDG) targets by 2030 will be challenging. Thus, the impact of socioeconomic status, parity, and living in rural areas , as well as political upheavals, insurgencies, and economic crises [ – ], famine, and epidemics , as well as the fact that most births occur outside of health facilities, that few women receive antenatal care (ANC), and that less than half of births are attended by skilled birth attendants in Ethiopia, are some of the reasons for this. Maternal health service utilization, such as family planning, preconception care, ANC, institutional delivery, postnatal care, and vaccination reduce maternal deaths significantly . An institutional delivery service utilization, in particular, is one of the key strategies to reduce maternal mortality and ensure safe births. However, there is a lack of access to medical care during pregnancy in SSA , which resulted in low utilization of facility delivery services and the majority of pregnant women across SSA including Ethiopia, still deliver at home. Hence, maternal health service coverage in all areas of the country needs to be ensured to achieve the ambitious goals of SDGs. As a result, Ethiopia is working with partners to address access to quality maternal and newborn healthcare services inequalities, ensuring universal health coverage, tackling all causes of maternal mortality, strengthening health information systems, and ensuring accountability . To this date, the recent pooled analysis reported a varying proportion of institutional delivery service utilization, with studies showing 23.09% by Dessie et al. , 31% by Nugusie et al. , and 21% by Tenaw et al. compared to the mini Ethiopian demographic health survey (EDHS) 2019 and the 2019 Performance Monitoring for Action survey data set reported the same outcome as 48% and 54.49% , respectively. Likewise, there is inconclusive reporting about the effects of different predisposing, enabling, and need factors on institutional delivery service utilization. Dessie et al., for example, found that ANC attendance, having information about maternal health service fee exemption, short distance to the health facility, and attending formal education were predictors of a health facility delivery in Ethiopia, while Tenaw et al. reported husband involvement in the decision of where to deliver, good knowledge of MCH services among women, having a positive attitude toward MCH services, and availability of health institutions . Moreover, Nugusie et al. reported maternal age at first pregnancy, place of residence, frequency of ANC follow-up, knowledge of danger signs during pregnancy, advantages of institutional delivery, and the place of birth of the elder child , and Kebede et al. reported experiencing a problem during pregnancy as predictors of a health facility delivery in Ethiopia. Hence, it is tiresome for information users to design appropriate interventions and decision-making. The existing systematic review and meta-analysis (SRMA) studies were mainly conducted using different primary studies in Ethiopia. To the best of our knowledge, despite the importance of predictors of institutional delivery service utilization, no synthesis of reviews to date has systematically investigated institutional delivery service utilization and its associated factors and no comprehensive review has yet synthesized the relevant evidence. Since there are multiple reviews, it is essential to perform an umbrella review to compile them by examining the current systematic review and meta-analysis on the use of institutional delivery services in order to find any gaps and build solid evidence. Thus, the present umbrella review allows summarizing all available data on predictors of institutional delivery service utilization into a concise study, making it the source of evidence-based knowledge to the highest degree. To inform policy and decision-making, identify potential solutions, and contribute to developing better strategies to address challenges, a comprehensive understanding of predictors is needed. Hence, the objective of this umbrella review is to identify predictors of institutional delivery service utilization in Ethiopia, its prevalence, and research gaps.
An umbrella review of multiple systematic review methodology was used to conduct this umbrella review . A systematic synthesis of eligible systematic review and meta-analysis studies on institutional delivery service utilization and its associated factors in Ethiopia was undertaken. We conducted this umbrella review to cover the different areas and manage the large volume of research that has been conducted on the predictors of institutional delivery service utilization. The protocol of this umbrella review was prepared, but not registered. Information sources As many databases as possible were searched, especially the main ones such as Medline or PubMed, Embase, Web of Science, CINAHL, Scopus, Google Scholar, and databases specific to systematic review such as the Cochrane Database of Systematic Reviews and the Database of Abstracts of Reviews of Effects, were conducted for SRMA studies on institutional delivery service utilization in Ethiopia as it exploits the chance of gathering all relevant studies. A comprehensive search for an umbrella review encompassed a search for gray literature, and reports using key terms in the title or abstract fields and PICO questions that developed from the following search keywords and/or Medical Subject Headings (MeSH), which combined using the ‘OR’ and ‘AND’ Boolean operators. Population pregnant women, laboring women, postpartum women, intrapartum women, women in labor, reproductive age women, women of reproductive age, 15–49 year women. Outcome institutional delivery, institutional delivery service utilization, place of delivery, health facility delivery, facility-based delivery, maternal health service utilization, use of skilled birth attendants, skilled delivery, birth at a health facility, predictors, determinants, associated factors, correlates, risk factors, influencing factors, related factors. Study design systematic review and meta-analysis. Setting/Context Ethiopia. Literature searches of published and unpublished studies were conducted from June 2024 until July 2024. Manual searches through the references of the submitted studies to find more research that may not be found by the database’s algorithms were performed. Two independent researchers conducted the literature search, and discrepancies were resolved by discussion and consensus. The detailed search filters were employed sequentially in the appendix for all the databases searched using a combination of MeSH terms and free texts along with the search dates (Additional file ). Eligibility criteria A systematic review and meta-analysis were deemed eligible if it (a) presented a defined literature search strategy, (b) appraised its included studies using a relevant tool, c) reported either the prevalence of institutional delivery service utilization or predictors of institutional delivery service utilization, (d) followed a standard approach in pooling studies and providing summaries estimates, and e) included research synthesis of all quantitative design systematic reviews, and meta-analysis. Publications from January 1990–July 2024 were eligible for inclusion. On the other hand, narrative reviews, editorials, abstracts, and methodological studies were not eligible. Study selection Endnote version VIII was used to download and remove duplicates. To select studies, the following steps were used: (a) the studies screened using title and abstract for full-text reviewing and (b) full-text reviews were conducted to identify potentially eligible studies. Independent researchers (KS and GT) conducted these two steps. A third researcher (HB) reviewed and resolved the disagreement where a consensus could not be reached between the researchers. Data extraction To reduce the risk of bias in the umbrella review process, two reviewers independently extracted the required data from eligible SRMA using a JBI standardized data extraction form for systematic review and research synthesis . This was developed in an Excel spreadsheet. Once the SRMAs to be included were agreed; disagreement on data extraction was resolved by consensus. Guided by the data extraction tool, the researcher independently extracted the following information: (a) author’s name and publication year, (b) aim, (c) proportion or coverage of institutional delivery service utilization, (d) predictors of institutional delivery service utilization, (e) odds ratio or relative risk with 95% confidence intervals for predictors, (f) number of primary studies included within each SRMA study and their respective design type, (g) total number of sample size included, (h) publication bias assessment methods and scores, (i) quality assessment methods and scores, (j) method of data synthesis and (k) the authors’ main conclusion (see Table ). Data items Institutional delivery service utilization is the outcome variable. It is defined as the proportion of index births delivered in health facilities by skilled birth attendants preceding the surveys/studies. Risk of bias assessment To ensure the methodological and evidence quality of the studies, SRMAs that are eligible for inclusion in an umbrella review are critically appraised for validity scoring of their results using the Assessment of Multiple System Review (AMSTAR) tool . The SRMA pooled empirical studies and their summary estimates were evaluated using the tool consisting of 11 questions that measure the quality of the approach used. Thus, quality scores 8 to 11, 4 to 7, and less than 3 indicate high, medium, and low, respectively (see Table ). Two independent reviewers conducted the appraisal, and the decision whether to include a review was made based on these quality scores. Statistical analysis Overall, statistical analysis and data synthesis were conducted using STATA version 16.1 software. Researchers presented a range of estimates, including calculating a pooled estimate when two or more estimates were provided on the prevalence and predictors of institutional delivery service utilization. The between-studies heterogeneity that was assessed using Higgin’s I 2 statistics guided the choice of the meta-analysis model. Since there was a high level of difference between studies, the DerSimonian-laird random-effect model was used to pool the estimates . A summary list of the predictors of institutional delivery service utilization (i.e., dichotomous outcome) with their respective odds ratios and 95% confidence interval was prepared. Only five SRMAs were included in this umbrella review; it was not possible to assess publication bias since a minimum of ten studies is needed to evaluate publication bias . When two SRMA included the same primary studies, we calculated corrected covered area (CCA) suggested by Pieper et al. to quantify the degree of overlap in umbrella review; we assessed and documented primary studies’ degree of overlapping as guidelines recommend .
As many databases as possible were searched, especially the main ones such as Medline or PubMed, Embase, Web of Science, CINAHL, Scopus, Google Scholar, and databases specific to systematic review such as the Cochrane Database of Systematic Reviews and the Database of Abstracts of Reviews of Effects, were conducted for SRMA studies on institutional delivery service utilization in Ethiopia as it exploits the chance of gathering all relevant studies. A comprehensive search for an umbrella review encompassed a search for gray literature, and reports using key terms in the title or abstract fields and PICO questions that developed from the following search keywords and/or Medical Subject Headings (MeSH), which combined using the ‘OR’ and ‘AND’ Boolean operators. Population pregnant women, laboring women, postpartum women, intrapartum women, women in labor, reproductive age women, women of reproductive age, 15–49 year women. Outcome institutional delivery, institutional delivery service utilization, place of delivery, health facility delivery, facility-based delivery, maternal health service utilization, use of skilled birth attendants, skilled delivery, birth at a health facility, predictors, determinants, associated factors, correlates, risk factors, influencing factors, related factors. Study design systematic review and meta-analysis. Setting/Context Ethiopia. Literature searches of published and unpublished studies were conducted from June 2024 until July 2024. Manual searches through the references of the submitted studies to find more research that may not be found by the database’s algorithms were performed. Two independent researchers conducted the literature search, and discrepancies were resolved by discussion and consensus. The detailed search filters were employed sequentially in the appendix for all the databases searched using a combination of MeSH terms and free texts along with the search dates (Additional file ).
pregnant women, laboring women, postpartum women, intrapartum women, women in labor, reproductive age women, women of reproductive age, 15–49 year women.
institutional delivery, institutional delivery service utilization, place of delivery, health facility delivery, facility-based delivery, maternal health service utilization, use of skilled birth attendants, skilled delivery, birth at a health facility, predictors, determinants, associated factors, correlates, risk factors, influencing factors, related factors.
systematic review and meta-analysis.
Ethiopia. Literature searches of published and unpublished studies were conducted from June 2024 until July 2024. Manual searches through the references of the submitted studies to find more research that may not be found by the database’s algorithms were performed. Two independent researchers conducted the literature search, and discrepancies were resolved by discussion and consensus. The detailed search filters were employed sequentially in the appendix for all the databases searched using a combination of MeSH terms and free texts along with the search dates (Additional file ).
A systematic review and meta-analysis were deemed eligible if it (a) presented a defined literature search strategy, (b) appraised its included studies using a relevant tool, c) reported either the prevalence of institutional delivery service utilization or predictors of institutional delivery service utilization, (d) followed a standard approach in pooling studies and providing summaries estimates, and e) included research synthesis of all quantitative design systematic reviews, and meta-analysis. Publications from January 1990–July 2024 were eligible for inclusion. On the other hand, narrative reviews, editorials, abstracts, and methodological studies were not eligible.
Endnote version VIII was used to download and remove duplicates. To select studies, the following steps were used: (a) the studies screened using title and abstract for full-text reviewing and (b) full-text reviews were conducted to identify potentially eligible studies. Independent researchers (KS and GT) conducted these two steps. A third researcher (HB) reviewed and resolved the disagreement where a consensus could not be reached between the researchers.
To reduce the risk of bias in the umbrella review process, two reviewers independently extracted the required data from eligible SRMA using a JBI standardized data extraction form for systematic review and research synthesis . This was developed in an Excel spreadsheet. Once the SRMAs to be included were agreed; disagreement on data extraction was resolved by consensus. Guided by the data extraction tool, the researcher independently extracted the following information: (a) author’s name and publication year, (b) aim, (c) proportion or coverage of institutional delivery service utilization, (d) predictors of institutional delivery service utilization, (e) odds ratio or relative risk with 95% confidence intervals for predictors, (f) number of primary studies included within each SRMA study and their respective design type, (g) total number of sample size included, (h) publication bias assessment methods and scores, (i) quality assessment methods and scores, (j) method of data synthesis and (k) the authors’ main conclusion (see Table ).
Institutional delivery service utilization is the outcome variable. It is defined as the proportion of index births delivered in health facilities by skilled birth attendants preceding the surveys/studies.
To ensure the methodological and evidence quality of the studies, SRMAs that are eligible for inclusion in an umbrella review are critically appraised for validity scoring of their results using the Assessment of Multiple System Review (AMSTAR) tool . The SRMA pooled empirical studies and their summary estimates were evaluated using the tool consisting of 11 questions that measure the quality of the approach used. Thus, quality scores 8 to 11, 4 to 7, and less than 3 indicate high, medium, and low, respectively (see Table ). Two independent reviewers conducted the appraisal, and the decision whether to include a review was made based on these quality scores.
Overall, statistical analysis and data synthesis were conducted using STATA version 16.1 software. Researchers presented a range of estimates, including calculating a pooled estimate when two or more estimates were provided on the prevalence and predictors of institutional delivery service utilization. The between-studies heterogeneity that was assessed using Higgin’s I 2 statistics guided the choice of the meta-analysis model. Since there was a high level of difference between studies, the DerSimonian-laird random-effect model was used to pool the estimates . A summary list of the predictors of institutional delivery service utilization (i.e., dichotomous outcome) with their respective odds ratios and 95% confidence interval was prepared. Only five SRMAs were included in this umbrella review; it was not possible to assess publication bias since a minimum of ten studies is needed to evaluate publication bias . When two SRMA included the same primary studies, we calculated corrected covered area (CCA) suggested by Pieper et al. to quantify the degree of overlap in umbrella review; we assessed and documented primary studies’ degree of overlapping as guidelines recommend .
Literature search result We retrieved 1190 records after searching PubMed, Embase, CINAHL, Scopus, Web of Science and other sources. After removing duplicates, 1080 and 20 articles were screened using title/abstract and full-text, respectively. Among the twenty articles that were identified using full-text review, only five were checked for eligibility. Thus, a total of five SRMA studies [ – , , ] that summarized in Fig. were included in this umbrella review. Characteristics of SRMA studies These SRMA studies were based on observational primary studies (i.e., cross-sectional, case-control, and cohort designs). The included primary studies ranged from nine to thirty-four per SRMA study. Even though two SRMA studies didn’t mention their sample size , it ranged from 7660 to 26,350 , among others. Kebede et al. and Fekadu et al. assessed factors associated with institutional delivery service utilization whereas the rest of the SRMA studies assessed both prevalence and factors associated with institutional delivery service utilization [ – ]. The reported prevalence estimates of institutional delivery service utilization ranged from 21.2% to 31% . Table illustrates the general characteristics of the included SRMA studies. Primary studies In five SRMA studies 64 primary studies have been cited 107 times regardless of overlap. Of these, thirty-seven primary studies were included in only one SRMA study, while the remaining studies were incorporated in at least two SRMA studies or based on the same primary evidence. For instance, Wako et al. cited in four SRMA studies [ – , ], nine primary studies cited in three SRMA studies, and sixty primary studies cited in two SRMA studies. The corrected coverage area was 49% indicating very high overlap. We included all SRMA studies as they provided important information either for overall results or specific primary studies; deletion of some did not decrease overlap (Additional file ). Methodological quality of SRMA studies According to an evaluation of the methodological quality of SRMA studies using the AMSTAR tool, the quality score ranged from 5 to 11 points, with a mean score of 9.2 points, which is high quality. All SRMA studies had a comprehensive search, used appropriate methods to combine the studies’ findings, and acknowledged potential sources of support clearly in both the systematic review and the included studies (Table ). Systematic review Both women’s [ – , ] and husband’s education , and pregnant women’s positive attitude toward MCH service , knowledge of dangerous signs of pregnancy and the benefits of institutional delivery service utilization and MCH services favored institutional delivery service utilization. Maternal age at first pregnancy of 15–24 years and husband’s involvement in decision-making of place of delivery were associated with institutional delivery service utilization. Rural women and distant women from health facilities [ , , ] have low institutional delivery service utilization. Even though the autonomy of women did not show any influence , the availability of information sources and having information on the exemption of MCH service improved the utilization of institutional delivery services. Both women having at least one ANC follow-up [ – , ] and the frequency of ANC follow-up , encountering problems during pregnancy and facility delivery of the elderly child also increased odds of institutional delivery service utilization. Meta-analysis Prevalence of health facility delivery According to three SRMA studies, the pooled estimate of institutional delivery services utilization ranged from 21.2% (95% CI: 16.2, 26.1) to 31% (95% CI: 30, 31.2). The umbrella review based on these SRMA studies (18–20) revealed that 24% (95% CI: 14 to 34) of pregnant women utilized institutional delivery services in Ethiopia. As indicated in Table , the variation in effect size attributable to heterogeneity (I 2 ) was 99.77%, which is significant. There was no publication bias according to Egger’s test for the small-study effect ( P -value = 0.45). Predictors of health facility delivery Predisposing factors Women’s education [ – , ](18–20, 23) and pregnant women’s positive attitude toward maternal and child health (MCH) service favored institutional delivery service utilization (Fig. ). Pregnant women who attended primary and above education had increased odds of the utilization of institutional delivery services compared to those who had no formal education (OR = 3.54, 95% CI: 3.04, 4.12). Pregnant women’s positive attitude toward MCH services favored increased odds of the utilization of institutional delivery service compared to those with a negative attitude (OR = 2.20, 95% CI: 1.30, 3.74). Enabling factors In Fig. , place of residence and distance from a health facility [ , , ] influenced institutional delivery service utilization. Thus, urban residents had more than three times the increased odds of utilizing institutional delivery service compared to their rural resident counterparts (OR = 3.29, 95% CI: 2.02, 5.34). Those women who lived less than 5 km away from the health facility had increased odds of delivering at the health facility than those who lived more than 35 km away from the health facility (OR = 3.48, 95% CI: 2.58, 4.71). Need factors Women having at least one ANC follow-up [ – , ] have shown a significant effect on giving birth at a health facility in Ethiopia (Fig. ). Pregnant women who had at least one ANC visit had increased odds of institutional delivery service utilization compared to those who had no ANC follow-up (OR = 3.62, 95% CI: 3.03, 4.33).
We retrieved 1190 records after searching PubMed, Embase, CINAHL, Scopus, Web of Science and other sources. After removing duplicates, 1080 and 20 articles were screened using title/abstract and full-text, respectively. Among the twenty articles that were identified using full-text review, only five were checked for eligibility. Thus, a total of five SRMA studies [ – , , ] that summarized in Fig. were included in this umbrella review.
These SRMA studies were based on observational primary studies (i.e., cross-sectional, case-control, and cohort designs). The included primary studies ranged from nine to thirty-four per SRMA study. Even though two SRMA studies didn’t mention their sample size , it ranged from 7660 to 26,350 , among others. Kebede et al. and Fekadu et al. assessed factors associated with institutional delivery service utilization whereas the rest of the SRMA studies assessed both prevalence and factors associated with institutional delivery service utilization [ – ]. The reported prevalence estimates of institutional delivery service utilization ranged from 21.2% to 31% . Table illustrates the general characteristics of the included SRMA studies.
In five SRMA studies 64 primary studies have been cited 107 times regardless of overlap. Of these, thirty-seven primary studies were included in only one SRMA study, while the remaining studies were incorporated in at least two SRMA studies or based on the same primary evidence. For instance, Wako et al. cited in four SRMA studies [ – , ], nine primary studies cited in three SRMA studies, and sixty primary studies cited in two SRMA studies. The corrected coverage area was 49% indicating very high overlap. We included all SRMA studies as they provided important information either for overall results or specific primary studies; deletion of some did not decrease overlap (Additional file ).
According to an evaluation of the methodological quality of SRMA studies using the AMSTAR tool, the quality score ranged from 5 to 11 points, with a mean score of 9.2 points, which is high quality. All SRMA studies had a comprehensive search, used appropriate methods to combine the studies’ findings, and acknowledged potential sources of support clearly in both the systematic review and the included studies (Table ).
Both women’s [ – , ] and husband’s education , and pregnant women’s positive attitude toward MCH service , knowledge of dangerous signs of pregnancy and the benefits of institutional delivery service utilization and MCH services favored institutional delivery service utilization. Maternal age at first pregnancy of 15–24 years and husband’s involvement in decision-making of place of delivery were associated with institutional delivery service utilization. Rural women and distant women from health facilities [ , , ] have low institutional delivery service utilization. Even though the autonomy of women did not show any influence , the availability of information sources and having information on the exemption of MCH service improved the utilization of institutional delivery services. Both women having at least one ANC follow-up [ – , ] and the frequency of ANC follow-up , encountering problems during pregnancy and facility delivery of the elderly child also increased odds of institutional delivery service utilization.
Prevalence of health facility delivery According to three SRMA studies, the pooled estimate of institutional delivery services utilization ranged from 21.2% (95% CI: 16.2, 26.1) to 31% (95% CI: 30, 31.2). The umbrella review based on these SRMA studies (18–20) revealed that 24% (95% CI: 14 to 34) of pregnant women utilized institutional delivery services in Ethiopia. As indicated in Table , the variation in effect size attributable to heterogeneity (I 2 ) was 99.77%, which is significant. There was no publication bias according to Egger’s test for the small-study effect ( P -value = 0.45). Predictors of health facility delivery Predisposing factors Women’s education [ – , ](18–20, 23) and pregnant women’s positive attitude toward maternal and child health (MCH) service favored institutional delivery service utilization (Fig. ). Pregnant women who attended primary and above education had increased odds of the utilization of institutional delivery services compared to those who had no formal education (OR = 3.54, 95% CI: 3.04, 4.12). Pregnant women’s positive attitude toward MCH services favored increased odds of the utilization of institutional delivery service compared to those with a negative attitude (OR = 2.20, 95% CI: 1.30, 3.74). Enabling factors In Fig. , place of residence and distance from a health facility [ , , ] influenced institutional delivery service utilization. Thus, urban residents had more than three times the increased odds of utilizing institutional delivery service compared to their rural resident counterparts (OR = 3.29, 95% CI: 2.02, 5.34). Those women who lived less than 5 km away from the health facility had increased odds of delivering at the health facility than those who lived more than 35 km away from the health facility (OR = 3.48, 95% CI: 2.58, 4.71). Need factors Women having at least one ANC follow-up [ – , ] have shown a significant effect on giving birth at a health facility in Ethiopia (Fig. ). Pregnant women who had at least one ANC visit had increased odds of institutional delivery service utilization compared to those who had no ANC follow-up (OR = 3.62, 95% CI: 3.03, 4.33).
According to three SRMA studies, the pooled estimate of institutional delivery services utilization ranged from 21.2% (95% CI: 16.2, 26.1) to 31% (95% CI: 30, 31.2). The umbrella review based on these SRMA studies (18–20) revealed that 24% (95% CI: 14 to 34) of pregnant women utilized institutional delivery services in Ethiopia. As indicated in Table , the variation in effect size attributable to heterogeneity (I 2 ) was 99.77%, which is significant. There was no publication bias according to Egger’s test for the small-study effect ( P -value = 0.45).
Predisposing factors Women’s education [ – , ](18–20, 23) and pregnant women’s positive attitude toward maternal and child health (MCH) service favored institutional delivery service utilization (Fig. ). Pregnant women who attended primary and above education had increased odds of the utilization of institutional delivery services compared to those who had no formal education (OR = 3.54, 95% CI: 3.04, 4.12). Pregnant women’s positive attitude toward MCH services favored increased odds of the utilization of institutional delivery service compared to those with a negative attitude (OR = 2.20, 95% CI: 1.30, 3.74). Enabling factors In Fig. , place of residence and distance from a health facility [ , , ] influenced institutional delivery service utilization. Thus, urban residents had more than three times the increased odds of utilizing institutional delivery service compared to their rural resident counterparts (OR = 3.29, 95% CI: 2.02, 5.34). Those women who lived less than 5 km away from the health facility had increased odds of delivering at the health facility than those who lived more than 35 km away from the health facility (OR = 3.48, 95% CI: 2.58, 4.71). Need factors Women having at least one ANC follow-up [ – , ] have shown a significant effect on giving birth at a health facility in Ethiopia (Fig. ). Pregnant women who had at least one ANC visit had increased odds of institutional delivery service utilization compared to those who had no ANC follow-up (OR = 3.62, 95% CI: 3.03, 4.33).
Women’s education [ – , ](18–20, 23) and pregnant women’s positive attitude toward maternal and child health (MCH) service favored institutional delivery service utilization (Fig. ). Pregnant women who attended primary and above education had increased odds of the utilization of institutional delivery services compared to those who had no formal education (OR = 3.54, 95% CI: 3.04, 4.12). Pregnant women’s positive attitude toward MCH services favored increased odds of the utilization of institutional delivery service compared to those with a negative attitude (OR = 2.20, 95% CI: 1.30, 3.74).
In Fig. , place of residence and distance from a health facility [ , , ] influenced institutional delivery service utilization. Thus, urban residents had more than three times the increased odds of utilizing institutional delivery service compared to their rural resident counterparts (OR = 3.29, 95% CI: 2.02, 5.34). Those women who lived less than 5 km away from the health facility had increased odds of delivering at the health facility than those who lived more than 35 km away from the health facility (OR = 3.48, 95% CI: 2.58, 4.71).
Women having at least one ANC follow-up [ – , ] have shown a significant effect on giving birth at a health facility in Ethiopia (Fig. ). Pregnant women who had at least one ANC visit had increased odds of institutional delivery service utilization compared to those who had no ANC follow-up (OR = 3.62, 95% CI: 3.03, 4.33).
The purpose of the present review is to identify predictors of institutional delivery service utilization and the pooled prevalence in Ethiopia. Predisposing factors such as women’s education, the attitude of women toward MCH services; enabling factors, i.e., place of residence, distance from the health facility, and need factors (having at least one ANC follow-up), predicted institutional delivery service utilization in Ethiopia. The present umbrella review indicates that the pooled prevalence of institutional delivery service utilization in Ethiopia is 24%, which is in line with a review done in Ethiopia that indicated health facility-based delivery of 26.5% . However, it is lower than the SSA (53%) and global (76%) reports in 2017 . This could possibly be attributable to distance, lower education, no ANC visit, non-exposure to media, rural residence, and poverty that hinder Ethiopian women from accessing the majority of healthcare services. Thus, husband education, knowledge of dangerous signs of pregnancy and the benefit of health facility delivery and MCH services, younger age during first pregnancy, husband’s involvement in decision-making of place of delivery, availability of information sources and being informed about MCH service exemption, elder child’s place of birth favor health facility delivery. Women’s education plays a crucial role in the increased odds of institutional delivery service utilization compared to those who didn’t attend formal education. Similarly, primary, secondary, and higher-educated women were two times more likely to deliver in health institutes than non-educated women in SSA . Maternal education was the factor most consistently associated with facility-based delivery and the odds of non-use of facility-based delivery increased for women who had no education in Nigeria . The largest increase in health facility delivery did not occur among less educated and rural Cambodian women . Vital information about healthcare utilization may be more often accessible to well-educated women, and they are less likely to subscribe to some notions in the community that impede maternal service utilization. It implies the benefit of facility-based delivery should be advocated among those who have formal education or strengthening female education across the nation. Women’s positive/favorable attitude of women toward MCH services increases the odds of institutional delivery service utilization. This is in line with the review in SSA; the belief, behavior, and attitude ascribed to the nomad in particular are identified as barriers to health service utilization at fixed-post or outreach . Women and their families believe childbirth has become medicalized and dehumanized due to the emphasis placed on increasing health facility delivery by public health entities; women in LMICs may fear several undesirable procedures and prefer home delivery with support from traditional birth attendants . It could be due to demand-side barriers such as a lack of information on healthcare services, cultural beliefs/practices, stigma, women’s self-esteem/assertiveness, and ignorance about required obstetric health services . Further, women’s subjection to disrespectful and abusive behavior and enduring psychological humiliation may also hinder the use of facility-based childbirth . Since maternal and neonatal healthcare-related cultural beliefs and practices are intergenerational, community-specific education to enhance changes of behavior and adopting practical approaches such as involving husband/partners and communities in maternal health services are required . This implies a health workforce that is knowledgeable, skillful, and companionable, respectful and performance-based financing, community-based health insurance, and better governance and leadership are needed. An establishment of a welcoming environment for women coming for MCH services (i.e., improved the quality of MCH services) and tailored interventions on women’s attitudes are also among the implications. Urban dwellers have increased odds of institutional delivery service utilization compared to their rural-dweller counterparts. A systematic review in SSA revealed that urban/rural residence was most consistently associated with facility-based delivery . The use of health facilities was relatively high due to the availability of health centers for city dwellers . Home delivery persists in rural areas due to social and economic issues and cultural meanings attached to childbirth, including challenges related to the accessibility and affordability of respectful and culturally acceptable services in rural settings . Having home delivery is more convenient for a particular group of families in rural areas due to the strong religious and cultural beliefs that have been embedded in families’ beliefs and families’ decisions that affected women’s place of delivery [ – ]. The finding highlights addressing inequalities associated with maternal education, improving access to health facilities, and narrowing gaps between urban and rural resident women to achieve the SDG agenda of leaving no one behind by 2030. Residing a short distance from a health facility compared to a long distance has increased the odds of institutional delivery service utilization, which is in line with a study in SSA where a short distance from home to a health facility increased the odds of institutional delivery service utilization and the distance to the nearest health facility was the factor most consistently associated with health facility delivery . The review is also in line with a study in Tanzania identifying maternal deaths that are associated with distance to health facilities. Despite a different health and cultural setting, women who live more than 35 km away from health facilities find it difficult to access maternal services compared to those who live less than 5 km from health facilities, hence having a higher mortality ratio . Sociocultural, economic, health facility accessibility, and healthcare delivery-related factors hinder ANC and health facility delivery . Similarly, physical distance between health facilities and service users’ residences is among the supply-side barriers that could be worsened by demand-side barriers such as limited income, non-availability of means of transportation, and indirect cost of transportation . Moreover, distance from health services is a factor that influences reproductive healthcare-seeking behavior in Bangladesh . Thus, they receive maternal health services from unskilled people, which potentially creates negative impacts on maternal health. Empowering women via education, access to transportation, capacity building and staffing can improve institutional delivery service utilization. Delivery at health facilities increased among women who received incentives and information about the incentive program in Nepal that impacted the use of maternal health services positively. It may focus on women who live in rural, far distances and have low socio-economic status . This implies revising Ethiopia’s healthcare availability, facilitating transportation incentives, or improving maternity waiting home services for those in need. Having at least one ANC follow-up increases the odds of institutional delivery service utilization. In line with this, the review indicated that those women who had ANC visits were more likely to deliver in a health facility compared to those who had no ANC visits . ANC and the primary and secondary prevention of complications during delivery by skilled attendants avert the risk of maternal and child death that increases with home delivery . This could be contributed by health education during ANC that informs women about the importance of facility birth to timely diagnose, prevent, or manage complications. In contrast, some women do not need to seek out skilled birth attendants during labor and delivery, thinking attending ANC guarantees health during pregnancy and childbirth and despite high ANC sessions, there was a low rate of health facility delivery, an indication that there is the need to improve health education in timely ANC visits, a sign of true labor and benefit of institutional delivery service utilization . This could be due to healthcare providers’ training and supervision gap, workload, limited staff numbers, lack of incentives, poor infrastructure in the health facility, and absence of teamwork and communication . Hence, the review highlights strengthening ANC contact, advocating an importance of facility-based delivery during ANC contact and tailored intervention for those who had no ANC contact to contribute to an institutional delivery service utilization. The review implies tailored interventions incorporating health extension workers, mHealth (i.e., text messages), incentives, and implementation of community-level initiatives may increase the use of institutional delivery services to reduce labor and delivery-related mortality . Interventions that target improving primary health care (PHC) services (i.e., community engagement, improving an effectiveness of the health extension program using community scorecard, managerial accountability for PHC, network of care, establishing health extension program unit at each health facility, and improving health post capacity and functionalities) thorough research on its effectiveness to improve maternal health service utilization, health facility birth in particular are needed. Strength and limitation Multiple databases were searched to minimize risk of selection bias independently. The AMSTAR tool was used to assess SRMA studies’ risk of bias. In contrast, even though an overlap of the data among primary studies that were included in SRMA studies were mapped, there is a potential to overestimate the strength of the findings due to summarizing multiple meta-analysis data that included overlapping primary studies with increased sample size. Since aggregated group data was used to identify confounding factors that were not possible, the readers should be mindful while interpreting and using this finding in the context of both the inherent limitations of primary studies and umbrella review analysis.
Multiple databases were searched to minimize risk of selection bias independently. The AMSTAR tool was used to assess SRMA studies’ risk of bias. In contrast, even though an overlap of the data among primary studies that were included in SRMA studies were mapped, there is a potential to overestimate the strength of the findings due to summarizing multiple meta-analysis data that included overlapping primary studies with increased sample size. Since aggregated group data was used to identify confounding factors that were not possible, the readers should be mindful while interpreting and using this finding in the context of both the inherent limitations of primary studies and umbrella review analysis.
Only about one in four pregnant women in Ethiopia give birth in a health facility. The findings highlight that improving women’s education, applying targeted intervention to change women’s attitudes towards MCH services, supporting rural residents, improving the access and availability of health facilities, and promoting ANC utilization are key implications for enhancing health facility’s childbirths.
Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2
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Skeletal muscle atrophy induced by aging and disuse atrophy are strongly associated with the upregulation of the endoplasmic stress protein CHOP in rats | 3f130238-9c24-4b84-9db0-e0c2deb555d8 | 11919930 | Musculoskeletal System[mh] | Skeletal muscle is the largest tissue by weight in the human body and exhibits remarkable plasticity . This hallmark of skeletal muscle is exemplified by its ability to respond to different stimuli . Common adaptations include hypertrophy in response to mechanical overload and atrophy in response to whole body disuse or limb unloading [ – ]. Aging along with disuse are other potent drivers of skeletal muscle (mal)adaptation , often manifesting in dysregulated proteostasis, increased senescent burden, and increased endoplasmic reticulum (ER) stress (ERS) . While alterations to muscle protein synthesis (MPS) and muscle protein breakdown (MPB) are well studied in the context of aging, the impact of ERS is an underappreciated mechanism of skeletal muscle regulation . The ER is a primary site of protein folding and the release of proteins from the ER is a highly stringent process. Indeed, release of proteins from the ER is thought to be the rate limiting step in the arrival of mature proteins at their subsequent destinations (e.g. at the plasma membrane or in circulation; reviewed in ). This phenomenon is indicative of the proper structure conformity that proteins must acquire within the ER prior to release and performance of canonical actions. ERS refers to the stress imposed upon the ER in response to an increase in truncated, improperly translated, or otherwise misfolded proteins, and the resultant pathway that becomes active in response to sustained ERS is the unfolded protein response (UPR) . The UPR is a signaling cascade triggered by three main effectors including protein kinase R (PKR) like ER kinase (PERK), inositol requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6). The UPR serves to blunt global protein synthesis via phosphorylation of eukaryotic initiation factor 2α (eIF2α) and preferentially translate a set of basic leucine zipper motif (bZIP) transcription factors (e.g. Activating Transcription Factor 4 (ATF4), spliced form of X-box binding protein 1 (XBP1s)). These bZIP transcription factors enhance the translation of chaperones, folding enzymes, and ER-associated protein degradation (ERAD) enzymes that are beneficial in mitigating the burden of errant proteins . The UPR can be beneficial for skeletal muscle adaptation by effectively serving as a brake on translation during periods of high concentrations of misfolded proteins, thus allowing the cell to regain translational control and produce properly functioning proteins. This response can, however, become maladaptive if the unfolded protein burden is not remediated, as sustained activation of this system can trigger apoptosis and cell death via the upregulation of proapoptotic proteins such as the terminal effector C/EBP homologous protein (CHOP) . Indeed, the downstream induction of CHOP has been shown to play a major role in the proapoptotic function of the UPR [ – ]. ERS is often observed in the context of disease and/or intentional provocation (e.g. cancer models, tunicamycin treatment, high glucose or type 2 diabetes mellitus models) and can interplay with other cell outcomes such as increases in senescence or increased proteolysis [ – ]. Given that ERS can enhance the burden imposed by other markers typically associated with dysfunction in aging it has become clear that ERS can be harmful to longevity [ – ]. While ERS burden and subsequent activation of the UPR is often elevated in aged (22 + months of age) versus young (3–6 months of age) rodents, UPR markers at different ages do not appear to have been examined . Therefore, the purpose of this study was to examine the UPR response in skeletal muscle in the basal state from a cohort of Fischer 344 rats that were collected from young to old age. As a secondary aim, we sought to examine similar markers in response to hindlimb immobilization (HLI) induced skeletal muscle atrophy in a cohort of adult Wistar rats to determine if HLI elicited similar effects.
Animal experimental procedures All procedures involving animal husbandry and experimentation for rats collected at different ages were approved by Auburn University’s Institutional Animal Care and Use Committee (protocol #2015–2790). Male, Fischer 344 rats (300–600 g) aged 3, 6, 12, 18, and 24 months ( n = 9–10 rats per group) were purchased from Envigo Laboratories (Indianapolis, IN, USA), and housed two per cage on the week prior to and during experimentation. During this time, animal quarters were maintained on a constant 12 h light: 12 h dark cycle at ambient room temperature and tap water and standard rodent chow (24% protein, 58% carbohydrate, 18% fat; Teklad Global #2018 Diet, Envigo Laboratories) were provided to animals ad libitum. The morning of experimentation, animals were removed from their quarters between 0600 and 0700, transported to the Molecular and Applied Sciences Laboratory in the School of Kinesiology building and euthanized under CO2 gas induction in a 2 L chamber (VetEquip, Inc., Pleasanton, CA, USA). Following euthanasia, body masses were recorded, right-leg plantaris and soleus muscles were dissected out, and muscles were weighed using an analytical scale with a sensitivity of 0.0001 g (Mettler-Toledo; Columbus, OH, USA). During dissection muscles were cut in very close proximity at the origin and insertion sites and visible connective tissue at the insertion site was removed. Muscles were then processed for biochemical assays on the day of euthanasia as described in the following paragraphs. All procedures involving animal husbandry and experimentation for rats undergoing hindlimb immobilization were approved by the University of Missouri Animal Care and Use Committee (MU ACUC) (ACUC protocol #35961). Three-month-old female Wistar rats ( n = 12 per group) were ordered from Charles River Laboratories for this study. Prior to the start of the study, all rats were housed until ~ 15 weeks of age when groups were chosen at random. For rats undergoing hindlimb immobilization (HLI), veterinary casts were applied (BSN Medical Delta-Lite Plus Casting Tape). Briefly, rats were anesthetized using isoflurane and casting material was applied to the extended hindlimbs with feet positioned in plantarflexion and knee at or near full extension. Rats in the CTL group were either completely cast-free or had the same casting material wrapped around the abdomen to mimic casting variables without physiological disuse. Notably, these CTL groups were collapsed due to no differences in downstream experiments between the two control groups. Rats were housed within-group in large cages to maintain socialization and mobility. More details regarding general animal husbandry can be found in Kerr et al. Details regarding euthanasia procedures can be found in Kerr et al. Briefly, upon the tenth day of hindlimb disuse, a ketamine/xylazine cocktail was administered to facilitate cast removal and sacrifice. Then the 4-month-old rats were euthanized via decapitation whereafter the gastrocnemius was dissected out and weighed on an analytical scale, then immediately flash-frozen in liquid nitrogen. Gastrocnemius muscles were then stored at -80 °C for subsequent analysis. Critically, we were limited to the use of male Fischer 344 rats for aging experiments and female Wistar rats for disuse-induced atrophy experiments. We acknowledge that there is likely an effect of sex and strain of these rats of the results reported herein, however it has been reported that both male and female rats experience significant atrophy in response to aging and hindlimb disuse regardless of strain. Wet laboratory analyses Real-time qPCR Frozen muscle foils were removed from − 80ºC storage and crushed on a liquid nitrogen-cooled ceramic mortar and pestle. Approximately 10 mg of muscle was used to isolate RNA via the Trizol (Thermo Scientific, Waltham, MA, USA) method coupled with the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA, USA) per the manufacturer’s recommendations. Following RNA isolation, the RNA pellet was reconstituted in 20 µL of RNase-free water and RNA concentrations were determined in duplicate at an absorbance of 260 nm by using a NanoDrop Lite (Thermo Scientific, Waltham, MA, USA). Thereafter, cDNA (2 µg) was synthesized using a commercial qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) per the manufacturer’s recommendations. qPCR was performed with gene-specific primers and SYBR-green-based methods (Quanta Biosciences) using a real-time PCR thermal cycler (Bio-Rad). Gene-specific primers were designed with primer design software (Primer3Plus, Cambridge, MA, USA). The final volume of qPCR reactions was 20 µL, which contained a final concentration of 2 µM of forward and reverse primers and 25 ng of cDNA. All reactions were performed in duplicate. Forward and reverse sequences for all primers are shown in Table . Fold-change values from CTL were performed using the 2 ΔΔCq method where 2 ΔCq = 2^ [housekeeping gene (HKG) Cq– gene of interest Cq], and 2 ΔΔCq (or fold-change) = [2 ΔCq individual CTL or HLI value/2 ΔCq average of all PRE values]. Primer sets for valosin-containing protein (VCP) were used for mRNA normalization given that this gene did not differ between rat groups ( P > 0.05). Western blotting During tissue extraction for RNA isolation a portion of skeletal muscle tissue was also allocated for protein isolation. Approximately 20 mg of tissue was placed in 1.7 mL microcentrifuge tubes and homogenized in general cell lysate buffer (VWR; Cat #: 97063-130) using tight-fitting pestles. Tissues were then centrifuged at 500 x g for 10 min, whereafter supernatants were collected for further analysis. Protein lysates were prepared via homogenization with tight fitting pestles in 500 µL of general cell lysis buffer (catalog #: 9803 S; Cell Signaling Technologies) followed by centrifugation at 500 G at 4 °C. Lysates were then batch process-assayed for total protein content using a BCA Protein Assay Kit (Thermo Fisher; Waltham, MA, USA). Lysates were then prepared for western blotting using 4x Laemmli buffer at 1 µg/µL. Thereafter, 15 µL of prepped samples were loaded onto 4–15% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and subjected to electrophoresis (180 V for 50 min) using pre-made 1x SDS-PAGE running buffer (VWR; Cat #: 0783). Proteins were then transferred (200 mA for 2 h) to polyvinylidene difluoride membranes (Bio-Rad), Ponceau stained and imaged to ensure equal protein loading between lanes. Membranes were then blocked for 1 h at room temperature with 5% nonfat milk powder in Tris-buffered saline with 0.1% Tween-20 (TBST; VWR). Membranes containing 1-hour treated samples were incubated with the antibodies described in Table at a 1:1000 dilution in TBST with 5% bovine serum albumin (BSA) overnight. The following day, membranes were incubated with horseradish peroxidase-conjugated HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, Cat No. 7074), HRP-conjugated anti-mouse IgG (Cell Signaling Technology, Cat No. 7076), or HRP-conjugated anti-goat IgG (Genetex, Irvine, CA, USA Cat No. GTX628547-01) in TBST with 5% BSA at room temperature for 1 h. Membrane development was performed using an enhanced chemiluminescent reagent (Luminata Forte HRP substrate; Millipore Sigma), and band densitometry was performed using a gel documentation system and associated densitometry software (ChemiDoc Touch, Bio-Rad). Densitometry values of protein targets were normalized to Ponceau densities. These values were then normalized to the grand mean of the appropriate control to obtain relative protein expression. These values were then expressed as fold change from control, where control was set to 1.00. Phosphorylated target band densities were divided by pan densities of the same target to obtain a ratio. Nuclear protein fraction isolation Nuclear and sarcoplasmic fractions were separated from HLI and CTL rats via a commercially available kit (ab113474; Abcam; Cambridge, UK) by adhering to manufacturer’s instructions. After isolations, BCA assays were performed to determine protein concentrations in each fraction and western blotting was performed as described above. Statistics Statistical analyses were performed using GraphPad Prism (Version 10.1.1; GraphPad Software, San Diego, CA, USA). All data are presented as means and standard deviation (SD), and individual respondent data are also presented. Rat aging data were checked for normality using the Shapiro-Wilk test. For normally distributed data, one-way repeated measures ANOVAs were performed. In cases where the ANOVA showed significance ( P < 0.05), Tukey post hoc tests were performed between age groups. For non-normally distributed data, Friedman’s tests were performed. In cases where the Friedman’s test demonstrated significance, a Wilcoxon’s signed rank test was performed between age groups. Hindlimb disuse rat data were checked for normality using the Shapiro-Wilk test. For normally distributed data, unpaired t-tests were performed with significance being established as P < 0.05. For non-normally distributed data, Mann-Whitney tests were performed with significance level again set at P < 0.05. Given the substantial findings suggesting that CHOP plays a role in the proapoptotic response, we opted to further investigate this protein in the context of aging- and disuse-atrophy. Therefore, correlations were performed to examine the relationship between CHOP expression and muscle size changes in all rats examined herein. Data for both aging and hindlimb immobilization rats were checked for normality and since all failed to meet normality assumptions, Spearman’s correlations were performed between CHOP protein expression and normalized muscle weight ((muscle of interest/Body weight)*100). Associations were considered significant if P < 0.05.
All procedures involving animal husbandry and experimentation for rats collected at different ages were approved by Auburn University’s Institutional Animal Care and Use Committee (protocol #2015–2790). Male, Fischer 344 rats (300–600 g) aged 3, 6, 12, 18, and 24 months ( n = 9–10 rats per group) were purchased from Envigo Laboratories (Indianapolis, IN, USA), and housed two per cage on the week prior to and during experimentation. During this time, animal quarters were maintained on a constant 12 h light: 12 h dark cycle at ambient room temperature and tap water and standard rodent chow (24% protein, 58% carbohydrate, 18% fat; Teklad Global #2018 Diet, Envigo Laboratories) were provided to animals ad libitum. The morning of experimentation, animals were removed from their quarters between 0600 and 0700, transported to the Molecular and Applied Sciences Laboratory in the School of Kinesiology building and euthanized under CO2 gas induction in a 2 L chamber (VetEquip, Inc., Pleasanton, CA, USA). Following euthanasia, body masses were recorded, right-leg plantaris and soleus muscles were dissected out, and muscles were weighed using an analytical scale with a sensitivity of 0.0001 g (Mettler-Toledo; Columbus, OH, USA). During dissection muscles were cut in very close proximity at the origin and insertion sites and visible connective tissue at the insertion site was removed. Muscles were then processed for biochemical assays on the day of euthanasia as described in the following paragraphs. All procedures involving animal husbandry and experimentation for rats undergoing hindlimb immobilization were approved by the University of Missouri Animal Care and Use Committee (MU ACUC) (ACUC protocol #35961). Three-month-old female Wistar rats ( n = 12 per group) were ordered from Charles River Laboratories for this study. Prior to the start of the study, all rats were housed until ~ 15 weeks of age when groups were chosen at random. For rats undergoing hindlimb immobilization (HLI), veterinary casts were applied (BSN Medical Delta-Lite Plus Casting Tape). Briefly, rats were anesthetized using isoflurane and casting material was applied to the extended hindlimbs with feet positioned in plantarflexion and knee at or near full extension. Rats in the CTL group were either completely cast-free or had the same casting material wrapped around the abdomen to mimic casting variables without physiological disuse. Notably, these CTL groups were collapsed due to no differences in downstream experiments between the two control groups. Rats were housed within-group in large cages to maintain socialization and mobility. More details regarding general animal husbandry can be found in Kerr et al. Details regarding euthanasia procedures can be found in Kerr et al. Briefly, upon the tenth day of hindlimb disuse, a ketamine/xylazine cocktail was administered to facilitate cast removal and sacrifice. Then the 4-month-old rats were euthanized via decapitation whereafter the gastrocnemius was dissected out and weighed on an analytical scale, then immediately flash-frozen in liquid nitrogen. Gastrocnemius muscles were then stored at -80 °C for subsequent analysis. Critically, we were limited to the use of male Fischer 344 rats for aging experiments and female Wistar rats for disuse-induced atrophy experiments. We acknowledge that there is likely an effect of sex and strain of these rats of the results reported herein, however it has been reported that both male and female rats experience significant atrophy in response to aging and hindlimb disuse regardless of strain.
Real-time qPCR Frozen muscle foils were removed from − 80ºC storage and crushed on a liquid nitrogen-cooled ceramic mortar and pestle. Approximately 10 mg of muscle was used to isolate RNA via the Trizol (Thermo Scientific, Waltham, MA, USA) method coupled with the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA, USA) per the manufacturer’s recommendations. Following RNA isolation, the RNA pellet was reconstituted in 20 µL of RNase-free water and RNA concentrations were determined in duplicate at an absorbance of 260 nm by using a NanoDrop Lite (Thermo Scientific, Waltham, MA, USA). Thereafter, cDNA (2 µg) was synthesized using a commercial qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) per the manufacturer’s recommendations. qPCR was performed with gene-specific primers and SYBR-green-based methods (Quanta Biosciences) using a real-time PCR thermal cycler (Bio-Rad). Gene-specific primers were designed with primer design software (Primer3Plus, Cambridge, MA, USA). The final volume of qPCR reactions was 20 µL, which contained a final concentration of 2 µM of forward and reverse primers and 25 ng of cDNA. All reactions were performed in duplicate. Forward and reverse sequences for all primers are shown in Table . Fold-change values from CTL were performed using the 2 ΔΔCq method where 2 ΔCq = 2^ [housekeeping gene (HKG) Cq– gene of interest Cq], and 2 ΔΔCq (or fold-change) = [2 ΔCq individual CTL or HLI value/2 ΔCq average of all PRE values]. Primer sets for valosin-containing protein (VCP) were used for mRNA normalization given that this gene did not differ between rat groups ( P > 0.05).
Frozen muscle foils were removed from − 80ºC storage and crushed on a liquid nitrogen-cooled ceramic mortar and pestle. Approximately 10 mg of muscle was used to isolate RNA via the Trizol (Thermo Scientific, Waltham, MA, USA) method coupled with the Direct-zol RNA miniprep kit (Zymo Research, Irvine, CA, USA) per the manufacturer’s recommendations. Following RNA isolation, the RNA pellet was reconstituted in 20 µL of RNase-free water and RNA concentrations were determined in duplicate at an absorbance of 260 nm by using a NanoDrop Lite (Thermo Scientific, Waltham, MA, USA). Thereafter, cDNA (2 µg) was synthesized using a commercial qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) per the manufacturer’s recommendations. qPCR was performed with gene-specific primers and SYBR-green-based methods (Quanta Biosciences) using a real-time PCR thermal cycler (Bio-Rad). Gene-specific primers were designed with primer design software (Primer3Plus, Cambridge, MA, USA). The final volume of qPCR reactions was 20 µL, which contained a final concentration of 2 µM of forward and reverse primers and 25 ng of cDNA. All reactions were performed in duplicate. Forward and reverse sequences for all primers are shown in Table . Fold-change values from CTL were performed using the 2 ΔΔCq method where 2 ΔCq = 2^ [housekeeping gene (HKG) Cq– gene of interest Cq], and 2 ΔΔCq (or fold-change) = [2 ΔCq individual CTL or HLI value/2 ΔCq average of all PRE values]. Primer sets for valosin-containing protein (VCP) were used for mRNA normalization given that this gene did not differ between rat groups ( P > 0.05).
During tissue extraction for RNA isolation a portion of skeletal muscle tissue was also allocated for protein isolation. Approximately 20 mg of tissue was placed in 1.7 mL microcentrifuge tubes and homogenized in general cell lysate buffer (VWR; Cat #: 97063-130) using tight-fitting pestles. Tissues were then centrifuged at 500 x g for 10 min, whereafter supernatants were collected for further analysis. Protein lysates were prepared via homogenization with tight fitting pestles in 500 µL of general cell lysis buffer (catalog #: 9803 S; Cell Signaling Technologies) followed by centrifugation at 500 G at 4 °C. Lysates were then batch process-assayed for total protein content using a BCA Protein Assay Kit (Thermo Fisher; Waltham, MA, USA). Lysates were then prepared for western blotting using 4x Laemmli buffer at 1 µg/µL. Thereafter, 15 µL of prepped samples were loaded onto 4–15% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and subjected to electrophoresis (180 V for 50 min) using pre-made 1x SDS-PAGE running buffer (VWR; Cat #: 0783). Proteins were then transferred (200 mA for 2 h) to polyvinylidene difluoride membranes (Bio-Rad), Ponceau stained and imaged to ensure equal protein loading between lanes. Membranes were then blocked for 1 h at room temperature with 5% nonfat milk powder in Tris-buffered saline with 0.1% Tween-20 (TBST; VWR). Membranes containing 1-hour treated samples were incubated with the antibodies described in Table at a 1:1000 dilution in TBST with 5% bovine serum albumin (BSA) overnight. The following day, membranes were incubated with horseradish peroxidase-conjugated HRP-conjugated anti-rabbit IgG (Cell Signaling Technology, Cat No. 7074), HRP-conjugated anti-mouse IgG (Cell Signaling Technology, Cat No. 7076), or HRP-conjugated anti-goat IgG (Genetex, Irvine, CA, USA Cat No. GTX628547-01) in TBST with 5% BSA at room temperature for 1 h. Membrane development was performed using an enhanced chemiluminescent reagent (Luminata Forte HRP substrate; Millipore Sigma), and band densitometry was performed using a gel documentation system and associated densitometry software (ChemiDoc Touch, Bio-Rad). Densitometry values of protein targets were normalized to Ponceau densities. These values were then normalized to the grand mean of the appropriate control to obtain relative protein expression. These values were then expressed as fold change from control, where control was set to 1.00. Phosphorylated target band densities were divided by pan densities of the same target to obtain a ratio.
Nuclear and sarcoplasmic fractions were separated from HLI and CTL rats via a commercially available kit (ab113474; Abcam; Cambridge, UK) by adhering to manufacturer’s instructions. After isolations, BCA assays were performed to determine protein concentrations in each fraction and western blotting was performed as described above.
Statistical analyses were performed using GraphPad Prism (Version 10.1.1; GraphPad Software, San Diego, CA, USA). All data are presented as means and standard deviation (SD), and individual respondent data are also presented. Rat aging data were checked for normality using the Shapiro-Wilk test. For normally distributed data, one-way repeated measures ANOVAs were performed. In cases where the ANOVA showed significance ( P < 0.05), Tukey post hoc tests were performed between age groups. For non-normally distributed data, Friedman’s tests were performed. In cases where the Friedman’s test demonstrated significance, a Wilcoxon’s signed rank test was performed between age groups. Hindlimb disuse rat data were checked for normality using the Shapiro-Wilk test. For normally distributed data, unpaired t-tests were performed with significance being established as P < 0.05. For non-normally distributed data, Mann-Whitney tests were performed with significance level again set at P < 0.05. Given the substantial findings suggesting that CHOP plays a role in the proapoptotic response, we opted to further investigate this protein in the context of aging- and disuse-atrophy. Therefore, correlations were performed to examine the relationship between CHOP expression and muscle size changes in all rats examined herein. Data for both aging and hindlimb immobilization rats were checked for normality and since all failed to meet normality assumptions, Spearman’s correlations were performed between CHOP protein expression and normalized muscle weight ((muscle of interest/Body weight)*100). Associations were considered significant if P < 0.05.
Rodent characteristics For male Fischer 344 rats analyzed in aging experiments, right PLT weight (in mg) normalized to body weight (in g) was 0.86 ± 0.03 mg/g for 3 mo rats, 0.90 ± 0.03 mg/g for 6 mo rats, 0.79 ± 0.04 mg/g for 12 mo rats, 0.73 ± 0.04 mg/g for 18 mo rats, and 0.70 ± 0.05 mg/g for 24 mo rats. Significant differences ( P < 0.05) in values were noted whereby 3 mo and 6 mo were greater than other age groups and 12 mo were greater than 18 mo and 24 mo. In these same rats, the right SOL weight (in mg) normalized to body weight (in g) showed a similar pattern, where values were 0.36 ± 0.03 mg/g for 3 mo, 0.32 ± 0.05 mg/g for 6 mo, 0.34 ± 0.02 mg/g for 12 mo, 0.28 ± 0.03 mg/g for 18 mo, and 0.30 ± 0.04 for 24 mo rats. Again, significant differences ( P < 0.05) in values were noted whereby 3 mo were greater than 6 mo, and 18 and 24 mo values were lower than other age groups. For female Wistar rats in the disuse experiment, gastrocnemius weight (in mg) normalized to body weight (in g) was 3.52 ± 0.33 mg/g for HLI rats and 5.30 ± 0.27 mg/g for CTL rats. This 33.6% difference reached statistical significance ( P < 0.001). Readers are referred to Mobley et al. and Kerr et al. for more detailed phenotypic data for rat aging and hindlimb immobilization analyses, respectively. ERS markers with aging in the plantaris muscle BiP protein expression in the plantaris muscle was greater in 3 mo versus all other age cohorts except 12 mo ( P ≤ 0.008; Fig. A), and 24 mo had lower BiP protein expression than 3 mo and 12 mo ( P < 0.002). Cleaved ATF6 and ATF4 failed to reach ANOVA significance ( P ≥ 0.062, Fig. B-C). Phospho/pan eIF2α was greater in 24 mo versus 3 mo and 18 mo rats (P ≤ 0.006, Fig. D). CHOP protein was greater in 18 mo and 24 mo versus all other age groups ( P < 0.001), and 12 mo was greater than 3 mo and 6 mo ( P ≤ 0.022, Fig. E). Phospho/pan JNK was greater in 18 and 24 mo versus all other age groups ( P ≤ 0.002, Fig. F), and 12 mo was greater than 3 mo ( P = 0.009). Finally, CHOP protein expression demonstrated a significant, moderate negative correlation with plantaris weight normalized to body weight ( P < 0.001; Spearman’s R =-0.702, Fig. G). ERS markers with aging in the soleus muscle BiP expression in soleus was greater in 3 mo versus 18 mo and 24 mo ( P ≤ 0.008, Fig. A), and 12 mo was greater than 24 mo ( P < 0.002). Cleaved ATF6 was greater in 18 mo and 6 mo ( P = 0.023, Fig. B). ATF4 failed to reach ANOVA significance ( P ≥ 0.073, Fig. C). Phospho/pan eIF2α was greater in 3 mo versus all other age groups ( P ≤ 0.004) except for 24 mo ( P = 0.126, Fig. D). CHOP protein was greater in 18 mo and 24 mo versus other age groups ( P < 0.001, Fig. E), and 12 mo showed greater protein expression than 3 mo ( P = 0.017). Phospho/pan JNK was greater in 18 and 24 mo versus other age groups ( P < 0.001, Fig. F). Finally, CHOP protein expression demonstrated a significant, moderate negative correlation with normalized soleus weight normalized to body weight ( P < 0.001; Spearman’s R =-0.658, Fig. G). ERS markers in gastrocnemius muscle with rat hindlimb immobilization Based on findings from the aging rats presented above, we opted to examine proteins that showed a consistent pattern with age, or proteins that are critical to the UPR pathway as a whole. ATF4 protein expression was higher in the gastrocnemius muscle of CTL than HLI rats ( P = 0.014, Fig. A). Similarly, phospho/pan eIF2α phosphorylated protein ratio was higher in CTL than HLI rats ( P ≤ 0.001, Fig. B). Phospho/pan JNK phosphorylated protein ratio was higher in HLI than CTL rats ( P ≤ 0.002, Fig. C). Similarly, CHOP protein expression was higher in HLI than CTL rats ( P ≤ 0.001, Fig. D). CHOP associations with hindlimb immobilization Given the robust increase in skeletal muscle CHOP protein levels with disuse (Fig. D) and aging (Figs. F and F), further interrogations were performed into CHOP protein and disuse atrophy. CHOP protein expression demonstrated a significant, strong negative correlation with gastrocnemius weight normalized to body weight ( P < 0.001; Spearman’s R =-0.814, Fig. A). Upon separation of nuclear and sarcoplasmic protein fractions in HLI and CTL rats, it was found that CHOP protein expression demonstrated a main effect of protein fraction ( P < 0.001), a main effect of treatment group ( P < 0.001), and a protein fraction by treatment group interaction ( P < 0.001, Fig. B). Subsequent unpaired t-tests were then performed to compare between treatment groups, but within protein fraction. In both the sarcoplasmic and nuclear fraction, CHOP protein expression was upregulated in HLI as compared to CTL rats (P≤, Fig. C-D). After finding that CHOP protein expression was higher in the nuclear fraction of the HLI rats as compared to CTL rats, targeted qPCR was performed to assess CHOP’s potential role as a transcriptional regulator. The hallmark downstream target gene of CHOP, Gadd34 was upregulated ~ 35-fold in HLI versus CTL rats. Similarly, Bax , a pro-apoptotic gene, was upregulated ~ 20-fold in HLI versus CTL rats ( P < 0.001, Fig. E-F). A validation western blot of the nuclear protein histone H3 is included to confirm that nuclear fractionation was successful (Fig. G).
For male Fischer 344 rats analyzed in aging experiments, right PLT weight (in mg) normalized to body weight (in g) was 0.86 ± 0.03 mg/g for 3 mo rats, 0.90 ± 0.03 mg/g for 6 mo rats, 0.79 ± 0.04 mg/g for 12 mo rats, 0.73 ± 0.04 mg/g for 18 mo rats, and 0.70 ± 0.05 mg/g for 24 mo rats. Significant differences ( P < 0.05) in values were noted whereby 3 mo and 6 mo were greater than other age groups and 12 mo were greater than 18 mo and 24 mo. In these same rats, the right SOL weight (in mg) normalized to body weight (in g) showed a similar pattern, where values were 0.36 ± 0.03 mg/g for 3 mo, 0.32 ± 0.05 mg/g for 6 mo, 0.34 ± 0.02 mg/g for 12 mo, 0.28 ± 0.03 mg/g for 18 mo, and 0.30 ± 0.04 for 24 mo rats. Again, significant differences ( P < 0.05) in values were noted whereby 3 mo were greater than 6 mo, and 18 and 24 mo values were lower than other age groups. For female Wistar rats in the disuse experiment, gastrocnemius weight (in mg) normalized to body weight (in g) was 3.52 ± 0.33 mg/g for HLI rats and 5.30 ± 0.27 mg/g for CTL rats. This 33.6% difference reached statistical significance ( P < 0.001). Readers are referred to Mobley et al. and Kerr et al. for more detailed phenotypic data for rat aging and hindlimb immobilization analyses, respectively.
BiP protein expression in the plantaris muscle was greater in 3 mo versus all other age cohorts except 12 mo ( P ≤ 0.008; Fig. A), and 24 mo had lower BiP protein expression than 3 mo and 12 mo ( P < 0.002). Cleaved ATF6 and ATF4 failed to reach ANOVA significance ( P ≥ 0.062, Fig. B-C). Phospho/pan eIF2α was greater in 24 mo versus 3 mo and 18 mo rats (P ≤ 0.006, Fig. D). CHOP protein was greater in 18 mo and 24 mo versus all other age groups ( P < 0.001), and 12 mo was greater than 3 mo and 6 mo ( P ≤ 0.022, Fig. E). Phospho/pan JNK was greater in 18 and 24 mo versus all other age groups ( P ≤ 0.002, Fig. F), and 12 mo was greater than 3 mo ( P = 0.009). Finally, CHOP protein expression demonstrated a significant, moderate negative correlation with plantaris weight normalized to body weight ( P < 0.001; Spearman’s R =-0.702, Fig. G).
BiP expression in soleus was greater in 3 mo versus 18 mo and 24 mo ( P ≤ 0.008, Fig. A), and 12 mo was greater than 24 mo ( P < 0.002). Cleaved ATF6 was greater in 18 mo and 6 mo ( P = 0.023, Fig. B). ATF4 failed to reach ANOVA significance ( P ≥ 0.073, Fig. C). Phospho/pan eIF2α was greater in 3 mo versus all other age groups ( P ≤ 0.004) except for 24 mo ( P = 0.126, Fig. D). CHOP protein was greater in 18 mo and 24 mo versus other age groups ( P < 0.001, Fig. E), and 12 mo showed greater protein expression than 3 mo ( P = 0.017). Phospho/pan JNK was greater in 18 and 24 mo versus other age groups ( P < 0.001, Fig. F). Finally, CHOP protein expression demonstrated a significant, moderate negative correlation with normalized soleus weight normalized to body weight ( P < 0.001; Spearman’s R =-0.658, Fig. G).
Based on findings from the aging rats presented above, we opted to examine proteins that showed a consistent pattern with age, or proteins that are critical to the UPR pathway as a whole. ATF4 protein expression was higher in the gastrocnemius muscle of CTL than HLI rats ( P = 0.014, Fig. A). Similarly, phospho/pan eIF2α phosphorylated protein ratio was higher in CTL than HLI rats ( P ≤ 0.001, Fig. B). Phospho/pan JNK phosphorylated protein ratio was higher in HLI than CTL rats ( P ≤ 0.002, Fig. C). Similarly, CHOP protein expression was higher in HLI than CTL rats ( P ≤ 0.001, Fig. D).
Given the robust increase in skeletal muscle CHOP protein levels with disuse (Fig. D) and aging (Figs. F and F), further interrogations were performed into CHOP protein and disuse atrophy. CHOP protein expression demonstrated a significant, strong negative correlation with gastrocnemius weight normalized to body weight ( P < 0.001; Spearman’s R =-0.814, Fig. A). Upon separation of nuclear and sarcoplasmic protein fractions in HLI and CTL rats, it was found that CHOP protein expression demonstrated a main effect of protein fraction ( P < 0.001), a main effect of treatment group ( P < 0.001), and a protein fraction by treatment group interaction ( P < 0.001, Fig. B). Subsequent unpaired t-tests were then performed to compare between treatment groups, but within protein fraction. In both the sarcoplasmic and nuclear fraction, CHOP protein expression was upregulated in HLI as compared to CTL rats (P≤, Fig. C-D). After finding that CHOP protein expression was higher in the nuclear fraction of the HLI rats as compared to CTL rats, targeted qPCR was performed to assess CHOP’s potential role as a transcriptional regulator. The hallmark downstream target gene of CHOP, Gadd34 was upregulated ~ 35-fold in HLI versus CTL rats. Similarly, Bax , a pro-apoptotic gene, was upregulated ~ 20-fold in HLI versus CTL rats ( P < 0.001, Fig. E-F). A validation western blot of the nuclear protein histone H3 is included to confirm that nuclear fractionation was successful (Fig. G).
The contributions of ERS and the UPR have been overlooked regarding the consequences of aging and disuse in skeletal muscle. The current findings demonstrate a robust upregulation of ERS and the UPR (as evidenced by upregulation of the UPR marker CHOP) at older ages in both the plantaris and soleus muscles of rats. Furthermore, these findings were recapitulated using a disuse-induced skeletal muscle atrophy model of hindlimb immobilization (HLI) in the gastrocnemius muscle of 4-month-old rats. While UPR activation was shown via CHOP upregulation, other canonical targets within the UPR pathway did not show as clear a pattern of regulation. However, it is notable that the chaperone protein BiP showed general downregulation with aging. This could serve to diminish protein folding capacity at the ER lumen and lead to enhanced UPR activation due to dissociation from the primary stress sensors PERK, IRE1α, and ATF6 . This remains speculative, however, as the remaining portions of these pathways that were measured did not respond in kind. It is of note that the phosphorylated/pan protein ratio of the protein JNK responded in a similar manner to CHOP, further implicating proapoptotic cell activity in response to aging and disuse . Notably, while JNK regulation can be influenced by the UPR, JNK resides in the family of mitogen activated protein kinases (MAPK) referred to as stress-activated protein kinases (SAPK) and as such have a much broader regulatory system than the UPR alone . Given the broad regulation of JNK through various forms of cell stress induction, we opted to focus on the more ERS associated effector CHOP. Therefore, we posit that the novel and primary finding herein is that the UPR effector CHOP is upregulated with aging and disuse in skeletal muscle. CHOP protein expression was higher in both the soleus and plantaris muscles of 18- and 24-month-old rats versus their younger counterparts. It is intriguing that CHOP was upregulated in 18 mo rats given that others have observed an upregulation later in life . To this point, the Bodine laboratory has previously demonstrated that CHOP is upregulated following disuse and subsequent reloading in aged rats , and in the basal state of 24 month old wild type mice . In both instances, the upregulation in CHOP was linked to depressed proteasome function and an accumulation of ubiquitin tagged proteins. CHOP was additionally upregulated in the mixed gastrocnemius muscle following 10-days of HLI in rats. This finding is notable, given the marked disagreement with Hunter et al. who found no upregulation of CHOP with 1- or 7-days of hindlimb unloading (HU) in Wistar rats. This investigation did, however, show expression differences between the predominantly slow-fiber soleus and the predominantly fast-fiber extensor digitorum longus (EDL), with the EDL demonstrating greater values with or without HU. Conversely, we report no difference in relative expression patterns between the slow-twitch SOL or the fast-twitch PLT with aging, and an upregulation in expression with HLI in the mixed gastrocnemius. Additionally, we found that CHOP expression negatively correlates to normalized muscle weight in all of the aforementioned muscle groups. This is a potential avenue for further investigation, as unraveling proteolytic mechanisms between fiber types is an emerging area of current research . Nonetheless, UPR activation via an upregulation in CHOP has been observed amongst late-age and disuse-atrophy models . While the exact cause for CHOP upregulation in these models is unclear, it is likely that CHOP induction is a function of an accumulation of protein aggregates due to impaired proteostasis in disuse and/or aging. To this end, it has been shown that protein aggregates and impaired proteostasis are seen in both aging and disuse models by way of impaired proteolytic systems, altered protein synthesis rates, or both [ – ]. In a model taking both aging and disuse in to account, Fuqua et al. report that collagen protein synthesis was not different between 10-month and 28-month F344BN rats after 14 days of HU and 60 days of reloading. These investigators did, however, observe higher collagen concentration and an accumulation of advanced glycation end products which are indicative of impaired protein clearance (e.g. proteolysis) and overall proteostasis. This is certainly a possible mechanism by which the protein folding machinery is stressed to the point that the UPR is activated, and the end-effector CHOP is induced. Canonically, CHOP primarily serves as an apoptotic regulator and contributing to cellular dysfunction . To this end, various tissue models have observed an apoptotic response when CHOP is overexpressed and a reduction/resistance to apoptosis when CHOP or its major dimerization partner C/EBPβ is knocked down or depleted . While these examples are convincing, most arise from non-skeletal muscle models and tissue types. Indeed, the mechanism and existence of apoptosis in skeletal muscle (a largely post-mitotic tissue) has been debated and the true function of apoptosis in skeletal muscle remains elusive (reviewed in ). Notwithstanding, the present data suggest a role for CHOP in apoptotic signaling with HLI in Wistar rats. Notably, after 10-days of HLI, CHOP localization in the nuclear fraction was higher versus CTL rats, and the downstream transcriptional target of CHOP, Gadd34 , along with the canonical apoptotic gene Bax , were both ~ 25-fold greater in HLI versus CTL rats. The GADD34 protein has been shown to increase in atrophic murine models of sepsis and cancer cachexia , as well as in an in vitro model of atrophic sepsis (LPS administration) . Critically however, when ERS was globally blocked with 4-phenyl butyric acid (PBA), atrophy was exacerbated in wildtype and tumor bearing mice, suggesting that there is a beneficial role of ERS in the maintenance of skeletal muscle in certain conditions . In a murine model of exercise preconditioning to combat HU-induced atrophy, Gadd34 mRNA was elevated upon 1- and 3-days of HU in the control group. Interestingly, 7 days of exercise preconditioning blunted this increase at 1-day of HU but numerically exacerbated this increase at 3 days. Further, 7 days of exercise preconditioning abrogated the atrophy that was seen with 3 days of HU. Although the model of atrophy differs drastically, the peculiar effects of ERS, particularly by way of GADD34 accumulation at the protein and/or mRNA level, on skeletal muscle remodeling remain at the forefront. While the current study does not delineate between nuclear subpopulations (myonuclei versus resident stromal cells), our data imply that CHOP is operative as a transcriptional regulator with HLI. However, given the results yielded from the aforementioned studies, whether CHOP and its downstream effectors are mechanistic drivers, responsively induced, or have a compensatory role in such models of disuse atrophy remains to be determined. The elevation in CHOP protein expression observed with aging muscle (plantaris and soleus) and HLI muscle (gastrocnemius), and the negative correlations in CHOP protein expression with muscle masses, also warrant discussion. A similar relationship has also been reported whereby the mRNA expression of several key factors in the ERS pathway were significantly correlated with skeletal muscle mass following disuse atrophy in mice . These investigators also demonstrated that a global blockade of ERS (via Tauroursodeoxycholic Acid, TUDCA) mitigates skeletal muscle atrophy following 14 days of HU. While these findings implicate a role for ERS and the UPR in unloading-induced skeletal muscle atrophy, the precise signaling mechanism by which atrophy is alleviated remains unclear. Given the current data showing an upregulation of CHOP expression at later ages and in disuse atrophy, an increased nuclear localization of CHOP following HLI, and the upregulation of known downstream genes affected by CHOP with HLI, we posit that CHOP could play a key role in aging- and disuse-induced skeletal muscle atrophy. However, further mechanistic investigations into the direct role of CHOP are likely needed to fully elucidate this hypothesis. Limitations We are limited in this study by the sole reliance on rodent findings and therefore cannot be certain that the findings presented herein are recapitulated in humans, but the findings could be used for the development of hypotheses to test in humans. Therefore, future research involving human disuse and/or aging models aiming to measure ERS related proteins and responses are warranted. Moreover, given that we lack data from very old rodents (e.g. 28 + months old), we cannot firmly conclude that observed responses are further affected with advanced aging in rodents. In addition to this, we were limited to the use of male rats to examine aging and female rats to examine hindlimb unloading. While sex differences likely exist and play a role in the signaling responses observed herein, it has been reported that both male and female rats experience significant atrophy in response to aging and hindlimb disuse . Finally, we did not perform experiments related to the experimental knockdown of CHOP in vitro or in vivo. Thus, further mechanistic investigation will be needed to determine whether CHOP is a key mechanistic driver of skeletal muscle atrophy.
We are limited in this study by the sole reliance on rodent findings and therefore cannot be certain that the findings presented herein are recapitulated in humans, but the findings could be used for the development of hypotheses to test in humans. Therefore, future research involving human disuse and/or aging models aiming to measure ERS related proteins and responses are warranted. Moreover, given that we lack data from very old rodents (e.g. 28 + months old), we cannot firmly conclude that observed responses are further affected with advanced aging in rodents. In addition to this, we were limited to the use of male rats to examine aging and female rats to examine hindlimb unloading. While sex differences likely exist and play a role in the signaling responses observed herein, it has been reported that both male and female rats experience significant atrophy in response to aging and hindlimb disuse . Finally, we did not perform experiments related to the experimental knockdown of CHOP in vitro or in vivo. Thus, further mechanistic investigation will be needed to determine whether CHOP is a key mechanistic driver of skeletal muscle atrophy.
ERS and the UPR are upregulated with aging in rats and this response was recapitulated at a young age after 10 days of HLI in 4-month-old adult rats as evidenced by an upregulation in the end-effector protein, CHOP. In addition to global upregulation, nuclear CHOP localization and the production of mRNAs induced by CHOP’s transcriptional regulation are heightened during hindlimb immobilization induced skeletal muscle atrophy. Finally, CHOP protein expression is negatively correlated with muscle masses in aging and disuse models of atrophy. Taking these findings in total, it appears that the regulation of CHOP is sensitive to multiple atrophy inducing models. Within these systems CHOP could be atrophy responsive or atrophy inducing, however further interrogation using viral or genetic manipulation of CHOP is needed to determine this. In sum, these findings warrant additional investigation to determine if CHOP is a key mechanistic player in these two different types of skeletal muscle atrophy.
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Potential Efficacy of Herbal Medicine-Derived Carbon Dots in the Treatment of Diseases: From Mechanism to Clinic | 64b80e2b-c596-4369-b295-3624ec6fcf54 | 10642355 | Pharmacology[mh] | As an emerging carbon material after carbon nanotubes, graphene, fullerenes, and nano-diamonds, carbon dots (CDs) have superior properties than other nanomaterials, including ultra-fine size, favorable photoluminescence performance, low toxicity, strong biocompatibility, and excellent electron transfer ability. , The bioactivity of CDs has also been investigated and discovered recently, which were used as mitochondrial oxidative stress amplifiers for targeted therapy of cancer, as well as for fluorescent biosensing and imaging through additional properties. , Moreover, researchers from RMIT University stated that CDs can be utilized as a therapeutic platform to study drug delivery, distribution, metabolism, excretion, and toxicity. These investigations have established a solid foundation for the application of nano carbon dots in numerous research fields. However, the research on carbon dots (CDs) is mostly focused on the optimization of preparation methods and the expansion of application fields. The exploration of chemical and natural substances in synthetic raw materials also deserves considerable attention. The use of “green” substances as raw materials is turning into a hot topic in production research as the notion of green chemistry steadily gains acceptance with societal development. For the synthesis of CDs, numerous green precursors, which have significant advantages such as abundant and renewable raw materials, free of chemical pollution as well as environmentally benign, can be employed as natural carbon sources. Various green carbon precursors have been studied and applied, including fruits, vegetables, and a variety of food and beverage products, to achieve the synthesis of materials with excellent properties, high cost-effectiveness, economic, and environmental protection. Herbal medicine (HM), one of these green precursors, has gained substantial attention due to its distinctive medical efficacy. Numerous clinical studies have demonstrated that herbal medicine has shown exceptional efficacy on various specific diseases such as SARS and COVID-19. However, the medicinal efficacy of herbal medicines is failing to be adequately expressed due to their complex composition. As a result, researchers have initiated attempts to prepare various nanoscale substances of herbal medicines. Interestingly, as one of the most distinctive drugs in clinical applications, herbal medicines can be used as raw materials under elevated temperature conditions to produce novel nanomaterials, which are called herbal medicine-derived carbon dots (HM-CDs), with a diameter of less than 10 nm. Compared to general carbon dots, HM-CDs are synthesized using the medicinal components of herbs, thereby retaining the medicinal value and biological activities of the herbs. HM-CDs may contain active ingredients and chemical substances derived from herbs, such as polysaccharides, phenolic compounds, and alkaloids. , These components confer specific medicinal properties and biological activities to HM-CDs. Due to the preservation of medicinal value and biological activities of herbs, HM-CDs find wider applications in areas such as herbal extraction, drug delivery, and targeted therapy. The structural characteristics, physicochemical qualities, and biological functions of HM-CDs vary amongst different herbal medicines. At present, multiple pharmacological experiments have demonstrated the biological effect of carbon dots in herbal medicines, which revealed the pharmacodynamic basis of herbal medicines in various diseases from a fresh perspective. The purpose of this paper is to exhibit a comparative summary of the synthesis strategies and the main properties of HM-CDs. Special attention is also given to the latest trends in the management of multiple human diseases (including bleeding disorders, inflammatory diseases, cancer, pain of various forms and causes, gastrointestinal disorders, etc.) based on HM-CDs. The intrinsic pharmacological activities and mechanisms of these HM-CDs are also discussed to further advance their clinical applications. To date, numerous techniques have been developed for the synthesis of CDs, such as pyrolysis, microwave-mediated synthesis, chemical oxidation, and hydrothermal treatment. For the preparation of different types of HM-CDs, each of these methods has distinct properties in terms of particle size, quantum yield, and pharmacological activity . However, most of these methods are not environmentally friendly, which require copious volumes of strong acids, harsh synthesis conditions, and complex processes, making large-scale manufacturing challenging. From the perspective of environmental protection, this raises concerns about the toxicity and environmental impact of CDs, necessitating the urgent need for green, eco-friendly, and straightforward synthesis processes. The hydrothermal and calcination methods, which are the most commonly used methods for the preparation of HM-CDs, have the advantages of inexpensive instrumentation, environmental friendliness, and ease of operation, and will be a crucial strategy for further research on the bioactivity of HM-CDs. Hydrothermal Method Hydrothermal synthesis is environmentally friendly without the addition of organic solvents. The surface of CDs does not require additional passivation to maximize safety and minimize toxicity. Prior to preparation, the dried herbs are cut into tiny pieces or ground into a powder in purified water. After the ultrasonic treatment, the mixture is transferred to a PTFE-lined stainless steel autoclave and heated at a specific temperature. To purify the CDs, the suspension needed to be further filtered through a 0.22 μm cellulose membrane and dialyzed for several days using a dialysis bag . The reaction temperature would affect the properties of HM-CDs. The hydrothermal reaction temperature is generally 100–200°C. Li et al synthesized nitrogen-doped CDs of ginkgo fruits (H-N-CDs) at different temperatures. These CDs had the best fluorescence intensity and maximum quantum yield (QY) when the temperature was set to 200°C. In a separate study, the corresponding carbon dots were prepared based on Mentha haplocalyx Briq . When the temperature was below 120°C, electron microscopy revealed a significant amount of polymers, suggesting that the carbonization was incomplete and the CDs were difficult to form. However, no polymers were found by electron microscopy when the temperature reached 180°C, indicating that all compounds were carbonized. This phenomenon has also been observed in coix seed-CDs. The fluorescence intensity of coix seed-CDs decreased with the increase in temperature. In addition, Li et al used Salvia miltiorrhiza Bunge as a carbon source and synthesized three CDs (CDs-100, CDs-150, and CDs-180) by hydrothermal method at different temperatures (100, 150, and 180°C) for 6 h. The average diameters were 16.94, 1.53, and 2.03 nm, respectively. Similarly, the reaction time affects the performance of the CDs. The QY of CDs generated from orange peel ( Citrus reticulata Blanco .) at different time points at the same temperature decreases with increasing reaction time, while the particle size increases slightly. , In another study, honey-CDs prepared by hydrothermal heating at 100°C for 2 h were only stable at 4°C for 3 months. The difference in fluorescence intensity of honey-CDs ceased to be significant when the synthesis time increased to 12 or even 16 hours, suggesting that the fluorescence intensity may have reached saturation. This phenomenon was also observed in aloe vera ( Aloe L .) CDs, where the fluorescence intensity progressively increased with reaction time up to 11 h but decreased afterward. Therefore, the synthesis of CDs by the hydrothermal method needs to be adequately considered and validated with respect to the reaction temperature and time. The hydrothermal temperature should preferably be higher than 100°C. The required time can be determined by the color shift in the precursor solution, which is commonly yellow, orange, or brown. Pyrolysis Method High-temperature pyrolysis is a typical process in addition to hydrothermal synthesis. Natural organic materials are gradually transformed into CDs under vacuum or inert gas by high-temperature processes such as heating, dehydration, degradation, and carbonization. The process is easy to operate, solvent-free, inexpensive, and appropriate for mass manufacturing. The herbs are first placed in a crucible and heated at a specific temperature in a muffle furnace until they are charred. The charred medicine is then crushed and boiled in ultrapure water, and the upper liquid layer is collected. The solution was filtered through a 0.22 μm microporous membrane and dialyzed for several days using dialysis bags to purify the CDs . Carbonization is one of the main elements that affect the success rate of the preparation process through high-temperature calcination. There exist two main traditional methods of carbonation: carbonizing by stir-frying and carbonizing by calcining (also known as wok-covering calcining). Both techniques are applicable to drugs in general. Carbonizing by stir-frying means heating the drug in a preheated vessel over high or moderate heat until the drug turns reddish-brown inside and burns black on the outside, mainly used for root drugs such as carbonized rhubarb, carbonized ginger, and carbonized cortical peony. Carbonization by calcination implies heating and carbonizing the drug under high temperature and anoxic conditions. It is appropriate for loose or light medications that can be easily carbonized (such as Juncus efsus, Radix Rehmanniae , and Nodus Nelumbinis Rhizomatis ). Unfortunately, the limitations of these conventional charring techniques make it challenging to regulate the charring of charcoal-based pharmaceuticals. For example, (i) non-uniform heating can lead to ashing, carbonization, or raw blanks; (ii) it is difficult to control the duration and degree of heating for light-weight drugs, resulting in excessive waste rate; (iii) the root drug is not dry inside, so it cannot be entirely charred; (iv) the operation is cumbersome, time-consuming, and smoke-filled. It is worth noting that the two most popular instrumental means for the production of HM-CDs are calcination in a muffle furnace and carbonization in a drying oven. This type of carbonization can solve the problem of uncontrollable temperature and time. Compared to hydrothermal synthesis, high-temperature pyrolysis generally requires higher reaction temperatures (300°C-400°C) and shorter heating times. The optimal pyrolysis temperature varies when different carbon sources are used to synthesize CDs. Dager et al prepared fennel seed-CDs at 500°C for 3 h, which is the maximum calcination temperature recorded in the current study. These fennel seed-CDs with excellent properties can be preserved for up to 15 months. In contrast, the present HM-CDs made by high-temperature pyrolysis have heating temperatures as low as 220°C. At this temperature, Blue-light CDs were prepared using watermelon peel as the carbon source. However, the reality is that the reaction mechanism for the synthesis of HM-CDs is extremely complex and is affected by various factors such as the temperature, time, and pH of the reaction system. Zhang et al prepared PCC-CDs based on Phellodendri Chinensis Cortex (PCC) under different conditions. PCC-CDs produced at 400°C were a novel carbon-based nanomaterial with exceptional bioactivity, and their antipsoriatic activity was superior to those prepared at alternative temperatures (325°C and 475°C). Another study used pyrolysis to prepare Zingiberis rhizoma -based carbon dots (ZR-CDs) and examined their analgesic activity when carbonized at different temperatures (300, 350, and 400°C) for 1 h or at 350°C for varying periods of time (0.5, 1, and 1.5 h). Ultimately, ZR-CDs were found to have the optimum outcome when prepared at 350°C for 1 h. One hypothesis suggests that the multiple properties of CDs, such as rich chemical groups, size, and solubility, play an important role in their biological applications, , which would lead to small or significant changes in the physicochemical properties of PCC-CDs prepared at different temperatures, resulting in large changes in the biological activity of the obtained CDs. Alternative Methods In view of the drawbacks of the aforementioned techniques, two novel carbonization techniques have been developed: heating with sand and microwave carbonization . Due to its excellent thermal conductivity, highly heated sand prevents inhomogeneous heating of the drug, quickly achieves the desired energy, and is low-cost and simple to manufacture. The majority of charcoal-based medicines, including Nodus Nelumbinis Rhizomatis, Sanguisorba officinalis , and Fructus Crataegi , can be prepared using this approach, while light, friable, or non-separable medicines are not applicable. Yet, the idea behind microwave carbonization is to use energy transmission to cause the breaking of chemical bonds. The response time is drastically decreased and preparation efficacy is increased because of its simpler operation. This method has the advantages of high processing accuracy, minimum contamination, and a wide range of applications for light-textured charcoal herbs. Moreover, a substitute for traditional hydrothermal synthesis has been reported: microwave-assisted hydrothermal synthesis. Li et al prepared two ginkgo fruit-CDs (H-CDs/M-CDs) by hydrothermal (H) and microwave methods (M), respectively. The time required for M-CDs was 5–15 min, which was significantly shorter than that of the hydrothermal method and the particle size was relatively smaller. However, the performance of H-CDs is significantly superior to that of M-CDs. This is due, in part, to the more regular and homogeneous morphology of H-CDs, as well as to the fact that their quantum yields and lifetimes are larger than those of M-CDs and their fluorescence intensity is higher. Interestingly, the microwave technique can even prepare orange peel-CDs in 1 min with a yield of up to 16.20%. In terms of reflection time and efficiency, the microwave synthesis method is undoubtedly superior to hydrothermal and pyrolysis methods, and its time-saving, inexpensive, and easy-to-operate features are particularly attractive for environmentally friendly synthesis of HM-CDs from renewable herbs. Currently, more and more HM-CDs are being prepared by applying microwave carbonization , but it is still far from being a completely developed technique. Another technique for creating HM-CDs, in addition to the ones mentioned above, is heating extraction using different solvents. , For example, Wang et al prepared ethanol-papaya CDs (E-CDs) using 90% ethanol. Although the size of the E-CDs increased with the amount of organic macromolecules in 90% ethanol, alternative solvents may produce the best preparation of CDs when corresponding to various herbal species. Sugarcane ( Saccharum sinensis Roxb .) has also been employed as a carbon source for the synthesis of herbal CDs via the solvothermal method. , The use of organic solvents facilitated the carbonization process during the synthesis of CDs, which significantly altered the photophysical characteristics of the carbon nanoparticles. In another study, environmentally friendly CDs of the Codonopsis pilosula were prepared at room temperature using a one-step solvothermal method. The obtained codonopsis pilosula -derived CDs (CP-CDs) exhibited excellent fluorescence properties (QY up to 12.8%) and strong photostability without any passivation or functionalization on the CP-CDs surface. Therefore, the preparation methods for HM-CDs are diverse, sharing similarities with general carbon dot synthesis methods. The common feature of these methods is that they mainly control the carbon source and reaction conditions to achieve the preparation of carbon dots. However, the distinctive aspect lies in the incorporation of herbal materials as the carbon source in the preparation of HM-CDs. Herbal materials contain abundant organic substances, such as polysaccharides, proteins, and polyphenols, which can be decomposed into carbon dots at high temperatures. The preparation methods of HM-CDs also consider the characteristics and medicinal effects of herbal materials. This includes selecting appropriate extraction methods, solvents, and reaction conditions to retain the effective components of herbal materials and convert them into carbon dots. Furthermore, the preparation methods of HM-CDs can be combined with traditional herbal processing techniques, such as decocting and frying, to further regulate the morphology and properties of carbon dots. These special preparation methods can endow HM-CDs with enhanced biocompatibility and drug release performance, making them suitable for applications in the field of biomedicine. Overall, hydrothermal and pyrolysis technologies are the most popular methods for producing HM-CDs due to their practicality, economy, simplicity of usage, and environmental friendliness. However, the synthesis of HM-CDs by the hydrothermal method is normally considered as a time-consuming process. Although microwave-assisted methods are not as frequently used as hydrothermal and pyrolysis methods, their time-saving, low-cost, easy-to-operate, and efficient features are ideal for the synthesis of HM-CDs. Hydrothermal synthesis is environmentally friendly without the addition of organic solvents. The surface of CDs does not require additional passivation to maximize safety and minimize toxicity. Prior to preparation, the dried herbs are cut into tiny pieces or ground into a powder in purified water. After the ultrasonic treatment, the mixture is transferred to a PTFE-lined stainless steel autoclave and heated at a specific temperature. To purify the CDs, the suspension needed to be further filtered through a 0.22 μm cellulose membrane and dialyzed for several days using a dialysis bag . The reaction temperature would affect the properties of HM-CDs. The hydrothermal reaction temperature is generally 100–200°C. Li et al synthesized nitrogen-doped CDs of ginkgo fruits (H-N-CDs) at different temperatures. These CDs had the best fluorescence intensity and maximum quantum yield (QY) when the temperature was set to 200°C. In a separate study, the corresponding carbon dots were prepared based on Mentha haplocalyx Briq . When the temperature was below 120°C, electron microscopy revealed a significant amount of polymers, suggesting that the carbonization was incomplete and the CDs were difficult to form. However, no polymers were found by electron microscopy when the temperature reached 180°C, indicating that all compounds were carbonized. This phenomenon has also been observed in coix seed-CDs. The fluorescence intensity of coix seed-CDs decreased with the increase in temperature. In addition, Li et al used Salvia miltiorrhiza Bunge as a carbon source and synthesized three CDs (CDs-100, CDs-150, and CDs-180) by hydrothermal method at different temperatures (100, 150, and 180°C) for 6 h. The average diameters were 16.94, 1.53, and 2.03 nm, respectively. Similarly, the reaction time affects the performance of the CDs. The QY of CDs generated from orange peel ( Citrus reticulata Blanco .) at different time points at the same temperature decreases with increasing reaction time, while the particle size increases slightly. , In another study, honey-CDs prepared by hydrothermal heating at 100°C for 2 h were only stable at 4°C for 3 months. The difference in fluorescence intensity of honey-CDs ceased to be significant when the synthesis time increased to 12 or even 16 hours, suggesting that the fluorescence intensity may have reached saturation. This phenomenon was also observed in aloe vera ( Aloe L .) CDs, where the fluorescence intensity progressively increased with reaction time up to 11 h but decreased afterward. Therefore, the synthesis of CDs by the hydrothermal method needs to be adequately considered and validated with respect to the reaction temperature and time. The hydrothermal temperature should preferably be higher than 100°C. The required time can be determined by the color shift in the precursor solution, which is commonly yellow, orange, or brown. High-temperature pyrolysis is a typical process in addition to hydrothermal synthesis. Natural organic materials are gradually transformed into CDs under vacuum or inert gas by high-temperature processes such as heating, dehydration, degradation, and carbonization. The process is easy to operate, solvent-free, inexpensive, and appropriate for mass manufacturing. The herbs are first placed in a crucible and heated at a specific temperature in a muffle furnace until they are charred. The charred medicine is then crushed and boiled in ultrapure water, and the upper liquid layer is collected. The solution was filtered through a 0.22 μm microporous membrane and dialyzed for several days using dialysis bags to purify the CDs . Carbonization is one of the main elements that affect the success rate of the preparation process through high-temperature calcination. There exist two main traditional methods of carbonation: carbonizing by stir-frying and carbonizing by calcining (also known as wok-covering calcining). Both techniques are applicable to drugs in general. Carbonizing by stir-frying means heating the drug in a preheated vessel over high or moderate heat until the drug turns reddish-brown inside and burns black on the outside, mainly used for root drugs such as carbonized rhubarb, carbonized ginger, and carbonized cortical peony. Carbonization by calcination implies heating and carbonizing the drug under high temperature and anoxic conditions. It is appropriate for loose or light medications that can be easily carbonized (such as Juncus efsus, Radix Rehmanniae , and Nodus Nelumbinis Rhizomatis ). Unfortunately, the limitations of these conventional charring techniques make it challenging to regulate the charring of charcoal-based pharmaceuticals. For example, (i) non-uniform heating can lead to ashing, carbonization, or raw blanks; (ii) it is difficult to control the duration and degree of heating for light-weight drugs, resulting in excessive waste rate; (iii) the root drug is not dry inside, so it cannot be entirely charred; (iv) the operation is cumbersome, time-consuming, and smoke-filled. It is worth noting that the two most popular instrumental means for the production of HM-CDs are calcination in a muffle furnace and carbonization in a drying oven. This type of carbonization can solve the problem of uncontrollable temperature and time. Compared to hydrothermal synthesis, high-temperature pyrolysis generally requires higher reaction temperatures (300°C-400°C) and shorter heating times. The optimal pyrolysis temperature varies when different carbon sources are used to synthesize CDs. Dager et al prepared fennel seed-CDs at 500°C for 3 h, which is the maximum calcination temperature recorded in the current study. These fennel seed-CDs with excellent properties can be preserved for up to 15 months. In contrast, the present HM-CDs made by high-temperature pyrolysis have heating temperatures as low as 220°C. At this temperature, Blue-light CDs were prepared using watermelon peel as the carbon source. However, the reality is that the reaction mechanism for the synthesis of HM-CDs is extremely complex and is affected by various factors such as the temperature, time, and pH of the reaction system. Zhang et al prepared PCC-CDs based on Phellodendri Chinensis Cortex (PCC) under different conditions. PCC-CDs produced at 400°C were a novel carbon-based nanomaterial with exceptional bioactivity, and their antipsoriatic activity was superior to those prepared at alternative temperatures (325°C and 475°C). Another study used pyrolysis to prepare Zingiberis rhizoma -based carbon dots (ZR-CDs) and examined their analgesic activity when carbonized at different temperatures (300, 350, and 400°C) for 1 h or at 350°C for varying periods of time (0.5, 1, and 1.5 h). Ultimately, ZR-CDs were found to have the optimum outcome when prepared at 350°C for 1 h. One hypothesis suggests that the multiple properties of CDs, such as rich chemical groups, size, and solubility, play an important role in their biological applications, , which would lead to small or significant changes in the physicochemical properties of PCC-CDs prepared at different temperatures, resulting in large changes in the biological activity of the obtained CDs. In view of the drawbacks of the aforementioned techniques, two novel carbonization techniques have been developed: heating with sand and microwave carbonization . Due to its excellent thermal conductivity, highly heated sand prevents inhomogeneous heating of the drug, quickly achieves the desired energy, and is low-cost and simple to manufacture. The majority of charcoal-based medicines, including Nodus Nelumbinis Rhizomatis, Sanguisorba officinalis , and Fructus Crataegi , can be prepared using this approach, while light, friable, or non-separable medicines are not applicable. Yet, the idea behind microwave carbonization is to use energy transmission to cause the breaking of chemical bonds. The response time is drastically decreased and preparation efficacy is increased because of its simpler operation. This method has the advantages of high processing accuracy, minimum contamination, and a wide range of applications for light-textured charcoal herbs. Moreover, a substitute for traditional hydrothermal synthesis has been reported: microwave-assisted hydrothermal synthesis. Li et al prepared two ginkgo fruit-CDs (H-CDs/M-CDs) by hydrothermal (H) and microwave methods (M), respectively. The time required for M-CDs was 5–15 min, which was significantly shorter than that of the hydrothermal method and the particle size was relatively smaller. However, the performance of H-CDs is significantly superior to that of M-CDs. This is due, in part, to the more regular and homogeneous morphology of H-CDs, as well as to the fact that their quantum yields and lifetimes are larger than those of M-CDs and their fluorescence intensity is higher. Interestingly, the microwave technique can even prepare orange peel-CDs in 1 min with a yield of up to 16.20%. In terms of reflection time and efficiency, the microwave synthesis method is undoubtedly superior to hydrothermal and pyrolysis methods, and its time-saving, inexpensive, and easy-to-operate features are particularly attractive for environmentally friendly synthesis of HM-CDs from renewable herbs. Currently, more and more HM-CDs are being prepared by applying microwave carbonization , but it is still far from being a completely developed technique. Another technique for creating HM-CDs, in addition to the ones mentioned above, is heating extraction using different solvents. , For example, Wang et al prepared ethanol-papaya CDs (E-CDs) using 90% ethanol. Although the size of the E-CDs increased with the amount of organic macromolecules in 90% ethanol, alternative solvents may produce the best preparation of CDs when corresponding to various herbal species. Sugarcane ( Saccharum sinensis Roxb .) has also been employed as a carbon source for the synthesis of herbal CDs via the solvothermal method. , The use of organic solvents facilitated the carbonization process during the synthesis of CDs, which significantly altered the photophysical characteristics of the carbon nanoparticles. In another study, environmentally friendly CDs of the Codonopsis pilosula were prepared at room temperature using a one-step solvothermal method. The obtained codonopsis pilosula -derived CDs (CP-CDs) exhibited excellent fluorescence properties (QY up to 12.8%) and strong photostability without any passivation or functionalization on the CP-CDs surface. Therefore, the preparation methods for HM-CDs are diverse, sharing similarities with general carbon dot synthesis methods. The common feature of these methods is that they mainly control the carbon source and reaction conditions to achieve the preparation of carbon dots. However, the distinctive aspect lies in the incorporation of herbal materials as the carbon source in the preparation of HM-CDs. Herbal materials contain abundant organic substances, such as polysaccharides, proteins, and polyphenols, which can be decomposed into carbon dots at high temperatures. The preparation methods of HM-CDs also consider the characteristics and medicinal effects of herbal materials. This includes selecting appropriate extraction methods, solvents, and reaction conditions to retain the effective components of herbal materials and convert them into carbon dots. Furthermore, the preparation methods of HM-CDs can be combined with traditional herbal processing techniques, such as decocting and frying, to further regulate the morphology and properties of carbon dots. These special preparation methods can endow HM-CDs with enhanced biocompatibility and drug release performance, making them suitable for applications in the field of biomedicine. Overall, hydrothermal and pyrolysis technologies are the most popular methods for producing HM-CDs due to their practicality, economy, simplicity of usage, and environmental friendliness. However, the synthesis of HM-CDs by the hydrothermal method is normally considered as a time-consuming process. Although microwave-assisted methods are not as frequently used as hydrothermal and pyrolysis methods, their time-saving, low-cost, easy-to-operate, and efficient features are ideal for the synthesis of HM-CDs. Particle Size It is well known that nanoscale HM-CDs have received considerable attention worldwide. The bulk of HM-CDs have an average particle size of less than 10 nm, while the average diameter of the smallest one is 1.12 nm. Pyrolysis can produce lower particle sizes, despite the fact that hydrothermal synthesis is more effective at achieving narrow particle size distributions of CDs. The particle size of HM-CDs prepared by pyrolysis was approximately 5 nm under the current synthesis conditions, which seems to represent no discernible difference in the particle sizes of HM-CDs synthesized by the two methods and merits additional exploration. Wang et al isolated a novel carbon dots (PT-CDs) derived from Pollen Typhae by pyrolysis to ameliorate acute kidney injury. The particle size of PT-CDs prepared at different temperatures of 250, 300, 350, and 400°C was less than 20 nm, and the average particle size tended to increase and then decrease, with the lowest value at 400°C (4.85 ± 2.06 nm). The BUN index (250°C, 32.46 ± 2.93 mmol/L; 300°C, 31.98 ± 3.29 mmol/L; 350°C, 31.13 ± 3.11 mmol/L; and 400°C, 31.05 ± 2.70 mmol/L) and CRE level (250°C, 241.95 ± 21.56 μmol/L; 300°C, 242.85 ± 18.79 μmol/L; 350°C, 231.75 ± 19.58 μmol/L; and 400°C, 223.42 ± 17.90 μmol/L) of rats decreased with the increase in preparation temperature after PT-CDs prepared under various conditions were used for treatment, and the best anti-AKI effect was observed at 400°C, according to the results of the renal function evaluation. It is thus speculated that particle size is also one of the elements that regulate the pharmacological activity of HM-CDs. Nanoscale HM-CDs significantly improved membrane permeability and exerted a greater effect than herbal medicines . Pn-CDs can cross the blood-brain barrier (BBB), which may be due to the ultra-tiny size of Pn-CDs, the abundance of functional groups on the surface, and the strong affinity for the BBB endothelial cell membrane. Drugs are also covalently attached to CDs, which facilitates carrier-mediated macromolecular transport. These features enable CDs to enhance BBB permeability through passive transport. Ashrafizadeh et al summarized innovative drug delivery systems using functionalized CDs as carriers for the treatment of various neurological diseases. However, the costly modified ligands constrain their wide range of applications. Although some herbal drugs have been utilized for a long time for the treatment of neurological diseases, the BBB hinders the infiltration of herbal macromolecules. HM-CDs can enhance the BBB permeability of certain macromolecules under non-functionalized conditions. Therefore, this strategy has the potential to be a current breakthrough for herbal medicines to overcome biological barriers. Quantum Yield The quantum yield (QY) of pyrolytic synthesis was discovered to be lower than that of hydrothermal synthesis as a result of the diversity of carbon sources. Most CDs synthesized by pyrolysis had an average QY of less than 10%. However, two investigations produced different results for the synthesis of Schizonepetae Herba Carbonisata -CDs (SHC-CDs) under the same circumstances. One of the SHC-CDs had an average particle size of 0.8–4.0 nm and a QY of 2.26%, whereas those from the another had an average particle size of 1.29–6.87 nm and a QY of 6.31%. These findings illustrate the instability of the method. Nevertheless, Zhang et al created hair CDs with a higher QY (86.06%), which was significantly greater than that of citric acid CDs (19.73%), by combining pyrolysis and microwave. The fusion of the two synthetic techniques could offer potential benefits in addition to the differences in carbon sources. They also produced skin CDs with higher QY (51.35%), indicating that protein-rich materials are more suitable as precursors for CDs preparation. Thus, animal-derived herbs may be the most promising high-yielding drugs for future synthetic of CDs. The carbon dots originating from the same part of different herbs have different properties. Researchers developed CDs from 14 different orange peels under the same preparation conditions with significant differences in QY, possibly related to the volatile oil content. In addition, there are differences in the properties of CDs extracted from different parts of the same herb. For instance, Jiang et al prepared ginkgo leaves-CDs with a high QY (22.80%) using a hydrothermal synthesis technique. However, ginkgo fruit-CDs had a QY of only 3.33%. Evidence suggests that herbs from various portions of the same plant produce distinct HM-CDs, presumably due to compositional variations. It is well known that nanoscale HM-CDs have received considerable attention worldwide. The bulk of HM-CDs have an average particle size of less than 10 nm, while the average diameter of the smallest one is 1.12 nm. Pyrolysis can produce lower particle sizes, despite the fact that hydrothermal synthesis is more effective at achieving narrow particle size distributions of CDs. The particle size of HM-CDs prepared by pyrolysis was approximately 5 nm under the current synthesis conditions, which seems to represent no discernible difference in the particle sizes of HM-CDs synthesized by the two methods and merits additional exploration. Wang et al isolated a novel carbon dots (PT-CDs) derived from Pollen Typhae by pyrolysis to ameliorate acute kidney injury. The particle size of PT-CDs prepared at different temperatures of 250, 300, 350, and 400°C was less than 20 nm, and the average particle size tended to increase and then decrease, with the lowest value at 400°C (4.85 ± 2.06 nm). The BUN index (250°C, 32.46 ± 2.93 mmol/L; 300°C, 31.98 ± 3.29 mmol/L; 350°C, 31.13 ± 3.11 mmol/L; and 400°C, 31.05 ± 2.70 mmol/L) and CRE level (250°C, 241.95 ± 21.56 μmol/L; 300°C, 242.85 ± 18.79 μmol/L; 350°C, 231.75 ± 19.58 μmol/L; and 400°C, 223.42 ± 17.90 μmol/L) of rats decreased with the increase in preparation temperature after PT-CDs prepared under various conditions were used for treatment, and the best anti-AKI effect was observed at 400°C, according to the results of the renal function evaluation. It is thus speculated that particle size is also one of the elements that regulate the pharmacological activity of HM-CDs. Nanoscale HM-CDs significantly improved membrane permeability and exerted a greater effect than herbal medicines . Pn-CDs can cross the blood-brain barrier (BBB), which may be due to the ultra-tiny size of Pn-CDs, the abundance of functional groups on the surface, and the strong affinity for the BBB endothelial cell membrane. Drugs are also covalently attached to CDs, which facilitates carrier-mediated macromolecular transport. These features enable CDs to enhance BBB permeability through passive transport. Ashrafizadeh et al summarized innovative drug delivery systems using functionalized CDs as carriers for the treatment of various neurological diseases. However, the costly modified ligands constrain their wide range of applications. Although some herbal drugs have been utilized for a long time for the treatment of neurological diseases, the BBB hinders the infiltration of herbal macromolecules. HM-CDs can enhance the BBB permeability of certain macromolecules under non-functionalized conditions. Therefore, this strategy has the potential to be a current breakthrough for herbal medicines to overcome biological barriers. The quantum yield (QY) of pyrolytic synthesis was discovered to be lower than that of hydrothermal synthesis as a result of the diversity of carbon sources. Most CDs synthesized by pyrolysis had an average QY of less than 10%. However, two investigations produced different results for the synthesis of Schizonepetae Herba Carbonisata -CDs (SHC-CDs) under the same circumstances. One of the SHC-CDs had an average particle size of 0.8–4.0 nm and a QY of 2.26%, whereas those from the another had an average particle size of 1.29–6.87 nm and a QY of 6.31%. These findings illustrate the instability of the method. Nevertheless, Zhang et al created hair CDs with a higher QY (86.06%), which was significantly greater than that of citric acid CDs (19.73%), by combining pyrolysis and microwave. The fusion of the two synthetic techniques could offer potential benefits in addition to the differences in carbon sources. They also produced skin CDs with higher QY (51.35%), indicating that protein-rich materials are more suitable as precursors for CDs preparation. Thus, animal-derived herbs may be the most promising high-yielding drugs for future synthetic of CDs. The carbon dots originating from the same part of different herbs have different properties. Researchers developed CDs from 14 different orange peels under the same preparation conditions with significant differences in QY, possibly related to the volatile oil content. In addition, there are differences in the properties of CDs extracted from different parts of the same herb. For instance, Jiang et al prepared ginkgo leaves-CDs with a high QY (22.80%) using a hydrothermal synthesis technique. However, ginkgo fruit-CDs had a QY of only 3.33%. Evidence suggests that herbs from various portions of the same plant produce distinct HM-CDs, presumably due to compositional variations. As a novel constituent of the “nanoparticle universe”, HM-CDs have garnered significant attention, prompting extensive investigations into their inherent characteristics using diverse analytical techniques. Distinct spectroscopic methodologies, such as FTIR and UV-Vis spectroscopy, have been judiciously employed to probe the nuanced attributes of herbal CDs. Furthermore, the crystal structure, elemental composition, morphology, and sundry properties of CDs extracted from natural products have been meticulously elucidated via electron microscopy, zeta potential analysis, and X-ray techniques. Spectrographic Techniques Spectroscopic techniques such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, and Raman spectroscopy are employed to analyze the optical properties and electronic structure of HM-CDs. Interestingly, UV-Vis spectroscopy is commonly recommended to evaluate the optical properties of HM-CDs as they typically exhibit strong UV absorption, although the absorption peaks may vary. High-performance liquid chromatography and gel electrophoresis are utilized to separate HM-CDs, allowing for the isolation of CDs with different sizes and shapes. It has been confirmed that CDs with sizes of 1.2, 1.5–3, and 3.8 nm emit light in the visible (400–700 nm), UV (350 nm), and near-infrared (NIR) regions, respectively. Therefore, the absorption band peak centered around the UV region of 250–300 nm is often referred to as the typical π-π* transition peak in most CDs. For instance, Lycii fructus CDs synthesized through hydrothermal treatment exhibit a strong absorption peak at 271 nm in the UV region. CDs derived from Borassus flabellifer flower via thermal decomposition exhibit a UV absorption peak at 282 nm, which is attributed to the π-π* transition of aromatic C=C bonds. Moreover, the surface of HM-CDs is typically composed of various functional groups, such as hydroxyl, carboxyl, carbonyl, ether, or epoxy groups, depending on the synthesis techniques used. Fourier-transform infrared spectroscopy (FTIR) can be utilized to determine the surface functional groups of HM-CDs. For instance, the peaks appearing in FTIR spectra of CDs prepared by ultrasonication irradiation of crab shells were at 3398 cm −1 , 2930 cm −1 , 1640 cm −1 , 1563 cm −1 and 1415 cm −1 , which correspond to the stretching vibration of -H stretching, N-H stretching, C-H stretching, C=O stretching, N-H bending and C=C stretching. The advantage of FTIR in characterizing the surface functionalization of carbon dots lies in its affordability, ease of sample preparation, and rapidity. Electron Microscopy Techniques Electron microscopy techniques play a crucial role in the characterization of nanoparticles. Researchers have widely employed scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to visualize HM-CDs and gain insights into their morphology, size, and formation mechanism. SEM involves scanning the surface of HM-CDs with a focused electron beam to generate images. However, since the particle size of HM-CDs is typically smaller than 10 nm, TEM, which utilizes high-energy electron beams to obtain images through the herbal CD samples, offers higher resolution and is more suitable for identifying small-sized particles compared to SEM. For instance, Dager et al utilized TEM to determine the size of HM-CDs synthesized from microwave irradiation of Fenugreek seeds, revealing an average diameter of 4.25 ± 0.56 nm. Moreover, high-resolution transmission electron microscopy (HRTEM) has proven effective in the structural analysis and detection of lattice defects in HM-CDs. HRTEM analysis demonstrated that green CDs derived from tomatoes exhibit a spherical shape, with a size distribution ranging from 5 to 10 nm. X-Ray Techniques X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) are valuable techniques for analyzing the crystal structure and elemental composition of HM-CDs. XRD, as an important structural tool, is commonly used for effective characterization of CDs as it provides crucial information about their size and purity. The obtained XRD patterns are unique and serve as fingerprints of the periodic atomic arrangement, which can be determined by analyzing the distribution of atoms within the lattice. For instance, XRD analysis of CDs isolated from Actinidia deliciosa ( kiwi) fruit extract through hydrothermal treatment revealed strong broad peaks around 2θ = 28.5° and a weak peak at 2θ = 40.3°, which can be attributed to the diffraction patterns of graphite carbon (002) and (001). The crystal size can then be calculated using the Scherer formula (D = kλ/βcos) by selecting the highest peak displayed in the XRD pattern. However, it is important to note that XRD is not suitable for characterizing amorphous CDs, as it is primarily used for determining key features of CDs with a crystalline structure. Zeta Potential Zeta potential, an essential measurement for evaluating the effective surface charge and quantifying the charge of nanoparticles, plays a crucial role in analyzing the stability of colloidal systems and the surface effects of nanoparticles. This measurement method is particularly important in assessing the toxicity of nanoparticles and their initial absorption by cell membranes. The magnitude of the zeta potential provides valuable insights into the electrical stability of the colloidal system. Research has shown that higher values of zeta potential indicate system stability, while the positive or negative sign of the zeta potential represents the surface charge of the nanoparticles. Nanoparticles with low zeta potential values tend to aggregate together. In a study conducted by the Ramanan group, carbon dots (CDs) were synthesized from algal blooms using microwave irradiation. The researchers successfully obtained highly negative zeta potential values (−22.3±8.39 mV), indicating that the synthesized CDs are negatively charged and rich in carboxyl functional groups. Thus, the measurement of zeta potential provides valuable insights into the stability and aggregation of HM-CDs. Therefore, the characterization of HM-CDs is essential for a deeper understanding of their distinctive properties and behavior. Through analysis of the structural characteristics, one can elucidate their optical, electronic, and chemical properties. The optical properties and surface characteristics of HM-CDs play a crucial role in determining their efficacy in biological systems. Moreover, the morphology and size information of HM-CDs hold significant importance in comprehending their dispersibility and stability in herbal formulations. Spectroscopic techniques such as UV-Vis absorption spectroscopy, fluorescence spectroscopy, and Raman spectroscopy are employed to analyze the optical properties and electronic structure of HM-CDs. Interestingly, UV-Vis spectroscopy is commonly recommended to evaluate the optical properties of HM-CDs as they typically exhibit strong UV absorption, although the absorption peaks may vary. High-performance liquid chromatography and gel electrophoresis are utilized to separate HM-CDs, allowing for the isolation of CDs with different sizes and shapes. It has been confirmed that CDs with sizes of 1.2, 1.5–3, and 3.8 nm emit light in the visible (400–700 nm), UV (350 nm), and near-infrared (NIR) regions, respectively. Therefore, the absorption band peak centered around the UV region of 250–300 nm is often referred to as the typical π-π* transition peak in most CDs. For instance, Lycii fructus CDs synthesized through hydrothermal treatment exhibit a strong absorption peak at 271 nm in the UV region. CDs derived from Borassus flabellifer flower via thermal decomposition exhibit a UV absorption peak at 282 nm, which is attributed to the π-π* transition of aromatic C=C bonds. Moreover, the surface of HM-CDs is typically composed of various functional groups, such as hydroxyl, carboxyl, carbonyl, ether, or epoxy groups, depending on the synthesis techniques used. Fourier-transform infrared spectroscopy (FTIR) can be utilized to determine the surface functional groups of HM-CDs. For instance, the peaks appearing in FTIR spectra of CDs prepared by ultrasonication irradiation of crab shells were at 3398 cm −1 , 2930 cm −1 , 1640 cm −1 , 1563 cm −1 and 1415 cm −1 , which correspond to the stretching vibration of -H stretching, N-H stretching, C-H stretching, C=O stretching, N-H bending and C=C stretching. The advantage of FTIR in characterizing the surface functionalization of carbon dots lies in its affordability, ease of sample preparation, and rapidity. Electron microscopy techniques play a crucial role in the characterization of nanoparticles. Researchers have widely employed scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to visualize HM-CDs and gain insights into their morphology, size, and formation mechanism. SEM involves scanning the surface of HM-CDs with a focused electron beam to generate images. However, since the particle size of HM-CDs is typically smaller than 10 nm, TEM, which utilizes high-energy electron beams to obtain images through the herbal CD samples, offers higher resolution and is more suitable for identifying small-sized particles compared to SEM. For instance, Dager et al utilized TEM to determine the size of HM-CDs synthesized from microwave irradiation of Fenugreek seeds, revealing an average diameter of 4.25 ± 0.56 nm. Moreover, high-resolution transmission electron microscopy (HRTEM) has proven effective in the structural analysis and detection of lattice defects in HM-CDs. HRTEM analysis demonstrated that green CDs derived from tomatoes exhibit a spherical shape, with a size distribution ranging from 5 to 10 nm. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) are valuable techniques for analyzing the crystal structure and elemental composition of HM-CDs. XRD, as an important structural tool, is commonly used for effective characterization of CDs as it provides crucial information about their size and purity. The obtained XRD patterns are unique and serve as fingerprints of the periodic atomic arrangement, which can be determined by analyzing the distribution of atoms within the lattice. For instance, XRD analysis of CDs isolated from Actinidia deliciosa ( kiwi) fruit extract through hydrothermal treatment revealed strong broad peaks around 2θ = 28.5° and a weak peak at 2θ = 40.3°, which can be attributed to the diffraction patterns of graphite carbon (002) and (001). The crystal size can then be calculated using the Scherer formula (D = kλ/βcos) by selecting the highest peak displayed in the XRD pattern. However, it is important to note that XRD is not suitable for characterizing amorphous CDs, as it is primarily used for determining key features of CDs with a crystalline structure. Zeta potential, an essential measurement for evaluating the effective surface charge and quantifying the charge of nanoparticles, plays a crucial role in analyzing the stability of colloidal systems and the surface effects of nanoparticles. This measurement method is particularly important in assessing the toxicity of nanoparticles and their initial absorption by cell membranes. The magnitude of the zeta potential provides valuable insights into the electrical stability of the colloidal system. Research has shown that higher values of zeta potential indicate system stability, while the positive or negative sign of the zeta potential represents the surface charge of the nanoparticles. Nanoparticles with low zeta potential values tend to aggregate together. In a study conducted by the Ramanan group, carbon dots (CDs) were synthesized from algal blooms using microwave irradiation. The researchers successfully obtained highly negative zeta potential values (−22.3±8.39 mV), indicating that the synthesized CDs are negatively charged and rich in carboxyl functional groups. Thus, the measurement of zeta potential provides valuable insights into the stability and aggregation of HM-CDs. Therefore, the characterization of HM-CDs is essential for a deeper understanding of their distinctive properties and behavior. Through analysis of the structural characteristics, one can elucidate their optical, electronic, and chemical properties. The optical properties and surface characteristics of HM-CDs play a crucial role in determining their efficacy in biological systems. Moreover, the morphology and size information of HM-CDs hold significant importance in comprehending their dispersibility and stability in herbal formulations. Existing CDs typically can only be used to cure diseases by loading pharmacophores or as drug delivery vehicles, requiring expensive chemical materials and sophisticated modification techniques. Interestingly, the ability of herbal medicines as precursors to overcome these limitations through their specific efficacy has naturally attracted the attention of researchers. The medical therapeutic effects of HM-CDs and their specific functional mechanisms are mainly reflected in the following aspects . Hematological System Hemostasis For the treatment of hemorrhagic disorders, the carbonization of herbal medicines has a lengthy history and a wealth of clinical evidence. Through the study of CDs in herbal medicines such as Schizonepetae Spica Carbonisata , Cirsii Japonici Herba Carbonisata , and Pollen Typhae Carbonisata , it was found that CDs were present in most herbal medicines with low toxicity, excellent water-solubility, and biocompatibility. Most extracted pure CDs showed positive hemostatic effects . However, hemostasis is a complex system involving the interaction of endothelial cells, platelets, coagulation, and fibrinolytic systems . Routinely used coagulation parameters include activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). The PT value is related to the overall efficiency of the extrinsic coagulation pathway, while the APTT is tied to the intrinsic coagulation pathway. The common coagulation pathway and the activities that promote the conversion of FIB to fibrin in plasma are associated with TT and FIB levels. Extrinsic Coagulation Pathway and Activation of the FIB System Cirsium Setosum Carbonisata (CSC) has cooling, pain-relieving, and heat-clearing properties in traditional medicine. CSC-CDs synthesized based on CSC exhibited moderate hemostatic activity in a mouse model of tail amputation and liver scratch. The hemostatic effect of CSC-CDs may be related to the stimulation of extrinsic coagulation activity and activation of the FIB system, according to the researchers who evaluated coagulation parameters in mice and observed that mice treated with the CSC-CDs group had lower PT values and higher FIB values. Another study created novel water-soluble CDs using Schizonepetae Herba Carbonisata (SHC) as the sole precursor, and pharmacodynamic experiments revealed that SHC-CDs significantly inhibited hemorrhaging in a rat model of tail amputation and liver scratch. Based on the assessment of coagulation parameters in mice, these effects may be related to extrinsic coagulation activity and activation of the FIB system. Junci Medulla Carbonisata (JMC), a herbal medicine for the treatment of bleeding disorders, has not yet been identified as to its potential bioactive components or its mechanisms of action. Cheng’s group first identified novel CDs (JMC-CDs) in JMC and explored the hemostatic mechanism of JMC-CDs by measuring coagulation parameters in rats. JMC-CDs exhibited excellent hemostatic effects through extrinsic coagulation pathways and activation of the FIB system. Intrinsic Coagulation Pathway and Activation of the FIB System Ptollen Typhae Carbonisata (PTC) has been used as a hemostatic drug. To investigate its hemostatic pharmacological effects and mechanism, Yan et al identified and isolated novel water-soluble CDs (PTC-CDs) from the aqueous solution of PTC. The mouse model of tail amputation and liver scratch demonstrated that PTC-CDs exerted hemostatic effects by stimulating intrinsic coagulation pathways and activating the FIB system. After therapy, the high (15.05 s) and low (16.25 s) dose of PTC-CDs decreased APTT significantly (P < 0.01). The PTC-CDs (high, medium, and low concentration) and hemocoagulase groups showed a significant increase (P < 0.01) in the FIB (2.35, 2.30, 2.18, and 2.35 g/L, respectively) compared with that of the control group (2.08 g/L). Meanwhile, all doses of PTC-CDs and hemocoagulase increased PLT significantly to 1201, 1137, 1140, and 1040 × 10 9 /L, which is in agreement with the results of the bleeding times. Zhao et al extracted a novel chemical from Egg yolk oil (EYO) and obtained EYO-CDs through pyrolysis in another study. EYO alone has no hemostatic effect and is regularly used in clinics to treat both acute and chronic eczema, as well as a variety of burns. Nonetheless, the experimental results point to the stimulation and activation of the intrinsic coagulation and FIB systems as the primary hemostatic mechanisms of the novel drug EYO-CDs. The reason for this phenomenon may be the enhanced absorbency and astringency of the HM-CDs generated after the carbonization of herbal medicines. Numerous loose pores are generated in their structure, which can cause accelerated hemostasis by physical adsorption. In addition, due to the peculiar structure of the carbon surface, it can activate plasma clotting factors and split platelets, releasing platelet factors to promote clotting. Intrinsic and Extrinsic Coagulation Pathways To compare the pharmacodynamic basis of plant and animal materials, PHT-CDs, OJT-CDs, DJT-CDs, and XYT-CDs (prepared from the dried pollen of T. angustifolia L ., dry rhizome node of N. nucifera Gaertn ., dry rhizome node of Cirsium japonicum Fisch. ex DC ., and healthy human hair) were investigated for their hemostatic and anti-inflammatory activities in the pre-trauma phase. The four CDs were found to exhibit similar hemostatic effects and mechanisms. By enhancing the activity of pertinent coagulation components in the plasma via extrinsic coagulation routes, the different concentrations of CDs can drastically reduce the PT values. Meanwhile, different concentrations of CDs also shortened the APTT values, indicating that CDs can also affect the coagulation factors to transform the blood into a hypercoagulable state via the intrinsic coagulation pathway. This study serves as a reference for the development of current hemostatic materials and hemostatic drugs. Notably, the anti-inflammatory effect of XYT-CD prepared from human hair was stronger than the remaining three CDs. Common Coagulation Pathway and Activation of the FIB System Although Cirsii Japonici’s (CJ) hemostatic activity is obvious, its active ingredients and underlying mechanisms are yet unknown. Wang et al synthesized novel Cirsii Japonici -derived CDs (CJ-CDs) and evaluated their pharmacological activity and coagulation parameters in rats. The findings demonstrated that CJ-CDs dramatically reduced bleeding in mice caused by liver scratch or tail amputation, and suggested that the hemostatic effect may involve the common coagulation pathway, the FIB system. Similarly, scholars identified the presence of a different substance, Phellodendri Cortex -derived carbon dots (PCC-CDs), from the aqueous extract of Phellodendri Cortex and investigated the hemostatic activity of PCC-CDs by mouse models of tail amputation and liver scratch. The PCC-CDs group-treated mice exhibited satisfactory hemostatic effects (comparable to hemostatic agents), and for the first time, the hemostatic mechanism was found to be through the activation of the FIB system, thus exerting hemostatic efficacy. Acute trauma hemorrhage may benefit from synthetic PCC-CDs due to their outstanding stability, suitability for long-term storage, and potential as a complementary and alternative therapy. Other In addition to the aforementioned coagulation pathways, CDs can increase drug solubility, thereby facilitating drug absorption to indirectly improve the hemostatic effect. For instance, CDs in charcoal-based medications can increase the solubility of glycosides in water by affecting glycosidic acid. Luo et al investigated the effect of novel water-soluble CDs on baicalin, the main component of Radix Puerariae Carbonisata (RPC), and discovered that pure CDs considerably increased the solubility of baicalin in water. The oral bioavailability of RPC-CD was confirmed to be 1.7 times higher than that of pure baicalin. Furthermore, baicalin in Scutellaria baicalensis undergoes carbonization to become easily absorbed charcoal baicalin, which has a potent hemostatic effect. CDs obtained by high-temperature charring are among the key substances that play the role of hemostats and can be directly applied in the treatment of hemorrhagic symptoms of blood fever. It can also promote the absorption of glycosides and indirectly enhance the hemostatic effect. Tail amputation and liver scratch models are common tools to study the hemostatic activity of drugs. However, Sun et al prepared Schizonepetae Spica Carbonisata (SSC)-derived CDs using an improved pyrolysis method and noted that the original SSC-CDs exhibited favorable hemostatic properties via PLT enhancement. More importantly, this is the first evaluation of the hemostatic bioactivity of SSC using the Deinagkistrodon acutus (D. acutus) venom model. Scholars have investigated the pharmacodynamic basis of the hemostatic effect of charcoal-based drugs by introducing transdisciplinary characterization techniques to herbal medicines. It was found that CDs, present in numerous herbal medicines, hold specific structural characteristics, physicochemical properties, and biological activities. These CDs are derived from a variety of natural products with different biological activities and deserve further investigation. Hypoglycemic and Blood Enrichment In addition to hemostasis, HM-CDs are also suitable for additional hematological diseases. Sun et al developed Jiaosanxian -derived CDs (JSX-CDs) with an average diameter of 4.4–6.4 nm by pyrolysis. JSX-CDs have a large number of surface groups, which contributes to their strong solubility and biological activity. The pharmacodynamic findings indicated that JSX-CDs, a promising new type of hypoglycemic agents, have excellent hypoglycemic efficacy and safe hypoglycemic activity. For hemopoietic effects, Xu et al successfully prepared Jujube-CDs (J-CDs) with excellent anemia therapeutic effects. In both in vitro and in vivo experiments, the synthesized J-CDs were able to promote the self-renewal of erythroid progenitor cells. They also specifically increased the proliferation of erythroid cells by modulating the hypoxia response pathway and increasing the phosphorylation levels of STAT5. Therefore, they have great potential as therapeutic agents for cancer-related anemia. Bacterial Infection Infections caused by fungi, bacteria, parasites, or viruses can cause numerous serious diseases. The identification and inactivation of several bacterial species in photosensitizers (PS) has been done using CDs as a possible fluorescent nanomaterial. , HM-CDs also exhibit powerful photodynamic effects due to their optical properties and have been utilized to destroy bacteria under visible light irradiation. Yoon et al prepared mushroom CDs (MCDs) with intense blue fluorescence under the excitation of 360 nm UV light. Under LED visible light illumination, MCDs can produce ROS (such as OH- and O2-) that can adhere directly to the surface of Escherichia coli (E. coli) and induce cell membrane damage. Lin et al prepared four fluorescent CDs using various herbs (onion, ginger, garlic) and additional natural products (fish) as carbon sources. Onion CDs (O-CDs) demonstrated the strongest antibacterial efficacy against Pseudomonas fragilis of all of them. Persistent endodontic infections (PEIs) associated with Enterococcus faecalis (E. faecalis) biofilms are one of the most common dental lesions, and a study was conducted to prepare Fucoidan (FD)-derived CDs for the treatment of PEIs. By causing the development of both intracellular and extracellular reactive oxygen species and modifying the permeability of the bacteria, in vitro tests have shown that FD-CDs have a favorable inhibitory impact on Enterococcus faecalis and its biofilms. Importantly, FD-CDs penetrated root canals and dentin tubules, and removed E. faecalis biofilms, which has great potential for the treatment of PEIs. In addition, some of the HM-CDs alone could not significantly inhibit bacterial growth. However, as drug delivery systems, when loaded with herbal monomers that likewise failed to appreciably reduce bacterial growth, they demonstrated remarkable dose-dependent antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus pathogens. Inflammation-Related Diseases HM-CDs have also gained extensive research attention in the treatment of inflammatory diseases due to their distinctive advantages, such as great biocompatibility, photostability, and inherent targeting of functional groups. Wang et al synthesized a novel Mulberry Silkworm Cocoon -CDs (MSC- CDs) based on MSC. To assess the anti-inflammatory bioactivity of MSC-CDs, the authors of this work creatively applied three conventional experimental models of inflammation. The results showed that MSC-CDs possess significant anti-inflammatory activity, which may be related to the inhibition of inflammatory factors IL-6 and TNF-α expression, providing a reference for further investigation of the potential pharmacodynamic basis of MSC-CDs. In addition to anti-inflammation, the main aspects of HM-CDs for the treatment of inflammation-related diseases are as follows. Arthritis Arthritis is broadly defined as an inflammatory disease that occurs in the human joints and their surrounding tissues. The charcoal-processed drug AFIC of Aurantii fructus ymulturus (AFI) has long been used to treat inflammatory and metabolic diseases. However, the pharmacodynamic basis and action mechanism of AFIC remain unclear. Wang’s group produced a novel type of carbon dots (AFIC-CDs) through pyrolysis and carbonization. AFIC-CDs effectively attenuate the monosodium urate (MSU) crystal-induced inflammatory response by inhibiting the production of inflammatory factors (IL-1β and TNF-α), playing an influential role in the pathophysiology of acute gouty arthritis. Meanwhile, PLR-CDs reduced IL-1 and TNF levels in a dose-dependent manner, which reduced the severity of joint swelling in gouty arthritis. Epidermal Inflammation The emergence of HM-CDs offers hope for the treatment of psoriasis, a chronic inflammatory skin disease. Zhang et al prepared novel non-toxic Phellodendri Cortex CDs (PCC-CDs). The considerable anti-psoriatic action of PCC-CDs was first demonstrated using a mouse model of psoriasis-like skin. The underlying mechanism may be related to the suppression of M1 polarization of macrophages and the relative promotion of M2 polarization. Systemic inflammatory reactions are generally accompanied by fever or hypothermia, and lipopolysaccharide (LPS)-induced fever is caused by inflammation. Therefore, Wu et al explored the effects of synthetic Lonicerae japonicae Flos (LJF) Carbonisata-CDs on LPS-induced fever and hypothermia models in rats. The experimental results showed that LJFC-CDs significantly attenuated the LPS-induced inflammatory response, as evidenced by the expression of TNF-α, IL-1β, IL-6 and the restoration of normal body temperature. Consequently, LJFC-CDs may have some anti-inflammatory properties and alleviate inflammation-induced fever and hypothermia. Frostbite induced by cold conditions triggers varying degrees of tissue damage, but interventions are lacking. To bridge this gap, Kong et al synthesized Artemisiae Argyi Folium (AAF) Carbonisata-CDs (AAFC-CDs) by pyrolysis. AAFC-CDs ameliorate local inflammation by mediating IL-1β and TNF-α and provide the body with energy to alleviate the fall in blood glucose level caused by frostbite, so as to achieve anti-frostbite effects. In contrast to conventional AAF, isochlorogenic acid is no longer present in AAFC-CDs, but its specific composition has not been identified. The conventional AAF is not suitable for treating frostbite. Therefore, the emergence of AAFC-CDs may extend the practical applications of AAF. Allergic Inflammation Allergies are also frequently linked to inflammation. Scutellariae Radix Carbonisata (SRC) is a traditional medicine that can be used to treat allergic diseases. To elucidate the function and mechanism of the carbonized fraction in SRC, Kong et al isolated novel water-soluble SRC-CDs with particle sizes ranging from 2 to 9 nm from aqueous extracts of Scutellariae Radix Carbonisata . Their anti-inflammatory effects are directly related to their stabilization of mast cell agonism, which may be associated with the reduction of mast cell functional agonism, inhibition of RBL-2H3 cell degranulation, and reduction of histamine and inflammatory factor levels. SRC-CDs are therefore effective in reducing allergic responses. By demonstrating the anti-allergic action of SRC-CDs and the associated mechanisms, researchers have filled a research void and laid the groundwork for future innovative drug development. SRC-CDs may then be used as possible medications to treat allergic conditions. Organ Damage Inflammation The organ damage is accompanied by infiltration of inflammatory factors. Zhao et al found that ASAC-CDs synthesized by Armeniacae Semen Amarum could effectively inhibit the expression levels of inflammatory factors (IL-6, IL-1β, and TNF-α) and exhibited satisfactory anti-inflammatory effects, particularly the high-dose group. Compared to the model group (20.56 ± 1.41 pg/mL, 21.07 ± 2.26 pg/mL, and 69.49 ± 9.62 pg/mL, respectively), treatment with high concentrations of ASAC-CDs (8.13 ± 1.40 pg/mL, 8.53 ± 0.82 pg/mL, and 32.03 ± 5.20 pg/mL) significantly reduced the levels of IL-6, IL-1β, and TNF-α (p < 0.01). To a certain degree, they are able to reduce the increase of neutrophils in the blood and decrease the chemotaxis of neutrophils to inflammatory sites, thereby reducing the release of inflammatory mediators and inhibiting LPS-induced damage and deterioration of lung tissue. In a model of acute kidney injury, another study found that PCC-CDs had a direct renoprotective impact by reversing the rise in serum creatinine (SCR), blood urea nitrogen (BUN), urinary total protein (UTP), and microalbuminuria (MALB). PCC-CDs also attenuated the inflammatory response and thrombocytopenia associated with acute kidney injury, thus exerting a multifaceted effect. Inspired by the above, Wang et al isolated a novel carbon dots (PT-CDs) from Pollen Typhae . Using a rat model of rhabdomyolysis (RM)-induced acute kidney injury (AKI), the authors demonstrated that PT-CDs had significant activity in improving BUN and CRE levels, urine volume, renal index, and histopathological morphology in rats with RM-induced AKI. The intervention of PT-CDs dramatically reduced the degree of inflammatory response and oxidative stress, which may be related to the basal potential mechanism of anti-AKI activity. Additionally, cytotoxicity assays and biosafety assessments demonstrated the high biocompatibility of PT-CDs. Herbal medicines are normally considered to be only for chronic diseases but slow to respond or ineffective for acute injuries. However, in addition to achieving protection of organs such as liver, kidney and lung through anti-inflammation, HM-CDs have confirmed the therapeutic effects of herbs on acute injuries. In a recent study, Paeoniae Radix Alba -derived CDs (PRAC-CDs) can inhibit alanine transaminase (ALT) and acetone transaminase (AST) levels and have a mitigating effect on the rise in TBA and TBIL in a mouse model of acute liver injury. By eliminating free oxygen, preventing lipid peroxidation of hepatocytes, controlling bile acid metabolism, reducing malondialdehyde (MDA) levels and increasing superoxide dismutase (SOD) levels, PRAC-CDs exhibit excellent hepatoprotective effects. The Junci Medulla Carbonisata carbon dots (JMC-CDs) also achieved a similar hepatoprotective effect. In animal models of trauma hemorrhagic and internal hemorrhage caused by Deinagkistrodon acutus venom, the researchers showed that JMC-CDs not only had significant hemostatic effects, but also prevented hemorrhagic liver injury with reduced levels of biochemical indicators of liver injury such as aspartate aminotransferase, alanine amino transferase, alkaline phosphatase, total bilirubin, and direct bilirubin. Inflammation of the Gastrointestinal Tract The pharmacological effects of HM-CDs are also involved in the gastrointestinal system for the treatment of various ulcers, which may also be associated with the inflammatory responses. Recently, Hu et al showed that Radix Sophorae Flavescentis carbonisata (RSFC)-CDs could inhibit ethanol-induced acute gastric ulcers in rats by suppressing the release of TNF-α and IL-6 through downregulation of the NF-κB pathway. Most notably, RSFC has been widely used for the treatment of systemic ulcerative diseases. The authors hypothesized that HM-CDs produced by high-temperature pyrolysis may have inherent biological activity, though the active ingredients were not disclosed. Another study synthesized GRR-CDs using Glycyrrhizae Radix et Rhizoma (GRR) as precursors by an environment-friendly one-step pyrolysis process. GRR-CDs significantly reduced the oxidative damage to the gastric mucosa and tissues caused by alcohol, as well as restored the expression of malondialdehyde, superoxide dismutase, and nitric oxide in the serum and tissues of mice. This suggests that the explicit anti-ulcer activity of GRR-CDs, which provides a fresh perspective on how to investigate the pharmacodynamic basis of GRR. Cancer Herbal medicines offer more potent and distinctive anti-tumor effects, and some of them can be combined with radiotherapy to lessen toxicity and boost efficacy. Similarly, CDs prepared by herbal medicine have great potential for oncology treatments. The strategy of combining herbal medicine and CDs is also expected to reduce anti-cancer side effects, increase tumor accumulation, and enhance therapeutic effectiveness. Inspired by curcumin, Li et al prepared novel CDs (G-CDs) based on Ginger and found that G-CDs could have an extremely strong inhibitory effect on the growth of HepG2 cells by up-regulating the expression of the p53 gene in cancer cells and inducing the level of intracellular ROS. G-CDs also exhibited significant anti-hepatocellular carcinoma activity in vivo, which was able to accumulate at the tumor site through enhanced permeation retention (EPR) effect in solid tumors. Ginsenoside Re -based carbon dots (Re-CDs) with a particle size of 4.6 nm were created in another investigation. Re-CDs have demonstrated reduced toxicity to normal cells and higher efficacy in preventing cancer cell proliferation when compared to APIs. Their cancer-fighting effects were coupled with high levels of ROS and the creation of apoptosis associated with caspase-3. Furthermore, Arul et al prepared nitrogen-doped CDs (N-CDs) by a simple hydrothermal method using Actinidia deliciosa (A. deliciosa) fruit extract as a carbonized precursor and aqueous ammonia as a nitrogen dopant. When tested on mouse fibroblast (L-929) cells and human breast cancer (MCF7) cells, the N-CDs also exhibited some anticancer activity. Diseases Related to Oxidative Stress Normal levels of ROS play a decisive role in cell signaling and homeostasis, but excessive ROS accumulation may lead to oxidative damage, inflammation, various diseases, and cancer. Some HM-CDs also have potent antioxidant activities. Among them, natural gynostemma fluorescent CDs can protect zebrafish from oxidative stress by increasing ROS-related enzymes, thus reducing ROS levels through a compensatory mechanism. As a result, as antioxidants, they are effective in reducing ROS damage in Hela cells and zebrafish. Additionally, utilizing Salvia miltiorrhiza Bunge as a carbon source, Li et al created multifunctional antioxidant CDs. Compared to natural Salvia extracts, the resulting CDs had higher antioxidant capacity and greater ability to scavenge ROS, attenuating abiotic stress in plants and opening up a wide range of potential applications in botany. Subsequently, the group synthesized a Salvia miltiorrhiza Bunge -derived CDs. Under conditions of salt and nutrient deficiency, the abundance of functional groups (-OH and -COOH) on the surface encourages Ca2+ signaling and environmental adaptation in plants, which in turn causes ROS-independent Ca2+ activation in the root system. As such, the CDs can be utilized for crop enhancement as both a ROS scavenger and a simultaneous Ca2+ signaling amplifier. Additional Diseases Antinociceptive Effects Ginger has been used as an analgesic with notable results for more than a thousand years, while its material basis is still unknown. With Zingiber officinale Roscoe (ZR) as the raw material, Zhang et al prepared a revolutionary environmentally friendly CDs (ZR-CDs) utilizing direct pyrolysis. The authors confirmed the significant analgesic activity of ZR-CDs using classical hot-plate, tail-immersion, and acetic acid writhing methods, and demonstrated for the first time that the analgesic effect of ZR-CDs was mediated by an opioid-like mechanism and the regulation of 5-hydroxytryptamine levels in serum. In addition to ZR, a study has prepared non-toxic nanocarrier GRR-CDs using Glycyrrhizae Radix et Rhizoma as the only material and an environmentally friendly, simple and low-cost calcination method, which increased the glycyrrhizic acid (GA) solubility significantly by 27-fold. In both the hot-plate model and the acetic acid-induced writhing model, the GRR-CDs-GA complex showed significantly higher antinociceptive activity compared to the unprocessed GRR-CDs and GA. These results support the promising application of GRR-CDs as a technique to improve the solubility and antinociceptive properties of poorly water-soluble drugs (such as GA). Menopausal Syndrome Glycyrrhizae Radix et Rhizoma (GRR) is frequently used in the treatment of menopausal syndrome (MPS) and other gynecological disorders in addition to the antinociceptive activity. Zhang et al successfully synthesized GRR into GRR-CDs by pyrolysis. The study is the first to demonstrate that GRR-CDs can alleviate MPS by elevating the estradiol (E2) level, decreasing follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, and raising the degree of uterine atrophy. This not only indicates the potential of GRR-CDs as a drug to alleviate menopausal syndrome and its associated symptoms, but also provides possibility for nanomedicines to treat hormonal disorders. Anxiolytic Effects People are susceptible to developing depression and anxiety disorders in response to stress. Os Draconis (OD) has gained recognition as a medication that has been used for a long time to treat neurological diseases. In order to elucidate the biological basis of the anxiolytic effects of OD, a study isolated the novel OD-CDs obtained from Os Draconis. Interestingly, OD-CDs significantly reduced anxiety in four behavioral tests, including the Open Field Test (OFT), Light/Dark Box Test (LDT), Elevated Plus Maze Test (EPMT), and Novelty-Suppressed Feeding Test (NSFT). The results also imply that OD-CDs mediate the modulation of monoaminergic neurotransmitters and the HPA axis to a certain extent, although additional research is required to pinpoint the precise processes. Given that OD-CDs exhibit observable anxiolytic effects, this supports their development as novel anxiolytic agents that merit additional study. In summary, HM-CDs, as an emerging nanomaterial, have been widely used in the medical field for their remarkable therapeutic effects due to their excellent photoluminescence capabilities, superior chemical stability and low toxicity, water dispersibility, and biocompatibility. It is noteworthy that the study of the auto-biological activity of HM-CDs has received increasing attention, which is anticipated to reveal their various pharmacological and active effects. However, the therapeutic mechanisms of HM-CDs have not been thoroughly investigated yet, which need to be further explored. In addition, as nanomaterials, elucidating the metabolic processes of HM-CDs in vivo is another major challenge. Hemostasis For the treatment of hemorrhagic disorders, the carbonization of herbal medicines has a lengthy history and a wealth of clinical evidence. Through the study of CDs in herbal medicines such as Schizonepetae Spica Carbonisata , Cirsii Japonici Herba Carbonisata , and Pollen Typhae Carbonisata , it was found that CDs were present in most herbal medicines with low toxicity, excellent water-solubility, and biocompatibility. Most extracted pure CDs showed positive hemostatic effects . However, hemostasis is a complex system involving the interaction of endothelial cells, platelets, coagulation, and fibrinolytic systems . Routinely used coagulation parameters include activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). The PT value is related to the overall efficiency of the extrinsic coagulation pathway, while the APTT is tied to the intrinsic coagulation pathway. The common coagulation pathway and the activities that promote the conversion of FIB to fibrin in plasma are associated with TT and FIB levels. Extrinsic Coagulation Pathway and Activation of the FIB System Cirsium Setosum Carbonisata (CSC) has cooling, pain-relieving, and heat-clearing properties in traditional medicine. CSC-CDs synthesized based on CSC exhibited moderate hemostatic activity in a mouse model of tail amputation and liver scratch. The hemostatic effect of CSC-CDs may be related to the stimulation of extrinsic coagulation activity and activation of the FIB system, according to the researchers who evaluated coagulation parameters in mice and observed that mice treated with the CSC-CDs group had lower PT values and higher FIB values. Another study created novel water-soluble CDs using Schizonepetae Herba Carbonisata (SHC) as the sole precursor, and pharmacodynamic experiments revealed that SHC-CDs significantly inhibited hemorrhaging in a rat model of tail amputation and liver scratch. Based on the assessment of coagulation parameters in mice, these effects may be related to extrinsic coagulation activity and activation of the FIB system. Junci Medulla Carbonisata (JMC), a herbal medicine for the treatment of bleeding disorders, has not yet been identified as to its potential bioactive components or its mechanisms of action. Cheng’s group first identified novel CDs (JMC-CDs) in JMC and explored the hemostatic mechanism of JMC-CDs by measuring coagulation parameters in rats. JMC-CDs exhibited excellent hemostatic effects through extrinsic coagulation pathways and activation of the FIB system. Intrinsic Coagulation Pathway and Activation of the FIB System Ptollen Typhae Carbonisata (PTC) has been used as a hemostatic drug. To investigate its hemostatic pharmacological effects and mechanism, Yan et al identified and isolated novel water-soluble CDs (PTC-CDs) from the aqueous solution of PTC. The mouse model of tail amputation and liver scratch demonstrated that PTC-CDs exerted hemostatic effects by stimulating intrinsic coagulation pathways and activating the FIB system. After therapy, the high (15.05 s) and low (16.25 s) dose of PTC-CDs decreased APTT significantly (P < 0.01). The PTC-CDs (high, medium, and low concentration) and hemocoagulase groups showed a significant increase (P < 0.01) in the FIB (2.35, 2.30, 2.18, and 2.35 g/L, respectively) compared with that of the control group (2.08 g/L). Meanwhile, all doses of PTC-CDs and hemocoagulase increased PLT significantly to 1201, 1137, 1140, and 1040 × 10 9 /L, which is in agreement with the results of the bleeding times. Zhao et al extracted a novel chemical from Egg yolk oil (EYO) and obtained EYO-CDs through pyrolysis in another study. EYO alone has no hemostatic effect and is regularly used in clinics to treat both acute and chronic eczema, as well as a variety of burns. Nonetheless, the experimental results point to the stimulation and activation of the intrinsic coagulation and FIB systems as the primary hemostatic mechanisms of the novel drug EYO-CDs. The reason for this phenomenon may be the enhanced absorbency and astringency of the HM-CDs generated after the carbonization of herbal medicines. Numerous loose pores are generated in their structure, which can cause accelerated hemostasis by physical adsorption. In addition, due to the peculiar structure of the carbon surface, it can activate plasma clotting factors and split platelets, releasing platelet factors to promote clotting. Intrinsic and Extrinsic Coagulation Pathways To compare the pharmacodynamic basis of plant and animal materials, PHT-CDs, OJT-CDs, DJT-CDs, and XYT-CDs (prepared from the dried pollen of T. angustifolia L ., dry rhizome node of N. nucifera Gaertn ., dry rhizome node of Cirsium japonicum Fisch. ex DC ., and healthy human hair) were investigated for their hemostatic and anti-inflammatory activities in the pre-trauma phase. The four CDs were found to exhibit similar hemostatic effects and mechanisms. By enhancing the activity of pertinent coagulation components in the plasma via extrinsic coagulation routes, the different concentrations of CDs can drastically reduce the PT values. Meanwhile, different concentrations of CDs also shortened the APTT values, indicating that CDs can also affect the coagulation factors to transform the blood into a hypercoagulable state via the intrinsic coagulation pathway. This study serves as a reference for the development of current hemostatic materials and hemostatic drugs. Notably, the anti-inflammatory effect of XYT-CD prepared from human hair was stronger than the remaining three CDs. Common Coagulation Pathway and Activation of the FIB System Although Cirsii Japonici’s (CJ) hemostatic activity is obvious, its active ingredients and underlying mechanisms are yet unknown. Wang et al synthesized novel Cirsii Japonici -derived CDs (CJ-CDs) and evaluated their pharmacological activity and coagulation parameters in rats. The findings demonstrated that CJ-CDs dramatically reduced bleeding in mice caused by liver scratch or tail amputation, and suggested that the hemostatic effect may involve the common coagulation pathway, the FIB system. Similarly, scholars identified the presence of a different substance, Phellodendri Cortex -derived carbon dots (PCC-CDs), from the aqueous extract of Phellodendri Cortex and investigated the hemostatic activity of PCC-CDs by mouse models of tail amputation and liver scratch. The PCC-CDs group-treated mice exhibited satisfactory hemostatic effects (comparable to hemostatic agents), and for the first time, the hemostatic mechanism was found to be through the activation of the FIB system, thus exerting hemostatic efficacy. Acute trauma hemorrhage may benefit from synthetic PCC-CDs due to their outstanding stability, suitability for long-term storage, and potential as a complementary and alternative therapy. Other In addition to the aforementioned coagulation pathways, CDs can increase drug solubility, thereby facilitating drug absorption to indirectly improve the hemostatic effect. For instance, CDs in charcoal-based medications can increase the solubility of glycosides in water by affecting glycosidic acid. Luo et al investigated the effect of novel water-soluble CDs on baicalin, the main component of Radix Puerariae Carbonisata (RPC), and discovered that pure CDs considerably increased the solubility of baicalin in water. The oral bioavailability of RPC-CD was confirmed to be 1.7 times higher than that of pure baicalin. Furthermore, baicalin in Scutellaria baicalensis undergoes carbonization to become easily absorbed charcoal baicalin, which has a potent hemostatic effect. CDs obtained by high-temperature charring are among the key substances that play the role of hemostats and can be directly applied in the treatment of hemorrhagic symptoms of blood fever. It can also promote the absorption of glycosides and indirectly enhance the hemostatic effect. Tail amputation and liver scratch models are common tools to study the hemostatic activity of drugs. However, Sun et al prepared Schizonepetae Spica Carbonisata (SSC)-derived CDs using an improved pyrolysis method and noted that the original SSC-CDs exhibited favorable hemostatic properties via PLT enhancement. More importantly, this is the first evaluation of the hemostatic bioactivity of SSC using the Deinagkistrodon acutus (D. acutus) venom model. Scholars have investigated the pharmacodynamic basis of the hemostatic effect of charcoal-based drugs by introducing transdisciplinary characterization techniques to herbal medicines. It was found that CDs, present in numerous herbal medicines, hold specific structural characteristics, physicochemical properties, and biological activities. These CDs are derived from a variety of natural products with different biological activities and deserve further investigation. Hypoglycemic and Blood Enrichment In addition to hemostasis, HM-CDs are also suitable for additional hematological diseases. Sun et al developed Jiaosanxian -derived CDs (JSX-CDs) with an average diameter of 4.4–6.4 nm by pyrolysis. JSX-CDs have a large number of surface groups, which contributes to their strong solubility and biological activity. The pharmacodynamic findings indicated that JSX-CDs, a promising new type of hypoglycemic agents, have excellent hypoglycemic efficacy and safe hypoglycemic activity. For hemopoietic effects, Xu et al successfully prepared Jujube-CDs (J-CDs) with excellent anemia therapeutic effects. In both in vitro and in vivo experiments, the synthesized J-CDs were able to promote the self-renewal of erythroid progenitor cells. They also specifically increased the proliferation of erythroid cells by modulating the hypoxia response pathway and increasing the phosphorylation levels of STAT5. Therefore, they have great potential as therapeutic agents for cancer-related anemia. For the treatment of hemorrhagic disorders, the carbonization of herbal medicines has a lengthy history and a wealth of clinical evidence. Through the study of CDs in herbal medicines such as Schizonepetae Spica Carbonisata , Cirsii Japonici Herba Carbonisata , and Pollen Typhae Carbonisata , it was found that CDs were present in most herbal medicines with low toxicity, excellent water-solubility, and biocompatibility. Most extracted pure CDs showed positive hemostatic effects . However, hemostasis is a complex system involving the interaction of endothelial cells, platelets, coagulation, and fibrinolytic systems . Routinely used coagulation parameters include activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). The PT value is related to the overall efficiency of the extrinsic coagulation pathway, while the APTT is tied to the intrinsic coagulation pathway. The common coagulation pathway and the activities that promote the conversion of FIB to fibrin in plasma are associated with TT and FIB levels. Extrinsic Coagulation Pathway and Activation of the FIB System Cirsium Setosum Carbonisata (CSC) has cooling, pain-relieving, and heat-clearing properties in traditional medicine. CSC-CDs synthesized based on CSC exhibited moderate hemostatic activity in a mouse model of tail amputation and liver scratch. The hemostatic effect of CSC-CDs may be related to the stimulation of extrinsic coagulation activity and activation of the FIB system, according to the researchers who evaluated coagulation parameters in mice and observed that mice treated with the CSC-CDs group had lower PT values and higher FIB values. Another study created novel water-soluble CDs using Schizonepetae Herba Carbonisata (SHC) as the sole precursor, and pharmacodynamic experiments revealed that SHC-CDs significantly inhibited hemorrhaging in a rat model of tail amputation and liver scratch. Based on the assessment of coagulation parameters in mice, these effects may be related to extrinsic coagulation activity and activation of the FIB system. Junci Medulla Carbonisata (JMC), a herbal medicine for the treatment of bleeding disorders, has not yet been identified as to its potential bioactive components or its mechanisms of action. Cheng’s group first identified novel CDs (JMC-CDs) in JMC and explored the hemostatic mechanism of JMC-CDs by measuring coagulation parameters in rats. JMC-CDs exhibited excellent hemostatic effects through extrinsic coagulation pathways and activation of the FIB system. Intrinsic Coagulation Pathway and Activation of the FIB System Ptollen Typhae Carbonisata (PTC) has been used as a hemostatic drug. To investigate its hemostatic pharmacological effects and mechanism, Yan et al identified and isolated novel water-soluble CDs (PTC-CDs) from the aqueous solution of PTC. The mouse model of tail amputation and liver scratch demonstrated that PTC-CDs exerted hemostatic effects by stimulating intrinsic coagulation pathways and activating the FIB system. After therapy, the high (15.05 s) and low (16.25 s) dose of PTC-CDs decreased APTT significantly (P < 0.01). The PTC-CDs (high, medium, and low concentration) and hemocoagulase groups showed a significant increase (P < 0.01) in the FIB (2.35, 2.30, 2.18, and 2.35 g/L, respectively) compared with that of the control group (2.08 g/L). Meanwhile, all doses of PTC-CDs and hemocoagulase increased PLT significantly to 1201, 1137, 1140, and 1040 × 10 9 /L, which is in agreement with the results of the bleeding times. Zhao et al extracted a novel chemical from Egg yolk oil (EYO) and obtained EYO-CDs through pyrolysis in another study. EYO alone has no hemostatic effect and is regularly used in clinics to treat both acute and chronic eczema, as well as a variety of burns. Nonetheless, the experimental results point to the stimulation and activation of the intrinsic coagulation and FIB systems as the primary hemostatic mechanisms of the novel drug EYO-CDs. The reason for this phenomenon may be the enhanced absorbency and astringency of the HM-CDs generated after the carbonization of herbal medicines. Numerous loose pores are generated in their structure, which can cause accelerated hemostasis by physical adsorption. In addition, due to the peculiar structure of the carbon surface, it can activate plasma clotting factors and split platelets, releasing platelet factors to promote clotting. Intrinsic and Extrinsic Coagulation Pathways To compare the pharmacodynamic basis of plant and animal materials, PHT-CDs, OJT-CDs, DJT-CDs, and XYT-CDs (prepared from the dried pollen of T. angustifolia L ., dry rhizome node of N. nucifera Gaertn ., dry rhizome node of Cirsium japonicum Fisch. ex DC ., and healthy human hair) were investigated for their hemostatic and anti-inflammatory activities in the pre-trauma phase. The four CDs were found to exhibit similar hemostatic effects and mechanisms. By enhancing the activity of pertinent coagulation components in the plasma via extrinsic coagulation routes, the different concentrations of CDs can drastically reduce the PT values. Meanwhile, different concentrations of CDs also shortened the APTT values, indicating that CDs can also affect the coagulation factors to transform the blood into a hypercoagulable state via the intrinsic coagulation pathway. This study serves as a reference for the development of current hemostatic materials and hemostatic drugs. Notably, the anti-inflammatory effect of XYT-CD prepared from human hair was stronger than the remaining three CDs. Common Coagulation Pathway and Activation of the FIB System Although Cirsii Japonici’s (CJ) hemostatic activity is obvious, its active ingredients and underlying mechanisms are yet unknown. Wang et al synthesized novel Cirsii Japonici -derived CDs (CJ-CDs) and evaluated their pharmacological activity and coagulation parameters in rats. The findings demonstrated that CJ-CDs dramatically reduced bleeding in mice caused by liver scratch or tail amputation, and suggested that the hemostatic effect may involve the common coagulation pathway, the FIB system. Similarly, scholars identified the presence of a different substance, Phellodendri Cortex -derived carbon dots (PCC-CDs), from the aqueous extract of Phellodendri Cortex and investigated the hemostatic activity of PCC-CDs by mouse models of tail amputation and liver scratch. The PCC-CDs group-treated mice exhibited satisfactory hemostatic effects (comparable to hemostatic agents), and for the first time, the hemostatic mechanism was found to be through the activation of the FIB system, thus exerting hemostatic efficacy. Acute trauma hemorrhage may benefit from synthetic PCC-CDs due to their outstanding stability, suitability for long-term storage, and potential as a complementary and alternative therapy. Other In addition to the aforementioned coagulation pathways, CDs can increase drug solubility, thereby facilitating drug absorption to indirectly improve the hemostatic effect. For instance, CDs in charcoal-based medications can increase the solubility of glycosides in water by affecting glycosidic acid. Luo et al investigated the effect of novel water-soluble CDs on baicalin, the main component of Radix Puerariae Carbonisata (RPC), and discovered that pure CDs considerably increased the solubility of baicalin in water. The oral bioavailability of RPC-CD was confirmed to be 1.7 times higher than that of pure baicalin. Furthermore, baicalin in Scutellaria baicalensis undergoes carbonization to become easily absorbed charcoal baicalin, which has a potent hemostatic effect. CDs obtained by high-temperature charring are among the key substances that play the role of hemostats and can be directly applied in the treatment of hemorrhagic symptoms of blood fever. It can also promote the absorption of glycosides and indirectly enhance the hemostatic effect. Tail amputation and liver scratch models are common tools to study the hemostatic activity of drugs. However, Sun et al prepared Schizonepetae Spica Carbonisata (SSC)-derived CDs using an improved pyrolysis method and noted that the original SSC-CDs exhibited favorable hemostatic properties via PLT enhancement. More importantly, this is the first evaluation of the hemostatic bioactivity of SSC using the Deinagkistrodon acutus (D. acutus) venom model. Scholars have investigated the pharmacodynamic basis of the hemostatic effect of charcoal-based drugs by introducing transdisciplinary characterization techniques to herbal medicines. It was found that CDs, present in numerous herbal medicines, hold specific structural characteristics, physicochemical properties, and biological activities. These CDs are derived from a variety of natural products with different biological activities and deserve further investigation. Cirsium Setosum Carbonisata (CSC) has cooling, pain-relieving, and heat-clearing properties in traditional medicine. CSC-CDs synthesized based on CSC exhibited moderate hemostatic activity in a mouse model of tail amputation and liver scratch. The hemostatic effect of CSC-CDs may be related to the stimulation of extrinsic coagulation activity and activation of the FIB system, according to the researchers who evaluated coagulation parameters in mice and observed that mice treated with the CSC-CDs group had lower PT values and higher FIB values. Another study created novel water-soluble CDs using Schizonepetae Herba Carbonisata (SHC) as the sole precursor, and pharmacodynamic experiments revealed that SHC-CDs significantly inhibited hemorrhaging in a rat model of tail amputation and liver scratch. Based on the assessment of coagulation parameters in mice, these effects may be related to extrinsic coagulation activity and activation of the FIB system. Junci Medulla Carbonisata (JMC), a herbal medicine for the treatment of bleeding disorders, has not yet been identified as to its potential bioactive components or its mechanisms of action. Cheng’s group first identified novel CDs (JMC-CDs) in JMC and explored the hemostatic mechanism of JMC-CDs by measuring coagulation parameters in rats. JMC-CDs exhibited excellent hemostatic effects through extrinsic coagulation pathways and activation of the FIB system. Ptollen Typhae Carbonisata (PTC) has been used as a hemostatic drug. To investigate its hemostatic pharmacological effects and mechanism, Yan et al identified and isolated novel water-soluble CDs (PTC-CDs) from the aqueous solution of PTC. The mouse model of tail amputation and liver scratch demonstrated that PTC-CDs exerted hemostatic effects by stimulating intrinsic coagulation pathways and activating the FIB system. After therapy, the high (15.05 s) and low (16.25 s) dose of PTC-CDs decreased APTT significantly (P < 0.01). The PTC-CDs (high, medium, and low concentration) and hemocoagulase groups showed a significant increase (P < 0.01) in the FIB (2.35, 2.30, 2.18, and 2.35 g/L, respectively) compared with that of the control group (2.08 g/L). Meanwhile, all doses of PTC-CDs and hemocoagulase increased PLT significantly to 1201, 1137, 1140, and 1040 × 10 9 /L, which is in agreement with the results of the bleeding times. Zhao et al extracted a novel chemical from Egg yolk oil (EYO) and obtained EYO-CDs through pyrolysis in another study. EYO alone has no hemostatic effect and is regularly used in clinics to treat both acute and chronic eczema, as well as a variety of burns. Nonetheless, the experimental results point to the stimulation and activation of the intrinsic coagulation and FIB systems as the primary hemostatic mechanisms of the novel drug EYO-CDs. The reason for this phenomenon may be the enhanced absorbency and astringency of the HM-CDs generated after the carbonization of herbal medicines. Numerous loose pores are generated in their structure, which can cause accelerated hemostasis by physical adsorption. In addition, due to the peculiar structure of the carbon surface, it can activate plasma clotting factors and split platelets, releasing platelet factors to promote clotting. To compare the pharmacodynamic basis of plant and animal materials, PHT-CDs, OJT-CDs, DJT-CDs, and XYT-CDs (prepared from the dried pollen of T. angustifolia L ., dry rhizome node of N. nucifera Gaertn ., dry rhizome node of Cirsium japonicum Fisch. ex DC ., and healthy human hair) were investigated for their hemostatic and anti-inflammatory activities in the pre-trauma phase. The four CDs were found to exhibit similar hemostatic effects and mechanisms. By enhancing the activity of pertinent coagulation components in the plasma via extrinsic coagulation routes, the different concentrations of CDs can drastically reduce the PT values. Meanwhile, different concentrations of CDs also shortened the APTT values, indicating that CDs can also affect the coagulation factors to transform the blood into a hypercoagulable state via the intrinsic coagulation pathway. This study serves as a reference for the development of current hemostatic materials and hemostatic drugs. Notably, the anti-inflammatory effect of XYT-CD prepared from human hair was stronger than the remaining three CDs. Although Cirsii Japonici’s (CJ) hemostatic activity is obvious, its active ingredients and underlying mechanisms are yet unknown. Wang et al synthesized novel Cirsii Japonici -derived CDs (CJ-CDs) and evaluated their pharmacological activity and coagulation parameters in rats. The findings demonstrated that CJ-CDs dramatically reduced bleeding in mice caused by liver scratch or tail amputation, and suggested that the hemostatic effect may involve the common coagulation pathway, the FIB system. Similarly, scholars identified the presence of a different substance, Phellodendri Cortex -derived carbon dots (PCC-CDs), from the aqueous extract of Phellodendri Cortex and investigated the hemostatic activity of PCC-CDs by mouse models of tail amputation and liver scratch. The PCC-CDs group-treated mice exhibited satisfactory hemostatic effects (comparable to hemostatic agents), and for the first time, the hemostatic mechanism was found to be through the activation of the FIB system, thus exerting hemostatic efficacy. Acute trauma hemorrhage may benefit from synthetic PCC-CDs due to their outstanding stability, suitability for long-term storage, and potential as a complementary and alternative therapy. In addition to the aforementioned coagulation pathways, CDs can increase drug solubility, thereby facilitating drug absorption to indirectly improve the hemostatic effect. For instance, CDs in charcoal-based medications can increase the solubility of glycosides in water by affecting glycosidic acid. Luo et al investigated the effect of novel water-soluble CDs on baicalin, the main component of Radix Puerariae Carbonisata (RPC), and discovered that pure CDs considerably increased the solubility of baicalin in water. The oral bioavailability of RPC-CD was confirmed to be 1.7 times higher than that of pure baicalin. Furthermore, baicalin in Scutellaria baicalensis undergoes carbonization to become easily absorbed charcoal baicalin, which has a potent hemostatic effect. CDs obtained by high-temperature charring are among the key substances that play the role of hemostats and can be directly applied in the treatment of hemorrhagic symptoms of blood fever. It can also promote the absorption of glycosides and indirectly enhance the hemostatic effect. Tail amputation and liver scratch models are common tools to study the hemostatic activity of drugs. However, Sun et al prepared Schizonepetae Spica Carbonisata (SSC)-derived CDs using an improved pyrolysis method and noted that the original SSC-CDs exhibited favorable hemostatic properties via PLT enhancement. More importantly, this is the first evaluation of the hemostatic bioactivity of SSC using the Deinagkistrodon acutus (D. acutus) venom model. Scholars have investigated the pharmacodynamic basis of the hemostatic effect of charcoal-based drugs by introducing transdisciplinary characterization techniques to herbal medicines. It was found that CDs, present in numerous herbal medicines, hold specific structural characteristics, physicochemical properties, and biological activities. These CDs are derived from a variety of natural products with different biological activities and deserve further investigation. In addition to hemostasis, HM-CDs are also suitable for additional hematological diseases. Sun et al developed Jiaosanxian -derived CDs (JSX-CDs) with an average diameter of 4.4–6.4 nm by pyrolysis. JSX-CDs have a large number of surface groups, which contributes to their strong solubility and biological activity. The pharmacodynamic findings indicated that JSX-CDs, a promising new type of hypoglycemic agents, have excellent hypoglycemic efficacy and safe hypoglycemic activity. For hemopoietic effects, Xu et al successfully prepared Jujube-CDs (J-CDs) with excellent anemia therapeutic effects. In both in vitro and in vivo experiments, the synthesized J-CDs were able to promote the self-renewal of erythroid progenitor cells. They also specifically increased the proliferation of erythroid cells by modulating the hypoxia response pathway and increasing the phosphorylation levels of STAT5. Therefore, they have great potential as therapeutic agents for cancer-related anemia. Infections caused by fungi, bacteria, parasites, or viruses can cause numerous serious diseases. The identification and inactivation of several bacterial species in photosensitizers (PS) has been done using CDs as a possible fluorescent nanomaterial. , HM-CDs also exhibit powerful photodynamic effects due to their optical properties and have been utilized to destroy bacteria under visible light irradiation. Yoon et al prepared mushroom CDs (MCDs) with intense blue fluorescence under the excitation of 360 nm UV light. Under LED visible light illumination, MCDs can produce ROS (such as OH- and O2-) that can adhere directly to the surface of Escherichia coli (E. coli) and induce cell membrane damage. Lin et al prepared four fluorescent CDs using various herbs (onion, ginger, garlic) and additional natural products (fish) as carbon sources. Onion CDs (O-CDs) demonstrated the strongest antibacterial efficacy against Pseudomonas fragilis of all of them. Persistent endodontic infections (PEIs) associated with Enterococcus faecalis (E. faecalis) biofilms are one of the most common dental lesions, and a study was conducted to prepare Fucoidan (FD)-derived CDs for the treatment of PEIs. By causing the development of both intracellular and extracellular reactive oxygen species and modifying the permeability of the bacteria, in vitro tests have shown that FD-CDs have a favorable inhibitory impact on Enterococcus faecalis and its biofilms. Importantly, FD-CDs penetrated root canals and dentin tubules, and removed E. faecalis biofilms, which has great potential for the treatment of PEIs. In addition, some of the HM-CDs alone could not significantly inhibit bacterial growth. However, as drug delivery systems, when loaded with herbal monomers that likewise failed to appreciably reduce bacterial growth, they demonstrated remarkable dose-dependent antibacterial activity against Gram-negative E. coli and Gram-positive S. aureus pathogens. HM-CDs have also gained extensive research attention in the treatment of inflammatory diseases due to their distinctive advantages, such as great biocompatibility, photostability, and inherent targeting of functional groups. Wang et al synthesized a novel Mulberry Silkworm Cocoon -CDs (MSC- CDs) based on MSC. To assess the anti-inflammatory bioactivity of MSC-CDs, the authors of this work creatively applied three conventional experimental models of inflammation. The results showed that MSC-CDs possess significant anti-inflammatory activity, which may be related to the inhibition of inflammatory factors IL-6 and TNF-α expression, providing a reference for further investigation of the potential pharmacodynamic basis of MSC-CDs. In addition to anti-inflammation, the main aspects of HM-CDs for the treatment of inflammation-related diseases are as follows. Arthritis Arthritis is broadly defined as an inflammatory disease that occurs in the human joints and their surrounding tissues. The charcoal-processed drug AFIC of Aurantii fructus ymulturus (AFI) has long been used to treat inflammatory and metabolic diseases. However, the pharmacodynamic basis and action mechanism of AFIC remain unclear. Wang’s group produced a novel type of carbon dots (AFIC-CDs) through pyrolysis and carbonization. AFIC-CDs effectively attenuate the monosodium urate (MSU) crystal-induced inflammatory response by inhibiting the production of inflammatory factors (IL-1β and TNF-α), playing an influential role in the pathophysiology of acute gouty arthritis. Meanwhile, PLR-CDs reduced IL-1 and TNF levels in a dose-dependent manner, which reduced the severity of joint swelling in gouty arthritis. Epidermal Inflammation The emergence of HM-CDs offers hope for the treatment of psoriasis, a chronic inflammatory skin disease. Zhang et al prepared novel non-toxic Phellodendri Cortex CDs (PCC-CDs). The considerable anti-psoriatic action of PCC-CDs was first demonstrated using a mouse model of psoriasis-like skin. The underlying mechanism may be related to the suppression of M1 polarization of macrophages and the relative promotion of M2 polarization. Systemic inflammatory reactions are generally accompanied by fever or hypothermia, and lipopolysaccharide (LPS)-induced fever is caused by inflammation. Therefore, Wu et al explored the effects of synthetic Lonicerae japonicae Flos (LJF) Carbonisata-CDs on LPS-induced fever and hypothermia models in rats. The experimental results showed that LJFC-CDs significantly attenuated the LPS-induced inflammatory response, as evidenced by the expression of TNF-α, IL-1β, IL-6 and the restoration of normal body temperature. Consequently, LJFC-CDs may have some anti-inflammatory properties and alleviate inflammation-induced fever and hypothermia. Frostbite induced by cold conditions triggers varying degrees of tissue damage, but interventions are lacking. To bridge this gap, Kong et al synthesized Artemisiae Argyi Folium (AAF) Carbonisata-CDs (AAFC-CDs) by pyrolysis. AAFC-CDs ameliorate local inflammation by mediating IL-1β and TNF-α and provide the body with energy to alleviate the fall in blood glucose level caused by frostbite, so as to achieve anti-frostbite effects. In contrast to conventional AAF, isochlorogenic acid is no longer present in AAFC-CDs, but its specific composition has not been identified. The conventional AAF is not suitable for treating frostbite. Therefore, the emergence of AAFC-CDs may extend the practical applications of AAF. Allergic Inflammation Allergies are also frequently linked to inflammation. Scutellariae Radix Carbonisata (SRC) is a traditional medicine that can be used to treat allergic diseases. To elucidate the function and mechanism of the carbonized fraction in SRC, Kong et al isolated novel water-soluble SRC-CDs with particle sizes ranging from 2 to 9 nm from aqueous extracts of Scutellariae Radix Carbonisata . Their anti-inflammatory effects are directly related to their stabilization of mast cell agonism, which may be associated with the reduction of mast cell functional agonism, inhibition of RBL-2H3 cell degranulation, and reduction of histamine and inflammatory factor levels. SRC-CDs are therefore effective in reducing allergic responses. By demonstrating the anti-allergic action of SRC-CDs and the associated mechanisms, researchers have filled a research void and laid the groundwork for future innovative drug development. SRC-CDs may then be used as possible medications to treat allergic conditions. Organ Damage Inflammation The organ damage is accompanied by infiltration of inflammatory factors. Zhao et al found that ASAC-CDs synthesized by Armeniacae Semen Amarum could effectively inhibit the expression levels of inflammatory factors (IL-6, IL-1β, and TNF-α) and exhibited satisfactory anti-inflammatory effects, particularly the high-dose group. Compared to the model group (20.56 ± 1.41 pg/mL, 21.07 ± 2.26 pg/mL, and 69.49 ± 9.62 pg/mL, respectively), treatment with high concentrations of ASAC-CDs (8.13 ± 1.40 pg/mL, 8.53 ± 0.82 pg/mL, and 32.03 ± 5.20 pg/mL) significantly reduced the levels of IL-6, IL-1β, and TNF-α (p < 0.01). To a certain degree, they are able to reduce the increase of neutrophils in the blood and decrease the chemotaxis of neutrophils to inflammatory sites, thereby reducing the release of inflammatory mediators and inhibiting LPS-induced damage and deterioration of lung tissue. In a model of acute kidney injury, another study found that PCC-CDs had a direct renoprotective impact by reversing the rise in serum creatinine (SCR), blood urea nitrogen (BUN), urinary total protein (UTP), and microalbuminuria (MALB). PCC-CDs also attenuated the inflammatory response and thrombocytopenia associated with acute kidney injury, thus exerting a multifaceted effect. Inspired by the above, Wang et al isolated a novel carbon dots (PT-CDs) from Pollen Typhae . Using a rat model of rhabdomyolysis (RM)-induced acute kidney injury (AKI), the authors demonstrated that PT-CDs had significant activity in improving BUN and CRE levels, urine volume, renal index, and histopathological morphology in rats with RM-induced AKI. The intervention of PT-CDs dramatically reduced the degree of inflammatory response and oxidative stress, which may be related to the basal potential mechanism of anti-AKI activity. Additionally, cytotoxicity assays and biosafety assessments demonstrated the high biocompatibility of PT-CDs. Herbal medicines are normally considered to be only for chronic diseases but slow to respond or ineffective for acute injuries. However, in addition to achieving protection of organs such as liver, kidney and lung through anti-inflammation, HM-CDs have confirmed the therapeutic effects of herbs on acute injuries. In a recent study, Paeoniae Radix Alba -derived CDs (PRAC-CDs) can inhibit alanine transaminase (ALT) and acetone transaminase (AST) levels and have a mitigating effect on the rise in TBA and TBIL in a mouse model of acute liver injury. By eliminating free oxygen, preventing lipid peroxidation of hepatocytes, controlling bile acid metabolism, reducing malondialdehyde (MDA) levels and increasing superoxide dismutase (SOD) levels, PRAC-CDs exhibit excellent hepatoprotective effects. The Junci Medulla Carbonisata carbon dots (JMC-CDs) also achieved a similar hepatoprotective effect. In animal models of trauma hemorrhagic and internal hemorrhage caused by Deinagkistrodon acutus venom, the researchers showed that JMC-CDs not only had significant hemostatic effects, but also prevented hemorrhagic liver injury with reduced levels of biochemical indicators of liver injury such as aspartate aminotransferase, alanine amino transferase, alkaline phosphatase, total bilirubin, and direct bilirubin. Inflammation of the Gastrointestinal Tract The pharmacological effects of HM-CDs are also involved in the gastrointestinal system for the treatment of various ulcers, which may also be associated with the inflammatory responses. Recently, Hu et al showed that Radix Sophorae Flavescentis carbonisata (RSFC)-CDs could inhibit ethanol-induced acute gastric ulcers in rats by suppressing the release of TNF-α and IL-6 through downregulation of the NF-κB pathway. Most notably, RSFC has been widely used for the treatment of systemic ulcerative diseases. The authors hypothesized that HM-CDs produced by high-temperature pyrolysis may have inherent biological activity, though the active ingredients were not disclosed. Another study synthesized GRR-CDs using Glycyrrhizae Radix et Rhizoma (GRR) as precursors by an environment-friendly one-step pyrolysis process. GRR-CDs significantly reduced the oxidative damage to the gastric mucosa and tissues caused by alcohol, as well as restored the expression of malondialdehyde, superoxide dismutase, and nitric oxide in the serum and tissues of mice. This suggests that the explicit anti-ulcer activity of GRR-CDs, which provides a fresh perspective on how to investigate the pharmacodynamic basis of GRR. Arthritis is broadly defined as an inflammatory disease that occurs in the human joints and their surrounding tissues. The charcoal-processed drug AFIC of Aurantii fructus ymulturus (AFI) has long been used to treat inflammatory and metabolic diseases. However, the pharmacodynamic basis and action mechanism of AFIC remain unclear. Wang’s group produced a novel type of carbon dots (AFIC-CDs) through pyrolysis and carbonization. AFIC-CDs effectively attenuate the monosodium urate (MSU) crystal-induced inflammatory response by inhibiting the production of inflammatory factors (IL-1β and TNF-α), playing an influential role in the pathophysiology of acute gouty arthritis. Meanwhile, PLR-CDs reduced IL-1 and TNF levels in a dose-dependent manner, which reduced the severity of joint swelling in gouty arthritis. The emergence of HM-CDs offers hope for the treatment of psoriasis, a chronic inflammatory skin disease. Zhang et al prepared novel non-toxic Phellodendri Cortex CDs (PCC-CDs). The considerable anti-psoriatic action of PCC-CDs was first demonstrated using a mouse model of psoriasis-like skin. The underlying mechanism may be related to the suppression of M1 polarization of macrophages and the relative promotion of M2 polarization. Systemic inflammatory reactions are generally accompanied by fever or hypothermia, and lipopolysaccharide (LPS)-induced fever is caused by inflammation. Therefore, Wu et al explored the effects of synthetic Lonicerae japonicae Flos (LJF) Carbonisata-CDs on LPS-induced fever and hypothermia models in rats. The experimental results showed that LJFC-CDs significantly attenuated the LPS-induced inflammatory response, as evidenced by the expression of TNF-α, IL-1β, IL-6 and the restoration of normal body temperature. Consequently, LJFC-CDs may have some anti-inflammatory properties and alleviate inflammation-induced fever and hypothermia. Frostbite induced by cold conditions triggers varying degrees of tissue damage, but interventions are lacking. To bridge this gap, Kong et al synthesized Artemisiae Argyi Folium (AAF) Carbonisata-CDs (AAFC-CDs) by pyrolysis. AAFC-CDs ameliorate local inflammation by mediating IL-1β and TNF-α and provide the body with energy to alleviate the fall in blood glucose level caused by frostbite, so as to achieve anti-frostbite effects. In contrast to conventional AAF, isochlorogenic acid is no longer present in AAFC-CDs, but its specific composition has not been identified. The conventional AAF is not suitable for treating frostbite. Therefore, the emergence of AAFC-CDs may extend the practical applications of AAF. Allergies are also frequently linked to inflammation. Scutellariae Radix Carbonisata (SRC) is a traditional medicine that can be used to treat allergic diseases. To elucidate the function and mechanism of the carbonized fraction in SRC, Kong et al isolated novel water-soluble SRC-CDs with particle sizes ranging from 2 to 9 nm from aqueous extracts of Scutellariae Radix Carbonisata . Their anti-inflammatory effects are directly related to their stabilization of mast cell agonism, which may be associated with the reduction of mast cell functional agonism, inhibition of RBL-2H3 cell degranulation, and reduction of histamine and inflammatory factor levels. SRC-CDs are therefore effective in reducing allergic responses. By demonstrating the anti-allergic action of SRC-CDs and the associated mechanisms, researchers have filled a research void and laid the groundwork for future innovative drug development. SRC-CDs may then be used as possible medications to treat allergic conditions. The organ damage is accompanied by infiltration of inflammatory factors. Zhao et al found that ASAC-CDs synthesized by Armeniacae Semen Amarum could effectively inhibit the expression levels of inflammatory factors (IL-6, IL-1β, and TNF-α) and exhibited satisfactory anti-inflammatory effects, particularly the high-dose group. Compared to the model group (20.56 ± 1.41 pg/mL, 21.07 ± 2.26 pg/mL, and 69.49 ± 9.62 pg/mL, respectively), treatment with high concentrations of ASAC-CDs (8.13 ± 1.40 pg/mL, 8.53 ± 0.82 pg/mL, and 32.03 ± 5.20 pg/mL) significantly reduced the levels of IL-6, IL-1β, and TNF-α (p < 0.01). To a certain degree, they are able to reduce the increase of neutrophils in the blood and decrease the chemotaxis of neutrophils to inflammatory sites, thereby reducing the release of inflammatory mediators and inhibiting LPS-induced damage and deterioration of lung tissue. In a model of acute kidney injury, another study found that PCC-CDs had a direct renoprotective impact by reversing the rise in serum creatinine (SCR), blood urea nitrogen (BUN), urinary total protein (UTP), and microalbuminuria (MALB). PCC-CDs also attenuated the inflammatory response and thrombocytopenia associated with acute kidney injury, thus exerting a multifaceted effect. Inspired by the above, Wang et al isolated a novel carbon dots (PT-CDs) from Pollen Typhae . Using a rat model of rhabdomyolysis (RM)-induced acute kidney injury (AKI), the authors demonstrated that PT-CDs had significant activity in improving BUN and CRE levels, urine volume, renal index, and histopathological morphology in rats with RM-induced AKI. The intervention of PT-CDs dramatically reduced the degree of inflammatory response and oxidative stress, which may be related to the basal potential mechanism of anti-AKI activity. Additionally, cytotoxicity assays and biosafety assessments demonstrated the high biocompatibility of PT-CDs. Herbal medicines are normally considered to be only for chronic diseases but slow to respond or ineffective for acute injuries. However, in addition to achieving protection of organs such as liver, kidney and lung through anti-inflammation, HM-CDs have confirmed the therapeutic effects of herbs on acute injuries. In a recent study, Paeoniae Radix Alba -derived CDs (PRAC-CDs) can inhibit alanine transaminase (ALT) and acetone transaminase (AST) levels and have a mitigating effect on the rise in TBA and TBIL in a mouse model of acute liver injury. By eliminating free oxygen, preventing lipid peroxidation of hepatocytes, controlling bile acid metabolism, reducing malondialdehyde (MDA) levels and increasing superoxide dismutase (SOD) levels, PRAC-CDs exhibit excellent hepatoprotective effects. The Junci Medulla Carbonisata carbon dots (JMC-CDs) also achieved a similar hepatoprotective effect. In animal models of trauma hemorrhagic and internal hemorrhage caused by Deinagkistrodon acutus venom, the researchers showed that JMC-CDs not only had significant hemostatic effects, but also prevented hemorrhagic liver injury with reduced levels of biochemical indicators of liver injury such as aspartate aminotransferase, alanine amino transferase, alkaline phosphatase, total bilirubin, and direct bilirubin. The pharmacological effects of HM-CDs are also involved in the gastrointestinal system for the treatment of various ulcers, which may also be associated with the inflammatory responses. Recently, Hu et al showed that Radix Sophorae Flavescentis carbonisata (RSFC)-CDs could inhibit ethanol-induced acute gastric ulcers in rats by suppressing the release of TNF-α and IL-6 through downregulation of the NF-κB pathway. Most notably, RSFC has been widely used for the treatment of systemic ulcerative diseases. The authors hypothesized that HM-CDs produced by high-temperature pyrolysis may have inherent biological activity, though the active ingredients were not disclosed. Another study synthesized GRR-CDs using Glycyrrhizae Radix et Rhizoma (GRR) as precursors by an environment-friendly one-step pyrolysis process. GRR-CDs significantly reduced the oxidative damage to the gastric mucosa and tissues caused by alcohol, as well as restored the expression of malondialdehyde, superoxide dismutase, and nitric oxide in the serum and tissues of mice. This suggests that the explicit anti-ulcer activity of GRR-CDs, which provides a fresh perspective on how to investigate the pharmacodynamic basis of GRR. Herbal medicines offer more potent and distinctive anti-tumor effects, and some of them can be combined with radiotherapy to lessen toxicity and boost efficacy. Similarly, CDs prepared by herbal medicine have great potential for oncology treatments. The strategy of combining herbal medicine and CDs is also expected to reduce anti-cancer side effects, increase tumor accumulation, and enhance therapeutic effectiveness. Inspired by curcumin, Li et al prepared novel CDs (G-CDs) based on Ginger and found that G-CDs could have an extremely strong inhibitory effect on the growth of HepG2 cells by up-regulating the expression of the p53 gene in cancer cells and inducing the level of intracellular ROS. G-CDs also exhibited significant anti-hepatocellular carcinoma activity in vivo, which was able to accumulate at the tumor site through enhanced permeation retention (EPR) effect in solid tumors. Ginsenoside Re -based carbon dots (Re-CDs) with a particle size of 4.6 nm were created in another investigation. Re-CDs have demonstrated reduced toxicity to normal cells and higher efficacy in preventing cancer cell proliferation when compared to APIs. Their cancer-fighting effects were coupled with high levels of ROS and the creation of apoptosis associated with caspase-3. Furthermore, Arul et al prepared nitrogen-doped CDs (N-CDs) by a simple hydrothermal method using Actinidia deliciosa (A. deliciosa) fruit extract as a carbonized precursor and aqueous ammonia as a nitrogen dopant. When tested on mouse fibroblast (L-929) cells and human breast cancer (MCF7) cells, the N-CDs also exhibited some anticancer activity. Normal levels of ROS play a decisive role in cell signaling and homeostasis, but excessive ROS accumulation may lead to oxidative damage, inflammation, various diseases, and cancer. Some HM-CDs also have potent antioxidant activities. Among them, natural gynostemma fluorescent CDs can protect zebrafish from oxidative stress by increasing ROS-related enzymes, thus reducing ROS levels through a compensatory mechanism. As a result, as antioxidants, they are effective in reducing ROS damage in Hela cells and zebrafish. Additionally, utilizing Salvia miltiorrhiza Bunge as a carbon source, Li et al created multifunctional antioxidant CDs. Compared to natural Salvia extracts, the resulting CDs had higher antioxidant capacity and greater ability to scavenge ROS, attenuating abiotic stress in plants and opening up a wide range of potential applications in botany. Subsequently, the group synthesized a Salvia miltiorrhiza Bunge -derived CDs. Under conditions of salt and nutrient deficiency, the abundance of functional groups (-OH and -COOH) on the surface encourages Ca2+ signaling and environmental adaptation in plants, which in turn causes ROS-independent Ca2+ activation in the root system. As such, the CDs can be utilized for crop enhancement as both a ROS scavenger and a simultaneous Ca2+ signaling amplifier. Antinociceptive Effects Ginger has been used as an analgesic with notable results for more than a thousand years, while its material basis is still unknown. With Zingiber officinale Roscoe (ZR) as the raw material, Zhang et al prepared a revolutionary environmentally friendly CDs (ZR-CDs) utilizing direct pyrolysis. The authors confirmed the significant analgesic activity of ZR-CDs using classical hot-plate, tail-immersion, and acetic acid writhing methods, and demonstrated for the first time that the analgesic effect of ZR-CDs was mediated by an opioid-like mechanism and the regulation of 5-hydroxytryptamine levels in serum. In addition to ZR, a study has prepared non-toxic nanocarrier GRR-CDs using Glycyrrhizae Radix et Rhizoma as the only material and an environmentally friendly, simple and low-cost calcination method, which increased the glycyrrhizic acid (GA) solubility significantly by 27-fold. In both the hot-plate model and the acetic acid-induced writhing model, the GRR-CDs-GA complex showed significantly higher antinociceptive activity compared to the unprocessed GRR-CDs and GA. These results support the promising application of GRR-CDs as a technique to improve the solubility and antinociceptive properties of poorly water-soluble drugs (such as GA). Menopausal Syndrome Glycyrrhizae Radix et Rhizoma (GRR) is frequently used in the treatment of menopausal syndrome (MPS) and other gynecological disorders in addition to the antinociceptive activity. Zhang et al successfully synthesized GRR into GRR-CDs by pyrolysis. The study is the first to demonstrate that GRR-CDs can alleviate MPS by elevating the estradiol (E2) level, decreasing follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, and raising the degree of uterine atrophy. This not only indicates the potential of GRR-CDs as a drug to alleviate menopausal syndrome and its associated symptoms, but also provides possibility for nanomedicines to treat hormonal disorders. Anxiolytic Effects People are susceptible to developing depression and anxiety disorders in response to stress. Os Draconis (OD) has gained recognition as a medication that has been used for a long time to treat neurological diseases. In order to elucidate the biological basis of the anxiolytic effects of OD, a study isolated the novel OD-CDs obtained from Os Draconis. Interestingly, OD-CDs significantly reduced anxiety in four behavioral tests, including the Open Field Test (OFT), Light/Dark Box Test (LDT), Elevated Plus Maze Test (EPMT), and Novelty-Suppressed Feeding Test (NSFT). The results also imply that OD-CDs mediate the modulation of monoaminergic neurotransmitters and the HPA axis to a certain extent, although additional research is required to pinpoint the precise processes. Given that OD-CDs exhibit observable anxiolytic effects, this supports their development as novel anxiolytic agents that merit additional study. In summary, HM-CDs, as an emerging nanomaterial, have been widely used in the medical field for their remarkable therapeutic effects due to their excellent photoluminescence capabilities, superior chemical stability and low toxicity, water dispersibility, and biocompatibility. It is noteworthy that the study of the auto-biological activity of HM-CDs has received increasing attention, which is anticipated to reveal their various pharmacological and active effects. However, the therapeutic mechanisms of HM-CDs have not been thoroughly investigated yet, which need to be further explored. In addition, as nanomaterials, elucidating the metabolic processes of HM-CDs in vivo is another major challenge. Ginger has been used as an analgesic with notable results for more than a thousand years, while its material basis is still unknown. With Zingiber officinale Roscoe (ZR) as the raw material, Zhang et al prepared a revolutionary environmentally friendly CDs (ZR-CDs) utilizing direct pyrolysis. The authors confirmed the significant analgesic activity of ZR-CDs using classical hot-plate, tail-immersion, and acetic acid writhing methods, and demonstrated for the first time that the analgesic effect of ZR-CDs was mediated by an opioid-like mechanism and the regulation of 5-hydroxytryptamine levels in serum. In addition to ZR, a study has prepared non-toxic nanocarrier GRR-CDs using Glycyrrhizae Radix et Rhizoma as the only material and an environmentally friendly, simple and low-cost calcination method, which increased the glycyrrhizic acid (GA) solubility significantly by 27-fold. In both the hot-plate model and the acetic acid-induced writhing model, the GRR-CDs-GA complex showed significantly higher antinociceptive activity compared to the unprocessed GRR-CDs and GA. These results support the promising application of GRR-CDs as a technique to improve the solubility and antinociceptive properties of poorly water-soluble drugs (such as GA). Glycyrrhizae Radix et Rhizoma (GRR) is frequently used in the treatment of menopausal syndrome (MPS) and other gynecological disorders in addition to the antinociceptive activity. Zhang et al successfully synthesized GRR into GRR-CDs by pyrolysis. The study is the first to demonstrate that GRR-CDs can alleviate MPS by elevating the estradiol (E2) level, decreasing follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels, and raising the degree of uterine atrophy. This not only indicates the potential of GRR-CDs as a drug to alleviate menopausal syndrome and its associated symptoms, but also provides possibility for nanomedicines to treat hormonal disorders. People are susceptible to developing depression and anxiety disorders in response to stress. Os Draconis (OD) has gained recognition as a medication that has been used for a long time to treat neurological diseases. In order to elucidate the biological basis of the anxiolytic effects of OD, a study isolated the novel OD-CDs obtained from Os Draconis. Interestingly, OD-CDs significantly reduced anxiety in four behavioral tests, including the Open Field Test (OFT), Light/Dark Box Test (LDT), Elevated Plus Maze Test (EPMT), and Novelty-Suppressed Feeding Test (NSFT). The results also imply that OD-CDs mediate the modulation of monoaminergic neurotransmitters and the HPA axis to a certain extent, although additional research is required to pinpoint the precise processes. Given that OD-CDs exhibit observable anxiolytic effects, this supports their development as novel anxiolytic agents that merit additional study. In summary, HM-CDs, as an emerging nanomaterial, have been widely used in the medical field for their remarkable therapeutic effects due to their excellent photoluminescence capabilities, superior chemical stability and low toxicity, water dispersibility, and biocompatibility. It is noteworthy that the study of the auto-biological activity of HM-CDs has received increasing attention, which is anticipated to reveal their various pharmacological and active effects. However, the therapeutic mechanisms of HM-CDs have not been thoroughly investigated yet, which need to be further explored. In addition, as nanomaterials, elucidating the metabolic processes of HM-CDs in vivo is another major challenge. CDs, as a novel class of fluorescent carbon nanomaterials, have made numerous significant breakthroughs from their fundamental optical features to potential applications. As a fresh branch of CDs, HM-CDs have been extensively applied in disease treatment. The potential therapeutic efficacy and fluorescence properties are significant markers to distinguish HM-CDs from ordinary CDs. HM-CDs have a higher pharmacological activity than raw material products, which may lead to altered therapeutic efficacy. Their removal of the requirement for drug loading can successfully prevent negative effects, promising significant advances in the near future. Despite the rapid advancement of CDs in herbal medicine, there are still numerous issues that remain to be resolved. First, although CDs show low toxicity, their potential effects on humans are unknown. The tiny molecule compounds produced by the photodegradation of CDs cause some toxicity. On the other hand, some CDs have novel cytotoxic properties, including ROS-generating toxicity and dose-dependent toxicity. Therefore, the risks of HM-CDs in clinical therapeutic applications still require further attention and research. Second, the preparation and processing of CDs are not subject to a unified objective quantitative assessment. It is challenging to guarantee the stable and uniform quality of CDs under different preparation circumstances (temperature, time, etc.), which raises the possibility of variations in the composition and pharmacological properties of HM-CDs. The most popular procedures for preparation, hydrothermal synthesis and high-temperature pyrolysis, produce CDs with unstable QY, particle size, and fluorescence intensity. Third, the intrinsic active ingredient of HM-CDs remains uncertain, while the active ingredient is crucial for the treatment of diseases. The identification of active substances is direct evidence to elucidate the mechanism of action of HM-CDs. Under high temperatures, current synthesis techniques may lead to the decomposition or even disappearance of some actual components of herbal medicines. Therefore, the remaining fraction of active compounds in HM-CDs under different synthesis methods and conditions is an important direction for future research. High performance liquid chromatography and tandem mass spectrometry (HPLC-MS) may be able to offer some answers. Fourth, further research is needed on the in vivo distribution and metabolism of HM-CDs. In contrast to classical medicine, the circulation of HM-CDs in living bodies and organs is unclear and the interaction with living molecules is complicated, leading to the limitations of CDs in clinical applications. Last but not least, the mechanism of the luminescence of HM-CDs is still unclear. The lack of a universally applicable luminescence mechanism, generally due to the difficulty in determining the structure of the synthesized HM-CDs, has limited the structural modification of HM-CDs and the improvement of luminescence properties to meet clinical needs. As a consequence, the structural composition of CDs synthesized based on herbal medicine and the luminescence mechanisms associated with them remain to be further explored. In order to expedite the translation of HM-CDs from the laboratory to clinical applications, there are currently challenges such as complexity of composition, quality control, individual variations, clinical validation, and ethical and regulatory issues. HM-CDs are prepared from herbal extracts, which contain multiple complex components. Understanding the effects and interactions of these components on the human body is a difficult task that requires detailed analysis and research. Ensuring consistent quality of HM-CDs is another challenge due to the diversity and variability of herbal sources. To overcome this, it is important to establish standardized preparation methods and implement quality control measures. Moreover, the response and effects of HM-CDs may vary among different patients, influenced by factors such as genetic background and metabolic differences. Therefore, individualized studies and evaluations are necessary to determine the appropriate application methods and dosages for specific patient populations. Clinical trials are essential to validate the efficacy and safety of HM-CDs. These trials will provide the necessary evidence to support their use in clinical settings. However, it is crucial to comply with ethical principles and legal regulations in order to ensure patient safety and privacy during these trials and in the eventual market adoption of HM-CDs. Considering HM-CDs as a novel nanomaterial, it is important to subject them to additional ethical and regulatory scrutiny to address any potential risks or concerns. The integration of modern technologies such as proteomics, genomics, and metabolomics is conducive to promoting the industrial development of HM-CDs. The primary focus of our future research will be on systematic studies of the toxicity and metabolic pathways of HM-CDs in animal models, optimization of HM-CDs preparation methods, biodistribution and active ingredient analysis of HM-CDs, and the precise mechanism of interaction between the human system and HM-CDs. |
Phytochemical Profiles and Biological Activities of | 5c3617b8-6da0-4468-bb7e-fb3a9a55edb2 | 10933895 | Pharmacology[mh] | According to a very recent report from the World Health Organization (WHO), ‘traditional medicine has a long history of contributing to conventional medicine and continues to hold promise’ . Indeed, since early times, human beings have learned how to address their health problems and various so-called traditional medicines have emerged all over the world and are still used, according to the 2019 WHO global report on traditional and complementary medicine . These traditional medicines usually rely on natural products and mixtures of them, issued mostly from plants , but also from animals and microorganisms . Marine environments are known for their high biodiversity. Among them, coastal environments exhibit specific plants able to grow in highly saline areas, often under severe variations in temperature, light intensity and drought. These plants, named halophytes, are not limited to such coastal areas but can be found in a diverse array of highly saline soils . To withstand such severe conditions, these plants have developed several ways to control and/or take away salt, but they also exhibit strong antioxidant systems composed of enzymes and highly bioactive secondary metabolites, such as phenolic compounds and alkaloids . Probably for these reasons, halophytes are traditionally used in folk medicine for their curative properties against infectious diseases . Hence, halophytes are currently gaining interest due to their nutraceutical potential, powerful antioxidant abilities and therapeutic significance in treating a variety of pathologies . Among the halophytes, the Frankeniaceae family constitutes a relatively small family with originally 2 to 4–5 genera, but a recent taxonomic revision based on molecular phylogenetic studies retained the Frankeniaceae as a single genus, Frankenia . The latter contains between 70 and 82 species that are found in deserts and sandy coastal locations with dry areas . Similarly, the Frankeniaceae and Tamaricaceae families were considered as a pair of families that together made up the order Tamaricales, however, genetic studies have allowed them to be distinguished . The shrubby and herbaceous species of Frankenia are known to mainly grow in arid and semi-arid climates in extremely saline, alkaline or calcareous soils. They can be found on all continents but are most common in the Western Hemisphere, particularly in the Mediterranean region up to the Middle East . Indeed, Frankenia species have been recorded in North Africa, especially in Algeria and Tunisia, as well as in Egypt, Portugal, Spain and France, but also in Turkey, Syria, Lebanon, Jordan and Palestine. They can also be found in Iraq and neighboring regions, such as Qatar, Kuwait and Iran . Despite their relevance in medicine and industry, studies about the Frankenia genus are unexpectedly limited, probably due to the scarcity of these plant species. Nonetheless, only a few species have been investigated in some detail . Their chemical profile and/or their biological properties have been explored, revealing a wide variety of natural products and bioactivities. The purpose of this study was to collect and systematically review the published phytochemical compositions and biological activities of the medicinal Frankenia species. Frankenia Plants From the studies mentioned above, more than 200 phytocompounds obtained from Frankenia extracts have already been identified. Among them, polyphenols, such as phenolic acids and flavonoids, are the major constituents and essential chemotaxonomic indicators. Further isolated compounds include alkaloids, terpenoids, steroids, fatty acids and other molecules. 2.1. Polyphenols 2.1.1. Phenolics Phenolics are a broad category of chemical compounds that have one or more hydroxyl groups linked to at least one aromatic hydrocarbon ring . Due to their hydroxylated conjugated structure, phenolic compounds have considerable potential as antioxidants . Furthermore, their stability could allow their use as therapeutic agents. Phenolic natural products are abundant in the Frankenia genus, especially in F. laevis , and exhibit a large variability . They were mainly represented by gallic acid ( 1 – 9 ), hydroxybenzoic acid ( 10 – 13 ), ellagitannins ( 14 – 27 ) and hydroxycinnamic acid ( 28 – 41 ) derivatives . Other phenolic compounds ( 42 – 53 ) were also identified . The dihydroxybenzenes ( 44 ) and ( 45 ) were present in significant amounts in F. pulverulenta . Additionally, compounds ( 10 ), ( 28 ) and ( 31 ) were the most representative compounds in F. thymifolia , followed by ( 29 ) . 2.1.2. Flavonoids The Frankenia species, especially F. laevis , F. pulverulenta and F. thymifolia , contain a wide range of flavonoids , mainly represented by flavonols, such as kaempferol ( 54 – 61 ), quercetin ( 62 – 73 ), catechin ( 74 – 78 ) and isorhamnetin ( 79 – 81 ) derivatives. The compounds ( 74 ) and ( 75 ) were the most representative flavonoids in F. thymifolia and F. pulverulenta , respectively . Additionally, the flavanone ( 82 ) and the O -glycosylated flavone ( 83 ) were described in F. thymifolia . 2.1.3. Lignans Lignans do not appear to be extremely prevalent in Frankenia . The arylated tetralin derivative ( 84 ) is the most common lignan found in these plants. A few tetrahydrofuranic lignans ( 85 – 87 ) have also been isolated and identified . 2.1.4. Coumarins So far, a single example of coumarin, the simplest one ( 88 ), has been isolated and characterized from a Frankenia species ( and ). 2.2. Alkaloids Surprisingly, only a few alkaloids could be found in Frankenia species, and they were mainly detected in F. pulverulenta . The phytochemical investigation of this species has led to the identification of several compounds with a wide variety of structures ( 89 – 101 ). The alkaloid dihydrotecomanine ( 102 ) was detected in both F. pulverulenta and F. hirsuta . In F. aucheri(hirsuta) , an α-amino acid metabolite, the N -acetyl serine ( 103 ), could also be observed and characterized. The same species also contains another peculiar amino acid with a heterocyclic core, the pterin-6-carboxylic acid ( 104 ), but an indoloquinolizine derivative ( 105 ) was also identified. 2.3. Terpenoids As important building blocks in biosynthesis, terpenoids are widely represented in living species. This large class of natural compounds is extremely prevalent in the plant kingdom, where they play key roles in plant defense and communication . They are frequently found in plant essential oils. Therefore, it is natural to find them in plants of the Frankenia genus. However, in Frankenia species, most of them have been identified as sesquiterpenes. Nevertheless, some mono- and diterpenes have also been found . 2.3.1. Monoterpenes Monoterpenes are not common among Frankenia species. It was only in 2021 and 2022 that a few monoterpenes were identified in, respectively, F. pulverulenta and laevis . Indeed, the phytochemical analysis of F. laevis extracts revealed the presence of three tetrahydrobenzofuran-2(4 H )-ones ( 106 – 108 ). In F. pulverulenta extracts, the bicyclic α-pinene ( 109 ) was detected . 2.3.2. Diterpenes A few compounds have been reported as diterpenes from Frankenia species. The acyclic diterpenoids ( 110 – 112 ) were isolated from F. laevis , while ( 113 ) and its derivative gibberellic acid ( 114 ) were present in the essential oil (EO) and methanolic leaf extract of F. pulverulenta , respectively . 2.3.3. Sesquiterpenes Sesquiterpenes are abundant in the Frankenia genus. Overall, 22 compounds belonging to this subclass of terpenoids were identified in the EO of F. laevis and F. pulverulenta , including eleven oxygenated sesquiterpenes ( 115 – 125 ) and eleven sesquiterpene hydrocarbons ( 126 – 136 ) . Nerolidol ( 116 ) and farnesyl acetate ( 119 ) were the most widespread sesquiterpenes present in F. laevis . Furthermore, β-caryophyllene ( 135 ) was the major compound detected in F. pulverulenta . The second major compounds were cadinene ( 134 ), allo-aromadendrene ( 136 ), copaene ( 128 ) and ledol ( 121 ) . However, the later terpenes ( 134 ), ( 135 ) and ( 136 ) were found to be present at a much lower amount in F. laevis . 2.4. Steroids Although they do not seem to be common in Frankenia species, steroids have nevertheless been isolated and characterized ( and ). The majority of them have been isolated from F. foliosa and identified as secosteroids ( 137 – 141 ) . It is worth noticing that the latter included vitamin D ( 139 ) as well as the unusual eringiacetal A ( 141 ). In addition, the two steroids ( 142 ) and ( 143 ) have been identified in F. pulverulenta , while the steroid ( 144 ) has been detected in F. hirsuta . 2.5. Alkanes and Alkenes Long-chain alkanes are common in terrestrial plants, especially as part of their cuticular leaf wax. Therefore, alkanes are quite common in Frankenia species . Overall, 15 alkane chemicals ( 145 – 158 ) were reported in both F. laevis and F. pulverulenta . Most of these alkanes exhibit linear and long carbon chains, containing from 17 to 35 carbons. So far, a single example of an α-methylated chain alkane ( 156 ) has been reported in F. laevis . Similarly, the C20 linear alkane eicosane ( 159 ) has so far only been characterized in F. hirsuta . In contrast, long-chain alkenes seem to be quite rare in Frankenia species. Indeed, only two alkenes ( 160 ) and ( 161 ) have been reported as the only alkenes in the genus . Both exhibit a terminal vinyl group within a linear chain (C19 and C22, respectively). 2.6. Fatty Acids and Esters Fatty acids and esters are ubiquitous in all living organisms and are essential to them, as they serve as membrane constituents, modulators in glycerolipids and as carbon and energy reserves in triacylglycerols, but also as signal molecules . The Frankenia plants contain various fatty acids and esters. At least 20 different fatty acids and fatty acid esters were found within members of the genus Frankenia , and grouped as saturated ( 162–171 ), monounsaturated ( 172 – 173 ) and polyunsaturated ( 174 – 178 ) fatty acids, saturated fatty acid methyl esters ( 179 – 180 ) and unsaturated fatty acid methyl esters ( 181 – 183 ). Palmitic acid ( 167 ) was the major compound of F. laevis , followed by ( 181 ) . In addition, ( 167 ), ( 173 ) and ( 174 ) were reported as the major fatty acids in the oil of F. thymifolia . 2.7. Other Compounds In addition to the large well-known natural product families mentioned above, compounds from other classes of natural chemicals have also been detected ( and ). A large variety of compounds was identified, such as alcohols ( 184 – 185 ), glycosides ( 186 – 191 ), aromatic compounds ( 192 – 195 ), heterocyclic compounds ( 196 – 198 ), aldehydes ( 199 – 200 ), ketones ( 201 – 202 ), organic acids ( 203 – 205 ) and esters ( 206 – 208 ). Long-chain alkyl alcohols, unsaturated or not, ( 184 – 185 ) were detected in F. pulverulenta and F. laevis , respectively . Surprisingly, the same hexadecane-1-ol ( 185 ) found in F. laevis was also observed in F. hirsuta but as its glycidyl ether ( 197 ) . Highly abundant in organisms, especially in plants, glycosides were only scarcely found in Frankenia species. The common monosaccharides, glucose and mannose, were both detected in F. pulverulenta and F. hirsuta , but as, respectively, their 3- O -methylated or 6- O -acetylated derivatives ( 186 – 188 ) . A desulfonylated allyl glucosinolate was also detected in F. hirsuta , ( 191 ) . Such a sinigrin derivative is usually found in the Brassicaceae family. A di- and a trisaccharide were also detected in F. pulverulenta . The disaccharide was unexpectedly characterized as lactose ( 190 ) , while the trisaccharide was assigned as a β-analog of melezitose ( 189 ) . Interestingly, the aromatized form of glucose, i.e., hydroxymethylfurfural (HMF) ( 199 ), was also detected in F. hirsuta . Among the other aromatic compounds found ( 192 – 195 ), a demetallated chlorophyll, i.e., pheophytin A, was observed in Frankenia species, and more precisely in F. laevis . Alternatively, the F. hirsuta species seems relatively rich in aromatic compounds, since the simple 1,3,5-trimethylbenzene ( 194 ), phthalate esters ( 206 – 207 ) and various benzyl or phenyl derivatives ( 192 – 193 and 208 – 209 ) have been observed . A hexamethoxylated naphthalene derivative, i.e., ( 195 ), was detected in F. thymifolia . A few jasmonoids, i.e., ( 204 – 205 ), were found in F. laevis , as well as a related cyclopentenyl bicyclic lactone in F. pulverulenta . Interestingly, the macrolactone ( 201 ) , related to the methyl ester ( 183 ) , was also detected in F. pulverulenta . Furthermore, the hydrazine ( 209 ) and the stable peroxide ( 210 ) were both reported from the F. hirsuta species . 2.8. Phytochemical Outcome The phytochemical compositions of the various Frankenia species collected above reveal the rich chemical content of these plants and the variety of chemicals that have been detected, or isolated and characterized. These plants mainly produce phenol derivatives, which represent around one-quarter of all the so far identified chemicals. The other chemicals mostly observed belong to the flavonoid and terpenoid families (14–15% each), while alkaloids, fatty acids and esters represent approximately 10% of all Frankenia phytochemicals . Overall, Frankenia species might be regarded as potentially rich sources of phenolics, flavonoids, sesquiterpenes and fatty acids or esters. Nevertheless, the phytochemical repartition is quite different from one species to another, as revealed in . Indeed, F. laevis and F. thymifolia are particularly rich in phenol derivatives, while F. pulverulenta and F. hirsuta exhibit less than 10% of such chemicals. Similarly, fatty acids or esters are mostly present in F. hirsuta and F. thymifolia , while they represent less than 10% of the phytochemical content of F. pulverulenta and F. laevis . Flavonoids are mostly present in F. thymifolia and F. pulverulenta, and to a lesser extent in F. laevis . However, they are surprisingly almost absent in other species. The same surprising repartition can be observed for terpenoids, which are mostly present in F. pulverulenta and F. laevis , but also to a small extent in F. hirsuta and almost absent in other species. Among Frankenia species, F. hirsuta seems to present the largest diversity. Indeed, in addition to the large and ubiquitous classes of compounds mentioned above, F. hirsuta also contains alkaloids, steroids, (oligo)saccharides and aromatic derivatives, as well as unexpected hydrazine and peroxide derivatives. Such different repartitions may be useful as chemotaxonomic tools, complementing others. Indeed, previous research demonstrated that plants of the genus Frankenia may produce sulfated chemicals, where they serve as an indirect chemotaxonomic marker. Furthermore, their presence has been correlated to their affinity for saline environments . 2.1.1. Phenolics Phenolics are a broad category of chemical compounds that have one or more hydroxyl groups linked to at least one aromatic hydrocarbon ring . Due to their hydroxylated conjugated structure, phenolic compounds have considerable potential as antioxidants . Furthermore, their stability could allow their use as therapeutic agents. Phenolic natural products are abundant in the Frankenia genus, especially in F. laevis , and exhibit a large variability . They were mainly represented by gallic acid ( 1 – 9 ), hydroxybenzoic acid ( 10 – 13 ), ellagitannins ( 14 – 27 ) and hydroxycinnamic acid ( 28 – 41 ) derivatives . Other phenolic compounds ( 42 – 53 ) were also identified . The dihydroxybenzenes ( 44 ) and ( 45 ) were present in significant amounts in F. pulverulenta . Additionally, compounds ( 10 ), ( 28 ) and ( 31 ) were the most representative compounds in F. thymifolia , followed by ( 29 ) . 2.1.2. Flavonoids The Frankenia species, especially F. laevis , F. pulverulenta and F. thymifolia , contain a wide range of flavonoids , mainly represented by flavonols, such as kaempferol ( 54 – 61 ), quercetin ( 62 – 73 ), catechin ( 74 – 78 ) and isorhamnetin ( 79 – 81 ) derivatives. The compounds ( 74 ) and ( 75 ) were the most representative flavonoids in F. thymifolia and F. pulverulenta , respectively . Additionally, the flavanone ( 82 ) and the O -glycosylated flavone ( 83 ) were described in F. thymifolia . 2.1.3. Lignans Lignans do not appear to be extremely prevalent in Frankenia . The arylated tetralin derivative ( 84 ) is the most common lignan found in these plants. A few tetrahydrofuranic lignans ( 85 – 87 ) have also been isolated and identified . 2.1.4. Coumarins So far, a single example of coumarin, the simplest one ( 88 ), has been isolated and characterized from a Frankenia species ( and ). Phenolics are a broad category of chemical compounds that have one or more hydroxyl groups linked to at least one aromatic hydrocarbon ring . Due to their hydroxylated conjugated structure, phenolic compounds have considerable potential as antioxidants . Furthermore, their stability could allow their use as therapeutic agents. Phenolic natural products are abundant in the Frankenia genus, especially in F. laevis , and exhibit a large variability . They were mainly represented by gallic acid ( 1 – 9 ), hydroxybenzoic acid ( 10 – 13 ), ellagitannins ( 14 – 27 ) and hydroxycinnamic acid ( 28 – 41 ) derivatives . Other phenolic compounds ( 42 – 53 ) were also identified . The dihydroxybenzenes ( 44 ) and ( 45 ) were present in significant amounts in F. pulverulenta . Additionally, compounds ( 10 ), ( 28 ) and ( 31 ) were the most representative compounds in F. thymifolia , followed by ( 29 ) . The Frankenia species, especially F. laevis , F. pulverulenta and F. thymifolia , contain a wide range of flavonoids , mainly represented by flavonols, such as kaempferol ( 54 – 61 ), quercetin ( 62 – 73 ), catechin ( 74 – 78 ) and isorhamnetin ( 79 – 81 ) derivatives. The compounds ( 74 ) and ( 75 ) were the most representative flavonoids in F. thymifolia and F. pulverulenta , respectively . Additionally, the flavanone ( 82 ) and the O -glycosylated flavone ( 83 ) were described in F. thymifolia . Lignans do not appear to be extremely prevalent in Frankenia . The arylated tetralin derivative ( 84 ) is the most common lignan found in these plants. A few tetrahydrofuranic lignans ( 85 – 87 ) have also been isolated and identified . So far, a single example of coumarin, the simplest one ( 88 ), has been isolated and characterized from a Frankenia species ( and ). Surprisingly, only a few alkaloids could be found in Frankenia species, and they were mainly detected in F. pulverulenta . The phytochemical investigation of this species has led to the identification of several compounds with a wide variety of structures ( 89 – 101 ). The alkaloid dihydrotecomanine ( 102 ) was detected in both F. pulverulenta and F. hirsuta . In F. aucheri(hirsuta) , an α-amino acid metabolite, the N -acetyl serine ( 103 ), could also be observed and characterized. The same species also contains another peculiar amino acid with a heterocyclic core, the pterin-6-carboxylic acid ( 104 ), but an indoloquinolizine derivative ( 105 ) was also identified. As important building blocks in biosynthesis, terpenoids are widely represented in living species. This large class of natural compounds is extremely prevalent in the plant kingdom, where they play key roles in plant defense and communication . They are frequently found in plant essential oils. Therefore, it is natural to find them in plants of the Frankenia genus. However, in Frankenia species, most of them have been identified as sesquiterpenes. Nevertheless, some mono- and diterpenes have also been found . 2.3.1. Monoterpenes Monoterpenes are not common among Frankenia species. It was only in 2021 and 2022 that a few monoterpenes were identified in, respectively, F. pulverulenta and laevis . Indeed, the phytochemical analysis of F. laevis extracts revealed the presence of three tetrahydrobenzofuran-2(4 H )-ones ( 106 – 108 ). In F. pulverulenta extracts, the bicyclic α-pinene ( 109 ) was detected . 2.3.2. Diterpenes A few compounds have been reported as diterpenes from Frankenia species. The acyclic diterpenoids ( 110 – 112 ) were isolated from F. laevis , while ( 113 ) and its derivative gibberellic acid ( 114 ) were present in the essential oil (EO) and methanolic leaf extract of F. pulverulenta , respectively . 2.3.3. Sesquiterpenes Sesquiterpenes are abundant in the Frankenia genus. Overall, 22 compounds belonging to this subclass of terpenoids were identified in the EO of F. laevis and F. pulverulenta , including eleven oxygenated sesquiterpenes ( 115 – 125 ) and eleven sesquiterpene hydrocarbons ( 126 – 136 ) . Nerolidol ( 116 ) and farnesyl acetate ( 119 ) were the most widespread sesquiterpenes present in F. laevis . Furthermore, β-caryophyllene ( 135 ) was the major compound detected in F. pulverulenta . The second major compounds were cadinene ( 134 ), allo-aromadendrene ( 136 ), copaene ( 128 ) and ledol ( 121 ) . However, the later terpenes ( 134 ), ( 135 ) and ( 136 ) were found to be present at a much lower amount in F. laevis . Monoterpenes are not common among Frankenia species. It was only in 2021 and 2022 that a few monoterpenes were identified in, respectively, F. pulverulenta and laevis . Indeed, the phytochemical analysis of F. laevis extracts revealed the presence of three tetrahydrobenzofuran-2(4 H )-ones ( 106 – 108 ). In F. pulverulenta extracts, the bicyclic α-pinene ( 109 ) was detected . A few compounds have been reported as diterpenes from Frankenia species. The acyclic diterpenoids ( 110 – 112 ) were isolated from F. laevis , while ( 113 ) and its derivative gibberellic acid ( 114 ) were present in the essential oil (EO) and methanolic leaf extract of F. pulverulenta , respectively . Sesquiterpenes are abundant in the Frankenia genus. Overall, 22 compounds belonging to this subclass of terpenoids were identified in the EO of F. laevis and F. pulverulenta , including eleven oxygenated sesquiterpenes ( 115 – 125 ) and eleven sesquiterpene hydrocarbons ( 126 – 136 ) . Nerolidol ( 116 ) and farnesyl acetate ( 119 ) were the most widespread sesquiterpenes present in F. laevis . Furthermore, β-caryophyllene ( 135 ) was the major compound detected in F. pulverulenta . The second major compounds were cadinene ( 134 ), allo-aromadendrene ( 136 ), copaene ( 128 ) and ledol ( 121 ) . However, the later terpenes ( 134 ), ( 135 ) and ( 136 ) were found to be present at a much lower amount in F. laevis . Although they do not seem to be common in Frankenia species, steroids have nevertheless been isolated and characterized ( and ). The majority of them have been isolated from F. foliosa and identified as secosteroids ( 137 – 141 ) . It is worth noticing that the latter included vitamin D ( 139 ) as well as the unusual eringiacetal A ( 141 ). In addition, the two steroids ( 142 ) and ( 143 ) have been identified in F. pulverulenta , while the steroid ( 144 ) has been detected in F. hirsuta . Long-chain alkanes are common in terrestrial plants, especially as part of their cuticular leaf wax. Therefore, alkanes are quite common in Frankenia species . Overall, 15 alkane chemicals ( 145 – 158 ) were reported in both F. laevis and F. pulverulenta . Most of these alkanes exhibit linear and long carbon chains, containing from 17 to 35 carbons. So far, a single example of an α-methylated chain alkane ( 156 ) has been reported in F. laevis . Similarly, the C20 linear alkane eicosane ( 159 ) has so far only been characterized in F. hirsuta . In contrast, long-chain alkenes seem to be quite rare in Frankenia species. Indeed, only two alkenes ( 160 ) and ( 161 ) have been reported as the only alkenes in the genus . Both exhibit a terminal vinyl group within a linear chain (C19 and C22, respectively). Fatty acids and esters are ubiquitous in all living organisms and are essential to them, as they serve as membrane constituents, modulators in glycerolipids and as carbon and energy reserves in triacylglycerols, but also as signal molecules . The Frankenia plants contain various fatty acids and esters. At least 20 different fatty acids and fatty acid esters were found within members of the genus Frankenia , and grouped as saturated ( 162–171 ), monounsaturated ( 172 – 173 ) and polyunsaturated ( 174 – 178 ) fatty acids, saturated fatty acid methyl esters ( 179 – 180 ) and unsaturated fatty acid methyl esters ( 181 – 183 ). Palmitic acid ( 167 ) was the major compound of F. laevis , followed by ( 181 ) . In addition, ( 167 ), ( 173 ) and ( 174 ) were reported as the major fatty acids in the oil of F. thymifolia . In addition to the large well-known natural product families mentioned above, compounds from other classes of natural chemicals have also been detected ( and ). A large variety of compounds was identified, such as alcohols ( 184 – 185 ), glycosides ( 186 – 191 ), aromatic compounds ( 192 – 195 ), heterocyclic compounds ( 196 – 198 ), aldehydes ( 199 – 200 ), ketones ( 201 – 202 ), organic acids ( 203 – 205 ) and esters ( 206 – 208 ). Long-chain alkyl alcohols, unsaturated or not, ( 184 – 185 ) were detected in F. pulverulenta and F. laevis , respectively . Surprisingly, the same hexadecane-1-ol ( 185 ) found in F. laevis was also observed in F. hirsuta but as its glycidyl ether ( 197 ) . Highly abundant in organisms, especially in plants, glycosides were only scarcely found in Frankenia species. The common monosaccharides, glucose and mannose, were both detected in F. pulverulenta and F. hirsuta , but as, respectively, their 3- O -methylated or 6- O -acetylated derivatives ( 186 – 188 ) . A desulfonylated allyl glucosinolate was also detected in F. hirsuta , ( 191 ) . Such a sinigrin derivative is usually found in the Brassicaceae family. A di- and a trisaccharide were also detected in F. pulverulenta . The disaccharide was unexpectedly characterized as lactose ( 190 ) , while the trisaccharide was assigned as a β-analog of melezitose ( 189 ) . Interestingly, the aromatized form of glucose, i.e., hydroxymethylfurfural (HMF) ( 199 ), was also detected in F. hirsuta . Among the other aromatic compounds found ( 192 – 195 ), a demetallated chlorophyll, i.e., pheophytin A, was observed in Frankenia species, and more precisely in F. laevis . Alternatively, the F. hirsuta species seems relatively rich in aromatic compounds, since the simple 1,3,5-trimethylbenzene ( 194 ), phthalate esters ( 206 – 207 ) and various benzyl or phenyl derivatives ( 192 – 193 and 208 – 209 ) have been observed . A hexamethoxylated naphthalene derivative, i.e., ( 195 ), was detected in F. thymifolia . A few jasmonoids, i.e., ( 204 – 205 ), were found in F. laevis , as well as a related cyclopentenyl bicyclic lactone in F. pulverulenta . Interestingly, the macrolactone ( 201 ) , related to the methyl ester ( 183 ) , was also detected in F. pulverulenta . Furthermore, the hydrazine ( 209 ) and the stable peroxide ( 210 ) were both reported from the F. hirsuta species . The phytochemical compositions of the various Frankenia species collected above reveal the rich chemical content of these plants and the variety of chemicals that have been detected, or isolated and characterized. These plants mainly produce phenol derivatives, which represent around one-quarter of all the so far identified chemicals. The other chemicals mostly observed belong to the flavonoid and terpenoid families (14–15% each), while alkaloids, fatty acids and esters represent approximately 10% of all Frankenia phytochemicals . Overall, Frankenia species might be regarded as potentially rich sources of phenolics, flavonoids, sesquiterpenes and fatty acids or esters. Nevertheless, the phytochemical repartition is quite different from one species to another, as revealed in . Indeed, F. laevis and F. thymifolia are particularly rich in phenol derivatives, while F. pulverulenta and F. hirsuta exhibit less than 10% of such chemicals. Similarly, fatty acids or esters are mostly present in F. hirsuta and F. thymifolia , while they represent less than 10% of the phytochemical content of F. pulverulenta and F. laevis . Flavonoids are mostly present in F. thymifolia and F. pulverulenta, and to a lesser extent in F. laevis . However, they are surprisingly almost absent in other species. The same surprising repartition can be observed for terpenoids, which are mostly present in F. pulverulenta and F. laevis , but also to a small extent in F. hirsuta and almost absent in other species. Among Frankenia species, F. hirsuta seems to present the largest diversity. Indeed, in addition to the large and ubiquitous classes of compounds mentioned above, F. hirsuta also contains alkaloids, steroids, (oligo)saccharides and aromatic derivatives, as well as unexpected hydrazine and peroxide derivatives. Such different repartitions may be useful as chemotaxonomic tools, complementing others. Indeed, previous research demonstrated that plants of the genus Frankenia may produce sulfated chemicals, where they serve as an indirect chemotaxonomic marker. Furthermore, their presence has been correlated to their affinity for saline environments . Frankenia Plants 3.1. In Traditional Medicine As was reminded in the introduction, traditional medicine has a long history in human health and various variants have been developed around the world and are still practiced nowadays. In countries with limited access to modern therapy, traditional medicine is frequently the major source of primary healthcare requirements . Used as traditional medicinal plants, Frankenia species appear to play a prominent role in the treatment of various diseases. Due to their astringent properties, Frankenia species are utilized in Asian and African (especially in Morocco) folk medicine for gargling or for topical application, either as tinctures or as herbal tea, e.g., with F. laevis or F. thymifolia . They are also used in these countries to treat a variety of clinical disorders, such as dysentery, diarrhea, gonorrhea, vaginal leucorrhea, mucus releases from the nose and catarrh-induced infections, again as plant infusions, with, e.g., F. pulverulenta , or as stupe, depending on the localization . Gargle and decoction generated from the entire plant of F. pulverulenta are widely used in local medicine by the inhabitants of the Onaizah province in Saudi Arabia and are mostly used orally for their analgesic and carminative properties . Also in Saudi Arabia, the powdered rhizome of F. aucheri (hirsuta) combined with milk is used to stimulate lactation in cows and camels, particularly in the winter . It has also been reported that Puna inhabitants in South America used F. triandra as a forage but also as antiseptic in folk medicine . Additionally, some Frankenia species can be converted into sticky glue mixtures, due to their specific natural product contents, e.g., kaempferol, quercetin and tannin. Therefore, they are used in totally different applications, notably to stick blade cutting edges and to seal stoneware (e.g., F. hirsuta ) . Overall, Frankenia plant species may thus be viewed as promising prospects for different applications in industry, and mostly in pharmaceutical applications. 3.2. In Vitro Biological Activities Secondary plant metabolites, which are produced in large amounts by plant species, are crucial components for supporting human health. They contribute to the medicinal properties of plant species as antioxidants, anti-inflammatory, anti-carcinogenic and antibacterial agents , along with other capacities . Because the large diversity of natural compounds discovered in Frankenia species mostly belong to well-known families of bioactive compounds, it is expected that these plants exhibit the corresponding biological activities. Therefore, several works have been performed to check these bioactivities. They are listed below. 3.2.1. Antioxidant Activity The antioxidant activity of Frankenia species has been assessed using several methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging analysis, 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radical trapping, ferric ion reducing antioxidant power (FRAP), metal chelating activity (MCA), including copper (CCA)- and iron (ICA)-chelating activities, oxygen radical absorbance capacity (ORAC) assay and β-carotene oxidation test. The corresponding details have been collected in . The aqueous acetone , methanol and ethanol extracts of F. laevis exhibited spectacular in vitro radical scavenging and copper chelating properties. However, the dichloromethane extract from this species was only able to chelate iron, probably due to the presence of various phenolics and flavonoids that can act as phytochelators , such as gallic acid, kaempferol and quercetin derivatives (see and and and ). Likewise, F. pulverulenta ethyl acetate and methanolic extracts were investigated using DPPH, ABTS and ORAC assays, and potent antioxidant activity was reported. The antioxidant activity of the aqueous acetone extract of F. pulverulenta was also assessed . The antioxidant potency can be linked to the well-known antioxidant compounds gallic acid ( 1 ), p -coumaric acid ( 34 ), quercetin ( 62 ) and catechin ( 74 ) which are abundantly present in this species. Ben Mansour et al. demonstrated, in 2016, that F. thymifolia ethyl acetate extracts exhibited strong antioxidant activity in both shoots and roots. Furthermore, this extract has the highest TPC and antioxidant capacities . Other studies investigated the methanolic and chloroformic extracts of F. thymifolia and demonstrated that the methanolic extract exhibited better antioxidant activity, again linked to the high level of phenolic compounds, including salicylic acid ( 10 ), cinnamic acid ( 28 ), 2-hydroxycinnamic acid ( 29 ), chlorogenic acid ( 31 ) and catechin (74) . Torres Carro et al. showed, in 2016, the significant antioxidant activity of the ethanolic and soxhlet of F. triandra evaluated by the β-carotene assay . Overall, plants from the Frankenia family are rich in polyphenols, and this richness is often, if not always, correlated to the strong antioxidant properties these plants exhibit . 3.2.2. Antimicrobial Activity Bacteria were involved in many of the most devastating diseases and massive epidemics in human history, before the discovery of antibiotics. Due to the misuse of the latter, bacteria have now developed resistance to the commonly used antibiotics . Therefore, it is imperative to identify new and advanced chemical agents in order to have more productive resistance to microorganisms . Since synthetic chemicals are related to adverse effects and harmful residues, novel antibacterial, antifungal, antiviral and antiparasitic drugs from plant sources must be developed worldwide . Accordingly, a few Frankenia species were collected and screened for their antimicrobial activities. The corresponding details have been collected in . Jdey et al. showed, in 2017, that the ethanolic extract of F. laevis significantly inhibited the development of both Gram-positive (Gram+) and Gram-negative (Gram−) bacteria engaged in their study . All these strains were indeed inhibited by more than 55%, and the best inhibitions were observed for Micrococcus luteus (83%) and Salmonella enterica (77%) at a concentration of 1 mg/mL. This antibacterial effect may be attributed to the chlorogenic acid ( 31 ) and catechin ( 74 ) contained in this species, probably by inducing structural or functional damage to the bacterial cell membranes . Similarly, Saïdana et al. demonstrated, in 2010, that EO from the aerial parts of F. laevis was efficient against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Escherichia coli and Salmonella typhimurium . According to the authors, the antimicrobial activity of the EO can be attributed to the presence of fatty acids such as palmitic acid ( 167 ), fatty acid esters like methyl linoleate ( 181 ), sesquiterpenes such as farnesyl acetate ( 119 ) , aromatic compounds such as benzyl benzoate ( 192 ) and benzyl cinnamate ( 193 ), and, to a lesser extent, eugenol ( 49 ), β-caryophyllene ( 135 ), phytol ( 110 ), isophytol ( 112 ), ( E , E )-farnesol ( 117 ) and hexadecanol ( 185 ). However, no significant effect on Pseudomonas aeruginosa was detected . This Gram− bacteria has already been demonstrated to be less susceptible to the action of several other plant EOs . The antifungal activity of the EO was also investigated. Despite the presence of eugenol ( 49 ) and β-caryophyllene ( 135 ) in the oil composition, known to have antifungal effects , none of the tested fungi were successfully inhibited by the EO at the tested doses. This may be explained by the low amounts of such chemicals in the EO . The antibacterial and antifungal activity of EO from F. pulverulenta was also investigated . Despite being rich in β-caryophyllene ( 135 ), which represents its main constituent (32%), this EO did not prevent bacterial growth. This finding appears contradictory to previous research, which showed that the presence of β-caryophyllene enhances the biological activities of EO, including their antibacterial activity . Furthermore, the F. pulverulenta EO displayed poor antifungal activity and was exclusively efficient against the basidiomycete Rhizoctonia solani . In 2011, Megdiche-Ksouri et al. investigated the activity of methanolic (polar) and chloroformic (less polar) extracts from F. thymifolia against five bacteria and one fungus . The chloroformic extract provided the best performance, being active against all the evaluated bacterial strains. Similar inhibition results have been observed with other halophytes (e.g., sea holly, sea fennel) . Such an outcome has been correlated to the polarity of the extracting solvent and could be attributed to the presence of lipophilic compounds in these extracts. Indeed, it has been demonstrated that long-chain unsaturated fatty acids, notably oleic ( 172 ) and linoleic ( 174 ), exhibit a strong inhibiting activity against mycobacteria . Furthermore, it has been reported that the relatively lipophilic flavonoids catechin ( 74 ) and epigallocatechino-3-gallate ( 76 ) exhibit protective and antibacterial effects . Likewise, the n -butanol fraction from F. thymifolia exhibited a stronger antibacterial effect against all tested bacterial strains compared to the ethyl acetate fraction ( Pseudomonas aeruginosa was the most vulnerable strain) . The extracts investigated also presented anti-leishmanial and antiamoebic effects against Leishmania amazonensis and Acanthamoeba castellanii , respectively. The antiparasitic capacities of these extracts may be related to the presence of quercetin ( 62 ) . Canli et al. demonstrated the antibacterial and antifungal activity of F. hirsuta ethanolic extract against seventeen bacteria and one fungus . Except for the Gram ˗ bacteria Enterobacter aerogenes and Escherichia coli , all of the examined strains were sensitive to the antimicrobial action of the F. hirsuta extract. The most sensitive strains were Gram+ bacteria, especially Staphylococcus epidermidis and Enterecoccus faecium , compared to Gram ˗ bacteria. Such antibiotic activity was again associated with the presence in this extract of oleic and linoleic acid in high amounts . The concomitant presence of mesitylene ( 194 ) , eudesmic acid ( 8 ) and stearic acid ( 169 ) also suggested a possible role in the F. hirsuta antibacterial activity, because some mesitylene derivatives , eudesmic acid and stearic acid analogs are known as antibacterial agents. The difference in sensitivity to plant extracts between Gram+ and Gram− bacteria observed for F. hirsuta ethanolic extract could be generalized according to Canli et al. in other studies . It is worth reminding here that the antibacterial properties of certain unsaturated fatty acids (oleic ( 172 ) and linoleic ( 174 ) acids) and, to some extent, of palmitic and stearic acids ( 167 , 169 ) are linked to their ability to inhibit enoyl-acyl carrier protein reductase (FabI) activity . The antibacterial activity of an ethanolic extract of F. triandra was also investigated . The antischistosomal action of the methanol extract from F. hirsuta can also be found . Interestingly, the acetonic and methanolic extracts derived from the aerial part of F. pulverulenta exhibited antiviral activity against Herpes simplex virus type 1 (HSV-1) at a dose of 500 μg/mL . F. pulverulenta is known to contain flavonoids, including the 7-bisulfate-3-glucuronide of kaempferol ( 61 ), isorhamnetins ( 79 – 80 ) and quercetin ( 62 ) . As some flavonoids, notably quercetin and, to a lesser extent, catechin and hesperetin, have been reported to possess antiviral capacities against a number of viruses, including HSV-1, the antiviral activity of F. pulverulenta extracts may be linked to its content of flavonoids . These investigations and their results clearly suggest that Frankenia plants might be a valuable source of antimicrobial substances. 3.2.3. Neuroprotective Activity Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive and irreversible memory loss and other cognitive impairments. At the cellular level, AD is characterized by synaptic and neuronal loss, deposition of plaques made of β-amyloid peptide (Aβ) and the formation of fibrils in the brain made of tau-protein. Several data issued from genetic, neuropathological and biochemical studies have established the central role of the β-amyloid peptide (Aβ), which results from the cleavage of the so-called amyloid precursor protein (APP), a membrane glycoprotein . However, its precise role in AD pathogenesis is still unclear. One hypothesis suggests that oxidative damage in the brain may cause ROS generation in neurons, which in turn could potentiate the Aβ neurotoxicity and metabolism perturbation . Therefore, limiting or inhibiting oxidative stress could be a way to treat AD. As various plants contain various antioxidant natural products, especially those of the Frankenia family (see .1 and ), the neuroprotective properties of plants have been, and are, still being explored . In order to determine whether Frankenia plant species may prevent Aβ-induced neuronal cell toxicity, neuroprotection tests were carried out. The neuroprotective potential of ethyl acetate fractions from F. pulverulenta shoots and roots was evaluated . An Aβ(25–35)-induced cytotoxicity assay using pheochromocytoma-derived (PC12) cells was assessed. Both fractions remarkably prevented the cytotoxic response of Aβ(25–35) at levels around 57% and 80% at 100 and 200 μg/mL, respectively, compared with non-treated cells. At a higher concentration (300 μg/mL), the root fraction entirely counteracted the toxic effect of Aβ(25–35). Using the same process, the neuroprotective capacities of methanolic extracts from F. thymifolia and F. pulverulenta aerial parts were demonstrated in another study . Both species exerted a powerful neuroprotective effect in a dose-dependent manner, and about 80% of the cell viability was restored at 100 μg/mL. Additionally, the ethyl acetate fractions from F. thymifolia shoots and roots demonstrated a strong neuroprotective effect on neuronal PC12 cells and totally counterbalanced the damaging effect of Aβ(25–35) at 25 and 50 μg/mL, respectively . Most phenolics isolated from F. pulverulenta ethyl acetate fractions were shown to exhibit potent neuroprotective activities, particularly procyanidin dimers ( 78 ), which prevented Aβ-induced toxicity at levels close to 100% at 50 μM, while catechin ( 74 ) prevented it only at 70% at the same concentration, and quercetin ( 62 ) did not . The strong capacity of F. thymifolia and F. pulverulenta extracts to inhibit Aβ(25–35) aggregation could be attributed to their significant antioxidant activities and phenolic contents. Various reports have indeed shown that phenolic substances may prevent neurodegenerative disorders, either by directly preventing the formation of Aβ fibril deposits in the brain or by exhibiting protective effects through scavenging ROS . Furthermore, a two-to-one complex between a polyphenol and the full Aβ peptide was observed by ESI-MS . It was also reported that gallic acid ( 1 ), found in F. thymifolia roots, in its glucosylated form and the corresponding gallotannins effectively suppressed Aβ(25–35) aggregation in vitro . Another study revealed that kaempferol-3- O -glucoside ( 56 ) presented a modest inhibitory effect on Aβ(25–35) aggregation, whereas kaempferol itself had a moderate effect. However, the reverse situation was observed with quercetin ( 62 ) and its 3′- O -glucoside ( 72 ), the latter exhibiting a good activity while the former had a modest one . These results are quite surprising due to the structural similarity between these compounds (see ). Interestingly, the very similar hyperoside ( 73 ) significantly diminished Aβ-induced cytotoxicity and apoptosis by restoring Aβ-induced mitochondrial dysfunction . Alternatively, it has been shown that caffeic acid ( 30 ), epigallocatechin ( 75 ) and its 3- O -gallate ( 76 ) exhibit a modest aggregation inhibition, and p -hydroxybenzoic acid ( 11 ) presents a moderate one, while the hydroxy derivatives of benzyl benzoate ( 192 ) exhibit interesting inhibition . However, the latter have so far not been detected in Frankenia species. Another approach to facing AD is to attempt to treat the synaptic and neuronal loss associated with AD. During the progression of AD, different types of neurons deteriorate, but the main loss occurs in forebrain cholinergic neurons, which play an important role in cognition. Therapies have thus been, and are still being, designed to reverse this cholinergic deficit. Cholinergic neurons rely on acetylcholine (ACh) as a neurotransmitter, which is hydrolyzed by acetylcholinesterase (AChE) in the synapse and to a lesser extent by the non-specific butyrylcholinesterase (BuChE) . Furthermore, several studies have suggested that AChE can modulate APP processing in a way that enhances β-amyloid plaque deposition . As a consequence, the inhibition of these enzymes is actively pursued. Various inhibitors have proven beneficial as a curative approach to AD, and a few are commercially available . As some of the earliest inhibitors discovered were alkaloids issued from plants, plant extracts are now often evaluated as cholinesterase inhibitors. In Frankenia species, only a few have so far been evaluated. Interestingly, methanol extracts from F. laevis demonstrated significant AChE and BuChE inhibition (about 80% at 1 mg/mL) . 3.2.4. Tyrosinase Inhibition Activity Tyrosinase is a multipurpose copper-containing oxidase that participates in melanin production and enzymatic browning processes that happen in damaged fruits during post-harvest processing . Natural substances are widely utilized in cosmetic formulations as tyrosinase inhibitors to cure skin hyperpigmentation, melasma and post-inflammatory hyperpigmentation . They are also applied in the food industry to prohibit enzymatic browning action in injured vegetables . The inhibition of tyrosinase by F. laevis shoot extracts (50% ethanol) was conducted by performing both the inhibition of l -tyrosine hydroxylation to l -3,4-dihydroxyphenylalanine (L-DOPA) (monophenolase) and that of L-DOPA oxidation to dopaquinone (diphenolase) . A strong inhibition of monophenolase and diphenolase functions was achieved (IC 50 = 730.43 and 123.62 μg/mL, respectively). In agreement with previous studies , the high levels of phenolic compounds, such as chlorogenic acid ( 31 ) and quercetin ( 62 ), in F. laevis extracts are probably responsible for the anti-tyrosinase effect, making this species a prospective source of natural skin-lightening agents and conservatives . 3.2.5. Anti-Inflammatory Activity Inflammation is induced by either external or internal causes. In the former, inflammation occurs in response to infection caused by microorganisms or to tissue injury. In the latter, cell death, cancer and other dysfunctions initiate a cascade of events leading to inflammation. In turn, various inflammatory mediators are produced, such as cytokines, chemokines, polyunsaturated fatty acids, etc., some acting as pro- and/or anti-inflammatory agents. The enzymes that are responsible for the generation of these inflammatory mediators, such as cyclooxygenase (COX), lipoxygenase (LOX) and hyaluronidase, are the major targets of anti-inflammatory therapies and a number of drugs have been developed For such common anti-inflammatory activity, the use of plants has been known since antiquity and is still applied. Although traditional medicines provide numerous anti-inflammatory extracts or plant parts, this activity is still being explored and remains one of the most sought-after bioactivities from plants . The anti-inflammatory capacity of ethanolic and soxhlet extracts obtained from F. triandra aerial parts was evaluated . The inhibition of LOX and COX2 capacities was assessed on the basis of the enzymatic oxidation of linoleic acid to the corresponding hydroperoxide and prostaglandin measurement, respectively. The extracts displayed a satisfactory ability to prevent LOX (IC 50 = 134.5 ± 12.9 and 117.8 ± 1.8 μg/mL, respectively) and COX2 (54% and 50% inhibition, respectively) actions. Hence, it is thought that these inhibition values are high for a crude extract . The authors have also examined the hyaluronidase activity by measuring the quantity of generated N -acetyl glucosamine (NAGA) . Both soxhlet and ethanolic extracts demonstrated a high degree of inhibition, but the soxhlet extract was three times more effective than the ethanolic extract (IC 50 = 146.3 ± 4.3 and 412.2 ± 8.9 μg/mL, respectively) as compared to the commercial anti-inflammatory, indomethacin (IC 50 = 502.0 ± 7.1 μg/mL), and the control sample, quercetin (IC 50 = 340.0 ± 12.0 μg/mL). Numerous studies have shown a strong correlation between inflammation and oxidative species production. Consequently, plants with antioxidant capabilities frequently have anti-inflammatory characteristics . 3.2.6. Carbonic Anhydrase II Inhibition Activity Carbonic anhydrase II (CA-II) belongs to the carbonic anhydrase family of enzymes, which are zinc metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 ) and a proton (H + ) . In addition to their key roles in transporting CO 2 and maintaining acid–base balance, the 16 human carbonic anhydrases are also involved in several essential physiological processes, and, thus, their dysregulated expression and/or abnormal activity have important pathological consequences. For example, CA-II is mainly involved in the regulation of bicarbonate concentration in the eyes, and is thus linked to glaucoma, but also expressed in malignant brain tumors and renal, gastritis and pancreatic carcinomas. CA-II and other CAs are therefore interesting therapeutic targets for the treatment of related diseases. CA-II inhibitors are, for example, used in the treatment of several illnesses, including glaucoma, idiopathic intracranial hypertension, altitude sickness, congestive heart failure and epilepsy . In order to look for some activity in such health problems, the EO extracted from the aerial parts of F. pulverulenta was screened against the CA-II enzyme. The experiment was done at a micromolar level using acetazolamide as a standard inhibitor (IC 50 = 18.2 ± 1.2 μM). The EO demonstrated a substantial and spectacular CA-II inhibition effect (IC 50 = 101.5 ± 2.35%) and might have application in the management of CA-related disorders . 3.2.7. Antidiabetic Activity In type 2 diabetic patients postprandial hyperglycemia occurs because the peak insulin release is delayed, and levels are thus insufficient to control the accelerated blood glucose elevation. Such hyperglycemic spikes induce inflammatory reactions, oxidative stress and endothelial dysfunction, which in turn increase the occurrence of cardiovascular diseases. To reduce postprandial hyperglycemia, the most common type 2 diabetes preventive therapy involves decreasing carbohydrate digestibility by blocking two important hydrolyzing enzymes, specifically, α-amylase and α-glucosidase . The methanol and dichloromethane extracts of F. laevis were investigated for their capacity to inhibit α-glucosidase and α-amylase enzymes using a standard in vitro inhibition assay . The extracts showed a marked α-glucosidase inhibition (EC 50 = 1.02 ± 0.01 mg/mL and 0.52 ± 0.04 mg/mL, respectively) compared to the positive control, acarbose (EC 50 = 3.14 ± 0.23 mg/mL). On the other hand, the extracts had no significant effect on α-amylase activity. Abundant in F. laevis extracts, linoleic acid ( 174 ) and its derivatives, as well as loliolide ( 107 ), isololiolide ( 106 ) and dihydroactinidiolide ( 108 ), were found to have a strong inhibitory effect on α-glucosidase. Their higher abundance in the dichloromethane extract may explain the anti-α-glucosidase activity of the F. laevis extracts. In addition, the antioxidant properties of these extracts may also help to decrease the incidence of diabetes complications related to oxidative stress, specifically microvascular and cardiovascular issues . 3.2.8. Anticancer Activity Prior to human usage, substances or chemicals must undergo rigorous safety evaluations. Cytotoxic tests using various human cell lines are often performed to assess the potential toxicity of different substances in vitro . The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test, a colorimetric approach that measures cell metabolic activity, is one of the most frequently used methods to determine how a substance affects cellular viability . On the other hand, cytotoxicity could be useful to control tumor cell proliferation and thus treat cancers. For the latter purpose, the anticancer and antiproliferative activities of extracts from the aerial parts of F. laevis were investigated against human hepatocarcinoma cells (HepG2) . Sea heath dichloromethane extract showed potential anti-HepG2 activity (EC 50 = 52.1 μg/mL). In contrast, methanol extract did not present significant cytotoxicity. This difference could be ascribed to the high content of certain metabolites and fatty acids in the dichloromethane extract. It has indeed been reported that fatty acids, especially linoleic acid ( 174 ) which is abundant in this extract, have shown chemoprotective effects . Furthermore, the monoterpenes loliolide ( 107 ) and isololiolide ( 106 ), also abundant in this extract, are known for their strong cytotoxic activities on HepG2 cells , as is dihydroactinidiolide ( 108 ) on human lung carcinoma cells (A549) and human breast cancer cells . The phytohormone oxophytodienoic acid ( 204 ) also present in this extract is also known for its cytotoxic activity on human breast cancer cells . A similar study on a large series of halophyte plants, including both F. laevis and F. pulverulenta, has been performed . The viability of four cancer cell types, including the same HepG2 cell line, was evaluated, and the F. laevis extract was found to significantly decrease it (71%), while F. pulverulenta did not. Due to the abundance of the active compounds mentioned above, the F. laevis dichloromethane extract may represent an interesting natural alternative for treating some cancers. Furthermore, the natural products probably responsible for these antitumor activities could become promising candidates for new antitumor drugs. 3.2.9. Insecticidal Activity The control of insect proliferation and of the so-induced destruction of agricultural plants is usually achieved with synthetic insecticides. However, their intensive and uncontrolled utilization has led to the development of resistance in insects and to various environmental damages. Although a few insecticides are issued from plants, such as pyrethrenoids, plants may provide potentially safer alternatives to the currently used insect-control agents. In this context, petroleum ether and chloroformic and ethyl acetate extracts obtained from the aerial parts of F. laevis were evaluated for their antifeedant, toxic and insect growth inhibition activities against larvae and adults of the confused flour beetle Tribolium confusum . At a concentration of 1%, the petroleum ether extract demonstrated moderate antifeedant properties. At the same concentration, the tested extracts considerably induced larval mortality (up to 97% inhibition with the ethyl acetate extract), while adult toxicity did not surpass 33%. Furthermore, the F. laevis extracts inhibited feeding, exhibited high toxicity and greatly affected the development of Tribolium confusum larvae when used at a dose of 1%. Therefore, this halophyte plant seems to have great potential for pest control; it would be worth identifying the compound(s) responsible for the interesting insecticidal activity of these extracts even at low concentrations . As was reminded in the introduction, traditional medicine has a long history in human health and various variants have been developed around the world and are still practiced nowadays. In countries with limited access to modern therapy, traditional medicine is frequently the major source of primary healthcare requirements . Used as traditional medicinal plants, Frankenia species appear to play a prominent role in the treatment of various diseases. Due to their astringent properties, Frankenia species are utilized in Asian and African (especially in Morocco) folk medicine for gargling or for topical application, either as tinctures or as herbal tea, e.g., with F. laevis or F. thymifolia . They are also used in these countries to treat a variety of clinical disorders, such as dysentery, diarrhea, gonorrhea, vaginal leucorrhea, mucus releases from the nose and catarrh-induced infections, again as plant infusions, with, e.g., F. pulverulenta , or as stupe, depending on the localization . Gargle and decoction generated from the entire plant of F. pulverulenta are widely used in local medicine by the inhabitants of the Onaizah province in Saudi Arabia and are mostly used orally for their analgesic and carminative properties . Also in Saudi Arabia, the powdered rhizome of F. aucheri (hirsuta) combined with milk is used to stimulate lactation in cows and camels, particularly in the winter . It has also been reported that Puna inhabitants in South America used F. triandra as a forage but also as antiseptic in folk medicine . Additionally, some Frankenia species can be converted into sticky glue mixtures, due to their specific natural product contents, e.g., kaempferol, quercetin and tannin. Therefore, they are used in totally different applications, notably to stick blade cutting edges and to seal stoneware (e.g., F. hirsuta ) . Overall, Frankenia plant species may thus be viewed as promising prospects for different applications in industry, and mostly in pharmaceutical applications. Secondary plant metabolites, which are produced in large amounts by plant species, are crucial components for supporting human health. They contribute to the medicinal properties of plant species as antioxidants, anti-inflammatory, anti-carcinogenic and antibacterial agents , along with other capacities . Because the large diversity of natural compounds discovered in Frankenia species mostly belong to well-known families of bioactive compounds, it is expected that these plants exhibit the corresponding biological activities. Therefore, several works have been performed to check these bioactivities. They are listed below. 3.2.1. Antioxidant Activity The antioxidant activity of Frankenia species has been assessed using several methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging analysis, 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radical trapping, ferric ion reducing antioxidant power (FRAP), metal chelating activity (MCA), including copper (CCA)- and iron (ICA)-chelating activities, oxygen radical absorbance capacity (ORAC) assay and β-carotene oxidation test. The corresponding details have been collected in . The aqueous acetone , methanol and ethanol extracts of F. laevis exhibited spectacular in vitro radical scavenging and copper chelating properties. However, the dichloromethane extract from this species was only able to chelate iron, probably due to the presence of various phenolics and flavonoids that can act as phytochelators , such as gallic acid, kaempferol and quercetin derivatives (see and and and ). Likewise, F. pulverulenta ethyl acetate and methanolic extracts were investigated using DPPH, ABTS and ORAC assays, and potent antioxidant activity was reported. The antioxidant activity of the aqueous acetone extract of F. pulverulenta was also assessed . The antioxidant potency can be linked to the well-known antioxidant compounds gallic acid ( 1 ), p -coumaric acid ( 34 ), quercetin ( 62 ) and catechin ( 74 ) which are abundantly present in this species. Ben Mansour et al. demonstrated, in 2016, that F. thymifolia ethyl acetate extracts exhibited strong antioxidant activity in both shoots and roots. Furthermore, this extract has the highest TPC and antioxidant capacities . Other studies investigated the methanolic and chloroformic extracts of F. thymifolia and demonstrated that the methanolic extract exhibited better antioxidant activity, again linked to the high level of phenolic compounds, including salicylic acid ( 10 ), cinnamic acid ( 28 ), 2-hydroxycinnamic acid ( 29 ), chlorogenic acid ( 31 ) and catechin (74) . Torres Carro et al. showed, in 2016, the significant antioxidant activity of the ethanolic and soxhlet of F. triandra evaluated by the β-carotene assay . Overall, plants from the Frankenia family are rich in polyphenols, and this richness is often, if not always, correlated to the strong antioxidant properties these plants exhibit . 3.2.2. Antimicrobial Activity Bacteria were involved in many of the most devastating diseases and massive epidemics in human history, before the discovery of antibiotics. Due to the misuse of the latter, bacteria have now developed resistance to the commonly used antibiotics . Therefore, it is imperative to identify new and advanced chemical agents in order to have more productive resistance to microorganisms . Since synthetic chemicals are related to adverse effects and harmful residues, novel antibacterial, antifungal, antiviral and antiparasitic drugs from plant sources must be developed worldwide . Accordingly, a few Frankenia species were collected and screened for their antimicrobial activities. The corresponding details have been collected in . Jdey et al. showed, in 2017, that the ethanolic extract of F. laevis significantly inhibited the development of both Gram-positive (Gram+) and Gram-negative (Gram−) bacteria engaged in their study . All these strains were indeed inhibited by more than 55%, and the best inhibitions were observed for Micrococcus luteus (83%) and Salmonella enterica (77%) at a concentration of 1 mg/mL. This antibacterial effect may be attributed to the chlorogenic acid ( 31 ) and catechin ( 74 ) contained in this species, probably by inducing structural or functional damage to the bacterial cell membranes . Similarly, Saïdana et al. demonstrated, in 2010, that EO from the aerial parts of F. laevis was efficient against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Escherichia coli and Salmonella typhimurium . According to the authors, the antimicrobial activity of the EO can be attributed to the presence of fatty acids such as palmitic acid ( 167 ), fatty acid esters like methyl linoleate ( 181 ), sesquiterpenes such as farnesyl acetate ( 119 ) , aromatic compounds such as benzyl benzoate ( 192 ) and benzyl cinnamate ( 193 ), and, to a lesser extent, eugenol ( 49 ), β-caryophyllene ( 135 ), phytol ( 110 ), isophytol ( 112 ), ( E , E )-farnesol ( 117 ) and hexadecanol ( 185 ). However, no significant effect on Pseudomonas aeruginosa was detected . This Gram− bacteria has already been demonstrated to be less susceptible to the action of several other plant EOs . The antifungal activity of the EO was also investigated. Despite the presence of eugenol ( 49 ) and β-caryophyllene ( 135 ) in the oil composition, known to have antifungal effects , none of the tested fungi were successfully inhibited by the EO at the tested doses. This may be explained by the low amounts of such chemicals in the EO . The antibacterial and antifungal activity of EO from F. pulverulenta was also investigated . Despite being rich in β-caryophyllene ( 135 ), which represents its main constituent (32%), this EO did not prevent bacterial growth. This finding appears contradictory to previous research, which showed that the presence of β-caryophyllene enhances the biological activities of EO, including their antibacterial activity . Furthermore, the F. pulverulenta EO displayed poor antifungal activity and was exclusively efficient against the basidiomycete Rhizoctonia solani . In 2011, Megdiche-Ksouri et al. investigated the activity of methanolic (polar) and chloroformic (less polar) extracts from F. thymifolia against five bacteria and one fungus . The chloroformic extract provided the best performance, being active against all the evaluated bacterial strains. Similar inhibition results have been observed with other halophytes (e.g., sea holly, sea fennel) . Such an outcome has been correlated to the polarity of the extracting solvent and could be attributed to the presence of lipophilic compounds in these extracts. Indeed, it has been demonstrated that long-chain unsaturated fatty acids, notably oleic ( 172 ) and linoleic ( 174 ), exhibit a strong inhibiting activity against mycobacteria . Furthermore, it has been reported that the relatively lipophilic flavonoids catechin ( 74 ) and epigallocatechino-3-gallate ( 76 ) exhibit protective and antibacterial effects . Likewise, the n -butanol fraction from F. thymifolia exhibited a stronger antibacterial effect against all tested bacterial strains compared to the ethyl acetate fraction ( Pseudomonas aeruginosa was the most vulnerable strain) . The extracts investigated also presented anti-leishmanial and antiamoebic effects against Leishmania amazonensis and Acanthamoeba castellanii , respectively. The antiparasitic capacities of these extracts may be related to the presence of quercetin ( 62 ) . Canli et al. demonstrated the antibacterial and antifungal activity of F. hirsuta ethanolic extract against seventeen bacteria and one fungus . Except for the Gram ˗ bacteria Enterobacter aerogenes and Escherichia coli , all of the examined strains were sensitive to the antimicrobial action of the F. hirsuta extract. The most sensitive strains were Gram+ bacteria, especially Staphylococcus epidermidis and Enterecoccus faecium , compared to Gram ˗ bacteria. Such antibiotic activity was again associated with the presence in this extract of oleic and linoleic acid in high amounts . The concomitant presence of mesitylene ( 194 ) , eudesmic acid ( 8 ) and stearic acid ( 169 ) also suggested a possible role in the F. hirsuta antibacterial activity, because some mesitylene derivatives , eudesmic acid and stearic acid analogs are known as antibacterial agents. The difference in sensitivity to plant extracts between Gram+ and Gram− bacteria observed for F. hirsuta ethanolic extract could be generalized according to Canli et al. in other studies . It is worth reminding here that the antibacterial properties of certain unsaturated fatty acids (oleic ( 172 ) and linoleic ( 174 ) acids) and, to some extent, of palmitic and stearic acids ( 167 , 169 ) are linked to their ability to inhibit enoyl-acyl carrier protein reductase (FabI) activity . The antibacterial activity of an ethanolic extract of F. triandra was also investigated . The antischistosomal action of the methanol extract from F. hirsuta can also be found . Interestingly, the acetonic and methanolic extracts derived from the aerial part of F. pulverulenta exhibited antiviral activity against Herpes simplex virus type 1 (HSV-1) at a dose of 500 μg/mL . F. pulverulenta is known to contain flavonoids, including the 7-bisulfate-3-glucuronide of kaempferol ( 61 ), isorhamnetins ( 79 – 80 ) and quercetin ( 62 ) . As some flavonoids, notably quercetin and, to a lesser extent, catechin and hesperetin, have been reported to possess antiviral capacities against a number of viruses, including HSV-1, the antiviral activity of F. pulverulenta extracts may be linked to its content of flavonoids . These investigations and their results clearly suggest that Frankenia plants might be a valuable source of antimicrobial substances. 3.2.3. Neuroprotective Activity Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive and irreversible memory loss and other cognitive impairments. At the cellular level, AD is characterized by synaptic and neuronal loss, deposition of plaques made of β-amyloid peptide (Aβ) and the formation of fibrils in the brain made of tau-protein. Several data issued from genetic, neuropathological and biochemical studies have established the central role of the β-amyloid peptide (Aβ), which results from the cleavage of the so-called amyloid precursor protein (APP), a membrane glycoprotein . However, its precise role in AD pathogenesis is still unclear. One hypothesis suggests that oxidative damage in the brain may cause ROS generation in neurons, which in turn could potentiate the Aβ neurotoxicity and metabolism perturbation . Therefore, limiting or inhibiting oxidative stress could be a way to treat AD. As various plants contain various antioxidant natural products, especially those of the Frankenia family (see .1 and ), the neuroprotective properties of plants have been, and are, still being explored . In order to determine whether Frankenia plant species may prevent Aβ-induced neuronal cell toxicity, neuroprotection tests were carried out. The neuroprotective potential of ethyl acetate fractions from F. pulverulenta shoots and roots was evaluated . An Aβ(25–35)-induced cytotoxicity assay using pheochromocytoma-derived (PC12) cells was assessed. Both fractions remarkably prevented the cytotoxic response of Aβ(25–35) at levels around 57% and 80% at 100 and 200 μg/mL, respectively, compared with non-treated cells. At a higher concentration (300 μg/mL), the root fraction entirely counteracted the toxic effect of Aβ(25–35). Using the same process, the neuroprotective capacities of methanolic extracts from F. thymifolia and F. pulverulenta aerial parts were demonstrated in another study . Both species exerted a powerful neuroprotective effect in a dose-dependent manner, and about 80% of the cell viability was restored at 100 μg/mL. Additionally, the ethyl acetate fractions from F. thymifolia shoots and roots demonstrated a strong neuroprotective effect on neuronal PC12 cells and totally counterbalanced the damaging effect of Aβ(25–35) at 25 and 50 μg/mL, respectively . Most phenolics isolated from F. pulverulenta ethyl acetate fractions were shown to exhibit potent neuroprotective activities, particularly procyanidin dimers ( 78 ), which prevented Aβ-induced toxicity at levels close to 100% at 50 μM, while catechin ( 74 ) prevented it only at 70% at the same concentration, and quercetin ( 62 ) did not . The strong capacity of F. thymifolia and F. pulverulenta extracts to inhibit Aβ(25–35) aggregation could be attributed to their significant antioxidant activities and phenolic contents. Various reports have indeed shown that phenolic substances may prevent neurodegenerative disorders, either by directly preventing the formation of Aβ fibril deposits in the brain or by exhibiting protective effects through scavenging ROS . Furthermore, a two-to-one complex between a polyphenol and the full Aβ peptide was observed by ESI-MS . It was also reported that gallic acid ( 1 ), found in F. thymifolia roots, in its glucosylated form and the corresponding gallotannins effectively suppressed Aβ(25–35) aggregation in vitro . Another study revealed that kaempferol-3- O -glucoside ( 56 ) presented a modest inhibitory effect on Aβ(25–35) aggregation, whereas kaempferol itself had a moderate effect. However, the reverse situation was observed with quercetin ( 62 ) and its 3′- O -glucoside ( 72 ), the latter exhibiting a good activity while the former had a modest one . These results are quite surprising due to the structural similarity between these compounds (see ). Interestingly, the very similar hyperoside ( 73 ) significantly diminished Aβ-induced cytotoxicity and apoptosis by restoring Aβ-induced mitochondrial dysfunction . Alternatively, it has been shown that caffeic acid ( 30 ), epigallocatechin ( 75 ) and its 3- O -gallate ( 76 ) exhibit a modest aggregation inhibition, and p -hydroxybenzoic acid ( 11 ) presents a moderate one, while the hydroxy derivatives of benzyl benzoate ( 192 ) exhibit interesting inhibition . However, the latter have so far not been detected in Frankenia species. Another approach to facing AD is to attempt to treat the synaptic and neuronal loss associated with AD. During the progression of AD, different types of neurons deteriorate, but the main loss occurs in forebrain cholinergic neurons, which play an important role in cognition. Therapies have thus been, and are still being, designed to reverse this cholinergic deficit. Cholinergic neurons rely on acetylcholine (ACh) as a neurotransmitter, which is hydrolyzed by acetylcholinesterase (AChE) in the synapse and to a lesser extent by the non-specific butyrylcholinesterase (BuChE) . Furthermore, several studies have suggested that AChE can modulate APP processing in a way that enhances β-amyloid plaque deposition . As a consequence, the inhibition of these enzymes is actively pursued. Various inhibitors have proven beneficial as a curative approach to AD, and a few are commercially available . As some of the earliest inhibitors discovered were alkaloids issued from plants, plant extracts are now often evaluated as cholinesterase inhibitors. In Frankenia species, only a few have so far been evaluated. Interestingly, methanol extracts from F. laevis demonstrated significant AChE and BuChE inhibition (about 80% at 1 mg/mL) . 3.2.4. Tyrosinase Inhibition Activity Tyrosinase is a multipurpose copper-containing oxidase that participates in melanin production and enzymatic browning processes that happen in damaged fruits during post-harvest processing . Natural substances are widely utilized in cosmetic formulations as tyrosinase inhibitors to cure skin hyperpigmentation, melasma and post-inflammatory hyperpigmentation . They are also applied in the food industry to prohibit enzymatic browning action in injured vegetables . The inhibition of tyrosinase by F. laevis shoot extracts (50% ethanol) was conducted by performing both the inhibition of l -tyrosine hydroxylation to l -3,4-dihydroxyphenylalanine (L-DOPA) (monophenolase) and that of L-DOPA oxidation to dopaquinone (diphenolase) . A strong inhibition of monophenolase and diphenolase functions was achieved (IC 50 = 730.43 and 123.62 μg/mL, respectively). In agreement with previous studies , the high levels of phenolic compounds, such as chlorogenic acid ( 31 ) and quercetin ( 62 ), in F. laevis extracts are probably responsible for the anti-tyrosinase effect, making this species a prospective source of natural skin-lightening agents and conservatives . 3.2.5. Anti-Inflammatory Activity Inflammation is induced by either external or internal causes. In the former, inflammation occurs in response to infection caused by microorganisms or to tissue injury. In the latter, cell death, cancer and other dysfunctions initiate a cascade of events leading to inflammation. In turn, various inflammatory mediators are produced, such as cytokines, chemokines, polyunsaturated fatty acids, etc., some acting as pro- and/or anti-inflammatory agents. The enzymes that are responsible for the generation of these inflammatory mediators, such as cyclooxygenase (COX), lipoxygenase (LOX) and hyaluronidase, are the major targets of anti-inflammatory therapies and a number of drugs have been developed For such common anti-inflammatory activity, the use of plants has been known since antiquity and is still applied. Although traditional medicines provide numerous anti-inflammatory extracts or plant parts, this activity is still being explored and remains one of the most sought-after bioactivities from plants . The anti-inflammatory capacity of ethanolic and soxhlet extracts obtained from F. triandra aerial parts was evaluated . The inhibition of LOX and COX2 capacities was assessed on the basis of the enzymatic oxidation of linoleic acid to the corresponding hydroperoxide and prostaglandin measurement, respectively. The extracts displayed a satisfactory ability to prevent LOX (IC 50 = 134.5 ± 12.9 and 117.8 ± 1.8 μg/mL, respectively) and COX2 (54% and 50% inhibition, respectively) actions. Hence, it is thought that these inhibition values are high for a crude extract . The authors have also examined the hyaluronidase activity by measuring the quantity of generated N -acetyl glucosamine (NAGA) . Both soxhlet and ethanolic extracts demonstrated a high degree of inhibition, but the soxhlet extract was three times more effective than the ethanolic extract (IC 50 = 146.3 ± 4.3 and 412.2 ± 8.9 μg/mL, respectively) as compared to the commercial anti-inflammatory, indomethacin (IC 50 = 502.0 ± 7.1 μg/mL), and the control sample, quercetin (IC 50 = 340.0 ± 12.0 μg/mL). Numerous studies have shown a strong correlation between inflammation and oxidative species production. Consequently, plants with antioxidant capabilities frequently have anti-inflammatory characteristics . 3.2.6. Carbonic Anhydrase II Inhibition Activity Carbonic anhydrase II (CA-II) belongs to the carbonic anhydrase family of enzymes, which are zinc metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 ) and a proton (H + ) . In addition to their key roles in transporting CO 2 and maintaining acid–base balance, the 16 human carbonic anhydrases are also involved in several essential physiological processes, and, thus, their dysregulated expression and/or abnormal activity have important pathological consequences. For example, CA-II is mainly involved in the regulation of bicarbonate concentration in the eyes, and is thus linked to glaucoma, but also expressed in malignant brain tumors and renal, gastritis and pancreatic carcinomas. CA-II and other CAs are therefore interesting therapeutic targets for the treatment of related diseases. CA-II inhibitors are, for example, used in the treatment of several illnesses, including glaucoma, idiopathic intracranial hypertension, altitude sickness, congestive heart failure and epilepsy . In order to look for some activity in such health problems, the EO extracted from the aerial parts of F. pulverulenta was screened against the CA-II enzyme. The experiment was done at a micromolar level using acetazolamide as a standard inhibitor (IC 50 = 18.2 ± 1.2 μM). The EO demonstrated a substantial and spectacular CA-II inhibition effect (IC 50 = 101.5 ± 2.35%) and might have application in the management of CA-related disorders . 3.2.7. Antidiabetic Activity In type 2 diabetic patients postprandial hyperglycemia occurs because the peak insulin release is delayed, and levels are thus insufficient to control the accelerated blood glucose elevation. Such hyperglycemic spikes induce inflammatory reactions, oxidative stress and endothelial dysfunction, which in turn increase the occurrence of cardiovascular diseases. To reduce postprandial hyperglycemia, the most common type 2 diabetes preventive therapy involves decreasing carbohydrate digestibility by blocking two important hydrolyzing enzymes, specifically, α-amylase and α-glucosidase . The methanol and dichloromethane extracts of F. laevis were investigated for their capacity to inhibit α-glucosidase and α-amylase enzymes using a standard in vitro inhibition assay . The extracts showed a marked α-glucosidase inhibition (EC 50 = 1.02 ± 0.01 mg/mL and 0.52 ± 0.04 mg/mL, respectively) compared to the positive control, acarbose (EC 50 = 3.14 ± 0.23 mg/mL). On the other hand, the extracts had no significant effect on α-amylase activity. Abundant in F. laevis extracts, linoleic acid ( 174 ) and its derivatives, as well as loliolide ( 107 ), isololiolide ( 106 ) and dihydroactinidiolide ( 108 ), were found to have a strong inhibitory effect on α-glucosidase. Their higher abundance in the dichloromethane extract may explain the anti-α-glucosidase activity of the F. laevis extracts. In addition, the antioxidant properties of these extracts may also help to decrease the incidence of diabetes complications related to oxidative stress, specifically microvascular and cardiovascular issues . 3.2.8. Anticancer Activity Prior to human usage, substances or chemicals must undergo rigorous safety evaluations. Cytotoxic tests using various human cell lines are often performed to assess the potential toxicity of different substances in vitro . The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test, a colorimetric approach that measures cell metabolic activity, is one of the most frequently used methods to determine how a substance affects cellular viability . On the other hand, cytotoxicity could be useful to control tumor cell proliferation and thus treat cancers. For the latter purpose, the anticancer and antiproliferative activities of extracts from the aerial parts of F. laevis were investigated against human hepatocarcinoma cells (HepG2) . Sea heath dichloromethane extract showed potential anti-HepG2 activity (EC 50 = 52.1 μg/mL). In contrast, methanol extract did not present significant cytotoxicity. This difference could be ascribed to the high content of certain metabolites and fatty acids in the dichloromethane extract. It has indeed been reported that fatty acids, especially linoleic acid ( 174 ) which is abundant in this extract, have shown chemoprotective effects . Furthermore, the monoterpenes loliolide ( 107 ) and isololiolide ( 106 ), also abundant in this extract, are known for their strong cytotoxic activities on HepG2 cells , as is dihydroactinidiolide ( 108 ) on human lung carcinoma cells (A549) and human breast cancer cells . The phytohormone oxophytodienoic acid ( 204 ) also present in this extract is also known for its cytotoxic activity on human breast cancer cells . A similar study on a large series of halophyte plants, including both F. laevis and F. pulverulenta, has been performed . The viability of four cancer cell types, including the same HepG2 cell line, was evaluated, and the F. laevis extract was found to significantly decrease it (71%), while F. pulverulenta did not. Due to the abundance of the active compounds mentioned above, the F. laevis dichloromethane extract may represent an interesting natural alternative for treating some cancers. Furthermore, the natural products probably responsible for these antitumor activities could become promising candidates for new antitumor drugs. 3.2.9. Insecticidal Activity The control of insect proliferation and of the so-induced destruction of agricultural plants is usually achieved with synthetic insecticides. However, their intensive and uncontrolled utilization has led to the development of resistance in insects and to various environmental damages. Although a few insecticides are issued from plants, such as pyrethrenoids, plants may provide potentially safer alternatives to the currently used insect-control agents. In this context, petroleum ether and chloroformic and ethyl acetate extracts obtained from the aerial parts of F. laevis were evaluated for their antifeedant, toxic and insect growth inhibition activities against larvae and adults of the confused flour beetle Tribolium confusum . At a concentration of 1%, the petroleum ether extract demonstrated moderate antifeedant properties. At the same concentration, the tested extracts considerably induced larval mortality (up to 97% inhibition with the ethyl acetate extract), while adult toxicity did not surpass 33%. Furthermore, the F. laevis extracts inhibited feeding, exhibited high toxicity and greatly affected the development of Tribolium confusum larvae when used at a dose of 1%. Therefore, this halophyte plant seems to have great potential for pest control; it would be worth identifying the compound(s) responsible for the interesting insecticidal activity of these extracts even at low concentrations . The antioxidant activity of Frankenia species has been assessed using several methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical-scavenging analysis, 2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) cation radical trapping, ferric ion reducing antioxidant power (FRAP), metal chelating activity (MCA), including copper (CCA)- and iron (ICA)-chelating activities, oxygen radical absorbance capacity (ORAC) assay and β-carotene oxidation test. The corresponding details have been collected in . The aqueous acetone , methanol and ethanol extracts of F. laevis exhibited spectacular in vitro radical scavenging and copper chelating properties. However, the dichloromethane extract from this species was only able to chelate iron, probably due to the presence of various phenolics and flavonoids that can act as phytochelators , such as gallic acid, kaempferol and quercetin derivatives (see and and and ). Likewise, F. pulverulenta ethyl acetate and methanolic extracts were investigated using DPPH, ABTS and ORAC assays, and potent antioxidant activity was reported. The antioxidant activity of the aqueous acetone extract of F. pulverulenta was also assessed . The antioxidant potency can be linked to the well-known antioxidant compounds gallic acid ( 1 ), p -coumaric acid ( 34 ), quercetin ( 62 ) and catechin ( 74 ) which are abundantly present in this species. Ben Mansour et al. demonstrated, in 2016, that F. thymifolia ethyl acetate extracts exhibited strong antioxidant activity in both shoots and roots. Furthermore, this extract has the highest TPC and antioxidant capacities . Other studies investigated the methanolic and chloroformic extracts of F. thymifolia and demonstrated that the methanolic extract exhibited better antioxidant activity, again linked to the high level of phenolic compounds, including salicylic acid ( 10 ), cinnamic acid ( 28 ), 2-hydroxycinnamic acid ( 29 ), chlorogenic acid ( 31 ) and catechin (74) . Torres Carro et al. showed, in 2016, the significant antioxidant activity of the ethanolic and soxhlet of F. triandra evaluated by the β-carotene assay . Overall, plants from the Frankenia family are rich in polyphenols, and this richness is often, if not always, correlated to the strong antioxidant properties these plants exhibit . Bacteria were involved in many of the most devastating diseases and massive epidemics in human history, before the discovery of antibiotics. Due to the misuse of the latter, bacteria have now developed resistance to the commonly used antibiotics . Therefore, it is imperative to identify new and advanced chemical agents in order to have more productive resistance to microorganisms . Since synthetic chemicals are related to adverse effects and harmful residues, novel antibacterial, antifungal, antiviral and antiparasitic drugs from plant sources must be developed worldwide . Accordingly, a few Frankenia species were collected and screened for their antimicrobial activities. The corresponding details have been collected in . Jdey et al. showed, in 2017, that the ethanolic extract of F. laevis significantly inhibited the development of both Gram-positive (Gram+) and Gram-negative (Gram−) bacteria engaged in their study . All these strains were indeed inhibited by more than 55%, and the best inhibitions were observed for Micrococcus luteus (83%) and Salmonella enterica (77%) at a concentration of 1 mg/mL. This antibacterial effect may be attributed to the chlorogenic acid ( 31 ) and catechin ( 74 ) contained in this species, probably by inducing structural or functional damage to the bacterial cell membranes . Similarly, Saïdana et al. demonstrated, in 2010, that EO from the aerial parts of F. laevis was efficient against Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus, Escherichia coli and Salmonella typhimurium . According to the authors, the antimicrobial activity of the EO can be attributed to the presence of fatty acids such as palmitic acid ( 167 ), fatty acid esters like methyl linoleate ( 181 ), sesquiterpenes such as farnesyl acetate ( 119 ) , aromatic compounds such as benzyl benzoate ( 192 ) and benzyl cinnamate ( 193 ), and, to a lesser extent, eugenol ( 49 ), β-caryophyllene ( 135 ), phytol ( 110 ), isophytol ( 112 ), ( E , E )-farnesol ( 117 ) and hexadecanol ( 185 ). However, no significant effect on Pseudomonas aeruginosa was detected . This Gram− bacteria has already been demonstrated to be less susceptible to the action of several other plant EOs . The antifungal activity of the EO was also investigated. Despite the presence of eugenol ( 49 ) and β-caryophyllene ( 135 ) in the oil composition, known to have antifungal effects , none of the tested fungi were successfully inhibited by the EO at the tested doses. This may be explained by the low amounts of such chemicals in the EO . The antibacterial and antifungal activity of EO from F. pulverulenta was also investigated . Despite being rich in β-caryophyllene ( 135 ), which represents its main constituent (32%), this EO did not prevent bacterial growth. This finding appears contradictory to previous research, which showed that the presence of β-caryophyllene enhances the biological activities of EO, including their antibacterial activity . Furthermore, the F. pulverulenta EO displayed poor antifungal activity and was exclusively efficient against the basidiomycete Rhizoctonia solani . In 2011, Megdiche-Ksouri et al. investigated the activity of methanolic (polar) and chloroformic (less polar) extracts from F. thymifolia against five bacteria and one fungus . The chloroformic extract provided the best performance, being active against all the evaluated bacterial strains. Similar inhibition results have been observed with other halophytes (e.g., sea holly, sea fennel) . Such an outcome has been correlated to the polarity of the extracting solvent and could be attributed to the presence of lipophilic compounds in these extracts. Indeed, it has been demonstrated that long-chain unsaturated fatty acids, notably oleic ( 172 ) and linoleic ( 174 ), exhibit a strong inhibiting activity against mycobacteria . Furthermore, it has been reported that the relatively lipophilic flavonoids catechin ( 74 ) and epigallocatechino-3-gallate ( 76 ) exhibit protective and antibacterial effects . Likewise, the n -butanol fraction from F. thymifolia exhibited a stronger antibacterial effect against all tested bacterial strains compared to the ethyl acetate fraction ( Pseudomonas aeruginosa was the most vulnerable strain) . The extracts investigated also presented anti-leishmanial and antiamoebic effects against Leishmania amazonensis and Acanthamoeba castellanii , respectively. The antiparasitic capacities of these extracts may be related to the presence of quercetin ( 62 ) . Canli et al. demonstrated the antibacterial and antifungal activity of F. hirsuta ethanolic extract against seventeen bacteria and one fungus . Except for the Gram ˗ bacteria Enterobacter aerogenes and Escherichia coli , all of the examined strains were sensitive to the antimicrobial action of the F. hirsuta extract. The most sensitive strains were Gram+ bacteria, especially Staphylococcus epidermidis and Enterecoccus faecium , compared to Gram ˗ bacteria. Such antibiotic activity was again associated with the presence in this extract of oleic and linoleic acid in high amounts . The concomitant presence of mesitylene ( 194 ) , eudesmic acid ( 8 ) and stearic acid ( 169 ) also suggested a possible role in the F. hirsuta antibacterial activity, because some mesitylene derivatives , eudesmic acid and stearic acid analogs are known as antibacterial agents. The difference in sensitivity to plant extracts between Gram+ and Gram− bacteria observed for F. hirsuta ethanolic extract could be generalized according to Canli et al. in other studies . It is worth reminding here that the antibacterial properties of certain unsaturated fatty acids (oleic ( 172 ) and linoleic ( 174 ) acids) and, to some extent, of palmitic and stearic acids ( 167 , 169 ) are linked to their ability to inhibit enoyl-acyl carrier protein reductase (FabI) activity . The antibacterial activity of an ethanolic extract of F. triandra was also investigated . The antischistosomal action of the methanol extract from F. hirsuta can also be found . Interestingly, the acetonic and methanolic extracts derived from the aerial part of F. pulverulenta exhibited antiviral activity against Herpes simplex virus type 1 (HSV-1) at a dose of 500 μg/mL . F. pulverulenta is known to contain flavonoids, including the 7-bisulfate-3-glucuronide of kaempferol ( 61 ), isorhamnetins ( 79 – 80 ) and quercetin ( 62 ) . As some flavonoids, notably quercetin and, to a lesser extent, catechin and hesperetin, have been reported to possess antiviral capacities against a number of viruses, including HSV-1, the antiviral activity of F. pulverulenta extracts may be linked to its content of flavonoids . These investigations and their results clearly suggest that Frankenia plants might be a valuable source of antimicrobial substances. Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive and irreversible memory loss and other cognitive impairments. At the cellular level, AD is characterized by synaptic and neuronal loss, deposition of plaques made of β-amyloid peptide (Aβ) and the formation of fibrils in the brain made of tau-protein. Several data issued from genetic, neuropathological and biochemical studies have established the central role of the β-amyloid peptide (Aβ), which results from the cleavage of the so-called amyloid precursor protein (APP), a membrane glycoprotein . However, its precise role in AD pathogenesis is still unclear. One hypothesis suggests that oxidative damage in the brain may cause ROS generation in neurons, which in turn could potentiate the Aβ neurotoxicity and metabolism perturbation . Therefore, limiting or inhibiting oxidative stress could be a way to treat AD. As various plants contain various antioxidant natural products, especially those of the Frankenia family (see .1 and ), the neuroprotective properties of plants have been, and are, still being explored . In order to determine whether Frankenia plant species may prevent Aβ-induced neuronal cell toxicity, neuroprotection tests were carried out. The neuroprotective potential of ethyl acetate fractions from F. pulverulenta shoots and roots was evaluated . An Aβ(25–35)-induced cytotoxicity assay using pheochromocytoma-derived (PC12) cells was assessed. Both fractions remarkably prevented the cytotoxic response of Aβ(25–35) at levels around 57% and 80% at 100 and 200 μg/mL, respectively, compared with non-treated cells. At a higher concentration (300 μg/mL), the root fraction entirely counteracted the toxic effect of Aβ(25–35). Using the same process, the neuroprotective capacities of methanolic extracts from F. thymifolia and F. pulverulenta aerial parts were demonstrated in another study . Both species exerted a powerful neuroprotective effect in a dose-dependent manner, and about 80% of the cell viability was restored at 100 μg/mL. Additionally, the ethyl acetate fractions from F. thymifolia shoots and roots demonstrated a strong neuroprotective effect on neuronal PC12 cells and totally counterbalanced the damaging effect of Aβ(25–35) at 25 and 50 μg/mL, respectively . Most phenolics isolated from F. pulverulenta ethyl acetate fractions were shown to exhibit potent neuroprotective activities, particularly procyanidin dimers ( 78 ), which prevented Aβ-induced toxicity at levels close to 100% at 50 μM, while catechin ( 74 ) prevented it only at 70% at the same concentration, and quercetin ( 62 ) did not . The strong capacity of F. thymifolia and F. pulverulenta extracts to inhibit Aβ(25–35) aggregation could be attributed to their significant antioxidant activities and phenolic contents. Various reports have indeed shown that phenolic substances may prevent neurodegenerative disorders, either by directly preventing the formation of Aβ fibril deposits in the brain or by exhibiting protective effects through scavenging ROS . Furthermore, a two-to-one complex between a polyphenol and the full Aβ peptide was observed by ESI-MS . It was also reported that gallic acid ( 1 ), found in F. thymifolia roots, in its glucosylated form and the corresponding gallotannins effectively suppressed Aβ(25–35) aggregation in vitro . Another study revealed that kaempferol-3- O -glucoside ( 56 ) presented a modest inhibitory effect on Aβ(25–35) aggregation, whereas kaempferol itself had a moderate effect. However, the reverse situation was observed with quercetin ( 62 ) and its 3′- O -glucoside ( 72 ), the latter exhibiting a good activity while the former had a modest one . These results are quite surprising due to the structural similarity between these compounds (see ). Interestingly, the very similar hyperoside ( 73 ) significantly diminished Aβ-induced cytotoxicity and apoptosis by restoring Aβ-induced mitochondrial dysfunction . Alternatively, it has been shown that caffeic acid ( 30 ), epigallocatechin ( 75 ) and its 3- O -gallate ( 76 ) exhibit a modest aggregation inhibition, and p -hydroxybenzoic acid ( 11 ) presents a moderate one, while the hydroxy derivatives of benzyl benzoate ( 192 ) exhibit interesting inhibition . However, the latter have so far not been detected in Frankenia species. Another approach to facing AD is to attempt to treat the synaptic and neuronal loss associated with AD. During the progression of AD, different types of neurons deteriorate, but the main loss occurs in forebrain cholinergic neurons, which play an important role in cognition. Therapies have thus been, and are still being, designed to reverse this cholinergic deficit. Cholinergic neurons rely on acetylcholine (ACh) as a neurotransmitter, which is hydrolyzed by acetylcholinesterase (AChE) in the synapse and to a lesser extent by the non-specific butyrylcholinesterase (BuChE) . Furthermore, several studies have suggested that AChE can modulate APP processing in a way that enhances β-amyloid plaque deposition . As a consequence, the inhibition of these enzymes is actively pursued. Various inhibitors have proven beneficial as a curative approach to AD, and a few are commercially available . As some of the earliest inhibitors discovered were alkaloids issued from plants, plant extracts are now often evaluated as cholinesterase inhibitors. In Frankenia species, only a few have so far been evaluated. Interestingly, methanol extracts from F. laevis demonstrated significant AChE and BuChE inhibition (about 80% at 1 mg/mL) . Tyrosinase is a multipurpose copper-containing oxidase that participates in melanin production and enzymatic browning processes that happen in damaged fruits during post-harvest processing . Natural substances are widely utilized in cosmetic formulations as tyrosinase inhibitors to cure skin hyperpigmentation, melasma and post-inflammatory hyperpigmentation . They are also applied in the food industry to prohibit enzymatic browning action in injured vegetables . The inhibition of tyrosinase by F. laevis shoot extracts (50% ethanol) was conducted by performing both the inhibition of l -tyrosine hydroxylation to l -3,4-dihydroxyphenylalanine (L-DOPA) (monophenolase) and that of L-DOPA oxidation to dopaquinone (diphenolase) . A strong inhibition of monophenolase and diphenolase functions was achieved (IC 50 = 730.43 and 123.62 μg/mL, respectively). In agreement with previous studies , the high levels of phenolic compounds, such as chlorogenic acid ( 31 ) and quercetin ( 62 ), in F. laevis extracts are probably responsible for the anti-tyrosinase effect, making this species a prospective source of natural skin-lightening agents and conservatives . Inflammation is induced by either external or internal causes. In the former, inflammation occurs in response to infection caused by microorganisms or to tissue injury. In the latter, cell death, cancer and other dysfunctions initiate a cascade of events leading to inflammation. In turn, various inflammatory mediators are produced, such as cytokines, chemokines, polyunsaturated fatty acids, etc., some acting as pro- and/or anti-inflammatory agents. The enzymes that are responsible for the generation of these inflammatory mediators, such as cyclooxygenase (COX), lipoxygenase (LOX) and hyaluronidase, are the major targets of anti-inflammatory therapies and a number of drugs have been developed For such common anti-inflammatory activity, the use of plants has been known since antiquity and is still applied. Although traditional medicines provide numerous anti-inflammatory extracts or plant parts, this activity is still being explored and remains one of the most sought-after bioactivities from plants . The anti-inflammatory capacity of ethanolic and soxhlet extracts obtained from F. triandra aerial parts was evaluated . The inhibition of LOX and COX2 capacities was assessed on the basis of the enzymatic oxidation of linoleic acid to the corresponding hydroperoxide and prostaglandin measurement, respectively. The extracts displayed a satisfactory ability to prevent LOX (IC 50 = 134.5 ± 12.9 and 117.8 ± 1.8 μg/mL, respectively) and COX2 (54% and 50% inhibition, respectively) actions. Hence, it is thought that these inhibition values are high for a crude extract . The authors have also examined the hyaluronidase activity by measuring the quantity of generated N -acetyl glucosamine (NAGA) . Both soxhlet and ethanolic extracts demonstrated a high degree of inhibition, but the soxhlet extract was three times more effective than the ethanolic extract (IC 50 = 146.3 ± 4.3 and 412.2 ± 8.9 μg/mL, respectively) as compared to the commercial anti-inflammatory, indomethacin (IC 50 = 502.0 ± 7.1 μg/mL), and the control sample, quercetin (IC 50 = 340.0 ± 12.0 μg/mL). Numerous studies have shown a strong correlation between inflammation and oxidative species production. Consequently, plants with antioxidant capabilities frequently have anti-inflammatory characteristics . Carbonic anhydrase II (CA-II) belongs to the carbonic anhydrase family of enzymes, which are zinc metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 ) and a proton (H + ) . In addition to their key roles in transporting CO 2 and maintaining acid–base balance, the 16 human carbonic anhydrases are also involved in several essential physiological processes, and, thus, their dysregulated expression and/or abnormal activity have important pathological consequences. For example, CA-II is mainly involved in the regulation of bicarbonate concentration in the eyes, and is thus linked to glaucoma, but also expressed in malignant brain tumors and renal, gastritis and pancreatic carcinomas. CA-II and other CAs are therefore interesting therapeutic targets for the treatment of related diseases. CA-II inhibitors are, for example, used in the treatment of several illnesses, including glaucoma, idiopathic intracranial hypertension, altitude sickness, congestive heart failure and epilepsy . In order to look for some activity in such health problems, the EO extracted from the aerial parts of F. pulverulenta was screened against the CA-II enzyme. The experiment was done at a micromolar level using acetazolamide as a standard inhibitor (IC 50 = 18.2 ± 1.2 μM). The EO demonstrated a substantial and spectacular CA-II inhibition effect (IC 50 = 101.5 ± 2.35%) and might have application in the management of CA-related disorders . In type 2 diabetic patients postprandial hyperglycemia occurs because the peak insulin release is delayed, and levels are thus insufficient to control the accelerated blood glucose elevation. Such hyperglycemic spikes induce inflammatory reactions, oxidative stress and endothelial dysfunction, which in turn increase the occurrence of cardiovascular diseases. To reduce postprandial hyperglycemia, the most common type 2 diabetes preventive therapy involves decreasing carbohydrate digestibility by blocking two important hydrolyzing enzymes, specifically, α-amylase and α-glucosidase . The methanol and dichloromethane extracts of F. laevis were investigated for their capacity to inhibit α-glucosidase and α-amylase enzymes using a standard in vitro inhibition assay . The extracts showed a marked α-glucosidase inhibition (EC 50 = 1.02 ± 0.01 mg/mL and 0.52 ± 0.04 mg/mL, respectively) compared to the positive control, acarbose (EC 50 = 3.14 ± 0.23 mg/mL). On the other hand, the extracts had no significant effect on α-amylase activity. Abundant in F. laevis extracts, linoleic acid ( 174 ) and its derivatives, as well as loliolide ( 107 ), isololiolide ( 106 ) and dihydroactinidiolide ( 108 ), were found to have a strong inhibitory effect on α-glucosidase. Their higher abundance in the dichloromethane extract may explain the anti-α-glucosidase activity of the F. laevis extracts. In addition, the antioxidant properties of these extracts may also help to decrease the incidence of diabetes complications related to oxidative stress, specifically microvascular and cardiovascular issues . Prior to human usage, substances or chemicals must undergo rigorous safety evaluations. Cytotoxic tests using various human cell lines are often performed to assess the potential toxicity of different substances in vitro . The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) test, a colorimetric approach that measures cell metabolic activity, is one of the most frequently used methods to determine how a substance affects cellular viability . On the other hand, cytotoxicity could be useful to control tumor cell proliferation and thus treat cancers. For the latter purpose, the anticancer and antiproliferative activities of extracts from the aerial parts of F. laevis were investigated against human hepatocarcinoma cells (HepG2) . Sea heath dichloromethane extract showed potential anti-HepG2 activity (EC 50 = 52.1 μg/mL). In contrast, methanol extract did not present significant cytotoxicity. This difference could be ascribed to the high content of certain metabolites and fatty acids in the dichloromethane extract. It has indeed been reported that fatty acids, especially linoleic acid ( 174 ) which is abundant in this extract, have shown chemoprotective effects . Furthermore, the monoterpenes loliolide ( 107 ) and isololiolide ( 106 ), also abundant in this extract, are known for their strong cytotoxic activities on HepG2 cells , as is dihydroactinidiolide ( 108 ) on human lung carcinoma cells (A549) and human breast cancer cells . The phytohormone oxophytodienoic acid ( 204 ) also present in this extract is also known for its cytotoxic activity on human breast cancer cells . A similar study on a large series of halophyte plants, including both F. laevis and F. pulverulenta, has been performed . The viability of four cancer cell types, including the same HepG2 cell line, was evaluated, and the F. laevis extract was found to significantly decrease it (71%), while F. pulverulenta did not. Due to the abundance of the active compounds mentioned above, the F. laevis dichloromethane extract may represent an interesting natural alternative for treating some cancers. Furthermore, the natural products probably responsible for these antitumor activities could become promising candidates for new antitumor drugs. The control of insect proliferation and of the so-induced destruction of agricultural plants is usually achieved with synthetic insecticides. However, their intensive and uncontrolled utilization has led to the development of resistance in insects and to various environmental damages. Although a few insecticides are issued from plants, such as pyrethrenoids, plants may provide potentially safer alternatives to the currently used insect-control agents. In this context, petroleum ether and chloroformic and ethyl acetate extracts obtained from the aerial parts of F. laevis were evaluated for their antifeedant, toxic and insect growth inhibition activities against larvae and adults of the confused flour beetle Tribolium confusum . At a concentration of 1%, the petroleum ether extract demonstrated moderate antifeedant properties. At the same concentration, the tested extracts considerably induced larval mortality (up to 97% inhibition with the ethyl acetate extract), while adult toxicity did not surpass 33%. Furthermore, the F. laevis extracts inhibited feeding, exhibited high toxicity and greatly affected the development of Tribolium confusum larvae when used at a dose of 1%. Therefore, this halophyte plant seems to have great potential for pest control; it would be worth identifying the compound(s) responsible for the interesting insecticidal activity of these extracts even at low concentrations . In this review, we have described a series of Frankenia plant species known for their role in traditional medicine. These plants are indicated for the treatment of a variety of illnesses, including diarrhea, respiratory issues and wounds. The corresponding phytochemical investigations have been collected here and analyzed. These data revealed that these Frankenia species produce a wide range of interesting metabolites. They contain relatively high levels of specific substances, such as phenolics, flavonoids and terpenoids, as well as various fatty acids, as such or as derivatives, and alkanes. Some alkaloids and steroids have also been identified, but only a few lignans and coumarins have so far been observed. Furthermore, the corresponding biological investigations have also been collected when available and the results have been interpreted as much as possible in terms of the chemical content of each extract or part of the plants. Interestingly, these in vitro studies revealed a variety of biological activities, from the classical antioxidant effects and the related anti-inflammatory activity to enzyme inhibitions, neuroprotection, anti-diabetic and anti-tumor activities, as well as insecticidal properties. All these bioactivities are obviously linked to the application of Frankenia plants in traditional medicine. However, the molecular mechanisms of these biological effects correlated to the chemicals recovered from Frankenia species remain unclear. As shown here, the value of plants as sources of bioactive natural substances resides not only in their insecticidal, pharmacological or chemotherapeutic effects but also in their roles in the development of novel drugs . However, the number of higher plant species on earth is estimated to be between 250,000 and 500,000, from which only 15% have been evaluated phytochemically and only 6–7% have been screened for biologic activity . It is thus worth looking at more plants for their chemical profile and their biological activity. Unfortunately, only six species of the Frankenia genus were investigated in detail for their chemical composition and/or pharmacological activities. In summary, research on Frankenia species is still in its infancy and needs to be developed further, in order to discover novel bioactive compounds and better understand the correlation between the identified natural substances and the corresponding biological activity. Additionally, future research should also expand on in vivo studies and clinical trials to learn more about the potential modes of action in human metabolic disorders and illnesses. |
Flow-Based Coronary Artery Bypass Graft Patency Metrics: Uncertainty Quantification Simulations to Guide Development | 7d2174b3-3c83-41a8-9ad7-05e5e6296beb | 11933184 | Thoracic Surgery[mh] | Coronary artery bypass grafting (CABG) is a surgical procedure with a high rate of success. Still, a small percentage of grafts is reported to fail in the period up to 12 months post-surgery [ – ]. Reasons for graft failure are multitudinous, but among them is technical error. Therefore, to detect and correct technical error before chest closure, and thus reduce the risk of subsequent graft failure, intraoperative graft patency assessment is generally considered a valuable addition to the CABG surgery procedure . The gold standard for detecting flaws in a graft and its anastomosis is coronary angiography (CAG) . Angiography provides a precise assessment of vascular constriction; a qualitative assessment of the graft’s capacity to carry flow. The measurement involves relatively costly surgical equipment, requiring a trained operator and added surgical time, and is therefore not widely used for intraoperative patency assessment . As a faster, simpler, and more affordable alternative, ultrasound transit time flow measurement (TTFM), as originally developed by Transonic Systems Inc. , is increasingly used for intraoperative CABG patency assessment [ – ]. By placing a hand-held flow probe around a newly created graft and holding it still for several seconds, the surgeon can accurately measure the time-varying flow rate through the graft. Deviations in the measured flow waveform can alert the surgeon of possible technical imperfections before the patient’s chest is closed. Presently in Japan, all CABG procedures include some form of flow-based patency assessment , while in the United States this percentage lies around 20% , the majority of which concerns off-pump CABG surgery. Also, the European 2018 ESC/EACTS Guidelines on Myocardial Revascularization recommend TTFM to confirm graft patency. The measured waveform is summarized in a number of patency metrics (see Sect. for details), which were established over the years, based on clinical experience. Ideally, a (combination of) metric(s) should identify correctable technical errors in the newly constructed graft, independent of surgical confounders and patient parameters. However, in practice flow-based patency assessment is complicated by the many factors influencing the CABG flow waveform, such as target coronary, graft type (e.g. arterial or venous, single or sequential), competitive flow (resulting from incomplete occlusion of native coronary), autoregulation, quality of coronary microvasculature (resistance, compliance), but also patient parameters such as disease history, heart rate, cardiac index, blood pressure, and BMI . As a result, the reliability of most flow-based patency metrics is high for the identification of severe stenoses, but decreases in case of subcritical stenoses . Accounting for these factors in the metrics displayed on a TTFM flow monitor, or developing a novel metric, would require years of carefully controlled clinical studies. Development of novel metrics is ongoing [ – ], and generally is an empirical process, based on experience and clinical studies. To better guide such developments, a basic understanding is needed of the mechanisms determining the graft flow waveform and the factors influencing it. At the same time, such basic understanding is also expected to facilitate the user’s interpretation of a measured waveform on existing flow monitors. Therefore, as a complement to knowledge gained from clinical studies, we use computational fluid dynamics models to improve our understanding of the graft flow waveform. The aim of our present study was to quantify uncertainty in the most commonly used patency metrics, and thus to assess their performance. To this end, we implemented a validated multi-scale numerical model of the coronary circulation [ – ], customized to include a CABG graft. To select parameters for uncertainty quantification using this model, Morris screening sensitivity analysis was performed using a simpler lumped-parameter model. Results were qualitatively verified against data from a recent clinical study by Takahashi et al. . By providing an indication of the epistemic uncertainty associated with a certain metric, and of the key physiological parameters influencing this uncertainty, our approach should facilitate the design of effective clinical studies, and thus accelerate and focus the ongoing development of improved CABG patency metrics. Sensitivity analysis and uncertainty quantification on a similar model, but applied to the pulmonary circulation, were reported by Colebank et al. , while Ge et al. did a more limited, one-at-a-time (OAT) sensitivity analysis on a similar model of the coronary circulation. To the authors’ knowledge, this study is the first to specifically target flow-based CABG patency assessment.
To study the effects of stenosis in a graft, we use a realistic multi-scale numerical model of the coronary circulation (Figs. and ), embedded in a closed-loop cardiovascular model (Fig. ), both developed by Mynard et al. and validated in-vivo . Because, depending on stenosis severity and other parameter values, simulations with this model take between 5 and 100 h, performing a global sensitivity analysis is not feasible with this model. Instead, generalized polynomial chaos expansion (gPCE) uncertainty quantification was performed, with a limited number of parameters. To guide parameter selection, Morris screening sensitivity analysis was performed with a simpler – and thus, faster – lumped-parameter model (Fig. ). The overall set-up our study is explained schematically in Fig. . Patency Metrics In this study, the four most commonly used TTFM CABG patency metrics were included: 1a \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Mean\, flow\, rate:}&\quad &Q_\text {mean} = \frac{1}{T}\int _0^T Q(t) \textrm{d}t, \end{aligned}$$\end{document} Mean\, flow\, rate: Q mean = 1 T ∫ 0 T Q ( t ) d t , 1b \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Pulsatility\, index:}&\quad &\text {PI} = \frac{Q_\text {max} - Q_\text {min}}{Q_\text {mean}}, \end{aligned}$$\end{document} Pulsatility\, index: PI = Q max - Q min Q mean , 1c \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Diastolic/systolic\, ratio:}&\quad &\text {D/S-ratio} = \frac{|V_\text {dia}|}{|V_\text {sys}|}, \end{aligned}$$\end{document} Diastolic/systolic\, ratio: D/S-ratio = | V dia | | V sys | , 1d \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Diastolic\, filling\, percentage:}&\quad &\text {DF\%} = \frac{|V_\text {dia}|}{|V_\text {tot}|}. \end{aligned}$$\end{document} Diastolic\, filling\, percentage: DF\% = | V dia | | V tot | . Here, Q is flow rate, T is cardiac period, V is delivered blood volume (based on absolute flow rate), and subscripts max, min, tot, sys, and dia indicate maximum, minimum, total, systolic, and diastolic, respectively. DF% and D/S-ratio are directly related, as \documentclass[12pt]{minimal}
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\begin{document}$$\text {DF\%} = 100 \times \text {D/S-ratio}/(1 + \text {D/S-ratio})$$\end{document} DF\% = 100 × D/S-ratio / ( 1 + D/S-ratio ) . Therefore, DF% was not included in the figures presented here. Additionally, diastolic resistance index (DRI) was included: 2 \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Diastolic\, resistance\, index:}&\quad &\text {DRI} = \frac{\overline{p}_\text {dia}/|\overline{Q}_\text {dia}|}{\overline{p}_\text {sys}/|\overline{Q}_\text {sys}|}, \end{aligned}$$\end{document} Diastolic\, resistance\, index: DRI = p ¯ dia / | Q ¯ dia | p ¯ sys / | Q ¯ sys | , where p is (central) pressure. This novel metric, developed by Transonic Systems Inc. (Ithaca, NY), was recently evaluated in a first clinical study by Takahashi et al. . It incorporates both flow rate and pressure, and is related to D/S-ratio as: \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \text {DRI} = \frac{\overline{p}_\text {dia}}{\overline{p}_\text {sys}}\frac{T_\text {dia}}{T_\text {sys}}\frac{1}{\text {D/S-ratio}}. \end{aligned}$$\end{document} DRI = p ¯ dia p ¯ sys T dia T sys 1 D/S-ratio . It should be noted that, because DF% and D/S-ratio are computed using the absolute value of the flow rate, the same is done for DRI. As explained in more detail in Appendix A, these patency metrics quantify different aspects of how the flow waveform changes in the presence of stenosis: \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean quantifies the overall decrease in flow rate, while D/S-ratio, DF% and DRI capture the decrease in diastolic dominance. Finally, PI quantifies the combined effect of increasing capacitive flow proximal to the stenosis, and decreasing mean flow rate. 1D/0D Multiscale Model The high-accuracy multiscale model that was used for gPCE uncertainty quantification consists of a model of the coronary circulation (Fig. ), embedded in a closed-loop cardiovascular model (Fig. ). Both the coronary tree model and the closed-loop cardiovascular model are made up of 1D segments representing the coronary arteries, and main systemic and pulmonary arteries and veins. The coronary artery segments are terminated with 0D lumped parameter intramyocardial perfusion models, and also the pulmonary and systemic vascular beds, and the heart were represented by 0D lumped parameter models. In the lumped-parameter intramyocardial perfusion model (Fig. ), the resistances in the sub-epicardial, mid-wall, and sub-endocardial layers depend on vascular volume. Incorporating volume-dependence of the compliances in these layers, as outlined by Algranati et al. , did not significantly alter the results, while it did increase the number of uncertain parameters in the model. Therefore, the compliances were assumed to be fixed in our study. Following Suga et al. , maximum elastance in the heart model was set to be independent of cardiac period, while for diastole duration as a function of cardiac period the relation determined by Salvi et al. was implemented. To this model a left internal thoracic artery (LITA) graft was added (indicated in grey in Fig. ). Length and diameter of the LITA graft were based on data from the anatomically detailed arterial network (ADAN) model , resulting in a 20 cm long graft, with a radius tapering linearly from 0.142 cm to 0.0837 cm. This graft was anastomosed onto the left anterior descending (LAD) coronary artery, 0.3 cm distal to its origin from the left main (LM) coronary artery. Proximal to the anastomosis, the LAD was fully occluded. The origin of the LITA was placed on the systemic artery segment, 8 cm from the coronary ostia, so that the delay and distortion of its pressure waveform matched the ADAN data. To the LITA graft, a stenosis was added at 1 cm from its distal end (model structure did not allow for stenosis to coincide with the anastomosis). Graft flow rate and pressure were monitored at two sites: 1 cm and 5 cm proximal of this stenosis. The former position corresponds with the flow probe position that is recommended by flow probe manufacturers, whereas the latter seems to better agree with clinical practice (based on informal discussions). Stenosis was modeled following Young and Tsai : 3 \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \Delta p_\text {s} = \frac{4 K_\text {v} \mu }{\pi D_0^3} Q_\text {s} + \frac{\rho K_\text {t}}{2 A_0^2}\left( \frac{A_0}{A_\text {s}} - 1\right) ^2 Q_\text {s}^2 + \frac{\rho K_\text {u} l_\text {s}}{A_0} \frac{\textrm{d}Q_\text {s}}{\textrm{d}t}, \end{aligned}$$\end{document} Δ p s = 4 K v μ π D 0 3 Q s + ρ K t 2 A 0 2 A 0 A s - 1 2 Q s 2 + ρ K u l s A 0 d Q s d t , where \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} K_\text {v} = 32 \frac{0.83 l_\text {s} + 1.64 D_\text {s}}{D_0}\left( \frac{A_0}{A_\text {s}}\right) ^2, \end{aligned}$$\end{document} K v = 32 0.83 l s + 1.64 D s D 0 A 0 A s 2 , with \documentclass[12pt]{minimal}
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\begin{document}$$D_\text {s}$$\end{document} D s the diameter of the vena contracta. To study the effects of stenosis on graft flow, preliminary simulations were carried out, with 0, 50, 75, and 90% graft diameter reduction (see Appendix A). Even though arterial pressure is known to be influenced by manipulation of the heart during off-pump CABG surgery [ – ], insufficient quantitative data were available to reliably include these changes in our simulations. Finally, to limit model complexity, chamber interaction, as implemented by Mynard et al. , and autoregulation, as implemented by Ge et al. , were not included in most simulations (however, see Sect. ). Full model details and parameter values can be found in Appendix B. The model was implemented in Python (https://www.python.org), using a finite difference flux-limited MacCormack scheme to discretize the 1D flow equations. After checking for grid independence, a grid size of 0.1 cm was selected, with a time step of \documentclass[12pt]{minimal}
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\begin{document}$$2\times 10^{-5}$$\end{document} 2 × 10 - 5 s for simulations without stenosis, and progressively smaller for increasing stenosis severity. To simulate 10 cardiac periods, this resulted in simulation duration ranging from around 5 to 100 h (Dell Optiplex-7070 with Intel Core i7 processor). 0D Lumped Parameter Model Because, based on earlier, exploratory simulations, most of the changes in the flow waveform as a result of stenosis were observed to originate from the lumped parameter stenosis model and the coronary microvasculature, a simplified model was created by connecting a single instance of the lumped parameter model of the intramyocardial vessels (Fig. ) to the closed-loop lumped parameter cardiovascular circulation model of Avanzolini et al. (Fig. ). In a lumped parameter model, the geometric and mechanical properties of blood vessels are represented by lumped resistance, compliance, and (in larger vessels) inductance components. This way, a model comparable to that of Mantero et al. was obtained, with which all essential features of the coronary flow waveform could be reproduced (see e.g. Fig. in appendix C), including changes in myocardial perfusion as a function of diastolic time fraction and perfusion pressure . This model, with 39 parameters (including stenosis percentage), was implemented in OpenModelica (https://openmodelica.org/), and then exported as a functional mock-up unit (FMU), to be called from Python. With around 0.5 s to simulate 10 cardiac periods, this model allowed for global sensitivity analysis using Morris screening . Sensitivity Analysis For the Morris screening sensitivity analysis, the Python package SALib was used, in which the efficient sampling strategy proposed by Ruano et al. is implemented. For two parameters – stenosis percentage and subendocardial-subepicardial volume ratio – a uniform distribution was assumed. For all other parameters, a normal distribution was assumed, truncated to mean \documentclass[12pt]{minimal}
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\begin{document}$$\pm 2\times $$\end{document} ± 2 × standard deviation, with mean and standard deviation values guided by literature, such as the work of Charlton et al. . If no standard deviation could be found in literature, a value of 25% of the mean of that parameter was assumed. For Morris screening, the parameter value distributions were sampled at \documentclass[12pt]{minimal}
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\begin{document}$$r = 50$$\end{document} r = 50 trajectories were computed, resulting in a total of 2000 simulations ( r was determined by increasing it in steps of 10, until the resulting 3 most influential parameters stopped changing). The patency metrics presented in the previous subsection, \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean , PI, D/S-ratio, and DRI, were used as response variables. The influence of a parameter, quantified by the sensitivity measures \documentclass[12pt]{minimal}
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\begin{document}$$\sqrt{\mu ^{*2} + \sigma ^2}$$\end{document} μ ∗ 2 + σ 2 . To ensure that the variables with uniform distribution did not have more influence just because the uniform distribution has higher entropy than the normal distribution, the sensitivity analysis was repeated with uniform distribution for all variables (with values in same range as in the corresponding truncated normal distribution). The influence of using all uniform distributions was found to be limited, and did not change the ranking of the most influential parameters. Uncertainty Quantification Apart from stenosis severity, the six most influential parameters resulting from Morris screening were selected for uncertainty quantification of the multiscale model. In addition to these parameters, the cross-sectional area and pulse wave speed of the systemic artery and graft were included. These were not part of the simplified lumped parameter model, but are known to influence the graft flow waveform and systemic pressure, respectively . To limit the number of required simulations, only a model with LITA-LAD graft was analyzed, as this is the most commonly applied graft. For the resulting parameters, a generalized polynomial chaos expansion (gPCE) was computed using Dakota . Normal distributions were assumed for all parameter values but percentage stenosis, for which a uniform distribution was assumed. To prevent unrealistic values, normally distributed parameters were constrained to [mean value \documentclass[12pt]{minimal}
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\begin{document}$$\pm 1.96\times $$\end{document} ± 1.96 × standard deviation] (i.e. 95% of probability density function). Using a maximum polynomial order of 2 (based on exploratory, coarse grid UQ with maximum polynomial order 4), and least-squares regression with a collocation factor of 2, this resulted in 110 simulations. As a part of the analysis, Dakota tested the quality of the gPCE using k -fold cross-validation, with 10 folds. To quantify the sensitivity of the flow-based metrics to each parameter, Sobol indices were computed directly from the resulting best-fitting gPCE . Because parameters were assumed to vary independently, not all resulting flow and pressure waveforms were physiologically realistic. Using healthy subject values as a reference, as done by Charlton et al. , seemed too restrictive, as such values can hardly be expected to be representative of patients undergoing CABG surgery. Also, the availability of detailed physiological data of CABG surgery patients is insufficient to filter out unrealistic results. Therefore, all Morris screening results were retained, while from the gPCE results only the obviously erroneous results, with maximum pressure during diastole, were removed. This resulted in the removal of 6 out of 110 simulations (5%). Autoregulation In a normally functioning heart, autoregulation dilates or contracts the vessels in the myocardial wall, depending on flow demand . As a result, blood flow to the myocardium, and subendocardial-to-subepicardial flow ratio, are not significantly affected in the presence of a coronary stenosis up to about 68% (90% area reduction, see e.g. Buss et al. ). Therefore, we expect the patency metrics to be affected by autoregulation. As mentioned earlier, autoregulation was not included in the sensitivity analysis and uncertainty quantification simulations, to restrict computational cost. To still get an impression of the effects of autoregulation, four simulations with a stenosed LITA graft were run separately, in which intramyocardial resistance was tuned iteratively to maintain intramyocardial flow rate and endo-epi ratio at the un-stenosed level. These simulations were run with 20, 40, 50, and 60% stenosis, respectively. Verification with Clinical Data Dr. Takahashi of Nippon Medical School, Tokyo, Japan, kindly shared the raw data from his recent clinical study , which consisted of graft flow rate, radial artery pressure, and ECG waveforms, measured intraoperatively in 123 grafts, in 41 patients undergoing off-pump CABG surgery. From these data, \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean , PI, D/S-ratio, DF%, and DRI were determined. Additionally, in all grafts percentage stenosis was measured postoperatively, using coronary computed tomography (CCT, for correspondence between measured stenosis grades, patency classes, and percentage diameter reduction, see Appendix, Table ). The ECG signal was used for systole and diastole detection. Central systolic and diastolic pressure were estimated from the radial artery pressure waveform using the N -point moving average method proposed by Xiao et al. . For measurement protocol, see Appendix, Sect. D.1. For each patency metric, the median and interquartile range (IQR) for three different patency classes were compared between the clinical and simulated (1D/0D model) results. Because of the lack of detailed physiological data in CABG surgery patients, the 1D/0D model results were not expected to show quantitative agreement with the clinical results. Therefore, although the nonparametric Wilcoxon’s rank sum test was used to test for the significance of any observed differences, comparison was mostly qualitative, focusing more on trends than on exact agreement.
In this study, the four most commonly used TTFM CABG patency metrics were included: 1a \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Diastolic/systolic\, ratio:}&\quad &\text {D/S-ratio} = \frac{|V_\text {dia}|}{|V_\text {sys}|}, \end{aligned}$$\end{document} Diastolic/systolic\, ratio: D/S-ratio = | V dia | | V sys | , 1d \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Diastolic\, filling\, percentage:}&\quad &\text {DF\%} = \frac{|V_\text {dia}|}{|V_\text {tot}|}. \end{aligned}$$\end{document} Diastolic\, filling\, percentage: DF\% = | V dia | | V tot | . Here, Q is flow rate, T is cardiac period, V is delivered blood volume (based on absolute flow rate), and subscripts max, min, tot, sys, and dia indicate maximum, minimum, total, systolic, and diastolic, respectively. DF% and D/S-ratio are directly related, as \documentclass[12pt]{minimal}
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\begin{document}$$\text {DF\%} = 100 \times \text {D/S-ratio}/(1 + \text {D/S-ratio})$$\end{document} DF\% = 100 × D/S-ratio / ( 1 + D/S-ratio ) . Therefore, DF% was not included in the figures presented here. Additionally, diastolic resistance index (DRI) was included: 2 \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned}&\text {Diastolic\, resistance\, index:}&\quad &\text {DRI} = \frac{\overline{p}_\text {dia}/|\overline{Q}_\text {dia}|}{\overline{p}_\text {sys}/|\overline{Q}_\text {sys}|}, \end{aligned}$$\end{document} Diastolic\, resistance\, index: DRI = p ¯ dia / | Q ¯ dia | p ¯ sys / | Q ¯ sys | , where p is (central) pressure. This novel metric, developed by Transonic Systems Inc. (Ithaca, NY), was recently evaluated in a first clinical study by Takahashi et al. . It incorporates both flow rate and pressure, and is related to D/S-ratio as: \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \text {DRI} = \frac{\overline{p}_\text {dia}}{\overline{p}_\text {sys}}\frac{T_\text {dia}}{T_\text {sys}}\frac{1}{\text {D/S-ratio}}. \end{aligned}$$\end{document} DRI = p ¯ dia p ¯ sys T dia T sys 1 D/S-ratio . It should be noted that, because DF% and D/S-ratio are computed using the absolute value of the flow rate, the same is done for DRI. As explained in more detail in Appendix A, these patency metrics quantify different aspects of how the flow waveform changes in the presence of stenosis: \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean quantifies the overall decrease in flow rate, while D/S-ratio, DF% and DRI capture the decrease in diastolic dominance. Finally, PI quantifies the combined effect of increasing capacitive flow proximal to the stenosis, and decreasing mean flow rate.
The high-accuracy multiscale model that was used for gPCE uncertainty quantification consists of a model of the coronary circulation (Fig. ), embedded in a closed-loop cardiovascular model (Fig. ). Both the coronary tree model and the closed-loop cardiovascular model are made up of 1D segments representing the coronary arteries, and main systemic and pulmonary arteries and veins. The coronary artery segments are terminated with 0D lumped parameter intramyocardial perfusion models, and also the pulmonary and systemic vascular beds, and the heart were represented by 0D lumped parameter models. In the lumped-parameter intramyocardial perfusion model (Fig. ), the resistances in the sub-epicardial, mid-wall, and sub-endocardial layers depend on vascular volume. Incorporating volume-dependence of the compliances in these layers, as outlined by Algranati et al. , did not significantly alter the results, while it did increase the number of uncertain parameters in the model. Therefore, the compliances were assumed to be fixed in our study. Following Suga et al. , maximum elastance in the heart model was set to be independent of cardiac period, while for diastole duration as a function of cardiac period the relation determined by Salvi et al. was implemented. To this model a left internal thoracic artery (LITA) graft was added (indicated in grey in Fig. ). Length and diameter of the LITA graft were based on data from the anatomically detailed arterial network (ADAN) model , resulting in a 20 cm long graft, with a radius tapering linearly from 0.142 cm to 0.0837 cm. This graft was anastomosed onto the left anterior descending (LAD) coronary artery, 0.3 cm distal to its origin from the left main (LM) coronary artery. Proximal to the anastomosis, the LAD was fully occluded. The origin of the LITA was placed on the systemic artery segment, 8 cm from the coronary ostia, so that the delay and distortion of its pressure waveform matched the ADAN data. To the LITA graft, a stenosis was added at 1 cm from its distal end (model structure did not allow for stenosis to coincide with the anastomosis). Graft flow rate and pressure were monitored at two sites: 1 cm and 5 cm proximal of this stenosis. The former position corresponds with the flow probe position that is recommended by flow probe manufacturers, whereas the latter seems to better agree with clinical practice (based on informal discussions). Stenosis was modeled following Young and Tsai : 3 \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} \Delta p_\text {s} = \frac{4 K_\text {v} \mu }{\pi D_0^3} Q_\text {s} + \frac{\rho K_\text {t}}{2 A_0^2}\left( \frac{A_0}{A_\text {s}} - 1\right) ^2 Q_\text {s}^2 + \frac{\rho K_\text {u} l_\text {s}}{A_0} \frac{\textrm{d}Q_\text {s}}{\textrm{d}t}, \end{aligned}$$\end{document} Δ p s = 4 K v μ π D 0 3 Q s + ρ K t 2 A 0 2 A 0 A s - 1 2 Q s 2 + ρ K u l s A 0 d Q s d t , where \documentclass[12pt]{minimal}
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\begin{document}$$\Delta p_\text {s}$$\end{document} Δ p s is the pressure drop over the stenosis, \documentclass[12pt]{minimal}
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\begin{document}$$l_\text {s}$$\end{document} l s are the cross-sectional area and effective length of the stenosis, respectively, and \documentclass[12pt]{minimal}
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\begin{document}$$K_\text {u}$$\end{document} K u are empirical constants related to viscous, turbulent, and inertial effects, respectively. The values of \documentclass[12pt]{minimal}
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\begin{document}$$K_\text {v}$$\end{document} K v depends on stenosis geometry as \documentclass[12pt]{minimal}
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\begin{document}$$\begin{aligned} K_\text {v} = 32 \frac{0.83 l_\text {s} + 1.64 D_\text {s}}{D_0}\left( \frac{A_0}{A_\text {s}}\right) ^2, \end{aligned}$$\end{document} K v = 32 0.83 l s + 1.64 D s D 0 A 0 A s 2 , with \documentclass[12pt]{minimal}
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\begin{document}$$D_\text {s}$$\end{document} D s the diameter of the vena contracta. To study the effects of stenosis on graft flow, preliminary simulations were carried out, with 0, 50, 75, and 90% graft diameter reduction (see Appendix A). Even though arterial pressure is known to be influenced by manipulation of the heart during off-pump CABG surgery [ – ], insufficient quantitative data were available to reliably include these changes in our simulations. Finally, to limit model complexity, chamber interaction, as implemented by Mynard et al. , and autoregulation, as implemented by Ge et al. , were not included in most simulations (however, see Sect. ). Full model details and parameter values can be found in Appendix B. The model was implemented in Python (https://www.python.org), using a finite difference flux-limited MacCormack scheme to discretize the 1D flow equations. After checking for grid independence, a grid size of 0.1 cm was selected, with a time step of \documentclass[12pt]{minimal}
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\begin{document}$$2\times 10^{-5}$$\end{document} 2 × 10 - 5 s for simulations without stenosis, and progressively smaller for increasing stenosis severity. To simulate 10 cardiac periods, this resulted in simulation duration ranging from around 5 to 100 h (Dell Optiplex-7070 with Intel Core i7 processor).
Because, based on earlier, exploratory simulations, most of the changes in the flow waveform as a result of stenosis were observed to originate from the lumped parameter stenosis model and the coronary microvasculature, a simplified model was created by connecting a single instance of the lumped parameter model of the intramyocardial vessels (Fig. ) to the closed-loop lumped parameter cardiovascular circulation model of Avanzolini et al. (Fig. ). In a lumped parameter model, the geometric and mechanical properties of blood vessels are represented by lumped resistance, compliance, and (in larger vessels) inductance components. This way, a model comparable to that of Mantero et al. was obtained, with which all essential features of the coronary flow waveform could be reproduced (see e.g. Fig. in appendix C), including changes in myocardial perfusion as a function of diastolic time fraction and perfusion pressure . This model, with 39 parameters (including stenosis percentage), was implemented in OpenModelica (https://openmodelica.org/), and then exported as a functional mock-up unit (FMU), to be called from Python. With around 0.5 s to simulate 10 cardiac periods, this model allowed for global sensitivity analysis using Morris screening .
For the Morris screening sensitivity analysis, the Python package SALib was used, in which the efficient sampling strategy proposed by Ruano et al. is implemented. For two parameters – stenosis percentage and subendocardial-subepicardial volume ratio – a uniform distribution was assumed. For all other parameters, a normal distribution was assumed, truncated to mean \documentclass[12pt]{minimal}
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\begin{document}$$\pm 2\times $$\end{document} ± 2 × standard deviation, with mean and standard deviation values guided by literature, such as the work of Charlton et al. . If no standard deviation could be found in literature, a value of 25% of the mean of that parameter was assumed. For Morris screening, the parameter value distributions were sampled at \documentclass[12pt]{minimal}
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\begin{document}$$r = 50$$\end{document} r = 50 trajectories were computed, resulting in a total of 2000 simulations ( r was determined by increasing it in steps of 10, until the resulting 3 most influential parameters stopped changing). The patency metrics presented in the previous subsection, \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean , PI, D/S-ratio, and DRI, were used as response variables. The influence of a parameter, quantified by the sensitivity measures \documentclass[12pt]{minimal}
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\begin{document}$$\mu ^*$$\end{document} μ ∗ (mean of absolute elementary effects) and \documentclass[12pt]{minimal}
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\begin{document}$$\sqrt{\mu ^{*2} + \sigma ^2}$$\end{document} μ ∗ 2 + σ 2 . To ensure that the variables with uniform distribution did not have more influence just because the uniform distribution has higher entropy than the normal distribution, the sensitivity analysis was repeated with uniform distribution for all variables (with values in same range as in the corresponding truncated normal distribution). The influence of using all uniform distributions was found to be limited, and did not change the ranking of the most influential parameters.
Apart from stenosis severity, the six most influential parameters resulting from Morris screening were selected for uncertainty quantification of the multiscale model. In addition to these parameters, the cross-sectional area and pulse wave speed of the systemic artery and graft were included. These were not part of the simplified lumped parameter model, but are known to influence the graft flow waveform and systemic pressure, respectively . To limit the number of required simulations, only a model with LITA-LAD graft was analyzed, as this is the most commonly applied graft. For the resulting parameters, a generalized polynomial chaos expansion (gPCE) was computed using Dakota . Normal distributions were assumed for all parameter values but percentage stenosis, for which a uniform distribution was assumed. To prevent unrealistic values, normally distributed parameters were constrained to [mean value \documentclass[12pt]{minimal}
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\begin{document}$$\pm 1.96\times $$\end{document} ± 1.96 × standard deviation] (i.e. 95% of probability density function). Using a maximum polynomial order of 2 (based on exploratory, coarse grid UQ with maximum polynomial order 4), and least-squares regression with a collocation factor of 2, this resulted in 110 simulations. As a part of the analysis, Dakota tested the quality of the gPCE using k -fold cross-validation, with 10 folds. To quantify the sensitivity of the flow-based metrics to each parameter, Sobol indices were computed directly from the resulting best-fitting gPCE . Because parameters were assumed to vary independently, not all resulting flow and pressure waveforms were physiologically realistic. Using healthy subject values as a reference, as done by Charlton et al. , seemed too restrictive, as such values can hardly be expected to be representative of patients undergoing CABG surgery. Also, the availability of detailed physiological data of CABG surgery patients is insufficient to filter out unrealistic results. Therefore, all Morris screening results were retained, while from the gPCE results only the obviously erroneous results, with maximum pressure during diastole, were removed. This resulted in the removal of 6 out of 110 simulations (5%).
In a normally functioning heart, autoregulation dilates or contracts the vessels in the myocardial wall, depending on flow demand . As a result, blood flow to the myocardium, and subendocardial-to-subepicardial flow ratio, are not significantly affected in the presence of a coronary stenosis up to about 68% (90% area reduction, see e.g. Buss et al. ). Therefore, we expect the patency metrics to be affected by autoregulation. As mentioned earlier, autoregulation was not included in the sensitivity analysis and uncertainty quantification simulations, to restrict computational cost. To still get an impression of the effects of autoregulation, four simulations with a stenosed LITA graft were run separately, in which intramyocardial resistance was tuned iteratively to maintain intramyocardial flow rate and endo-epi ratio at the un-stenosed level. These simulations were run with 20, 40, 50, and 60% stenosis, respectively.
Dr. Takahashi of Nippon Medical School, Tokyo, Japan, kindly shared the raw data from his recent clinical study , which consisted of graft flow rate, radial artery pressure, and ECG waveforms, measured intraoperatively in 123 grafts, in 41 patients undergoing off-pump CABG surgery. From these data, \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean , PI, D/S-ratio, DF%, and DRI were determined. Additionally, in all grafts percentage stenosis was measured postoperatively, using coronary computed tomography (CCT, for correspondence between measured stenosis grades, patency classes, and percentage diameter reduction, see Appendix, Table ). The ECG signal was used for systole and diastole detection. Central systolic and diastolic pressure were estimated from the radial artery pressure waveform using the N -point moving average method proposed by Xiao et al. . For measurement protocol, see Appendix, Sect. D.1. For each patency metric, the median and interquartile range (IQR) for three different patency classes were compared between the clinical and simulated (1D/0D model) results. Because of the lack of detailed physiological data in CABG surgery patients, the 1D/0D model results were not expected to show quantitative agreement with the clinical results. Therefore, although the nonparametric Wilcoxon’s rank sum test was used to test for the significance of any observed differences, comparison was mostly qualitative, focusing more on trends than on exact agreement.
Morris Screening and Uncertainty Quantification Based on the results of the Morris screening analysis (see Appendix E), systemic vascular bed reference resistance \documentclass[12pt]{minimal}
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\begin{document}$$V_{0,1}$$\end{document} V 0 , 1 , and cardiac period T were selected for uncertainty quantification of the multi-scale model. Two additional parameters, maximum and minimum left ventricular elastance, were not selected for uncertainty quantification, as they were observed to mainly influence systemic pressure. Because systemic pressure is generally more influenced by cross-sectional area \documentclass[12pt]{minimal}
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\begin{document}$$c_{0,\text {sa}}$$\end{document} c 0 , sa of the systemic arteries (not included in the lumped-parameter model), these parameters were selected instead. Finally, also the cross-sectional area and pulse wave velocity of the LITA graft, \documentclass[12pt]{minimal}
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\begin{document}$$c_\text {0,LITA}$$\end{document} c 0,LITA , respectively, were included. Scatter plots of the resulting patency metrics’ values vs. percentage stenosis, as monitored at 5 cm and 1 cm from the stenosis, are shown in Fig. . The observation by Jelenc et al. , that diastolic dominance is consistently lower when measured on the proximal side of the graft, is confirmed by our simulation results: D/S-ratio is on average 1.4% lower at 5 cm than at 1 cm from the stenosis (Wilcoxon signed rank test: significant difference, \documentclass[12pt]{minimal}
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\begin{document}$$p < 0.001$$\end{document} p < 0.001 ). Significant differences were similarly seen for DF% (0.4% lower), DRI (1.6% higher), and PI (2.7% lower). Based on 10-fold cross-validation, linear expansion (i.e. order 1) was found to give the smallest error for all metrics. That is, even though the number of simulations allowed for second-order expansion, linear expansion was used for the gPCE. The Sobol indices computed from the gPCE, monitored at 5 cm from the stenosis, are listed in Table , with the most influential parameters printed in bold (see Appendix, Table for results at 1 cm). Because linear expansion was used in all cases, the main and total Sobol indices are equal, so that only one value is given in the table. Autoregulation The results of the four autoregulation simulations, together with their reference results (i.e. same parameters, without autoregulation) are superimposed onto the raw uncertainty quantification results in Fig. . As intended, mean flow rate remains practically unchanged, while PI decreases slightly with stenosis severity, and both D/S-ratio and DRI are not noticeably affected by autoregulation. Verification Against Clinical Study Results Box plots of hemodynamic parameters (systolic, diastolic, and mean arterial pressure in ascending aorta, with corresponding pulse pressure), and cardiac parameters (cardiac period, diastolic time fraction) are shown in Fig. . To quantify the variability of these parameters, the value of the median absolute deviation (MAD) relative to the median is given under each box. The corresponding flow-based metrics’ values are shown in Fig. . The clinical study values of SAP, DAP and MAP are significantly lower than those in the simulation results (Wilcoxon’s rank-sum test), while PP and T are significantly higher. The variability of all hemodynamic and cardiac parameters is slightly higher in the clinical study results, or even much higher, as in the case of diastolic time fraction. The 1D/0D simulation results for \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean , D/S-ratio, and DRI show the same trend as the clinical results, but for PI, the trend is in opposite direction, and only the outliers suggest an increase with increasing stenosis severity. Furthermore, the clinical study values of \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean are much lower than those in the simulations. All metrics’ values show a larger variability in the clinical study results.
Based on the results of the Morris screening analysis (see Appendix E), systemic vascular bed reference resistance \documentclass[12pt]{minimal}
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\begin{document}$$V_{0,1}$$\end{document} V 0 , 1 , and cardiac period T were selected for uncertainty quantification of the multi-scale model. Two additional parameters, maximum and minimum left ventricular elastance, were not selected for uncertainty quantification, as they were observed to mainly influence systemic pressure. Because systemic pressure is generally more influenced by cross-sectional area \documentclass[12pt]{minimal}
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\begin{document}$$c_\text {0,LITA}$$\end{document} c 0,LITA , respectively, were included. Scatter plots of the resulting patency metrics’ values vs. percentage stenosis, as monitored at 5 cm and 1 cm from the stenosis, are shown in Fig. . The observation by Jelenc et al. , that diastolic dominance is consistently lower when measured on the proximal side of the graft, is confirmed by our simulation results: D/S-ratio is on average 1.4% lower at 5 cm than at 1 cm from the stenosis (Wilcoxon signed rank test: significant difference, \documentclass[12pt]{minimal}
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\begin{document}$$p < 0.001$$\end{document} p < 0.001 ). Significant differences were similarly seen for DF% (0.4% lower), DRI (1.6% higher), and PI (2.7% lower). Based on 10-fold cross-validation, linear expansion (i.e. order 1) was found to give the smallest error for all metrics. That is, even though the number of simulations allowed for second-order expansion, linear expansion was used for the gPCE. The Sobol indices computed from the gPCE, monitored at 5 cm from the stenosis, are listed in Table , with the most influential parameters printed in bold (see Appendix, Table for results at 1 cm). Because linear expansion was used in all cases, the main and total Sobol indices are equal, so that only one value is given in the table.
The results of the four autoregulation simulations, together with their reference results (i.e. same parameters, without autoregulation) are superimposed onto the raw uncertainty quantification results in Fig. . As intended, mean flow rate remains practically unchanged, while PI decreases slightly with stenosis severity, and both D/S-ratio and DRI are not noticeably affected by autoregulation.
Box plots of hemodynamic parameters (systolic, diastolic, and mean arterial pressure in ascending aorta, with corresponding pulse pressure), and cardiac parameters (cardiac period, diastolic time fraction) are shown in Fig. . To quantify the variability of these parameters, the value of the median absolute deviation (MAD) relative to the median is given under each box. The corresponding flow-based metrics’ values are shown in Fig. . The clinical study values of SAP, DAP and MAP are significantly lower than those in the simulation results (Wilcoxon’s rank-sum test), while PP and T are significantly higher. The variability of all hemodynamic and cardiac parameters is slightly higher in the clinical study results, or even much higher, as in the case of diastolic time fraction. The 1D/0D simulation results for \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean , D/S-ratio, and DRI show the same trend as the clinical results, but for PI, the trend is in opposite direction, and only the outliers suggest an increase with increasing stenosis severity. Furthermore, the clinical study values of \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean are much lower than those in the simulations. All metrics’ values show a larger variability in the clinical study results.
Uncertainty quantification of the multiscale model, using generalized polynomial chaos expansion (gPCE), showed that for D/S-ratio and DRI, percentage stenosis is the most influential parameter, while for \documentclass[12pt]{minimal}
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\begin{document}$$A_\text {0,lita}$$\end{document} A 0,lita and T are most important, respectively. Considering the inverse square relation between graft resistance and \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean is understandable. The low sensitivity of PI to pct \documentclass[12pt]{minimal}
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\begin{document}$$_\text {s}$$\end{document} s found here might be explained by the fact that stenosis severity was limited to a maximum of 75% in our simulations, while PI only seems to start consistently increasing at pct \documentclass[12pt]{minimal}
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\begin{document}$$_\text {s}$$\end{document} s even higher than that (e.g. Fig. ). For a metric to reliably detect stenosis, a high Sobol index for pct \documentclass[12pt]{minimal}
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\begin{document}$$_\text {s}$$\end{document} s is needed, preferably combined with low Sobol indices for other parameters of influence. During CABG surgery, \documentclass[12pt]{minimal}
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\begin{document}$$A_\text {0,lita}$$\end{document} A 0,lita and T are routinely monitored so that it should be possible to account for their influence. In the presence of autoregulation, intramyocardial arterioles will dilate to compensate for a reduction in perfusion pressure. As a result, stenoses with less than 90% area reduction (i.e. 68% diameter reduction) hardly affect mean flow rate and endo/epi ratio . Therefore, in clinical practice, \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean is generally observed to be useful for the detection of severe stenosis only. Our preliminary autoregulation simulations showed that also the utility of PI decreases. The diastolic dominance-based metrics D/S-ratio, DF% and DRI are hardly affected at all, suggesting that the influence of a shift in endo-epi ratio is only minor, and that the reduction in diastolic dominance with increasing stenosis is mainly caused by the flow rate dependence of stenosis pressure drop ( ). This is further illustrated in appendix A. The variability of most hemodynamic and cardiac parameter values in the uncertainty quantification results is slightly smaller, but reasonably close to that in the clinical study results used for comparison. Only for diastolic time fraction the variability is much smaller in the simulations than in the clinical study results. Systolic, diastolic and mean arterial pressure in the clinical study results are significantly lower than those in the simulation results, while pulse pressure and cardiac period are significantly higher. This may in part be caused by inaccuracies in estimating central pressure from peripheral pressure in the clinical study, but also the changes in hemodynamics during CABG surgery, due to anesthetics and manipulation of the heart, are expected to be of influence [ – ]. The much lower values of \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean in the clinical study results are expected to be related to these lower pressure values, and perhaps the relatively small patient size in the (Japanese) clinical study. The combination of lower SAP, DAP, and MAP, with higher PP, may also explain the higher PI values in the clinical data. Overall, the metrics’ values show reasonable correspondence between clinical and simulation results, especially considering the fact that healthy subject parameter values were used in the simulation models. Plausible explanations for the larger variability and weaker correlation with stenosis severity of the clinical study results may be the variation in graft types and targets (single and sequential, arterial and venous grafts, to different target coronaries, possibly not all fully occluded). Additionally, measurement inaccuracy may have played a role, for example, in estimating central pressure from peripheral measurements, but also because of the sometimes large time lapse between surgery and postoperative CCT scan (median time interval from CABG surgery to CCT examination was 33 days; IQR 19–89 days; range 6–160 days). More patient data are needed to reliably adapt the 1D/0D model to represent CABG surgery patients, and to estimate realistic ranges and distributions for the different model parameters.
Among the most commonly used patency metrics, D/S-ratio (and hence, DF%) displayed the highest sensitivity to stenosis severity, and the lowest sensitivity to the remaining physiological parameters in the study. The novel DRI showed even better performance in the simulation study, but not in the clinical results, suggesting that a more accurate central pressure estimate or measurement would be needed. All diastolic dominance-based metrics appear to be relatively insensitive to autoregulation. As pointed out by Jelenc et al. , all metrics’ values except \documentclass[12pt]{minimal}
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\begin{document}$$Q_\text {mean}$$\end{document} Q mean depend on flow probe position. However, in our results the difference between values measured proximally and distally was only \documentclass[12pt]{minimal}
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\begin{document}$$1-2$$\end{document} 1 - 2 % on average, and the corresponding Sobol indices are not significantly affected. To further increase the practical relevance of the multiscale model used in this study, it may be extended to include different graft types and targets, autoregulation, and competitive flow. This would also allow further honing of flow-based patency metrics and their associated measurement protocol. Extending the model would require more computing power, further restriction of UQ complexity, or a more efficient model implementation. Additionally, a better understanding of hemodynamic and physiological changes during CABG surgery is needed for a more realistic model, using CABG patient parameters rather than healthy subject parameters. To this end, data from existing and new clinical studies may be used, for example, in combination with the approach adopted by Tran et al. . Overall, from the angle of a company seeking to further improve and broaden the diagnostic strength of its flow-based CABG patency metrics and protocols, the method presented in this paper appears promising. Existing or newly-formulated metrics can be examined for their ability to separate the technical error conditions under control by the surgeon from the graft properties and patient conditions not under surgical control. Along with this, the statistical correlation between graft properties and patient conditions can be associated to the model variables, to define surgery/patient-specific modifiers to existing CABG patency metrics and thereby enhance their diagnostic precision. This can guide engineering development and ensuing clinical studies to validate efficacy and clinical outcomes.
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Stent-graft implantation for late postpancreatectomy hemorrhage after pancreatoduodenectomy | 11015547-c246-44b5-8a5b-5738b61621c3 | 11840307 | Surgical Procedures, Operative[mh] | Introduction Pancreatoduodenectomy (PD) is the standard surgical procedure for resectable pancreatic head lesions. With the improvement of surgical technique and perioperative management, the mortality rate of PD dropped significantly to less than 5% in high-volume centers. , Postpancreatectomy hemorrhage (PPH) is a life-threatening complication of PD. According to its time of onset, PPH was classified as early (within 24 h after the end of the index operation) or late (over 24 h) according to the International Study Group of Pancreatic Surgery (ISGPS) definition. The underlying pathophysiological process of late PPH was different from early PPH, possible mechanisms including enzymatic digestion of the blood vessel, intraabdominal infection with involvement of peripancreatic vessels, and vascular injury during resection that leads to a pseudoaneurysm. The incidence of late PPH ranged from 3% to 16%, while its mortality rate was up to 21%. Invasive treatment options for late PPH includes relaparotomy, endoscopic intervention, and endovascular intervention. Relaparotomy is recommended for hemodynamically unstable patients, and endoscopy is recommended for hemodynamically stable patients in certain settings. Endovascular intervention is the recommended treatment option for initial management of late PPH in both hemodynamically stable and unstable patients. Transarterial embolization (TAE) and stent-graft implantation are the 2 major methods of endovascular intervention. , TAE raises the concern of impaired perfusion of distal organ, especially when hepatic artery is embolized, the physiological function of the liver could be influenced. With the development of covered stent-graft, stent-graft implantation becomes the preferred technique to treat late PPH in our center. The advantages of covered stent-graft includes: (1) The procedure is minimally invasive; (2) angiography has high sensitivity in identifying the bleeding site; (3) the hemostasis is conclusive after excluding the bleeding site and could be reconfirmed with angiography; (4) a conduit is built with a covered stent-graft for perfusion of the distal organ. With this study, we aim to report the single-center experience in treating PPH with covered stent-graft implantation. Methods 2.1 Study population Between April 2020 and December 2023, a total of 1723 pancreatectomies were performed, including 1068 PD cases and 655 total pancreatectomy cases. Among those, 16 patients underwent endovascular intervention owing to late PPH, including 4 cases treated with TAE and 12 cases treated with covered stent-graft implantation. These patients were followed and outcomes were recorded. Written informed consent was obtained from each patient. This study conformed to the ethical principles of the Declaration of Helsinki and was approved by the institutional review board. An electronic, prospective database is regularly updated, collecting demographics, intra-operative data, and postoperative outcomes for all pancreatic resections performed at our institution. Data were retrieved from the database and retrospectively reviewed. 2.2 Endovascular procedure Local anesthesia was used in all cases. The left/right common femoral artery was incised as an access route based on previous contrast-enhanced CT. The guidewire was introduced from the access route together with a 6 or 7 Fr catheter (Destination, Terumo, Tokyo, Japan). After arriving at the plane above the celiac artery, a 5 Fr pigtail angiographic catheter (COOK, Bloomington, IN, USA) was exchanged to perform angiography. The bleeding site and anatomical pattern of relevant arteries were identified according to the distribution and leakage of the contrast medium. After the initial angiography, the 5 Fr pigtail angiographic catheter was exchanged to 0.035 (Microport, Shanghai, China) or 0.018 (Boston Scientific, MA, USA) guidewire together with suitable catheter (Cobra; Medikit, Tokyo, Japan) to select the celiac artery and advance to the bleeding artery (common hepatic artery, right hepatic artery, proper hepatic artery, splenic artery, gastroduodenal artery stump, and superior mesenteric artery). The guidewire was advanced to the distal region of the bleeding site to allow sufficient zone for stent-graft deployment, and sufficient proximal/distal landing zone. Self-expandable covered stent-graft Viabahn (TBE, WL Gore, Flagstaff, AZ, USA) was used in all cases, the diameter of which ranged from 5 to 8 mm. Angiography would be performed after the deployment of stent-graft. If persistent contrast extravasation, incomplete exclusion of pseudoaneurysm, or insufficient expansion of the stent-graft was detected, balloon dilatation or extra bridging stent-graft implantation would be performed. Final angiography was performed to reconfirm the complete exclusion of pseudoaneurysm, no leakage of contrast, normal distal organ perfusion, ideal stent-graft shape, and no sign of endoleak. 2.3 Perioperative routine After the endovascular intervention, all patients were sent to the intensive care unit and monitored with cardiac monitoring for at least 24 h. Complete blood count and liver function test were performed immediately after the operation and daily thereafter. Only after a comprehensive evaluation of the patients’ vital signs, laboratory findings, and volume and color of drainage, the patient would be transferred back to the general ward. If any sign of recurrent bleeding was observed, the contrast-enhanced CT would be immediately performed, and endovascular intervention or relaparotomy would be considered. Coagulation was not considered in concern of rebleeding or high risk of bleeding. After discharge, dual antiplatelet therapy with aspirin (100 mg) and clopidogrel (75 mg) was performed according to the judgment of individual physicians. 2.4 Definition PPH was recorded according to the ISGPS classification. Based on the severity of hemorrhage, PPH was defined as mild (small or medium volume blood loss; mild clinical volume impairment; therapeutic consequence; no need for reoperation or interventional angiographic embolization) or severe (large volume blood loss; clinically significant impairment; need for blood transfusion; need for invasive treatment). Postoperative pancreatic fistula (POPF) was defined as a drain output of any measurable volume of fluid with an amylase level over 3 times the upper limit of institutional normal serum amylase activity, associated with a clinically relevant development/condition related directly to the POPF. Technical success was defined as angiography showing complete cessation of bleeding, patent stent-graft, and normal end-organ perfusion of the affected aorta. Clinical success was defined as resolution of hemorrhagic symptoms (based on vital signs, laboratory tests, and hemorrhagic drainage) without additional endovascular or surgical intervention. Procedure-related complications were evaluated according to the Claviene-Dindo classification. Survival or any other adverse events were defined as those occurring during hospitalization (in-hospital mortality), within 90 days postoperatively (90-day mortality), and after 90 days postoperatively (follow-up mortality). The reason for death and adverse events were described in detail and recorded as indeterminate if not available. After discharge, patients were followed every 3 months at the outpatient. Contrast-enhanced CT was performed at 3-month intervals for 1 year and every 6 months thereafter. Stent-graft patency and its end-organ perfusion were evaluated with contrast-enhanced CT during follow-up. Primary patency was patent vessel without endovascular reintervention, and secondary patency was patent vessel after endovascular reintervention. 2.5 Statistical analysis Continuous data are reported as means ± standard deviation (SD) when normally distributed or as median (Q 1 , Q 3 ) when the assumption of normality was not met according to Shapiro-Wilk tests. Categorical data are reported as n . A p < 0.05 was considered statistically significant. Kaplan-Meier estimates were used for stent patency and patients’ survival. The data were analyzed using SPSS version 21 (SPSS Inc, Chicago, IL, USA) and GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA). Study population Between April 2020 and December 2023, a total of 1723 pancreatectomies were performed, including 1068 PD cases and 655 total pancreatectomy cases. Among those, 16 patients underwent endovascular intervention owing to late PPH, including 4 cases treated with TAE and 12 cases treated with covered stent-graft implantation. These patients were followed and outcomes were recorded. Written informed consent was obtained from each patient. This study conformed to the ethical principles of the Declaration of Helsinki and was approved by the institutional review board. An electronic, prospective database is regularly updated, collecting demographics, intra-operative data, and postoperative outcomes for all pancreatic resections performed at our institution. Data were retrieved from the database and retrospectively reviewed. Endovascular procedure Local anesthesia was used in all cases. The left/right common femoral artery was incised as an access route based on previous contrast-enhanced CT. The guidewire was introduced from the access route together with a 6 or 7 Fr catheter (Destination, Terumo, Tokyo, Japan). After arriving at the plane above the celiac artery, a 5 Fr pigtail angiographic catheter (COOK, Bloomington, IN, USA) was exchanged to perform angiography. The bleeding site and anatomical pattern of relevant arteries were identified according to the distribution and leakage of the contrast medium. After the initial angiography, the 5 Fr pigtail angiographic catheter was exchanged to 0.035 (Microport, Shanghai, China) or 0.018 (Boston Scientific, MA, USA) guidewire together with suitable catheter (Cobra; Medikit, Tokyo, Japan) to select the celiac artery and advance to the bleeding artery (common hepatic artery, right hepatic artery, proper hepatic artery, splenic artery, gastroduodenal artery stump, and superior mesenteric artery). The guidewire was advanced to the distal region of the bleeding site to allow sufficient zone for stent-graft deployment, and sufficient proximal/distal landing zone. Self-expandable covered stent-graft Viabahn (TBE, WL Gore, Flagstaff, AZ, USA) was used in all cases, the diameter of which ranged from 5 to 8 mm. Angiography would be performed after the deployment of stent-graft. If persistent contrast extravasation, incomplete exclusion of pseudoaneurysm, or insufficient expansion of the stent-graft was detected, balloon dilatation or extra bridging stent-graft implantation would be performed. Final angiography was performed to reconfirm the complete exclusion of pseudoaneurysm, no leakage of contrast, normal distal organ perfusion, ideal stent-graft shape, and no sign of endoleak. Perioperative routine After the endovascular intervention, all patients were sent to the intensive care unit and monitored with cardiac monitoring for at least 24 h. Complete blood count and liver function test were performed immediately after the operation and daily thereafter. Only after a comprehensive evaluation of the patients’ vital signs, laboratory findings, and volume and color of drainage, the patient would be transferred back to the general ward. If any sign of recurrent bleeding was observed, the contrast-enhanced CT would be immediately performed, and endovascular intervention or relaparotomy would be considered. Coagulation was not considered in concern of rebleeding or high risk of bleeding. After discharge, dual antiplatelet therapy with aspirin (100 mg) and clopidogrel (75 mg) was performed according to the judgment of individual physicians. Definition PPH was recorded according to the ISGPS classification. Based on the severity of hemorrhage, PPH was defined as mild (small or medium volume blood loss; mild clinical volume impairment; therapeutic consequence; no need for reoperation or interventional angiographic embolization) or severe (large volume blood loss; clinically significant impairment; need for blood transfusion; need for invasive treatment). Postoperative pancreatic fistula (POPF) was defined as a drain output of any measurable volume of fluid with an amylase level over 3 times the upper limit of institutional normal serum amylase activity, associated with a clinically relevant development/condition related directly to the POPF. Technical success was defined as angiography showing complete cessation of bleeding, patent stent-graft, and normal end-organ perfusion of the affected aorta. Clinical success was defined as resolution of hemorrhagic symptoms (based on vital signs, laboratory tests, and hemorrhagic drainage) without additional endovascular or surgical intervention. Procedure-related complications were evaluated according to the Claviene-Dindo classification. Survival or any other adverse events were defined as those occurring during hospitalization (in-hospital mortality), within 90 days postoperatively (90-day mortality), and after 90 days postoperatively (follow-up mortality). The reason for death and adverse events were described in detail and recorded as indeterminate if not available. After discharge, patients were followed every 3 months at the outpatient. Contrast-enhanced CT was performed at 3-month intervals for 1 year and every 6 months thereafter. Stent-graft patency and its end-organ perfusion were evaluated with contrast-enhanced CT during follow-up. Primary patency was patent vessel without endovascular reintervention, and secondary patency was patent vessel after endovascular reintervention. Statistical analysis Continuous data are reported as means ± standard deviation (SD) when normally distributed or as median (Q 1 , Q 3 ) when the assumption of normality was not met according to Shapiro-Wilk tests. Categorical data are reported as n . A p < 0.05 was considered statistically significant. Kaplan-Meier estimates were used for stent patency and patients’ survival. The data were analyzed using SPSS version 21 (SPSS Inc, Chicago, IL, USA) and GraphPad Prism (GraphPad Software Inc, La Jolla, CA, USA). Results 3.1 Baseline characteristics There were 12 patients with 9 male and 3 female, aged 63 (44, 80) years who had Viabahn implantation owing to late PPH been involved. Patients’ characteristics are summarized in . The most common underlying disease was pancreatic cancer ( n = 8, 66.7%). Eleven patients underwent open PD, and 1 patient underwent robot-assisted PD. All patients had 3−4 drain tubes placed in the abdomen cavity during the operation. The median onset of hemorrhage was 24.9 (10.0, 50.0) days after surgery. All patients had late, extraluminal, severe PPH, which was Grade C according to the ISGPS grading. All patients underwent contrast-enhanced CT before the procedure, and all had positive findings revealing contrast extravasation or pseudoaneurysm at the bleeding site. 3.2 Procedure outcomes The median duration of the operation was 68.3 (30.0, 120.0) min. A pseudoaneurysm and/or contrast extravasation was identified in all patients. The pancreatic fistula was identified in 6 (50.0%) cases, and pseudoaneurysm was identified in 3 (25.0%) cases, including pancreatic fistula together with pseudoaneurysm in 1 case. The common hepatic artery was the most common bleeding site ( n = 6), after which were splenic artery ( n = 3), gastroduodenal artery (GDA) stump ( n = 2), and superior mesenteric artery ( n = 1). In 3 cases, 2 Viabahns were implanted for complete hemostasis. The diameter of the stent-graft was 5 mm ( n = 1), 6 mm ( n = 4), 7 mm ( n = 4), and 8 mm ( n = 5). In 1 case, post-stent balloon dilatation was required. In another case with pseudoaneurysm in the hepatic artery, the GDA stump was embolized with coils, and the pseudoaneurysm was excluded with Viabahn stent-graft. In 2 cases, Pulsar stent-graft was deployed distal to the Viabahn stent-graft, with several segments overlapping due to lack of Viabahn of appropriate size. Technical success was achieved in all cases, which is, bleeding successfully was treated with covered stent-graft, and distal organ perfusion remained patent . 3.3 Clinical outcomes The median hospital stay was 40.0 (18.0, 92.0) days, and the median ICU stay was 10.8 (1.0, 29.0) days. During hospitalization, 1 patient developed pulmonary embolism after the operation and was considered as contraindicated to coagulation. The patient underwent inferior vena-cava filter implantation to prevent disease progression. One patient had herniation of the small intestine into the thoracic cavity, which caused broad thoracic and abdominal infection. The patient refused further surgical intervention and died of septicemia . The median follow-up was 316.5 (156.3, 682.0) days. During the follow-up, 2 patients died of malignancy recurrence at 10 and 12 months after the operation, whose stent-grafts’ patency was indetermined. Rebleeding occurred in 1 case after stent-graft implantation for the splenic artery. The patient was sent to the hospital because of abdominal pain and hemorrhagic shock 2 months after the initial operation. The newly-occurred bleeding site was GDA stump and another Viabahn was implanted and achieved hemostasis. At the latest image follow-up (27 months after PD), both stent-grafts were patent. Asymptomatic stent-graft occlusion was confirmed with follow-up CT at 26.3 and 24.6 months after the operation, respectively . Baseline characteristics There were 12 patients with 9 male and 3 female, aged 63 (44, 80) years who had Viabahn implantation owing to late PPH been involved. Patients’ characteristics are summarized in . The most common underlying disease was pancreatic cancer ( n = 8, 66.7%). Eleven patients underwent open PD, and 1 patient underwent robot-assisted PD. All patients had 3−4 drain tubes placed in the abdomen cavity during the operation. The median onset of hemorrhage was 24.9 (10.0, 50.0) days after surgery. All patients had late, extraluminal, severe PPH, which was Grade C according to the ISGPS grading. All patients underwent contrast-enhanced CT before the procedure, and all had positive findings revealing contrast extravasation or pseudoaneurysm at the bleeding site. Procedure outcomes The median duration of the operation was 68.3 (30.0, 120.0) min. A pseudoaneurysm and/or contrast extravasation was identified in all patients. The pancreatic fistula was identified in 6 (50.0%) cases, and pseudoaneurysm was identified in 3 (25.0%) cases, including pancreatic fistula together with pseudoaneurysm in 1 case. The common hepatic artery was the most common bleeding site ( n = 6), after which were splenic artery ( n = 3), gastroduodenal artery (GDA) stump ( n = 2), and superior mesenteric artery ( n = 1). In 3 cases, 2 Viabahns were implanted for complete hemostasis. The diameter of the stent-graft was 5 mm ( n = 1), 6 mm ( n = 4), 7 mm ( n = 4), and 8 mm ( n = 5). In 1 case, post-stent balloon dilatation was required. In another case with pseudoaneurysm in the hepatic artery, the GDA stump was embolized with coils, and the pseudoaneurysm was excluded with Viabahn stent-graft. In 2 cases, Pulsar stent-graft was deployed distal to the Viabahn stent-graft, with several segments overlapping due to lack of Viabahn of appropriate size. Technical success was achieved in all cases, which is, bleeding successfully was treated with covered stent-graft, and distal organ perfusion remained patent . Clinical outcomes The median hospital stay was 40.0 (18.0, 92.0) days, and the median ICU stay was 10.8 (1.0, 29.0) days. During hospitalization, 1 patient developed pulmonary embolism after the operation and was considered as contraindicated to coagulation. The patient underwent inferior vena-cava filter implantation to prevent disease progression. One patient had herniation of the small intestine into the thoracic cavity, which caused broad thoracic and abdominal infection. The patient refused further surgical intervention and died of septicemia . The median follow-up was 316.5 (156.3, 682.0) days. During the follow-up, 2 patients died of malignancy recurrence at 10 and 12 months after the operation, whose stent-grafts’ patency was indetermined. Rebleeding occurred in 1 case after stent-graft implantation for the splenic artery. The patient was sent to the hospital because of abdominal pain and hemorrhagic shock 2 months after the initial operation. The newly-occurred bleeding site was GDA stump and another Viabahn was implanted and achieved hemostasis. At the latest image follow-up (27 months after PD), both stent-grafts were patent. Asymptomatic stent-graft occlusion was confirmed with follow-up CT at 26.3 and 24.6 months after the operation, respectively . Discussion PPH is a life-threatening complication of PD. With the improvement in surgical technique and perioperative management, the incidence of PPH has decreased significantly. The incidence of PPH varies in different centers, which could be as low as 3% in high-volume centers, and up to 10% in several reports. , , , Despite timely diagnosis and treatment, the mortality rate of PPH remains high, ranging from 21% to 34% in previous reports. According to the timing of onset, PPH was classified as early and late. The etiology of early and late PPH was different. Early PPH was mostly caused by technical failure of hemostasis during the index operation or coagulopathy. Late PPH is more complicated and multifactorial. During operation, ligation of the arteries, lymphadenectomy, and manipulation of peripancreatic vessels could lead to injury and damage to the protective tissues, rendering the vessel more vulnerable to subsequent damage. POPF was associated with leakage of enzyme rich fluid into the abdomen, causing vessel erosion and formation of pseudoaneurysm. In our series, POPF was identified in 6 late PPH cases, which was the major cause of PPH. Pseudoaneurysm was another main cause of late PPH. The reason for pseudoaneurysm formation was commonly believed to be associated with pancreatic fistula or anastomotic dehiscence, while its pathological process was not fully understood. For 3 cases with pseudoaneurysms, only 1 case had both POPF and splenic artery pseudoaneurysm, where POPF was considered to cause pseudoaneurysm. Continuous vessel erosion by the enzyme rich fluid eventually led to rupture of pseudoaneurysm, causing major hemorrhage. Early stratification of PPH risk was beneficial for patients’ management. Birgin et al. developed and validated a prediction model for PPH. PPH was associated with sentinel bleeding, drain fluid culture positive for Candida species, and radiologic proof of rim enhancement of or gas within a peripancreatic fluid collection. Similarly, Palumbo et al. found postoperative CT evidence of fluid collections, air bubbles, and posterior pancreaticojejunostomy defect associated with PPH. Robust prediction models could identify PPH risk at an early stage, and allow different management strategies for patients with different risks. Management for late PPH included relaparotomy, endoscopic intervention, and endovascular intervention. Immediate relaparotomy used to be the mainstream treatment was now recommended for those with negative findings, insufficient hemostasis in angiography and endoscopy, and hemodynamically unstable patients. Adhesion within the abdominal cavity after the initial PD increased the difficulty in exposure and identification of the bleeding site. Relaparotomy was highly invasive, requiring a longer time for full recovery and higher costs. Survival after relaparotomy was also a concern. A systematic review reported the survival rate after primary relaparotomy as 37%. The indication of endoscopic intervention was limited, which was mostly used in hemodynamically stable patients with intra-luminal hemorrhage. Endovascular intervention was composed of TAE and stent-graft implantation. At the early stage, TAE was the most used approach in our center. However, after embolization of the hepatic artery, liver infarction, liver abscess, and impaired liver function had been observed in early cases. The blood flow was impaired and caused distal organ malperfusion. Hassold et al. compared TAE and covered stent-graft implantation for delayed PPH. In the TAE group, infarction distal to the embolized vessel occurred in 6/11 cases; in the covered stent-graft group, ischemia distal to the occluded stent-graft was observed in 2/14 cases. The 1- and 2-year survival rate was also higher in the covered stent-graft group. After Viabahn stent-graft became available, stent-graft implantation has become the preferred treatment option in our center. The major limitation of stent-graft implantation was its requirement for suitable anatomy. Torturous access artery increases the difficulty in the selection of bleeding arteries, and some bleeding artery was too angulated for stent-graft implantation. For those with unsuitable anatomy, TAE would be considered to achieve hemostasis. A major concern of stent-graft implantation was rebleeding. Maccabe et al. summarized outcomes of PPH stratified according to ISGPS grading. For Grade B PPH, the reintervention rate after initial endovascular treatment was higher than initial surgical intervention (9.7% (3/31) vs . 0 (0/9)). For Grade C PPH, the reintervention rate after initial endovascular treatment was 9.2% (11/119), similar to that of Grade B PPH. In our series, rebleeding occurred in 1 case. The initial bleeding site was a splenic artery, and the rebleeding site was a GDA stump. Another Viabahn was implanted, and hemostasis was achieved after the deployment of the stent-graft. From our experience, Viabahn stent-graft was effective in hemostasis. Endovascular intervention allowed precise adjustment within the artery, and its technical and clinical success rate was high. In cases where rebleeding occurs, another endovascular or surgical intervention could be considered, providing more optimal options compared with initial surgical intervention. , Another concern after stent-graft implantation was its long-term patency. Occlusion or stenosis of stent-graft would impair the distal organ perfusion. In the previous reports, Viabahn stent-graft patency at 1 year varied significantly (40%−80%), while all data were from retrospective studies with small sample sizes. , , Interestingly, Min et al. reported no liver ischemia despite a high stent failure rate (50%). The explanation was that the process of stent-graft occlusion was thrombotic, which was long enough for the formation of collateral blood vessels. Antiplatelet therapy is recommended after implantation of stent-graft with a small diameter to prevent thrombosis, including all that used in this series (5−8 mm). Anticoagulation or antiplatelet therapy was not routinely used during the perioperative period considering the rebleeding risk after PPH, and antiplatelet therapy was recommended after discharge in our center. For those with available follow-up contrast-enhanced CT, stent-grafts (4 cases, 5 stent-grafts) were all patent. Izumi et al. suggested that anticoagulation was not a necessity since there was no association between the administration of anticoagulation or antiplatelet and stent-graft patency. Instead, recurrence of primary malignancy was associated with stent failure, suggesting that physical compression of the tumor was the major reason for thrombotic occlusion. Current evidence for the relation between rebleeding, coagulation, stent-graft patency, and distal organ perfusion was low level, and further randomized study was needed to guide practice. This was a single-center, retrospective study with a small sample size, where selection bias was inevitable. Owing to the short survival period of pancreatic cancer, the follow-up period was limited. Only patients with suitable anatomy would be considered for stent-graft implantation, while the other would be treated with TAE, thus the technical success rate could be over-evaluated. The bleeding site varies, and preoperative evaluation was performed by an experienced physician. A randomized, multicenter study with longer follow-up was needed to further verify the safety and feasibility of Viabahn implantation to treat PPH with different bleeding sites. In conclusion, covered stent-graft implantation with Viabahn is an effective and safe treatment option for PPH with various bleeding sites and causes. Once the covered stent-graft was successfully deployed at the bleeding site, immediate hemostasis would be achieved, and distal organ perfusion was preserved with a low risk of rebleeding. The technical and clinical success rate was high. The major reason for death was a recurrence of primary malignancy during follow-up. Xiaoye Li: Conceptualization, Data curation, Formal analysis, Writing - original draft, Writing - review & editing. Shibo Xia: Data curation, Formal analysis. Liangxi Yuan: Data curation, Formal analysis, Investigation, Methodology, Project administration. Lei Zhang: Data curation, Formal analysis, Methodology, Project administration. Chao Song: Data curation, Formal analysis, Supervision. Xiaolong Wei: Data curation, Project administration. Qingsheng Lu: Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing–original draft. Written informed consent was obtained from each patient. This study conformed to the ethical principles of the Declaration of Helsinki and was approved by the institutional review board. This study was supported by the 234 Discipline Climbing Program, A multicenter study of robotic treatment of dilated aortic disease by endovascular repair, 2019YXK048. There is no conflict of interest or funding to disclosure. |
The Role of Optometry in the Delivery of Eye Care via Telehealth: A Systematic Literature Review | 25bce657-b173-443b-a537-94bfe4745a2e | 9805855 | Ophthalmology[mh] | In March 2020, the World Health Organization declared coronavirus disease 2019 (COVID-19) a pandemic. As a result, the capacity of health systems to continue to deliver optometry services in an office-based face-to-face experience has been restricted to ensure the safety of both the patient and the provider. In response to the need to maintain essential optometry services throughout the COVID-19 pandemic, there has been a dramatic rise in the utilization of telehealth services, and the profession has moved rapidly toward the adoption and delivery of teleoptometry. The increase in the uptake of telehealth to deliver optometry care during COVID-19 has caused the optometry profession to reimagine the role of telehealth. Telehealth is a promising and well-received approach to connect practitioners and patients to deliver health care to individuals where access and resources may be limited. The utilization of telehealth is becoming increasingly widespread in health care, with the many applications extending to numerous medical specialties. A recent systematic review highlighted the effectiveness of telemedicine in specialties, including telepsychiatry, teleradiology, and telecardiology. The review demonstrated the efficacy of consultations between primary and secondary level health care providers and described the criticisms of the limited and inconsistent evidence surrounding efficacy and cost-effectiveness in telemedicine. , Other investigations that relate to the cost-effectiveness of telemedicine compared to conventional health care delivery has been found to be inconclusive at this time. , , While there is evidence that showed telemedicine to be cost-effective when delivered in rural and remote areas, conclusions shared in the systematic review by Ekeland et al. called for more extensive studies to address the limited and inconsistent results regarding impact and cost in the literature. The terms telehealth and telemedicine are at times used interchangeably. However, in an evaluation of peer-reviewed definitions of these terms, Sood et al. demonstrated that these terms are not synonymous, and telemedicine is a subset of telehealth. Telemedicine is limited in scope to the delivery of the clinical service aspects of health care, whereas telehealth is more expansive and covers the preventive, promotive, and curative aspects of health care. For the purposes of this review, the term “telehealth” will be used when referring to telehealth or telemedicine. There are two primary forms of telehealth: synchronous and asynchronous telehealth. Synchronous telehealth refers to the delivery of consultations in real-time. For example, video consultations commonly included prescribing medications, reassurance, or escalating the need for an in-person appointment. In contrast, asynchronous telehealth involves transmitting health information in a store-and-forward approach that does not require real-time communication, and applications include screening for diabetic retinopathy and retinopathy of prematurity. The telehealth model has been applied across many health domains. Teleophthalmology is a well-researched discipline in telemedicine that is highly regarded by practitioners and patients. Teleophthalmology is an evidence-based intervention where ophthalmologists deliver specialty care in hospital and outpatient settings to unserved or underserved populations. Teleophthalmology is often dependent on optometrists and other skilled health workers to capture and transmit patient information. Applications of teleophthalmology are feasible for triage, screening, consultation, and remote supervision. Screening for diabetic retinopathy is one such service commonly delivered by teleophthalmology, and in the United States, teleophthalmology screening services for diabetic retinopathy include optometrists. Numerous systematic reviews support the use of teleophthalmology, citing teleophthalmology to be cost-efficient, reliable, and valid. Teleoptometry care models delivered during the COVID-19 pandemic include urgent care for acute eye health concerns, deterioration of chronic eye conditions, and other consultations, including contact lens follow-up. In recent years, there has been increasing reports of optometry services being provided via telehealth. Teleoptometry can be defined as the application of optometrist-provided care via telehealth. The increasing utilization of teleoptometry is partly due to the emergence of novel and innovative approaches to eye care delivery supported by high-speed internet and continual software and equipment innovation in the eye care sector. During the initial phases of the lockdown, only 5.54% of optometric services were delivered in optometry practices in Pakistan compared to prelockdown. The impact of the COVID-19 pandemic disproportionately affected vulnerable populations such as those living in remote areas, elderly people, socially disadvantaged people, children, and people living with disabilities to access optometric care. The inability for these at-risk patients to present in-person contributed to an increase in inequity in accessing eye care services. Telehealth can increase access to health care to vulnerable groups by reducing barriers, including reduced travel time and cost. , The COVID-19 pandemic has further highlighted the need to create innovative approaches to provide primary eye care to vulnerable populations who experience barriers to access eye care. , RATIONALE A number of studies detail the application of teleophthalmology programs in underserved areas and populations. However, despite growing interest in adopting teleoptometry during the pandemic, there is a paucity of published literature regarding the role of optometry in telehealth. Further, there is a lack of policies and protocols to guide the delivery of optometry services via telehealth platforms. The U.S. Department of Veterans Affairs (VA) is leading the expansion of optometry-facilitated telehealth services with the introduction of protocols and services, including low-vision rehabilitation, teleretinal screening services in primary care clinics, and Technology-based Eye Care Services (TECS). Globally, there is an urgent need to collate current evidence on the use of optometric care delivered by telehealth. OBJECTIVES The purpose of this report is to conduct a literature review to seek all of the peer-reviewed evidence surrounding the application of optometry services when delivered via telehealth models.
A number of studies detail the application of teleophthalmology programs in underserved areas and populations. However, despite growing interest in adopting teleoptometry during the pandemic, there is a paucity of published literature regarding the role of optometry in telehealth. Further, there is a lack of policies and protocols to guide the delivery of optometry services via telehealth platforms. The U.S. Department of Veterans Affairs (VA) is leading the expansion of optometry-facilitated telehealth services with the introduction of protocols and services, including low-vision rehabilitation, teleretinal screening services in primary care clinics, and Technology-based Eye Care Services (TECS). Globally, there is an urgent need to collate current evidence on the use of optometric care delivered by telehealth.
The purpose of this report is to conduct a literature review to seek all of the peer-reviewed evidence surrounding the application of optometry services when delivered via telehealth models.
This review includes only peer-reviewed publications that were in English. Publications that were limited to a review of the literature were excluded. All peer-reviewed publications that included optometrists in the telehealth service were included in this review. Due to the scoping nature of the literature review, abstract presentations were included in the eligibility criteria. SEARCH STRATEGY A comprehensive database search was undertaken using MEDLINE, Global Health and Web of Science in October 2020. Reference lists were hand-searched for other relevant articles by the primary author. MEDLINE was searched using the following search string: “teleoptometry” OR “tele-optometry” OR “telemedicine” OR “telehealth” OR “teleophthalmology” OR “ehealth” OR “telecare” Or “video consultation” OR “electronic consultation” OR “e consultation” OR “virtual consultation” OR “remote consultation” OR “videoconferencing” OR “mhealth” AND “optom*”. Relevant MeSH terms were included in the search strategy. Analogous search terms were used for Global Health and Web of Science. No limits on study design or intervention were imposed. No restrictions were applied to publication date or location. INCLUDED STUDIES In total, 206 abstracts were identified via database searching. Nine additional studies were included that were identified through other sources. Following the removal of duplicates, 212 references were screened. Fifty-one full-text articles and four articles limited to abstracts only were accessed and included by the first author. Of the remaining articles, 27 met the inclusion criteria for analysis . DATA EXTRACTION AND SYNTHESIS Following the review of full-text and abstract articles, a framework for data extraction and synthesis was developed, and the first author, (J.M.) independently extracted relevant data into Microsoft Excel. The framework listed the variables to be extracted from each article and included year, study design, country, study focus, mode of telehealth, population, collaboration type, and study summary.
A comprehensive database search was undertaken using MEDLINE, Global Health and Web of Science in October 2020. Reference lists were hand-searched for other relevant articles by the primary author. MEDLINE was searched using the following search string: “teleoptometry” OR “tele-optometry” OR “telemedicine” OR “telehealth” OR “teleophthalmology” OR “ehealth” OR “telecare” Or “video consultation” OR “electronic consultation” OR “e consultation” OR “virtual consultation” OR “remote consultation” OR “videoconferencing” OR “mhealth” AND “optom*”. Relevant MeSH terms were included in the search strategy. Analogous search terms were used for Global Health and Web of Science. No limits on study design or intervention were imposed. No restrictions were applied to publication date or location.
In total, 206 abstracts were identified via database searching. Nine additional studies were included that were identified through other sources. Following the removal of duplicates, 212 references were screened. Fifty-one full-text articles and four articles limited to abstracts only were accessed and included by the first author. Of the remaining articles, 27 met the inclusion criteria for analysis .
Following the review of full-text and abstract articles, a framework for data extraction and synthesis was developed, and the first author, (J.M.) independently extracted relevant data into Microsoft Excel. The framework listed the variables to be extracted from each article and included year, study design, country, study focus, mode of telehealth, population, collaboration type, and study summary.
In total, 27 articles met the inclusion criteria for the review. The first published study in optometry-facilitated teleoptometry emerged in 1999, where Smythe reported on a study exploring teleoptometry to facilitate contact lens fittings via asynchronous video. Since 1999, there has been a surge in studies examining optometry-facilitated telehealth in recent years, with most publications emerging between 2015 and 2021 ( n = 20). provides a summary of the 27 publications relating the optometric care in telehealth. Publication dates of all studies included in this review ranged between 1999 and 2020. The location of the studies reviewed include seven studies conducted in Australia, , seven in the United States, , three in Canada, two in India, , two in The Netherlands, , three in the United Kingdom, one study was conducted in an undisclosed location, and single studies were conducted in Ethiopia and Spain. STUDY CHARACTERISTICS Prospective study designs represent the largest number of included studies, including four cohort studies , , , and four prospective audits. , , , Other frequently utilized study designs included retrospective audits ( n = 4), , , , interobserver reliability studies ( n = 2), , questionnaires ( n = 3), , , and mixed methods studies ( n = 2). , Less frequently utilized study designs included retrospective cohorts ( n = 2), , retrospective case series ( n = 2), , one clinical protocol, a prospective case series, a clinical protocol for a pilot study, and a cross-sectional pre–post study . Of the 27 studies reporting the involvement of an optometrist to deliver telehealth, only 11 studies included the role of optometrists as a member of the telehealth team providing eye care services. , , , , That is, the optometrist had a role in the care of the patient beyond providing and receiving telehealth referrals, being present for the teleconsultation, collecting clinical data required for asynchronous teleophthalmology consultations, and providing the patient with continuing care following telehealth consultation with an ophthalmologist. MODE OF TELEHEALTH USED TO DELIVER EYE CARE SERVICES outlines the inclusion of optometry in the delivery of eye care services via different models of telehealth. Of the 27 studies that included optometrists in the telehealth model of care, 12 studies utilized asynchronous telehealth, , , , , , , , , where the optometrist collected clinical data from the patient in an in-person consultation that usually accompanied a referral to a teleophthalmology service. Eleven studies used synchronous telehealth, , , , , , , , where optometrists used video consultations to deliver primary care, including low-vision rehabilitation, consulted with general and subspecialty ophthalmologists to improve the efficiency of the referral process, and delivered comprehensive eye examinations with the assistance of an in-person technician. The remaining four studies utilized both synchronous and asynchronous methods of delivering eye care via telehealth. , , , The role of optometrists in these hybrid telehealth services involved optometrists consulting with the patients to collect the clinical data required for the ophthalmologist to asynchronously review, followed by the optometrist supporting the video consultation between the patient and the ophthalmologist. TELEHEALTH COLLABORATION TYPE The most common form of collaboration involving optometrists is the delivery of eye care via teleophthalmology. In 19 studies, the optometrist facilitates the referral, communication, and management plans between the patient and the teleophthalmology service. , , , , , , In this collaboration type, optometrists are often consulting in-person with the patient. Eight studies described the independent application of teleoptometry between the patient and the optometrist. , , , , The scope of practice of independent teleoptometry included optometrists delivering comprehensive eye examinations assisted by a technician, performing subjective refractions via a digital platform that included videoconferencing, seeking advice from other optometry colleagues via teleoptometry, supporting low-vision rehabilitation via teleconference (often supported by a technician who is present with the patient), and providing primary eye care during the COVID-19 pandemic. The format and geographical areas where eye care via telehealth is being delivered Single reports of other forms of collaborations were observed, including technicians working between the optometrist and ophthalmologist, where the optometrist collected retinal photos to send to the ophthalmology clinic for interpretation by the hospital technician. In this study, the scope of practice of optometrists was limited to refraction care. Optometry-facilitated telehealth was most frequently observed in general teleophthalmology services, and accounted for nine of the included studies. , , , Optometrists have a role in other teleophthalmology subspecialties, including anterior and orbital disease, and triaging for medical retina. , , The role of optometrists in glaucoma management via telehealth was explored in six studies. , , , , , The scope of practice of optometrists reported in these publications included organizing referrals to teleophthalmology services, and facilitating videoconference calls between patients and specialists. The role of optometrists in these comanagement arrangements also included initiating management plans made by ophthalmologists, booking patients for surgery during the consultation, and in asynchronous telehealth services, the optometrist communicated the ophthalmology findings to the patient. Two studies reported on the level of agreement between optometrist clinical findings as rated by an ophthalmologist or optometrist. , Furthermore, telerehabilitation emerged as an area where optometrists can deliver teleoptometry to patients who have low vision (usually supported by technicians who are with the patient in-person), or as part of a collaboration with other optometrists and ophthalmologists. Teleoptometry was utilized by optometrists to remotely conduct comprehensive eye examinations using a platform supported by technicians present with the patients. Teleoptometry was reportedly utilized during the COVID-19 pandemic to support the delivery of primary eye care, however, the details of how teleoptometry was conducted were not reported. Thirteen of the included studies exclusively examined optometry-facilitated telehealth in rural settings. , , , , , In these rural settings, synchronous telehealth was most commonly utilized ( n = 8), as opposed to asynchronous ( n = 2) or mixed-format telehealth ( n = 3).
Prospective study designs represent the largest number of included studies, including four cohort studies , , , and four prospective audits. , , , Other frequently utilized study designs included retrospective audits ( n = 4), , , , interobserver reliability studies ( n = 2), , questionnaires ( n = 3), , , and mixed methods studies ( n = 2). , Less frequently utilized study designs included retrospective cohorts ( n = 2), , retrospective case series ( n = 2), , one clinical protocol, a prospective case series, a clinical protocol for a pilot study, and a cross-sectional pre–post study . Of the 27 studies reporting the involvement of an optometrist to deliver telehealth, only 11 studies included the role of optometrists as a member of the telehealth team providing eye care services. , , , , That is, the optometrist had a role in the care of the patient beyond providing and receiving telehealth referrals, being present for the teleconsultation, collecting clinical data required for asynchronous teleophthalmology consultations, and providing the patient with continuing care following telehealth consultation with an ophthalmologist.
outlines the inclusion of optometry in the delivery of eye care services via different models of telehealth. Of the 27 studies that included optometrists in the telehealth model of care, 12 studies utilized asynchronous telehealth, , , , , , , , , where the optometrist collected clinical data from the patient in an in-person consultation that usually accompanied a referral to a teleophthalmology service. Eleven studies used synchronous telehealth, , , , , , , , where optometrists used video consultations to deliver primary care, including low-vision rehabilitation, consulted with general and subspecialty ophthalmologists to improve the efficiency of the referral process, and delivered comprehensive eye examinations with the assistance of an in-person technician. The remaining four studies utilized both synchronous and asynchronous methods of delivering eye care via telehealth. , , , The role of optometrists in these hybrid telehealth services involved optometrists consulting with the patients to collect the clinical data required for the ophthalmologist to asynchronously review, followed by the optometrist supporting the video consultation between the patient and the ophthalmologist.
The most common form of collaboration involving optometrists is the delivery of eye care via teleophthalmology. In 19 studies, the optometrist facilitates the referral, communication, and management plans between the patient and the teleophthalmology service. , , , , , , In this collaboration type, optometrists are often consulting in-person with the patient. Eight studies described the independent application of teleoptometry between the patient and the optometrist. , , , , The scope of practice of independent teleoptometry included optometrists delivering comprehensive eye examinations assisted by a technician, performing subjective refractions via a digital platform that included videoconferencing, seeking advice from other optometry colleagues via teleoptometry, supporting low-vision rehabilitation via teleconference (often supported by a technician who is present with the patient), and providing primary eye care during the COVID-19 pandemic.
Single reports of other forms of collaborations were observed, including technicians working between the optometrist and ophthalmologist, where the optometrist collected retinal photos to send to the ophthalmology clinic for interpretation by the hospital technician. In this study, the scope of practice of optometrists was limited to refraction care. Optometry-facilitated telehealth was most frequently observed in general teleophthalmology services, and accounted for nine of the included studies. , , , Optometrists have a role in other teleophthalmology subspecialties, including anterior and orbital disease, and triaging for medical retina. , , The role of optometrists in glaucoma management via telehealth was explored in six studies. , , , , , The scope of practice of optometrists reported in these publications included organizing referrals to teleophthalmology services, and facilitating videoconference calls between patients and specialists. The role of optometrists in these comanagement arrangements also included initiating management plans made by ophthalmologists, booking patients for surgery during the consultation, and in asynchronous telehealth services, the optometrist communicated the ophthalmology findings to the patient. Two studies reported on the level of agreement between optometrist clinical findings as rated by an ophthalmologist or optometrist. , Furthermore, telerehabilitation emerged as an area where optometrists can deliver teleoptometry to patients who have low vision (usually supported by technicians who are with the patient in-person), or as part of a collaboration with other optometrists and ophthalmologists. Teleoptometry was utilized by optometrists to remotely conduct comprehensive eye examinations using a platform supported by technicians present with the patients. Teleoptometry was reportedly utilized during the COVID-19 pandemic to support the delivery of primary eye care, however, the details of how teleoptometry was conducted were not reported. Thirteen of the included studies exclusively examined optometry-facilitated telehealth in rural settings. , , , , , In these rural settings, synchronous telehealth was most commonly utilized ( n = 8), as opposed to asynchronous ( n = 2) or mixed-format telehealth ( n = 3).
This review highlights the unique and important role optometrists have in meeting the eye care needs of populations at different levels of the health care system. A strength of this study is that it is the first review to synthesize what is known about how telehealth is being utilized to provide optometric care in the form of teleoptometry. The studies exploring optometry-facilitated telehealth varied by country, population, and mode of telehealth. The common utilization of comanagement arrangements between optometrists and ophthalmologists emerged as an area with great potential that is already highly utilized. In delivering optometric care via telehealth independent of ophthalmology, teleoptometry emerged as an area where contact lens consultations can be conducted, subjective refractions may be performed, and low-vision services can be delivered to patients. Of the 215 studies identified in the search, only 27 studies were deemed relevant to the research question. This review highlights a paucity of published research in teleoptometry, and more broadly, research supporting optometry's role in telehealth. Although the delivery of optometry has drastically changed during the COVID-19 pandemic to improve patient access to eye care ; only 11 studies published in the area of telehealth expanded the role of optometrists in the telehealth service beyond sending referrals, collecting data required for asynchronous teleophthalmology, and supporting the telehealth consultation between the ophthalmologist and the patient. , , , , The role of optometrists in the remaining 16 publications is as an indirect facilitator in the delivery of telehealth, , , , , , , primarily referring patients into a teleopthalmology service. , , , , , Other indirect roles of optometrists in telehealth services include the following: collecting clinical data required for the teleophthalmology consultation , , , , , , , ; providing support to the patient during the video consultation , , , ; suggest a management plan to accompany the referral , ; and initiate the management plan recommended by the ophthalmologist if the patient is referred back to the care of the optometrist. , , , , ACCEPTABILITY OF OPTOMETRY-FACILITATED TELEHEALTH The published literature exploring practitioner and patient acceptability of teleoptometry is limited; however, the literature that does exist in the area reports high levels of satisfaction. Two studies examining synchronous teleophthalmology services in rural Western Australia report an overall acceptance of teleoptometry by patients. In addition, patients in rural locations highly value the role of optometrists when attending teleophthalmology consultations. , Furthermore, Patel et al. report that most patients were highly satisfied with the quality of care they received when attending a comprehensive teleoptometric eye examination. Interestingly, one study identified older patients as having a greater acceptance of optometric care delivered via telehealth. Research by Verma et al. also highlights increased management continuity and patient engagement via the role of collaboration with optometrists to deliver teleophthalmology services. In the area of telerehabilitation, Bittner et al. found that all patients were satisfied and comfortable receiving low-vision evaluation and rehabilitation via teleoptometry. Patients who received low-vision services via teleoptometry perceived the accuracy of the evaluation as equal to an in-person consultation. Providing low-vision services via teleoptometry allowed additional follow-up consultations that would not usually occur at in-person services. Similarly, Ihrig states veterans who attended the low-vision telerehabilitation service report being highly satisfied with the care they received. Vision aids significantly improve quality of life ; the area of telehrehabilitation is an area with great potential to assist patients who cannot access low-vision rehabilitation at in-person services. THE ROLE OF COLLABORATIVE PARTNERSHIPS The literature highlights the significance of the collaboration between optometrists and ophthalmologists in the delivery of teleophthalmology. Despite the lack of financial reimbursement, optometrists have the greatest utilization of telehealth consultations with ophthalmologists of any of health care provider. At present, only a single study explores the positive impact of introducing financial incentives for optometrists to deliver telehealth services. Furthermore, Karthikeyan et al. reported that in India, 50.94% of optometrists offered teleoptometry services at the beginning of the COVID-19 pandemic; however, 85.19% of those optometrists did not charge for the service. Studies report increased efficiency and access to ophthalmology surgical care when optometrists facilitate teleophthalmology services between the patient and the ophthalmologist. , , , , Improved referral refinement and triaging of specialist referrals has also been demonstrated to reduce unnecessary in-person consultations, thereby reducing the workload of tertiary services, and increase case detection. Keenan et al. highlights how the skills of optometrists can be effectively and safely utilised in colloborations with ophthalmologists to evaluate glaucoma referrals at the community level via telehealth in the form of comanagement arrangements. Comanagement plans were determined by the ophthalmologists and implemented by the optometrist. This application of task sharing increases the capacity of tertiary ophthalmology services. However, Verma et al. reported limitations in comanagement arrangements where there was a lack of infrastructure with respect to data collection needed to understand optometrist adherence to management plans outlined by the ophthalmologist. The application of teleoptometry in primary eye care reduces some of the logistical barriers vulnerable patients face. , Utilizing optometrists to virtually comanage glaucoma reduces the need for some patients to attend in-person consultations with ophthalmologists and reduces the indirect costs of care. In contexts where optometrists have a limited scope of practice, optometrists are involved in telehealth comanaged screening. In addition, utilizing optometry-facilitated telehealth to prebook in-person ophthalmology procedures via telehealth reduces the number of practice visits and increases the efficiency of in-person consultations. This is further highlighted by Turner et al. who reported that 44% of patients who attended an optometrist-facilitated telehealth consultation were directly booked for surgery. Optometrists who provide teleoptometry services and are involved in comanagement arrangements benefit from the learning opportunities that arise from continuous feedback given by ophthalmologists; these collaborative arrangements foster upskilling. , In addition, optometrists in rural locations who are involved in teleophthalmology delivery directly benefit by having greater access to teleophthalmology services available when seeking timely specialist consultation. A study examining interobserver agreement is favorable. A meeting abstract published by Randhawa et al. examining telehealth-delivered subjective refraction found no statistically significant difference between in-person and telehealth-delivered subjective refraction. A single study has been published on the cost-effectiveness of optometry-facilitated telehealth services; Ihrig demonstrated the delivery of low-vision telerehabilitation significantly reduced the cost to the individual and increased access to the low-vision service compared to in-person services. LIMITATIONS The literature review has several limitations. This review represents peer-reviewed literature published in English only. Another flaw in the review is the fact that many of the studies included assessed the role of optometry in telehealth as a secondary research outcome. Furthermore, the dates of publication vary widely and extend back to 1999. Technological innovation in telehealth has rapidly evolved over the past two decades, and therefore the results from the studies should be considered in the context of publication date. The scope of practice in optometry varies between countries. At present, comanagement between ophthalmology and optometry is commonplace in many countries. As a result, the studies included presented recommendations about the role of optometry in telehealth that varied depending on the location and may not reflect what is available globally. This variability reflects a paucity of studies in the area of teleoptometry and the need for further research.
The published literature exploring practitioner and patient acceptability of teleoptometry is limited; however, the literature that does exist in the area reports high levels of satisfaction. Two studies examining synchronous teleophthalmology services in rural Western Australia report an overall acceptance of teleoptometry by patients. In addition, patients in rural locations highly value the role of optometrists when attending teleophthalmology consultations. , Furthermore, Patel et al. report that most patients were highly satisfied with the quality of care they received when attending a comprehensive teleoptometric eye examination. Interestingly, one study identified older patients as having a greater acceptance of optometric care delivered via telehealth. Research by Verma et al. also highlights increased management continuity and patient engagement via the role of collaboration with optometrists to deliver teleophthalmology services. In the area of telerehabilitation, Bittner et al. found that all patients were satisfied and comfortable receiving low-vision evaluation and rehabilitation via teleoptometry. Patients who received low-vision services via teleoptometry perceived the accuracy of the evaluation as equal to an in-person consultation. Providing low-vision services via teleoptometry allowed additional follow-up consultations that would not usually occur at in-person services. Similarly, Ihrig states veterans who attended the low-vision telerehabilitation service report being highly satisfied with the care they received. Vision aids significantly improve quality of life ; the area of telehrehabilitation is an area with great potential to assist patients who cannot access low-vision rehabilitation at in-person services.
The literature highlights the significance of the collaboration between optometrists and ophthalmologists in the delivery of teleophthalmology. Despite the lack of financial reimbursement, optometrists have the greatest utilization of telehealth consultations with ophthalmologists of any of health care provider. At present, only a single study explores the positive impact of introducing financial incentives for optometrists to deliver telehealth services. Furthermore, Karthikeyan et al. reported that in India, 50.94% of optometrists offered teleoptometry services at the beginning of the COVID-19 pandemic; however, 85.19% of those optometrists did not charge for the service. Studies report increased efficiency and access to ophthalmology surgical care when optometrists facilitate teleophthalmology services between the patient and the ophthalmologist. , , , , Improved referral refinement and triaging of specialist referrals has also been demonstrated to reduce unnecessary in-person consultations, thereby reducing the workload of tertiary services, and increase case detection. Keenan et al. highlights how the skills of optometrists can be effectively and safely utilised in colloborations with ophthalmologists to evaluate glaucoma referrals at the community level via telehealth in the form of comanagement arrangements. Comanagement plans were determined by the ophthalmologists and implemented by the optometrist. This application of task sharing increases the capacity of tertiary ophthalmology services. However, Verma et al. reported limitations in comanagement arrangements where there was a lack of infrastructure with respect to data collection needed to understand optometrist adherence to management plans outlined by the ophthalmologist. The application of teleoptometry in primary eye care reduces some of the logistical barriers vulnerable patients face. , Utilizing optometrists to virtually comanage glaucoma reduces the need for some patients to attend in-person consultations with ophthalmologists and reduces the indirect costs of care. In contexts where optometrists have a limited scope of practice, optometrists are involved in telehealth comanaged screening. In addition, utilizing optometry-facilitated telehealth to prebook in-person ophthalmology procedures via telehealth reduces the number of practice visits and increases the efficiency of in-person consultations. This is further highlighted by Turner et al. who reported that 44% of patients who attended an optometrist-facilitated telehealth consultation were directly booked for surgery. Optometrists who provide teleoptometry services and are involved in comanagement arrangements benefit from the learning opportunities that arise from continuous feedback given by ophthalmologists; these collaborative arrangements foster upskilling. , In addition, optometrists in rural locations who are involved in teleophthalmology delivery directly benefit by having greater access to teleophthalmology services available when seeking timely specialist consultation. A study examining interobserver agreement is favorable. A meeting abstract published by Randhawa et al. examining telehealth-delivered subjective refraction found no statistically significant difference between in-person and telehealth-delivered subjective refraction. A single study has been published on the cost-effectiveness of optometry-facilitated telehealth services; Ihrig demonstrated the delivery of low-vision telerehabilitation significantly reduced the cost to the individual and increased access to the low-vision service compared to in-person services.
The literature review has several limitations. This review represents peer-reviewed literature published in English only. Another flaw in the review is the fact that many of the studies included assessed the role of optometry in telehealth as a secondary research outcome. Furthermore, the dates of publication vary widely and extend back to 1999. Technological innovation in telehealth has rapidly evolved over the past two decades, and therefore the results from the studies should be considered in the context of publication date. The scope of practice in optometry varies between countries. At present, comanagement between ophthalmology and optometry is commonplace in many countries. As a result, the studies included presented recommendations about the role of optometry in telehealth that varied depending on the location and may not reflect what is available globally. This variability reflects a paucity of studies in the area of teleoptometry and the need for further research.
The role of optometrists in telehealth is rapidly emerging, and the field has great potential. Teleoptometry appears to be a viable adjunct to the delivery of eye care, or where necessary, an alternative to in-person optometric services. The literature highlights the multiple applications of optometry-facilitated telehealth, including care delivered as part of a collaboration with ophthalmology, or independently through telerehabilitation and teleoptometry. The suitability of teleoptometry as an adjunct to face-to-face optometric care using telehealth is feasible. The literature demonstrates teleoptometry to be highly acceptable to patients and practitioners. The role of optometrists in providing eye care services via telehealth has value for many individuals, including the elderly, people living with a disability, and people living in rural locations. However, there is a scarcity of evidence regarding clinical benefits, safety, and outcomes of optometry-facilitated teleoptometry. More significant research is required to determine safe and effective models of integrating optometry into the delivery of telehealth services. Furthermore, there is only a small number of peer-reviewed policies and protocols published at present to guide the delivery of optometry services via telehealth platforms. This review of the current evidence base in optometry services delivered via telehealth will guide future research and policy creation. The continued creation of focused, inclusive teleoptometry strategies and policies will support achieving equitable access to optometry services. Further, additional advocacy surrounding increased research that supports policy development and appropriate reimbursement for optometric services is needed to address financial barriers to uptake. Policymakers should heed caution in recommending ongoing and increased use of teleoptometry applications pending further research. This review demonstrates the severe paucity of evidence-based research in teleoptometry. Providers of teleoptometry should be mindful that none of the studies included an economic analysis of optometric telehealth. No studies included a safety analysis, highlighting the urgent need for research in the area. As the field of teleoptometry is in its infancy, outside of the area of low-vision telerehabilitation, there is limited peer-reviewed evidence to support optometry services delivered via telehealth as a safe, acceptable and cost-effective means to provide health care. Findings of the review did not constitute evidence of the safety of optometric care via telehealth, unless under the supervision of an ophthalmologist where a comanagement plan is in place.
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Developing a Machine Learning ‘Smart’ Polymerase Chain Reaction Thermocycler Part 2: Putting the Theoretical Framework into Practice | d2c05beb-799f-4dd6-86f5-16ab7bc3b0bd | 11431514 | Forensic Medicine[mh] | Since its conceptualisation, the polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world of deoxyribonucleic acid (DNA) around us. Despite the PCR having a wide range of applications across the fields of biology and medicine, the basic formula it uses for amplification has undergone little change since it was first introduced to forensic science in the early 1990s . The development of a PCR method that enabled simultaneous amplification of four hypervariable regions, known as short tandem repeats (STRs), in the human genome marked the beginning of a transformative era in forensic science, an era where the value of DNA evidence skyrocketed . Since then, the number of regions targeted for amplification has expanded from 6 in 1995 to 25 in 2018 , and there has been a significant decrease in the amount of DNA required to produce informative DNA profiles, which has been demonstrated on a single cell (0.006 ng , down from 2 ng in 1994) . These developments have allowed DNA profiling to become a highly sensitive technique that affords high powers of discrimination between individuals. However, despite these advancements, the PCR cycling conditions used today are largely the same as those first used in the 1990s, with uniform conditions used in most forensic applications . This uniform approach does not adjust or account for the variations in enzyme activity and amounts of target DNA across the PCR cycles, meaning there is potential for further optimisation in the system. Trace DNA is commonly defined as low levels of DNA from which the biological source is unknown. DNA deposited from a touch is usually considered trace DNA. While trace DNA is one of the most common sample types encountered by operational forensic laboratories , it remains a sample type that has little success in producing informative DNA profiles . Previous efforts have concentrated on pre-amplification of the whole genome , direct PCR of the sample , single-cell amplification or reduced reaction volumes . Despite the many advancements in the PCR for forensic science outside of cycling conditions, the limits of the technology are continually being pushed in an attempt to process smaller quantities, in more compromised conditions, and in shorter timeframes. One exception to the general lack of experimentation in PCR cycling conditions is in the area of speeding up reaction time within specialised equipment. Altered PCR protocols have been successfully integrated into forensic casework (such as in Rapid DNA); however, when these protocols are used with sub-optimal samples, the DNA profiles often contain undesirable characteristics, for example, small peak heights, heterozygote imbalance and dropout. Thus, there is still a need to overcome the challenges that trace samples present for DNA profiling . Part 1 of this publication proposed a framework to produce a smart PCR system that uses machine learning to identify PCR program adjustments that can improve DNA profiling on a per-sample basis using real-time fluorescence feedback produced as the PCR proceeds. Theoretically, this system could be used to generate optimal DNA profiles from a range of challenging sample types (such as trace or degraded types and/or samples containing inhibitors) by amplifying each using individually tailored cycling conditions. A range of goals could be set for the system, such as to improve the quality of a profile (as just mentioned), or to reduce the cost (by altering PCR components or reducing volume while changing cycling conditions to maintain quality), or to improve efficiency (such as reducing the time taken to complete a PCR, again without a practically relevant reduction in profile quality). In this paper, we adjust PCR cycling conditions and apply the profile scoring metric for DNA profile comparison as outlined in the smart PCR framework in part 1 as a proof-of-concept implementation of the proposed framework. We initially chose to target PCR time reduction for the proof-of-concept implementation, for the following reasons: The variable that is tied directly to the goal (time) is an easily measurable quantity for which there can be no dispute (unlike a goal such as improved quality, in which there is subjectivity over the relative importance of different features, such as peak height vs. balance). There is a current interest in producing quick PCR programs, evident in the rise in the number and popularity of Rapid DNA instruments. There are existing alternate protocols being used in Rapid DNA instruments that can provide some guidance on which components of PCR programs can be altered and in what way. In later work, we intend to explore other goals, such as improving DNA profile quality from low-level or degraded profiles or overcoming inhibition. In part 1, we provided an outline of all the components theoretically required to build a smart PCR system. In this study, we carried out a pilot study to demonstrate the performance of the theory to achieve the specific goal of reducing PCR runtime through a large-scale PCR cycling condition alteration experiment. Although we did trial the machine learning optimisation method proposed, the main aspect we did not test was the full training of an ANN-based statistical model (digital twin) of the PCR process. This was due to the absence of a large database upon which to train such a system. We intend to revisit this aspect later. As well as trialling some of the foundational components of the larger project in a manual way, the purpose of this study was to begin to build up a bank of real-time fluorescence data that could be used to train machine learning algorithms for PCR optimisation. 2.1. Ethics Approval Ethics approval for this project was obtained from the Low-Risk Human Research panel of the Social and Behavioural Research Ethics Committee at Flinders University, Australia (reference number 4915). All DNA samples provided by a volunteer were obtained with informed consent. 2.2. Setting the Goal We initially set the goal of reducing the time a PCR program takes to run with minimal loss of DNA profile quality. We chose this goal as it is relatively easy to achieve. By this, we mean PCR programs can certainly be changed in ways that guarantee they take less time. There is also literature relating to reduced PCR program times from use in Rapid DNA instruments and other quick PCR program research that could be consulted. A goal to reduce PCR program time also meant that the experiments could be conducted on optimal amounts of pristine DNA, which remained constant across all experiments. 2.3. Setting the Real-Time Feedback Mechanism We trialled the use of a combined STR PCR and qPCR reaction mix as described by McDonald et al. . The control DNA obtained and used in part 1 was also used as control DNA for all experiments in this study. The standard GlobalFiler (ThermoFisher Scientific, Scoresby, VIC, Australia) PCR components comprised 7.5 µL of master mix, 2.5 µL of primer mix and 15 µL of DNA in solution (made up to a concentration so that 500 pg of DNA was used in the PCR). The combined kit used 7.5 µL of GlobalFiler master mix, 2.5 µL of GlobalFiler primer mix, 4.5 µL of Investigator Quantiplex Pro master mix, 4.5 µL of Investigator Quantiplex Pro reaction mix and 6 µL of DNA in solution (made up to a concentration so that 500 pg of DNA was used in the PCR). We compared the results obtained from the combined quantitation/STR PCR system described above with those of a two-tube system where Quantiplex Pro reaction mixture was set as per the manufacturer’s instruction (but at half volume so as to align with the half volume in the combined kit and allow direct comparison of performance), and a GlobalFiler reaction was set up as per the manufacturer’s instructions. These two reactions were amplified separately but at the same time, both using the manufacturer’s recommended 30-cycle GlobalFiler PCR protocol. The PCR products were separated on a 3500 Genetic Analyzer TM (ThermoFisher Scientific) using 8.5 μL Hi-Di Formamide, 0.5 μL 600 LIZ ® Size Standard (ThermoFisher Scientific) and 1 μL of amplified PCR product. The 3500 settings were 1.2 kV/15 s injection, 13 kV/1550 s runtime, dye set J6. 2.4. Calculating the Profile Score Metric In part 1, we defined a score, f ( P ), for a DNA profile P with allele peak heights x 1 , …, x n by (1) f P = K 0 [ log 10 p x ¯ | μ a , σ a − C + K 1 log 10 p c v | λ + K 2 n a + K 3 t where the following definitions apply: x ¯ = 1 N ∑ i = 1 n x i is the mean of all observed allelic peaks; p x ¯ | μ a , σ a ∼ N μ a , σ a is the probability density function at the point x ¯ for the distribution with mean μ a (the ideal or reference mean value) and standard deviation σ a (the value for weighting variations from the ideal); C = log 10 p μ a | μ a , σ a + l o g 10 λ is the constant offset to ensure an ideal peak height achieved a score of 0; p c v | λ ∼ exp λ is the exponential distribution at the point c v with coefficient λ ; c v = 1 N ∑ i = 1 n x i − x ¯ 2 X ¯ is the coefficient of variation in the peaks; n a is the number of observed artefact peaks; t is the time (in minutes) the PCR program takes to complete; K parameters weight the importance of each element of the score. All calculations and production of graphs were carried out in R . With reference to Equation (1), we used values K 0 = 1 , μ a = 5000 , σ a = 1000 , λ = 1 and K 2 = − 2 The choice of parameter values for the target peak height ( μ a ) and standard deviation ( σ a ) was subjective and based on the personal experience of the authors. Based on the selection of μ a and σ a , the peak height offset value (that resulted in a baseline value of 0) C was calculated to be −3.399. The value for the COV ( λ ) was largely irrelevant as it scaled linearly by the gain value ( K 1 ). During the pilot parameter tests, the average score for the peak height component was found to be −2.047. Using this as a target, K 1 was set to 5 to ensure, on average, an equal contribution from the COV component of the score based on the pilot study (−2.083). The value chosen to penalise any artefacts present in the profiles ( K 2 ) was subjectively set to −2 to ensure that profiles containing artefacts were harshly penalised. Three different values were used for the time component, K 3 = 0 , − 0.01 , − 0.05 , representing no, small and large weightings. The value of −0.05 gave, on average, from the pilot study, a score of −2.191, which was approximately equal to the height and COV components of the score. This meant that, when time had a high weighting, the three main quality components (allele peak height, allele peak height variation and running time) were approximately equally weighted, while a single artefact in the data would contribute approximately equally to any one of the other components. 2.5. Trialling Different PCR Program Alterations PCR program alterations were either carried out on the denaturation step or the annealing/extension step of the standard GlobalFiler PCR program. In addition, changes were either made in a way that altered each cycle in the same way (referred to as ‘bulk’ changes) or made in a way that was incremental across the program (referred to as ‘gradient’ changes). shows the different set-ups of PCR programs in parameterised form. Note that the one-step changes (altering only one aspect of the PCR program) were carried out first, and then the final two-, three- or four-step changes (altering multiple aspects of the PCR program together) were carried out after review of the initial one-step change results. Note that denaturation temperature program changes were trialled even though they did not change the timing of the PCR program. This was carried out as the interaction between temperature and timing changes was unknown, and it was hypothesised that a poor-quality profile produced using a quicker PCR program may be improved by a temperature change. A previous study explored the viability of using published Rapid DNA programs for non-rapid PCR kits and found that the STR profiles generated were of very poor quality . It is hypothesised that the components within Rapid DNA PCR kits are optimised for these rapid programs; thus, when non-rapid kits are used with Rapid DNA PCR programs, they produce poor DNA profiles. As a result, we did not use cycling conditions from published Rapid DNA PCR programs for this study; instead, we made small, controlled changes in the PCR cycling conditions from a standard program. This allowed us to acquire knowledge of how each of these small changes influenced DNA profile quality, which can be used as training data in subsequent projects. 2.6. Weighing Time against Profile Quality Profiles’ scores were considered in three ways: unadjusted, considering time as mildly important and considering time as highly important. The unadjusted values were the raw profile quality scores obtained by inspection of the DNA profiles ( K 3 = 0 in Equation (1)). When considering time as being mildly important, the raw profile quality score was adjusted by subtracting 0.01 for each minute the program ran ( K 3 = −0.01 in Equation (1)). When considering time as being highly important, the raw profile quality score was adjusted by subtracting 0.05 for each minute the program ran ( K 3 = −0.05 in Equation (1)). 2.7. Comparing Profile Quality between PCR Programs The quality of DNA profiles generated using the various altered PCR programs was compared to the quality of the profiles generated using the standard GlobalFiler conditions. The quality of DNA profiles produced using the combination kit was compared to those produced using GlobalFiler only for each of the altered PCR programs and the standard GlobalFiler program. All comparisons were conducted using t -testing in R , where p -values less than 0.05 were considered significant. 2.8. Using Experimental Results to Suggest New PCR Cycling Conditions At the beginning of the experiment, there was limited information about how changes in PCR cycling conditions may affect profile quality. Therefore, the PCR cycling condition changes were made by parameterising the conditions of the cycling program, and these were altered in a controlled manner to build a bank of data which could be used to inform further choices. shows typical PCR program cycling conditions, parameterised so that each time, t , and each temperature, C , for each step of a cycle (i.e., denaturation, annealing and extension) have their own terms. At the most configurable, this leads to 6 N parameters for an N cycle program. Such a large number of parameters is likely to take too many experiments to optimise individually and so parameter values can be tied to each other to reduce the dimensionality of the problem. At the most extreme, for the task of reducing PCR program time, the problem could be reduced to a single dimension by scaling the time of each step down proportionally. In , for step X in cycle N , the temperature is C X,N and is held for t X,N seconds. We parameterised the times and temperatures using the following linear equations: (2) Denaturation time : t D , n = β t , D , 0 + β D , 1 N (3) Denaturation temperature : C D , n = β C , D , 0 + β C , D , 1 N (4) Annealing / extension time : t A E , n = β t , A E , 0 + β t , A E , 1 N (5) Annealing / extension temperature : C A E , n = β C , A E , 0 + β C , A E , 1 N Note that we combined the annealing and extension into a single term compared to given that the GlobalFiler PCR cycling conditions only have two steps, a denaturation and an annealing/extension step. Using the linear equations, the time and temperature could be changed so that the same time (or temperature) is used in each step (referred to a as bulk change) by setting the slope terms, β . , . , 1 = 0 . Alternatively, gradient changes can be achieved by setting a non-zero value for the slope term. For example, if the denaturation step time is being considered, then Equation (2) is used. If the starting time is 10 s, then β t , D , 0 = 10 . If no change to this timing is desired across the PCR program, then the value for β D , 1 would be set to 0, and then, for all cycles, t D , n = 10 . If a gradient is desired, such as a 1 s decrease every 5 cycles, then β D , 1 = − 0.2 . This would lead to the GC1 denaturing timing conditions seen in . For each of the programs listed in , we had five replicate profiles. We ignored the annealing/extension temperature parameters as they were not altered in any of the PCR program changes trailled. Therefore, we had 85 profiles to preliminarily explore the six-variable parameter space. Once initial information about the system performance over the parameters’ space (as detailed in ) was collected, it was necessary to use that information to perform optimisation to discover the optimal set of parameters for a given situation. The method chosen to perform this was based on the firefly algorithm . Conceptually, each set of parameters could be thought of as a firefly, flying around in six-dimensional space. Each set of parameters leads to a DNA profile produced in a specific time, and, ultimately, a score, which, in the firefly analogy, is related to the fly’s brightness. Flies are attracted to brighter areas (high scores) and repelled from darker areas (low scores), and so, for each iteration, each fly will move according to the brightness of the fireflies around it. The more distant the brightness, the weaker the effect. For each set of parameters (e.g., a single firefly) going into the algorithm, a set of parameters will be obtained after an iteration of movement (firefly at a new location). See . Specifically for our application, the following process was undertaken: (1) Sensible bounds were placed on each parameter so that the algorithm did not suggest values that would clearly not work. This step was only required due to the low number of datapoints, and, in larger datasets, the bounds on parameter values would be self-imposed due to lower scores obtained as the parameter values become more extreme. The bounds placed on the parameters were 1 ≤ β t , D , 0 ≤ 15 , − 0.4 ≤ β t , D , 1 ≤ 0.4 , 87 ≤ β C , D , 0 ≤ 95 , − 0.3 ≤ β C , D , 1 ≤ 0.3 , 25 ≤ β t , A E , 0 ≤ 100 , − 1.5 ≤ β t , A E , 1 ≤ 1.5 . These limits were put in place to prevent parameters such as negative times or excessive temperatures that could damage the DNA. (2) The supplied parameters were used in the PCR process. Performance scores were generated from the results based on Equation (1). (3) Each parameter value was scaled (normalised) to be between 0 and 1 so that each parameter had the same level of optimisation within the algorithm. (4) The performance score for each parameter set was normalised between −1 and 1 to ensure high values were attractive and low values repulsive. (5) A learning rate (which dictates the size of the move that can be made in each iteration) of 0.5 was set. If a very large number of repetitions were undertaken, this level could be reduced after a number of rounds to better hone in on the optimal parameters. (6) For each point of focus, the parametric distance to each other point was calculated. (7) The movement of the point of focus from each other point was set as the difference between the current parameter position of the point of focus and other parameter sets, multiplied by the learning rate, multiplied by the score, and scaled by one less than the number of datapoints. (8) The new positions were then rescaled back to their natural form and represented new sets of parameter values. (9) The new parameter values were tested, performance scores determined, and the cycle of the process could be started again with the new parameters. When many points exist, they can be ranked based on the theoretical time the program would take to run; however, we chose to run one iteration of the firefly algorithm to produce a full set of new values that could be trialled. Ethics approval for this project was obtained from the Low-Risk Human Research panel of the Social and Behavioural Research Ethics Committee at Flinders University, Australia (reference number 4915). All DNA samples provided by a volunteer were obtained with informed consent. We initially set the goal of reducing the time a PCR program takes to run with minimal loss of DNA profile quality. We chose this goal as it is relatively easy to achieve. By this, we mean PCR programs can certainly be changed in ways that guarantee they take less time. There is also literature relating to reduced PCR program times from use in Rapid DNA instruments and other quick PCR program research that could be consulted. A goal to reduce PCR program time also meant that the experiments could be conducted on optimal amounts of pristine DNA, which remained constant across all experiments. We trialled the use of a combined STR PCR and qPCR reaction mix as described by McDonald et al. . The control DNA obtained and used in part 1 was also used as control DNA for all experiments in this study. The standard GlobalFiler (ThermoFisher Scientific, Scoresby, VIC, Australia) PCR components comprised 7.5 µL of master mix, 2.5 µL of primer mix and 15 µL of DNA in solution (made up to a concentration so that 500 pg of DNA was used in the PCR). The combined kit used 7.5 µL of GlobalFiler master mix, 2.5 µL of GlobalFiler primer mix, 4.5 µL of Investigator Quantiplex Pro master mix, 4.5 µL of Investigator Quantiplex Pro reaction mix and 6 µL of DNA in solution (made up to a concentration so that 500 pg of DNA was used in the PCR). We compared the results obtained from the combined quantitation/STR PCR system described above with those of a two-tube system where Quantiplex Pro reaction mixture was set as per the manufacturer’s instruction (but at half volume so as to align with the half volume in the combined kit and allow direct comparison of performance), and a GlobalFiler reaction was set up as per the manufacturer’s instructions. These two reactions were amplified separately but at the same time, both using the manufacturer’s recommended 30-cycle GlobalFiler PCR protocol. The PCR products were separated on a 3500 Genetic Analyzer TM (ThermoFisher Scientific) using 8.5 μL Hi-Di Formamide, 0.5 μL 600 LIZ ® Size Standard (ThermoFisher Scientific) and 1 μL of amplified PCR product. The 3500 settings were 1.2 kV/15 s injection, 13 kV/1550 s runtime, dye set J6. In part 1, we defined a score, f ( P ), for a DNA profile P with allele peak heights x 1 , …, x n by (1) f P = K 0 [ log 10 p x ¯ | μ a , σ a − C + K 1 log 10 p c v | λ + K 2 n a + K 3 t where the following definitions apply: x ¯ = 1 N ∑ i = 1 n x i is the mean of all observed allelic peaks; p x ¯ | μ a , σ a ∼ N μ a , σ a is the probability density function at the point x ¯ for the distribution with mean μ a (the ideal or reference mean value) and standard deviation σ a (the value for weighting variations from the ideal); C = log 10 p μ a | μ a , σ a + l o g 10 λ is the constant offset to ensure an ideal peak height achieved a score of 0; p c v | λ ∼ exp λ is the exponential distribution at the point c v with coefficient λ ; c v = 1 N ∑ i = 1 n x i − x ¯ 2 X ¯ is the coefficient of variation in the peaks; n a is the number of observed artefact peaks; t is the time (in minutes) the PCR program takes to complete; K parameters weight the importance of each element of the score. All calculations and production of graphs were carried out in R . With reference to Equation (1), we used values K 0 = 1 , μ a = 5000 , σ a = 1000 , λ = 1 and K 2 = − 2 The choice of parameter values for the target peak height ( μ a ) and standard deviation ( σ a ) was subjective and based on the personal experience of the authors. Based on the selection of μ a and σ a , the peak height offset value (that resulted in a baseline value of 0) C was calculated to be −3.399. The value for the COV ( λ ) was largely irrelevant as it scaled linearly by the gain value ( K 1 ). During the pilot parameter tests, the average score for the peak height component was found to be −2.047. Using this as a target, K 1 was set to 5 to ensure, on average, an equal contribution from the COV component of the score based on the pilot study (−2.083). The value chosen to penalise any artefacts present in the profiles ( K 2 ) was subjectively set to −2 to ensure that profiles containing artefacts were harshly penalised. Three different values were used for the time component, K 3 = 0 , − 0.01 , − 0.05 , representing no, small and large weightings. The value of −0.05 gave, on average, from the pilot study, a score of −2.191, which was approximately equal to the height and COV components of the score. This meant that, when time had a high weighting, the three main quality components (allele peak height, allele peak height variation and running time) were approximately equally weighted, while a single artefact in the data would contribute approximately equally to any one of the other components. PCR program alterations were either carried out on the denaturation step or the annealing/extension step of the standard GlobalFiler PCR program. In addition, changes were either made in a way that altered each cycle in the same way (referred to as ‘bulk’ changes) or made in a way that was incremental across the program (referred to as ‘gradient’ changes). shows the different set-ups of PCR programs in parameterised form. Note that the one-step changes (altering only one aspect of the PCR program) were carried out first, and then the final two-, three- or four-step changes (altering multiple aspects of the PCR program together) were carried out after review of the initial one-step change results. Note that denaturation temperature program changes were trialled even though they did not change the timing of the PCR program. This was carried out as the interaction between temperature and timing changes was unknown, and it was hypothesised that a poor-quality profile produced using a quicker PCR program may be improved by a temperature change. A previous study explored the viability of using published Rapid DNA programs for non-rapid PCR kits and found that the STR profiles generated were of very poor quality . It is hypothesised that the components within Rapid DNA PCR kits are optimised for these rapid programs; thus, when non-rapid kits are used with Rapid DNA PCR programs, they produce poor DNA profiles. As a result, we did not use cycling conditions from published Rapid DNA PCR programs for this study; instead, we made small, controlled changes in the PCR cycling conditions from a standard program. This allowed us to acquire knowledge of how each of these small changes influenced DNA profile quality, which can be used as training data in subsequent projects. Profiles’ scores were considered in three ways: unadjusted, considering time as mildly important and considering time as highly important. The unadjusted values were the raw profile quality scores obtained by inspection of the DNA profiles ( K 3 = 0 in Equation (1)). When considering time as being mildly important, the raw profile quality score was adjusted by subtracting 0.01 for each minute the program ran ( K 3 = −0.01 in Equation (1)). When considering time as being highly important, the raw profile quality score was adjusted by subtracting 0.05 for each minute the program ran ( K 3 = −0.05 in Equation (1)). The quality of DNA profiles generated using the various altered PCR programs was compared to the quality of the profiles generated using the standard GlobalFiler conditions. The quality of DNA profiles produced using the combination kit was compared to those produced using GlobalFiler only for each of the altered PCR programs and the standard GlobalFiler program. All comparisons were conducted using t -testing in R , where p -values less than 0.05 were considered significant. At the beginning of the experiment, there was limited information about how changes in PCR cycling conditions may affect profile quality. Therefore, the PCR cycling condition changes were made by parameterising the conditions of the cycling program, and these were altered in a controlled manner to build a bank of data which could be used to inform further choices. shows typical PCR program cycling conditions, parameterised so that each time, t , and each temperature, C , for each step of a cycle (i.e., denaturation, annealing and extension) have their own terms. At the most configurable, this leads to 6 N parameters for an N cycle program. Such a large number of parameters is likely to take too many experiments to optimise individually and so parameter values can be tied to each other to reduce the dimensionality of the problem. At the most extreme, for the task of reducing PCR program time, the problem could be reduced to a single dimension by scaling the time of each step down proportionally. In , for step X in cycle N , the temperature is C X,N and is held for t X,N seconds. We parameterised the times and temperatures using the following linear equations: (2) Denaturation time : t D , n = β t , D , 0 + β D , 1 N (3) Denaturation temperature : C D , n = β C , D , 0 + β C , D , 1 N (4) Annealing / extension time : t A E , n = β t , A E , 0 + β t , A E , 1 N (5) Annealing / extension temperature : C A E , n = β C , A E , 0 + β C , A E , 1 N Note that we combined the annealing and extension into a single term compared to given that the GlobalFiler PCR cycling conditions only have two steps, a denaturation and an annealing/extension step. Using the linear equations, the time and temperature could be changed so that the same time (or temperature) is used in each step (referred to a as bulk change) by setting the slope terms, β . , . , 1 = 0 . Alternatively, gradient changes can be achieved by setting a non-zero value for the slope term. For example, if the denaturation step time is being considered, then Equation (2) is used. If the starting time is 10 s, then β t , D , 0 = 10 . If no change to this timing is desired across the PCR program, then the value for β D , 1 would be set to 0, and then, for all cycles, t D , n = 10 . If a gradient is desired, such as a 1 s decrease every 5 cycles, then β D , 1 = − 0.2 . This would lead to the GC1 denaturing timing conditions seen in . For each of the programs listed in , we had five replicate profiles. We ignored the annealing/extension temperature parameters as they were not altered in any of the PCR program changes trailled. Therefore, we had 85 profiles to preliminarily explore the six-variable parameter space. Once initial information about the system performance over the parameters’ space (as detailed in ) was collected, it was necessary to use that information to perform optimisation to discover the optimal set of parameters for a given situation. The method chosen to perform this was based on the firefly algorithm . Conceptually, each set of parameters could be thought of as a firefly, flying around in six-dimensional space. Each set of parameters leads to a DNA profile produced in a specific time, and, ultimately, a score, which, in the firefly analogy, is related to the fly’s brightness. Flies are attracted to brighter areas (high scores) and repelled from darker areas (low scores), and so, for each iteration, each fly will move according to the brightness of the fireflies around it. The more distant the brightness, the weaker the effect. For each set of parameters (e.g., a single firefly) going into the algorithm, a set of parameters will be obtained after an iteration of movement (firefly at a new location). See . Specifically for our application, the following process was undertaken: (1) Sensible bounds were placed on each parameter so that the algorithm did not suggest values that would clearly not work. This step was only required due to the low number of datapoints, and, in larger datasets, the bounds on parameter values would be self-imposed due to lower scores obtained as the parameter values become more extreme. The bounds placed on the parameters were 1 ≤ β t , D , 0 ≤ 15 , − 0.4 ≤ β t , D , 1 ≤ 0.4 , 87 ≤ β C , D , 0 ≤ 95 , − 0.3 ≤ β C , D , 1 ≤ 0.3 , 25 ≤ β t , A E , 0 ≤ 100 , − 1.5 ≤ β t , A E , 1 ≤ 1.5 . These limits were put in place to prevent parameters such as negative times or excessive temperatures that could damage the DNA. (2) The supplied parameters were used in the PCR process. Performance scores were generated from the results based on Equation (1). (3) Each parameter value was scaled (normalised) to be between 0 and 1 so that each parameter had the same level of optimisation within the algorithm. (4) The performance score for each parameter set was normalised between −1 and 1 to ensure high values were attractive and low values repulsive. (5) A learning rate (which dictates the size of the move that can be made in each iteration) of 0.5 was set. If a very large number of repetitions were undertaken, this level could be reduced after a number of rounds to better hone in on the optimal parameters. (6) For each point of focus, the parametric distance to each other point was calculated. (7) The movement of the point of focus from each other point was set as the difference between the current parameter position of the point of focus and other parameter sets, multiplied by the learning rate, multiplied by the score, and scaled by one less than the number of datapoints. (8) The new positions were then rescaled back to their natural form and represented new sets of parameter values. (9) The new parameter values were tested, performance scores determined, and the cycle of the process could be started again with the new parameters. When many points exist, they can be ranked based on the theoretical time the program would take to run; however, we chose to run one iteration of the firefly algorithm to produce a full set of new values that could be trialled. 3.1. Combined GlobalFiler and Investigator Quantiplex Pro Results DNA profiles were generated from the standard GlobalFiler-only PCR and the combined PCR mixture (the qPCR and PCR combined kit) when separated by capillary electrophoresis. In general, there was a loss of peak height for the combined kit. shows the distribution of height differences between the combined kit and standard kit. The distribution is modelled on the per-profile average peak height of the DNA profiles produced by the GlobalFiler-only kit divided by the average peak height of the DNA profiles produced by the combined kit. While the combined kit was found to produce DNA profiles with lower average peak heights than the profiles produced using GlobalFiler only (approximately 1.6 times or log 10 [0.21]), this variation was found to be insignificant ( p = 0.077, shaded area in ). The Standard Quantiplex Pro reaction was run for 40 cycles. The red dashed point in shows the 30-cycle mark, which corresponds to the endpoint of a standard GlobalFiler PCR program. It can be seen that the inflection point of the quantification reaction has not been reached. There is a risk that even using a two-tube system (where the quantification and STR amplification reactions are in separate vessels) without 10 cycles of pre-amplification of the quantification reaction prior to the start of the accompanying GlobalFiler PCR will not provide useable real-time information. This fact is shown in , where the quantification reaction is depicted under standard qPCR conditions, and the curve only just starts to visibly increase in the final cycles (>23). It is yet to be seen whether there is sufficient information in the real-time fluorescence in this combined system to facilitate learning and, eventually, real-time program adaption. However, what is clear is that the PCR system developed using a combined GlobalFiler/Quantiplex Pro reaction did provide some real-time fluorescence feedback that could be exploited. Furthermore, the fluorescence data obtained from the GlobalFiler/Quantiplex Pro combination were compared to the fluorescence data obtained using the two-tube method, where the GlobalFiler and Quantiplex Pro reactions were conducted separately. The resulting amplification curves indicate that the STR kit does produce some detectable fluorescence when used independently of the qPCR reagents. However, as the primers in the STR kit will fluoresce regardless of if they are incorporated or unincorporated, the amount of fluorescence produced by them should remain constant across the PCR. This means there is the potential to correct for any fluorescence produced by the STR primers in the first few cycles to significantly reduce if not eliminate the interference of the STR kit in the real-time fluorescence feedback we aim to acquire. In , it can be seen that the Quantiplex Pro system alone produced very little fluorescence (which was smoothed to a flat line by the model). Despite this, the fluorescence from the combined Quantiplex Pro and GlobalFiler system was markedly more intense than the GlobalFiler alone. It is not currently clear whether there was some interaction occurring to cause this unexpected jump in fluorescence in the combined system. The performance of the real-time fluorescent feedback under varying conditions is the next phase of research to be conducted in our work. 3.2. Results of Altering PCR Program Conditions 3.2.1. One-Step Changes to Denaturation Timing When profile quality was considered in isolation, the PCR protocols involving bulk changes to denaturation timing using the combination kit produced DNA profiles that were significantly lower in quality than profiles produced following the standard GlobalFiler protocol . The PCR protocol involving a gradually decreasing denaturation timing (gradient change 1 protocol) with the combination kit produced profiles of significantly lower quality than the standard protocol, but generally higher quality than the other altered protocols. Consideration of the quality of the DNA profile, and incorporating the timing penalty, led to a ‘score’ (based on Equation (1)). When the PCR runtime was considered mildly important in quality assessment, the programs with altered denaturation times using the combination kit were up to 29 min faster. The bulk change programs, and one gradient program (gradient change 2 protocol), still produced profiles with a significantly lower score than the standard. Notably, the gradient change 1 program produced profiles that were approximately equal in quality to those produced using the standard protocol . Considering the PCR runtime as highly important when scoring found that profiles produced using both bulk change programs and the gradient change 2 program still yielded significantly lower scores than the standard profiles. However, the profiles produced using the gradient change 1 program were now of significantly higher quality than the profiles produced following the standard protocol. DNA profiles produced using a denaturation time of 5 s (bulk change 1 protocol) with only the GlobalFiler kit were not of significantly different quality to those produced using the combination kit for the same protocol across all scores (see ). Similarly, the profiles produced using a gradually decreasing denaturation timing (gradient change 1 protocol) or gradually increasing denaturation time (gradient change 2 protocol) with GlobalFiler only were of approximately equal quality to those produced using the combination kit under the same conditions. However, the profiles generated using a denaturation time of 2 s (bulk change 2 protocol) with the GlobalFiler kit were notably lower in quality than the profiles produced using the same PCR program with the combination kit across all scores (see ). However, when the scores were standardised, the DNA profiles produced using the GlobalFiler kit were not significantly different in quality to those produced using the combination kit for the same protocol across all standardised scores (see ). 3.2.2. One-Step Changes to Annealing/Extension Timing The DNA profiles generated using an annealing/extension time of 60 s (bulk change 1 protocol) and 30 s (bulk change 2 protocol) with the combination kit were significantly lower in comparison to the profiles produced under the standard GlobalFiler conditions ( p = 0.0216 and p = 0.0122, respectively) . The profiles generated with a PCR program involving a gradual increase or decrease in annealing/extension timing (gradient change protocols 1 and 2) using the combination kit were not found to have a significant variation in raw quality score when compared to the standard profiles (see ). When considering the PCR runtime as mildly important, the influence of the bulk and gradient changes to annealing/extension timing on overall profile score was largely conserved despite the altered protocols being up to 50 min faster than the standard protocol. The DNA profiles generated using the two gradient change programs were still approximately equal in score to those generated using the standard program. While the profiles produced using the bulk change 2 protocol were still of significantly lower score than the standard profiles ( p = 0.0122), the profiles produced using the bulk change 1 protocol were found to be of approximately equal score to the standard . When PCR runtime was considered to be highly important, the bulk change 1 protocol produced DNA profiles that were of significantly higher score than the profiles generated using the standard GlobalFiler protocol . The profiles generated using the gradient change protocols were still found to be of approximately equal score to the standards, and the profiles generated using the bulk change 2 protocol had a significantly lower score than the standards ( p = 0.0122). The profiles generated using a gradually decreasing annealing/extension time (gradient change 1 protocol) with the combination kit were found to be of approximately equal quality to those produced using GlobalFiler only for the same protocols (see ). However, the DNA profiles produced using an annealing/extension time of 60 s (bulk change 1 protocol), an annealing/extension time of 30 s (bulk change 2 protocol) and a gradually increasing annealing/extension time (gradient change 2 protocol) with only the GlobalFiler kit were found to be of significantly lower quality than those produced using the combination kit for the same protocol across all scores ( p = 0.033, p = 0.00038 and p = 0.0316). When the scores were standardised, the DNA profiles produced using the gradient change programs and the GlobalFiler kit were not significantly different in quality to those produced using the combination kit for the same protocol across all standardised scores (see ). However, despite standardisation, the profiles produced using a reduced annealing/extension time of 30 s with GlobalFiler only were still found to be of significantly lower quality than those produced using the combination kit for the same protocol across all scores ( p = 0.0117). 3.2.3. One-Step Changes to Denaturation Temperature All DNA profiles generated using an altered denaturation temperature with the combination kit were found to be significantly lower in quality than the profiles produced under the standard GlobalFiler conditions . However, while the DNA profiles produced using a gradient increase in denaturation temperature (gradient change 2 protocol) were significantly lower in quality than the standard profiles, of all the altered protocols trialled, these were closest in quality to the standards. The DNA profiles generated using the combination kit were not significantly different in overall quality than the DNA profiles generated using only the GlobalFiler kit for all programs involving an altered denaturation temperature. The DNA profiles generated using a gradually increasing denaturation temperature with the combination kit were found to be of approximately equal quality to the profiles produced under the standard GlobalFiler conditions . The DNA profiles generated using a denaturation temperature of 90 °C (bulk change protocol 1) and 88 °C (bulk change 2 protocol) with the combination kit were significantly lower in comparison to the profiles produced under the standard GlobalFiler conditions ( p = 0.0122 for both) . The profiles generated using a gradually decreasing denaturation temperature with the combination kit were also found to be significantly lower in quality when compared to the standards ( p = 0.0122) . DNA profiles generated using the combination kit were not significantly different in overall quality to the DNA profiles generated using only the GlobalFiler kit for all programs involving an altered denaturation temperature (see ). When the quality scores were standardised, the profiles produced using the bulk change programs and gradient change 1 program with the combination kit were of significantly lower quality than the profiles produced using the same protocols with GlobalFiler only across all scores . However, the profiles generated using a gradually increasing denaturation temperature (gradient change 2 protocol) with the combination kit were of significantly higher quality than the profiles generated using GlobalFiler only for the same protocol across all standardised scores . 3.2.4. Two-Step Changes When total runtime was not considered, the profiles generated using the optimised protocols with the combination kit were lower quality than those produced following the standard GlobalFiler protocol . When the profile scores were adjusted to include the PCR runtime as mildly important, the variation in quality between profiles produced using the optimised protocols and the standard GlobalFiler protocol with the combination kit was maintained. However, out of all the optimised protocols trialled, the profiles generated using protocol version 3 were closest in quality to the profiles generated using the standard protocol . This can be attributed to optimised protocol version 3 being 30 min faster than the standard GlobalFiler protocol. Lastly, when the scores were adjusted to make the runtime highly important, the profiles generated using optimised protocols 1, 2 and 3 were all of approximately equal quality to the profiles produced following the standard GlobalFiler protocol. This can be attributed to these optimised PCR programs having runtimes that were 28–31 min faster than the standard. Importantly, the DNA profiles produced using the combination kit were also found to be of significantly higher quality than the profiles generated using only GlobalFiler for all of the optimised PCR programs trialled (see ). When standardised, the DNA profiles produced using optimised protocols 2, 3 and 4 with the combination kit were all still of significantly higher quality than the profiles produced using GlobalFiler only, but profiles produced using optimised protocol 1 with the combination kit and GlobalFiler were found to be of approximately equal quality (see ). It is important to note that all optimised PCR programs trialled produced some DNA profiles with instances of dropout; however, the profiles still contained information sufficient for downstream interpretation and analysis. The potential exists to alter the profile metric to make the peak height component more dominant than in this trial. Given a database now exists of all the profiles generated, it is possible to re-evaluate the dataset and trial various combinations of peak height and peak variability model parameters to identify which combination is most intuitively correct. 3.3. Suggesting New PCR Cycling Conditions Using Machine Learning Overall, this provided 85 profiles’ worth of real-time fluorescence data, trialling 17 different combinations of PCR cycling conditions (five repeats of each of the combinations), that could be used in the training of a machine learning algorithm. Before the real-time data were used, we demonstrated the use of machine learning to suggest new parameters based on the DNA profile scoring results obtained. The firefly algorithm was used, incorporating all 17 combinations. Initial trials showed a sub-optimal performance of the firefly algorithm due to the fact that not enough sample space had been explored in the dataset of 17 points. To rectify this, additional parameter sets were developed by generating 60 random values for each of the six parameters ( β t , D , 0 , β t , D , 1 , β C , D , 0 , β C , D , 1 , β t , A E , 0 , β t , A E , 1 ) from truncated normal distributions. The constraints used for each parameter aligned with the bounds for that specific parameter, as identified in . From the 60 random sets of parameters generated, the combinations that pushed the cycling parameters outside of the acceptable ranges were removed. This led to the identification of six additional PCR programs using the randomly generated parameter values shown in . These six randomised parameter sets were then trialled to generate DNA profiles that could be scored and fed back into the system. This increased the sample space explored and thus worked to improve the performance of the algorithm. The results of these randomised PCR programs can be seen in . As expected, all randomised PCR programs gave significantly lower scores than the standard; however, the point of producing these programs was not to obtain high scores, but rather to fill out some of the parameter space to inform the firefly algorithm (in this case, with very dim flies). Hence, the system knew what parameters not to use. Using the 17 original sets of parameter values and the 6 random sets of values, the firefly algorithm was used and suggested the values shown in . To complete the proof of concept, there were four sets of conditions suggested by the firefly algorithm that were trialled (those rows bolded in and marked with an asterisk). The four sets of parameters were chosen to be trialled based on their theoretical success, previous experimental results and the projected impact of the time differences on the scores. Suggested programs S9 and S17 were chosen as they were expected to produce good-quality DNA profiles, while suggested programs S2 and S7 were chosen as they were expected to produce lower-quality DNA profiles. The results of these suggested methods can be seen in . DNA profiles were generated from the standard GlobalFiler-only PCR and the combined PCR mixture (the qPCR and PCR combined kit) when separated by capillary electrophoresis. In general, there was a loss of peak height for the combined kit. shows the distribution of height differences between the combined kit and standard kit. The distribution is modelled on the per-profile average peak height of the DNA profiles produced by the GlobalFiler-only kit divided by the average peak height of the DNA profiles produced by the combined kit. While the combined kit was found to produce DNA profiles with lower average peak heights than the profiles produced using GlobalFiler only (approximately 1.6 times or log 10 [0.21]), this variation was found to be insignificant ( p = 0.077, shaded area in ). The Standard Quantiplex Pro reaction was run for 40 cycles. The red dashed point in shows the 30-cycle mark, which corresponds to the endpoint of a standard GlobalFiler PCR program. It can be seen that the inflection point of the quantification reaction has not been reached. There is a risk that even using a two-tube system (where the quantification and STR amplification reactions are in separate vessels) without 10 cycles of pre-amplification of the quantification reaction prior to the start of the accompanying GlobalFiler PCR will not provide useable real-time information. This fact is shown in , where the quantification reaction is depicted under standard qPCR conditions, and the curve only just starts to visibly increase in the final cycles (>23). It is yet to be seen whether there is sufficient information in the real-time fluorescence in this combined system to facilitate learning and, eventually, real-time program adaption. However, what is clear is that the PCR system developed using a combined GlobalFiler/Quantiplex Pro reaction did provide some real-time fluorescence feedback that could be exploited. Furthermore, the fluorescence data obtained from the GlobalFiler/Quantiplex Pro combination were compared to the fluorescence data obtained using the two-tube method, where the GlobalFiler and Quantiplex Pro reactions were conducted separately. The resulting amplification curves indicate that the STR kit does produce some detectable fluorescence when used independently of the qPCR reagents. However, as the primers in the STR kit will fluoresce regardless of if they are incorporated or unincorporated, the amount of fluorescence produced by them should remain constant across the PCR. This means there is the potential to correct for any fluorescence produced by the STR primers in the first few cycles to significantly reduce if not eliminate the interference of the STR kit in the real-time fluorescence feedback we aim to acquire. In , it can be seen that the Quantiplex Pro system alone produced very little fluorescence (which was smoothed to a flat line by the model). Despite this, the fluorescence from the combined Quantiplex Pro and GlobalFiler system was markedly more intense than the GlobalFiler alone. It is not currently clear whether there was some interaction occurring to cause this unexpected jump in fluorescence in the combined system. The performance of the real-time fluorescent feedback under varying conditions is the next phase of research to be conducted in our work. 3.2.1. One-Step Changes to Denaturation Timing When profile quality was considered in isolation, the PCR protocols involving bulk changes to denaturation timing using the combination kit produced DNA profiles that were significantly lower in quality than profiles produced following the standard GlobalFiler protocol . The PCR protocol involving a gradually decreasing denaturation timing (gradient change 1 protocol) with the combination kit produced profiles of significantly lower quality than the standard protocol, but generally higher quality than the other altered protocols. Consideration of the quality of the DNA profile, and incorporating the timing penalty, led to a ‘score’ (based on Equation (1)). When the PCR runtime was considered mildly important in quality assessment, the programs with altered denaturation times using the combination kit were up to 29 min faster. The bulk change programs, and one gradient program (gradient change 2 protocol), still produced profiles with a significantly lower score than the standard. Notably, the gradient change 1 program produced profiles that were approximately equal in quality to those produced using the standard protocol . Considering the PCR runtime as highly important when scoring found that profiles produced using both bulk change programs and the gradient change 2 program still yielded significantly lower scores than the standard profiles. However, the profiles produced using the gradient change 1 program were now of significantly higher quality than the profiles produced following the standard protocol. DNA profiles produced using a denaturation time of 5 s (bulk change 1 protocol) with only the GlobalFiler kit were not of significantly different quality to those produced using the combination kit for the same protocol across all scores (see ). Similarly, the profiles produced using a gradually decreasing denaturation timing (gradient change 1 protocol) or gradually increasing denaturation time (gradient change 2 protocol) with GlobalFiler only were of approximately equal quality to those produced using the combination kit under the same conditions. However, the profiles generated using a denaturation time of 2 s (bulk change 2 protocol) with the GlobalFiler kit were notably lower in quality than the profiles produced using the same PCR program with the combination kit across all scores (see ). However, when the scores were standardised, the DNA profiles produced using the GlobalFiler kit were not significantly different in quality to those produced using the combination kit for the same protocol across all standardised scores (see ). 3.2.2. One-Step Changes to Annealing/Extension Timing The DNA profiles generated using an annealing/extension time of 60 s (bulk change 1 protocol) and 30 s (bulk change 2 protocol) with the combination kit were significantly lower in comparison to the profiles produced under the standard GlobalFiler conditions ( p = 0.0216 and p = 0.0122, respectively) . The profiles generated with a PCR program involving a gradual increase or decrease in annealing/extension timing (gradient change protocols 1 and 2) using the combination kit were not found to have a significant variation in raw quality score when compared to the standard profiles (see ). When considering the PCR runtime as mildly important, the influence of the bulk and gradient changes to annealing/extension timing on overall profile score was largely conserved despite the altered protocols being up to 50 min faster than the standard protocol. The DNA profiles generated using the two gradient change programs were still approximately equal in score to those generated using the standard program. While the profiles produced using the bulk change 2 protocol were still of significantly lower score than the standard profiles ( p = 0.0122), the profiles produced using the bulk change 1 protocol were found to be of approximately equal score to the standard . When PCR runtime was considered to be highly important, the bulk change 1 protocol produced DNA profiles that were of significantly higher score than the profiles generated using the standard GlobalFiler protocol . The profiles generated using the gradient change protocols were still found to be of approximately equal score to the standards, and the profiles generated using the bulk change 2 protocol had a significantly lower score than the standards ( p = 0.0122). The profiles generated using a gradually decreasing annealing/extension time (gradient change 1 protocol) with the combination kit were found to be of approximately equal quality to those produced using GlobalFiler only for the same protocols (see ). However, the DNA profiles produced using an annealing/extension time of 60 s (bulk change 1 protocol), an annealing/extension time of 30 s (bulk change 2 protocol) and a gradually increasing annealing/extension time (gradient change 2 protocol) with only the GlobalFiler kit were found to be of significantly lower quality than those produced using the combination kit for the same protocol across all scores ( p = 0.033, p = 0.00038 and p = 0.0316). When the scores were standardised, the DNA profiles produced using the gradient change programs and the GlobalFiler kit were not significantly different in quality to those produced using the combination kit for the same protocol across all standardised scores (see ). However, despite standardisation, the profiles produced using a reduced annealing/extension time of 30 s with GlobalFiler only were still found to be of significantly lower quality than those produced using the combination kit for the same protocol across all scores ( p = 0.0117). 3.2.3. One-Step Changes to Denaturation Temperature All DNA profiles generated using an altered denaturation temperature with the combination kit were found to be significantly lower in quality than the profiles produced under the standard GlobalFiler conditions . However, while the DNA profiles produced using a gradient increase in denaturation temperature (gradient change 2 protocol) were significantly lower in quality than the standard profiles, of all the altered protocols trialled, these were closest in quality to the standards. The DNA profiles generated using the combination kit were not significantly different in overall quality than the DNA profiles generated using only the GlobalFiler kit for all programs involving an altered denaturation temperature. The DNA profiles generated using a gradually increasing denaturation temperature with the combination kit were found to be of approximately equal quality to the profiles produced under the standard GlobalFiler conditions . The DNA profiles generated using a denaturation temperature of 90 °C (bulk change protocol 1) and 88 °C (bulk change 2 protocol) with the combination kit were significantly lower in comparison to the profiles produced under the standard GlobalFiler conditions ( p = 0.0122 for both) . The profiles generated using a gradually decreasing denaturation temperature with the combination kit were also found to be significantly lower in quality when compared to the standards ( p = 0.0122) . DNA profiles generated using the combination kit were not significantly different in overall quality to the DNA profiles generated using only the GlobalFiler kit for all programs involving an altered denaturation temperature (see ). When the quality scores were standardised, the profiles produced using the bulk change programs and gradient change 1 program with the combination kit were of significantly lower quality than the profiles produced using the same protocols with GlobalFiler only across all scores . However, the profiles generated using a gradually increasing denaturation temperature (gradient change 2 protocol) with the combination kit were of significantly higher quality than the profiles generated using GlobalFiler only for the same protocol across all standardised scores . 3.2.4. Two-Step Changes When total runtime was not considered, the profiles generated using the optimised protocols with the combination kit were lower quality than those produced following the standard GlobalFiler protocol . When the profile scores were adjusted to include the PCR runtime as mildly important, the variation in quality between profiles produced using the optimised protocols and the standard GlobalFiler protocol with the combination kit was maintained. However, out of all the optimised protocols trialled, the profiles generated using protocol version 3 were closest in quality to the profiles generated using the standard protocol . This can be attributed to optimised protocol version 3 being 30 min faster than the standard GlobalFiler protocol. Lastly, when the scores were adjusted to make the runtime highly important, the profiles generated using optimised protocols 1, 2 and 3 were all of approximately equal quality to the profiles produced following the standard GlobalFiler protocol. This can be attributed to these optimised PCR programs having runtimes that were 28–31 min faster than the standard. Importantly, the DNA profiles produced using the combination kit were also found to be of significantly higher quality than the profiles generated using only GlobalFiler for all of the optimised PCR programs trialled (see ). When standardised, the DNA profiles produced using optimised protocols 2, 3 and 4 with the combination kit were all still of significantly higher quality than the profiles produced using GlobalFiler only, but profiles produced using optimised protocol 1 with the combination kit and GlobalFiler were found to be of approximately equal quality (see ). It is important to note that all optimised PCR programs trialled produced some DNA profiles with instances of dropout; however, the profiles still contained information sufficient for downstream interpretation and analysis. The potential exists to alter the profile metric to make the peak height component more dominant than in this trial. Given a database now exists of all the profiles generated, it is possible to re-evaluate the dataset and trial various combinations of peak height and peak variability model parameters to identify which combination is most intuitively correct. When profile quality was considered in isolation, the PCR protocols involving bulk changes to denaturation timing using the combination kit produced DNA profiles that were significantly lower in quality than profiles produced following the standard GlobalFiler protocol . The PCR protocol involving a gradually decreasing denaturation timing (gradient change 1 protocol) with the combination kit produced profiles of significantly lower quality than the standard protocol, but generally higher quality than the other altered protocols. Consideration of the quality of the DNA profile, and incorporating the timing penalty, led to a ‘score’ (based on Equation (1)). When the PCR runtime was considered mildly important in quality assessment, the programs with altered denaturation times using the combination kit were up to 29 min faster. The bulk change programs, and one gradient program (gradient change 2 protocol), still produced profiles with a significantly lower score than the standard. Notably, the gradient change 1 program produced profiles that were approximately equal in quality to those produced using the standard protocol . Considering the PCR runtime as highly important when scoring found that profiles produced using both bulk change programs and the gradient change 2 program still yielded significantly lower scores than the standard profiles. However, the profiles produced using the gradient change 1 program were now of significantly higher quality than the profiles produced following the standard protocol. DNA profiles produced using a denaturation time of 5 s (bulk change 1 protocol) with only the GlobalFiler kit were not of significantly different quality to those produced using the combination kit for the same protocol across all scores (see ). Similarly, the profiles produced using a gradually decreasing denaturation timing (gradient change 1 protocol) or gradually increasing denaturation time (gradient change 2 protocol) with GlobalFiler only were of approximately equal quality to those produced using the combination kit under the same conditions. However, the profiles generated using a denaturation time of 2 s (bulk change 2 protocol) with the GlobalFiler kit were notably lower in quality than the profiles produced using the same PCR program with the combination kit across all scores (see ). However, when the scores were standardised, the DNA profiles produced using the GlobalFiler kit were not significantly different in quality to those produced using the combination kit for the same protocol across all standardised scores (see ). The DNA profiles generated using an annealing/extension time of 60 s (bulk change 1 protocol) and 30 s (bulk change 2 protocol) with the combination kit were significantly lower in comparison to the profiles produced under the standard GlobalFiler conditions ( p = 0.0216 and p = 0.0122, respectively) . The profiles generated with a PCR program involving a gradual increase or decrease in annealing/extension timing (gradient change protocols 1 and 2) using the combination kit were not found to have a significant variation in raw quality score when compared to the standard profiles (see ). When considering the PCR runtime as mildly important, the influence of the bulk and gradient changes to annealing/extension timing on overall profile score was largely conserved despite the altered protocols being up to 50 min faster than the standard protocol. The DNA profiles generated using the two gradient change programs were still approximately equal in score to those generated using the standard program. While the profiles produced using the bulk change 2 protocol were still of significantly lower score than the standard profiles ( p = 0.0122), the profiles produced using the bulk change 1 protocol were found to be of approximately equal score to the standard . When PCR runtime was considered to be highly important, the bulk change 1 protocol produced DNA profiles that were of significantly higher score than the profiles generated using the standard GlobalFiler protocol . The profiles generated using the gradient change protocols were still found to be of approximately equal score to the standards, and the profiles generated using the bulk change 2 protocol had a significantly lower score than the standards ( p = 0.0122). The profiles generated using a gradually decreasing annealing/extension time (gradient change 1 protocol) with the combination kit were found to be of approximately equal quality to those produced using GlobalFiler only for the same protocols (see ). However, the DNA profiles produced using an annealing/extension time of 60 s (bulk change 1 protocol), an annealing/extension time of 30 s (bulk change 2 protocol) and a gradually increasing annealing/extension time (gradient change 2 protocol) with only the GlobalFiler kit were found to be of significantly lower quality than those produced using the combination kit for the same protocol across all scores ( p = 0.033, p = 0.00038 and p = 0.0316). When the scores were standardised, the DNA profiles produced using the gradient change programs and the GlobalFiler kit were not significantly different in quality to those produced using the combination kit for the same protocol across all standardised scores (see ). However, despite standardisation, the profiles produced using a reduced annealing/extension time of 30 s with GlobalFiler only were still found to be of significantly lower quality than those produced using the combination kit for the same protocol across all scores ( p = 0.0117). All DNA profiles generated using an altered denaturation temperature with the combination kit were found to be significantly lower in quality than the profiles produced under the standard GlobalFiler conditions . However, while the DNA profiles produced using a gradient increase in denaturation temperature (gradient change 2 protocol) were significantly lower in quality than the standard profiles, of all the altered protocols trialled, these were closest in quality to the standards. The DNA profiles generated using the combination kit were not significantly different in overall quality than the DNA profiles generated using only the GlobalFiler kit for all programs involving an altered denaturation temperature. The DNA profiles generated using a gradually increasing denaturation temperature with the combination kit were found to be of approximately equal quality to the profiles produced under the standard GlobalFiler conditions . The DNA profiles generated using a denaturation temperature of 90 °C (bulk change protocol 1) and 88 °C (bulk change 2 protocol) with the combination kit were significantly lower in comparison to the profiles produced under the standard GlobalFiler conditions ( p = 0.0122 for both) . The profiles generated using a gradually decreasing denaturation temperature with the combination kit were also found to be significantly lower in quality when compared to the standards ( p = 0.0122) . DNA profiles generated using the combination kit were not significantly different in overall quality to the DNA profiles generated using only the GlobalFiler kit for all programs involving an altered denaturation temperature (see ). When the quality scores were standardised, the profiles produced using the bulk change programs and gradient change 1 program with the combination kit were of significantly lower quality than the profiles produced using the same protocols with GlobalFiler only across all scores . However, the profiles generated using a gradually increasing denaturation temperature (gradient change 2 protocol) with the combination kit were of significantly higher quality than the profiles generated using GlobalFiler only for the same protocol across all standardised scores . When total runtime was not considered, the profiles generated using the optimised protocols with the combination kit were lower quality than those produced following the standard GlobalFiler protocol . When the profile scores were adjusted to include the PCR runtime as mildly important, the variation in quality between profiles produced using the optimised protocols and the standard GlobalFiler protocol with the combination kit was maintained. However, out of all the optimised protocols trialled, the profiles generated using protocol version 3 were closest in quality to the profiles generated using the standard protocol . This can be attributed to optimised protocol version 3 being 30 min faster than the standard GlobalFiler protocol. Lastly, when the scores were adjusted to make the runtime highly important, the profiles generated using optimised protocols 1, 2 and 3 were all of approximately equal quality to the profiles produced following the standard GlobalFiler protocol. This can be attributed to these optimised PCR programs having runtimes that were 28–31 min faster than the standard. Importantly, the DNA profiles produced using the combination kit were also found to be of significantly higher quality than the profiles generated using only GlobalFiler for all of the optimised PCR programs trialled (see ). When standardised, the DNA profiles produced using optimised protocols 2, 3 and 4 with the combination kit were all still of significantly higher quality than the profiles produced using GlobalFiler only, but profiles produced using optimised protocol 1 with the combination kit and GlobalFiler were found to be of approximately equal quality (see ). It is important to note that all optimised PCR programs trialled produced some DNA profiles with instances of dropout; however, the profiles still contained information sufficient for downstream interpretation and analysis. The potential exists to alter the profile metric to make the peak height component more dominant than in this trial. Given a database now exists of all the profiles generated, it is possible to re-evaluate the dataset and trial various combinations of peak height and peak variability model parameters to identify which combination is most intuitively correct. Overall, this provided 85 profiles’ worth of real-time fluorescence data, trialling 17 different combinations of PCR cycling conditions (five repeats of each of the combinations), that could be used in the training of a machine learning algorithm. Before the real-time data were used, we demonstrated the use of machine learning to suggest new parameters based on the DNA profile scoring results obtained. The firefly algorithm was used, incorporating all 17 combinations. Initial trials showed a sub-optimal performance of the firefly algorithm due to the fact that not enough sample space had been explored in the dataset of 17 points. To rectify this, additional parameter sets were developed by generating 60 random values for each of the six parameters ( β t , D , 0 , β t , D , 1 , β C , D , 0 , β C , D , 1 , β t , A E , 0 , β t , A E , 1 ) from truncated normal distributions. The constraints used for each parameter aligned with the bounds for that specific parameter, as identified in . From the 60 random sets of parameters generated, the combinations that pushed the cycling parameters outside of the acceptable ranges were removed. This led to the identification of six additional PCR programs using the randomly generated parameter values shown in . These six randomised parameter sets were then trialled to generate DNA profiles that could be scored and fed back into the system. This increased the sample space explored and thus worked to improve the performance of the algorithm. The results of these randomised PCR programs can be seen in . As expected, all randomised PCR programs gave significantly lower scores than the standard; however, the point of producing these programs was not to obtain high scores, but rather to fill out some of the parameter space to inform the firefly algorithm (in this case, with very dim flies). Hence, the system knew what parameters not to use. Using the 17 original sets of parameter values and the 6 random sets of values, the firefly algorithm was used and suggested the values shown in . To complete the proof of concept, there were four sets of conditions suggested by the firefly algorithm that were trialled (those rows bolded in and marked with an asterisk). The four sets of parameters were chosen to be trialled based on their theoretical success, previous experimental results and the projected impact of the time differences on the scores. Suggested programs S9 and S17 were chosen as they were expected to produce good-quality DNA profiles, while suggested programs S2 and S7 were chosen as they were expected to produce lower-quality DNA profiles. The results of these suggested methods can be seen in . The goal for the PCR program alterations was to reduce PCR time without significantly reducing profile quality. While the practical outcome of the experiment was not hugely successful (i.e., in producing a program that ran quicker than the standard and with higher-quality profiles), this is secondary, as the main goal of the study was to start practically applying the theoretical steps outlined in part 1 of the publication to train a smart PCR system. In addressing these theoretical points, we were able to achieve the following: Create a combined quantification/STR PCR reaction that still produced high-quality DNA profiles; Carry out complex PCR program manipulations using an open qPCR instrument; Utilise a profile-scoring equation to assess the outcome of the PCR, and weigh it against a goal (in this case, PCR program time); Take a set of experiments and build a statistical framework for predicting new PCR program parameter values to trial; Trial new predicted PCR parameters. Given the results of our pilot study, we found that a combined kit system does produce profiles of reasonable quality and can provide limited real-time fluorescence feedback. Depending on the quality of the real-time fluorescence data provided by the combined kit, it might be necessary to use a two-tube system. In this set-up, a quantitative PCR and STR PCR would be performed simultaneously but in separate tubes. The quantitative PCR should be primed with a number of initial cycles to ensure it provides meaningful real-time data at the beginning of the STR amplification process. There are limitations to using a split-tube system, the main one being that there is a possibility that the fluorescence data obtained from the qPCR tube would not exactly reflect the amplification efficiency within the STR PCR tube. Thus, the capability to monitor the exact amplification kinetics that are afforded by use of the combination kit would be lost. In our pilot study, we ‘unsmartly’ altered PCR program conditions (in other words, not reacting to real-time feedback). We found that, when using a combined kit system, the goal of shortening PCR program time, without compromising DNA profile quality, can be approached. Of the programs trialled, the best altered performance was obtained using a gradually decreasing denaturation time (94 °C for 10–5 s) and a gradually increasing annealing/extension time (59 °C for 90–115 s). Depending on the weight given to the timing component, these modifications produced DNA profiles with higher scores than those produced using the standard GlobalFiler PCR protocol, with a time saving of 30 min. Using these results, we were able to use a machine learning firefly algorithm to suggest new conditions for reaching the goal of a quicker PCR program. The success of this final step was limited due to the fact that none of the suggested parameter set values produced profiles with higher scores than the originally trialled sets. Nevertheless, it showed the conceptual ability to use machine learning to achieve the desired goal, and it is likely that the issue in our proof of concept was merely one of low sample size and limited rounds of suggesting new parameter values. This is particularly true given that machine learning/optimisation algorithms typically require many iterations (generations) before yielding good results . This study focused on optimisation using standard forensic profiling kits and laboratory hardware. This was to ensure the optimised PCR programs could be directly applied to casework in operational forensic laboratories. However, the findings of this study could be trialled with a range of other commonly used forensic profiling kits (e.g., VeriFiler Plus), quantification kits (e.g., Quantifiler Trio), detectors, fluorophores, DNA polymerases and buffer compositions. When considered alongside the findings of other recent studies , there is the potential for additional optimisation to complement the PCR programs by altering the make-up of these forensic profiling kits. Whilst the combination kit was found to be successful, if it is not possible to deconvolute the fluorescence data obtained from it, the composition of the kit may need to be further refined, or an entirely new bespoke kit with dual quantification and STR amplification capabilities may need to be designed. There is also the possibility to make changes to a qPCR instrument in future studies, such as in the wavelength of the detectors being used. Optimisation of STR data from trace samples is core to the concept of a smart PCR machine rather than the analyses of reference samples. Trace DNA, while the most common sample type submitted to a forensic science laboratory , generates poor genetic information . Increasing the genetic data from trace DNA inevitably will provide greater DNA data in criminal case investigations. It is only the timing at stages within the amplification that is being modified and not an overall increase in the number of cycles, with the inherent introduction of stochastic effects , none of which was recorded in this study. An increase in alleles amplified yet with no stochastic effects increases the opportunity to transfer into operational use the concept described in this study. As a parallel example, the RapidHIT ® ID system was developed for reference samples but more recently has been applied to trace samples typical of those encountered in forensic science . A difference between the technologies is that the chemistry and cycling parameters in RapidHIT ® ID remain constant whether the sample added to the cartridge is a buccal swab or a swab from an item containing trace DNA , whereas the smart PCR concept allows modifications to the cycling parameters. In this experiment, we were able to complete what would be considered a single round of training a machine learning algorithm. Given the complexity of the system on which this system is being applied, it is likely that multiple rounds of training would be required before significant advances are observed. It may also be the case that the current standard conditions do in fact happen to be the best cycling conditions (at least without also adjusting the reaction chemistry, as is performed for the Rapid DNA PCR kits). With increased data from the performance of PCR cycling conditions, it may be that different, or more sophisticated, methods of machine learning could be used. One possibility is to model the performance of the PCR system with an algorithm such as an artificial neural network, building up a digital twin that can then be probed for new cycling conditions. Another possibility is that the optimal settings for DNA replication vary based on the current concentration and that a single evaluation of a quality score at the end of the process is not the best way to evaluate the system. Instead, a better system may look at the rate of change in fluorescent feedback obtained during DNA replication by using a qPCR machine. This real-time feedback will likely give more insight and lead to better, and more variable, parameter sets for use. We outline in this work the basic considerations for a smart PCR system that could have wide-reaching impacts on genetic laboratory work. The ultimate goal would be to produce a PCR system that could respond on a cycle-by-cycle basis as the program progresses in order to amplify each sample to its optimum. This is a lofty goal, and there are many fundamental aspects to producing such a system that need to be developed along the way. In part 1 of the publication, we outlined the theory for addressing some of these fundamental aspects, and, in part 2, we have shown how those theoretical aspects can be put into practice. Like almost any machine learning task, the key is to have a large database of data to train a system. In the initial proof of concept, we chose a simplified version of the main goal, that is, to train an open-loop system (rather than a system that changes in response to real-time feedback), and to employ a relatively simple machine learning algorithm, the firefly algorithm. We were successful in showing how an open-loop smart PCR system could be developed and refined over time and plan to expand the sophistication of the machine learning approach as more data are collected from these initial experiments. |
Effect of dietary | 91302d8c-48a3-4ec4-940a-8f5f7f3a4dd0 | 11897522 | Digestive System[mh] | Introduction It is well known that gut is an important organ of fish that performs the absorption and metabolism of nutrients and the essential maintenance of gut health and homeostasis of fish in the healthy state ( ) as a matter of gut microbiota contributions ( , ). For instance, gut microbiota participate in the development and repair of the host’s gut epithelia and maintain the integrity of the gut barrier ( ), resisting the invasion of pathogenic bacteria via inhibiting the proliferation of pathogenic bacteria ( – ). In contrast, unhealthy gut is linked to the imbalance of gut flora, accompanied with disruption of gut mucosal barrier function and the colonization and proliferation of pathogenic microbiota, eventually leading to diarrhea or enteritis ( ). The practice of intensive farming has become an important factor contributing to the unhealthy conditions of farmed animals, resulting in the development of many diseases ( ). Therefore, how to manipulate gut microbiota to achieve a more favorable balance received widespread attention, from a perspective view of improving health by feed measures ( ). Historically, antibiotics have been used extensively for routine disease prevention and control ( ). However, there is still an urgent shift to use environmentally friendly and safe measures to replace antibiotics. This is not only a drug resistance issue, but also a major concern for human health ( , ). Probiotics are live bacteria that confer a health benefit on animals as their consumption is sufficient ( ). As a functional substance, they can regulate gut microbiota by lowering gut pH, providing nutrients, secreting antibacterial substances, adhering to pathogenic bacteria and regulating host immune responses ( , ). Numerous feeding experiments have shown that the addition of probiotics to feed can improve growth performance and health status of fish and shellfish, enhancing resistance to pathogen infections ( – ). With these potential health benefits of probiotics on aquatic animals, the use of probiotics in aquaculture has gained increasing attention in recent years. Clostridium butyricum ( C. butyricum ) is a specific anaerobic, Gram-positive, which is widely present in animals and soil ( , ). It not only has excellent environmental adaptability to heat, acidity, alkalinity, and bile salts, but can also regulate intestinal function by secreting bioactive substances such as short chain fatty acids, B vitamins, digestive enzymes, etc. in the intestine ( ). It is precisely because of many excellent characteristics mentioned above that C. butyricum has attracted widespread attention from aquaculture as a highly promising feed probiotic ( , , – ). At present, many studies have emphasized the positive effects of adding C. butyricum to feed, particularly on aquatic animals ( , ). However, limited direct comparative studies have been conducted the effects of C. butyricum strains derived from different sources. The different origins of C. butyricum strains may also have varying effects on fish growth and intestinal health. Over the past three decades, probiotic microorganisms have been widely used in aquaculture, sourced from both host and non-host-derived organisms ( , ). Host-derived probiotics have demonstrated significant potential for application in feed due to their unique biochemical characteristics ( ). When selecting suitable probiotics, various factors should be evaluated, including microbial type, source, strain type, biological activity, and spore formation, all of which are crucial for their successful application in aquaculture ( , ). Therefore, selecting the appropriate C. butyricum strains remains a critical step for its effective application in aquaculture. The hybrid grouper ( E. lanceolatus ♂ × E. fuscoguttatus ♀ ) is a predatory marine fish that is extensively cultivated in China and other Southeast Asian coastal nations due to its good flavor and benefits for intensive farming ( ). However, the frequency of disease occurrence is increasing with the continuous promotion of intensive farming of hybrid grouper, which brings about difficulties in disease prevention and control, and has become one of the major issues that need to be addressed in the healthy culture of grouper. In particular, three types of enteric pathogen such as Vibrio anguillarum , Edwardsiella tarda and Aeromonas hydrophila pose a major threat to grouper enteritis ( ). Recent studies reported that C. butyricum addition in feed can improve growth performance and intestinal health in hybrid grouper fed with commercial feed or the feed with fish meal replacement by cottonseed protein concentrate via promoting digestive ability, gut morphometry, and disease resistance ( , ). However, it remains unknown how C. butyricum exerts the beneficial effects on growth and gut health improvement in this species. Therefore, with the use of microbiome, this study explored the mechanisms by which C. butyricum improves growth and intestinal health of hybrid grouper. The present study will provide a scientific basis for the rational application of C. butyricum in the diet of hybrid grouper.
Materials and methods 2.1 Experimental diets Three distinct strains of C. butyricum were purchased as powdered products for this experiment. The KM strain ( C. butyricum strain number GDMCC NO 1.4721, with a viable bacterial concentration of 1 × 10⁹ cfu/g) was isolated from the intestinal tract of fish in natural aquatic environments and obtained from Guangzhou Kemu Biotechnology Co., Ltd. The DZN strain ( C. butyricum strain number GDMCC NO M2016421, with a viable bacterial concentration of 1 × 10⁹ cfu/g) was sourced from Guangdong Dazhe Agricultural Biotechnology Co., Ltd., and isolated from the intestines of healthy terrestrial animals. The CLH strain ( C. butyricum strain number GDMCC NO 1.676, with a viable bacterial concentration of 1 × 10⁹ cfu/g) was isolated from natural aquatic environments and obtained from Zhanjiang Hengxing Aquaculture Technology Service Co., Ltd. The control diet (CON) was formulated using fish meal and gelatin as the protein sources and fish oil, soybean oil, and soybean lecithin as the fat sources ( ). Based on this formulation, three strains of C. butyricum (KM (CB1), DZN (CB2), and CLH (CB3), respectively) were evenly sprayed onto the diets to achieve a viable bacterial count of 1 × 10 7 cfu/g. The feed ingredients were ground using a ZFJ-300 grinder (Jiangyin Ruizong Machinery Manufacturing Co., Ltd., Jiangyin, Jiangsu, China) passed through a 60-mesh sieve, weighed, and homogenized. Liquid ingredients were then added to the dry feed ingredients to prepare malt syrup. The dough was shaped into strips using a double-helix feed extruder (F-76, Guangzhou Huagong Optoelectronic Technology Co., Ltd., Guangzhou, Guangdong, China) and a feed pellet-forming machine (GY-500, Changzhou Beicheng Drying Equipment Engineering Co., Ltd., Changzhou, Jiangsu, China), and then pelletized using 2.5 mm and 5 mm dies. The feed was dried in a ventilated oven at 55°C for 24 hours to reduce its moisture content to below 100 g/kg. The feed was then ventilated at room temperature for 24 hours, sealed in plastic bags, and stored in a refrigerator at -20°C. 2.2 Feeding experiment The experiment was conducted at the fishery base of Dabeinong Group, located in Zhaoan County, Zhangzhou City, Fujian Province, China. Prior to the experiment, juvenile hybrid groupers were raised in blue polypropylene tanks and fed with the control diet (CON) for two weeks. At the commencement of the experiment, 300 juvenile hybrid groupers, with an initial average weight of 24.91 ± 0.01 g, were randomly distributed into 12 tanks (500 L/tank), with a density of 25 fish per tank. Under the natural photoperiod, triplicate groups of tanks were hand-fed one of the diets to satiation twice daily, at 8:00 AM and 5:00 PM,for a duration of 56 days. Any excess feed was collected 30 minutes after each meal, dried at 65℃ for 24 hours, and then weighed to calculate the feed intake. 2.3 Sample collection and index determination Upon completion of the culture experiment, the total weight of the fish in each tank was measured, and the number of fish in each tank was recorded. The fish were subsequently returned to their respective tanks and subjected to a 2-day feeding cessation to mitigate the stress effects induced by the weighing process, prior to sampling. At the time of sampling, 10 fish were randomly selected from each tank, anesthetized with eugenol and then weighed, measured, and recorded. Blood was drawn from the tail vein using a 1 mL syringe and collected in a 1.5 mL centrifuge tube. The blood allowed to stand for 12 h at 3000 rpm, centrifuged at 4°C for 10 min, and then combined with serum by tank. The sample was stored in a refrigerator at -80°C for serum biochemical index determination. The liver of each fish was then excised, weighed and recorded for the calculation of the hepatosomatic index (HSI). Intestinal tissues (proximal intestine, middle intestine and distal intestine) from the same portion of each fish were collected and stored in Bonn solution for paraffin sectioning. Intestinal tissues from four fish were collected, quickly placed in a freezing tube, frozen in liquid nitrogen, and stored in a refrigerator at -80°C. Three fish were then randomly selected and stored in a refrigerator at -20°C for determination of whole fish body composition. Whole fish samples were processed following the method described by Ye et al. ( ). Specifically, after thawing the whole fish sample, its surface was wiped with gauze, followed by weighing. The fish was then placed in a weighed aluminum box, autoclave at 121°C for 30 min, cooled, cut into pieces, dried in an oven at 65°C for 24 h, and reweighed. After 24 h at room temperature, the sample was reweighed and then crushed with a small grinder, packed in a self-sealing bag and stored in a refrigerator at -20°C. Measurements of moisture, crude protein, crude lipid and ash content in experimental diets and whole fish samples were made according to AOAC method ( ). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), C4, IgM, and LZM and total antioxidant capacity (T-AOC), MDA, GSH-Px, catalase (CAT) and SOD in the intestine were measured using the kit from Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China according to the manufacturer’s instructions ( , , , , ). 2.4 Intestinal histology observation Intestinal morphology was visualized using hematoxylin and eosin (H&E) staining. Following standard fixation, intestinal tissue samples were embedded in paraffin and sectioned into 5 μm-thick slices. After dewaxing in xylene I and xylene II for 20 min each, the intestinal slices underwent gradient dehydration in ethanol baths of decreasing concentrations, with 5 min per bath. The intestinal slices were washed three times with distilled water, followed by incubation with hematoxylin for 3 min, and then rinsed again with distilled water. Gradient dehydration of the intestinal slices was also performed in ethanol baths of increasing concentrations for 3 min per bath, followed by incubation with eosin for 3 min. The slices were then transferred to xylene and immersed for 3 min to render them transparent, prior to sealing with neutral resin. Images were examined under a light microscope (Leica DM5500B), and digital photographs were captured using a digital camera (Leica DFC450) equipped with LAS AF imaging software (Version 4.3.0 Leica). 2.5 Intestinal microbiota analysis Intestinal samples were collected for microbiological analysis. The E.Z.N.A.TM kit (Omega Bio-Tek, Norcross, GA, USA) was used to extract total DNA from the microbial genome of the intestinal samples. The instructions provided with the kit were followed for specific steps. A 1% agarose gel was employed to assess the concentration and purity of the extracted genomic DNA. Based on the concentration, the DNA was uniformly diluted to 15 ng/μL with sterile water. Amplification primers (338F: 5’-ACTCCTACGGGAGGCAGCAG-3’; 806R: 5’-GTGGACTACHVGGGTWTCTAAT-3’) were used to amplify total DNA using an ABI 9700 PCR instrument (Applied Biosystems, Inc., USA). An 8-bp barcode sequence was added to the 5’ end of the upstream and downstream primers to distinguish between different samples. All PCR reactions were conducted in 25 μL reaction volumes, using 12.5 μL of 2xTaq Plus Master Mix. 0.2 μM forward and reverse primers and approximately 30 ng of template DNA. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes; followed by 40 s denaturation at 94°C, 50 s of annealing at 50°C, 60 s extension at 72°C, repeated for 30 cycles, and a final extension at 72°C for 7 min. The PCR product was visualized using 1% agarose gel electrophoresis (170V 30 min). followed by automatic purification using the Agencourtam Pure XP nucleic acid purification kit (Beckman Coulter, Inc., USA). PCR libraries were constructed using the NebNext Ultra II DNA Library PrepKit (New England Biolabs, Inc., USA). The constructed library was purified using the AM Pure XP nucleic acid purification kit (Beckman Coulter, Inc., USA). The library was then sequenced on the Nextseq 2000 platform (Illumina, Inc., USA) using the PE300 sequencing strategy. The off-board data were classified according to the barcode sequence. Pear software was employed to filter and splice the sequencing data. After splicing, low-quality and chimera sequences were removed using Vsearch software, and the sequences were clustered into operational taxonomic units (OTUs) using the UPARSE algorithm, with a sequence similarity threshold of 97%. The OTU was then compared with the Silva138 database using BLAST algorithm, with an e-value threshold set to 1e-5 to obtain species classification information for each OTU. Based on the results of OTU abundance, the α diversity index and β diversity distance matrix were calculated using QIIME software, plotted with R software. 2.6 RNA extraction and gene expression Total RNA was extracted from individual livers using the SYBR Premix Ex Taq Kit (Takara, Dalian, China), followed by quantification of RNA concentration and purity via spectrophotometry and quality assessment using agarose gel electrophoresis. Reverse transcription was performed with 1 μg of total RNA using a reverse transcription kit (Thermo). Target gene expression was quantified by quantitative real-time PCR (qRT-PCR) using an ABI 7500 real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Green Real-time PCR Master Mix (Toyobo, Shanghai, China). Primers for the amplification of gene-specific PCR products were designed using Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), and primer details for qRT-PCR are provided in . All primers were commercially supplied by Integrated DNA Technologies (Hunan Accurate Biological Engineering Co., Ltd., Changsha, China). The real-time PCR procedure involved an initial denaturation step at 95°C for 30 s, 40 cycles at 95°C for 5 s, annealing and extension temperature at 60°C for 30 s, and the final dissociation step. The final step was performed to confirm amplification of a single product. qRT-PCR efficiency ( E ) was achieved using the equation E =10 (−1/slope) -1. Gene expression results were analyzed using the 2 -ΔΔCt method only after primers were verified to have an efficiency of approximately 100% through amplification, with data from all treatments compared to the control group. 2.7 Statistical analysis All data were presented as mean and standard error of the mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) to assess differences between treatments, followed by the Student-Newman-Keuls multiple comparison test. Normality and homogeneity of variance were confirmed using the Kolmogorov-Smirnov and Levene’s tests, and all analyses were conducted in SPSS Statistics 25.0 (SPSS, Michigan Avenue, Chicago, IL, USA). Data expressed as percentages or ratios underwent data conversion before statistical analysis. P -values < 0.05 were considered statistically significant.
Experimental diets Three distinct strains of C. butyricum were purchased as powdered products for this experiment. The KM strain ( C. butyricum strain number GDMCC NO 1.4721, with a viable bacterial concentration of 1 × 10⁹ cfu/g) was isolated from the intestinal tract of fish in natural aquatic environments and obtained from Guangzhou Kemu Biotechnology Co., Ltd. The DZN strain ( C. butyricum strain number GDMCC NO M2016421, with a viable bacterial concentration of 1 × 10⁹ cfu/g) was sourced from Guangdong Dazhe Agricultural Biotechnology Co., Ltd., and isolated from the intestines of healthy terrestrial animals. The CLH strain ( C. butyricum strain number GDMCC NO 1.676, with a viable bacterial concentration of 1 × 10⁹ cfu/g) was isolated from natural aquatic environments and obtained from Zhanjiang Hengxing Aquaculture Technology Service Co., Ltd. The control diet (CON) was formulated using fish meal and gelatin as the protein sources and fish oil, soybean oil, and soybean lecithin as the fat sources ( ). Based on this formulation, three strains of C. butyricum (KM (CB1), DZN (CB2), and CLH (CB3), respectively) were evenly sprayed onto the diets to achieve a viable bacterial count of 1 × 10 7 cfu/g. The feed ingredients were ground using a ZFJ-300 grinder (Jiangyin Ruizong Machinery Manufacturing Co., Ltd., Jiangyin, Jiangsu, China) passed through a 60-mesh sieve, weighed, and homogenized. Liquid ingredients were then added to the dry feed ingredients to prepare malt syrup. The dough was shaped into strips using a double-helix feed extruder (F-76, Guangzhou Huagong Optoelectronic Technology Co., Ltd., Guangzhou, Guangdong, China) and a feed pellet-forming machine (GY-500, Changzhou Beicheng Drying Equipment Engineering Co., Ltd., Changzhou, Jiangsu, China), and then pelletized using 2.5 mm and 5 mm dies. The feed was dried in a ventilated oven at 55°C for 24 hours to reduce its moisture content to below 100 g/kg. The feed was then ventilated at room temperature for 24 hours, sealed in plastic bags, and stored in a refrigerator at -20°C.
Feeding experiment The experiment was conducted at the fishery base of Dabeinong Group, located in Zhaoan County, Zhangzhou City, Fujian Province, China. Prior to the experiment, juvenile hybrid groupers were raised in blue polypropylene tanks and fed with the control diet (CON) for two weeks. At the commencement of the experiment, 300 juvenile hybrid groupers, with an initial average weight of 24.91 ± 0.01 g, were randomly distributed into 12 tanks (500 L/tank), with a density of 25 fish per tank. Under the natural photoperiod, triplicate groups of tanks were hand-fed one of the diets to satiation twice daily, at 8:00 AM and 5:00 PM,for a duration of 56 days. Any excess feed was collected 30 minutes after each meal, dried at 65℃ for 24 hours, and then weighed to calculate the feed intake.
Sample collection and index determination Upon completion of the culture experiment, the total weight of the fish in each tank was measured, and the number of fish in each tank was recorded. The fish were subsequently returned to their respective tanks and subjected to a 2-day feeding cessation to mitigate the stress effects induced by the weighing process, prior to sampling. At the time of sampling, 10 fish were randomly selected from each tank, anesthetized with eugenol and then weighed, measured, and recorded. Blood was drawn from the tail vein using a 1 mL syringe and collected in a 1.5 mL centrifuge tube. The blood allowed to stand for 12 h at 3000 rpm, centrifuged at 4°C for 10 min, and then combined with serum by tank. The sample was stored in a refrigerator at -80°C for serum biochemical index determination. The liver of each fish was then excised, weighed and recorded for the calculation of the hepatosomatic index (HSI). Intestinal tissues (proximal intestine, middle intestine and distal intestine) from the same portion of each fish were collected and stored in Bonn solution for paraffin sectioning. Intestinal tissues from four fish were collected, quickly placed in a freezing tube, frozen in liquid nitrogen, and stored in a refrigerator at -80°C. Three fish were then randomly selected and stored in a refrigerator at -20°C for determination of whole fish body composition. Whole fish samples were processed following the method described by Ye et al. ( ). Specifically, after thawing the whole fish sample, its surface was wiped with gauze, followed by weighing. The fish was then placed in a weighed aluminum box, autoclave at 121°C for 30 min, cooled, cut into pieces, dried in an oven at 65°C for 24 h, and reweighed. After 24 h at room temperature, the sample was reweighed and then crushed with a small grinder, packed in a self-sealing bag and stored in a refrigerator at -20°C. Measurements of moisture, crude protein, crude lipid and ash content in experimental diets and whole fish samples were made according to AOAC method ( ). Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), C4, IgM, and LZM and total antioxidant capacity (T-AOC), MDA, GSH-Px, catalase (CAT) and SOD in the intestine were measured using the kit from Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China according to the manufacturer’s instructions ( , , , , ).
Intestinal histology observation Intestinal morphology was visualized using hematoxylin and eosin (H&E) staining. Following standard fixation, intestinal tissue samples were embedded in paraffin and sectioned into 5 μm-thick slices. After dewaxing in xylene I and xylene II for 20 min each, the intestinal slices underwent gradient dehydration in ethanol baths of decreasing concentrations, with 5 min per bath. The intestinal slices were washed three times with distilled water, followed by incubation with hematoxylin for 3 min, and then rinsed again with distilled water. Gradient dehydration of the intestinal slices was also performed in ethanol baths of increasing concentrations for 3 min per bath, followed by incubation with eosin for 3 min. The slices were then transferred to xylene and immersed for 3 min to render them transparent, prior to sealing with neutral resin. Images were examined under a light microscope (Leica DM5500B), and digital photographs were captured using a digital camera (Leica DFC450) equipped with LAS AF imaging software (Version 4.3.0 Leica).
Intestinal microbiota analysis Intestinal samples were collected for microbiological analysis. The E.Z.N.A.TM kit (Omega Bio-Tek, Norcross, GA, USA) was used to extract total DNA from the microbial genome of the intestinal samples. The instructions provided with the kit were followed for specific steps. A 1% agarose gel was employed to assess the concentration and purity of the extracted genomic DNA. Based on the concentration, the DNA was uniformly diluted to 15 ng/μL with sterile water. Amplification primers (338F: 5’-ACTCCTACGGGAGGCAGCAG-3’; 806R: 5’-GTGGACTACHVGGGTWTCTAAT-3’) were used to amplify total DNA using an ABI 9700 PCR instrument (Applied Biosystems, Inc., USA). An 8-bp barcode sequence was added to the 5’ end of the upstream and downstream primers to distinguish between different samples. All PCR reactions were conducted in 25 μL reaction volumes, using 12.5 μL of 2xTaq Plus Master Mix. 0.2 μM forward and reverse primers and approximately 30 ng of template DNA. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 minutes; followed by 40 s denaturation at 94°C, 50 s of annealing at 50°C, 60 s extension at 72°C, repeated for 30 cycles, and a final extension at 72°C for 7 min. The PCR product was visualized using 1% agarose gel electrophoresis (170V 30 min). followed by automatic purification using the Agencourtam Pure XP nucleic acid purification kit (Beckman Coulter, Inc., USA). PCR libraries were constructed using the NebNext Ultra II DNA Library PrepKit (New England Biolabs, Inc., USA). The constructed library was purified using the AM Pure XP nucleic acid purification kit (Beckman Coulter, Inc., USA). The library was then sequenced on the Nextseq 2000 platform (Illumina, Inc., USA) using the PE300 sequencing strategy. The off-board data were classified according to the barcode sequence. Pear software was employed to filter and splice the sequencing data. After splicing, low-quality and chimera sequences were removed using Vsearch software, and the sequences were clustered into operational taxonomic units (OTUs) using the UPARSE algorithm, with a sequence similarity threshold of 97%. The OTU was then compared with the Silva138 database using BLAST algorithm, with an e-value threshold set to 1e-5 to obtain species classification information for each OTU. Based on the results of OTU abundance, the α diversity index and β diversity distance matrix were calculated using QIIME software, plotted with R software.
RNA extraction and gene expression Total RNA was extracted from individual livers using the SYBR Premix Ex Taq Kit (Takara, Dalian, China), followed by quantification of RNA concentration and purity via spectrophotometry and quality assessment using agarose gel electrophoresis. Reverse transcription was performed with 1 μg of total RNA using a reverse transcription kit (Thermo). Target gene expression was quantified by quantitative real-time PCR (qRT-PCR) using an ABI 7500 real-time PCR Detection System (Applied Biosystems, Foster City, CA, USA) with SYBR Green Real-time PCR Master Mix (Toyobo, Shanghai, China). Primers for the amplification of gene-specific PCR products were designed using Primer-BLAST ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), and primer details for qRT-PCR are provided in . All primers were commercially supplied by Integrated DNA Technologies (Hunan Accurate Biological Engineering Co., Ltd., Changsha, China). The real-time PCR procedure involved an initial denaturation step at 95°C for 30 s, 40 cycles at 95°C for 5 s, annealing and extension temperature at 60°C for 30 s, and the final dissociation step. The final step was performed to confirm amplification of a single product. qRT-PCR efficiency ( E ) was achieved using the equation E =10 (−1/slope) -1. Gene expression results were analyzed using the 2 -ΔΔCt method only after primers were verified to have an efficiency of approximately 100% through amplification, with data from all treatments compared to the control group.
Statistical analysis All data were presented as mean and standard error of the mean (SEM). The data were analyzed using one-way analysis of variance (ANOVA) to assess differences between treatments, followed by the Student-Newman-Keuls multiple comparison test. Normality and homogeneity of variance were confirmed using the Kolmogorov-Smirnov and Levene’s tests, and all analyses were conducted in SPSS Statistics 25.0 (SPSS, Michigan Avenue, Chicago, IL, USA). Data expressed as percentages or ratios underwent data conversion before statistical analysis. P -values < 0.05 were considered statistically significant.
Results 3.1 Growth performance and whole-body proximate composition shows that the addition of C. butyricum to the diet did not have no adverse effects on the growth performance and whole-body proximate composition of hybrid grouper. No significant differences were observed in WG, FE, FR, HSI, CF, SR, SGR, or whole-body proximate composition (moisture, crude protein, crude lipid and ash) among the groups ( P > 0.05). 3.2 Intestinal antioxidant capacity demonstrates the effect of adding C. butyricum to the feed on the intestinal antioxidant capacity of the hybrid grouper. The activities of GSH-Px and SOD in the experimental group were significantly higher than those in the control group ( P < 0.05). In the CON group, the CB3 group exhibited the highest values ( P < 0.05). The addition of CB1 and CB3 to the feed significantly decreased MDA content in the intestine ( P < 0.05), while no significant difference was observed between CB2 and CON ( P > 0.05). No statistically significant differences were observed in the activities of CAT and T-AOC among the groups ( P > 0.05). 3.3 Plasma components illustrates that dietary C. butyricum influenced the plasma composition of hybrid grouper. Compared to the CON group, the C4 content and LZM activity were significantly elevated in the other groups ( P < 0.05). Furthermore, the activities of AST and ALT were unaffected by the dietary treatments ( P > 0.05). 3.4 Intestinal histomorphology As shown in , the muscle layer thickness (MT) and mucosal fold height (HMF) of the midgut were significantly increased in the C. butyricum group compared to the CON group ( P < 0.05). The inclusion of C. butyricum in the feed had no effect on the morphological indicators of the anterior and posterior intestinal tissues of hybrid grouper ( P > 0.05). The observations of intestinal tissue slice are presented in . As shown in , the tissue structure of the anterior and middle intestines in the CB1, CB2, and CB3 groups appears clearer and more structurally intact than that of the CON group, with well-developed mucosal folds. The results indicated that adding C. butyricum to the feed significantly improved the tissue structure of the anterior and middle intestines of hybrid grouper, with the CB2 group demonstrating the most pronounced improvements. 3.5 Microbiological analysis By comparing the alpha diversity indices of microbial communities in different treatment groups, significant differences in species richness and diversity were observed among the groups ( ). Chao1 index: The CB1 group exhibited the highest estimated species richness, followed by the CON group, while the CB2 and CB3 groups exhibited significantly lower species richness. Observed species index: The CB1 group had the highest number of observed species, followed by the CON group, while the CB2 and CB3 groups had lower numbers of observed species. Shannon index: The Shannon diversity index of the CB1 group was the highest, indicating the highest community diversity and evenness. The CON group ranked second, while the CB2 and CB3 groups exhibited lower levels. Simpson index: The CB1 group had the highest Simpson diversity index, further demonstrating that the CB1 group exhibited the highest community diversity and stability. The CON group ranked second, while the CB2 and CB3 groups exhibited lower levels. Based on the heatmap and hierarchical clustering tree, β-diversity analysis revealed significant differences in microbial community structure among the treatment groups ( ). Samples within the same group (e.g., con_1, con_2, con_3 in the CON group) exhibited a predominance of blue coloration in the heatmap, indicating high similarity among these samples. In contrast, samples from different groups (e.g., the CON group compared with the CB1 group) displayed a predominance of red coloration in the heatmap, indicating lower similarity between these groups. The hierarchical clustering tree illustrated the relationships between samples, with highly similar samples clustering together. The results indicate that samples within the same group shared high similarity, while significant intergroup differences were observed. Specifically, the CON group exhibited low similarity with the CB1 and CB3 groups, while a higher similarity was observed with the CB2 group. These findings highlight the significant structural variations in microbial communities among the different treatment groups (CON, CB1, CB2, and CB3). The classification diagram of intestinal microorganisms at the phylum level among groups is shown in and . At the phylum level, the dominant microflora in the intestine of hybrid grouper were Proteobacteria, Firmicutes, and Bacteroidota, accounting for more than 90%. A higher relative abundance of Firmicutes and Bacteroidota and a lower relative abundance of Proteobacteria were observed in the CB1 and CB3 groups compared to the CON group, and CB2 exhibited the opposite trend. In addition, although not significantly different among groups ( P > 0.05), the relative abundance of Actinobacteriota was lower in the CON group than in any of the experimental groups. The classification diagram of intestinal microorganisms at the genus level among groups is shown in and . At the genus level, the bacterial communities in the dietary treatments included the following genera in particular: Bacteroides , Brevundimonas , Lactobacillus , Mitsuaria , Massilia , Pseudomonas , Stenotrophomonas and Subdoligranulum . Decreased abundances of the genera Massilia , Pseudomonas , Stenotrophomonas , and Subdoligranulum , and increased abundances of the genera Lactobacillus , Bacteroides and Mitsuaria were observed in the experimental groups compared to the CON group. 3.6 Expression of tight junction structural protein and intestinal inflammatory factor genes The effect of dietary C. butyricum supplementation on the expression levels of intestinal inflammatory factors and tight junction structural protein genes in hybrid grouper were illustrated in . Compared to the CON group, the gene expression levels of pro-inflammatory factors IL-1β and Ikk-β in the experimental groups generally exhibited a down-regulation trend ( P < 0.05), whereas the gene expression level of anti-inflammatory factor IL-10 was up-regulated, although no significant differences were observed ( P > 0.05). Regarding tight junction protein genes, the expression levels of the ZO-1 gene in the CB1 group were significantly higher than those in the CON group ( P < 0.05). However, no significant differences were found in the expression levels of claudin 15a and occludin proteins ( P > 0.05).
Growth performance and whole-body proximate composition shows that the addition of C. butyricum to the diet did not have no adverse effects on the growth performance and whole-body proximate composition of hybrid grouper. No significant differences were observed in WG, FE, FR, HSI, CF, SR, SGR, or whole-body proximate composition (moisture, crude protein, crude lipid and ash) among the groups ( P > 0.05).
Intestinal antioxidant capacity demonstrates the effect of adding C. butyricum to the feed on the intestinal antioxidant capacity of the hybrid grouper. The activities of GSH-Px and SOD in the experimental group were significantly higher than those in the control group ( P < 0.05). In the CON group, the CB3 group exhibited the highest values ( P < 0.05). The addition of CB1 and CB3 to the feed significantly decreased MDA content in the intestine ( P < 0.05), while no significant difference was observed between CB2 and CON ( P > 0.05). No statistically significant differences were observed in the activities of CAT and T-AOC among the groups ( P > 0.05).
Plasma components illustrates that dietary C. butyricum influenced the plasma composition of hybrid grouper. Compared to the CON group, the C4 content and LZM activity were significantly elevated in the other groups ( P < 0.05). Furthermore, the activities of AST and ALT were unaffected by the dietary treatments ( P > 0.05).
Intestinal histomorphology As shown in , the muscle layer thickness (MT) and mucosal fold height (HMF) of the midgut were significantly increased in the C. butyricum group compared to the CON group ( P < 0.05). The inclusion of C. butyricum in the feed had no effect on the morphological indicators of the anterior and posterior intestinal tissues of hybrid grouper ( P > 0.05). The observations of intestinal tissue slice are presented in . As shown in , the tissue structure of the anterior and middle intestines in the CB1, CB2, and CB3 groups appears clearer and more structurally intact than that of the CON group, with well-developed mucosal folds. The results indicated that adding C. butyricum to the feed significantly improved the tissue structure of the anterior and middle intestines of hybrid grouper, with the CB2 group demonstrating the most pronounced improvements.
Microbiological analysis By comparing the alpha diversity indices of microbial communities in different treatment groups, significant differences in species richness and diversity were observed among the groups ( ). Chao1 index: The CB1 group exhibited the highest estimated species richness, followed by the CON group, while the CB2 and CB3 groups exhibited significantly lower species richness. Observed species index: The CB1 group had the highest number of observed species, followed by the CON group, while the CB2 and CB3 groups had lower numbers of observed species. Shannon index: The Shannon diversity index of the CB1 group was the highest, indicating the highest community diversity and evenness. The CON group ranked second, while the CB2 and CB3 groups exhibited lower levels. Simpson index: The CB1 group had the highest Simpson diversity index, further demonstrating that the CB1 group exhibited the highest community diversity and stability. The CON group ranked second, while the CB2 and CB3 groups exhibited lower levels. Based on the heatmap and hierarchical clustering tree, β-diversity analysis revealed significant differences in microbial community structure among the treatment groups ( ). Samples within the same group (e.g., con_1, con_2, con_3 in the CON group) exhibited a predominance of blue coloration in the heatmap, indicating high similarity among these samples. In contrast, samples from different groups (e.g., the CON group compared with the CB1 group) displayed a predominance of red coloration in the heatmap, indicating lower similarity between these groups. The hierarchical clustering tree illustrated the relationships between samples, with highly similar samples clustering together. The results indicate that samples within the same group shared high similarity, while significant intergroup differences were observed. Specifically, the CON group exhibited low similarity with the CB1 and CB3 groups, while a higher similarity was observed with the CB2 group. These findings highlight the significant structural variations in microbial communities among the different treatment groups (CON, CB1, CB2, and CB3). The classification diagram of intestinal microorganisms at the phylum level among groups is shown in and . At the phylum level, the dominant microflora in the intestine of hybrid grouper were Proteobacteria, Firmicutes, and Bacteroidota, accounting for more than 90%. A higher relative abundance of Firmicutes and Bacteroidota and a lower relative abundance of Proteobacteria were observed in the CB1 and CB3 groups compared to the CON group, and CB2 exhibited the opposite trend. In addition, although not significantly different among groups ( P > 0.05), the relative abundance of Actinobacteriota was lower in the CON group than in any of the experimental groups. The classification diagram of intestinal microorganisms at the genus level among groups is shown in and . At the genus level, the bacterial communities in the dietary treatments included the following genera in particular: Bacteroides , Brevundimonas , Lactobacillus , Mitsuaria , Massilia , Pseudomonas , Stenotrophomonas and Subdoligranulum . Decreased abundances of the genera Massilia , Pseudomonas , Stenotrophomonas , and Subdoligranulum , and increased abundances of the genera Lactobacillus , Bacteroides and Mitsuaria were observed in the experimental groups compared to the CON group.
Expression of tight junction structural protein and intestinal inflammatory factor genes The effect of dietary C. butyricum supplementation on the expression levels of intestinal inflammatory factors and tight junction structural protein genes in hybrid grouper were illustrated in . Compared to the CON group, the gene expression levels of pro-inflammatory factors IL-1β and Ikk-β in the experimental groups generally exhibited a down-regulation trend ( P < 0.05), whereas the gene expression level of anti-inflammatory factor IL-10 was up-regulated, although no significant differences were observed ( P > 0.05). Regarding tight junction protein genes, the expression levels of the ZO-1 gene in the CB1 group were significantly higher than those in the CON group ( P < 0.05). However, no significant differences were found in the expression levels of claudin 15a and occludin proteins ( P > 0.05).
Discussion In this study, the addition of C. butyricum strains from different sources to the diets had no discernible effect on the growth performance of hybrid grouper. This outcome is consistent with the observations reported for other animals, including carp ( , ), Chinese drum ( ), and goats ( ). Incorporating C. butyricum into a dietary formulation comprising a low fish meal content has been shown to significantly promote fish growth ( , , ). This effect may be attributed to C. butyricum ’s ability to enhance intestinal metabolic function and facilitate nutrient digestion and absorption through the secretion of a multitude of short-chain fatty acids and digestive enzymes within the intestine ( , ). These disparities may be due to the fact that the protein and fat levels in the diets are tailored to meet the nutritional requirements of hybrid grouper in this study. Consequently, the incorporation of C. butyricum did not result in a pronounced growth-promoting effect in hybrid grouper, suggesting that the growth-promoting effects of C. butyricum cannot be fully realized by merely incorporating it into feeds with high-quality protein sources. The antioxidant capacity of the body serves as an indicator of overall health and well-being. During physiological stress or illness, the body generates an excess of free radicals, leading to oxidative damage ( ). The intestine, as a conduit between the internal and external environments, is constantly exposed to free radicals from both exogenous substances and endogenous metabolites, resulting in intestinal oxidative damage ( ). Previous studies have demonstrated that administering C. butyricum to animals enhances antioxidant enzyme activity and reduces MDA levels ( , , , , ), thus preventing damage to the body, which is consistent with the present study’s findings. The results of the present study demonstrated that dietary C. butyricum administration led to a significant increase in SOD and GSH-Px levels in the intestine of hybrid grouper, along with a decrease in MDA levels. The antioxidant effect of C. butyricum is linked to its production of short-chain fatty acids, such as butyric and propionic acids, in the intestinal lumen, providing an energy source for intestinal mucosal cells and supporting intestinal homeostasis ( ). Additionally, C. butyricum can produce nicotinamide adenine dinucleotide phosphate, scavenge reactive oxygen species, and enhance antioxidant potential, which may further contribute to the observed improvement in antioxidant capacity ( ). The present study demonstrated that dietary C. butyricum administration led to a significant increase in serum LZM levels, with the CB1 group showing a marked elevation in serum IgM. These findings indicate that dietary supplementation with C. butyricum enhances immune competence in fish. The observed outcomes were consistent across diverse species, including Miichthys miiuy ( ) silver pomfret ( ) large yellow croaker ( ), tilapia ( ), shrimp ( ), and gibel carp ( ). These results may be attributed to increased IgM and LZM levels, which are known to enhance the organism’s capacity to eliminate pathogens and pathogenic microorganisms ( , ). Furthermore, the incorporation of C. butyricum into the diet elevated the serum C4 concentration of tilapia ( ) and broilers ( , ), thereby reinforcing the immune system, findings corroborated by this study. The integrity of the intestinal tissue structure in fish is essential for the efficient maintenance of nutrient digestion and absorption ( ). Greater intestinal plica height corresponds to an increased intestinal surface area and improved nutrient absorption capacity ( ). Additionally, intestinal muscular layer thickness reflects its peristaltic capacity, which affects chyme digestion rate and intestinal emptying ( ). Short-chain fatty acids act as an energy source for epithelial cells, promoting the growth and development of intestinal villi, while mitigating mucosal injury and reducing intestinal cell apoptosis ( ). Our experimental findings, consistent with prior studies ( , ), demonstrate that the addition of C. butyricum to the feed effectively improves the intestinal structure in fish. Furthermore, C. butyricum enhances intestinal health by modulating the intestinal microbiota. This study identifies Proteobacteria, Firmicutes, and Bacteroidetes as the dominant intestinal flora in hybrid grouper, consistent with previous studies on grouper ( ). The addition of C. butyricum significantly altered the dominant intestinal flora, increasing the relative abundance of Firmicutes and Bacteroidetes while reducing that of Proteobacteria and Actinobacteria. This finding is consistent with studies on Epinephelus coioides ( ) and Procambarus clarkii ( ). Firmicutes and Bacteroidetes are known to produce substantial amounts of short-chain fatty acids ( , ), which may partly explain why C. butyricum increases short-chain fatty acid content in the intestine. Many bacteria within Proteobacteria and Actinobacteria are considered potential pathogens, and their increased relative abundance may disrupt microbial balance, increasing the risk of enteritis or pathogen invasion ( , ). In this study, C. butyricum significantly decreased the relative abundance of these two phyla, suggesting its potential to mitigate the risk of enteritis by improving microbial structure and enhancing intestinal health. This study evaluated the impact of C. butyricum supplementation on the expression of intestinal inflammatory cytokine and tight junction (TJ) protein genes in hybrid grouper ( E. lanceolatus × E. fuscoguttatus ). The results demonstrated that C. butyricum supplementation significantly regulated inflammation and improved intestinal barrier integrity. Pro-inflammatory cytokine gene expression (e.g., IL-1β and Ikk-β ) was significantly downregulated in the experimental group, consistent with findings reported by Lu et al. ( ). These cytokines are critical mediators of the inflammatory response, and their overexpression is closely linked to tissue damage and compromised intestinal function ( ). Beyond its anti-inflammatory effects, C. butyricum supplementation significantly upregulated ZO-1 expression levels. Tight junctions (TJs) are critical intercellular connections. Occludin, a key functional protein, interacts with tight junction proteins such as ZOs, playing an indispensable role in biological barrier function and tight junction formation. ZO proteins, particularly ZO-1, link occludin to the actin cytoskeleton and play a vital role in maintaining tight junction integrity, reducing the risk of intestinal tumors and related disease ( , ). This study revealed that C. butyricum supplementation significantly upregulated ZO-1 expression levels. This crucial TJ protein enhances intestinal barrier integrity and elasticity by regulating paracellular permeability, potentially reducing the risk of pathogen and endotoxin translocation into systemic circulation ( ). Notably, no significant changes were observed in claudin-15a and occludin expression levels, indicating that C. butyricum may selectively regulate specific TJ proteins.
Conclusions In this study, the addition of C. butyricum to the diet did not significant impact on the growth performance of hybrid grouper but notably enhanced intestinal antioxidant enzyme activity and improved intestinal morphology and structure. From the perspective of intestinal flora composition, tight junction proteins, and inflammatory gene expression, the addition of C. butyricum to the diet reduced intestinal inflammation, promoted the proliferation of beneficial bacteria, and suppress ed pathogenic bacteria. These findings highlighted its potential role in promoting intestinal structural integrity and health.
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Mapping between cognitive theories and psycho-physiological models of attention system performance | 0aea0518-e4be-4cd9-880d-f40d31da372b | 10502801 | Physiology[mh] | Situational awareness refers to the perception, comprehension, and projection of the threats within an environment across time and space . For example, network defense analysts establish and support situational awareness of cyber threat landscapes by closely and consistently paying attention to Security Event Information Management Systems (SEIMs) . SEIMs summarize the anomalous and potentially malicious patterns of network traffic as sets of alarms, or alerts, which analysts must individually investigate as potential cyber threats . Analysts’ capacity to sustain attention to their SEIM constrains their situational awareness of the cyber threat landscape and diminishes their protective capabilities . Situational awareness hinges on the capacity to sustain vigilant attention to threats distributed across abstract threat landscapes . For example, In network security, analysts use SEIMs to perceive and act on threats to protected cyber infrastructures . However, SEIM threat detection is a tedious, monotonous task requiring analysts to sustain high levels of attention for prolonged periods . Distinguishing between malicious and benign SEIM alerts is not dissimilar to the search for a needle in a haystack . Analysts sift through a substantial number of SEIM alerts, most of which are false positives, to identify and act on a small number of malicious threats . Although SEIM threat detection is initially easy to perform, analyst mistakes invariably begin to snowball with time spent distinguishing between malicious and benign element signals. This gradual decline in sustained attention is known as vigilance decrement ; it occurs when the brain is required to sustain a high level of workload processing activity for longer than its energy reserves can support . For example, drivers must sustain vigilance in attuning and responding to hazards on the road . However, a driver experiencing vigilance decrement will be less capable of responding to road hazards . Hence, failure to sustain attention to road hazards is the leading cause of thousands of road deaths yearly . Similarly, establishing and sustaining situational awareness in a cyber security operations center, or CSOC, requires that analysts sustain vigilant attention to their SEIM dashboards for prolonged periods . However, vigilance decrement has become an increasingly disruptive influence on operational CSOC analysts whose role requires the use of SEIM to hunt for threats in the cyber landscape . Norman , was commissioned by The Royal Air Force to study the problem in what would become seminal vigilance research. The following review explores parallels between contemporary cognitive and psycho-physiological accounts of vigilance decrement and sustained attention performance that has developed since Mackworth’s early work. Therefore, the gap in the literature that this review identifies is a map between cognitive theories of vigilance decrement and psycho-physiological models of cerebral systems associated with attention performance. Depending upon the context, vigilance decrement can manifest either as an increased reaction time to detect critical signals or as a reduction in their correct detection . For example, during World War II, British radar operators had to monitor their terminals for “blips” over prolonged periods, indicating Axis U-boats’ presence. Despite their training and motivation to avoid Axis invasion, these operators began to miss critical U-boat signals after only half an hour of monitoring . Norman , was commissioned by the Royal Air Force to study the problem in what would become seminal vigilance research. Mackworth devised a “Clock Test” that simulated the RAF radar displays, composed of a black pointer that traced along the circumference of a blank, featureless clock-type face in 0.3-inch increments per second . At random points during the task, the pointer would increment twice in a row to simulate the detection of a U-boat . illustrates an example of the Clock task’s critical signal. The pointer increments along the clock face for the first 3 s in single increment jumps. In the fourth second, the pointer randomly jumps across two consecutive points, which was the critical signal Mackworth used to symbolize the presence of an Axis U-boat. Mackworth tasked observers with detecting these double jumps by pressing a button when one was seen. Despite the clarity of , ) target signals, correct detections declined by 10% in the first 30 min of the 2-h-long task. This gradual drop in signal detection accuracy was the first laboratory demonstration of vigilance decrement. The phenomenon has since been demonstrated as one of the most ubiquitous and consistently replicated findings in the vigilance literature . Laboratory vigilance tasks require correctly identifying rare target stimuli in an array for a prolonged period . Vigilance decrement typically onsets within 15 min of sustained attention; however, it has been reported in as little as 8 min under particularly demanding situations . Note . Each number in refers to the order in which random “blips” were presented. Overload and underload Theoretical accounts of sustained attention task phenomena fall into two broad categories. “Underload,” or mindlessness, theories assume that sustaining attention is an underwhelming, monotonous experience, eventually redirecting attention from task-relevant to -irrelevant processes . Conversely, the premise of “Overload” or resource theories is that sustaining attention is an effortful experience that depletes a finite pool of information processing resources . Early underload accounts of vigilance decrement attributed the temporal decline in performance to reactive inhibition or arousal . Drive and arousal theories of vigilance decrement suggested that the tediously repetitive nature of sustained attention tasks inhibited brainstem and thalamic activation required to identify critical signals from an information stream . While arousal and drive theories of vigilance decrement accounted for the gradual decline in sustained attention task performance, or vigilance decrement, they could not explain why people ubiquitously experience vigilance tasks as effortful, tiring, and stressful . However, Mindlessness and Mind-Wandering Theories of vigilance decrement overcome this limitation of underload theories . Conversely, overload theories of vigilance decrement attributed subjective reports of effortful exertion as the result of depleted cognitive resources required to sustain attention to task-specific processes . Mindlessness theory of sustained attention task phenomenon Robertson’s Mindlessness Theory was an underload account of vigilance decrement that suggested task monotony and a lack of external support led to the “mindless” withdrawal of attention from task-relevant processes . Unlike drive and arousal theories, mindlessness theory accounted for the effort associated with vigilance tasks by drawing on idea that switching from a mindless to a mindful state is effortful. Despite this advancement in the explanatory power of the underload perspective, mindlessness theory remained hampered by its failure to account for task-unrelated thoughts. Vigilance decrement is not the only phenomenon associated with sustained attention tasks. Lapses in sustained attention task performance can also present as an increase in the frequency of task-unrelated-thoughts, or TUTs, as time-on-task increases . TUTs are internally manifested, stimulus-independent, spontaneous thoughts unrelated to performing the central task . As time progresses on a vigilance task, there is a corresponding increase in the frequency of TUTs that parallels the declines in performance known as vigilance decrement . While mindlessness theory suggested an explanation for the gradual decline in attention to vigilance task-relevant processing, it could not explain the link between vigilance decrement and TUTs. Put differently, the central premise of mindlessness theory implies that vigilance decrement affects global information processing rather than task-relevant processes alone. The association between sustained attention tasks and TUTs suggests that vigilance decrement does not lead to a “mindless” state. The mind-wandering theory was hence derived to explain the TUTs that manifest during sustained attention tasks. Mind-wandering theory of sustained attention task phenomenon The Mind-Wandering theory expanded on mindlessness theory by accounting for changes in the mind that coincide with the disengagement of attention from task-relevant to -irrelevant processes during vigilance decrement. Unlike the Mindlessness theory, Mind-Wandering is not based on the notion that vigilance decrement leads to a “mindless” state of decreased global processing . Instead, the Mind-wandering theory is premised on the notion that attention does not fade to mindlessness but grows increasingly re-directed away from task-relevant processes to TUTs during vigilance decrement . The premise of the Mindlessness and Mind-Wandering theories is that attentional resources withdraw gradually from underwhelming, task-relevant processes during sustained attention tasks . This is distinct from the resource depletion version of the overload theory, which is based on the notion that cognitive resources required to sustain attention to task-relevant processes are gradually drained until a shift in focus occurs . Parasuraman’s “unitary” resource depletion theory The resource depletion version of the overload theory has garnered far more support in the literature than the underload alternative . Brain imaging, mental workload, and behavioral studies have demonstrated lapses in attention function according to the difficulty associated with sustained task-relevant information processing . This association between task-specific cognitive load and cognitive resource utilization aligns with the depletion version of the overload theory. In addition, and demonstrated an association between sustained attention and the right prefrontal, parietal, and inferior regions of the cortex, as well as within the anterior cingulate cortex. and , ) cerebral blood flow velocity studies over these regions have suggested compelling support for the resource depletion theory. Hitchcock et al. and Shaw et al. associated vigilance decrement during sustained attention tasks with cerebral blood flow velocity decreases. Cerebral blood flow is the primary delivery mechanism of energetic resources (glucose) into the brain . and ; ), therefore, proposed a neurometabolic account of resource depletion and reductions in vigilance performance with time-on-task. presented a seminal case for the resource depletion theory in demonstrating a direct relationship between vigilance decrement and the event, or presentation, rate of non-critical sustained attention task information resource depletion theory suggested that neurons metabolized finite reserves of energy to sustain psycho-physiological processes that overload the energetic capacity of blood-based resources. Parasuraman’s Unitary Resource Depletion Theory, therefore, attributed vigilance decrement to the depletion of a singular neurometabolic resource required to psycho-physiologically sustain the processing of task-specific workloads . , derived the “unitary” component of his resource depletion theory from economic model of attention regulation, which accommodated the event-rate effect within a resource theory of vigilance decrement . The economic model Kahneman suggested that the human attentional system energetically regulates information processing demands that overload the energetic supply of blood-based reserves by metabolizing finite energetic cognitive resources homogenously distributed across the cortex. Kahneman grounded Parasuraman’s ideas in a neurometabolic account of sustained attention. , ) unitary resource account of vigilance decrement hence suggested that the temporal manifestation of vigilance decrement reflected the depletion of a single energetic resource distributed across the human attentional system . Astrocytes partner with neurons and act as natural energy reserves, which can supplement the metabolic requirements associated with action potential firing rates that exceed the energetic capacity of blood-based resources alone . However, at most, neurons can only metabolize 85% of glycogen reserved in their partnered astrocytes . Astrocytic glycogen is a metabolically finite reserve that depletes gradually under the psycho-physiological processing demands required to sustain the discrimination of critical task targets. Astrocytic glycogen hence reflects a finite, unitary resource that , ) suggested depletes during vigilance tasks. Parasuraman’s unitary resource depletion theory hence suggested that the rate astrocytic glycogen reserves deplete during a vigilance task is related to the rate at which new information is presented. For example, supported Parasuraman’s instantiation of the resource depletion theory by demonstrating a direct relationship between event rate and performance deficits in their cyber vigilance task. , Unitary Resource Depletion Theory of vigilance decrement therefore attributed increasingly frequent performance errors during sustained attention tasks to the systemic exhaustion of astrocytic glycogen in the human attentional system . Wickens et al.’s multiple resource theory Successive and simultaneous types of vigilance tasks are distinguished by what is required to identify targets: identifying targets in the former requires memory, whereas perceptual features distinguish targets in the latter compared the impact of concurrent memory use on vigilance performance across 42 vigilance tasks drawn from the literature. Parasuraman found that 14 of the 42 studies reported vigilance decrement in successive task types and suggested that the additional cognitive load associated with concurrent memory use may accelerate the depletion of supplementary energetic resources. However, meta-analysis suggested that , original, unitary conception of resource theory only held true for cognitive vigilance tasks. In addition, task targets can be cognitive or sensory; task-specific, symbolically encoded meanings distinguish cognitive vigilance tasks, whereas sensorially perceived attributes distinguish sensory vigilance tasks . For example, Desmond et al. compared performance on a simultaneous and successive form of a sensory vigilance task and showed steeper performance deficits in the former. Digit pairs presented with one element slightly smaller than its partner were the sensory features used to distinguish critical targets in the simultaneous form of Desmond et al.’s task. By contrast, participants had to remember the size of each previously presented digit pair and indicate when a new pair was smaller than its sequential predecessor in the successive form of Desmond et al.’s task. Under , resource theory, the added cognitive load associated with remembering each previously presented digit pair should have led to steeper performance deficits in successive vigilance task than in their simultaneous alternative. However, Desmond et al. did not support , resource theory because performance deficits in the simultaneous form of their sensory vigilance task were steeper than in the successive condition. Wickens et al. expanded upon , “unitary” paradigm with economic model of resource regulation in the human attentional system. Wickens et al.’s multiple resource theory of vigilance decrement attributed the temporal reductions in sustained attention performance to the number of task-specific workload factor dimensions needed to discriminate critical targets. Wickens et al. outlined four workload factor dimensions: information processing codes, visual channels, perceptual modalities, and processing stages. Wickens et al.’s version of multiple resource theory attributed greater cognitive demand, and a steeper performance decrement, to vigilance tasks characterized by information processes distributed along a single workload dimension. , version of The Depletion Theory suggested an account for the behavioral impact of astrocytic depletion on vigilance decrement during sustained attention task performance. However, this early version of the depletion theory did not recognize that astrocytic depletion occurs across multiple task-relevant cortical regions . Wickens et al.’s depletion theory extension better reflected astrocytic depletion’s impact on vigilance performance across multiple cortically regional processes. The resource depletion and mindlessness versions of the overload and underload accounts of vigilance task phenomenon contrast along four key lines of inquiry . These include how effortful vigilance tasks are, the impact of increasing task demands and engagement on performance, as well as the relationship between TUT frequency and time-on-task . The four lines of inquiry presented a clear bifurcation between the overload and underload accounts of vigilance task phenomenon. Moreover, resource control-failure theory resolved this bifurcation and unified the underload and overload accounts of vigilance decrement during sustained attention task performance along six central tenets. resource control theory unified the underload and overload accounts of sustained attention within the six tenets of their resource control-failure theory . Thomson et al. derived their theory from a combination of theory of attentional-resource allocation and , control-failure theory. Hence, Thomson et al.’s resource control-failure theory explained the temporal accumulation of vigilance performance errors and TUTs according to a breakdown in the controlled allocation of attentional resources between task-relevant and -irrelevant processes. illustrates resource control-failure account of vigilance performance. The dark horizontal line intercepting the left vertical axis represents the total volume of allocatable attentional resources. The dotted horizontal line represents the volume of resources constantly required to sustain the performance of task-relevant processes. The fifth tenet of resource control-failure theory holds that as time on task increases, executive control of attentional resource allocation to task-relevant processes decreases. Hence, the bold line depicts the relationship between time-on-task and executive control of attentional resource allocation, with a negative trend running along the top of . As time-on-task increases, executive control decreases, and an increasing volume of attentional resources become reallocated from task-relevant (represented in dark gray) to -irrelevant (represented by the union of white and light gray regions) processes. The decrease in attentional resources devoted to task-relevant processes corresponds to performance costs manifest behaviorally as vigilance decrement during sustained attention tasks. To be clear, TUTs do not increase because of failure in executive control under the tenets of resource control-failure theory. Instead, an executive control failure to allocate attentional resources to task-relevant processes causes unallocated resources to accumulate over time. Since the mind’s natural state is defined by task-irrelevant processes, the reallocation of this previously allocated volume of attentional resources is biased toward TUTs. The six tenets of resource control-failure theory presented a unified workload account of the four lines of inquiry . Resource control-failure theory first offered an account for the subjective reports that vigilance tasks are effortful and draining experiences. The first tenet of resource control-failure theory holds that sustained control of attentional resource allocation to task-relevant processes is an effortful experience. That effort is associated with the sustained executive control of attentional resource allocation to task-relevant and not -irrelevant processes. This effort aligned with reports of burnout and distress in operational network defense personnel. Secondly, how do increasing task demands impact vigilance decrement? Vigilance performance losses are more extensive and occur faster in more difficult sustained attention tasks. Resource control-failure theory holds that more difficult tasks demand a greater allocation of attentional resources to task-specific processes. Hence, time-related reductions in the control of attentional resource allocation will have a larger impact on performance than in simpler, less demanding tasks. Thirdly, how does increasing task engagement impact vigilance decrement? Task engagement refers to the range of resources required to sustain executive control of attention allocation to task-specific processes . The first tenet of resource control-failure theory holds that controlling attentional resource allocation is an effortful experience . Highly engaging tasks have access to a greater volume of resources available to sustain the active control of attention allocation to task-specific processes . By contrast, fewer resources are available to maintain executive control of attentional allocation to task-specific processes in less engaging tasks . Fourthly, what is the impact of increasing time-on-task on TUT frequency? The fifth tenet of theory holds that the executive controls that allocate attentional resources to task-specific processes fail with increasing frequency with time-on-task. Furthermore, the third tenet of resource control-failure theory holds that attention allocation is naturally biased toward TUTs . Therefore, as executive controls fail to allocate attentional resources to task-specific processes, they are increasingly misallocated to TUTs as time goes on. Reconciling the “control failure” and “resource depletion” hypotheses , resource depletion theory suggested vigilance decrement reflected the gradual metabolic exhaustion of a single, unitary reserve of energetic resources . Parasuraman derived the unitary component of the overload theory from model of the human attentional system. Kahneman modeled attention as an energetically draining process metabolically sustained by homogeneously allocatable but finite information processing reserves. Parasuraman based the ground truth of the unitary resource theory of vigilance decrement on Kahneman’s neurometabolic paradigm . That is, sustained attention tasks require task-specific cognitive processes, which metabolically “overload” the energetic capacity of blood-based glycogen alone . Wickens et al. rejected the unitary component of Parasuraman’s version of the overload theory. Instead, Wickens et al. suggested that vigilance decrement reflected the gradual metabolic depletion of multiple energetic resource caches distributed across multiple task-specific processes. , and Wickens et al.’s versions of the overload theory relied on the depletion of finite energetic resources to explain vigilance performance deficits during sustained attention tasks . Resource Control Failure Theory was based on their resource control failure theory. This theory stated that vigilance decrement and TUT accumulation stemmed from increasingly frequent failures to executively control the allocation of attentional resources between task-relevant and -irrelevant neuronal populations . , and Wickens et al. conception of resource depletion was explicitly rejected by . However, Resource Control Failure Theory can accommodate the idea that sustained task-specific processing by neurons distributed in executive-function-specific regions, or populations, of the cortex can metabolically deplete local caches of neuro-energetic resources required to sustain vigilance performance. For example, demonstrated that regional cortical activation was paralleled by anatomically segregated resource metabolization. In addition, Emmerling et al. suggested the notion that “resource depletion” reflected an accumulation of multiple metabolically costly cognitive “acts of self-regulation” (ASR). Emmerling et al. further suggested that ASRs incrementally exhausted metabolic resources distributed within neuronal populations in the right dorsolateral prefrontal cortex. However, Emmerling failed to demonstrate that metabolic resources can be depleted in anatomically segregated cortex regions, namely, the right dorsolateral and prefrontal lobes, using transcranial alternating current to stimulate activity. Emmerling’s results hence aligned with Thomson et al.’s rejection of the resource depletion theory. However, Wickens et al. suggested metabolic exhaustion occurred across “multiple” task-specific processing structures. Hence, it may have been the case that Emmerling et al.’s brain stimulation failed to induce a level of psycho-physiological information processing great enough to metabolically exhaust neuronal populations in the right dorsolateral prefrontal, task-relevant cortical region. Transcranial alternating current stimulation simultaneously enhances vigilance performance without inducing metabolic depletion in task-relevant regions of the cortex . This would undermine and rejection of depletion-based theories, as it would then follow that transcranial alternating current stimulation may serve as an energetic buffer against declines in vigilance performance associated with astrocytic glycogen exhaustion. That is, depletion may still be the underlying causal mechanism of vigilance decrement despite Thomson et al.’s rejection. further supported the depletion concept by directly demonstrating that sustained attention tasks can lead to the onset of an acute hypoglycemic episode. An acute hypoglycemic episode refers to the rapid blood glucose reduction from 7 mmol L −1 to 4 mmol L −1 or less . Glucose is the central neuro-energetic resource used by the brain to sustain information processing . Cox et al., therefore, directly demonstrated support for the reduction or depletion of a critical neuro-energetic resource required to sustain the processing of task-relevant information. and Wickens et al.’s depletion concept is reconcilable with the six tenets of resource control-failure theory of vigilance. According to Thomson et al., the first tenet states that individuals possess a finite volume of attentional resources for task-relevant processing. Blood-based glucose is the primary neuro-energetic resource that sustains information processing in the frontal lobes . Task-specific processing demands can require an action potential frequency that outpaces the energetic capacity of blood-based glucose. Neurons access glycogen reserved in astrocytes to supplement their energetic requirements when task-relevant processing demands outpace the metabolic capacity of blood-based glucose. However, astrocytic glycogen is finite. Once a neuron depletes its partnered astrocyte of glycogen, its firing rate necessarily reduces below the level required to sustain task-relevant information processing . Moreover, as demonstrated, this can deplete the blood-based glucose supply to hypoglycemic concentrations. The dilution of blood-based glycogen and depletion of astrocytic glycogen represent two finite resources required to sustain attention, decreasing availability with time-on-task. second tenet holds that task-irrelevant processes occupy unallocated attentional resources. Blood-based glucose and caches of astrocytic glycogen reserved in task-irrelevant cortex regions sustain TUT processes. Task-relevant neurons cannot access astrocytic glycogen reserved in task-irrelevant cortex regions . Hence, the TUT processes in task-irrelevant cortex regions occupy neuro-energetic attentional resources unallocated to task-specific processes. third tenet holds that the allocation of attention is internally biased toward TUTs and not task-relevant processes. The rate at which astrocytic glycogen is metabolized neuro-energetically explains why attention is biased. Beyond astrocytic depletion, blood glucose concentration can dilute to hypoglycemic levels for both task-relevant and -irrelevant neurons. Hence, the primary energy source used to sustain TUT processes falls to the glycogen stored in the astrocytes that partner with neurons distributed in task-irrelevant cortex regions. However, astrocytic glycogen is metabolized slower in task-irrelevant regions of the cortex, which are unburdened by the processing demands associated with task-relevant processes. It, therefore, follows that TUTs manifest with increasing frequency with time-on-task because their supply of supplementary astrocytic glycogen is not metabolized at the same rate as in task-relevant regions of the cortex . That is, the cortical origins of TUTs are less likely to be affected by astrocytic glycogen depletion in task-relevant regions of the cortex, as task-irrelevant processes will not metabolize local energetic reserves at the same rate . fourth tenet holds that allocating attention to task-relevant processes is controlled by an executive function. This tenet refers to a single executive function that controls the allocation of attentional resources. However, supplementary attentional resources (glycogen reserves) are allocated by astrocytes distributed across regions of the cortex that process multiple task-specific executive functions . For example, audible vigilance task information requires executive functions processed by neurons distributed in the auditory cortex and the frontal lobes. Astrocytes guide the allocation of attentional resources allocation among their partner neurons distributed within executive function-specific regions of the cortex . Astrocytes wrap around the synaptic cleft and use activity-dependent chemical signals to infer the metabolic needs of their partnered neurons . Supplementary glycogen is made available to neurons when their partnered astrocyte senses an action potential firing rate that exceeds the metabolic capacity of blood-based glucose. Supplementary attentional resource allocation is therefore mediated by the cognitive workload associated with processing task-specific executive functions across multiple cortex regions. Attentional resource allocation is a feature of the neurobiological systems used to process task-specific executive functions. That is, there is no single executive function that controls the attentional resource allocation of astrocytic glycogen. Instead, the controlled allocation of astrocytic glycogen is a neurochemical feature of the neuronal systems used to process task-specific executive functions . fifth tenet holds that executive control of attention allocation fails with time-on-task. Vigilance decrement will begin with the depletion of a single astrocyte–neuron system that is required to process a task-specific executive function . As time-on-task increases, so does the ratio of depleted to un-depleted astrocyte-neuron partners distributed in regions of the cortex specialized in processing task-specific information. In turn, processing errors accumulate between un-depleted and increasingly depleted populations of task-relevant neurons . sixth tenet holds that TUTs do not impact task-relevant processes that do not require the complete utilization of attentional resources. TUTs come at a neuro-energetic cost to the energetic capacity of blood-based glucose used to sustain task-relevant processes. If the metabolic cost associated with task-relevant and -irrelevant functions can be accommodated within the energetic capacity of blood-based glucose, TUTs will be unlikely to impact vigilance performance. TUTs will, however, impact vigilance performance if the glucose used to process them forces task-relevant neurons to supplement their energetic needs by metabolizing astrocytic glycogen. Theoretical accounts of sustained attention task phenomena fall into two broad categories. “Underload,” or mindlessness, theories assume that sustaining attention is an underwhelming, monotonous experience, eventually redirecting attention from task-relevant to -irrelevant processes . Conversely, the premise of “Overload” or resource theories is that sustaining attention is an effortful experience that depletes a finite pool of information processing resources . Early underload accounts of vigilance decrement attributed the temporal decline in performance to reactive inhibition or arousal . Drive and arousal theories of vigilance decrement suggested that the tediously repetitive nature of sustained attention tasks inhibited brainstem and thalamic activation required to identify critical signals from an information stream . While arousal and drive theories of vigilance decrement accounted for the gradual decline in sustained attention task performance, or vigilance decrement, they could not explain why people ubiquitously experience vigilance tasks as effortful, tiring, and stressful . However, Mindlessness and Mind-Wandering Theories of vigilance decrement overcome this limitation of underload theories . Conversely, overload theories of vigilance decrement attributed subjective reports of effortful exertion as the result of depleted cognitive resources required to sustain attention to task-specific processes . Robertson’s Mindlessness Theory was an underload account of vigilance decrement that suggested task monotony and a lack of external support led to the “mindless” withdrawal of attention from task-relevant processes . Unlike drive and arousal theories, mindlessness theory accounted for the effort associated with vigilance tasks by drawing on idea that switching from a mindless to a mindful state is effortful. Despite this advancement in the explanatory power of the underload perspective, mindlessness theory remained hampered by its failure to account for task-unrelated thoughts. Vigilance decrement is not the only phenomenon associated with sustained attention tasks. Lapses in sustained attention task performance can also present as an increase in the frequency of task-unrelated-thoughts, or TUTs, as time-on-task increases . TUTs are internally manifested, stimulus-independent, spontaneous thoughts unrelated to performing the central task . As time progresses on a vigilance task, there is a corresponding increase in the frequency of TUTs that parallels the declines in performance known as vigilance decrement . While mindlessness theory suggested an explanation for the gradual decline in attention to vigilance task-relevant processing, it could not explain the link between vigilance decrement and TUTs. Put differently, the central premise of mindlessness theory implies that vigilance decrement affects global information processing rather than task-relevant processes alone. The association between sustained attention tasks and TUTs suggests that vigilance decrement does not lead to a “mindless” state. The mind-wandering theory was hence derived to explain the TUTs that manifest during sustained attention tasks. The Mind-Wandering theory expanded on mindlessness theory by accounting for changes in the mind that coincide with the disengagement of attention from task-relevant to -irrelevant processes during vigilance decrement. Unlike the Mindlessness theory, Mind-Wandering is not based on the notion that vigilance decrement leads to a “mindless” state of decreased global processing . Instead, the Mind-wandering theory is premised on the notion that attention does not fade to mindlessness but grows increasingly re-directed away from task-relevant processes to TUTs during vigilance decrement . The premise of the Mindlessness and Mind-Wandering theories is that attentional resources withdraw gradually from underwhelming, task-relevant processes during sustained attention tasks . This is distinct from the resource depletion version of the overload theory, which is based on the notion that cognitive resources required to sustain attention to task-relevant processes are gradually drained until a shift in focus occurs “unitary” resource depletion theory The resource depletion version of the overload theory has garnered far more support in the literature than the underload alternative . Brain imaging, mental workload, and behavioral studies have demonstrated lapses in attention function according to the difficulty associated with sustained task-relevant information processing . This association between task-specific cognitive load and cognitive resource utilization aligns with the depletion version of the overload theory. In addition, and demonstrated an association between sustained attention and the right prefrontal, parietal, and inferior regions of the cortex, as well as within the anterior cingulate cortex. and , ) cerebral blood flow velocity studies over these regions have suggested compelling support for the resource depletion theory. Hitchcock et al. and Shaw et al. associated vigilance decrement during sustained attention tasks with cerebral blood flow velocity decreases. Cerebral blood flow is the primary delivery mechanism of energetic resources (glucose) into the brain . and ; ), therefore, proposed a neurometabolic account of resource depletion and reductions in vigilance performance with time-on-task. presented a seminal case for the resource depletion theory in demonstrating a direct relationship between vigilance decrement and the event, or presentation, rate of non-critical sustained attention task information resource depletion theory suggested that neurons metabolized finite reserves of energy to sustain psycho-physiological processes that overload the energetic capacity of blood-based resources. Parasuraman’s Unitary Resource Depletion Theory, therefore, attributed vigilance decrement to the depletion of a singular neurometabolic resource required to psycho-physiologically sustain the processing of task-specific workloads . , derived the “unitary” component of his resource depletion theory from economic model of attention regulation, which accommodated the event-rate effect within a resource theory of vigilance decrement . The economic model Kahneman suggested that the human attentional system energetically regulates information processing demands that overload the energetic supply of blood-based reserves by metabolizing finite energetic cognitive resources homogenously distributed across the cortex. Kahneman grounded Parasuraman’s ideas in a neurometabolic account of sustained attention. , ) unitary resource account of vigilance decrement hence suggested that the temporal manifestation of vigilance decrement reflected the depletion of a single energetic resource distributed across the human attentional system . Astrocytes partner with neurons and act as natural energy reserves, which can supplement the metabolic requirements associated with action potential firing rates that exceed the energetic capacity of blood-based resources alone . However, at most, neurons can only metabolize 85% of glycogen reserved in their partnered astrocytes . Astrocytic glycogen is a metabolically finite reserve that depletes gradually under the psycho-physiological processing demands required to sustain the discrimination of critical task targets. Astrocytic glycogen hence reflects a finite, unitary resource that , ) suggested depletes during vigilance tasks. Parasuraman’s unitary resource depletion theory hence suggested that the rate astrocytic glycogen reserves deplete during a vigilance task is related to the rate at which new information is presented. For example, supported Parasuraman’s instantiation of the resource depletion theory by demonstrating a direct relationship between event rate and performance deficits in their cyber vigilance task. , Unitary Resource Depletion Theory of vigilance decrement therefore attributed increasingly frequent performance errors during sustained attention tasks to the systemic exhaustion of astrocytic glycogen in the human attentional system multiple resource theory Successive and simultaneous types of vigilance tasks are distinguished by what is required to identify targets: identifying targets in the former requires memory, whereas perceptual features distinguish targets in the latter compared the impact of concurrent memory use on vigilance performance across 42 vigilance tasks drawn from the literature. Parasuraman found that 14 of the 42 studies reported vigilance decrement in successive task types and suggested that the additional cognitive load associated with concurrent memory use may accelerate the depletion of supplementary energetic resources. However, meta-analysis suggested that , original, unitary conception of resource theory only held true for cognitive vigilance tasks. In addition, task targets can be cognitive or sensory; task-specific, symbolically encoded meanings distinguish cognitive vigilance tasks, whereas sensorially perceived attributes distinguish sensory vigilance tasks . For example, Desmond et al. compared performance on a simultaneous and successive form of a sensory vigilance task and showed steeper performance deficits in the former. Digit pairs presented with one element slightly smaller than its partner were the sensory features used to distinguish critical targets in the simultaneous form of Desmond et al.’s task. By contrast, participants had to remember the size of each previously presented digit pair and indicate when a new pair was smaller than its sequential predecessor in the successive form of Desmond et al.’s task. Under , resource theory, the added cognitive load associated with remembering each previously presented digit pair should have led to steeper performance deficits in successive vigilance task than in their simultaneous alternative. However, Desmond et al. did not support , resource theory because performance deficits in the simultaneous form of their sensory vigilance task were steeper than in the successive condition. Wickens et al. expanded upon , “unitary” paradigm with economic model of resource regulation in the human attentional system. Wickens et al.’s multiple resource theory of vigilance decrement attributed the temporal reductions in sustained attention performance to the number of task-specific workload factor dimensions needed to discriminate critical targets. Wickens et al. outlined four workload factor dimensions: information processing codes, visual channels, perceptual modalities, and processing stages. Wickens et al.’s version of multiple resource theory attributed greater cognitive demand, and a steeper performance decrement, to vigilance tasks characterized by information processes distributed along a single workload dimension. , version of The Depletion Theory suggested an account for the behavioral impact of astrocytic depletion on vigilance decrement during sustained attention task performance. However, this early version of the depletion theory did not recognize that astrocytic depletion occurs across multiple task-relevant cortical regions . Wickens et al.’s depletion theory extension better reflected astrocytic depletion’s impact on vigilance performance across multiple cortically regional processes. The resource depletion and mindlessness versions of the overload and underload accounts of vigilance task phenomenon contrast along four key lines of inquiry . These include how effortful vigilance tasks are, the impact of increasing task demands and engagement on performance, as well as the relationship between TUT frequency and time-on-task . The four lines of inquiry presented a clear bifurcation between the overload and underload accounts of vigilance task phenomenon. Moreover, resource control-failure theory resolved this bifurcation and unified the underload and overload accounts of vigilance decrement during sustained attention task performance along six central tenets. unified the underload and overload accounts of sustained attention within the six tenets of their resource control-failure theory . Thomson et al. derived their theory from a combination of theory of attentional-resource allocation and , control-failure theory. Hence, Thomson et al.’s resource control-failure theory explained the temporal accumulation of vigilance performance errors and TUTs according to a breakdown in the controlled allocation of attentional resources between task-relevant and -irrelevant processes. illustrates resource control-failure account of vigilance performance. The dark horizontal line intercepting the left vertical axis represents the total volume of allocatable attentional resources. The dotted horizontal line represents the volume of resources constantly required to sustain the performance of task-relevant processes. The fifth tenet of resource control-failure theory holds that as time on task increases, executive control of attentional resource allocation to task-relevant processes decreases. Hence, the bold line depicts the relationship between time-on-task and executive control of attentional resource allocation, with a negative trend running along the top of . As time-on-task increases, executive control decreases, and an increasing volume of attentional resources become reallocated from task-relevant (represented in dark gray) to -irrelevant (represented by the union of white and light gray regions) processes. The decrease in attentional resources devoted to task-relevant processes corresponds to performance costs manifest behaviorally as vigilance decrement during sustained attention tasks. To be clear, TUTs do not increase because of failure in executive control under the tenets of resource control-failure theory. Instead, an executive control failure to allocate attentional resources to task-relevant processes causes unallocated resources to accumulate over time. Since the mind’s natural state is defined by task-irrelevant processes, the reallocation of this previously allocated volume of attentional resources is biased toward TUTs. The six tenets of resource control-failure theory presented a unified workload account of the four lines of inquiry . Resource control-failure theory first offered an account for the subjective reports that vigilance tasks are effortful and draining experiences. The first tenet of resource control-failure theory holds that sustained control of attentional resource allocation to task-relevant processes is an effortful experience. That effort is associated with the sustained executive control of attentional resource allocation to task-relevant and not -irrelevant processes. This effort aligned with reports of burnout and distress in operational network defense personnel. Secondly, how do increasing task demands impact vigilance decrement? Vigilance performance losses are more extensive and occur faster in more difficult sustained attention tasks. Resource control-failure theory holds that more difficult tasks demand a greater allocation of attentional resources to task-specific processes. Hence, time-related reductions in the control of attentional resource allocation will have a larger impact on performance than in simpler, less demanding tasks. Thirdly, how does increasing task engagement impact vigilance decrement? Task engagement refers to the range of resources required to sustain executive control of attention allocation to task-specific processes . The first tenet of resource control-failure theory holds that controlling attentional resource allocation is an effortful experience . Highly engaging tasks have access to a greater volume of resources available to sustain the active control of attention allocation to task-specific processes . By contrast, fewer resources are available to maintain executive control of attentional allocation to task-specific processes in less engaging tasks . Fourthly, what is the impact of increasing time-on-task on TUT frequency? The fifth tenet of theory holds that the executive controls that allocate attentional resources to task-specific processes fail with increasing frequency with time-on-task. Furthermore, the third tenet of resource control-failure theory holds that attention allocation is naturally biased toward TUTs . Therefore, as executive controls fail to allocate attentional resources to task-specific processes, they are increasingly misallocated to TUTs as time goes on. , resource depletion theory suggested vigilance decrement reflected the gradual metabolic exhaustion of a single, unitary reserve of energetic resources . Parasuraman derived the unitary component of the overload theory from model of the human attentional system. Kahneman modeled attention as an energetically draining process metabolically sustained by homogeneously allocatable but finite information processing reserves. Parasuraman based the ground truth of the unitary resource theory of vigilance decrement on Kahneman’s neurometabolic paradigm . That is, sustained attention tasks require task-specific cognitive processes, which metabolically “overload” the energetic capacity of blood-based glycogen alone . Wickens et al. rejected the unitary component of Parasuraman’s version of the overload theory. Instead, Wickens et al. suggested that vigilance decrement reflected the gradual metabolic depletion of multiple energetic resource caches distributed across multiple task-specific processes. , and Wickens et al.’s versions of the overload theory relied on the depletion of finite energetic resources to explain vigilance performance deficits during sustained attention tasks . Resource Control Failure Theory was based on their resource control failure theory. This theory stated that vigilance decrement and TUT accumulation stemmed from increasingly frequent failures to executively control the allocation of attentional resources between task-relevant and -irrelevant neuronal populations . , and Wickens et al. conception of resource depletion was explicitly rejected by . However, Resource Control Failure Theory can accommodate the idea that sustained task-specific processing by neurons distributed in executive-function-specific regions, or populations, of the cortex can metabolically deplete local caches of neuro-energetic resources required to sustain vigilance performance. For example, demonstrated that regional cortical activation was paralleled by anatomically segregated resource metabolization. In addition, Emmerling et al. suggested the notion that “resource depletion” reflected an accumulation of multiple metabolically costly cognitive “acts of self-regulation” (ASR). Emmerling et al. further suggested that ASRs incrementally exhausted metabolic resources distributed within neuronal populations in the right dorsolateral prefrontal cortex. However, Emmerling failed to demonstrate that metabolic resources can be depleted in anatomically segregated cortex regions, namely, the right dorsolateral and prefrontal lobes, using transcranial alternating current to stimulate activity. Emmerling’s results hence aligned with Thomson et al.’s rejection of the resource depletion theory. However, Wickens et al. suggested metabolic exhaustion occurred across “multiple” task-specific processing structures. Hence, it may have been the case that Emmerling et al.’s brain stimulation failed to induce a level of psycho-physiological information processing great enough to metabolically exhaust neuronal populations in the right dorsolateral prefrontal, task-relevant cortical region. Transcranial alternating current stimulation simultaneously enhances vigilance performance without inducing metabolic depletion in task-relevant regions of the cortex . This would undermine and rejection of depletion-based theories, as it would then follow that transcranial alternating current stimulation may serve as an energetic buffer against declines in vigilance performance associated with astrocytic glycogen exhaustion. That is, depletion may still be the underlying causal mechanism of vigilance decrement despite Thomson et al.’s rejection. further supported the depletion concept by directly demonstrating that sustained attention tasks can lead to the onset of an acute hypoglycemic episode. An acute hypoglycemic episode refers to the rapid blood glucose reduction from 7 mmol L −1 to 4 mmol L −1 or less . Glucose is the central neuro-energetic resource used by the brain to sustain information processing . Cox et al., therefore, directly demonstrated support for the reduction or depletion of a critical neuro-energetic resource required to sustain the processing of task-relevant information. and Wickens et al.’s depletion concept is reconcilable with the six tenets of resource control-failure theory of vigilance. According to Thomson et al., the first tenet states that individuals possess a finite volume of attentional resources for task-relevant processing. Blood-based glucose is the primary neuro-energetic resource that sustains information processing in the frontal lobes . Task-specific processing demands can require an action potential frequency that outpaces the energetic capacity of blood-based glucose. Neurons access glycogen reserved in astrocytes to supplement their energetic requirements when task-relevant processing demands outpace the metabolic capacity of blood-based glucose. However, astrocytic glycogen is finite. Once a neuron depletes its partnered astrocyte of glycogen, its firing rate necessarily reduces below the level required to sustain task-relevant information processing . Moreover, as demonstrated, this can deplete the blood-based glucose supply to hypoglycemic concentrations. The dilution of blood-based glycogen and depletion of astrocytic glycogen represent two finite resources required to sustain attention, decreasing availability with time-on-task. second tenet holds that task-irrelevant processes occupy unallocated attentional resources. Blood-based glucose and caches of astrocytic glycogen reserved in task-irrelevant cortex regions sustain TUT processes. Task-relevant neurons cannot access astrocytic glycogen reserved in task-irrelevant cortex regions . Hence, the TUT processes in task-irrelevant cortex regions occupy neuro-energetic attentional resources unallocated to task-specific processes. third tenet holds that the allocation of attention is internally biased toward TUTs and not task-relevant processes. The rate at which astrocytic glycogen is metabolized neuro-energetically explains why attention is biased. Beyond astrocytic depletion, blood glucose concentration can dilute to hypoglycemic levels for both task-relevant and -irrelevant neurons. Hence, the primary energy source used to sustain TUT processes falls to the glycogen stored in the astrocytes that partner with neurons distributed in task-irrelevant cortex regions. However, astrocytic glycogen is metabolized slower in task-irrelevant regions of the cortex, which are unburdened by the processing demands associated with task-relevant processes. It, therefore, follows that TUTs manifest with increasing frequency with time-on-task because their supply of supplementary astrocytic glycogen is not metabolized at the same rate as in task-relevant regions of the cortex . That is, the cortical origins of TUTs are less likely to be affected by astrocytic glycogen depletion in task-relevant regions of the cortex, as task-irrelevant processes will not metabolize local energetic reserves at the same rate . fourth tenet holds that allocating attention to task-relevant processes is controlled by an executive function. This tenet refers to a single executive function that controls the allocation of attentional resources. However, supplementary attentional resources (glycogen reserves) are allocated by astrocytes distributed across regions of the cortex that process multiple task-specific executive functions . For example, audible vigilance task information requires executive functions processed by neurons distributed in the auditory cortex and the frontal lobes. Astrocytes guide the allocation of attentional resources allocation among their partner neurons distributed within executive function-specific regions of the cortex . Astrocytes wrap around the synaptic cleft and use activity-dependent chemical signals to infer the metabolic needs of their partnered neurons . Supplementary glycogen is made available to neurons when their partnered astrocyte senses an action potential firing rate that exceeds the metabolic capacity of blood-based glucose. Supplementary attentional resource allocation is therefore mediated by the cognitive workload associated with processing task-specific executive functions across multiple cortex regions. Attentional resource allocation is a feature of the neurobiological systems used to process task-specific executive functions. That is, there is no single executive function that controls the attentional resource allocation of astrocytic glycogen. Instead, the controlled allocation of astrocytic glycogen is a neurochemical feature of the neuronal systems used to process task-specific executive functions . fifth tenet holds that executive control of attention allocation fails with time-on-task. Vigilance decrement will begin with the depletion of a single astrocyte–neuron system that is required to process a task-specific executive function . As time-on-task increases, so does the ratio of depleted to un-depleted astrocyte-neuron partners distributed in regions of the cortex specialized in processing task-specific information. In turn, processing errors accumulate between un-depleted and increasingly depleted populations of task-relevant neurons . sixth tenet holds that TUTs do not impact task-relevant processes that do not require the complete utilization of attentional resources. TUTs come at a neuro-energetic cost to the energetic capacity of blood-based glucose used to sustain task-relevant processes. If the metabolic cost associated with task-relevant and -irrelevant functions can be accommodated within the energetic capacity of blood-based glucose, TUTs will be unlikely to impact vigilance performance. TUTs will, however, impact vigilance performance if the glucose used to process them forces task-relevant neurons to supplement their energetic needs by metabolizing astrocytic glycogen. Resource Control Failure Theory is a cognitive theory of vigilance that describes sustained attention as the capacity to control energy allocation within task-relevant brain regions while simultaneously inhibiting the allocation of resources in task-irrelevant regions of the brain. Thomson et al. explain that vigilance decrement begins when task-specific cognitive demands outpace the controlled supply of cognitive resources to task-relevant regions of the brain. That is, vigilance decrement occurs due to a failure to control a sufficient allocation of cognitive resources to task-specific cortex regions. The “resources” that Resource Control Failure Theory refers to were cognitive analogs to physical biological resources, which modern psychophysiological models leverage in describing attentional system performance as a function of physical biological resources. These include Oscillatory Model and Optimal Control Model. Clayton et al.’s oscillatory model of cerebral vigilance systems provided a model for vigilance task performance phenomena based on optimal and suboptimal neural oscillation within and between populations of vigilance task-relevant and -irrelevant cortex regions. Firstly, task-specific regions, or populations, of neurons metabolize neuro-energetic attentional resources within cortically local regions of the cortex. Neuronal populations oscillate within distinct frequency bands, 1–4 Hz (delta), 4–8 Hz (theta), 8–14 Hz (alpha), 14–30 Hz (beta), and >30 Hz (gamma), that can be measured by electroencephalogram. Neuronal populations are said to be in or out of phase with one another if their oscillatory frequencies match or differ. Phase synchronization is the mechanism by which neuronal populations communicate. If an action potential arrives at a neuronal population in an excitation phase, this will subsequently trigger a postsynaptic action potential that facilitates in-phase communication between the two populations. If two regions are firing out of phase, however, the processing of information between the two breaks down and is said to have been deconstructed. As time spent performing a vigilance task increases, the ratio of depleted to un-depleted neurons in task-relevant regions of the cortex increases. Task-relevant information processing errors increase as the depleted to un-depleted neurons ratio increases until TUTS and vigilance decrement manifest . Brain region communication is optimized by phase synchronization at lower frequencies, as this overcomes conduction delays associated with long-range action potential transmission . The oscillatory model linked specific oscillations in populations of cortical neurons to functions of cognitive control required to sustain attention . Firstly, theta oscillations in the fronto-medial cortex mediate the function of cognitive control and monitoring needed to complete task goals. Secondly, gamma oscillations in task-relevant cortex regions guide the performance of processes relevant to the task. Thirdly, alpha oscillations in task-irrelevant cortical areas inhibit processes unrelated to task performance. This suggests that failure to control the allocation of attention between task-relevant and -irrelevant cortex regions explains increasingly frequent TUTs and vigilance decrement during sustained attention tasks. Clayton et al.’s oscillatory model therefore aligned with the “control-failure” theory in that vigilance decrement and increasing TUT frequency result from a failure to control executive resources within task-relevant cortex regions. Moreover, Clayton et al. based their control-failure theory on the depletion of astrocytic glycogen within task-relevant cortex regions. Clayton et al., therefore, supported the reconciliation of the “resource depletion” and “control-failure” versions of the overload hypotheses. attributed vigilance decrement to an undesirable increase in alpha power, which could be reversed by activation of theta oscillations in the frontal and posterior control regions. Moreover, Clayton et al. explicitly predicted vigilance task performance enhancement by transcranial alternating current stimulation (TACS) of theta waves over both the frontal and posterior cortex based on their oscillatory model. Two laboratory vigilance studies have affirmed Clayton et al.’s prediction. found that applying a 40 Hz (theta) TACS stimulation across the left frontal cortex improved vigilance performance. demonstrated that 40 Hz (theta) TACS stimulation of the posterior cortex enhanced vigilance performance. also demonstrated alpha power improvements in vigilance performance after a 10-min TACS battery. The performance-enhancing effects of Zaehle et al.’s stimulation were non-permanent and lasted for at least 30 min in the follow-up study conducted by . also supported Zaehle et al.’s results when they also showed that a TACS battery enhanced vigilance performance for up to 70 min. Zaehle et al. and hence reported a contrary result to by demonstrating support for cortical resource depletion localized to regions associated with task-specific executive functions. Oscillatory Model of Sustained Attention suggested further support for the unification of “control-failure” and Wickens et al.’s “multiple resource depletion” hypotheses. Taken together, Clayton et al. and Thomson et al. suggested that suboptimal astrocytic glycogen supplementation of task-relevant neurons leads to executive control failures in task-relevant cortex regions that accumulate with time-on-task. That is, The metabolic demands associated with task-relevant information processing require metabolic supplementation of astrocytic glycogen . Suboptimal glycogen supplementation leads to astrocytic depletion in task-relevant regions of the cortex . Neurons draw on astrocytic glycogen to sustain firing rates exceeding blood-based resources’ metabolic capacity . Neuronal activity levels necessarily drop to a level that can be energetically sustained by blood-based resource metabolization following astrocytic glycogen depletion . As time-on-task increases, the number of information processing errors between depleted and un-depleted neurons increases within task-relevant cortex regions . Moreover, TUTs do not decline with time on task, as they are associated with information processes within task-irrelevant cortex regions, unburdened by task-specific demands. Vigilance performance errors manifest as communication errors that accumulate with time-on-task between depleted and un-depleted neurons in regions of the cortex associated with task-relevant and -irrelevant processes . Furthermore, TUTs also accumulate with time-on-task, as they are processed by regions over the cortex that are unburdened by the neurometabolic costs associated with task-specific workload processing between un-depleted and task-irrelevant neurons. model provides an additional link between the depletion of astrocytic glycogen at the level of task-relevant neurons, to failures of the controlled allocation of attention between task-relevant and -irrelevant processes. In doing so, Clayton et al. further suggested that the “resource depletion” and “control failure” versions of the overload theory were reconcilable with theory of vigilance performance. Clayton et al.’s oscillatory model suggested that resource depletion of task-relevant neurons leads to communication errors within task-relevant regions of the cortex, which accumulate until they manifest as a failure to control the allocation of attentional resources between task-relevant and -irrelevant processes. Oscillatory accounts of TUTS and vigilance decrement Taken together, accounts of vigilance decrement put forward by and suggest the phenomenon may be due to suboptimal neurovascular regulation of metabolic resources. Low-frequency neuronal populations modulate higher-frequency neurons’ oscillatory amplitude, known as power, or phase-power, coupling. Cognitive control processes are reflected by low-frequency theta oscillations in the frontal medial cortex . When a task requires sustained attention, these control processes monitor for errors or lapses in attention. Fronto-posterior power-coupling supports these control processes by promoting gamma oscillations in the task-relevant regions and alpha oscillations in task-irrelevant regions. However, attentional control is impaired when this power-coupling is destabilized or decoupled by increased alpha oscillations in task-relevant cortical areas, including the posterior and frontal regions . Under the oscillatory model, vigilance decrement results from increased alpha power within cortical regions relevant to sustaining attention to the task . model also provides a resource account of the increasing manifestation of TUTs with time spent on task. Task-unrelated thoughts ubiquitously involve decoupling attentional resources from task-relevant to -irrelevant perceptual information processes . For example, demonstrated that cortical decoupling impaired information processing between task-relevant, localized cortex regions. Baird et al. hence suggested that attentive information processing was distributed between “multiple” networks of skill-specific neuronal populations that link together to process task-specific workloads, a notion which aligned with Wickens et al.’s as well as Clayton et al.’s model. further suggested that TUTs occurred with increasing frequency during sustained attention tasks due to relative differences in phase-power decoupling errors between task-relevant and -irrelevant cortex regions. The ratio of depleted to un-depleted neurons does not increase in task-irrelevant cortex regions that do not process task-specific processing demands. Therefore, phase-power communication errors occur less frequently between neurons distributed within task-irrelevant cortex regions. By contrast, phase-power communication errors arise with a greater frequency between task-relevant cortex regions, where the ratio of depleted to un-depleted neurons increases with time on task. Clayton et al., therefore, suggested that increasingly frequent TUTs reflected the relative proportion of phase-power communication errors between task-relevant and task-irrelevant regions of the cortex. Wickens et al.’s “multiple resource depletion” version of the overload theory aligned with the models of and , which raised further doubt for the validity of rejection of “resource depletion.” Clayton et al. provided a resource depletion account for the increasing frequency with which TUTs manifest during sustained attention tasks. also deviated from and rejection of the depletion theory when they demonstrated localized cortical resource depletion during a sustained attention task. Jeroski et al. psycho-physiologically explored the resource-control account of the overload theory by measuring changes in regional cortical oxygen saturation (rSO 2 ) over the anterior frontal lobes during a vigilance task. rSO 2 is a measure of neuronal activation, which encompasses the metabolization of glycogen and astrocytic glucose to produce energy through reactions that require oxygen delivered by blood . Although mammalian brains need substantial energy to function, only a small amount of glucose is reserved as supplementary astrocytic glycogen . Since astrocytic glycogen reserves are small and finite, this makes the brain highly dependent on the metabolization of blood-based energetic resources . For example, demonstrated that astrocytic glycogen served as a metabolic supplement when information processing demands outpace the energetic capacity of blood-based resources alone. Zielke et al. supported Wickens et al.’s suggestion that energetic cognitive reserves were distributed across multiple cortex regions and can be depleted by task-relevant information processes. Glycogen metabolites are circulated from astrocytic glycogen caches to its partnered neuron, through complex intercellular mechanisms, in part because cerebral metabolic regulation is a ubiquitously regionalized process . Astrocytic glycogen metabolically supplements neurons operating under processing demands that outpace the energetic capacity of blood-based resources . Firstly, glucose (C 6 H 12 O 6 ) crosses the blood–brain barrier by a glucose transporter protein, GLUT-1, at the capillary endothelial wall . Once glucose is inside the brain, it is taken up by two pathways . GLUT-1 transporter proteins bring the metabolites directly to neurons, while GLUT-3 transporters bring it to the astrocyte glial cells. In both the neuron and the astrocyte, this glucose is then oxidized to produce the adenosine triphosphate, or ATP (C 10 H 16 O 13 P 3 ), which maintains cellular activity . However, astrocytes only use up a portion of the glucose to sustain its cellular functions; the rest of the astrocyte’s glucose is stored as glycogen through glycogenesis . Neurons can use the glucose stored as astrocytic glycogen to support continuous processing demands that outpace the energetic capacity of blood-based resources . Neuronal access to astrocytic glycogen relies on activity-dependent signals, including noradrenaline, vasoactive intestinal peptide, adenosine, K + , glutamate, ammonium oxide, and nitric oxide . Astrocytes wrap around the synapse and the intracerebral blood vessels . Activity-dependent signals within the synapse can trigger metabolic glucose supplementation to task-relevant neurons across the lactate pipe. and suggested that neuron–astrocyte metabolic cooperation leads to a cortically regional vasomotor response that causes cerebral oxygen saturation levels to fluctuate during a sustained attention task. Triggering an activity-dependent signal during a vigilance task can signal the energetic supplementation of neuronal activity that cannot be sustained by metabolizing blood-based resources alone . However, blood-based glucose and astrocytic glycogen are both finite energetic resources. The depletion of astrocytic glycogen implies that blood-based glucose is metabolically insufficient in sustaining task-relevant information processes and replenishing depleted astrocyte reserves. Once depleted of supplementary astrocytic glycogen, neuronal activity drops to levels that can be sustained by metabolizing blood-based resources alone. The decline in task-relevant information processing manifests as a decrease in action potential firing rates and a subsequent decrease in CO 2 produced by reduced cerebral fuel metabolization. Once a neuron’s supply of astrocytic glycogen runs out, Glucose-6P metabolization decreases, reducing the amount of CO 2 produced . Decreasing the amount of available fuel (astrocytic glycogen) then decreases task-relevant neurons’ information processing capacity and causes a decline in CO 2 production. suggested that this decrease in CO 2 also decreased cerebral vasodilation. Decreased vasodilation would inhibit the replenishment of astrocytic glycogen and further limit the information processing capacity of task-relevant neurons. It follows those astrocytes, depleted of glycogen, cannot readily replenish their glycogen reserves while a vigilance task persists. demonstrated cortically regional fluctuations in cerebral tissue oxygenation, localized across task-relevant regions of the right dorsolateral prefrontal lobe. Jeroski et al.’s results aligned with demonstration of cerebral lateralization of vigilance executive functions. Jeroski et al. hence supported and Wickens et al.’s “resource depletion” versions of the overload theory. This did not align with rejection of “resource depletion” as the antecedent of a failure to control the allocation of attentional resources to task-relevant processes. Moreover, oscillatory model suggested that the six tenets of Thomson et al.’s resource control-failure theory could account for reductions in vigilance performance based on the depletion of astrocytic glycogen . Thomson et al.’s resource control-failure theory of vigilance performance is, therefore, neurochemically reconcilable with the depletion theory. In summary, idea that executive control of attentional resource allocation fails increasingly with time on task, therefore, hinges on the neuro-energetic depletion of astrocytic glycogen, induced by sustained task-specific processing . Firstly, glycogen depletion occurs within a single astrocyte–neuron system, thus forcing that system to process task-specific information at a suboptimal action potential rate in the alpha band . Phase-power decoupling errors can occur when depleted and un-depleted neurons communicate information relevant to task performance . This can occur within and between anatomically segregated regions activated in processing task-relevant information . Secondly, astrocytic depletion increases with time on task in a chain reaction of task-relevant astrocyte–neuron systems. Following depletion, astrocytes begin replenishing their glycogen reserves through glycogenesis. However, task-relevant neurons cannot access replenished glycogen reserves until task-specific processing demands cease. This is because, following depletion, task-relevant neurons fire action potentials at a rate sustainable by metabolizing blood-based glucose alone, which is slower than that required to process task-specific demands . This energetic reduction in the rate that task-relevant neurons fire action potentials post-depletion prevents the secretion of activity-based signals into the synapse required to trigger glycogen supplementation in recently replenished astrocytes. Christie and Schrater’s optimal control model of cerebral vigilance systems The “resources” that Resource Control Failure Theory refers to are cognitive. By contrast, Optimal Control Model refers to psycho-physiological resources. For example, the “resources” that refer to are caches of glycogen stored in astrocytes. Neurons access this energy reserve via the lactate pipe when the task-specific processing demands outpace that which can be sustained by the metabolization of blood-based glucose alone . Cerebral vigilance systems are thus highly dependent on blood-based resources supplied through circulation . Glucose is the primary resource used by the brain in times of high workload processing but also includes other additional energy substrates, such as lactate, pyruvate, glutamate, and glutamine . Energetic metabolites are circulated through the brain using complex intercellular chemical mechanisms, in part because cerebral metabolization is a cortically regionalized process . Astrocytic glycogen metabolization is the primary metabolite used to sustain neuronal activity that cannot be energetically sustained by blood-based reserves alone . Christie and Schrater’s optimal control model modeled the flow of energetic resources from astrocytic glycogen and blood-based reserves into neurons when information processing demands outstrip the energetic capacity of blood-based reserves to sustain. Therefore, Christie and Schrater provided a model to understand vigilance decrement, which begins at the astrocyte–neuron level in task-relevant cortex regions recruited during sustained attention to tasks. GLUT-1 and GLUT-3 proteins transport glucose (C 6 H 12 O 6 ) from capillary blood into astrocytes and neurons, respectively . GLUT-1 transports glucose from the blood into astrocytes; however, GLUT-1 and GLUT-3 proteins transport glucose into neurons . When the action potential firing rate exceeds the metabolic capacity of blood-based resources, energetic resources are also shuttled to neurons through the lactate pipe . Astrocytes wrap their end feet around the synapse and intracerebral blood vessels and utilize activity-dependent chemical signals, including noradrenaline, vasoactive intestinal peptide, adenosine, K+, glutamate, ammonium oxide, and nitric oxide, to sense the metabolic needs of neurons firing at different rates . When a neuron’s action potential firing rate exceeds what can be energetically sustained by metabolizing blood-based reserves alone, astrocytic glycogen is transported as glucose into the neuron via the lactate pipe to sustain activity . Astrocytic glycogen is oxidized into adenosine triphosphate, or ATP (C 10 H 16 O 13 P 3 ), to maintain information processing activity typified by vigilance tasks . Twenty-five percent of the glycogen astrocytes reserve preserves their own internal metabolic requirements . Hence, neurons can access up to 75% of an astrocyte’s glucose reserves; however, once astrocytic glycogen depletes, its activity level must decline to a level sustainable by metabolizing blood-based reserves alone . cognitive theory of vigilance decrement is paralleled by Optimal Control Model, which biologically described disruptions to cortical functioning that associated with vigilance decrement . For example, Christie and Schrater’s model predicted astrocytic glycogen depletion within 20 min of neuronal activity that outpaced the metabolic capacity of blood-based reserves, after which the neuron’s firing rate declined to a lower level. Christie and Schrater’s prediction aligned with Jeroski et al.’s demonstration of a decline in regional cortical oxygen saturation they observed during their 20-min-long vigilance task. Moreover, Jeroski et al. suggested their observations may be explained by the metabolic depletion of astrocytic glycogen by vigilance task-relevant astrocyte–neuron systems located over AF 4 . Christie and Schrater’s model and Thomson et al.’s control theory could explain Jeroski et al.’s observations from the neurochemical and cellular levels to observable behavioral levels. Similar to , Christie and Schrater’s model also suggested a reconciliation between the “resource depletion” and “control failure” versions of the overload hypotheses. optimal control model described energetic resource regulation in astrocyte–neuron systems operating under high action potential firing rates. The optimal control parameters in Christie and Schrater’s model were analytic proxies for the neurochemical mechanisms used to control the allocation of attentional reserves . Astrocytic depletion occurs as glycogen is transported into the neuron via the lactate pipe . However, two parameters in Christie and Schrater’s model [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ were parameterized as fixed constants, which does not robustly describe the neurochemical changes associated with vigilance decrement beyond the moment of astrocytic depletion, and they, therefore, may have been better parameterized as functions of time . [12pt]{minimal} ${a}_{}$ parameter describes energetic outflow from the astrocyte to the neuron. illustrates Christie and Schrater’s model before any cognitive load is placed on astrocyte–neuron system when their [12pt]{minimal} ${a}_{}$ would take a value of zero. Under the current version of Christie and Schrater’s model, [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ take fixed constants greater than zero once a neuron begins firing at a rate that exceeds the energetic capacity of blood-based reserves after t >0 . parameterization of [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ as constants did not capture the neurochemical changes that take place after the astrocyte’s reserves deplete to 25% of their original value, when vigilance decrement begins to manifest behaviorally . The onset of astrocytic depletion suggested that blood-based energetic reserves are an insufficient supplement for the metabolic demands required of task-relevant neurons. Beyond astrocytic depletion, the rate at which task-relevant neurons fire is energetically restricted to that sustainable by metabolizing blood-based reserves alone. That is, as the supply of astrocytic glucose dwindles, there is a subsequent increase in the metabolic load placed on blood-based reserves. Once a neuron’s supply of supplementary glycogen runs dry, there is a drop in the total amount of Glucose-6P metabolization, resulting in a reduction in the CO 2 produced within the astrocyte–neuron system . Less fuel (astrocyte glycogen) decreases the capacity of task-relevant neurons to fire during the vigilance task, leading to less CO 2 production and, importantly, reduced vasodilation . Once neurons deplete their supply of supplementary glycogen, the vasodilation dip also inhibits glucose replenishment in the now-depleted astrocyte and the neuron ( and ) . It follows that if task-relevant processes led to the depletion of astrocytic glycogen, then those reserves cannot readily be fully replenished or re-deployed while those task demands persist. This could explain the trend reversal in Jeroski et al.’s measurements of rSO 2 during a sustained attention task. At the outset of Jeroski et al.’s task, two oxygen-dependent processes that produce CO 2 as a by-product mediated cellular energy regulation over the cortex: glycogenolysis (of astrocytic glycogen) and glycolysis (of blood-based glucose). Hence, the initially positive rSO 2 trend observed may be a measure of dual oxygen-dependent processes. However, glycogenolysis would not have persisted in the absence of astrocytic glycogen post-depletion. Hence, the negative trend in rSO 2 observed in the latter half of their task may have reflected reduced demand for blood-based oxygen that had initially been required to sustain glycogenolysis of astrocytic glycogen. In addition, astrocytic depletion may have impacted CO 2 -induced vasoconstriction of the vasculature, contributing to the rSO 2 trend reversal Jeroski et al. observed. That is, between the first and second half of their vigilance task, two sources of CO 2 decreased to one when glycogenolysis ceased after astrocytic depletion. CO 2 -induced vasoconstriction might explain the trend reversal reported between their task’s first and second half. That is, while rSO 2 increased during the first half of the task, so did the volume of CO 2 , a by-product of dual metabolic processes. This accumulation of excess CO 2 from dual processes may have exacerbated vasoconstriction, through which blood passes into the brain. This increased vasoconstriction would have restricted the volume of blood that could pass into the brain and, so, would have reduced cerebral blood oxygen saturation in the second half of the task, as the vasoconstricting effects of the CO 2 would have slowly decreased from a neurochemical state of excessive abundance to depletion. Vigilance decrement is therefore associated with a shift in the metabolic supply of task-relevant neurons’ energetic resources away from supplementary astrocytic glycogen to entirely blood-based glucose supplied across the capillaries, implying [12pt]{minimal} ${a}_{}=0\, t>{t}_v$ . Vigilance task demands sustain the neuron’s depleting metabolic activity, even after astrocytic depletion at [12pt]{minimal} ${t}_v$ . Hence, the neuron’s energetic demands must be serviced entirely by blood-based resources. That is, beyond astrocytic depletion at [12pt]{minimal} ${t}_v$ , the neuron’s astrocytic fuel reserve will no longer supply the requisite glycogen level necessary to sustain vigilance performance. Since the neuron’s task demands do not decrease, astrocytic depletion must correspond to an increased demand for blood-based energy reserves to compensate for the lack of astrocytic glycogen supplied across the lactate pipe . Once depleted of astrocytic glycogen reserves, a neuron’s activity level will decline from over 30 Hz in the gamma oscillatory band to 8 to 14 Hz alpha band action potential firing rates, which are sustainable by blood-based glucose metabolization alone . However, because vigilance task demands persist regardless of depletion, it follows that task-relevant neurons’ energetic needs shift from two sources (blood-based glucose plus astrocytic glycogen) to just one (blood-based glucose). used their alpha and beta parameters to model the energetic load of astrocytes and neurons on blood-based resources. Once an astrocyte is depleted of glucose, its metabolic load on blood remains the same, as it subsists on the 25% of glucose it restricts from neuronal supplementation to sustain its own cellular functions. While the astrocyte can subsist off its normal blood glucose supply and reserves of restricted glycogen, the neuron that depleted it cannot. Therefore, once an astrocyte has depleted, task-relevant neurons’ energetic demands rest entirely on blood-based glucose, which might explain the acute hypoglycemia observed during driving vigilance tasks. Once astrocytes became depleted, task-relevant neurons would have been forced to draw increasingly on blood-based glucose to sustain the processing of task demands. However, astrocytic supplementation implies that blood-based glucose was an insufficient energetic source to sustain task-relevant processing demands. Hence, the steady decline in blood glucose concentration Cox et al. observed during their vigilance task may reflect the impact of astrocytic depletion on blood-based resource mobilization. It follows that the beta used to model the metabolic load of task-relevant neuron’s load on blood-based glucose changes after the depletion to a value that reflects the energetic deficit associated with astrocytic glycogen. Moreover, the value to which beta changes beyond depletion should reflect the total metabolic load on blood-based resources associated with task-specific processing demands in Christie and Schrater’s system, which is captured by the sum of their alpha and beta parameters . That is, [12pt]{minimal} $ > + =2 =, \, t>{t}_v$ . Therefore, by parameterizing [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ as constants, Christie and Schrater’s model did not robustly capture the neuron’s increase in blood-based glucose metabolization in [12pt]{minimal} $$ , or the drop in astrocytic glycogen metabolization across the lactate pipe in [12pt]{minimal} ${a}_{}$ . Therefore, [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ are not constant, but function according to the amount of time spent on a vigilance task . Resource depletion and control failure demonstrated that the individual information processing capacity of task-relevant neurons decreased following the depletion of their metabolic supply of astrocytic glycogen during an audible vigilance task. suggested that sustained attention task phenomena manifested phase-power decoupling communication errors within task-relevant cortex regions. Moreover, proposed a ground-up resource interpretation of Clayton et al.’s phase-power decoupling idea. Christie and Schrater’s model described the relationship between sustained attention task performance and their underlying psychophysiological processes. Taken together, Clayton et al. and Christie and Schrater suggested that task-relevant information processing declined with time on task as phase-power decoupling communication errors accumulate within and between task-relevant cortex regions. results aligned with this notion that communication errors accumulate within and between task-relevant cortex regions. For example, sustained attention to Pang et al.’s audible vigilance task required processing between the frontal lobes and auditory cortex. suggested that astrocytic depletion could have decreased task-relevant processing within the frontal lobes, the auditory cortex, or both. Clayton et al. further suggested that vigilance performance may also suffer from phase-power communication errors between depleted and un-depleted task-relevant cortex regions. Hence, vigilance performance on Pang et al.’s task may also have declined due to phase-power decoupling communication errors between depleted and un-depleted, task-relevant regions of the cortex. Pang et al., Clayton et al., and Christie and Schrater’s association of vigilance task performance deficits with astrocytic glycogen’s metabolic exhaustion did not align with rejection of the depletion hypothesis. undermined alternative explanation for the temporal accumulation of executive function errors in task-relevant cortex regions relative to task-irrelevant regions. Thomson et al. suggested that the gradual decline in task-specific executive processing behaviorally manifested the suboptimal control of attentional resources between task-relevant and -irrelevant processes. Thomson et al.’s rejection of the resource depletion version of the overload theory did not align with the claims of , , or . Moreover, Pang et al., Christie and Schrater, and Clayton et al. suggested phase-power decoupling communication errors temporally accumulated within and between task-relevant neuronal populations. Hence, under Pang et al., Christie and Schrater, and Clayton et al., vigilance decrement begins with the depletion of a single astrocyte–neuron system. As time-on-task increases, the ratio of depleted to un-depleted neurons also increases. As the ratio of depleted to un-depleted neurons grows within task-relevant regions of the cortex, phase-power decoupling communication errors accumulate until they behaviorally manifest as vigilance decrement. As time on task increases, neuron-to-neuron processing errors accumulate to become multiple population-to-population phase-power decoupling processing errors in sustaining task-relevant executive processes. Christie and Schrater, Clayton et al., and Pang et al. hence reconciled , and Wickens et al.’s resource depletion versions of the overload theory with Thomson et al.’s resource control failure theory of vigilance decrement. , , , , , and Wickens et al. therefore suggested that “resource depletion” was compatible within Resource Control Theory, despite rejection. Taken together, Pang et al., Christie and Schrater, Clayton et al., , , Wickens et al., and provided a comprehensive model of vigilance task performance that could be understood from neurochemical to behavioral levels. Implications and avenues of future research This review contributes to the literature a conceptual map between theoretical and accounts of vigilance performance phenomenon to contemporary psycho-physiological models. This review thus provides a comprehensive framework to understand the dynamics of human attention in novel situations. For example, this framework has informed an unpublished study of ours that explores the cerebral hemodynamics of vigilance performance in network defense analysts. Participants performed a task engineered to simulate the cognitive load associated with sustaining attention to cyber security command and control consoles, while regional oxygen tissue saturation, rSO 2 , was tracked. Preliminary results suggested that characteristic changes in rSO 2 reflected vigilance decrement, indicating that cortically regional metabolic activity may reflect support for the depletion theory. Additional avenues of future exploration suggested through this work include deriving psychological theories complemented by a biological component. For example, theoretical accounts of vigilance performance took an abstract perspective of what the “load” in overload theories reflected for approximately the first 70 years of research beyond , ) formative studies of vigilance. The fact that the psychophysiological models of vigilance performance that and proposed so closely parallel the tenets of modern overload theory provides the basis of this holistic and comprehensive review of vigilance performance. provided a model for vigilance task performance phenomena based on optimal and suboptimal neural oscillation within and between populations of vigilance task-relevant and -irrelevant cortex regions. Firstly, task-specific regions, or populations, of neurons metabolize neuro-energetic attentional resources within cortically local regions of the cortex. Neuronal populations oscillate within distinct frequency bands, 1–4 Hz (delta), 4–8 Hz (theta), 8–14 Hz (alpha), 14–30 Hz (beta), and >30 Hz (gamma), that can be measured by electroencephalogram. Neuronal populations are said to be in or out of phase with one another if their oscillatory frequencies match or differ. Phase synchronization is the mechanism by which neuronal populations communicate. If an action potential arrives at a neuronal population in an excitation phase, this will subsequently trigger a postsynaptic action potential that facilitates in-phase communication between the two populations. If two regions are firing out of phase, however, the processing of information between the two breaks down and is said to have been deconstructed. As time spent performing a vigilance task increases, the ratio of depleted to un-depleted neurons in task-relevant regions of the cortex increases. Task-relevant information processing errors increase as the depleted to un-depleted neurons ratio increases until TUTS and vigilance decrement manifest . Brain region communication is optimized by phase synchronization at lower frequencies, as this overcomes conduction delays associated with long-range action potential transmission . The oscillatory model linked specific oscillations in populations of cortical neurons to functions of cognitive control required to sustain attention . Firstly, theta oscillations in the fronto-medial cortex mediate the function of cognitive control and monitoring needed to complete task goals. Secondly, gamma oscillations in task-relevant cortex regions guide the performance of processes relevant to the task. Thirdly, alpha oscillations in task-irrelevant cortical areas inhibit processes unrelated to task performance. This suggests that failure to control the allocation of attention between task-relevant and -irrelevant cortex regions explains increasingly frequent TUTs and vigilance decrement during sustained attention tasks. Clayton et al.’s oscillatory model therefore aligned with the “control-failure” theory in that vigilance decrement and increasing TUT frequency result from a failure to control executive resources within task-relevant cortex regions. Moreover, Clayton et al. based their control-failure theory on the depletion of astrocytic glycogen within task-relevant cortex regions. Clayton et al., therefore, supported the reconciliation of the “resource depletion” and “control-failure” versions of the overload hypotheses. attributed vigilance decrement to an undesirable increase in alpha power, which could be reversed by activation of theta oscillations in the frontal and posterior control regions. Moreover, Clayton et al. explicitly predicted vigilance task performance enhancement by transcranial alternating current stimulation (TACS) of theta waves over both the frontal and posterior cortex based on their oscillatory model. Two laboratory vigilance studies have affirmed Clayton et al.’s prediction. found that applying a 40 Hz (theta) TACS stimulation across the left frontal cortex improved vigilance performance. demonstrated that 40 Hz (theta) TACS stimulation of the posterior cortex enhanced vigilance performance. also demonstrated alpha power improvements in vigilance performance after a 10-min TACS battery. The performance-enhancing effects of Zaehle et al.’s stimulation were non-permanent and lasted for at least 30 min in the follow-up study conducted by . also supported Zaehle et al.’s results when they also showed that a TACS battery enhanced vigilance performance for up to 70 min. Zaehle et al. and hence reported a contrary result to by demonstrating support for cortical resource depletion localized to regions associated with task-specific executive functions. Oscillatory Model of Sustained Attention suggested further support for the unification of “control-failure” and Wickens et al.’s “multiple resource depletion” hypotheses. Taken together, Clayton et al. and Thomson et al. suggested that suboptimal astrocytic glycogen supplementation of task-relevant neurons leads to executive control failures in task-relevant cortex regions that accumulate with time-on-task. That is, The metabolic demands associated with task-relevant information processing require metabolic supplementation of astrocytic glycogen . Suboptimal glycogen supplementation leads to astrocytic depletion in task-relevant regions of the cortex . Neurons draw on astrocytic glycogen to sustain firing rates exceeding blood-based resources’ metabolic capacity . Neuronal activity levels necessarily drop to a level that can be energetically sustained by blood-based resource metabolization following astrocytic glycogen depletion . As time-on-task increases, the number of information processing errors between depleted and un-depleted neurons increases within task-relevant cortex regions . Moreover, TUTs do not decline with time on task, as they are associated with information processes within task-irrelevant cortex regions, unburdened by task-specific demands. Vigilance performance errors manifest as communication errors that accumulate with time-on-task between depleted and un-depleted neurons in regions of the cortex associated with task-relevant and -irrelevant processes . Furthermore, TUTs also accumulate with time-on-task, as they are processed by regions over the cortex that are unburdened by the neurometabolic costs associated with task-specific workload processing between un-depleted and task-irrelevant neurons. model provides an additional link between the depletion of astrocytic glycogen at the level of task-relevant neurons, to failures of the controlled allocation of attention between task-relevant and -irrelevant processes. In doing so, Clayton et al. further suggested that the “resource depletion” and “control failure” versions of the overload theory were reconcilable with theory of vigilance performance. Clayton et al.’s oscillatory model suggested that resource depletion of task-relevant neurons leads to communication errors within task-relevant regions of the cortex, which accumulate until they manifest as a failure to control the allocation of attentional resources between task-relevant and -irrelevant processes. Taken together, accounts of vigilance decrement put forward by and suggest the phenomenon may be due to suboptimal neurovascular regulation of metabolic resources. Low-frequency neuronal populations modulate higher-frequency neurons’ oscillatory amplitude, known as power, or phase-power, coupling. Cognitive control processes are reflected by low-frequency theta oscillations in the frontal medial cortex . When a task requires sustained attention, these control processes monitor for errors or lapses in attention. Fronto-posterior power-coupling supports these control processes by promoting gamma oscillations in the task-relevant regions and alpha oscillations in task-irrelevant regions. However, attentional control is impaired when this power-coupling is destabilized or decoupled by increased alpha oscillations in task-relevant cortical areas, including the posterior and frontal regions . Under the oscillatory model, vigilance decrement results from increased alpha power within cortical regions relevant to sustaining attention to the task . model also provides a resource account of the increasing manifestation of TUTs with time spent on task. Task-unrelated thoughts ubiquitously involve decoupling attentional resources from task-relevant to -irrelevant perceptual information processes . For example, demonstrated that cortical decoupling impaired information processing between task-relevant, localized cortex regions. Baird et al. hence suggested that attentive information processing was distributed between “multiple” networks of skill-specific neuronal populations that link together to process task-specific workloads, a notion which aligned with Wickens et al.’s as well as Clayton et al.’s model. further suggested that TUTs occurred with increasing frequency during sustained attention tasks due to relative differences in phase-power decoupling errors between task-relevant and -irrelevant cortex regions. The ratio of depleted to un-depleted neurons does not increase in task-irrelevant cortex regions that do not process task-specific processing demands. Therefore, phase-power communication errors occur less frequently between neurons distributed within task-irrelevant cortex regions. By contrast, phase-power communication errors arise with a greater frequency between task-relevant cortex regions, where the ratio of depleted to un-depleted neurons increases with time on task. Clayton et al., therefore, suggested that increasingly frequent TUTs reflected the relative proportion of phase-power communication errors between task-relevant and task-irrelevant regions of the cortex. Wickens et al.’s “multiple resource depletion” version of the overload theory aligned with the models of and , which raised further doubt for the validity of rejection of “resource depletion.” Clayton et al. provided a resource depletion account for the increasing frequency with which TUTs manifest during sustained attention tasks. also deviated from and rejection of the depletion theory when they demonstrated localized cortical resource depletion during a sustained attention task. Jeroski et al. psycho-physiologically explored the resource-control account of the overload theory by measuring changes in regional cortical oxygen saturation (rSO 2 ) over the anterior frontal lobes during a vigilance task. rSO 2 is a measure of neuronal activation, which encompasses the metabolization of glycogen and astrocytic glucose to produce energy through reactions that require oxygen delivered by blood . Although mammalian brains need substantial energy to function, only a small amount of glucose is reserved as supplementary astrocytic glycogen . Since astrocytic glycogen reserves are small and finite, this makes the brain highly dependent on the metabolization of blood-based energetic resources . For example, demonstrated that astrocytic glycogen served as a metabolic supplement when information processing demands outpace the energetic capacity of blood-based resources alone. Zielke et al. supported Wickens et al.’s suggestion that energetic cognitive reserves were distributed across multiple cortex regions and can be depleted by task-relevant information processes. Glycogen metabolites are circulated from astrocytic glycogen caches to its partnered neuron, through complex intercellular mechanisms, in part because cerebral metabolic regulation is a ubiquitously regionalized process . Astrocytic glycogen metabolically supplements neurons operating under processing demands that outpace the energetic capacity of blood-based resources . Firstly, glucose (C 6 H 12 O 6 ) crosses the blood–brain barrier by a glucose transporter protein, GLUT-1, at the capillary endothelial wall . Once glucose is inside the brain, it is taken up by two pathways . GLUT-1 transporter proteins bring the metabolites directly to neurons, while GLUT-3 transporters bring it to the astrocyte glial cells. In both the neuron and the astrocyte, this glucose is then oxidized to produce the adenosine triphosphate, or ATP (C 10 H 16 O 13 P 3 ), which maintains cellular activity . However, astrocytes only use up a portion of the glucose to sustain its cellular functions; the rest of the astrocyte’s glucose is stored as glycogen through glycogenesis . Neurons can use the glucose stored as astrocytic glycogen to support continuous processing demands that outpace the energetic capacity of blood-based resources . Neuronal access to astrocytic glycogen relies on activity-dependent signals, including noradrenaline, vasoactive intestinal peptide, adenosine, K + , glutamate, ammonium oxide, and nitric oxide . Astrocytes wrap around the synapse and the intracerebral blood vessels . Activity-dependent signals within the synapse can trigger metabolic glucose supplementation to task-relevant neurons across the lactate pipe. and suggested that neuron–astrocyte metabolic cooperation leads to a cortically regional vasomotor response that causes cerebral oxygen saturation levels to fluctuate during a sustained attention task. Triggering an activity-dependent signal during a vigilance task can signal the energetic supplementation of neuronal activity that cannot be sustained by metabolizing blood-based resources alone . However, blood-based glucose and astrocytic glycogen are both finite energetic resources. The depletion of astrocytic glycogen implies that blood-based glucose is metabolically insufficient in sustaining task-relevant information processes and replenishing depleted astrocyte reserves. Once depleted of supplementary astrocytic glycogen, neuronal activity drops to levels that can be sustained by metabolizing blood-based resources alone. The decline in task-relevant information processing manifests as a decrease in action potential firing rates and a subsequent decrease in CO 2 produced by reduced cerebral fuel metabolization. Once a neuron’s supply of astrocytic glycogen runs out, Glucose-6P metabolization decreases, reducing the amount of CO 2 produced . Decreasing the amount of available fuel (astrocytic glycogen) then decreases task-relevant neurons’ information processing capacity and causes a decline in CO 2 production. suggested that this decrease in CO 2 also decreased cerebral vasodilation. Decreased vasodilation would inhibit the replenishment of astrocytic glycogen and further limit the information processing capacity of task-relevant neurons. It follows those astrocytes, depleted of glycogen, cannot readily replenish their glycogen reserves while a vigilance task persists. demonstrated cortically regional fluctuations in cerebral tissue oxygenation, localized across task-relevant regions of the right dorsolateral prefrontal lobe. Jeroski et al.’s results aligned with demonstration of cerebral lateralization of vigilance executive functions. Jeroski et al. hence supported and Wickens et al.’s “resource depletion” versions of the overload theory. This did not align with rejection of “resource depletion” as the antecedent of a failure to control the allocation of attentional resources to task-relevant processes. Moreover, oscillatory model suggested that the six tenets of Thomson et al.’s resource control-failure theory could account for reductions in vigilance performance based on the depletion of astrocytic glycogen . Thomson et al.’s resource control-failure theory of vigilance performance is, therefore, neurochemically reconcilable with the depletion theory. In summary, idea that executive control of attentional resource allocation fails increasingly with time on task, therefore, hinges on the neuro-energetic depletion of astrocytic glycogen, induced by sustained task-specific processing . Firstly, glycogen depletion occurs within a single astrocyte–neuron system, thus forcing that system to process task-specific information at a suboptimal action potential rate in the alpha band . Phase-power decoupling errors can occur when depleted and un-depleted neurons communicate information relevant to task performance . This can occur within and between anatomically segregated regions activated in processing task-relevant information . Secondly, astrocytic depletion increases with time on task in a chain reaction of task-relevant astrocyte–neuron systems. Following depletion, astrocytes begin replenishing their glycogen reserves through glycogenesis. However, task-relevant neurons cannot access replenished glycogen reserves until task-specific processing demands cease. This is because, following depletion, task-relevant neurons fire action potentials at a rate sustainable by metabolizing blood-based glucose alone, which is slower than that required to process task-specific demands . This energetic reduction in the rate that task-relevant neurons fire action potentials post-depletion prevents the secretion of activity-based signals into the synapse required to trigger glycogen supplementation in recently replenished astrocytes. The “resources” that Resource Control Failure Theory refers to are cognitive. By contrast, Optimal Control Model refers to psycho-physiological resources. For example, the “resources” that refer to are caches of glycogen stored in astrocytes. Neurons access this energy reserve via the lactate pipe when the task-specific processing demands outpace that which can be sustained by the metabolization of blood-based glucose alone . Cerebral vigilance systems are thus highly dependent on blood-based resources supplied through circulation . Glucose is the primary resource used by the brain in times of high workload processing but also includes other additional energy substrates, such as lactate, pyruvate, glutamate, and glutamine . Energetic metabolites are circulated through the brain using complex intercellular chemical mechanisms, in part because cerebral metabolization is a cortically regionalized process . Astrocytic glycogen metabolization is the primary metabolite used to sustain neuronal activity that cannot be energetically sustained by blood-based reserves alone . Christie and Schrater’s optimal control model modeled the flow of energetic resources from astrocytic glycogen and blood-based reserves into neurons when information processing demands outstrip the energetic capacity of blood-based reserves to sustain. Therefore, Christie and Schrater provided a model to understand vigilance decrement, which begins at the astrocyte–neuron level in task-relevant cortex regions recruited during sustained attention to tasks. GLUT-1 and GLUT-3 proteins transport glucose (C 6 H 12 O 6 ) from capillary blood into astrocytes and neurons, respectively . GLUT-1 transports glucose from the blood into astrocytes; however, GLUT-1 and GLUT-3 proteins transport glucose into neurons . When the action potential firing rate exceeds the metabolic capacity of blood-based resources, energetic resources are also shuttled to neurons through the lactate pipe . Astrocytes wrap their end feet around the synapse and intracerebral blood vessels and utilize activity-dependent chemical signals, including noradrenaline, vasoactive intestinal peptide, adenosine, K+, glutamate, ammonium oxide, and nitric oxide, to sense the metabolic needs of neurons firing at different rates . When a neuron’s action potential firing rate exceeds what can be energetically sustained by metabolizing blood-based reserves alone, astrocytic glycogen is transported as glucose into the neuron via the lactate pipe to sustain activity . Astrocytic glycogen is oxidized into adenosine triphosphate, or ATP (C 10 H 16 O 13 P 3 ), to maintain information processing activity typified by vigilance tasks . Twenty-five percent of the glycogen astrocytes reserve preserves their own internal metabolic requirements . Hence, neurons can access up to 75% of an astrocyte’s glucose reserves; however, once astrocytic glycogen depletes, its activity level must decline to a level sustainable by metabolizing blood-based reserves alone . cognitive theory of vigilance decrement is paralleled by Optimal Control Model, which biologically described disruptions to cortical functioning that associated with vigilance decrement . For example, Christie and Schrater’s model predicted astrocytic glycogen depletion within 20 min of neuronal activity that outpaced the metabolic capacity of blood-based reserves, after which the neuron’s firing rate declined to a lower level. Christie and Schrater’s prediction aligned with Jeroski et al.’s demonstration of a decline in regional cortical oxygen saturation they observed during their 20-min-long vigilance task. Moreover, Jeroski et al. suggested their observations may be explained by the metabolic depletion of astrocytic glycogen by vigilance task-relevant astrocyte–neuron systems located over AF 4 . Christie and Schrater’s model and Thomson et al.’s control theory could explain Jeroski et al.’s observations from the neurochemical and cellular levels to observable behavioral levels. Similar to , Christie and Schrater’s model also suggested a reconciliation between the “resource depletion” and “control failure” versions of the overload hypotheses. optimal control model described energetic resource regulation in astrocyte–neuron systems operating under high action potential firing rates. The optimal control parameters in Christie and Schrater’s model were analytic proxies for the neurochemical mechanisms used to control the allocation of attentional reserves . Astrocytic depletion occurs as glycogen is transported into the neuron via the lactate pipe . However, two parameters in Christie and Schrater’s model [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ were parameterized as fixed constants, which does not robustly describe the neurochemical changes associated with vigilance decrement beyond the moment of astrocytic depletion, and they, therefore, may have been better parameterized as functions of time . [12pt]{minimal} ${a}_{}$ parameter describes energetic outflow from the astrocyte to the neuron. illustrates Christie and Schrater’s model before any cognitive load is placed on astrocyte–neuron system when their [12pt]{minimal} ${a}_{}$ would take a value of zero. Under the current version of Christie and Schrater’s model, [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ take fixed constants greater than zero once a neuron begins firing at a rate that exceeds the energetic capacity of blood-based reserves after t >0 . parameterization of [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ as constants did not capture the neurochemical changes that take place after the astrocyte’s reserves deplete to 25% of their original value, when vigilance decrement begins to manifest behaviorally . The onset of astrocytic depletion suggested that blood-based energetic reserves are an insufficient supplement for the metabolic demands required of task-relevant neurons. Beyond astrocytic depletion, the rate at which task-relevant neurons fire is energetically restricted to that sustainable by metabolizing blood-based reserves alone. That is, as the supply of astrocytic glucose dwindles, there is a subsequent increase in the metabolic load placed on blood-based reserves. Once a neuron’s supply of supplementary glycogen runs dry, there is a drop in the total amount of Glucose-6P metabolization, resulting in a reduction in the CO 2 produced within the astrocyte–neuron system . Less fuel (astrocyte glycogen) decreases the capacity of task-relevant neurons to fire during the vigilance task, leading to less CO 2 production and, importantly, reduced vasodilation . Once neurons deplete their supply of supplementary glycogen, the vasodilation dip also inhibits glucose replenishment in the now-depleted astrocyte and the neuron ( and ) . It follows that if task-relevant processes led to the depletion of astrocytic glycogen, then those reserves cannot readily be fully replenished or re-deployed while those task demands persist. This could explain the trend reversal in Jeroski et al.’s measurements of rSO 2 during a sustained attention task. At the outset of Jeroski et al.’s task, two oxygen-dependent processes that produce CO 2 as a by-product mediated cellular energy regulation over the cortex: glycogenolysis (of astrocytic glycogen) and glycolysis (of blood-based glucose). Hence, the initially positive rSO 2 trend observed may be a measure of dual oxygen-dependent processes. However, glycogenolysis would not have persisted in the absence of astrocytic glycogen post-depletion. Hence, the negative trend in rSO 2 observed in the latter half of their task may have reflected reduced demand for blood-based oxygen that had initially been required to sustain glycogenolysis of astrocytic glycogen. In addition, astrocytic depletion may have impacted CO 2 -induced vasoconstriction of the vasculature, contributing to the rSO 2 trend reversal Jeroski et al. observed. That is, between the first and second half of their vigilance task, two sources of CO 2 decreased to one when glycogenolysis ceased after astrocytic depletion. CO 2 -induced vasoconstriction might explain the trend reversal reported between their task’s first and second half. That is, while rSO 2 increased during the first half of the task, so did the volume of CO 2 , a by-product of dual metabolic processes. This accumulation of excess CO 2 from dual processes may have exacerbated vasoconstriction, through which blood passes into the brain. This increased vasoconstriction would have restricted the volume of blood that could pass into the brain and, so, would have reduced cerebral blood oxygen saturation in the second half of the task, as the vasoconstricting effects of the CO 2 would have slowly decreased from a neurochemical state of excessive abundance to depletion. Vigilance decrement is therefore associated with a shift in the metabolic supply of task-relevant neurons’ energetic resources away from supplementary astrocytic glycogen to entirely blood-based glucose supplied across the capillaries, implying [12pt]{minimal} ${a}_{}=0\, t>{t}_v$ . Vigilance task demands sustain the neuron’s depleting metabolic activity, even after astrocytic depletion at [12pt]{minimal} ${t}_v$ . Hence, the neuron’s energetic demands must be serviced entirely by blood-based resources. That is, beyond astrocytic depletion at [12pt]{minimal} ${t}_v$ , the neuron’s astrocytic fuel reserve will no longer supply the requisite glycogen level necessary to sustain vigilance performance. Since the neuron’s task demands do not decrease, astrocytic depletion must correspond to an increased demand for blood-based energy reserves to compensate for the lack of astrocytic glycogen supplied across the lactate pipe . Once depleted of astrocytic glycogen reserves, a neuron’s activity level will decline from over 30 Hz in the gamma oscillatory band to 8 to 14 Hz alpha band action potential firing rates, which are sustainable by blood-based glucose metabolization alone . However, because vigilance task demands persist regardless of depletion, it follows that task-relevant neurons’ energetic needs shift from two sources (blood-based glucose plus astrocytic glycogen) to just one (blood-based glucose). used their alpha and beta parameters to model the energetic load of astrocytes and neurons on blood-based resources. Once an astrocyte is depleted of glucose, its metabolic load on blood remains the same, as it subsists on the 25% of glucose it restricts from neuronal supplementation to sustain its own cellular functions. While the astrocyte can subsist off its normal blood glucose supply and reserves of restricted glycogen, the neuron that depleted it cannot. Therefore, once an astrocyte has depleted, task-relevant neurons’ energetic demands rest entirely on blood-based glucose, which might explain the acute hypoglycemia observed during driving vigilance tasks. Once astrocytes became depleted, task-relevant neurons would have been forced to draw increasingly on blood-based glucose to sustain the processing of task demands. However, astrocytic supplementation implies that blood-based glucose was an insufficient energetic source to sustain task-relevant processing demands. Hence, the steady decline in blood glucose concentration Cox et al. observed during their vigilance task may reflect the impact of astrocytic depletion on blood-based resource mobilization. It follows that the beta used to model the metabolic load of task-relevant neuron’s load on blood-based glucose changes after the depletion to a value that reflects the energetic deficit associated with astrocytic glycogen. Moreover, the value to which beta changes beyond depletion should reflect the total metabolic load on blood-based resources associated with task-specific processing demands in Christie and Schrater’s system, which is captured by the sum of their alpha and beta parameters . That is, [12pt]{minimal} $ > + =2 =, \, t>{t}_v$ . Therefore, by parameterizing [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ as constants, Christie and Schrater’s model did not robustly capture the neuron’s increase in blood-based glucose metabolization in [12pt]{minimal} $$ , or the drop in astrocytic glycogen metabolization across the lactate pipe in [12pt]{minimal} ${a}_{}$ . Therefore, [12pt]{minimal} ${a}_{}$ and [12pt]{minimal} $$ are not constant, but function according to the amount of time spent on a vigilance task . demonstrated that the individual information processing capacity of task-relevant neurons decreased following the depletion of their metabolic supply of astrocytic glycogen during an audible vigilance task. suggested that sustained attention task phenomena manifested phase-power decoupling communication errors within task-relevant cortex regions. Moreover, proposed a ground-up resource interpretation of Clayton et al.’s phase-power decoupling idea. Christie and Schrater’s model described the relationship between sustained attention task performance and their underlying psychophysiological processes. Taken together, Clayton et al. and Christie and Schrater suggested that task-relevant information processing declined with time on task as phase-power decoupling communication errors accumulate within and between task-relevant cortex regions. results aligned with this notion that communication errors accumulate within and between task-relevant cortex regions. For example, sustained attention to Pang et al.’s audible vigilance task required processing between the frontal lobes and auditory cortex. suggested that astrocytic depletion could have decreased task-relevant processing within the frontal lobes, the auditory cortex, or both. Clayton et al. further suggested that vigilance performance may also suffer from phase-power communication errors between depleted and un-depleted task-relevant cortex regions. Hence, vigilance performance on Pang et al.’s task may also have declined due to phase-power decoupling communication errors between depleted and un-depleted, task-relevant regions of the cortex. Pang et al., Clayton et al., and Christie and Schrater’s association of vigilance task performance deficits with astrocytic glycogen’s metabolic exhaustion did not align with rejection of the depletion hypothesis. undermined alternative explanation for the temporal accumulation of executive function errors in task-relevant cortex regions relative to task-irrelevant regions. Thomson et al. suggested that the gradual decline in task-specific executive processing behaviorally manifested the suboptimal control of attentional resources between task-relevant and -irrelevant processes. Thomson et al.’s rejection of the resource depletion version of the overload theory did not align with the claims of , , or . Moreover, Pang et al., Christie and Schrater, and Clayton et al. suggested phase-power decoupling communication errors temporally accumulated within and between task-relevant neuronal populations. Hence, under Pang et al., Christie and Schrater, and Clayton et al., vigilance decrement begins with the depletion of a single astrocyte–neuron system. As time-on-task increases, the ratio of depleted to un-depleted neurons also increases. As the ratio of depleted to un-depleted neurons grows within task-relevant regions of the cortex, phase-power decoupling communication errors accumulate until they behaviorally manifest as vigilance decrement. As time on task increases, neuron-to-neuron processing errors accumulate to become multiple population-to-population phase-power decoupling processing errors in sustaining task-relevant executive processes. Christie and Schrater, Clayton et al., and Pang et al. hence reconciled , and Wickens et al.’s resource depletion versions of the overload theory with Thomson et al.’s resource control failure theory of vigilance decrement. , , , , , and Wickens et al. therefore suggested that “resource depletion” was compatible within Resource Control Theory, despite rejection. Taken together, Pang et al., Christie and Schrater, Clayton et al., , , Wickens et al., and provided a comprehensive model of vigilance task performance that could be understood from neurochemical to behavioral levels. This review contributes to the literature a conceptual map between theoretical and accounts of vigilance performance phenomenon to contemporary psycho-physiological models. This review thus provides a comprehensive framework to understand the dynamics of human attention in novel situations. For example, this framework has informed an unpublished study of ours that explores the cerebral hemodynamics of vigilance performance in network defense analysts. Participants performed a task engineered to simulate the cognitive load associated with sustaining attention to cyber security command and control consoles, while regional oxygen tissue saturation, rSO 2 , was tracked. Preliminary results suggested that characteristic changes in rSO 2 reflected vigilance decrement, indicating that cortically regional metabolic activity may reflect support for the depletion theory. Additional avenues of future exploration suggested through this work include deriving psychological theories complemented by a biological component. For example, theoretical accounts of vigilance performance took an abstract perspective of what the “load” in overload theories reflected for approximately the first 70 years of research beyond , ) formative studies of vigilance. The fact that the psychophysiological models of vigilance performance that and proposed so closely parallel the tenets of modern overload theory provides the basis of this holistic and comprehensive review of vigilance performance. The two driving paradigms currently used to understand vigilance decrement and sustained attention performance are, therefore, the cognitive theories of , , Wickens et al. , and , and the psycho-physiological models derived by and . This review identifies key parallels between modern cognitive and psycho-physiological accounts and identifies a gap in mapping between these different paradigms. There is, therefore, a gap in the literature manifested by a map from cognitive theories of vigilance decrement and psycho-physiological models of attention performance. Therefore, future research avenues should explore the link between “cognitive resource” theories of vigilance decrement and the psychophysiological models of attention performance. Understanding this link could help to better understand vigilance decrement and manage its influence in workplace vigilance tasks, as well as psychopathologies of the human attentional system. For example, tasks such as anomaly or error detection and critical system monitoring are workplace vigilance tasks that require the capacity to sustain attention for prolonged periods. In these contexts, vigilance decrement can pose serious operational risks that increase the risk of accidents and errors and decrease productivity. Interventions and strategies that more directly target the psycho-physiological basis of vigilance decrement in these workplace contexts may be more effective at mitigating its negative impact. More proactive measures that counteract the impact of vigilance decrement may be informed by this more comprehensive understanding of the functional psycho-physiological underpinnings of the phenomenon. One potential proactive approach would be to implement brief cognitive diversions into the workflow process to alleviate the demands associated with vigilance decrement. Technological advancements such as automation could also offload or augment the cognitive load contributing to vigilance decrement. Furthermore, attention-deficit hyper activity disorder (ADHD) is currently diagnosed based on a checklist of symptoms in The American Psychiatric Association’s (APA) Diagnostic And Statistics Manual . , however, critiqued this checklist diagnostic approach, as it is not based on any biological markers for ADHD and largely only describes a small amount of the variation in how the pathology manifests. Killeen posits that ADHD is a disorder of neuro-energetic regulation across regions of the cortex associated with attention. A psycho-physiological theory of vigilance and sustained attention performance that maps the cerebral changes associated with disordered attention may offer deeper, richer insights into ADHD symptomatology. For example, hyperfocus is a feature of ADHD that is distinct from obsession and refers to prolonged episodes of sustained attention to volitionally selected tasks, which are intense enough to diminish the perception of task-irrelevant stimuli . The model that posit, for instance, could suggest that hyperfocus reflects a critical difference in the way that energetic resources are distributed within ADHD as compared to a neurotypical case. ADHD psychopathology may not imply a lack of astrocytic supplementation but instead suboptimal control of astrocytic resources. For example, when a task is imposed on a person with ADHD, it may be harder to mobilize and control astrocytic glycogen within task-relevant cortex regions. However, when a task is volitionally selected, this may be the only time a person with ADHD can optimally control the regulation of astrocytic glycogen within task-relevant cortex regions. The capacity to hyperfocus on volitionally selected tasks may explain why ADHD has been associated with entrepreneurship . That is, since entrepreneurship is a volitionally selected task, people with ADHD may gravitate to the profession, as the nature of the task may allow their astrocytic resources to be optimally mobilized during hyperfocus . |
Heavy slow resistance training, radial extracorporeal shock wave therapy or advice for patients with tennis elbow in the Norwegian secondary care: a randomised controlled feasibility trial | b2e6798d-776e-4eaf-affb-030dddccb864 | 11667321 | Patient Education as Topic[mh] | Tennis elbow, also known as lateral epicondylalgia, is a common musculoskeletal condition with a prevalence of 1.3%, incidence 3.4 (95% CI, 3.3 to 3.5) per 1000 and a recurrence rate within 2 years of 8.5%. Tennis elbow is a painful condition that impacts quality of life. It causes functional disability with high costs due to productivity loss, healthcare use and absence from work. Although most cases of tennis elbow are self-limited, 66% report not fully recovered after 1 year and 54% after 2 years. Exercises are considered the first-line treatment for tennis elbow, and various physiotherapy treatments as well as radial extracorporeal shock wave therapy are often offered. However, there is no common agreement on the best management for tennis elbow. Recent systematic reviews report the beneficial effects of exercises for tennis elbow, but the lack of detailed descriptions of exercise components makes recommendations for the optimal exercise protocol difficult. Previous research on heavy slow resistance training for achilles and patellar tendinopathy reports clinical improvements, high patient satisfaction, good compliance rates and structural changes. To the best of our knowledge, there are no published trials testing heavy slow resistance training with dumbbells for tennis elbow. Previous studies suggest that radial extracorporeal shock wave therapy is effective in pain reduction and grip strength recovery for tennis elbow, but the evidence is sparse and inconsistent. Systematic reviews and meta-analyses of shock wave therapy on tennis elbow report low to moderate evidence of no benefit to sham shock wave therapy. More rigorously designed, large-sample, high-quality trials investigating shock wave therapy for tennis elbow are recommended to guide future clinical practice. Despite the pronounced use of physiotherapy, few studies with low risk of bias exist. One way to reduce bias and improve the quality of clinical trials is to work out the uncertainties of the trial design beforehand. A feasibility study is designed to inform about the process and management, help us understand resource requirements and to advance scientific inquiry. This may prevent research waste. The primary aim was to conduct a randomised controlled feasibility trial with heavy slow resistance training, radial extracorporeal shock wave therapy and information and advice in patients with tennis elbow. Specific aims were to evaluate the feasibility of recruitment, appointment adherence, intervention compliance, acceptance and comprehensibility, as well as retention rate and data completeness at follow-up. An ancillary aim was to describe within-group changes from baseline to follow-up in secondary outcome measures.
The current study was a single-centre, three-armed, randomised controlled feasibility trial with a parallel design, allocated 1:1:1, with follow-up after 3 and 6 months. Participants also took part in a study testing the psychometric properties of outcome measures. Hence, the actual enrolment on ClinicalTrials.gov is registered as 100 participants, 60 participants included in the feasibility study and another 40 participants only included to test the psychometric properties of the outcome measures. The final data collection was in December 2023. Patient and public involvement User involvement was embedded through the Patient Panel, consisting of user representatives with various musculoskeletal disorders, established at the Research and Communication Unit for Musculoskeletal Health at Oslo University Hospital. The Patient Panel participated in developing the study information and materials. The intervention protocols were presented to the Patient Panel, and adjustments were made accordingly. Study design The trial was designed as a randomised controlled feasibility trial and reported according to the 26-item checklist from The Consolidated Standards of Reporting Trials 2010 statement: extension to randomised pilot and feasibility trials. Approximately one week after inclusion, the participants were randomised by a co-investigator. The randomisation sequence was computer-generated with blocks of variable sizes (range 3–9), which were unknown to the treating physiotherapist. Data were collected on paper at an outpatient clinic at baseline, after the group allocation and after 3 months. Six months follow-up data were sent and collected by mail. If participants did not respond, they were followed up by phone and reminded to respond once. One postgraduate physiotherapist included, treated and followed up participants. Due to the nature of the interventions, neither the physiotherapist nor the participants could be blinded. Since this is not an effectiveness trial, crossover from the information and advice group was possible after the 3-month follow-up. Study population Participants were recruited from the outpatient clinic at the Department of Physical Medicine and Rehabilitation at Oslo University Hospital. Medical doctors screened participants prior to eligibility, as part of normal clinical practice at the outpatient clinic. Eligible participants were enrolled to the study after being assessed by the physiotherapist. The inclusion criteria were ≥18 years old. Two out of five clinical provocation tests had to be provocative of the symptoms on the lateral side of the elbow. (a) Pain on palpation, (b) pain on resisted wrist extension (Cozen test), (c) pain during power grip, (d) pain on resisted third finger extension (Maudsley test) and (e) pain on passive elbow extension combined with wrist palmar flexion (Mills test). The exclusion criteria were Insufficient language skills to participate. Contraindications to shock wave therapy (ie, pregnancy, coagulation disturbance, usage of anticoagulants, connective tissue disease, epilepsy or use of pacemaker). Any suspicion of other serious illness. All participants provided written informed consent before inclusion. Baseline assessments To describe the baseline demographics, participants filled out a set of sociodemographic variables such as age, sex, height, weight, education, works status and type of work. They also reported pain duration, usage of pain medication, previous treatments and number of pain sites. Interventions All groups received the same written and oral information about aetiology, pathogenesis, treatment options and the prognosis. Participants were informed that it is safe to use the arm despite pain and that pain does not equal harm. The importance of using the arm to regain and maintain function was emphasised. Load management with a gradual increase in load within tolerable pain was recommended. Heavy slow resistance training The participants received a written protocol on how to perform and progress the exercises based on previous protocols on heavy slow resistance training. A postgraduate physiotherapist instructed individually face-to-face, a 12-week home training programme (ie, totally 36 training sessions) consisting of two exercises. One exercise addressed wrist extension and flexion, and the other pronation and supination of the forearm holding a dumbbell. Details are reported after Consensus on Exercise Reporting Template . Participants were also instructed to stretch their extensor muscles with a straight elbow, pronated and palmar flexed wrist, three times a day, three sets for 30 s. Appointments for supervision with a physiotherapist during the intervention period were voluntary. Participants were offered supervision up to once per week and could choose between digital or physical consultations. They were provided with a workout diary and urged to report on fulfilled sessions. No restrictions were given, but a plan of action was provided if there was aggravation of pain from the training . Radial extracorporeal shock wave therapy Participants received written and oral information about the treatment. Shock wave therapy was given using an EMS-DolorClast with a Swiss DolorClast Evo blue handpiece with a 15 mm applicator able to deliver up to 0.18 mJ/mm 2 . A treatment protocol was followed as recommended by the manufacturer (EMS DolorClast). Participants had three sessions with approximately 1 week a part, receiving 2000 impulses at 10 Hz, with a low energy treatment between 1.5 and 3 BAR. To maximise potential outcomes, the treatment was given over the area of maximal pain during palpation of the lateral epicondyle. No restrictions were given after the treatment. Information and advice Participants received a single individual face-to-face session with a postgraduate physiotherapist providing information and advice according to the protocol. The session lasted up to 45 min. Included in this session was the same written and oral information as all participants. They were asked to specify their primary challenges and set their own recovery goals. Pacing and load management to accomplish their goals were discussed. Participants were asked about their pain-believes and behaviour. They were also assured about the robustness of the elbow despite the pain. The natural course of the condition and the good prognosis over time were emphasised. No restrictions were given, and participants were encouraged to use their arms as normal as possible regardless of pain. Feasibility outcomes To evaluate the feasibility of the trial, a priori criteria for success were determined . The first objective was to measure the process of recruitment. Second, the appointment adherence, intervention compliance, acceptability and comprehensiveness. Adherence was measured in attendance to scheduled physiotherapy appointments. The compliance was measured in a number of completed training sessions. The intervention acceptability and comprehensibility were measured directly after receiving the first session and at the 3-month follow-up. It was scored on a 19-point Likert scale from −9 (I do not accept/understand my treatment) to +9 (I completely accept/understand my treatment). The third objective was to measure the retention rate and completeness of data at 3 and 6 months. Results were interpreted accordingly: (a) go–proceed with Randomised Controlled Trial (RCT), (b) amend–proceed with changes and (c) stop–do not proceed unless changes are possible. Secondary outcome measures Participants filled in the Patient-rated tennis elbow evaluation, the Quick-Disabilities of the Arm, Shoulder and Hand (Quick-DASH), the 5-Level EuroQol-5D (EQ-5D-5L), a Numeric Rating Scale Pain (NRS-P) now and a 13-point Global Rating Of Change (GROC). Additionally, pain-free grip strength (kg) was measured and pain on gripping (0: no pain to 10: worst imaginable pain) was reported. The patient-rated tennis elbow evaluation consists of two subscales: (a) pain, which is scored from 0 (no pain) to 50 (worst imaginable) and (b) function, which is scored from 0 (no difficulty) to 50 (unable to do). A total score of 100 on the patient-rated tennis elbow evaluation can be computed, a higher score equals more disability. The Quick-DASH ranges from 0 to 100, a higher score indicates more disability. EQ-5D-5L is measured in five levels from 1 (no problems) to 5 (extreme problems). Results can also be presented as an index value from −0.59 to 1, where 1 represents the best possible health state. The EQ visual analogue scale is rated from 0 (worst imaginable health state) to 100 (best imaginable health state). NRS-P was scored from 0 (no pain) to 10 (worst imaginable pain). GROC was measured from −6 (maximal worsening) to +6 (completely recovered). Sample size As this was a feasibility study, a formal sample size calculation was not performed. Based on the total access of tennis elbow patients at the outpatient clinic in previous years, a total sample of 60 participants included at a rate of 3.75 participants per month was considered to be realistic. Hence we estimated an inclusion period of 16 months of inclusion, which would give the study a realistic impression of the process of recruitment. The sample size of 20 in each intervention arm was estimated as sufficient to capture the diversity in compliance, acceptability, comprehensiveness, adverse events and dropouts. Statistical analysis Statistical analysis was undertaken using IBM SPSS Statistics for Windows (V.29.0 Armonk, New York, USA: IBM Corp.) Descriptive statistics were used to assess the feasibility criteria and the patient-reported outcomes measures (secondary outcomes). Mean and SD, median and IQR, and frequency were reported according to the scale of the data.
User involvement was embedded through the Patient Panel, consisting of user representatives with various musculoskeletal disorders, established at the Research and Communication Unit for Musculoskeletal Health at Oslo University Hospital. The Patient Panel participated in developing the study information and materials. The intervention protocols were presented to the Patient Panel, and adjustments were made accordingly.
The trial was designed as a randomised controlled feasibility trial and reported according to the 26-item checklist from The Consolidated Standards of Reporting Trials 2010 statement: extension to randomised pilot and feasibility trials. Approximately one week after inclusion, the participants were randomised by a co-investigator. The randomisation sequence was computer-generated with blocks of variable sizes (range 3–9), which were unknown to the treating physiotherapist. Data were collected on paper at an outpatient clinic at baseline, after the group allocation and after 3 months. Six months follow-up data were sent and collected by mail. If participants did not respond, they were followed up by phone and reminded to respond once. One postgraduate physiotherapist included, treated and followed up participants. Due to the nature of the interventions, neither the physiotherapist nor the participants could be blinded. Since this is not an effectiveness trial, crossover from the information and advice group was possible after the 3-month follow-up.
Participants were recruited from the outpatient clinic at the Department of Physical Medicine and Rehabilitation at Oslo University Hospital. Medical doctors screened participants prior to eligibility, as part of normal clinical practice at the outpatient clinic. Eligible participants were enrolled to the study after being assessed by the physiotherapist. The inclusion criteria were ≥18 years old. Two out of five clinical provocation tests had to be provocative of the symptoms on the lateral side of the elbow. (a) Pain on palpation, (b) pain on resisted wrist extension (Cozen test), (c) pain during power grip, (d) pain on resisted third finger extension (Maudsley test) and (e) pain on passive elbow extension combined with wrist palmar flexion (Mills test). The exclusion criteria were Insufficient language skills to participate. Contraindications to shock wave therapy (ie, pregnancy, coagulation disturbance, usage of anticoagulants, connective tissue disease, epilepsy or use of pacemaker). Any suspicion of other serious illness. All participants provided written informed consent before inclusion.
To describe the baseline demographics, participants filled out a set of sociodemographic variables such as age, sex, height, weight, education, works status and type of work. They also reported pain duration, usage of pain medication, previous treatments and number of pain sites.
All groups received the same written and oral information about aetiology, pathogenesis, treatment options and the prognosis. Participants were informed that it is safe to use the arm despite pain and that pain does not equal harm. The importance of using the arm to regain and maintain function was emphasised. Load management with a gradual increase in load within tolerable pain was recommended. Heavy slow resistance training The participants received a written protocol on how to perform and progress the exercises based on previous protocols on heavy slow resistance training. A postgraduate physiotherapist instructed individually face-to-face, a 12-week home training programme (ie, totally 36 training sessions) consisting of two exercises. One exercise addressed wrist extension and flexion, and the other pronation and supination of the forearm holding a dumbbell. Details are reported after Consensus on Exercise Reporting Template . Participants were also instructed to stretch their extensor muscles with a straight elbow, pronated and palmar flexed wrist, three times a day, three sets for 30 s. Appointments for supervision with a physiotherapist during the intervention period were voluntary. Participants were offered supervision up to once per week and could choose between digital or physical consultations. They were provided with a workout diary and urged to report on fulfilled sessions. No restrictions were given, but a plan of action was provided if there was aggravation of pain from the training . Radial extracorporeal shock wave therapy Participants received written and oral information about the treatment. Shock wave therapy was given using an EMS-DolorClast with a Swiss DolorClast Evo blue handpiece with a 15 mm applicator able to deliver up to 0.18 mJ/mm 2 . A treatment protocol was followed as recommended by the manufacturer (EMS DolorClast). Participants had three sessions with approximately 1 week a part, receiving 2000 impulses at 10 Hz, with a low energy treatment between 1.5 and 3 BAR. To maximise potential outcomes, the treatment was given over the area of maximal pain during palpation of the lateral epicondyle. No restrictions were given after the treatment. Information and advice Participants received a single individual face-to-face session with a postgraduate physiotherapist providing information and advice according to the protocol. The session lasted up to 45 min. Included in this session was the same written and oral information as all participants. They were asked to specify their primary challenges and set their own recovery goals. Pacing and load management to accomplish their goals were discussed. Participants were asked about their pain-believes and behaviour. They were also assured about the robustness of the elbow despite the pain. The natural course of the condition and the good prognosis over time were emphasised. No restrictions were given, and participants were encouraged to use their arms as normal as possible regardless of pain.
The participants received a written protocol on how to perform and progress the exercises based on previous protocols on heavy slow resistance training. A postgraduate physiotherapist instructed individually face-to-face, a 12-week home training programme (ie, totally 36 training sessions) consisting of two exercises. One exercise addressed wrist extension and flexion, and the other pronation and supination of the forearm holding a dumbbell. Details are reported after Consensus on Exercise Reporting Template . Participants were also instructed to stretch their extensor muscles with a straight elbow, pronated and palmar flexed wrist, three times a day, three sets for 30 s. Appointments for supervision with a physiotherapist during the intervention period were voluntary. Participants were offered supervision up to once per week and could choose between digital or physical consultations. They were provided with a workout diary and urged to report on fulfilled sessions. No restrictions were given, but a plan of action was provided if there was aggravation of pain from the training .
Participants received written and oral information about the treatment. Shock wave therapy was given using an EMS-DolorClast with a Swiss DolorClast Evo blue handpiece with a 15 mm applicator able to deliver up to 0.18 mJ/mm 2 . A treatment protocol was followed as recommended by the manufacturer (EMS DolorClast). Participants had three sessions with approximately 1 week a part, receiving 2000 impulses at 10 Hz, with a low energy treatment between 1.5 and 3 BAR. To maximise potential outcomes, the treatment was given over the area of maximal pain during palpation of the lateral epicondyle. No restrictions were given after the treatment.
Participants received a single individual face-to-face session with a postgraduate physiotherapist providing information and advice according to the protocol. The session lasted up to 45 min. Included in this session was the same written and oral information as all participants. They were asked to specify their primary challenges and set their own recovery goals. Pacing and load management to accomplish their goals were discussed. Participants were asked about their pain-believes and behaviour. They were also assured about the robustness of the elbow despite the pain. The natural course of the condition and the good prognosis over time were emphasised. No restrictions were given, and participants were encouraged to use their arms as normal as possible regardless of pain.
To evaluate the feasibility of the trial, a priori criteria for success were determined . The first objective was to measure the process of recruitment. Second, the appointment adherence, intervention compliance, acceptability and comprehensiveness. Adherence was measured in attendance to scheduled physiotherapy appointments. The compliance was measured in a number of completed training sessions. The intervention acceptability and comprehensibility were measured directly after receiving the first session and at the 3-month follow-up. It was scored on a 19-point Likert scale from −9 (I do not accept/understand my treatment) to +9 (I completely accept/understand my treatment). The third objective was to measure the retention rate and completeness of data at 3 and 6 months. Results were interpreted accordingly: (a) go–proceed with Randomised Controlled Trial (RCT), (b) amend–proceed with changes and (c) stop–do not proceed unless changes are possible.
Participants filled in the Patient-rated tennis elbow evaluation, the Quick-Disabilities of the Arm, Shoulder and Hand (Quick-DASH), the 5-Level EuroQol-5D (EQ-5D-5L), a Numeric Rating Scale Pain (NRS-P) now and a 13-point Global Rating Of Change (GROC). Additionally, pain-free grip strength (kg) was measured and pain on gripping (0: no pain to 10: worst imaginable pain) was reported. The patient-rated tennis elbow evaluation consists of two subscales: (a) pain, which is scored from 0 (no pain) to 50 (worst imaginable) and (b) function, which is scored from 0 (no difficulty) to 50 (unable to do). A total score of 100 on the patient-rated tennis elbow evaluation can be computed, a higher score equals more disability. The Quick-DASH ranges from 0 to 100, a higher score indicates more disability. EQ-5D-5L is measured in five levels from 1 (no problems) to 5 (extreme problems). Results can also be presented as an index value from −0.59 to 1, where 1 represents the best possible health state. The EQ visual analogue scale is rated from 0 (worst imaginable health state) to 100 (best imaginable health state). NRS-P was scored from 0 (no pain) to 10 (worst imaginable pain). GROC was measured from −6 (maximal worsening) to +6 (completely recovered).
As this was a feasibility study, a formal sample size calculation was not performed. Based on the total access of tennis elbow patients at the outpatient clinic in previous years, a total sample of 60 participants included at a rate of 3.75 participants per month was considered to be realistic. Hence we estimated an inclusion period of 16 months of inclusion, which would give the study a realistic impression of the process of recruitment. The sample size of 20 in each intervention arm was estimated as sufficient to capture the diversity in compliance, acceptability, comprehensiveness, adverse events and dropouts.
Statistical analysis was undertaken using IBM SPSS Statistics for Windows (V.29.0 Armonk, New York, USA: IBM Corp.) Descriptive statistics were used to assess the feasibility criteria and the patient-reported outcomes measures (secondary outcomes). Mean and SD, median and IQR, and frequency were reported according to the scale of the data.
60 participants were included from August 2021 to February 2023. The final follow-up data collection for the study took place in September 2023. Participants were middle-aged women (68%) with a mean age of 47.8 (SD 9.3) years and a symptom duration of more than 3 months (90%). They were mainly university-educated (67%) and worked full or part time (63%). Most of the participants had previously received exercises for the affected elbow (67%) . The process of recruitment A total of 98 patients were registered with tennis elbow at the Department of Physical Medicine and Rehabilitation at Oslo University Hospital between 1 August 2020 and 31 January 2023. 89 patients were screened for eligibility, and 78% were eligible for randomisation . Of these, four did not meet the inclusion criteria, and five were not willing to be randomised. Thus, 92% were willing to be randomised which gave a recruitment rate of 3.4 participants per month . The main reasons for exclusion were the usage of anticoagulants and insufficient Norwegian language skills. Interventions All the interventions were above the criteria for success for acceptability and comprehensibility . Heavy slow resistance training The median number for follow-up appointments was 2 (range 0–5). Six participants used the opportunity for digital follow-up (ie, phone or video). The median number of performed self-training sessions within 12 weeks was 26 (range 6–35) of 36 possible. The compliance rate with 30 or more training sessions per participant was 32% (6/19). Eight participants reported increased pain from training as the reason for non-compliance. Five participants reported increased pain from training as an adverse event. 42 appointments for follow-up with the physiotherapist were scheduled among the 20 participants, whereas 38/42 (90%) were undertaken . Three participants reported seeking other treatment during the 3-month follow-up period. At 6 months, three participants reported seeking other treatments . Radial extracorporeal shock wave therapy The three shock wave therapy sessions were carried out for all 20 participants (100% adherence) . Two participants reported increased pain up to 12 hours after treatment. One participant reported seeking other treatment during the 3-month follow-up period. At 6 months, none of the participants reported seeking other treatment . Information and advice 10 participants wanted a crossover treatment after the 3-month follow-up. Of these, six chose shock wave therapy and four training. No adverse events were reported. One participant reported seeking other treatment during the 3-month follow-up period. At 6 months, two participants reported seeking other treatment . Feasibility of outcome measures At baseline and 3 months follow-up, the outcomes evaluating the retention rate and completeness of data were above the criteria for success (>75%). However, retention rate at 6 months was below the criteria for success with 68% . Within-group changes All groups improved in the patient-reported outcome measures and in the pain-free grip strength from baseline to 3 and 6 months. From baseline to 3 months, all groups were above the minimal detectable change for the patient-rated tennis elbow evaluation and Quick-DASH mean change scores of 8.9 and 11.2, respectively. At 3 months, 66% had improved and 10% were worse on the GROC scale among all participants .
A total of 98 patients were registered with tennis elbow at the Department of Physical Medicine and Rehabilitation at Oslo University Hospital between 1 August 2020 and 31 January 2023. 89 patients were screened for eligibility, and 78% were eligible for randomisation . Of these, four did not meet the inclusion criteria, and five were not willing to be randomised. Thus, 92% were willing to be randomised which gave a recruitment rate of 3.4 participants per month . The main reasons for exclusion were the usage of anticoagulants and insufficient Norwegian language skills.
All the interventions were above the criteria for success for acceptability and comprehensibility . Heavy slow resistance training The median number for follow-up appointments was 2 (range 0–5). Six participants used the opportunity for digital follow-up (ie, phone or video). The median number of performed self-training sessions within 12 weeks was 26 (range 6–35) of 36 possible. The compliance rate with 30 or more training sessions per participant was 32% (6/19). Eight participants reported increased pain from training as the reason for non-compliance. Five participants reported increased pain from training as an adverse event. 42 appointments for follow-up with the physiotherapist were scheduled among the 20 participants, whereas 38/42 (90%) were undertaken . Three participants reported seeking other treatment during the 3-month follow-up period. At 6 months, three participants reported seeking other treatments . Radial extracorporeal shock wave therapy The three shock wave therapy sessions were carried out for all 20 participants (100% adherence) . Two participants reported increased pain up to 12 hours after treatment. One participant reported seeking other treatment during the 3-month follow-up period. At 6 months, none of the participants reported seeking other treatment . Information and advice 10 participants wanted a crossover treatment after the 3-month follow-up. Of these, six chose shock wave therapy and four training. No adverse events were reported. One participant reported seeking other treatment during the 3-month follow-up period. At 6 months, two participants reported seeking other treatment .
The median number for follow-up appointments was 2 (range 0–5). Six participants used the opportunity for digital follow-up (ie, phone or video). The median number of performed self-training sessions within 12 weeks was 26 (range 6–35) of 36 possible. The compliance rate with 30 or more training sessions per participant was 32% (6/19). Eight participants reported increased pain from training as the reason for non-compliance. Five participants reported increased pain from training as an adverse event. 42 appointments for follow-up with the physiotherapist were scheduled among the 20 participants, whereas 38/42 (90%) were undertaken . Three participants reported seeking other treatment during the 3-month follow-up period. At 6 months, three participants reported seeking other treatments .
The three shock wave therapy sessions were carried out for all 20 participants (100% adherence) . Two participants reported increased pain up to 12 hours after treatment. One participant reported seeking other treatment during the 3-month follow-up period. At 6 months, none of the participants reported seeking other treatment .
10 participants wanted a crossover treatment after the 3-month follow-up. Of these, six chose shock wave therapy and four training. No adverse events were reported. One participant reported seeking other treatment during the 3-month follow-up period. At 6 months, two participants reported seeking other treatment .
At baseline and 3 months follow-up, the outcomes evaluating the retention rate and completeness of data were above the criteria for success (>75%). However, retention rate at 6 months was below the criteria for success with 68% .
All groups improved in the patient-reported outcome measures and in the pain-free grip strength from baseline to 3 and 6 months. From baseline to 3 months, all groups were above the minimal detectable change for the patient-rated tennis elbow evaluation and Quick-DASH mean change scores of 8.9 and 11.2, respectively. At 3 months, 66% had improved and 10% were worse on the GROC scale among all participants .
The aim of the study was to test the feasibility of an RCT comparing heavy slow resistance training, radial extracorporeal shock wave therapy and information and advice. The recruitment rate was 10% below the criteria for success. According to our predetermined criteria, all interventions were acceptable and comprehensible, but only 32% complied in the training group. The outcome measures showed a successful retention rate at 3 months, but not at 6 months. The completeness of data was above the criteria for success. The mean patient-reported and performance-based outcomes improved in all groups. Previous research on exercise therapy for the management of tendinopathy suggests that patients are generally satisfied. In the present study, the compliance with training was too low for recommendation of a full-scale RCT aiming to evaluate the effectiveness of heavy slow resistance training. One possible explanation is that 70% of participants in the training group had already tried some kind of exercise without success, suggesting that they had low expectations for the training intervention. The discrepancy between the acceptance rate of 68% of participants at 3 months and the low compliance to training of only 32% indicates that the 80% cut-off for compliance might have been set too high. However, adherence to exercise protocols is usually good and a recent scoping review found that among 97 studies, the median adherence was 82.5% (range: 45.3–100). Previous heavy slow resistance training research has reported good compliance and high patient satisfaction, unlike the present study. This research has been carried out on athletes, which represent a highly selected population with regard to exercise. 25% reported increased pain as the reason for not performing the exercises and 21% reported worsening at 3 months. This agrees with Vuvan et al reporting that 14% of their exercise group experienced worsening. The shock wave therapy group had 100% appointment adherence and was the only group without any dropouts, which indicates that this is a feasible intervention in a future study. However, to determine the effectiveness of shock wave therapy in a full-scale RCT, it must be compared with sham shock wave therapy. A systematic review found six studies comparing shock wave therapy to sham shock wave treatment for tennis elbow. Compliance and follow-up rates were high which suggests that both shock wave therapy and sham treatment were feasible. Recently, two large RCTs in the Norwegian secondary care successfully completed sham-controlled trials in patients with tendinopathies in the shoulder or in the heel and reported successful masking and low dropouts. Thus, sham shock wave therapy should also be feasible for a similar population with tennis elbow. The information and advice group was above the criteria for success for acceptability and comprehensibility. A recent consensus study on optimised physiotherapy for lateral elbow tendinopathy ranked advice and education as the most important part. Large RCTs including patients with shoulder pain have successfully carried out similar interventions. However, half of the included participants in this study wanted to cross over after the 3-month follow-up, thus not satisfied with the intervention alone. This may suggest that the criteria for success were set too low a priori. Patient expectations, contextual factors and communication with the therapist are important for a successful intervention and should be explored further before a full-scale RCT. The current study did not measure changes in the patient’s perception after the information and advice were given. Thus, the interventions effect on opinions and behaviour is unknown. The current study included mostly participants having a university education, and working on a computer, which is in line with characteristics Sanders et al found in their population-based study. Contrarily, most studies report that tennis elbow is more common among blue-collar workers exposed to hand or elbow movements. In agreement with previous research on tennis elbow conducted in the Norwegian primary healthcare, 30% of the participants were on sick leave. Health-related quality of life was below the Norwegian population norms at baseline, and about half the study cohort reported light symptoms of anxiety or depression which is in line with the general population. Among participants included in the present study, 90% had long-term symptoms (>3 months). This suggests that it is feasible to include only participants with long-term symptoms in a future full-scale RCT in the same setting. Baseline and change scores, for the patient-rated tennis elbow evaluation and Quick-DASH, are comparable to other RCTs on tennis elbow. From baseline to 3 months, all groups improved from moderate pain to mild pain according to the patient-rated tennis elbow evaluation pain subscale. Although the outcomes favoured the shock wave therapy group, the primary aim of the present study was not to compare improvements in symptoms. Limitations The present study discovered discrepancies between acceptance and the compliance rate in the training group, and acceptance and crossover treatment in the information and advice group, which suggests that the a priori criteria for success were set too low. A traffic light approach was used to evaluate the progression criteria, but the cut-off for amber light was not defined a priori. Thus, values below the cut-off were assessed based on empirical data. A recent recommendations for progression criteria endorse the use of the traffic light approach as a way to highlight, and draw attention to problems that have been faced, rather than use it as a firm rule. A guidance to decision-making if progression criteria results are below the cut-off could be a hierarchical prioritisation of outcomes based on their importance (eg, potential adverse events, compliance, important stakeholders and resources). Such prioritisation should be done a priori. A possible reason for the low retention rate at 6 months in our study is that follow-up only being questionnaires sent by postal mail. A physical attendance follow-up could have increased the retention-rate. Preferably, we should have explored this by asking participants why they did not respond to the follow-up questionnaires. Also, not excluding patients who recently have received shockwave treatment, exercises or other treatments might have influenced results. A future full-scale RCT measuring the effectiveness of treatment should try to reduce this. The study recruited participants with pragmatic exclusion criteria that reflect the real-world patient in secondary care, arguably increasing the external validity of the study. The exercise group did not have mandatory appointments for supervision as we wanted to evaluate the participant’s wishes for supervision, but this may have affected the intervention compliance. Mandatory appointments could have given another outcome, because supervision gives better adherence to exercises. Participants who experienced pain could potentially increase compliance if they were assured and guided through the training sessions. More appointments could also lead to better patient-practitioner relationship which is suggested to be an important predictor for adherence. There was no blinded assessor, thus the feasibility of the blinding was not evaluated. The participants answered questions about the acceptability and comprehensibility directly to the treating physiotherapist, who also gave the intervention, which might have created observer and reporting bias. The current study did not evaluate the feasibility of health economics questionnaires, which is an important part of a future full-scale RCT. Health economics questionnaires can be cumbersome to complete, and the lack of these might have led to an overestimation of the completeness of data.
The present study discovered discrepancies between acceptance and the compliance rate in the training group, and acceptance and crossover treatment in the information and advice group, which suggests that the a priori criteria for success were set too low. A traffic light approach was used to evaluate the progression criteria, but the cut-off for amber light was not defined a priori. Thus, values below the cut-off were assessed based on empirical data. A recent recommendations for progression criteria endorse the use of the traffic light approach as a way to highlight, and draw attention to problems that have been faced, rather than use it as a firm rule. A guidance to decision-making if progression criteria results are below the cut-off could be a hierarchical prioritisation of outcomes based on their importance (eg, potential adverse events, compliance, important stakeholders and resources). Such prioritisation should be done a priori. A possible reason for the low retention rate at 6 months in our study is that follow-up only being questionnaires sent by postal mail. A physical attendance follow-up could have increased the retention-rate. Preferably, we should have explored this by asking participants why they did not respond to the follow-up questionnaires. Also, not excluding patients who recently have received shockwave treatment, exercises or other treatments might have influenced results. A future full-scale RCT measuring the effectiveness of treatment should try to reduce this. The study recruited participants with pragmatic exclusion criteria that reflect the real-world patient in secondary care, arguably increasing the external validity of the study. The exercise group did not have mandatory appointments for supervision as we wanted to evaluate the participant’s wishes for supervision, but this may have affected the intervention compliance. Mandatory appointments could have given another outcome, because supervision gives better adherence to exercises. Participants who experienced pain could potentially increase compliance if they were assured and guided through the training sessions. More appointments could also lead to better patient-practitioner relationship which is suggested to be an important predictor for adherence. There was no blinded assessor, thus the feasibility of the blinding was not evaluated. The participants answered questions about the acceptability and comprehensibility directly to the treating physiotherapist, who also gave the intervention, which might have created observer and reporting bias. The current study did not evaluate the feasibility of health economics questionnaires, which is an important part of a future full-scale RCT. Health economics questionnaires can be cumbersome to complete, and the lack of these might have led to an overestimation of the completeness of data.
The current study shows that the process of recruitment and the retention rate at follow-up can be feasible with minor amendments. Participants had low compliance with heavy slow resistance training mainly due to pain aggravation, which suggests that this intervention was not suitable for patients with tennis elbow. Shock wave therapy and information and advice should be investigated further in a full-scale randomised controlled trial including sham shock wave therapy.
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A Virtual Cardiometabolic Health Program Among African Immigrants in the US | f3a470c4-e807-4fbc-87d4-b318bf265298 | 11880947 | Health Promotion[mh] | Black people, including immigrants, in the US bear a disproportionate burden of cardiometabolic conditions, such as hypertension and type 2 diabetes. , , Despite effective interventions to improve cardiometabolic health (CMH), these populations continue to experience poorer outcomes compared with their non-Hispanic White counterparts. Among non-Hispanic Black adults, including immigrants, more than half (56%) have hypertension, a significantly higher prevalence than the 48% observed among non-Hispanic White adults. Similarly, 12.7% of non-Hispanic Black individuals have type 2 diabetes, compared with 11.0% of non-Hispanic White adults, with a substantial proportion remaining undiagnosed. This disparity in cardiometabolic burden results in higher mortality rates, increased disability, and substantial health care costs, totaling nearly $320 annually among Black people. African immigrants represent a rapidly growing and heterogeneous minoritized group in the US that is also affected by adverse CMH outcomes. Between 2000 and 2015, the Black immigrant population increased by 137%, reaching 2.1 million, with approximately 30% originating from Ghana and Nigeria. Persons of African heritage, including African immigrants, are often aggregated and categorized as “Black/African American,” overlooking the cultural diversity within this group and the implications of implementing culturally tailored interventions. , A significant proportion of African immigrants experience a high burden of cardiometabolic disease risk. , Factors contributing to these disparities include the cumulative impact of acculturation stress, limited access to culturally competent health care services, dietary transitions, and lifestyle changes associated with migration. , , Furthermore, many African immigrants already face a disproportionate cardiometabolic disease burden before migrating to the US. Evidence-based programs, such as the Centers for Disease Control and Prevention’s National Diabetes Prevention Program (DPP), have shown benefits in promoting weight loss through dietary and exercise strategies. , However, participation among African immigrants and other ethnic minoritized populations remains low if the program is not tailored to incorporate relevant cultural beliefs, practices, and values. , Prior studies have demonstrated the potential of collaborating with faith-based and religious organizations to deliver culturally adapted, sustainable lifestyle interventions for underserved communities. , Integrating virtual platforms into health care delivery offers opportunities to facilitate self-monitoring of CMH indicators and disseminate health information. However, there is a paucity of rigorous research evaluating the effectiveness of culturally tailored, multicomponent virtual CMH programs designed specifically for African immigrant populations in the US. Addressing this gap is crucial to mitigate the disproportionate burden of cardiometabolic diseases in this rapidly growing population. This pilot clinical trial evaluated the effectiveness of a virtual, culturally tailored lifestyle intervention with remote blood pressure (BP) monitoring on CMH outcomes among African immigrants in Baltimore, Maryland, and Washington, DC metropolitan area. We hypothesized that a culturally tailored lifestyle intervention would improve BP and glycemic control among African immigrants with cardiometabolic risk factors.
Trial Design and Oversight The Afro-DPP was a pilot cluster-randomized, parallel-group, unmasked clinical trial conducted from January 1, 2022, to July 31, 2023, to evaluate the effectiveness of a culturally tailored, multicomponent virtual CMH intervention among African immigrants with CMH risk factors. The institutional review board of The Johns Hopkins University approved the trial protocol and statistical analysis plan ( ). The study followed the Consolidated Standards of Reporting Trials ( CONSORT ) guidelines (eFigure 1 in ), and an independent data and safety monitoring board provided oversight. All participants provided written informed consent. Participants Eligible participants were adults who were born in an African country and subsequently immigrated to the US, aged 25 to 75 years, and had at least 2 of the following CMH risk factors: (1) body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) of at least 25.0; (2) hemoglobin A 1c (HbA 1c ) level ranging from 5.7% to 6.5% (to convert to proportion of total hemoglobin, multiply by 0.01); and (3) systolic BP of at least 140 mm Hg or diastolic BP of at least 90 mm Hg. Smartphone ownership was a general inclusion criterion. Key exclusion criteria were cognitive impairment, severe chronic illness, inability to communicate in English, and nonmembership in the participating churches. We conducted targeted screenings with African churches, to improve enrollment of women in the study. Randomization and Masking Two churches with predominantly African immigrant congregations in the Baltimore–Washington, DC, metropolitan area were recruited and served as cluster units. The churches were assigned to either the first intervention group or the delayed intervention group. Due to the interactive nature of the intervention, participants were informed of their group assignment, and study staff and data collectors were unmasked. Interventions Lifestyle Intervention The multicomponent intervention was adapted from the National DPP curriculum and focused on intensive lifestyle modification delivered by a certified lifestyle coach of African heritage. Educational materials were culturally tailored to incorporate African cultural values, practices, beliefs, and traditional dietary choices. The adapted DPP curriculum is described briefly in the trial protocol in . First Intervention Group Participants randomized to the first intervention group immediately began the 6-month adapted DPP lifestyle intervention, which included weekly 60-minute virtual group sessions via videoconferencing. The sessions covered topics such as stress management, healthy nutritional choices, physical activity, sleep considerations, and strategies to reduce cardiometabolic risk. Participants received a copy of the curriculum to follow along during the sessions. Delayed Intervention Group Participants in the delayed intervention group received no lifestyle intervention during the first 6 months but underwent the same assessments as the first intervention group. After 6 months, they received the identical adapted DPP lifestyle intervention as the first group, including remote monitoring. Remote Monitoring All participants received a Bluetooth-enabled BP monitor (BP7250; Omron) and digital weight scale (BCM-500; Omron) at enrollment to enable remote monitoring of BP and body composition measures. The delayed intervention group served as an attention control with device monitoring only during the first 6 months. Participants were trained on using these devices for daily home measurements, which synchronized automatically to a smartphone app (Sphygmo) and a cloud-based platform accessible to the research team. This allowed for prompt feedback and reminders to enable participants to adhere to their prescribed medications and contact their clinicians for review of their treatment plan based on participants’ transmitted BP values. Details are included in the protocol ( ). Outcome Measures Consistent with the trial registration, the primary outcomes were (1) changes in clinic-measured systolic and diastolic BP from baseline to 6 months using a validated automated device (HEM-9210T [Omron] and the Omron 10 series) by trained research staff following standardized procedures and (2) changes in HbA 1c levels (measured using the A1CNow+ point of care system; PTS Diagnostics). Prespecified secondary outcomes included changes in body weight and BMI during the same period. Assessments and Follow-Up Participants attended in-person study visits at their respective churches for assessments at baseline, 1 month, 3 months, and 6 months. During these visits, trained staff followed standardized protocols to systematically measure BP, HbA 1c levels, body weight, and BMI. At each visit, trained staff shared and discussed the measurement results with each participant at every site. Statistical Analysis All analyses followed the intention-to-treat principle. This hypothesis-generating pilot study was designed to provide preliminary data and estimate effect sizes to inform a larger-scale trial. Analyses were performed using Stata, version 16 (StataCorp LLC). Baseline characteristics were summarized using descriptive statistics and compared between groups using χ 2 tests for categorical variables and 2-sample t tests for continuous variables. Complete data were available for age, educational level, employment status, and baseline physical measurements (30 per group). Other measures had varying numbers of participants with available data, as detailed in . Changes in primary (systolic and diastolic BP and HbA 1c levels) and secondary (body weight and BMI) outcomes over time were examined using linear mixed-effects models with fixed effects for time (baseline and 1, 3, and 6 months), intervention group (first or delayed), and their interaction. The linear mixed-effects models handled missing data under the missing at random assumption through maximum likelihood estimation. Random intercepts were included to account for clustering within churches. We estimated mean changes from baseline adjusting for age, sex, educational level, BP medication, and current alcohol use with 95% CIs. Effect sizes were calculated using Cohen d for continuous outcomes and number needed to treat for binary outcomes (eFigures 1-3 in ). Visual representations of the model-based means over time were created using line plots with separate lines for each study group. A 2-sided P < .05 was considered statistically significant for all tests. No adjustments for multiple comparisons were made in this exploratory pilot study.
The Afro-DPP was a pilot cluster-randomized, parallel-group, unmasked clinical trial conducted from January 1, 2022, to July 31, 2023, to evaluate the effectiveness of a culturally tailored, multicomponent virtual CMH intervention among African immigrants with CMH risk factors. The institutional review board of The Johns Hopkins University approved the trial protocol and statistical analysis plan ( ). The study followed the Consolidated Standards of Reporting Trials ( CONSORT ) guidelines (eFigure 1 in ), and an independent data and safety monitoring board provided oversight. All participants provided written informed consent.
Eligible participants were adults who were born in an African country and subsequently immigrated to the US, aged 25 to 75 years, and had at least 2 of the following CMH risk factors: (1) body mass index (BMI; calculated as weight in kilograms divided by height in meters squared) of at least 25.0; (2) hemoglobin A 1c (HbA 1c ) level ranging from 5.7% to 6.5% (to convert to proportion of total hemoglobin, multiply by 0.01); and (3) systolic BP of at least 140 mm Hg or diastolic BP of at least 90 mm Hg. Smartphone ownership was a general inclusion criterion. Key exclusion criteria were cognitive impairment, severe chronic illness, inability to communicate in English, and nonmembership in the participating churches. We conducted targeted screenings with African churches, to improve enrollment of women in the study.
Two churches with predominantly African immigrant congregations in the Baltimore–Washington, DC, metropolitan area were recruited and served as cluster units. The churches were assigned to either the first intervention group or the delayed intervention group. Due to the interactive nature of the intervention, participants were informed of their group assignment, and study staff and data collectors were unmasked.
Lifestyle Intervention The multicomponent intervention was adapted from the National DPP curriculum and focused on intensive lifestyle modification delivered by a certified lifestyle coach of African heritage. Educational materials were culturally tailored to incorporate African cultural values, practices, beliefs, and traditional dietary choices. The adapted DPP curriculum is described briefly in the trial protocol in . First Intervention Group Participants randomized to the first intervention group immediately began the 6-month adapted DPP lifestyle intervention, which included weekly 60-minute virtual group sessions via videoconferencing. The sessions covered topics such as stress management, healthy nutritional choices, physical activity, sleep considerations, and strategies to reduce cardiometabolic risk. Participants received a copy of the curriculum to follow along during the sessions. Delayed Intervention Group Participants in the delayed intervention group received no lifestyle intervention during the first 6 months but underwent the same assessments as the first intervention group. After 6 months, they received the identical adapted DPP lifestyle intervention as the first group, including remote monitoring.
The multicomponent intervention was adapted from the National DPP curriculum and focused on intensive lifestyle modification delivered by a certified lifestyle coach of African heritage. Educational materials were culturally tailored to incorporate African cultural values, practices, beliefs, and traditional dietary choices. The adapted DPP curriculum is described briefly in the trial protocol in .
Participants randomized to the first intervention group immediately began the 6-month adapted DPP lifestyle intervention, which included weekly 60-minute virtual group sessions via videoconferencing. The sessions covered topics such as stress management, healthy nutritional choices, physical activity, sleep considerations, and strategies to reduce cardiometabolic risk. Participants received a copy of the curriculum to follow along during the sessions.
Participants in the delayed intervention group received no lifestyle intervention during the first 6 months but underwent the same assessments as the first intervention group. After 6 months, they received the identical adapted DPP lifestyle intervention as the first group, including remote monitoring.
All participants received a Bluetooth-enabled BP monitor (BP7250; Omron) and digital weight scale (BCM-500; Omron) at enrollment to enable remote monitoring of BP and body composition measures. The delayed intervention group served as an attention control with device monitoring only during the first 6 months. Participants were trained on using these devices for daily home measurements, which synchronized automatically to a smartphone app (Sphygmo) and a cloud-based platform accessible to the research team. This allowed for prompt feedback and reminders to enable participants to adhere to their prescribed medications and contact their clinicians for review of their treatment plan based on participants’ transmitted BP values. Details are included in the protocol ( ).
Consistent with the trial registration, the primary outcomes were (1) changes in clinic-measured systolic and diastolic BP from baseline to 6 months using a validated automated device (HEM-9210T [Omron] and the Omron 10 series) by trained research staff following standardized procedures and (2) changes in HbA 1c levels (measured using the A1CNow+ point of care system; PTS Diagnostics). Prespecified secondary outcomes included changes in body weight and BMI during the same period.
Participants attended in-person study visits at their respective churches for assessments at baseline, 1 month, 3 months, and 6 months. During these visits, trained staff followed standardized protocols to systematically measure BP, HbA 1c levels, body weight, and BMI. At each visit, trained staff shared and discussed the measurement results with each participant at every site.
All analyses followed the intention-to-treat principle. This hypothesis-generating pilot study was designed to provide preliminary data and estimate effect sizes to inform a larger-scale trial. Analyses were performed using Stata, version 16 (StataCorp LLC). Baseline characteristics were summarized using descriptive statistics and compared between groups using χ 2 tests for categorical variables and 2-sample t tests for continuous variables. Complete data were available for age, educational level, employment status, and baseline physical measurements (30 per group). Other measures had varying numbers of participants with available data, as detailed in . Changes in primary (systolic and diastolic BP and HbA 1c levels) and secondary (body weight and BMI) outcomes over time were examined using linear mixed-effects models with fixed effects for time (baseline and 1, 3, and 6 months), intervention group (first or delayed), and their interaction. The linear mixed-effects models handled missing data under the missing at random assumption through maximum likelihood estimation. Random intercepts were included to account for clustering within churches. We estimated mean changes from baseline adjusting for age, sex, educational level, BP medication, and current alcohol use with 95% CIs. Effect sizes were calculated using Cohen d for continuous outcomes and number needed to treat for binary outcomes (eFigures 1-3 in ). Visual representations of the model-based means over time were created using line plots with separate lines for each study group. A 2-sided P < .05 was considered statistically significant for all tests. No adjustments for multiple comparisons were made in this exploratory pilot study.
Participant Enrollment and Sociodemographic and Clinical Characteristics A total of 60 participants were enrolled and randomized, 30 each to the first intervention and the delayed intervention groups ( ). The mean (SD) participant age was 50.6 (11.9) years; 40 participants (66.7%) were female and 20 (33.3%) were male. The mean (SD) length of stay in the US was 9.4 (1.4) years in the first intervention group and 20.9 (3.5) years in the delayed intervention group. The groups were well-balanced on most baseline characteristics ( ); the first intervention group had a higher proportion of participants with income less than $50 000 compared with the delayed intervention group (8 of 19 [42.1%] vs 2 of 22 [9.1%]). The first intervention group had a higher baseline mean (SD) HbA 1c level (5.6% [0.1%] vs 5.3% [0.1%]), body weight (220.1 [6.9] vs 192.4 [24.5] kg), BMI (35.8 [6.4] vs 31.2 [4.6]), and body fat percentage (44.6% [9.4%] vs 34.7% [9.8%]) compared with the delayed intervention group. Both groups had similar mean (SD) baseline systolic BP (144.4 [24.6] vs 148.3 [14.6] mm Hg) and diastolic BP (94.6 [15.7] vs 97.5 [14.3] mm Hg). The self-reported prevalences of hypertension, prediabetes, and hypercholesterolemia were also similar between the groups. The retention rate was 45 patients (75.0%) at 3 months and 43 patients (71.7%) at 6 months ( ). Primary Outcomes Systolic and Diastolic BP For systolic BP, there was a significant interaction between the study group and time ( P = .001), indicating that the change in systolic BP over time differed between the 2 groups. The first intervention group had a mean reduction in systolic BP of 9.2 (95% CI, 2.5-15.9) mm Hg ( P = .01) at 6 months, while the delayed intervention group had a mean reduction of 11.4 (95% CI, 2.4-20.5) mm Hg ( P = .01). The difference between groups at 6 months was not statistically significant (−2.3 mm Hg; 95% CI, −13.5 to 9.0 mm Hg; P = .69) ( , A, and A). Similarly, for diastolic BP, there was a significant interaction between study group and time ( P < .001). The first intervention group had a mean reduction in diastolic BP of 6.1 (95% CI, 2.1-10.0) mm Hg ( P = .003) at 6 months, while the delayed intervention group had a mean reduction of 10.3 (95% CI, 5.4-15.2) mm Hg ( P < .001). The difference between groups at 6 months was not statistically significant (−4.2 mm Hg; 95% CI, −10.5 to 2.1 mm Hg; P = .19) ( , B, and B). HbA 1c Level For HbA 1c level, there was evidence of a differential change over time between the 2 study groups, as indicated by a significant study group and time interaction ( P = .009). At 6 months, the first intervention group had a slight mean increase in HbA 1c level of 0.1% (95% CI, −0.05% to 0.4%; P = .18), while the delayed intervention group had a similarly slight mean increase of 0.4% (95% CI, 0.2%-0.6%; P < .001). The difference between groups at 6 months was statistically significant (0.3%; 95% CI, 0.03%-0.6%; P = .03) ( , C, and C). Secondary Outcomes The mixed-effects models for body weight and BMI showed significant interactions between study group and time ( P = .001 and P = .003, respectively). For BMI, at 6 months, the first intervention group had a mean reduction of 1.1 (95% CI, 0.4-1.7; P = .003), while the delayed intervention group had a mean increase of 0.3 (95% CI, −1.5 to 2.0; P = .75) ( , D, and D). For body weight, at 6 months, the first intervention group had a mean reduction of 4.9 (95% CI, 1.0-8.7) kg ( P = .01), while the delayed intervention group had a mean reduction of 3.3 (95% CI, 0.01-6.5) kg ( P = .051) ( , E, and E). For the supplemental analyses, parallel line plots for individual participants’ systolic BP, diastolic BP, HbA 1c levels, body weight, and BMI over time are shown in eFigures 1 to 5 in .
A total of 60 participants were enrolled and randomized, 30 each to the first intervention and the delayed intervention groups ( ). The mean (SD) participant age was 50.6 (11.9) years; 40 participants (66.7%) were female and 20 (33.3%) were male. The mean (SD) length of stay in the US was 9.4 (1.4) years in the first intervention group and 20.9 (3.5) years in the delayed intervention group. The groups were well-balanced on most baseline characteristics ( ); the first intervention group had a higher proportion of participants with income less than $50 000 compared with the delayed intervention group (8 of 19 [42.1%] vs 2 of 22 [9.1%]). The first intervention group had a higher baseline mean (SD) HbA 1c level (5.6% [0.1%] vs 5.3% [0.1%]), body weight (220.1 [6.9] vs 192.4 [24.5] kg), BMI (35.8 [6.4] vs 31.2 [4.6]), and body fat percentage (44.6% [9.4%] vs 34.7% [9.8%]) compared with the delayed intervention group. Both groups had similar mean (SD) baseline systolic BP (144.4 [24.6] vs 148.3 [14.6] mm Hg) and diastolic BP (94.6 [15.7] vs 97.5 [14.3] mm Hg). The self-reported prevalences of hypertension, prediabetes, and hypercholesterolemia were also similar between the groups. The retention rate was 45 patients (75.0%) at 3 months and 43 patients (71.7%) at 6 months ( ).
Systolic and Diastolic BP For systolic BP, there was a significant interaction between the study group and time ( P = .001), indicating that the change in systolic BP over time differed between the 2 groups. The first intervention group had a mean reduction in systolic BP of 9.2 (95% CI, 2.5-15.9) mm Hg ( P = .01) at 6 months, while the delayed intervention group had a mean reduction of 11.4 (95% CI, 2.4-20.5) mm Hg ( P = .01). The difference between groups at 6 months was not statistically significant (−2.3 mm Hg; 95% CI, −13.5 to 9.0 mm Hg; P = .69) ( , A, and A). Similarly, for diastolic BP, there was a significant interaction between study group and time ( P < .001). The first intervention group had a mean reduction in diastolic BP of 6.1 (95% CI, 2.1-10.0) mm Hg ( P = .003) at 6 months, while the delayed intervention group had a mean reduction of 10.3 (95% CI, 5.4-15.2) mm Hg ( P < .001). The difference between groups at 6 months was not statistically significant (−4.2 mm Hg; 95% CI, −10.5 to 2.1 mm Hg; P = .19) ( , B, and B). HbA 1c Level For HbA 1c level, there was evidence of a differential change over time between the 2 study groups, as indicated by a significant study group and time interaction ( P = .009). At 6 months, the first intervention group had a slight mean increase in HbA 1c level of 0.1% (95% CI, −0.05% to 0.4%; P = .18), while the delayed intervention group had a similarly slight mean increase of 0.4% (95% CI, 0.2%-0.6%; P < .001). The difference between groups at 6 months was statistically significant (0.3%; 95% CI, 0.03%-0.6%; P = .03) ( , C, and C).
For systolic BP, there was a significant interaction between the study group and time ( P = .001), indicating that the change in systolic BP over time differed between the 2 groups. The first intervention group had a mean reduction in systolic BP of 9.2 (95% CI, 2.5-15.9) mm Hg ( P = .01) at 6 months, while the delayed intervention group had a mean reduction of 11.4 (95% CI, 2.4-20.5) mm Hg ( P = .01). The difference between groups at 6 months was not statistically significant (−2.3 mm Hg; 95% CI, −13.5 to 9.0 mm Hg; P = .69) ( , A, and A). Similarly, for diastolic BP, there was a significant interaction between study group and time ( P < .001). The first intervention group had a mean reduction in diastolic BP of 6.1 (95% CI, 2.1-10.0) mm Hg ( P = .003) at 6 months, while the delayed intervention group had a mean reduction of 10.3 (95% CI, 5.4-15.2) mm Hg ( P < .001). The difference between groups at 6 months was not statistically significant (−4.2 mm Hg; 95% CI, −10.5 to 2.1 mm Hg; P = .19) ( , B, and B).
1c Level For HbA 1c level, there was evidence of a differential change over time between the 2 study groups, as indicated by a significant study group and time interaction ( P = .009). At 6 months, the first intervention group had a slight mean increase in HbA 1c level of 0.1% (95% CI, −0.05% to 0.4%; P = .18), while the delayed intervention group had a similarly slight mean increase of 0.4% (95% CI, 0.2%-0.6%; P < .001). The difference between groups at 6 months was statistically significant (0.3%; 95% CI, 0.03%-0.6%; P = .03) ( , C, and C).
The mixed-effects models for body weight and BMI showed significant interactions between study group and time ( P = .001 and P = .003, respectively). For BMI, at 6 months, the first intervention group had a mean reduction of 1.1 (95% CI, 0.4-1.7; P = .003), while the delayed intervention group had a mean increase of 0.3 (95% CI, −1.5 to 2.0; P = .75) ( , D, and D). For body weight, at 6 months, the first intervention group had a mean reduction of 4.9 (95% CI, 1.0-8.7) kg ( P = .01), while the delayed intervention group had a mean reduction of 3.3 (95% CI, 0.01-6.5) kg ( P = .051) ( , E, and E). For the supplemental analyses, parallel line plots for individual participants’ systolic BP, diastolic BP, HbA 1c levels, body weight, and BMI over time are shown in eFigures 1 to 5 in .
In this pilot cluster-randomized clinical trial among African immigrant adults with cardiometabolic risk factors, we found that a 6-month culturally tailored, virtual lifestyle intervention resulted in clinically meaningful improvements in systolic and diastolic BP, body weight, and BMI compared with the delayed intervention. The Afro-DPP program is one of the few pragmatic studies to adapt and test the effect of the National DPP program to improve the CMH of African immigrants. The observed reductions in BP and weight suggest that adapting evidence-based lifestyle programs, by incorporating cultural elements and leveraging virtual platforms, may be an effective strategy for improving CMH outcomes in this high-risk population. The findings of this pilot cluster-randomized trial provide preliminary evidence of the feasibility and effectiveness of adapting and delivering culturally sensitive, virtual multicomponent interventions that include lifestyle coaching, remote BP monitoring, and weight monitoring to improve the CMH of African immigrants. To our knowledge, this is the first CMH intervention that involved cultural tailoring of the DPP curriculum for African immigrants. Our findings are consistent with previous studies demonstrating the benefits of lifestyle interventions adapted from the DPP in improving cardiometabolic profiles among racial and ethnic minoritized groups. , A recent randomized clinical trial showed that a digital DPP produced significantly greater reductions in HbA 1c levels and body weight compared with enhanced standard care among participants with prediabetes, highlighting the potential for widespread dissemination and impact of digital health interventions in preventive health care services. A community-based translation of the DPP among underserved populations reported a mean weight reduction of 4.4% and modest improvements in HbA 1c levels. Similarly, a culturally tailored DPP intervention delivered virtually to low-income, Spanish-speaking Latino individuals significantly reduced weight and HbA 1c and cholesterol levels. However, unlike earlier community-based trials conducted predominantly among Black or Hispanic populations, our study uniquely focused on African immigrants and used a virtual delivery model augmented with remote monitoring of BP and body composition measures. While the changes in BP, weight, and BMI were promising, the Afro-DPP intervention did not result in statistically significant reductions in HbA 1c levels at 6 months. This result may be due to the intervention’s relatively short duration or the fact that a substantial proportion of participants did not have prediabetes at baseline. Longer-term follow-up may be warranted to fully evaluate the impact on glycemic control. Furthermore, integrating more intensive pharmacological management alongside lifestyle modifications could potentially yield greater improvements in HbA 1c levels. , Also, future studies should include systematic tracking of individual session attendance to better assess intervention dose effects. The positive outcomes observed in this study may be attributed to several factors. First, our intervention was developed through extensive community engagement and tailored to incorporate cultural values, beliefs, and dietary preferences specific to African immigrant communities. Engaging faith-based organizations and community leaders for the initial phases enhances trust, ownership, and commitment, likely contributing to the high retention and adherence rates observed. Second, using a lifestyle coach of African descent who could provide culturally congruent counseling and relatable examples may have facilitated a better understanding and adoption of the recommended lifestyle modifications. African immigrant populations face the challenge of navigating dietary choices in foreign environments and perceive available food options as unhealthy, while their food choices before migrating may be limited and expensive in the host country. Incorporating culturally appropriate dietary recommendations focused on familiar food choices, along with a culturally congruent coaching approach, may have enabled participants to make informed decisions about healthy food options. A unique aspect of our study was the incorporation of a virtual component, which significantly enhanced accessibility and participant engagement. While virtual sessions were initially prompted by COVID-19 pandemic restrictions, they effectively mitigated barriers related to transportation and scheduling for the coaching sessions. The virtual platform was complemented by one-on-one counseling sessions during the data collection, facilitating ongoing follow-up and clarification of concepts. At the core of the Afro-DPP program was the deployment of digital platforms and apps enabling remote monitoring of BP and weight, providing convenience and personalized feedback throughout the intervention. Despite initial connectivity challenges, periodic troubleshooting enabled participants to consistently monitor their health measures, likely contributing to the observed improvements in BP and weight. The virtual group sessions, complemented by individual remote monitoring, enabled continuous engagement, personalized feedback, and real-time adjustments, overcoming common barriers related to digital literacy, access, and transportation. Although earlier studies have adopted the National DPP program, particularly in faith-based settings, only a few have incorporated digital platforms despite their known convenience, accessibility, and positive impact on health outcomes. , , Our study provides valuable preliminary evidence supporting the feasibility and potential efficacy of a culturally tailored, virtual lifestyle intervention in improving key CMH indicators among African immigrants. These findings pave the way for larger, adequately powered definitive trials with extended follow-up to comprehensively evaluate such interventions’ long-term clinical impacts, cost-effectiveness, and scalability in mitigating cardiometabolic disparities in this rapidly growing minoritized population. Exploring strategies to enhance engagement and retention, such as incorporating mobile health technologies, peer support networks, and incentive-based approaches, is important. Strengths and Limitations This study has a number of strengths. The retention rate observed in our study was higher than that reported in earlier community-based studies, , suggesting that our intervention maintained participant engagement despite the limited in-person interaction. In addition, there was a high proportion of female participants in the study, highlighting the success of our efforts to improve participation and enrollment of women into trials. Our study also has several limitations that should be considered when interpreting the results. First, the relatively short follow-up period of 6 months limits our ability to determine whether the observed reductions in weight, HbA 1c levels, and BP would be sustained long term. Future studies with longer follow-up periods are needed to assess the long-term effectiveness of the intervention. Second, since the study was a pilot, the small sample size may have limited our power to detect significant differences in some outcomes. However, the sample size was appropriate for a pilot study designed to test a culturally tailored intervention’s feasibility and preliminary effectiveness among African immigrants. We acknowledge that the COVID-19 pandemic restrictions posed additional challenges for in-person meetings and home visit follow-ups, as reported in other DPP studies. In-person visits provide opportunities to enhance intervention retention, allow peer-to-peer interaction, and facilitate discussion-oriented activities that may have a greater impact on behavior change. We acknowledge that remote monitoring in the delayed intervention group may have influenced health behaviors even before their formal intervention began. This form of attention control allows us to better isolate the effects of the lifestyle intervention components while recognizing that monitoring itself may have therapeutic effects. Last, we note that the imbalance in key demographic characteristics, such as the length of stay in the US, may also impact the generalizability of our findings.
This study has a number of strengths. The retention rate observed in our study was higher than that reported in earlier community-based studies, , suggesting that our intervention maintained participant engagement despite the limited in-person interaction. In addition, there was a high proportion of female participants in the study, highlighting the success of our efforts to improve participation and enrollment of women into trials. Our study also has several limitations that should be considered when interpreting the results. First, the relatively short follow-up period of 6 months limits our ability to determine whether the observed reductions in weight, HbA 1c levels, and BP would be sustained long term. Future studies with longer follow-up periods are needed to assess the long-term effectiveness of the intervention. Second, since the study was a pilot, the small sample size may have limited our power to detect significant differences in some outcomes. However, the sample size was appropriate for a pilot study designed to test a culturally tailored intervention’s feasibility and preliminary effectiveness among African immigrants. We acknowledge that the COVID-19 pandemic restrictions posed additional challenges for in-person meetings and home visit follow-ups, as reported in other DPP studies. In-person visits provide opportunities to enhance intervention retention, allow peer-to-peer interaction, and facilitate discussion-oriented activities that may have a greater impact on behavior change. We acknowledge that remote monitoring in the delayed intervention group may have influenced health behaviors even before their formal intervention began. This form of attention control allows us to better isolate the effects of the lifestyle intervention components while recognizing that monitoring itself may have therapeutic effects. Last, we note that the imbalance in key demographic characteristics, such as the length of stay in the US, may also impact the generalizability of our findings.
The preliminary findings from the Afro-DPP for African immigrants at risk for cardiovascular disease demonstrate that a culturally tailored lifestyle intervention may effectively improve the CMH of this population. Integrating digital health solutions, remote monitoring, and culturally sensitive lifestyle coaching showed promise in promoting self-management and adherence to the intervention. These findings suggest potential avenues for further research and broader implementation of culturally sensitive interventions to mitigate cardiovascular risk factors among African immigrants.
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Single- versus multi-visit approach for fragment reattachment in complicated crown-root fractures: a cohort study | 49ecc80f-181c-4dd5-8752-a321650dc0bd | 11438244 | Dentistry[mh] | Dental injuries usually occur in children and adolescents aged 6–13 years, and the damages can range from enamel defects to tooth avulsion . Crown-root fracture (CRF) is a type of dental traumatic injury and is defined as a tooth fracture that involves the enamel, dentin, and cementum. They account for approximately 6.9% of all permanent tooth injuries . CRF could be classified as complicated or uncomplicated, depending on whether pulp exposure was involved . The majority of CRF affects the permanent maxillary anterior teeth before complete root formation, leading to deformities in both aesthetics and function. Usually, CRF has a fracture line that originates from the labial gingival margin and extends obliquely downward to the palatal alveolar crest. This fracture is caused by a horizontal impact resulting from the differences in shearing stress between the labial and palatal compression zones . The treatment of complicated CRF is always challenging due to difficulties in achieving a hermetic seal and periodontal health . The current International Association of Dental Trauma (IADT) 2020 guidelines for complicated CRF management include root canal treatment and restoration, orthodontic extrusion of the apical segment, surgical extrusion, root submergence, replantation, and extraction , in which the gingivectomy and osteotomy combined with the post-retained crown restoration were deleted from the IADT 2012 guidelines . It is noteworthy that most of the treatment options recommended by the IADT guidelines suggest the removal of the coronal fragment. However, these procedures may result in long-term tooth and phonetic defects, particularly for adolescent and pediatric patients. Additionally, these treatments may also require multiple visits and invasive surgical interventions, which can be expensive and time-consuming. However, with the advancements in acid-etch and dentinal adhesive techniques, fragment reattachment is well-known as a promising treatment option for patients with CRF, which may help avoid the need for complex restorative rehabilitation . Fragment reattachment was first described in the early 1960s , and it offers several advantages compared to traditional techniques, including immediate restoration of the tooth in terms of aesthetics and morphology, cost-effectiveness, and preservation of original occlusal contacts . Moreover, it could also serve as a temporary restoration for an adolescent patient until a permanent restoration becomes available. Recently, several studies have reported satisfactory results of fragment reattachment for patients with complicated CRF , the age of the included patients ranged from 10 to 54 years old, with more than half of them were adolescents. Additionally, a few systematic reviews have indicated that fragment reattachment in CRF teeth is associated with mild gingival inflammation, but it also provides satisfactory retention of function and esthetics. Therefore, it can be considered a viable interim treatment option, particularly for adolescent patients . In most of the published studies, root canal treatment was performed, and in approximately 85% of the cases, a post was inserted for restoration . However, this may not be applicable for immature permanent teeth with complicated CRF. Few studies have suggested that vital pulp treatment, including direct pulp capping and partial pulpotomy, should be preferred over root canal treatment combined with fragment reattachment in these cases . Besides, around 80.49% of the published studies reported that the fracture violated the biological width. In comparison, the remaining 19.51% of studies showed no involvement of the biologic width in their cases. Nevertheless, various concerns arise with fragment reattachment in complicated CRF, such as the formation of periodontal pockets due to violation of biological width, bonding failure and inadequate repositioning of the tooth crown . One long-term study pointed that fragment attachment in CRF treatment showed a high survival rate and occasionally compromised periodontal health. However, due to the high complication rate, it should be considered as a long-term temporary treatment to postpone other invasive therapy options . Thus, despite the current available evidence, there is still no consensus on the best tooth fragment reattachment procedures for patients with complicated CRF. For the best prognosis in treating patients with complicated CRF, a multidisciplinary approach involving pediatric, endodontic, and surgical dentistry should be implemented. In 63.89% of the published studies, patients with complicated CRF underwent single visit RCT, post-restoration. They accommodated fragment attachment, while 11.11% of the cases only underwent single-visit RCT and fragment attachment. In the remaining 25% of the studies, multi-visit RCTs were conducted, and post-restoration with fragment attachment was subsequently performed after RCT. Nevertheless, it is still not clear whether fragment attachment is a promising treatment strategy for CRF patients. And regarding our experience, we followed previous cases and conducted single-visit fragment reattachment combined with root canal therapy for patients suffering from complicated CRF. However, the treatment duration was long, and patients with insufficient compliance could not complete the treatment effectively. Therefore, in this study, we firstly reported two prospective complicated CRF cohorts with single- and multiple-visit multidisciplinary approach and compared the efficacy and reverse events. We compared the efficacy and adverse events of these approaches to determine if the multiple-visit approach has similar efficacy to the single-visit approach, but with fewer side effects. We aim to provide more treatment options for patients suffering from complicated CRF.
Study design and patients This prospective cohort study was approved by the Research Ethics Committee of West China Hospital of Stomatology, Sichuan University (Reference number WCHSIRB-D-2022–526). Informed consent forms were delivered and signed by each patient or their parents and legal guardians of any participant under the age of 16 that included in the study. The patients who suffered from permanent anterior tooth CRF and were initially treated at the Dental Emergency Department of West China Hospital of Stomatology, Sichuan University, from December 1, 2022, to March 30, 2023, were recruited for this study. To determine the sample size required for sufficient statistical validity, the formula, n = (Z α/2 + Z β ) 2 σ 2 /δ 2 , was used with IBM SPSS Version 21.0 (IBM Corp., Armonk, N.Y). The related parameters were established based on the references and data from a preliminary study: α value equal to 0.05, β value equal to 10%, σ value equal to 1.02, and δ equal to 1.1. The calculation indicated that nine patients in each group were required for a robust analysis. This observational study has been written according to Preferred Reporting items for Observational studies in Endodontics (PROBE) guidelines . Criteria for including patients All available treatment options, including fragment reattachment combined with root canal treatment, orthodontic extrusion of the apical segment, surgical extrusion, root submergence, replantation, and extraction, were provided. Informed consent forms were delivered and signed by each patient or their parents and legal guardians of any participant under the age of 16, and treatment was chosen based on their individual preferences. The patients who met the specified criteria and decided on fragment reattachment combined with root canal treatment were included in this study: Diagnosed with a complicated CRF via cone-beam computed tomography according to IADT classification ; There is no combination of tooth luxation and intrusion; The affected tooth was evaluated by radiographs using Nolla's classification, which classifies teeth into class 10; Patients were treated with single- or multiple-visit fragment reattachment and root canal treatment; The affected tooth has no previous history of trauma; Ages 10–18 years old. Patients suffering from complicated CRF with multiple fragments (e.g. dentin infractions) complicated CRF, or those suitable for pulpotomy were excluded. Treatment procedures Detailed single- or multiple-visit treatment was performed by two independent senior dentists at the dental emergency department, following standard local anesthesia. Single-visit fragment reattachment and root canal treatment: Fragment removal (stored in normal saline) and exposure of the fracture site with the preparation of a surgical flap and modified osteotomy (if necessary) (Fig. a, b); Pulp extirpation and complete root canal treatment were performed (Fig. c); Post-space preparation and post-restoration with a fiber post (Fig. c); Groove preparation was performed on the fractured fragment until it fit both on the post and the fractured tooth. Additionally, any remnants of pulp tissue from the fractured fragment were removed during this process (Fig. c); U200 self-adhesive resin cement (3 M Espe, St. Paul, MN, USA) was applied to the root canal, fiber post, and coronal fragment after acid etching with 37% phosphoric acid. The crown fragment was adapted and reattached to the fractured tooth and post carefully, and it was light-cured for 60 s after removing the excess composite (Fig. d); A sling suture for the palatal gingival papilla was performed after margin polishing using a composite polishing kit (Shofu Co., Kyoto, Japan) (Fig. d); Occlusion and occlusal interferences were checked and adjusted in the final stage. Multiple-visit fragment reattachment and root canal treatment: The removal of the fragment (stored in normal saline) and exposure of the fracture site with the preparation of a surgical flap and modified osteotomy (if necessary) (Fig. e, f); Root pulp extirpation and crown pulp chamber opening were performed, and remnants of pulp tissue from the fractured fragment were also removed during this process (Fig. g ) ; U200 self-adhesive resin cement (3 M Espe, St. Paul, MN, USA) was applied to both the root and crown fragments after acid etching with 37% phosphoric acid. The crown fragment was repositioned onto the fractured tooth using a gutta percha point inserted through the pulp-exposing hole (Fig. g); Gutta-percha point was removed after being light-cured for 60 s, and the root canal was filled with a calcium hydroxide paste and sealed with glass ionomer cement (Fig. g); A sling suture for the palatal gingival papilla was performed after margin polishing using a composite polishing kit (Shofu Co., Kyoto, Japan) (Fig. g); Occlusion and occlusal interferences were checked and adjusted in the final stage; Root canal preparation and filling were performed one week later after fragment reattachment (Fig. h); Post-space preparation and post-cementing with a fiber post were conducted 3 days after root canal therapy (Fig. h). Outcome measurements Intraoperative and post-treatment outcomes, including operating duration, patient cooperation during the procedure, wound infection, probing pocket depth (PPD), bleeding on probing (BoP), fragment detachment, radiographic findings, temporomandibular joint (TMJ) disorders, and patient satisfaction, were collected during the procedures and at a 1-year follow-up after treatment. The researcher who collected the post-treatment outcomes, including PPD, BoP, surgical complications, and patient satisfaction, were blinded from the interventions. Operating duration and operational cooperation Total operating duration and average operating duration per visit were measured. The patient's level of cooperation during the operation was assessed by measuring intraoperative pain and fatigue using a Visual Analog Scale (VAS, 0: no pain, 10: extreme pain) and a Numerical Rating Scale for fatigue (NRS-F, 0: energetic with no fatigue, 10: exhausted and unable to continue) . 2) PPD and BoP PPD was measured and recorded for the fractured tooth at six sites using a periodontal probe at 1-, 2-, 12-week and 1-year intervals after fragment reattachment. BoP was observed and recorded immediately after the PPD evaluation. 3) Fragment detachment and radiographical examination Tooth mobility and fragment detachment were measured at each visit during the follow-up. Cone-beam computed tomography was conducted before treatment for diagnosis, and the periapical film was conducted immediately, 3 months, and 1 year after root canal treatment. 4) TMJ disorders TMJ disorders, including pain in the facial muscles and jaw joints, as well as popping, clicking, or grating sounds when opening or closing the mouth, were recorded if they occurred during follow-up. 5) Patient Satisfaction Patient satisfaction regarding the aesthetics, function, and local pain of a fractured tooth was evaluated using a VAS at 1 year after the completion of treatment. 0: Not satisfactory at all, 5: Very satisfactory. Statistical analysis The collected data were converted, processed, and analyzed using SPSS software (version 17.0) and GraphPad Prism (version 7.0). The data analysis utilized the Students' t-test and Chi-square test, with a significance level of P < 0.05 indicating statistical significance.
This prospective cohort study was approved by the Research Ethics Committee of West China Hospital of Stomatology, Sichuan University (Reference number WCHSIRB-D-2022–526). Informed consent forms were delivered and signed by each patient or their parents and legal guardians of any participant under the age of 16 that included in the study. The patients who suffered from permanent anterior tooth CRF and were initially treated at the Dental Emergency Department of West China Hospital of Stomatology, Sichuan University, from December 1, 2022, to March 30, 2023, were recruited for this study. To determine the sample size required for sufficient statistical validity, the formula, n = (Z α/2 + Z β ) 2 σ 2 /δ 2 , was used with IBM SPSS Version 21.0 (IBM Corp., Armonk, N.Y). The related parameters were established based on the references and data from a preliminary study: α value equal to 0.05, β value equal to 10%, σ value equal to 1.02, and δ equal to 1.1. The calculation indicated that nine patients in each group were required for a robust analysis. This observational study has been written according to Preferred Reporting items for Observational studies in Endodontics (PROBE) guidelines .
All available treatment options, including fragment reattachment combined with root canal treatment, orthodontic extrusion of the apical segment, surgical extrusion, root submergence, replantation, and extraction, were provided. Informed consent forms were delivered and signed by each patient or their parents and legal guardians of any participant under the age of 16, and treatment was chosen based on their individual preferences. The patients who met the specified criteria and decided on fragment reattachment combined with root canal treatment were included in this study: Diagnosed with a complicated CRF via cone-beam computed tomography according to IADT classification ; There is no combination of tooth luxation and intrusion; The affected tooth was evaluated by radiographs using Nolla's classification, which classifies teeth into class 10; Patients were treated with single- or multiple-visit fragment reattachment and root canal treatment; The affected tooth has no previous history of trauma; Ages 10–18 years old. Patients suffering from complicated CRF with multiple fragments (e.g. dentin infractions) complicated CRF, or those suitable for pulpotomy were excluded.
Detailed single- or multiple-visit treatment was performed by two independent senior dentists at the dental emergency department, following standard local anesthesia. Single-visit fragment reattachment and root canal treatment: Fragment removal (stored in normal saline) and exposure of the fracture site with the preparation of a surgical flap and modified osteotomy (if necessary) (Fig. a, b); Pulp extirpation and complete root canal treatment were performed (Fig. c); Post-space preparation and post-restoration with a fiber post (Fig. c); Groove preparation was performed on the fractured fragment until it fit both on the post and the fractured tooth. Additionally, any remnants of pulp tissue from the fractured fragment were removed during this process (Fig. c); U200 self-adhesive resin cement (3 M Espe, St. Paul, MN, USA) was applied to the root canal, fiber post, and coronal fragment after acid etching with 37% phosphoric acid. The crown fragment was adapted and reattached to the fractured tooth and post carefully, and it was light-cured for 60 s after removing the excess composite (Fig. d); A sling suture for the palatal gingival papilla was performed after margin polishing using a composite polishing kit (Shofu Co., Kyoto, Japan) (Fig. d); Occlusion and occlusal interferences were checked and adjusted in the final stage. Multiple-visit fragment reattachment and root canal treatment: The removal of the fragment (stored in normal saline) and exposure of the fracture site with the preparation of a surgical flap and modified osteotomy (if necessary) (Fig. e, f); Root pulp extirpation and crown pulp chamber opening were performed, and remnants of pulp tissue from the fractured fragment were also removed during this process (Fig. g ) ; U200 self-adhesive resin cement (3 M Espe, St. Paul, MN, USA) was applied to both the root and crown fragments after acid etching with 37% phosphoric acid. The crown fragment was repositioned onto the fractured tooth using a gutta percha point inserted through the pulp-exposing hole (Fig. g); Gutta-percha point was removed after being light-cured for 60 s, and the root canal was filled with a calcium hydroxide paste and sealed with glass ionomer cement (Fig. g); A sling suture for the palatal gingival papilla was performed after margin polishing using a composite polishing kit (Shofu Co., Kyoto, Japan) (Fig. g); Occlusion and occlusal interferences were checked and adjusted in the final stage; Root canal preparation and filling were performed one week later after fragment reattachment (Fig. h); Post-space preparation and post-cementing with a fiber post were conducted 3 days after root canal therapy (Fig. h).
Intraoperative and post-treatment outcomes, including operating duration, patient cooperation during the procedure, wound infection, probing pocket depth (PPD), bleeding on probing (BoP), fragment detachment, radiographic findings, temporomandibular joint (TMJ) disorders, and patient satisfaction, were collected during the procedures and at a 1-year follow-up after treatment. The researcher who collected the post-treatment outcomes, including PPD, BoP, surgical complications, and patient satisfaction, were blinded from the interventions. Operating duration and operational cooperation Total operating duration and average operating duration per visit were measured. The patient's level of cooperation during the operation was assessed by measuring intraoperative pain and fatigue using a Visual Analog Scale (VAS, 0: no pain, 10: extreme pain) and a Numerical Rating Scale for fatigue (NRS-F, 0: energetic with no fatigue, 10: exhausted and unable to continue) . 2) PPD and BoP PPD was measured and recorded for the fractured tooth at six sites using a periodontal probe at 1-, 2-, 12-week and 1-year intervals after fragment reattachment. BoP was observed and recorded immediately after the PPD evaluation. 3) Fragment detachment and radiographical examination Tooth mobility and fragment detachment were measured at each visit during the follow-up. Cone-beam computed tomography was conducted before treatment for diagnosis, and the periapical film was conducted immediately, 3 months, and 1 year after root canal treatment. 4) TMJ disorders TMJ disorders, including pain in the facial muscles and jaw joints, as well as popping, clicking, or grating sounds when opening or closing the mouth, were recorded if they occurred during follow-up. 5) Patient Satisfaction Patient satisfaction regarding the aesthetics, function, and local pain of a fractured tooth was evaluated using a VAS at 1 year after the completion of treatment. 0: Not satisfactory at all, 5: Very satisfactory.
The collected data were converted, processed, and analyzed using SPSS software (version 17.0) and GraphPad Prism (version 7.0). The data analysis utilized the Students' t-test and Chi-square test, with a significance level of P < 0.05 indicating statistical significance.
Included patients’ characters A total of 20 patients and 22 teeth were included in this study. All of them finished the data collection and intro-operative or post-operative investigations during the 1-year follow-up. In the single-visit group, 10 patients with 11 complicated CRF teeth were included. All of the patients included in the study had complicated and were treated with single-visit fragment reattachment and root canal treatment. In the multiple-visit group, 10 patients with 11 teeth affected by complicated CRF were included. All of the patients included in the study had complicated CRF and underwent treatment with multiple-visit fragment reattachment and root canal treatment. No significant difference was found in terms of age, gender, etiology, and injured teeth between the single-visit and multi-visit fragment reattachment methods (Table ). All the patients included in the study had complicated CRF with a fracture line that originated at the supragingival margin on the labial side and ended subgingivally on the palatal side of the tooth. No significant difference was found between the groups in terms of the location of the fracture margin (Table ). Operating duration and operational cooperation In our study, we found that the total operative duration of single-visit fragment reattachment was significantly longer than the multiple-visit method (222.7 min vs. 159.9 min, p < 0.005 ) (Fig. a). Nevertheless, the results of the learning curve showed that the total operation time of the single-visit approach and the duration of the first visit of the multi-visit approach decreased with increasing surgical experience (Fig. b). As for operative cooperation, our results indicated that the patients who underwent single-visit fragment reattachment suffered significantly higher pain than those who underwent a multi-visit approach (2.00 vs. 0.90, p = 0.0094 ) (Fig. c). And not surprisingly, much stronger fatigue was observed in the patients in the single-visit group than those in the multi-visit group (3.90 vs. 1.90, p < 0.0001 ) (Fig. d). PPD and BoP In addition to intraoperative measurements, we also assessed the PPD and BoP at 1-, 2- and 12-week intervals following fragment reattachment in both the single-visit and multi-visit groups. Only one patient in the multi-visit group was not included in the PPD measurement and analysis due to fragment detachment on day 10 after surgery. The PPD in 3 points on the labial side of the injured tooth was measured at three points, as shown in Fig. a. Although no statistically significant difference was observed for PPD on the labial surface between the groups during the 3-month follow-up, the injured teeth that underwent multi-visit fragment reattachment showed a trend towards smaller labial PPD compared to those in the single-visit group at 1- and 2-weeks after surgery (Fig. b). Besides, the PPD at 3 points on the palatal side of the injured tooth was also measured, as shown in Fig. c. All the patients in either the single-visit or multi-visit group showed a decrease in PPD during the 1-year follow-up. Additionally, almost all of them achieved periodontal health with an approximate 2 mm PPD at 3 months and 1 year after fragment reattachment (Fig. d). Consistent with labial PPD, 8 patients in the single-visit group still tested positive for BOP. In comparison, only 6 patients in the multi-visit group were BOP positive at 1 week after surgery (Fig. e). Patients who underwent multi-visit fragment reattachment had slightly better periodontal health at an early stage. Fragment detachment and radiographic examination During the follow-up, one patient underwent multi-visit fragment reattachment occurred fragment detachment at day 10 after fragment reattachment (Fig. a). The coronal fragment was reattached after the restoration procedure was performed immediately. No significant tooth mobility was observed in any other patients. Radiographical examination was also performed at 3, 6 months, and 1 year after treatment. We did not find any newly formed or increased root fractures or periapical shadows in any of the included patients. TMJ disorders Two patients in the single-visit approach experienced intermittent pain in the TMJ area, which worsened when biting, on day 3 and day 7 after treatment, respectively (Fig. b). The symptoms gradually disappeared after 7–10 days with the help of local hot compresses. Patient satisfaction At 1 year after treatment, the majority of patients in both groups expressed satisfaction with the aesthetic and functional outcomes of the injured tooth. There was no significant difference observed in the satisfaction scores between the two groups (4.40 vs. 4.60, p = 0.3979 ) (Fig. c).
A total of 20 patients and 22 teeth were included in this study. All of them finished the data collection and intro-operative or post-operative investigations during the 1-year follow-up. In the single-visit group, 10 patients with 11 complicated CRF teeth were included. All of the patients included in the study had complicated and were treated with single-visit fragment reattachment and root canal treatment. In the multiple-visit group, 10 patients with 11 teeth affected by complicated CRF were included. All of the patients included in the study had complicated CRF and underwent treatment with multiple-visit fragment reattachment and root canal treatment. No significant difference was found in terms of age, gender, etiology, and injured teeth between the single-visit and multi-visit fragment reattachment methods (Table ). All the patients included in the study had complicated CRF with a fracture line that originated at the supragingival margin on the labial side and ended subgingivally on the palatal side of the tooth. No significant difference was found between the groups in terms of the location of the fracture margin (Table ).
In our study, we found that the total operative duration of single-visit fragment reattachment was significantly longer than the multiple-visit method (222.7 min vs. 159.9 min, p < 0.005 ) (Fig. a). Nevertheless, the results of the learning curve showed that the total operation time of the single-visit approach and the duration of the first visit of the multi-visit approach decreased with increasing surgical experience (Fig. b). As for operative cooperation, our results indicated that the patients who underwent single-visit fragment reattachment suffered significantly higher pain than those who underwent a multi-visit approach (2.00 vs. 0.90, p = 0.0094 ) (Fig. c). And not surprisingly, much stronger fatigue was observed in the patients in the single-visit group than those in the multi-visit group (3.90 vs. 1.90, p < 0.0001 ) (Fig. d).
In addition to intraoperative measurements, we also assessed the PPD and BoP at 1-, 2- and 12-week intervals following fragment reattachment in both the single-visit and multi-visit groups. Only one patient in the multi-visit group was not included in the PPD measurement and analysis due to fragment detachment on day 10 after surgery. The PPD in 3 points on the labial side of the injured tooth was measured at three points, as shown in Fig. a. Although no statistically significant difference was observed for PPD on the labial surface between the groups during the 3-month follow-up, the injured teeth that underwent multi-visit fragment reattachment showed a trend towards smaller labial PPD compared to those in the single-visit group at 1- and 2-weeks after surgery (Fig. b). Besides, the PPD at 3 points on the palatal side of the injured tooth was also measured, as shown in Fig. c. All the patients in either the single-visit or multi-visit group showed a decrease in PPD during the 1-year follow-up. Additionally, almost all of them achieved periodontal health with an approximate 2 mm PPD at 3 months and 1 year after fragment reattachment (Fig. d). Consistent with labial PPD, 8 patients in the single-visit group still tested positive for BOP. In comparison, only 6 patients in the multi-visit group were BOP positive at 1 week after surgery (Fig. e). Patients who underwent multi-visit fragment reattachment had slightly better periodontal health at an early stage.
During the follow-up, one patient underwent multi-visit fragment reattachment occurred fragment detachment at day 10 after fragment reattachment (Fig. a). The coronal fragment was reattached after the restoration procedure was performed immediately. No significant tooth mobility was observed in any other patients. Radiographical examination was also performed at 3, 6 months, and 1 year after treatment. We did not find any newly formed or increased root fractures or periapical shadows in any of the included patients.
Two patients in the single-visit approach experienced intermittent pain in the TMJ area, which worsened when biting, on day 3 and day 7 after treatment, respectively (Fig. b). The symptoms gradually disappeared after 7–10 days with the help of local hot compresses.
At 1 year after treatment, the majority of patients in both groups expressed satisfaction with the aesthetic and functional outcomes of the injured tooth. There was no significant difference observed in the satisfaction scores between the two groups (4.40 vs. 4.60, p = 0.3979 ) (Fig. c).
In our prospective cohort study, we first utilized two different methods to treat patients with complicated CFR to determine if the multiple-visit approach has similar efficacy to the single-visit approach, but with fewer side effects. Therefore providing an optimal methods with reduced operative duration and lower requirement of patient compliance. Our cohort study revealed that the total operative duration of the single-visit approach was significantly longer than the multiple-visit method, which may be because there is no requirement for the preparation of a retention box, bevel, or V-shaped groove to fit the post-restoration. It is much harder to perform complete root canal therapy and achieve sufficient sealing before coronal fragment reattachment. Not surprisingly, much stronger fatigue was observed in the patients underwent single-visit treatment, resulting from the longer operative duration and the relatively more complicated surgical and endodontic processes in the single-visit approach. Nevertheless, we found that both single-visit and multiple-visit fragment reattachment could achieve satisfactory aesthetic results 1 year after the fragment reattachment. Although the pocket probing depth was not satisfactory, and the BoP was mainly positive during the first two weeks after fragment reattachment, all cases in both groups achieved periodontal health 3 months and 1 year after surgery. However, patients who underwent multi-visit fragment reattachment had slightly better periodontal health at an early stage. This could be possibly due to a shorter duration of mucoperiosteal flap flipping and a lower requirement for hemostatic management of periodontal tissues during root canal therapy and post-restoration. With regard to the exposure of the fracture line, a mucoperiosteal flap was performed in 45.45% of published cases. In 21.21% of studies, a mucoperiosteal flap with osteoplasty or osteotomy was conducted to obtain sufficient biologic width. Although around 33.33% of the published studies showed that the flap preparation was not needed. In agreement with most previous studies , we only performed a mucoperiosteal flap and minimal osteotomy instead of crown lengthening to expose the fracture line in the included cases when necessary. This approach may result in a violation of the biological width. However, similar to the present case reports that have demonstrated successful therapy for complicated CRF in periodontally healthy teeth through adhesive fragment reattachment, we did not observe any cases of chronic periodontitis or gingivitis in any of the patients. This is not in agreement with widely accepted clinical concepts for the placement of restoration margins. Still, it was noteworthy that the violated biologic width-induced periodontal lesion is multifactorial and also mediated by plaque formation, host response, and the margin of restoration. As long as the patient maintains reasonable plaque control, the injured tooth could develop a newly established biologic width or adapt itself to a reattached crown fragment . Moreover, a few reported cases have applied crown lengthening during the treatment of complicated CRF patients. However, they also observed mild gingivitis and gingival atrophy in the short-term follow-up. Nevertheless, the gingival atrophy resulting from surgical crown lengthening or biologic width violation, which leads to alveolar crest absorption, may have a limited effect on the esthetic or functional outcomes of maxillary incisors. Furthermore, in recent years, the biocompatibility and therapeutic benefits of various cements have been thoroughly assessed in subgingival restorations. Clinical evidence so far indicates that properly cured and finished composite resins can be used in subgingival locations, the combination of acid etchant, bonding adhesive, and resin composite was the most frequently applied method (59.38%) for fragment cement in recently published studies. Additionally, sub-gingivally placed margins of composite resin restorations can significantly increase fracture resistance and show limited plaque-induced gingival inflammation . Therefore, dual-cure resin cement and resin composite could be used as a promising and valid intermediate material for fractured fragment reattachment. Nevertheless, in our study, we reported one fragment de-attachment case that underwent multi-visit fragment reattachment and root canal treatment. The fragment was fractured again due to biting ice lolly 17 days after fragment reattachment and 3 days prior to post-restoration. Although the multi-visit fragment reattachment method displayed significantly decreased total and per-visit operative duration, the patients suffered dramatically less pain and fatigue compared to the single-visit approach. Therefore, the authors suggested that an early-stage post-restoration may help stabilize the reattached fragment in CRF treatment, and the multi-visit fragment reattachment may increase the risk of fragment de-attachment prior to the post-insertion. As suggested by previous studies, 16.67% of reported cases used a splint for fragment fixation for 2–3 weeks. Therefore, a flexible splint could be applied to stabilize the tooth fragment. Still, two patients who underwent the single-visit method experienced transient pain in the temporomandibular joint area 5–7 days after surgery, and no similar symptom was observed in those who underwent the multi-visit fragment reattachment method. However, there were still two limitations in this study. On the one hand, 1-year follow-up is short for these two approaches, further investigation with longer follow-up was necessary to assess the long-term periodontal health as well as the functional outcomes. On the other hand, the fatigue scale used in this study was not specific for evaluating patient cooperation during dental treatment, which may bring potential measuring bias. Lastly, we excluded CRF patients presenting with multiple fragments and the patients that were suitable for pulpotomy from our study. We agree that pulpotomy would be the first choice whenever possible, especially in young patients with either open apices or completely formed teeth, but we still need to consider the method for the tooth restoration we might use in the future. According to the IADT guideline, the removal of the coronal fragment with subsequent endodontic treatment and restoration with a post-retained crown could be considered conducted in patients with mature apical development. This exclusion may limit the applicability of our findings to this specific patient population.
In conclusion, despite its limitations, fragment reattachment can be considered a reliable but temporary technique for treating complicated CRF in adolescent patients\ based on the data retrieved from this prospective cohort study. Our results indicated that single-visit fragment reattachment and root canal treatment could provide certain fracture resistance. Still, they had longer operative duration and a higher risk for transient TMJD compared to the multi-visit method. A proper way could be chosen depending on the specific patient situation and patient compliance.
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Population Pharmacogenomics for Health Equity | c297589c-df34-4526-9cd7-e85f51057f72 | 10606908 | Pharmacology[mh] | Population Pharmacogenomics Can and Should Be Leveraged for Health Equity—Improved Health Outcomes for All People Everywhere The United States (US) National Institute on Minority Health and Health Disparities (NIMHD) defines health equity as the assurance that “all individuals or populations have optimal opportunities to attain the best health possible” . The converse of health equity is health disparities, which are defined by NIMHD as differences in health outcomes that affect disadvantaged populations, and the elimination of health disparities is a key component of health equity . It should be stressed, however, that the health equity paradigm goes beyond just eliminating health disparities to include more aspirational goals of achieving optimal health for all populations. NIMHD states that the application of a health equity lens to biomedical research requires intentional efforts to “promote fairness, opportunity, quality, and social justice in treatments and outcomes” . This perspective article is focused on how population pharmacogenomics research can be deployed as one such intentional effort in support of health equity. We define population pharmacogenomics as the study of pharmacogenetic variation within and between populations, where pharmacogenetic variation refers to genetic differences associated with the response to medications, and populations are delineated as groups of people that share common characteristics, such as race, ethnicity, or ancestry. We explore how population pharmacogenomics research can and should be leveraged to facilitate health equity through a focus on medical treatments that consider the genetic variation of socially disadvantaged racial and ethnic groups that experience health disparities. We highlight examples from our own research on the relationship between race, ethnicity, ancestry, and pharmacogenomic variation in cosmopolitan countries of the global north, the US and the United Kingdom (UK), and in the South American country of Colombia, to illuminate the potential of pharmacogenomics to promote health equity. The United States (US) National Institute on Minority Health and Health Disparities (NIMHD) defines health equity as the assurance that “all individuals or populations have optimal opportunities to attain the best health possible” . The converse of health equity is health disparities, which are defined by NIMHD as differences in health outcomes that affect disadvantaged populations, and the elimination of health disparities is a key component of health equity . It should be stressed, however, that the health equity paradigm goes beyond just eliminating health disparities to include more aspirational goals of achieving optimal health for all populations. NIMHD states that the application of a health equity lens to biomedical research requires intentional efforts to “promote fairness, opportunity, quality, and social justice in treatments and outcomes” . This perspective article is focused on how population pharmacogenomics research can be deployed as one such intentional effort in support of health equity. We define population pharmacogenomics as the study of pharmacogenetic variation within and between populations, where pharmacogenetic variation refers to genetic differences associated with the response to medications, and populations are delineated as groups of people that share common characteristics, such as race, ethnicity, or ancestry. We explore how population pharmacogenomics research can and should be leveraged to facilitate health equity through a focus on medical treatments that consider the genetic variation of socially disadvantaged racial and ethnic groups that experience health disparities. We highlight examples from our own research on the relationship between race, ethnicity, ancestry, and pharmacogenomic variation in cosmopolitan countries of the global north, the US and the United Kingdom (UK), and in the South American country of Colombia, to illuminate the potential of pharmacogenomics to promote health equity. Socially Defined Racial and Ethnic Groups Exhibit Differences in the Frequencies of Pharmacogenomic Variants, with Direct Implications for Health Equity The US National Academies of Science Engineering and Medicine (NASEM) recently released a comprehensive and detailed report on the use of race, ethnicity, and ancestry as population descriptors in genomics research . The overarching recommendation of this report was that researchers should carefully consider and justify how and why they use race and ethnicity as population labels in their work, a recommendation with which we fully agree. The report emphasizes that race and ethnicity are social concepts, which are nevertheless often used as surrogates for describing human genetic differences. The use of race and ethnicity in this way can be problematic in the sense that it may reinforce the erroneous notion of discrete human groups with innate differences, while failing to capture complex patterns of global genetic variation. Our use of race and ethnicity population descriptors in this perspective, and the broader relevance of these group labels to pharmacogenomics, is rooted in both empirical and pragmatic considerations. With respect to the empirical justification for the use of race and ethnicity descriptors in pharmacogenomic research, we recognize that racial and ethnic groups are socially constructed. By this, we mean that the definitions of these groups, the boundaries around the groups which in turn define their composition, are determined by human actors. In the case of the US, the UK, and Colombia, government bureaucrats decide on and enumerate group labels and their definitions . Racial and ethnic groups differ across space and time, depending on the demographic composition of the societies in which they are used. The US uses a system that combines racial groups, defined by shared ancestral origins, and ethnic groups defined by shared culture. The UK defines ethnic groups based on individuals’ national origins, and Colombia uses the term etnia (Spanish for ethnicity) for ethnic minority groups and sin pertenencia étnica (Spanish for no ethnicity) for the majority population, which shows European and American admixture. In the US, the definitions of racial and ethnic groups have changed twenty times since they were first used in the late 18th-century census, and similar changes have occurred over time in the UK and Colombia . Nevertheless, the social and genetic dimensions of race and ethnicity are not mutually exclusive, and they can both be important for health outcomes. In particular, there is abundant evidence that socially defined racial and ethnic groups can show pronounced differences in the allele frequencies of genetic variants, including groups of variants that collectively influence polygenic traits, many of which may have clinical relevance . It should be emphasized that these are differences of quantity rather than differences of kind; alleles differ in relative frequencies between groups with almost no alleles found universally present in one group and universally absent in another group . Allele frequency differences between groups are a consequence of the fact that individuals’ racial and ethnic identities are tied to their ancestral origins, which are in turn correlated with genetic differences. This fact is underscored by the NASEM report’s definition of race, ethnicity, and ancestry as “descent-associated” groups composed of members who share characteristics based on common origins . Groups with ancestors that evolved in different parts of the world were once reproductively isolated, and they may remain so owing to cultural differences, and thus accumulated genetic differences over time . The resulting differences in the allele frequencies of pharmacogenomic variants among racial and ethnic groups have direct relevance to medical treatment decisions. As a more pragmatic consideration, race and ethnicity are readily available for use by clinicians, whereas patient genetic data is substantially harder to come by. Later in this perspective, we discuss how direct genetic assays capture pharmacogenomic variation far more reliably than race and ethnicity, which are poor proxies for individual-level genetic variants, but patients from socially disadvantaged groups are less likely to have access to these kinds of data than patients from majority groups. Finally, the use of race and ethnicity in pharmacogenomic research and clinical applications can help to avoid the kind of bias that arises when drug development and treatment decisions are made based on research conducted primarily on members of the majority group. If there are meaningful differences in the frequencies of pharmacogenomic variants between race and ethnicity groups, treatment strategies tailored to the majority group have the potential to do real harm to members of minority racial and ethnic groups. We believe that consideration of race and ethnicity when making treatment decisions, as we elaborate on below, can help to avoid these kinds of biases and their downstream consequences. Here, we briefly review examples of pharmacogenomic differences among racial and ethnic groups from our own work in the US, the UK, and Colombia . Most recently, we used population biobanks to interrogate the relationship between race, ethnicity, and pharmacogenomic variation in the US and the UK . Socially defined race and ethnicity were found to be highly correlated with genome-wide patterns of pharmacogenomic variation, in support of the relevance of these categories for patient stratification. The cohorts for this study were taken from the US All of Us Research Program ( All of Us ; n = 65,120; A) and the UK Biobank ( UKB ; n = 31,396; B). Machine learning classifiers were used to predict biobank participants’ self-identified race and ethnicity (SIRE) based on patterns of variation for 6311 pharmacogenomic variants in All of Us ( C) and 5966 pharmacogenomic variants in UKB ( D). Pharmacogenomic variation was found to predict participants’ SIRE with 92.1% accuracy for All of Us and 94.3% accuracy for UKB . The highest group-specific prediction accuracy was 99.0% for the White group in UKB , and the lowest group-specific prediction accuracy was 92.9% for the Hispanic group in All of Us . We also found numerous individual pharmacogenomic variants with large allele frequency differences between racial and ethnic groups in these cohorts, consistent with previous studies. In 2020, we analyzed a cohort of 8628 participants from the US Health and Retirement Study ( HRS ) to compare the ability of SIRE versus genetic ancestry (GA) to stratify pharmacogenomic variants . We hypothesized that GA would provide greater resolution for pharmacogenomic variant stratification compared to SIRE, given that SIRE is socially defined whereas GA is a characteristic of the genome, which can be characterized objectively and with precision. Nevertheless, GA was found to predict SIRE with >96% accuracy, and thus GA provided a negligible increase in resolution for pharmacogenomic variant stratification. This study also confirmed the long-held notion that the majority of human genetic variation is found within rather than between racial and ethnic groups, a fact that is often taken to argue against the relevance of race and ethnicity to human genetic variation. Despite this pattern of pharmacogenomic variance partitioning, numerous pharmacogenomic variants were found to show significant frequency differences among SIRE groups, and genome-wide patterns of pharmacogenomic variation were highly concordant with SIRE. This study underscored the clinical relevance of SIRE for the stratification of pharmacogenomic risk among groups, while also showing far less utility of SIRE as a proxy for individual-level pharmacogenomic stratification. We explore the distinction between individual-level versus population-level pharmacogenomic risk stratification further in the following section on precision medicine versus precision public health. In 2019, data from the ChocoGen research project was used to investigate population pharmacogenomics in the Colombian populations of Antioquia and Chocó in support of health equity in Colombia . Antioquia and Chocó are neighboring departments (states) with distinct ancestry profiles: European followed by American ancestry in Antioquia compared to primarily African ancestry in Chocó. We found numerous pharmacogenomic variants with highly divergent allele frequencies between these two Colombian populations, with differences that were correlated to their distinct ancestral backgrounds. We used these findings, working with local partners in Colombia, to develop and validate allele-specific PCR assays to test patients for population-specific pharmacogenomic variants. This approach allowed our Colombian partners to focus local resources on the populations where they are more likely to yield a return on investment, thereby serving as an example of how population pharmacogenomics can be leveraged to support precision medicine in resource-limited settings. The US National Academies of Science Engineering and Medicine (NASEM) recently released a comprehensive and detailed report on the use of race, ethnicity, and ancestry as population descriptors in genomics research . The overarching recommendation of this report was that researchers should carefully consider and justify how and why they use race and ethnicity as population labels in their work, a recommendation with which we fully agree. The report emphasizes that race and ethnicity are social concepts, which are nevertheless often used as surrogates for describing human genetic differences. The use of race and ethnicity in this way can be problematic in the sense that it may reinforce the erroneous notion of discrete human groups with innate differences, while failing to capture complex patterns of global genetic variation. Our use of race and ethnicity population descriptors in this perspective, and the broader relevance of these group labels to pharmacogenomics, is rooted in both empirical and pragmatic considerations. With respect to the empirical justification for the use of race and ethnicity descriptors in pharmacogenomic research, we recognize that racial and ethnic groups are socially constructed. By this, we mean that the definitions of these groups, the boundaries around the groups which in turn define their composition, are determined by human actors. In the case of the US, the UK, and Colombia, government bureaucrats decide on and enumerate group labels and their definitions . Racial and ethnic groups differ across space and time, depending on the demographic composition of the societies in which they are used. The US uses a system that combines racial groups, defined by shared ancestral origins, and ethnic groups defined by shared culture. The UK defines ethnic groups based on individuals’ national origins, and Colombia uses the term etnia (Spanish for ethnicity) for ethnic minority groups and sin pertenencia étnica (Spanish for no ethnicity) for the majority population, which shows European and American admixture. In the US, the definitions of racial and ethnic groups have changed twenty times since they were first used in the late 18th-century census, and similar changes have occurred over time in the UK and Colombia . Nevertheless, the social and genetic dimensions of race and ethnicity are not mutually exclusive, and they can both be important for health outcomes. In particular, there is abundant evidence that socially defined racial and ethnic groups can show pronounced differences in the allele frequencies of genetic variants, including groups of variants that collectively influence polygenic traits, many of which may have clinical relevance . It should be emphasized that these are differences of quantity rather than differences of kind; alleles differ in relative frequencies between groups with almost no alleles found universally present in one group and universally absent in another group . Allele frequency differences between groups are a consequence of the fact that individuals’ racial and ethnic identities are tied to their ancestral origins, which are in turn correlated with genetic differences. This fact is underscored by the NASEM report’s definition of race, ethnicity, and ancestry as “descent-associated” groups composed of members who share characteristics based on common origins . Groups with ancestors that evolved in different parts of the world were once reproductively isolated, and they may remain so owing to cultural differences, and thus accumulated genetic differences over time . The resulting differences in the allele frequencies of pharmacogenomic variants among racial and ethnic groups have direct relevance to medical treatment decisions. As a more pragmatic consideration, race and ethnicity are readily available for use by clinicians, whereas patient genetic data is substantially harder to come by. Later in this perspective, we discuss how direct genetic assays capture pharmacogenomic variation far more reliably than race and ethnicity, which are poor proxies for individual-level genetic variants, but patients from socially disadvantaged groups are less likely to have access to these kinds of data than patients from majority groups. Finally, the use of race and ethnicity in pharmacogenomic research and clinical applications can help to avoid the kind of bias that arises when drug development and treatment decisions are made based on research conducted primarily on members of the majority group. If there are meaningful differences in the frequencies of pharmacogenomic variants between race and ethnicity groups, treatment strategies tailored to the majority group have the potential to do real harm to members of minority racial and ethnic groups. We believe that consideration of race and ethnicity when making treatment decisions, as we elaborate on below, can help to avoid these kinds of biases and their downstream consequences. Here, we briefly review examples of pharmacogenomic differences among racial and ethnic groups from our own work in the US, the UK, and Colombia . Most recently, we used population biobanks to interrogate the relationship between race, ethnicity, and pharmacogenomic variation in the US and the UK . Socially defined race and ethnicity were found to be highly correlated with genome-wide patterns of pharmacogenomic variation, in support of the relevance of these categories for patient stratification. The cohorts for this study were taken from the US All of Us Research Program ( All of Us ; n = 65,120; A) and the UK Biobank ( UKB ; n = 31,396; B). Machine learning classifiers were used to predict biobank participants’ self-identified race and ethnicity (SIRE) based on patterns of variation for 6311 pharmacogenomic variants in All of Us ( C) and 5966 pharmacogenomic variants in UKB ( D). Pharmacogenomic variation was found to predict participants’ SIRE with 92.1% accuracy for All of Us and 94.3% accuracy for UKB . The highest group-specific prediction accuracy was 99.0% for the White group in UKB , and the lowest group-specific prediction accuracy was 92.9% for the Hispanic group in All of Us . We also found numerous individual pharmacogenomic variants with large allele frequency differences between racial and ethnic groups in these cohorts, consistent with previous studies. In 2020, we analyzed a cohort of 8628 participants from the US Health and Retirement Study ( HRS ) to compare the ability of SIRE versus genetic ancestry (GA) to stratify pharmacogenomic variants . We hypothesized that GA would provide greater resolution for pharmacogenomic variant stratification compared to SIRE, given that SIRE is socially defined whereas GA is a characteristic of the genome, which can be characterized objectively and with precision. Nevertheless, GA was found to predict SIRE with >96% accuracy, and thus GA provided a negligible increase in resolution for pharmacogenomic variant stratification. This study also confirmed the long-held notion that the majority of human genetic variation is found within rather than between racial and ethnic groups, a fact that is often taken to argue against the relevance of race and ethnicity to human genetic variation. Despite this pattern of pharmacogenomic variance partitioning, numerous pharmacogenomic variants were found to show significant frequency differences among SIRE groups, and genome-wide patterns of pharmacogenomic variation were highly concordant with SIRE. This study underscored the clinical relevance of SIRE for the stratification of pharmacogenomic risk among groups, while also showing far less utility of SIRE as a proxy for individual-level pharmacogenomic stratification. We explore the distinction between individual-level versus population-level pharmacogenomic risk stratification further in the following section on precision medicine versus precision public health. In 2019, data from the ChocoGen research project was used to investigate population pharmacogenomics in the Colombian populations of Antioquia and Chocó in support of health equity in Colombia . Antioquia and Chocó are neighboring departments (states) with distinct ancestry profiles: European followed by American ancestry in Antioquia compared to primarily African ancestry in Chocó. We found numerous pharmacogenomic variants with highly divergent allele frequencies between these two Colombian populations, with differences that were correlated to their distinct ancestral backgrounds. We used these findings, working with local partners in Colombia, to develop and validate allele-specific PCR assays to test patients for population-specific pharmacogenomic variants. This approach allowed our Colombian partners to focus local resources on the populations where they are more likely to yield a return on investment, thereby serving as an example of how population pharmacogenomics can be leveraged to support precision medicine in resource-limited settings. Race and Ethnicity Stratify Pharmacogenomic Variation at the Local Level but Do Not Represent Natural Groups That Correspond to Global Patterns of Human Genetic Variation The racial and ethnic stratification of pharmacogenomic variation described in the previous section does not negate the fact that these groups are social constructs, which do not map well to global patterns of human genetic variation. At the global level, human genetic variation is largely distributed as a continuum, based on reproductive isolation by distance, with discontinuities introduced by major geographic barriers, like oceans, mountain ranges, and deserts . Furthermore, the vast majority of human genetic variation can be found within the African continent, consistent with the fact that modern humans (Homo sapiens) evolved in Africa for more than 200,000 years before migrating out of Africa and populating the rest of the world . The deepest human lineages divide the Khoisan groups from all other human populations, with a divergence time of ~200,000 years, followed by the Rainforest Hunter-Gather split at ~150,000 years ago. West Africans and Eurasians who migrated out of Africa share a substantially more recent divergence ~75,000 years ago (note that all dates are approximate and subject to refinement). This means that people of the African diaspora in the US, UK, and Colombia, all of whom are descended from West African or Southwest African groups, are more genetically related to European descendants in these countries than either would be to Khoisan or Rainforest Hunter-Gatherers. This is despite the fact that Khoisan, Rainforest Hunter-Gathers, and West African individuals would all likely be considered as Black in these countries, based on their phenotype and geographical origins, whereas European descendants would be considered as White. The seeming contradiction of racially and ethnically stratified pharmacogenomic variation versus distinct global patterns of human genetic variation can be resolved by understanding how race and ethnicity are defined at the local level. As described in the previous section, different countries define race and/or ethnic groups based on the demographic characteristics of their own populations, which are in turn shaped by country-specific patterns of colonization and immigration . Definitions of race and ethnicity are also informed by political considerations, including advocacy by groups that seek official recognition, such as the Hispanic/Latino group in the US, which was only fully recognized as distinct ethnicity starting with the 1980 census . The distinct mix of native and immigrant groups that shape the demography of each country can be considered to represent an uneven sampling of (somewhat) continuous global genetic diversity, and it is this uneven sampling that yields meaningful genetic differences between socially constructed groups. This process can be illuminated using a number line analogy introduced by Joseph Graves . Considering human genetic variation as continuously distributed along a number line from one to ten, if a country’s population is generated by sampling discontinuously, around the values of one, five, and ten for instance, then this sampling would yield genetically distinct groups from an underlying continuous distribution of genetic variation. While this analogy is an oversimplification, it does reflect the kind of historical processes that generated the populations of modern, cosmopolitan countries and thereby explains how locally defined racial and ethnic groups can differ genetically in ways that are distinct from global patterns of human genetic variation. The racial and ethnic stratification of pharmacogenomic variation described in the previous section does not negate the fact that these groups are social constructs, which do not map well to global patterns of human genetic variation. At the global level, human genetic variation is largely distributed as a continuum, based on reproductive isolation by distance, with discontinuities introduced by major geographic barriers, like oceans, mountain ranges, and deserts . Furthermore, the vast majority of human genetic variation can be found within the African continent, consistent with the fact that modern humans (Homo sapiens) evolved in Africa for more than 200,000 years before migrating out of Africa and populating the rest of the world . The deepest human lineages divide the Khoisan groups from all other human populations, with a divergence time of ~200,000 years, followed by the Rainforest Hunter-Gather split at ~150,000 years ago. West Africans and Eurasians who migrated out of Africa share a substantially more recent divergence ~75,000 years ago (note that all dates are approximate and subject to refinement). This means that people of the African diaspora in the US, UK, and Colombia, all of whom are descended from West African or Southwest African groups, are more genetically related to European descendants in these countries than either would be to Khoisan or Rainforest Hunter-Gatherers. This is despite the fact that Khoisan, Rainforest Hunter-Gathers, and West African individuals would all likely be considered as Black in these countries, based on their phenotype and geographical origins, whereas European descendants would be considered as White. The seeming contradiction of racially and ethnically stratified pharmacogenomic variation versus distinct global patterns of human genetic variation can be resolved by understanding how race and ethnicity are defined at the local level. As described in the previous section, different countries define race and/or ethnic groups based on the demographic characteristics of their own populations, which are in turn shaped by country-specific patterns of colonization and immigration . Definitions of race and ethnicity are also informed by political considerations, including advocacy by groups that seek official recognition, such as the Hispanic/Latino group in the US, which was only fully recognized as distinct ethnicity starting with the 1980 census . The distinct mix of native and immigrant groups that shape the demography of each country can be considered to represent an uneven sampling of (somewhat) continuous global genetic diversity, and it is this uneven sampling that yields meaningful genetic differences between socially constructed groups. This process can be illuminated using a number line analogy introduced by Joseph Graves . Considering human genetic variation as continuously distributed along a number line from one to ten, if a country’s population is generated by sampling discontinuously, around the values of one, five, and ten for instance, then this sampling would yield genetically distinct groups from an underlying continuous distribution of genetic variation. While this analogy is an oversimplification, it does reflect the kind of historical processes that generated the populations of modern, cosmopolitan countries and thereby explains how locally defined racial and ethnic groups can differ genetically in ways that are distinct from global patterns of human genetic variation. The Characterization of Pharmacogenomic Variants Provides a Means to Elucidate Modifiable Risk Factors in Support of Health Equity Clinical applications of pharmacogenomics are closely tied to the concept of risk stratification. A risk factor is anything that increases the chance of developing a health condition or disease. The characterization and measurement of risk factors can be used to inform disease prevention strategies and treatment programs. Risk factors can be characterized as environmental, behavioral, physiological, demographic, or genetic. Environmental risk factors include chemical exposures and social relationships, behavioral risk factors include smoking, diet, and physical activity, and physiological risk factors include weight, blood pressure, and cholesterol. Demographic risk factors relate primarily to race, ethnicity, and sex, and genetic risk factors are based on individuals’ genetic makeup, i.e., their collection of genetic variants. Effective health interventions require modifiable risk factors. Environmental, behavioral, and physiological risk factors are all (potentially) modifiable, and it makes perfect sense for public health strategies and medical interventions to focus on modifiable risk factors of this kind. A corollary, and seemingly reasonable, critique of demographic and genetic risk factors is that they are not (readily) modifiable. Demographic characteristics are considered to be socially ascribed, meaning that they are assigned at birth or involuntarily later in life. Since socially ascribed characteristics are neither chosen nor earned, they are typically not considered to be modifiable. Genetic variants are inherited at birth and through the process of development manifest in the ~37 trillion cells of the human body. Thus, genetic risk factors are also non-modifiable. It should be noted that genetic risk factors may become partially modifiable in the not-too-distant future owing to developments in CRISPR or other gene editing approaches, but for the moment these technologies are not widely available in the healthcare setting. The key point with respect to epidemiology and health is that a focus on the discovery of non-modifiable demographic or genetic risk factors may not directly inform disease prevention and treatment. Pharmacogenomics provides a direct link between non-modifiable demographic or genetic risk factors and modifiable environmental risk factors. This is because pharmacogenomic variants are defined as a special case of gene-by-environment interactions, specifically the interaction between genetic variants and medications used to treat disease. And medications are of course a kind of chemical environmental exposure. The effects of medications—with respect to dosage, efficacy, and toxicity—vary based on the presence or absence of specific pharmacogenomic variants. In this sense, the presence of pharmacogenomic variants in individual patients, or their enrichment in certain demographic groups, can suggest environmental modifications, i.e., changing or modifying drug prescriptions, that lead to improved health outcomes. The way that pharmacogenomic variants are assayed—directly in the case of pharmacogenetic tests or indirectly in the case of variant population frequencies—will determine the level of intervention. Precision medicine approaches require pharmacogenomic information for individual patients, whereas precision public health relies on pharmacogenomic information gleaned at the population level. While demographic data, for race and ethnicity in particular, are not reliable proxies for individual-level pharmacogenetic variation, they can serve as valuable guides for population-level screening and as risk factors for patient group stratification. This crucial distinction is discussed at greater length in the following section on precision medicine versus precision public health. Clinical applications of pharmacogenomics are closely tied to the concept of risk stratification. A risk factor is anything that increases the chance of developing a health condition or disease. The characterization and measurement of risk factors can be used to inform disease prevention strategies and treatment programs. Risk factors can be characterized as environmental, behavioral, physiological, demographic, or genetic. Environmental risk factors include chemical exposures and social relationships, behavioral risk factors include smoking, diet, and physical activity, and physiological risk factors include weight, blood pressure, and cholesterol. Demographic risk factors relate primarily to race, ethnicity, and sex, and genetic risk factors are based on individuals’ genetic makeup, i.e., their collection of genetic variants. Effective health interventions require modifiable risk factors. Environmental, behavioral, and physiological risk factors are all (potentially) modifiable, and it makes perfect sense for public health strategies and medical interventions to focus on modifiable risk factors of this kind. A corollary, and seemingly reasonable, critique of demographic and genetic risk factors is that they are not (readily) modifiable. Demographic characteristics are considered to be socially ascribed, meaning that they are assigned at birth or involuntarily later in life. Since socially ascribed characteristics are neither chosen nor earned, they are typically not considered to be modifiable. Genetic variants are inherited at birth and through the process of development manifest in the ~37 trillion cells of the human body. Thus, genetic risk factors are also non-modifiable. It should be noted that genetic risk factors may become partially modifiable in the not-too-distant future owing to developments in CRISPR or other gene editing approaches, but for the moment these technologies are not widely available in the healthcare setting. The key point with respect to epidemiology and health is that a focus on the discovery of non-modifiable demographic or genetic risk factors may not directly inform disease prevention and treatment. Pharmacogenomics provides a direct link between non-modifiable demographic or genetic risk factors and modifiable environmental risk factors. This is because pharmacogenomic variants are defined as a special case of gene-by-environment interactions, specifically the interaction between genetic variants and medications used to treat disease. And medications are of course a kind of chemical environmental exposure. The effects of medications—with respect to dosage, efficacy, and toxicity—vary based on the presence or absence of specific pharmacogenomic variants. In this sense, the presence of pharmacogenomic variants in individual patients, or their enrichment in certain demographic groups, can suggest environmental modifications, i.e., changing or modifying drug prescriptions, that lead to improved health outcomes. The way that pharmacogenomic variants are assayed—directly in the case of pharmacogenetic tests or indirectly in the case of variant population frequencies—will determine the level of intervention. Precision medicine approaches require pharmacogenomic information for individual patients, whereas precision public health relies on pharmacogenomic information gleaned at the population level. While demographic data, for race and ethnicity in particular, are not reliable proxies for individual-level pharmacogenetic variation, they can serve as valuable guides for population-level screening and as risk factors for patient group stratification. This crucial distinction is discussed at greater length in the following section on precision medicine versus precision public health. Population Pharmacogenomics Is an Essential Component of Precision Public Health, Where the Focus Is on Population-Level Variation as Opposed to Individual-Level Differences Precision (genomic) medicine is an emergent medical discipline that involves the use of patients’ genomic information to inform their clinical care, with respect to disease diagnosis, prognosis, and treatment . Pharmacogenomic data is foundational to the treatment arm of precision medicine, which aims to deliver “the right medicine, to the right patient, at the right time”. In other words, precision medicine is squarely focused on individual-level pharmacogenomic information, i.e., the presence or absence of specific drug-associated variants in any given patient. Precision public health, on the other hand, relies on population-level data to inform public health strategies . As it relates to pharmacogenomics, precision public health aims for “the right intervention, to the right population, at the right time.” Population pharmacogenomic profiles—data on the relative frequencies of pharmacogenomic variants within and between populations of interest—can be used to guide precision public health initiatives. For example, as we demonstrated in our Colombian study, population pharmacogenomic profiles can be used to target the provisioning of resources and testing where they are more likely to lead to improved health outcomes . The distinction between precision medicine versus precision public health is key to understanding the utility, or lack thereof, of race and ethnicity as proxies for pharmacogenomic variation. Race and ethnicity are poor proxies for individual-level genetic variation and thus should not be used in place of direct pharmacogenomic testing for precision medicine. However, race and ethnicity do effectively stratify pharmacogenomic variation at the population level and therefore can be used to inform precision public health. This distinction is exemplified by the story of the nationwide rollout of the HIV medication efavirenz in Zimbabwe . In 2015, Zimbabwe followed the World Health Organization’s recommendation for HIV public health programs and switched from a three-drug cocktail to the single combination pill efavirenz. This change led to widespread adverse effects, including hallucinations, depression, and suicidal tendencies, with thousands of patients quickly abandoning treatment. It turns out that these adverse effects were associated with a pharmacogenomic variant that is found at anomalously high frequency in the Zimbabwe population. Approximately 20% of the population has two copies of the recessive efavirenz adverse effect associated variant, a fact which had been reported by Collen Masimirembwa, a local scientist working in the capital city Harare seven years earlier . If Masimirembwa’s advice on efavirenz dosing tailored to the local population had been heeded, the country could have avoided a public health crisis. This example underscores the distinction between the individual-level prediction accuracy of population labels compared to their utility for population-level stratification. If Zimbabwe is taken as a population label and used as a surrogate to predict individual patients’ adverse reactions to efavirenz, based on population pharmacogenomic data, it would have an extremely low prediction accuracy of 20% in the country. However, if the same pharmacogenomic data were used to inform public health interventions, they could prove to be extremely useful. Precision public health can leverage population pharmacogenomic data of this kind to inform population-wide treatment programs, directing pharmacogenomic testing where it is most needed and avoiding drugs that are predicted to cause adverse effects in a large fraction of the population. Race and ethnicity labels provide sufficient pharmacogenomic risk stratification to support the precision public health approach. We explore our own results on the relationship between race, ethnicity, and adverse drug reactions in the following section. Precision (genomic) medicine is an emergent medical discipline that involves the use of patients’ genomic information to inform their clinical care, with respect to disease diagnosis, prognosis, and treatment . Pharmacogenomic data is foundational to the treatment arm of precision medicine, which aims to deliver “the right medicine, to the right patient, at the right time”. In other words, precision medicine is squarely focused on individual-level pharmacogenomic information, i.e., the presence or absence of specific drug-associated variants in any given patient. Precision public health, on the other hand, relies on population-level data to inform public health strategies . As it relates to pharmacogenomics, precision public health aims for “the right intervention, to the right population, at the right time.” Population pharmacogenomic profiles—data on the relative frequencies of pharmacogenomic variants within and between populations of interest—can be used to guide precision public health initiatives. For example, as we demonstrated in our Colombian study, population pharmacogenomic profiles can be used to target the provisioning of resources and testing where they are more likely to lead to improved health outcomes . The distinction between precision medicine versus precision public health is key to understanding the utility, or lack thereof, of race and ethnicity as proxies for pharmacogenomic variation. Race and ethnicity are poor proxies for individual-level genetic variation and thus should not be used in place of direct pharmacogenomic testing for precision medicine. However, race and ethnicity do effectively stratify pharmacogenomic variation at the population level and therefore can be used to inform precision public health. This distinction is exemplified by the story of the nationwide rollout of the HIV medication efavirenz in Zimbabwe . In 2015, Zimbabwe followed the World Health Organization’s recommendation for HIV public health programs and switched from a three-drug cocktail to the single combination pill efavirenz. This change led to widespread adverse effects, including hallucinations, depression, and suicidal tendencies, with thousands of patients quickly abandoning treatment. It turns out that these adverse effects were associated with a pharmacogenomic variant that is found at anomalously high frequency in the Zimbabwe population. Approximately 20% of the population has two copies of the recessive efavirenz adverse effect associated variant, a fact which had been reported by Collen Masimirembwa, a local scientist working in the capital city Harare seven years earlier . If Masimirembwa’s advice on efavirenz dosing tailored to the local population had been heeded, the country could have avoided a public health crisis. This example underscores the distinction between the individual-level prediction accuracy of population labels compared to their utility for population-level stratification. If Zimbabwe is taken as a population label and used as a surrogate to predict individual patients’ adverse reactions to efavirenz, based on population pharmacogenomic data, it would have an extremely low prediction accuracy of 20% in the country. However, if the same pharmacogenomic data were used to inform public health interventions, they could prove to be extremely useful. Precision public health can leverage population pharmacogenomic data of this kind to inform population-wide treatment programs, directing pharmacogenomic testing where it is most needed and avoiding drugs that are predicted to cause adverse effects in a large fraction of the population. Race and ethnicity labels provide sufficient pharmacogenomic risk stratification to support the precision public health approach. We explore our own results on the relationship between race, ethnicity, and adverse drug reactions in the following section. The Mitigation of Adverse Drug Reactions Is an Area Where Population Pharmacogenomics Can Have a Direct and Immediate Impact on Public Health Toxicity-associated pharmacogenomic variants are linked to adverse reactions to numerous widely prescribed medications. As we have shown previously, differences in the frequencies of toxicity-associated pharmacogenomic variants between racial and ethnic groups have important implications for public health. Our approach to studying the implications of race and ethnicity for adverse drug reactions relies on the excess number of predicted adverse reactions per one thousand patients . Given the population frequency of toxicity-associated variants, along with their documented effect mode as either dominant requiring a single copy of the toxicity-associated allele or recessive requiring two copies of the toxicity-associated allele, the number of predicted adverse effects can be calculated. Population stratification, using race, ethnicity, or any other population grouping approach, can then be used to calculate group differences in the number of predicted adverse reactions for medications with toxicity-associated variants. Application of this approach to US racial and ethnic groups, using the All of Us and HRS cohorts, yields striking results . The Black group shows up to 726 predicted excess adverse drug reactions per 1000 patients compared to the majority White group for the dominant effect mode and up to 635 excess adverse reactions for the recessive effect mode. The Hispanic group, which is more ancestrally similar to the majority White group, shows up to 351 predicted adverse drug reactions per 1000 patients compared to the White group for the dominant effect mode and up to 286 excess adverse reactions for the recessive effect mode. By way of example, a pharmacogenomic variant in the CYP3A4 (Cytochrome P450 Family 3 Subfamily A Member 4) gene has been associated with adverse reactions to methadone, which is used to treat heroin-dependent patients. In the All of Us cohort, the toxicity-associated allele of this variant (dbSNP rs4646437) shows high frequency in the Black group (0.73) compared to the majority White group (0.11), and it exerts a dominant effect on adverse reactions to methadone. Given these allele frequency differences between groups, the dominant effect mode predicts 719 excess adverse drug reactions per 1000 patients in the Black group compared to the majority White group. A pharmacogenomic variant in the VKORC1 (Vitamin K Epoxide Reductase Complex Subunit 1) gene has been associated with anticoagulation and excess bleeding in patients treated with the blood thinners warfarin and phenprocoumon. In the All of Us cohort, the toxicity-associated allele of this variant (dbSNP rs9923231) shows high frequency in the Asian (0.67) group compared to the majority White group (0.34), and it exerts a dominant effect on adverse reactions to warfarin and phenprocoumon. This allele frequency difference, given the dominant effect mode, predicts 327 excess adverse drug reactions per 1000 patients in the Asian group compared to the majority White group. A pharmacogenomic variant in the DPYD (Dihydropyrimidine Dehydrogenase) gene has been associated with toxic side effects to the chemotherapeutic agents fluorouracil and capecitabine. In the All of Us cohort, the toxicity-associated allele of this variant (dbSNP rs1801265) shows high frequency in the Black (0.40) group compared to the majority White group (0.22), and it has been report to exert both recessive and dominant effects on toxicity. This allele frequency difference predicts 112 excess adverse drug reactions per 1000 patients in the Black group compared to the majority White group under the recessive effect mode and 248 excess adverse reactions under the dominant mode. A pharmacogenomic variant in the ERCC1 (Excision Repair 1, Endonuclease Non-Catalytic Subunit) gene has been associated with adverse reactions to nine different drugs, including cisplatin and oxaliplatin chemotherapy drugs. In the HRS cohort, the toxicity-associated allele of this variant (dbSNP rs11615) shows high frequency in the Black (0.88) and Hispanic (0.64) groups compared to the majority White group (0.37). These large allele frequency differences, given the recessive effect mode, yield predictions of up to 638 excess adverse drug reactions per 1000 patients in the Black group and up to 273 excess adverse reactions in the Hispanic group compared to the majority White group. These findings on race, ethnicity, and adverse drug reactions also have implications for the clinical relevance of human genetic variance components. In 1972, Richard Lewontin found that the majority of human genetic variation was found within (85%) rather than between (15%) racial groups . This fundamental result has been replicated many times since, and it is often taken to support the irrelevance of racial classification to human genetic variation . This is consistent with the notion that race and ethnicity are purely social constructs with little or no biological significance . Here, it must be reiterated that observed patterns of pharmacogenomic variation, particularly as they relate to adverse drug reactions, clearly support the clinical relevance of race and ethnicity . In fact, we previously showed that when pharmacogenomic variation is partitioned exactly in the way that Lewontin and others have described, 85% within-group variation and 15% between-group variation, there can be up to 700 excess adverse drug reactions per 1000 patients predicted for the recessive effect mode and as many as 300 adverse reactions predicted for the dominant mode . In other words, a high relative excess of within- versus between-group genetic variation, as is almost always seen for human populations, does not preclude the utility of race and ethnicity for pharmacogenomic risk stratification. Toxicity-associated pharmacogenomic variants are linked to adverse reactions to numerous widely prescribed medications. As we have shown previously, differences in the frequencies of toxicity-associated pharmacogenomic variants between racial and ethnic groups have important implications for public health. Our approach to studying the implications of race and ethnicity for adverse drug reactions relies on the excess number of predicted adverse reactions per one thousand patients . Given the population frequency of toxicity-associated variants, along with their documented effect mode as either dominant requiring a single copy of the toxicity-associated allele or recessive requiring two copies of the toxicity-associated allele, the number of predicted adverse effects can be calculated. Population stratification, using race, ethnicity, or any other population grouping approach, can then be used to calculate group differences in the number of predicted adverse reactions for medications with toxicity-associated variants. Application of this approach to US racial and ethnic groups, using the All of Us and HRS cohorts, yields striking results . The Black group shows up to 726 predicted excess adverse drug reactions per 1000 patients compared to the majority White group for the dominant effect mode and up to 635 excess adverse reactions for the recessive effect mode. The Hispanic group, which is more ancestrally similar to the majority White group, shows up to 351 predicted adverse drug reactions per 1000 patients compared to the White group for the dominant effect mode and up to 286 excess adverse reactions for the recessive effect mode. By way of example, a pharmacogenomic variant in the CYP3A4 (Cytochrome P450 Family 3 Subfamily A Member 4) gene has been associated with adverse reactions to methadone, which is used to treat heroin-dependent patients. In the All of Us cohort, the toxicity-associated allele of this variant (dbSNP rs4646437) shows high frequency in the Black group (0.73) compared to the majority White group (0.11), and it exerts a dominant effect on adverse reactions to methadone. Given these allele frequency differences between groups, the dominant effect mode predicts 719 excess adverse drug reactions per 1000 patients in the Black group compared to the majority White group. A pharmacogenomic variant in the VKORC1 (Vitamin K Epoxide Reductase Complex Subunit 1) gene has been associated with anticoagulation and excess bleeding in patients treated with the blood thinners warfarin and phenprocoumon. In the All of Us cohort, the toxicity-associated allele of this variant (dbSNP rs9923231) shows high frequency in the Asian (0.67) group compared to the majority White group (0.34), and it exerts a dominant effect on adverse reactions to warfarin and phenprocoumon. This allele frequency difference, given the dominant effect mode, predicts 327 excess adverse drug reactions per 1000 patients in the Asian group compared to the majority White group. A pharmacogenomic variant in the DPYD (Dihydropyrimidine Dehydrogenase) gene has been associated with toxic side effects to the chemotherapeutic agents fluorouracil and capecitabine. In the All of Us cohort, the toxicity-associated allele of this variant (dbSNP rs1801265) shows high frequency in the Black (0.40) group compared to the majority White group (0.22), and it has been report to exert both recessive and dominant effects on toxicity. This allele frequency difference predicts 112 excess adverse drug reactions per 1000 patients in the Black group compared to the majority White group under the recessive effect mode and 248 excess adverse reactions under the dominant mode. A pharmacogenomic variant in the ERCC1 (Excision Repair 1, Endonuclease Non-Catalytic Subunit) gene has been associated with adverse reactions to nine different drugs, including cisplatin and oxaliplatin chemotherapy drugs. In the HRS cohort, the toxicity-associated allele of this variant (dbSNP rs11615) shows high frequency in the Black (0.88) and Hispanic (0.64) groups compared to the majority White group (0.37). These large allele frequency differences, given the recessive effect mode, yield predictions of up to 638 excess adverse drug reactions per 1000 patients in the Black group and up to 273 excess adverse reactions in the Hispanic group compared to the majority White group. These findings on race, ethnicity, and adverse drug reactions also have implications for the clinical relevance of human genetic variance components. In 1972, Richard Lewontin found that the majority of human genetic variation was found within (85%) rather than between (15%) racial groups . This fundamental result has been replicated many times since, and it is often taken to support the irrelevance of racial classification to human genetic variation . This is consistent with the notion that race and ethnicity are purely social constructs with little or no biological significance . Here, it must be reiterated that observed patterns of pharmacogenomic variation, particularly as they relate to adverse drug reactions, clearly support the clinical relevance of race and ethnicity . In fact, we previously showed that when pharmacogenomic variation is partitioned exactly in the way that Lewontin and others have described, 85% within-group variation and 15% between-group variation, there can be up to 700 excess adverse drug reactions per 1000 patients predicted for the recessive effect mode and as many as 300 adverse reactions predicted for the dominant mode . In other words, a high relative excess of within- versus between-group genetic variation, as is almost always seen for human populations, does not preclude the utility of race and ethnicity for pharmacogenomic risk stratification. A Reckoning with the Implications of Population Pharmacogenomic Diversity Is a Prerequisite for Health Equity To conclude this perspective, we would like to emphasize once again the relevance of human genomic diversity for research efforts and clinical applications aimed at promoting health equity . The extent of actionable pharmacogenomic differences between racial and ethnic groups flies in the face of an emerging orthodoxy, which maintains that race and ethnicity are social constructs with no biological or clinical relevance . The ascendance of this strict social constructionist view of race and ethnicity points to a fundamental contradiction at the heart of genomics research efforts. On the one hand, there is an increasing awareness of the need to diversify genomics research cohorts in support of health equity, driven by the realization that the current bias towards European ancestry cohorts will limit the global reach and impact of genomic medicine . This view is exemplified by the National Human Genome Research Institute’s (NHGRI) pangenome initiative, which aims to expand the human reference genome to include representation from scores, and ultimately hundreds, of genome sequences from diverse human populations . On the other hand, more and more genomics researchers are simultaneously advocating for the elimination of race and ethnicity in genomics research and for developing methods that emphasize the genetic sameness among populations rather than their differences . This worldview stresses a continuum of human global diversity with population groups representing operationally defined abstractions rather than any real underlying structure in the data. There are even efforts to remove widely used terms like ancestry and admixture from the scientific lexicon, so as to avoid essentializing human genetic variation . This body of work is part of a larger trend, which Jerry Coyne and Luana Maroja recently described as “the ideological subversion of biology” . Much as we have done here, Coyne and Maroja soundly reject the ideologically motivated claim that there is no empirical value in studying genetic differences between races, ethnic groups, or populations, and they point out the threat that ideological research prerogatives of this kind pose to open scientific inquiry. We contend that scientists should be able to accommodate more than one view on the constitution of racial and ethnic groups. Clearly, these groups are socially constructed, given that their boundaries and composition are delineated by humans. Accordingly, they differ with respect to non-genetic factors that are relevant to health outcomes, such as diet, lifestyle, and socioenvironmental factors. And just as clearly, socially defined racial and ethnic groups can and do differ genetically in ways that are relevant to disease treatment decisions. It follows that efforts to eliminate race and ethnicity from genomics research and clinical considerations, however well-intentioned, will ultimately do more harm than good, exacerbating rather than ameliorating existing health disparities. A more nuanced approach to race and ethnicity, one which recognizes both the social and genetic dimensions of these groups, can be effectively leveraged to support health equity following the roadmap laid out by NIMHD, promoting fairness and opportunity in disease treatments and health outcomes. To conclude this perspective, we would like to emphasize once again the relevance of human genomic diversity for research efforts and clinical applications aimed at promoting health equity . The extent of actionable pharmacogenomic differences between racial and ethnic groups flies in the face of an emerging orthodoxy, which maintains that race and ethnicity are social constructs with no biological or clinical relevance . The ascendance of this strict social constructionist view of race and ethnicity points to a fundamental contradiction at the heart of genomics research efforts. On the one hand, there is an increasing awareness of the need to diversify genomics research cohorts in support of health equity, driven by the realization that the current bias towards European ancestry cohorts will limit the global reach and impact of genomic medicine . This view is exemplified by the National Human Genome Research Institute’s (NHGRI) pangenome initiative, which aims to expand the human reference genome to include representation from scores, and ultimately hundreds, of genome sequences from diverse human populations . On the other hand, more and more genomics researchers are simultaneously advocating for the elimination of race and ethnicity in genomics research and for developing methods that emphasize the genetic sameness among populations rather than their differences . This worldview stresses a continuum of human global diversity with population groups representing operationally defined abstractions rather than any real underlying structure in the data. There are even efforts to remove widely used terms like ancestry and admixture from the scientific lexicon, so as to avoid essentializing human genetic variation . This body of work is part of a larger trend, which Jerry Coyne and Luana Maroja recently described as “the ideological subversion of biology” . Much as we have done here, Coyne and Maroja soundly reject the ideologically motivated claim that there is no empirical value in studying genetic differences between races, ethnic groups, or populations, and they point out the threat that ideological research prerogatives of this kind pose to open scientific inquiry. We contend that scientists should be able to accommodate more than one view on the constitution of racial and ethnic groups. Clearly, these groups are socially constructed, given that their boundaries and composition are delineated by humans. Accordingly, they differ with respect to non-genetic factors that are relevant to health outcomes, such as diet, lifestyle, and socioenvironmental factors. And just as clearly, socially defined racial and ethnic groups can and do differ genetically in ways that are relevant to disease treatment decisions. It follows that efforts to eliminate race and ethnicity from genomics research and clinical considerations, however well-intentioned, will ultimately do more harm than good, exacerbating rather than ameliorating existing health disparities. A more nuanced approach to race and ethnicity, one which recognizes both the social and genetic dimensions of these groups, can be effectively leveraged to support health equity following the roadmap laid out by NIMHD, promoting fairness and opportunity in disease treatments and health outcomes. |
NLRC5 exerts anti-endometriosis effects through inhibiting ERβ-mediated inflammatory response | 9a06f5ca-9e1c-4510-aabd-dc3d01f2f02e | 11367751 | Anatomy[mh] | Endometriosis refers to the occurrence of endometrial glands and stroma with growth functions that are transplanted and implanted into other parts outside the uterine cavity . It can cause clinical symptoms such as chronic pelvic pain and infertility . Epidemiological studies have shown that the incidence of endometriosis in women of childbearing age is approximately 10%, and the incidence in infertile women can be as high as 30–40% . In addition, patients with endometriosis have a significantly increased risk of developing tumors . Notably, there is insensitivity to drug treatment in patients with endometriosis or patients with endometriosis have an average annual recurrence rate of 10% after treatment . Therefore, there is an urgent need to alleviate the related clinical symptoms caused by endometriosis and reduce the recurrence rates after treatment in clinical settings . Exploring the pathogenesis of endometriosis has become the key approach to improving the effectiveness of clinical treatment for endometriosis. In recent years, based on related clinical symptoms, endometriosis has been demonstrated to be strongly associated with an inflammatory response . Searching for specific molecules that impact the inflammatory response and exploring their underlying mechanisms is important for improving treatment outcomes for patients with endometriosis. Estrogen receptors (ERs) are key nuclear receptors involved in the pathophysiology of endometriosis . ERs include two subtypes, ERα and ERβ, which have been confirmed to play widely participating roles in a variety of human organ functions . ERs widely participated in reproductive diseases, including endometriosis . While compared to ERα, ERβ has been clearly recognized as a key molecular in the pathological process of endometriosis . When compared to normal human endometrial cells, human endometriotic lesions—whether ovarian or peritoneal—show higher expression of ERβ; ERα expression was not observed in these lesions. Moreover, elevated levels of ERβ contribute to the onset and progression of endometriosis by influencing inflammatory pathways . Therefore, conducting in-depth research into the mechanism underlying ERβ-mediated inflammation regulation in endometriosis and identifying potential therapeutic targets will have significant clinical relevance and treatment value . NOD-like receptors (NLRs), which are pattern recognition receptors (PRRs) protein family members found in the cytoplasm, have a significant involvement in the onset and progression of inflammation and immune diseases and they play a crucial role in the immune response and safeguarding of eukaryotic organisms by aiding in the quick elimination of invading pathogens . The innate immune molecule, NLRs family CARD domain-containing 5 (NLRC5), is a highly conserved member of the newly discovered NLR-like receptor family in recent years . In the inflammatory response, NLRC5 can act as a negative regulatory factor of the inflammatory response by inhibiting the nuclear factor-kappaB (NF-κB) inflammatory signaling pathway as well as the secretion of inflammatory factors to suppress the inflammatory response . In the immune response, NLRC5 activation can suppress tumor growth by promoting anti-tumor immune responses . Research has also revealed that NLRC5 suppresses inflammation by promoting autophagy in the secretory phase of ectopic endometrial stromal cells . According to the report, the NLRs can be targeted by ERs to regulate the Wnt/β-catenin signaling pathway in cancer . Therefore, examining whether the NLRC5-mediated inflammatory response is involved in ERβ-controlled endometriosis would offer valuable understanding of the development of endometriosis and improve treatment outcomes. Population sample collection This study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Anhui Medical University (No. SL-YX [YS] 2023-SZR010). Nine cases of secretory phase eutopic endometrium and ten cases of secretory phase ectopic endometrium were collected from patients with endometriosis who underwent laparoscopic surgery at the Second Affiliated Hospital of Anhui Medical University, and ten cases of secretory phase normal endometrial tissue were obtained from patients undergoing uterine resection for benign uterine diseases. The phase of the menstrual cycle was determined based on the endometrial histology and the expected day of the menstrual cycle provided by the women. Before surgery, none of the patients had undergone radiotherapy, chemotherapy, hormone therapy, or biological therapy. The age (34.07 ± 5.47) and BMI (24.83 ± 2.88) of the patients exhibited no statistically significant difference compared to the age (34.23 ± 5.85) and BMI (24.76 ± 3.19) of the control group ( t = 0.196, P = 0.845; t = − 0.141, P = 0.888). Cells and cell culture Immortalized human endometrial stromal cells (hESCs) were purchased from FENGHUISHENGWU (Hunan, China). The hESCs pellets were suspended again in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (PS) (Gibco, USA), plated on a cell culture dish, and cultured in an incubator (5% CO 2 , 37 °C). Lipopolysaccharide (LPS) (50 ng/ml) (Sigma, USA) was used to induce an inflammatory environment in hESCs. Adeno-associated virus and plasmid construction The recombinant adeno-associated virus (AAV) vector expressing short hairpin RNA (shRNA) against NLRC5 (shNLRC5) and its negative controls (NC) were synthesized by Shanghai GenePharma Co., Ltd. and injected into mice through the tail vein. Overexpression of ERβ (the ERβ group) and NLRC5 (the NLRC5 group) was achieved by plasmid construction and their negative controls (NC), which were respectively purchased from GenePharma (Shanghai, China) and GENERAL BIOL (Anhui, China). Mouse model of endometriosis From the Beijing Vital River Laboratory Animal Technology Co., Ltd., 5-week-old and 16–18 g C57BL/6 female mice were purchased. The animals were kept in a standard laboratory environment that was free from specific pathogens. For 3–5 days, the mice were subjected to a 12–12 h cycle of light and darkness at a temperature range of 20–24 °C and a humidity range of 60–65%. After 3 days of acclimatization, the mice were randomly divided into donor and recipient groups. Donor mice were euthanized by decapitation, and their abdomens were opened to remove the uterus. After removing the excess tissue, the uterus was cut open along the Y-shaped uterine midline and placed in separate saline solution containers. The uterine endometrium was exposed by longitudinally cutting open each uterus and then minced into small fragments of 1 mm 3 using ophthalmic scissors. The recipient mice were injected with a 1 ml saline solution containing the uterine fragments into their abdominal cavity using a sterile insulin syringe. An injection needle was inserted 5 mm into the left midline of the lower abdomen of the recipient mouse. Each uterus from the donor mouse was evenly distributed between the two recipient mice. Treatment The recombinant AAV vector expressing short hairpin RNA (shRNA) against NLRC5 (shNLRC5) and its negative controls (NC) were synthesized by Shanghai GenePharma Co., Ltd. and injected into mice through the tail vein. Overexpression of ERβ (the ERβ group) and NLRC5 (the NLRC5 group) was achieved by plasmid construction and their negative controls (NC), which were respectively purchased from GenePharma (Shanghai, China) and GENERAL BIOL (Anhui, China). The mice model of endometriosis were randomly divided into seven groups: the control group, the ERβ activator (ERB-041, purchased from MedChem Express, USA) group, the ERβ antagonist (PHTPP, purchased from MedChem Express, USA) group, the NC group, the NC + ERB-041 group, the shNLRC5 group, and the shNLRC5 + ERB-041 group. Each group has 10 mice. In the ERB-041 group and the PHTPP group (Fig. B), inject ERB-041 (2.5 mg/kg in 50 µl corn oil) and PHTPP (2.5 mg/kg in 50 µl corn oil) subcutaneously into mice separately. In the control group, an equal amount of corn oil was subcutaneously injected. The mice were euthanized after continuous injection (once every other day) for 3 weeks. In the shNLRC5 and shNLRC5 + ERB-041 groups (Fig. A), separately injected one dose of 1 × 10 10 TU/ml of shNLRC5 into the tail vein of mice, with a volume of 50 µl. In the NC and NC + ERB-041 groups, the NC AAV was injected into the mouse tail vein at a concentration of 1 × 10 10 TU/ml. After 3 weeks, mice in the NC + ERB-041 and shNLRC5 + ERB-041 groups were subcutaneously injected with 2.5 mg/kg ERB-041. The NC and shNLRC5 groups were subcutaneously injected with the same amount of corn oil on alternate days. After continuous injection for 3 weeks, the mice were euthanized. Open the abdominal cavity of the mice, eutopic and ectopic endometrial tissue were collected respectively. Then, we determined the size of ectopic lesions using the following method: V (mm 3 ) = 0.52 × length × width × height. Animal experiments were approved by the Ethics Review Committee of the Department of Laboratory Animal Science of Anhui Medical University (No. LLSC20231700). Anhui Medical University’s animal experimental guidelines were followed when conducting animal experiments and nursing procedures. Hematoxylin and eosin staining The lesions collected from mice with endometriosis were embedded in paraffin wax and prepared into multiple 5 mm paraffin sections. The paraffin sections were then dewaxed under gradient ethanol, followed by hematoxylin and eosin (H & E) staining, dehydration, penetration, and sealing. Images were collected using a 3D digital slicing scanner (Pannoramic MIDI, 3Dhistech, Hungary). Immunohistochemical staining Dewaxed and dehydrated paraffin-embedded sections of eutopic and ectopic endometria from mice with endometriosis underwent antigen retrieval, endogenous peroxidase blocking, and application of the primary antibody. The specific primary antibodies used in immunohistochemistry were as follows: NLRC5 (1:100, Affinity), ERβ (1:100, Affinity), tumor necrosis factor-alpha (TNF-α) (1:500, Abcam), interleukin (IL)-6 (1:1000, Abcam), and IL-10 (1:1000, Abcam). Next, an enzyme-labeled goat anti-mouse/rabbit IgG polymer was added, and the slices were dehydrated with alcohol, made transparent with xylene, and sealed with neutral gum. Images were collected and analyzed using a 3D digital slicing scanner (Pannoramic MIDI, 3Dhistech, Hungary). Western blotting The tissue proteins of mouse eutopic and ectopic endometria and cellular proteins of hESCs were extracted and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China). The proteins in these extracts were separated using 6% and 10% SDS-PAGE and then transferred to a 0.45-µm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking, the membranes were sequentially incubated with the corresponding primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. The primary antibodies used were ERβ (1:1000, Affinity) and NLRC5 (1:500, Bioworld). The membranes were exposed and developed after immersion in ECL reagent (Biosharp, China). The relative target protein levels were equal to the ratios of their gray values to GAPDH (1:10,000, Elabscience), which served as an internal reference. Quantitative real-time PCR The quality of total RNA was confirmed by Nanodrop 2000 (Thermo Fisher Scientific, USA), followed by extraction from eutopic endometrial tissue, ectopic endometrial tissue, and treated cells using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme, China). Total RNA (1000 ng) was reverse transcribed using HyperScriptTM III RT SuperMix for quantitative real-time PCR (qRT-PCR) with gDNA Remover (EnzyArtisan, China). qRT-PCR was conducted to quantify gene expression using the LightCycler 480 SYBR Green I Master (Roche, Switzerland). The primer sequences for each gene were designed and validated for specificity. The relative expression of genes of interest was calculated and normalized to that of β-actin using the 2 −ΔΔCt method . The PCR primers used are shown in Additional file 1: Table S1. Transfection hESCs were seeded at 1 × 10 5 cells/well into six-well plates and were transfected, respectively, with ERβ-overexpressing and NLRC5-overexpressing plasmids at approximately 80% confluence. The jetPRIME Transfection Reagent Kit (Polyplus-Transfection, France) was used to transfect the plasmid. Initially, a 2 µg DNA sample was diluted in 200 µl of jetPRIME buffer. Following a 10-s vortexing, 4 µl of jetPRIME reagent were introduced to the transfection mixture and vortexed for 1 s. Next, the cells were left to incubate at room temperature for 10 min. Ultimately, the mixture was incorporated into the culture medium. Transwell migration and invasion assay Chambers, either with or without Matrigel (BD Biosciences, USA), from Corning (USA), were utilized to assess cell migration and invasion, correspondingly. The transfected cells were seeded in the upper compartment in 100 µl serum-free medium. Afterwards, 600 µl of complete medium was added to the lower compartment. Cells on the upper side of the membranes were removed. Cells on the lower side of slide were stained with 0.1% crystal violet solution and then photographed under a microscope. hESCs that had migrated or invaded the lower surface of the membranes were counted using the ImageJ software (version 1.46, USA). Wound healing assay We conducted a comparative study of cell migration using a wound healing assay. First, the cells were seeded into a 12-well plate and cultured in a complete medium. When cell confluence reached 90–95%, we scratched a straight line using a 10-µl pipette tip. Subsequently, the original medium was discarded, and the cells were washed with PBS to remove the detached cells. Serum-free MEM medium was added to the wells. An inverted fluorescence microscope (IX73, Olympus, Japan) was used to photograph the scratched area at 0, 24, and 48 h after scratching. The wound width and migration area were analyzed using the ImageJ software. Enzyme-linked immunosorbent assay An enzyme-linked immunosorbent assay (ELISA) kit from China (ELISA LAB) was utilized to determine the concentrations of TNF-α, IL-6, and IL-10 by utilizing cell culture supernatant collected from control, control + LPS, NC, NC + LPS, ERβ, ERβ + LPS, NLRC5, NLRC5 + LPS, and ERβ + NLRC5 + LPS groups. A standard curve was used to determine the concentrations of TNF-α, IL-6, and IL-10. Statistical analysis The results are presented as the mean ± S.D. for continuous variables. Statistical data were analyzed by two-tailed Student’s t -test and one-way ANOVA, followed by Tukey’s post hoc analysis, using GraphPad Prism software (version 8.0, USA). The P value was set at less than 0.05 to determine statistical significance. This study was approved by the Ethics Review Committee of the Second Affiliated Hospital of Anhui Medical University (No. SL-YX [YS] 2023-SZR010). Nine cases of secretory phase eutopic endometrium and ten cases of secretory phase ectopic endometrium were collected from patients with endometriosis who underwent laparoscopic surgery at the Second Affiliated Hospital of Anhui Medical University, and ten cases of secretory phase normal endometrial tissue were obtained from patients undergoing uterine resection for benign uterine diseases. The phase of the menstrual cycle was determined based on the endometrial histology and the expected day of the menstrual cycle provided by the women. Before surgery, none of the patients had undergone radiotherapy, chemotherapy, hormone therapy, or biological therapy. The age (34.07 ± 5.47) and BMI (24.83 ± 2.88) of the patients exhibited no statistically significant difference compared to the age (34.23 ± 5.85) and BMI (24.76 ± 3.19) of the control group ( t = 0.196, P = 0.845; t = − 0.141, P = 0.888). Immortalized human endometrial stromal cells (hESCs) were purchased from FENGHUISHENGWU (Hunan, China). The hESCs pellets were suspended again in Minimum Essential Medium (MEM) containing 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin/streptomycin (PS) (Gibco, USA), plated on a cell culture dish, and cultured in an incubator (5% CO 2 , 37 °C). Lipopolysaccharide (LPS) (50 ng/ml) (Sigma, USA) was used to induce an inflammatory environment in hESCs. The recombinant adeno-associated virus (AAV) vector expressing short hairpin RNA (shRNA) against NLRC5 (shNLRC5) and its negative controls (NC) were synthesized by Shanghai GenePharma Co., Ltd. and injected into mice through the tail vein. Overexpression of ERβ (the ERβ group) and NLRC5 (the NLRC5 group) was achieved by plasmid construction and their negative controls (NC), which were respectively purchased from GenePharma (Shanghai, China) and GENERAL BIOL (Anhui, China). From the Beijing Vital River Laboratory Animal Technology Co., Ltd., 5-week-old and 16–18 g C57BL/6 female mice were purchased. The animals were kept in a standard laboratory environment that was free from specific pathogens. For 3–5 days, the mice were subjected to a 12–12 h cycle of light and darkness at a temperature range of 20–24 °C and a humidity range of 60–65%. After 3 days of acclimatization, the mice were randomly divided into donor and recipient groups. Donor mice were euthanized by decapitation, and their abdomens were opened to remove the uterus. After removing the excess tissue, the uterus was cut open along the Y-shaped uterine midline and placed in separate saline solution containers. The uterine endometrium was exposed by longitudinally cutting open each uterus and then minced into small fragments of 1 mm 3 using ophthalmic scissors. The recipient mice were injected with a 1 ml saline solution containing the uterine fragments into their abdominal cavity using a sterile insulin syringe. An injection needle was inserted 5 mm into the left midline of the lower abdomen of the recipient mouse. Each uterus from the donor mouse was evenly distributed between the two recipient mice. The recombinant AAV vector expressing short hairpin RNA (shRNA) against NLRC5 (shNLRC5) and its negative controls (NC) were synthesized by Shanghai GenePharma Co., Ltd. and injected into mice through the tail vein. Overexpression of ERβ (the ERβ group) and NLRC5 (the NLRC5 group) was achieved by plasmid construction and their negative controls (NC), which were respectively purchased from GenePharma (Shanghai, China) and GENERAL BIOL (Anhui, China). The mice model of endometriosis were randomly divided into seven groups: the control group, the ERβ activator (ERB-041, purchased from MedChem Express, USA) group, the ERβ antagonist (PHTPP, purchased from MedChem Express, USA) group, the NC group, the NC + ERB-041 group, the shNLRC5 group, and the shNLRC5 + ERB-041 group. Each group has 10 mice. In the ERB-041 group and the PHTPP group (Fig. B), inject ERB-041 (2.5 mg/kg in 50 µl corn oil) and PHTPP (2.5 mg/kg in 50 µl corn oil) subcutaneously into mice separately. In the control group, an equal amount of corn oil was subcutaneously injected. The mice were euthanized after continuous injection (once every other day) for 3 weeks. In the shNLRC5 and shNLRC5 + ERB-041 groups (Fig. A), separately injected one dose of 1 × 10 10 TU/ml of shNLRC5 into the tail vein of mice, with a volume of 50 µl. In the NC and NC + ERB-041 groups, the NC AAV was injected into the mouse tail vein at a concentration of 1 × 10 10 TU/ml. After 3 weeks, mice in the NC + ERB-041 and shNLRC5 + ERB-041 groups were subcutaneously injected with 2.5 mg/kg ERB-041. The NC and shNLRC5 groups were subcutaneously injected with the same amount of corn oil on alternate days. After continuous injection for 3 weeks, the mice were euthanized. Open the abdominal cavity of the mice, eutopic and ectopic endometrial tissue were collected respectively. Then, we determined the size of ectopic lesions using the following method: V (mm 3 ) = 0.52 × length × width × height. Animal experiments were approved by the Ethics Review Committee of the Department of Laboratory Animal Science of Anhui Medical University (No. LLSC20231700). Anhui Medical University’s animal experimental guidelines were followed when conducting animal experiments and nursing procedures. The lesions collected from mice with endometriosis were embedded in paraffin wax and prepared into multiple 5 mm paraffin sections. The paraffin sections were then dewaxed under gradient ethanol, followed by hematoxylin and eosin (H & E) staining, dehydration, penetration, and sealing. Images were collected using a 3D digital slicing scanner (Pannoramic MIDI, 3Dhistech, Hungary). Dewaxed and dehydrated paraffin-embedded sections of eutopic and ectopic endometria from mice with endometriosis underwent antigen retrieval, endogenous peroxidase blocking, and application of the primary antibody. The specific primary antibodies used in immunohistochemistry were as follows: NLRC5 (1:100, Affinity), ERβ (1:100, Affinity), tumor necrosis factor-alpha (TNF-α) (1:500, Abcam), interleukin (IL)-6 (1:1000, Abcam), and IL-10 (1:1000, Abcam). Next, an enzyme-labeled goat anti-mouse/rabbit IgG polymer was added, and the slices were dehydrated with alcohol, made transparent with xylene, and sealed with neutral gum. Images were collected and analyzed using a 3D digital slicing scanner (Pannoramic MIDI, 3Dhistech, Hungary). The tissue proteins of mouse eutopic and ectopic endometria and cellular proteins of hESCs were extracted and lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, China). The proteins in these extracts were separated using 6% and 10% SDS-PAGE and then transferred to a 0.45-µm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking, the membranes were sequentially incubated with the corresponding primary antibodies overnight at 4 °C and secondary antibodies for 1 h at room temperature. The primary antibodies used were ERβ (1:1000, Affinity) and NLRC5 (1:500, Bioworld). The membranes were exposed and developed after immersion in ECL reagent (Biosharp, China). The relative target protein levels were equal to the ratios of their gray values to GAPDH (1:10,000, Elabscience), which served as an internal reference. The quality of total RNA was confirmed by Nanodrop 2000 (Thermo Fisher Scientific, USA), followed by extraction from eutopic endometrial tissue, ectopic endometrial tissue, and treated cells using the FastPure® Cell/Tissue Total RNA Isolation Kit (Vazyme, China). Total RNA (1000 ng) was reverse transcribed using HyperScriptTM III RT SuperMix for quantitative real-time PCR (qRT-PCR) with gDNA Remover (EnzyArtisan, China). qRT-PCR was conducted to quantify gene expression using the LightCycler 480 SYBR Green I Master (Roche, Switzerland). The primer sequences for each gene were designed and validated for specificity. The relative expression of genes of interest was calculated and normalized to that of β-actin using the 2 −ΔΔCt method . The PCR primers used are shown in Additional file 1: Table S1. hESCs were seeded at 1 × 10 5 cells/well into six-well plates and were transfected, respectively, with ERβ-overexpressing and NLRC5-overexpressing plasmids at approximately 80% confluence. The jetPRIME Transfection Reagent Kit (Polyplus-Transfection, France) was used to transfect the plasmid. Initially, a 2 µg DNA sample was diluted in 200 µl of jetPRIME buffer. Following a 10-s vortexing, 4 µl of jetPRIME reagent were introduced to the transfection mixture and vortexed for 1 s. Next, the cells were left to incubate at room temperature for 10 min. Ultimately, the mixture was incorporated into the culture medium. Chambers, either with or without Matrigel (BD Biosciences, USA), from Corning (USA), were utilized to assess cell migration and invasion, correspondingly. The transfected cells were seeded in the upper compartment in 100 µl serum-free medium. Afterwards, 600 µl of complete medium was added to the lower compartment. Cells on the upper side of the membranes were removed. Cells on the lower side of slide were stained with 0.1% crystal violet solution and then photographed under a microscope. hESCs that had migrated or invaded the lower surface of the membranes were counted using the ImageJ software (version 1.46, USA). We conducted a comparative study of cell migration using a wound healing assay. First, the cells were seeded into a 12-well plate and cultured in a complete medium. When cell confluence reached 90–95%, we scratched a straight line using a 10-µl pipette tip. Subsequently, the original medium was discarded, and the cells were washed with PBS to remove the detached cells. Serum-free MEM medium was added to the wells. An inverted fluorescence microscope (IX73, Olympus, Japan) was used to photograph the scratched area at 0, 24, and 48 h after scratching. The wound width and migration area were analyzed using the ImageJ software. An enzyme-linked immunosorbent assay (ELISA) kit from China (ELISA LAB) was utilized to determine the concentrations of TNF-α, IL-6, and IL-10 by utilizing cell culture supernatant collected from control, control + LPS, NC, NC + LPS, ERβ, ERβ + LPS, NLRC5, NLRC5 + LPS, and ERβ + NLRC5 + LPS groups. A standard curve was used to determine the concentrations of TNF-α, IL-6, and IL-10. The results are presented as the mean ± S.D. for continuous variables. Statistical data were analyzed by two-tailed Student’s t -test and one-way ANOVA, followed by Tukey’s post hoc analysis, using GraphPad Prism software (version 8.0, USA). The P value was set at less than 0.05 to determine statistical significance. ERβ promoted the development of endometriosis In order to investigate the role of ERβ in the pathogenesis of endometriosis, we first detected the expression of ERβ in patients with endometriosis. Immunohistochemical results revealed that the expression of ERβ was significantly higher in the epithelium and stroma of endometriosis patients than in the epithelium and stroma of normal endometrium, and the expression of ERβ was also significantly higher in the epithelium and stroma of ectopic endometrium than in the epithelium and stroma of eutopic endometrium among women with endometriosis (Fig. A). We established an endometriosis mouse model to explore the role of ERβ in the development of endometriosis (Fig. B). The lesions were confirmed to be endometriosis using H & E staining that demonstrated growth of glandular and stromal endometrial tissue (Fig. C). Both in eutopic and ectopic endometrial tissue, immunohistochemical results revealed that ERB-041 could significantly increase ERβ expression in the epithelium and stroma of the endometrium in the ERB-041 group compared to the endometrium epithelium and stroma of the control group, and that PHTPP could significantly decrease ERβ expression in the epithelium and stroma of the endometrium in the PHTPP group compared to the endometrium epithelium and stroma of the control group (Fig. D, E). Additionally, we noted that the volume of ectopic lesions in the ERB-041 group was significantly larger than that in the control group, whereas the volume of ectopic lesions in the PHTPP group was significantly smaller than that in the control group, indicating that ERβ could promote the development of endometriosis (Fig. F). The ERβ overexpression in hESCs was confirmed through qRT-PCR and western blotting (Fig. G, H). Lastly, in in vitro, we noted that the overexpression of ERβ could promote the migration and invasion of hESCs (Fig. I, J). ERβ promoted the development of endometriosis by activating inflammatory response Inflammatory response is one of the most important factors contributing to the pathogenesis of endometriosis. We speculated whether ERβ contributed to the development of endometriosis by regulating the inflammatory response. In the endometriosis mouse model, both in the eutopic and ectopic endometrial tissue, immunohistochemical results that ERB-041 could increase the expression levels of pro-inflammatory factors TNF-α (Fig. A, B) and IL-6 (Fig. C, D) and decrease the expression level of anti-inflammatory factor IL-10 (Fig. E, F). Accordingly, PHTPP had the opposite effect (Fig. A–F). We observed that TNF-α was present in both the epithelium and stroma of eutopic and ectopic endometrial tissues in C57BL/6 J mice suffering from endometriosis (Fig. A, B). In the stroma of eutopic endometrial tissues, there is expression of IL-6 (Fig. C) and IL-10 (Fig. E), while the epithelium shows minimal expression of these cytokines. However, IL-6 (Fig. D) and IL-10 (Fig. F) are predominantly observed in the epithelium of ectopic endometrial tissues, with almost no expression in the stroma. The induction of inflammatory responses is a crucial aspect of Gram-negative bacteria’s virulence, and this is mainly carried out by LPS. Cell scratch test showed that LPS concentration of 50 ng/ml and 500 ng/ml did not affect the migration ability of hESCs. However, the migration ability of hESCs was inhibited when the concentration of LPS was 1000 ng/ml, suggesting that the concentration of LPS at 1000 ng/ml would impair cell function (Fig. G). Furthermore, the ELISA results demonstrated that 50 ng/ml LPS effectively created an inflammatory environment in hESCs (Fig. H). As a result, hESCs were exposed to 50 ng/ml LPS for future experiments. Importantly, we noted that overexpressing ERβ was unable to alter the levels of TNF-α, IL-6, and IL-10, whereas the addition of LPS to the hESCs to construct an inflammatory environment resulted in overexpressing ERβ being able to promote the secretion of pro-inflammatory factors TNF-α and IL-6 and inhibit the secretion of anti-inflammatory factor IL-10 (Fig. I). Furthermore, only LPS could not promote the migration of hESCs, whereas in the case of overexpression of ERβ, the migration and invasion abilities of hESCs significantly increased (Fig. J, K). These results indicated that in endometriosis, ERβ-mediated endometriosis development is partially dependent on the inflammatory environment, and that ERβ promotes the development of endometriosis by activating the inflammatory response. ERβ promoted NLRC5 expression in endometriosis Our previous study indicated that the innate immune molecule NLRC5 is upregulated in patients with endometriosis, and we believe that the inflammatory environment contributed to NLRC5 upregulation in endometriosis . In view of the decisive role of ERβ in the pathogenesis of endometriosis, we were curious about the role of ERβ in NLRC5 expression. In both eutopic and ectopic endometrial tissues of the endometriosis mouse model, the ERB-041 group showed a significant increase in the expression levels of NLRC5 mRNA and protein in the epithelium and stroma compared to the control group (Fig. A–D). Conversely, the PHTPP group displayed a significant decrease in the expression levels of NLRC5 mRNA and protein in the epithelium and stroma compared to the control group (Fig. A–D). In in vitro, we noted that the expression levels of NLRC5 mRNA and protein in the hESCs were significantly increased due to ERβ overexpression (Fig. E, F). These results indicate that ERβ could promote NLRC5 expression in endometriosis. NLRC5 affects the development of endometriosis In our previous study, we found evidence suggesting that NLRC5 has the ability to suppress inflammation in endometriosis. Nevertheless, we still lack understanding regarding the involvement of NLRC5 in the development of endometriosis. To address this, we conducted an investigation to determine the contribution of NLRC5 in the progression of endometriosis in a mouse model. Due to the excessive length of the NLRC5 overexpressed transcript from the mouse source, it is difficult to construct an NLRC5 overexpression AAV. Therefore, we constructed an shNLRC5 AAV vector (Fig. A). Immunohistochemistry results revealed that the shNLRC5 AAV inhibited the expression of NLRC5 in a mouse endometriosis model (Fig. B, C). Furthermore, the volume of ectopic lesions in the shNLRC5 group was significantly larger than that in the NC group (Fig. D). The overexpression of NLRC5 in hESCs was confirmed through qRT-PCR and western blotting (Fig. E–F). Moreover, NLRC5 overexpression was observed to suppress the migration and invasion of hESCs in vitro (Fig. G–H). NLRC5 inhibited the development of endometriosis by attenuating inflammatory response These results indicated that NLRC5 inhibits the development of endometriosis. We further investigated whether NLRC5 inhibited the development of endometriosis by regulating the inflammatory response. Immunohistochemical findings showed that there was an elevated expression of the pro-inflammatory factors TNF-α and IL-6 in both the eutopic and ectopic endometrial tissue of the shNLRC5 group as compared to the endometrial epithelium and stroma of the NC group (Fig. A–D). Additionally, a decrease in the expression of the anti-inflammatory factor IL-10 was observed in the endometrial epithelium and stroma of the shNLRC5 group in comparison to the NC group (Fig. E, F). The ELISA results showed that compared to its NC group, the NLRC5 group did not cause any significant changes in the levels of inflammatory factors (Fig. G). However, when LPS was added to the endometrial stromal cells to create an inflammatory environment, NLRC5 was able to suppress the release of pro-inflammatory factors TNF-α and IL-6 and stimulate the secretion of the anti-inflammatory factor IL-10 (Fig. G). Importantly, the inhibitory effect of NLRC5 overexpression on the migration and invasion of hESCs in an inflammatory environment was stronger than that of NLRC5 overexpression alone (Fig. H, I). NLRC5 inhibited ERβ-mediated development of endometriosis by attenuating inflammatory response The findings suggest that NLRC5 plays a role in suppressing the inflammatory response, thereby impeding the progression of endometriosis. We further detected whether NLRC5 could inhibit the ERβ-mediated development of endometriosis. In the mouse model of endometriosis, the shNLRC5 + ERB-041 group had significantly bigger volumes of ectopic lesions compared to the NC + ERB-041 group (Fig. A). Additionally, the ERB-041 group showed significantly larger volumes of ectopic lesions compared to the control group (Fig. A). Moreover, compared with the NC + LPS group, the ERβ + LPS group could promote the migration and invasion of hESCs (Fig. B, C). And overexpressed NLRC5 can inhibit the positive role of ERβ in promoting migration and invasion of hESCs (Fig. B, C). Lastly, we detect whether NLRC5-mediated anti-inflammation contributes to inhibiting the ERβ-mediated development of endometriosis. In both eutopic and ectopic endometrial tissue, the immunohistochemical results showed that the expression of pro-inflammatory factors TNF-α and IL-6 in both epithelium and stroma was increased in the shNLRC5 + ERB-041 group compared to the NC + ERB-041 group (Fig. D–G). Additionally, the expression of anti-inflammatory factor IL-10 in both epithelium and stroma was decreased in the shNLRC5 + ERB-041 group compared to the NC + ERB-041 group (Fig. H, I). Further in vitro experiments revealed that NLRC5 could inhibit the role of ERβ in promoting the secretion of pro-inflammatory factors TNF-α and IL-6 and inhibiting the secretion of anti-inflammatory factor IL-10 in an inflammatory environment (Fig. J). The above results indicate that NLRC5 can inhibit the ERβ-mediated development of endometriosis by attenuating the inflammatory response (Fig. ). In order to investigate the role of ERβ in the pathogenesis of endometriosis, we first detected the expression of ERβ in patients with endometriosis. Immunohistochemical results revealed that the expression of ERβ was significantly higher in the epithelium and stroma of endometriosis patients than in the epithelium and stroma of normal endometrium, and the expression of ERβ was also significantly higher in the epithelium and stroma of ectopic endometrium than in the epithelium and stroma of eutopic endometrium among women with endometriosis (Fig. A). We established an endometriosis mouse model to explore the role of ERβ in the development of endometriosis (Fig. B). The lesions were confirmed to be endometriosis using H & E staining that demonstrated growth of glandular and stromal endometrial tissue (Fig. C). Both in eutopic and ectopic endometrial tissue, immunohistochemical results revealed that ERB-041 could significantly increase ERβ expression in the epithelium and stroma of the endometrium in the ERB-041 group compared to the endometrium epithelium and stroma of the control group, and that PHTPP could significantly decrease ERβ expression in the epithelium and stroma of the endometrium in the PHTPP group compared to the endometrium epithelium and stroma of the control group (Fig. D, E). Additionally, we noted that the volume of ectopic lesions in the ERB-041 group was significantly larger than that in the control group, whereas the volume of ectopic lesions in the PHTPP group was significantly smaller than that in the control group, indicating that ERβ could promote the development of endometriosis (Fig. F). The ERβ overexpression in hESCs was confirmed through qRT-PCR and western blotting (Fig. G, H). Lastly, in in vitro, we noted that the overexpression of ERβ could promote the migration and invasion of hESCs (Fig. I, J). Inflammatory response is one of the most important factors contributing to the pathogenesis of endometriosis. We speculated whether ERβ contributed to the development of endometriosis by regulating the inflammatory response. In the endometriosis mouse model, both in the eutopic and ectopic endometrial tissue, immunohistochemical results that ERB-041 could increase the expression levels of pro-inflammatory factors TNF-α (Fig. A, B) and IL-6 (Fig. C, D) and decrease the expression level of anti-inflammatory factor IL-10 (Fig. E, F). Accordingly, PHTPP had the opposite effect (Fig. A–F). We observed that TNF-α was present in both the epithelium and stroma of eutopic and ectopic endometrial tissues in C57BL/6 J mice suffering from endometriosis (Fig. A, B). In the stroma of eutopic endometrial tissues, there is expression of IL-6 (Fig. C) and IL-10 (Fig. E), while the epithelium shows minimal expression of these cytokines. However, IL-6 (Fig. D) and IL-10 (Fig. F) are predominantly observed in the epithelium of ectopic endometrial tissues, with almost no expression in the stroma. The induction of inflammatory responses is a crucial aspect of Gram-negative bacteria’s virulence, and this is mainly carried out by LPS. Cell scratch test showed that LPS concentration of 50 ng/ml and 500 ng/ml did not affect the migration ability of hESCs. However, the migration ability of hESCs was inhibited when the concentration of LPS was 1000 ng/ml, suggesting that the concentration of LPS at 1000 ng/ml would impair cell function (Fig. G). Furthermore, the ELISA results demonstrated that 50 ng/ml LPS effectively created an inflammatory environment in hESCs (Fig. H). As a result, hESCs were exposed to 50 ng/ml LPS for future experiments. Importantly, we noted that overexpressing ERβ was unable to alter the levels of TNF-α, IL-6, and IL-10, whereas the addition of LPS to the hESCs to construct an inflammatory environment resulted in overexpressing ERβ being able to promote the secretion of pro-inflammatory factors TNF-α and IL-6 and inhibit the secretion of anti-inflammatory factor IL-10 (Fig. I). Furthermore, only LPS could not promote the migration of hESCs, whereas in the case of overexpression of ERβ, the migration and invasion abilities of hESCs significantly increased (Fig. J, K). These results indicated that in endometriosis, ERβ-mediated endometriosis development is partially dependent on the inflammatory environment, and that ERβ promotes the development of endometriosis by activating the inflammatory response. Our previous study indicated that the innate immune molecule NLRC5 is upregulated in patients with endometriosis, and we believe that the inflammatory environment contributed to NLRC5 upregulation in endometriosis . In view of the decisive role of ERβ in the pathogenesis of endometriosis, we were curious about the role of ERβ in NLRC5 expression. In both eutopic and ectopic endometrial tissues of the endometriosis mouse model, the ERB-041 group showed a significant increase in the expression levels of NLRC5 mRNA and protein in the epithelium and stroma compared to the control group (Fig. A–D). Conversely, the PHTPP group displayed a significant decrease in the expression levels of NLRC5 mRNA and protein in the epithelium and stroma compared to the control group (Fig. A–D). In in vitro, we noted that the expression levels of NLRC5 mRNA and protein in the hESCs were significantly increased due to ERβ overexpression (Fig. E, F). These results indicate that ERβ could promote NLRC5 expression in endometriosis. In our previous study, we found evidence suggesting that NLRC5 has the ability to suppress inflammation in endometriosis. Nevertheless, we still lack understanding regarding the involvement of NLRC5 in the development of endometriosis. To address this, we conducted an investigation to determine the contribution of NLRC5 in the progression of endometriosis in a mouse model. Due to the excessive length of the NLRC5 overexpressed transcript from the mouse source, it is difficult to construct an NLRC5 overexpression AAV. Therefore, we constructed an shNLRC5 AAV vector (Fig. A). Immunohistochemistry results revealed that the shNLRC5 AAV inhibited the expression of NLRC5 in a mouse endometriosis model (Fig. B, C). Furthermore, the volume of ectopic lesions in the shNLRC5 group was significantly larger than that in the NC group (Fig. D). The overexpression of NLRC5 in hESCs was confirmed through qRT-PCR and western blotting (Fig. E–F). Moreover, NLRC5 overexpression was observed to suppress the migration and invasion of hESCs in vitro (Fig. G–H). These results indicated that NLRC5 inhibits the development of endometriosis. We further investigated whether NLRC5 inhibited the development of endometriosis by regulating the inflammatory response. Immunohistochemical findings showed that there was an elevated expression of the pro-inflammatory factors TNF-α and IL-6 in both the eutopic and ectopic endometrial tissue of the shNLRC5 group as compared to the endometrial epithelium and stroma of the NC group (Fig. A–D). Additionally, a decrease in the expression of the anti-inflammatory factor IL-10 was observed in the endometrial epithelium and stroma of the shNLRC5 group in comparison to the NC group (Fig. E, F). The ELISA results showed that compared to its NC group, the NLRC5 group did not cause any significant changes in the levels of inflammatory factors (Fig. G). However, when LPS was added to the endometrial stromal cells to create an inflammatory environment, NLRC5 was able to suppress the release of pro-inflammatory factors TNF-α and IL-6 and stimulate the secretion of the anti-inflammatory factor IL-10 (Fig. G). Importantly, the inhibitory effect of NLRC5 overexpression on the migration and invasion of hESCs in an inflammatory environment was stronger than that of NLRC5 overexpression alone (Fig. H, I). The findings suggest that NLRC5 plays a role in suppressing the inflammatory response, thereby impeding the progression of endometriosis. We further detected whether NLRC5 could inhibit the ERβ-mediated development of endometriosis. In the mouse model of endometriosis, the shNLRC5 + ERB-041 group had significantly bigger volumes of ectopic lesions compared to the NC + ERB-041 group (Fig. A). Additionally, the ERB-041 group showed significantly larger volumes of ectopic lesions compared to the control group (Fig. A). Moreover, compared with the NC + LPS group, the ERβ + LPS group could promote the migration and invasion of hESCs (Fig. B, C). And overexpressed NLRC5 can inhibit the positive role of ERβ in promoting migration and invasion of hESCs (Fig. B, C). Lastly, we detect whether NLRC5-mediated anti-inflammation contributes to inhibiting the ERβ-mediated development of endometriosis. In both eutopic and ectopic endometrial tissue, the immunohistochemical results showed that the expression of pro-inflammatory factors TNF-α and IL-6 in both epithelium and stroma was increased in the shNLRC5 + ERB-041 group compared to the NC + ERB-041 group (Fig. D–G). Additionally, the expression of anti-inflammatory factor IL-10 in both epithelium and stroma was decreased in the shNLRC5 + ERB-041 group compared to the NC + ERB-041 group (Fig. H, I). Further in vitro experiments revealed that NLRC5 could inhibit the role of ERβ in promoting the secretion of pro-inflammatory factors TNF-α and IL-6 and inhibiting the secretion of anti-inflammatory factor IL-10 in an inflammatory environment (Fig. J). The above results indicate that NLRC5 can inhibit the ERβ-mediated development of endometriosis by attenuating the inflammatory response (Fig. ). Endometriosis is an estrogen-dependent inflammatory disease that has led to extensive research on the pathophysiology of inflammatory mediators as potential therapeutic targets . Previous studies had demonstrated that ERβ was recognized to be more highly expressed in ectopic endometriotic tissues than ERα and was noted to drive the progression of endometriosis . It has been reported that ERβ can activate the NF-κB inflammatory pathway and increase the expression of C–C motif chemokine ligand 2 (CCL2), leading to the recruitment of macrophages (M2). This, in turn, promotes the proliferation, invasion, adhesion, and metastasis of ectopic lesions in endometriosis . Thus, the significant presence of ERβ in abnormal growths of individuals with endometriosis is likely to contribute to the progression of this condition through its ability to enhance the inflammatory microenvironment within the endometrium. This subsequently results in the increased potential for proliferation, migration, and invasion of the abnormal endometrial tissue. The role of ERβ in individuals suffering from endometriosis remains intricate and necessitates additional investigation to fully comprehend its mechanism of action. Conducting a thorough investigation into the mechanism of ERβ-mediated inflammatory regulation in the development of endometriosis patients and exploring potential therapeutic targets will hold great clinical significance and offer valuable treatment possibilities. In this study, we have confirmed that the anti-endometriosis effect mediated by NLRC5 is achieved through the inhibition of the ERβ-mediated inflammatory response. ERβ and NLRC5 have limited expression in normal endometrium, whereas their expression is specifically observed in endometriotic tissue. Therefore, we believe that ERβ and NLRC5 may be the next generation of promising therapeutic targets compared with the current treatment of endometriosis. ERβ has emerged as an important player in the pathogenesis of endometriosis . Studies have revealed that human endometriotic lesions, either ovarian or deeply infiltrating endometriosis, display higher ERβ expression when compared to that of normal human tissues . The ERβ to ERα ratio was over 1 in both human and animal models of endometriosis lesions, while ERα was predominately expressed in normal endometrial cells . We proved that the level of ERβ expression in the epithelium and stroma of endometriosis patients was considerably greater compared to the level in the epithelium and stroma of normal endometrium. Studies have demonstrated that ERβ plays an important role in the progression of endometriosis by modulating inflammation . However, little is known about the targets that can be involved in the ERβ-mediated inflammatory response of endometriosis. In this study, we uncovered that NLRC5 inhibits the ERβ-mediated development of endometriosis by attenuating the inflammatory response both in vitro and in vivo. NLRs are pattern recognition receptor families in the cytoplasm that are implicated in inflammatory diseases and promote rapid removal of invasive pathogens . NLRC5 is the largest protein of the NLR family, contains a nucleotide-binding domain and leucine-rich repeats, and regulates the inflammatory response . Evidence has been steadily increasing, indicating that NLRC5 hinders the activation of NF-κB signaling caused by LPS, TNF-α, or IL-1β . By extracting secretory ectopic endometrial stromal cells, we noted that NLRC5 inhibited inflammation by promoting autophagy . Generally, the identified protein expression profile is helpful for studying its function. NLRC5 expression patterns have revealed that NLRC5 is expressed in various tissues and cells . Compared to the normal group, our previous study showed a significant increase in NLRC5 expression in both the eutopic and ectopic endometrium of patients with endometriosis . It has also been reported that NLRC5 expression levels in gastric cancer, lung cancer, and hepatocellular carcinoma are higher than in normal tissue . Another study revealed that, compared to healthy colorectal tissue, there is increased expression of NLRC5 in colorectal cancer (CRC), and the special high inflammatory state of this cancer type probably explains this phenomenon . Consistently, our previous research noted that NLRC5 was upregulated in ectopic endometrial stromal cells (EESCs) of patients with ovarian endometriosis in a highly inflammatory state . Since ERβ is a specific molecule of endometriosis, we observed the effect of ERβ on NLRC5. Our results confirmed that ERB-041 significantly upregulated the expression of NLRC5 in both eutopic and ectopic endometrial tissues of an endometriosis mouse model, as compared to the control group. And compared with the control group, PHTPP inhibited the expression of NLRC5. In vitro, we noted that the expression levels of NLRC5 in the hESCs were significantly increased due to ERβ overexpression. Further in vitro and in vivo experiments revealed that ERβ promotes the expression of pro-inflammatory factors TNF-α and IL-6 and inhibits the expression of anti-inflammatory factor IL-10 in the inflammatory environment. And NLRC5 can inhibit the ERβ-mediated inflammatory response in endometriosis. Therefore, we put forward our view that it may be a compensatory response in which NLRC5 is activated dependent on abundant ERβ in endometriosis in order to alleviate ERβ-mediated inflammatory response. One limitation of this study is that in the tissue analysis of population samples, the sample size of the organization was relatively insufficient. In this study, due to the excessive length of the NLRC5 overexpressed transcript from the mouse source, it is difficult to construct an NLRC5 overexpression AAV. Therefore, we constructed an shNLRC5 AAV vector. We believe that this is also one of the limitations of our present study. We noticed that the excessive length of the NLRC5 caused application constrained also drew great attention by other research groups. For example, the NLRC5/CIITA chimeric construct (DD-335-CIITA) generated by Neerincx et al. is considerably shorter than NLRC5 and shows improved activity to induce MHC I and MHC II promoters compared with NLRC5 and CIITA . Santharam et al. examined a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control . We thought these studies provided ideas for our future research. The lack of clarity regarding the specific mechanism of ERβ on NLRC5 is another limitation of the current study. It is critical to elucidate the specific mechanism of ERβ on NLRC5 to develop therapeutic strategies for patients with endometriosis. In endometriosis, ERβ acts on a molecular target through a specific mechanism of action, thereby promoting the development of endometriosis. Our study demonstrated that ERβ promotes NLRC5 expression in endometriosis in vitro and in vivo. We believe that further exploration of the specific mechanism of action of ERβ on NLRC5 is highly significant in understanding the pathogenesis of endometriosis and improving therapeutic effectiveness. To summarize, we have observed noteworthy increases in the expression of ERβ and NLRC5 in both the eutopic and ectopic endometrium of individuals with endometriosis, as compared to the control group. Importantly, our results also confirmed that the activation of NLRC5 in endometriosis is specifically dependent on abundant ERβ. And overexpression of NLRC5 inhibits the ERβ-mediated development of endometriosis by attenuating the inflammatory response. These findings indicate that NLRC5 is a novel therapeutic target for endometriosis. Additional file 1: Table S1. The PCR primers used in the study. |
Continuum of care for maternal and newborn health services in Nepal: An analysis from demographic and health survey 2022 | 056c9deb-33a2-487b-8f16-b4ea89538504 | 11932462 | Community Health Services[mh] | In 2020, approximately 800 women tragically lost their lives daily, with one death occurring every two minutes adding up to a total of 287,000 death globally due to preventable causes associated with pregnancy and childbirth . Similarly, in 2022, approximately 6,500 newborns lost their lives everyday summing up to a total of 2.3 million global deaths . One third of total neonatal deaths occur on the first day of birth and nearly three-quarters within the first week of life . Neonatal mortality constitutes approximately 47% of all deaths among children under five years of age . In Nepal, maternal mortality ratio (MMR) stands at 151 per 100,000 live births, with 66% of these deaths occurring in postpartum period, 33% during pregnancy, and 6% during delivery . Nepal has been committed to reduce MMR to 70 per 100,000 live births as per Sustainable Development Goals (SDGs) which requires accelerated progress towards coverage of quality maternal health services . Likewise, the present neonatal mortality rate (NMR) is 21 per 1,000 live births , and attaining the SDG target of 12 per 1,000 live births by 2030 necessitates an annual rate of reduction (ARR) of 4.8%, surpassing the existing ARR of 4.0% observed from 2000 to 2018 . The causes of most of these maternal and neonatal deaths are preventable . Among the leading causes of maternal deaths are non-obstetric complications, obstetric hemorrhage, hypertensive disorder, pregnancy related infections, self-harm, pregnancy with abortive outcomes, and complication of anesthesia management . The leading causes of neonatal death are premature birth, birth complications (such as birth asphyxia or trauma), neonatal infections, and congenital anomalies . In order to achieve a significant reduction in MMR and NMR and progress towards the elimination of preventable causes of maternal and newborn deaths, it is essential to not only expand the reach of healthcare services but also enhance the overall quality of care provided across the entire spectrum of services . Maternal mortality and newborn deaths largely stem from inadequate quality care during pregnancies, childbirth and immediately after birth, as well as during the early days of life [ , , ]. Maternal and newborn health (MNH) service utilization has shown gradual increase in Nepal. Institutional delivery (ID) was 19.0 in 2006 , which increased to 43.7% in 2011, 64.2% in 2016 and has reached 79.4% in 2022 . The proportion of women receiving any antenatal care (ANC) from a skilled provider increased from 45% in 2006 to 61% in 2011, 86% in 2016 and 94% in 2022 . The proportion of women receiving four or more ANC (≥4 ANC) visits was 31% in 2006 , 53% in 2011, 71% in 2016 , and 81% in 2022 . Similarly, 20% of births in 2006 , 44% in 2011 , 64% in 2016 and 79% in 2022 occurred in health facilities. In addition, 70% of women received postnatal care (PNC) during first two days of delivery in 2022 which is an increase from 57% in 2016 . Evidence suggest that effective linkage among ANC, delivery care and PNC could improve the health of mother and newborn . Despite policy documents highlighting the importance of a continuum of care , and an apparent upward trend in the utilization of specific services [ , – ], research indicates that a significant proportion of women do not complete the continuum of care . An analysis of the NDHS 2016 data reveals that only around 41% of women successfully completed all routine maternal care visits, and with notable discontinuation of the service utilization particularly around the time of childbirth . The continuum of care has been emphasized in recent decades as a fundamental principle in maternal, newborn, and child health programs in multiple documents at global level [ – ]. It is considered a crucial strategy for reducing the significant burden of maternal, neonatal and child health problems. Pregnant women must receive ANC integrated with safe childbirth services provided by skilled professionals. PNC is essential for both mothers and newborns during the critical first few weeks after birth, ensuring a seamless connection to newborn care. In cases of complications or illness among women and newborns, ensuring continuity of care—from the household to the hospital, with timely referrals and emergency management—is vital. This continuum of care has also been emphasized in national policy documents such as the Safe Motherhood and Newborn Health (SMNH) Road Map 2030 , the ANC and PNC Continuum of Care Guideline , and the Nepal’s Every Newborn Action Plan (NENAP), 2016 . Having evidence on determinants of continuum of care could be useful from policy and programme perspective in designing maternal and newborn health interventions ensuring that every woman and newborn receive maternal and newborn services as needed in Nepal. In this context, we identified determinants of ≥ 4 ANC visits, ID, PNC visit within two days of delivery, combined coverage of ≥ 4 ANC visits and ID and also the combined coverage of ≥ 4 ANC visits, ID and PNC visit within two days of delivery.
This study involves the analysis of data from the nationally representative Nepal Demographic and Health Survey (NDHS) 2022. The sampling frame used for NDHS 2022 is an updated version of the frame from the Nepal Population and Housing Census (NPHC) 2011, with 36,020 sub-wards which was adjusted for updated urban-rural classification. In NPHC 2011, there were 58 urban municipalities which was increased to 293 urban municipalities by the time of survey, with these urban settings housing around 65% of the population. The NDHS 2022 adopted an updated urban-rural classification system for the survey . Sampling Two stage stratified sampling technique was used in NDHS 2022. Seven provinces were divided into urban and rural settings leading to a total of 14 sampling stratum. A strategy of implicit stratification with proportionate allocation was adopted at the lowest administrative levels that included shorting the sampling frame inside each stratum prior to sample selection, including administrative units from various levels, and using a probability-proportional-to-size strategy during the first sampling step. A total of 476 primary sampling units (PSUs) were chosen using a probability-proportional-to-size of the PSU of which 248 PSUs were from urban settings, while 228 were from rural settings. A household listing was carried out in the selected PSUs, yielding a sampling frame. Segmentation was done in sub-wards when the projected number of homes exceeded 300, and only one segment was selected for the survey. The likelihood of selection was related to the size of the segment. A total of 14,243 households were chosen, and among them, 13,833 were confirmed as being inhabited. From these inhabited households, successful interviews were conducted with 13,786, resulting in a response rate exceeding 99%. All females aged 15 to 49 who were either permanent inhabitants of the designated houses or visitors who had spent the night before in those households, were eligible to participate in the interviews. In this study, we have analyzed the data from 1891 women who had live births two years preceding the survey ( ). Data collection tool Data collection for the survey was conducted between January 5 to June 22, 2022. The DHS Program’s model questionnaires were modified to address Nepal’s unique demographics and health problems in consultation with various stakeholders, including government departments, agencies, non-governmental organizations, and funders. After the English versions of the survey tools were completed, they were translated into Nepali, Maithili, and Bhojpuri, the three most common languages spoken in Nepal. The translated versions of the questionnaire (household, woman, and man) were then digitized to facilitate data collection using computer-assisted personal interviews (CAPI). Data for the NDHS 2022 was gathered by 19 teams. Each team comprised of a supervisor, one male interviewer, three female interviewers, and a biomarker specialist . Dependent variables The dependent variables for this study are maternal health service utilization variables, which includes (i) ≥ 4 ANC visits, (ii) ID, and (iii) PNC visit for mother and newborn the first two days of delivery, (iv) combined coverage of ≥ 4 ANC visits and ID, and (v) Continuum of care coverage: combined coverage of ≥ 4 ANC visits, ID, and PNC visit for mother and newborn during the first two days of delivery. Additional information on variables can be obtained from the full report . Independent variables Independent variables include place of residence (urban, rural), province (Koshi, Madhesh, Bagmati, Gandaki, Lumbini, Karnali, Sudurpashchim), age at delivery ( < 20 years, 20-34 years and ≥ 35 years), ethnicity (Brahmin/ Chhetri, Dalit, Janajati, Madhesi, other), wealth quintile (poorest, poorer, middle, richer, richest), education (no education, basic, secondary or higher), distance to facility ( < 30 min, 30-59 min, 1-2 hours, and ≥ 2 hours), birth order (one, two, three or more), insurance coverage (no, yes), media exposure-for example newspapers, TV, and radio (no, yes) and internet use (no, yes). To calculate wealth quintiles, households were assigned scores based on the variety of consumer goods they own, such as televisions, bicycles, or cars, as well as housing characteristics like drinking water sources, toilet facilities, and flooring materials. These scores were derived using principal component analysis. National wealth quintiles were then determined by assigning each household a score, ranking individuals within the household population according to these scores, and dividing the population into five equal groups, with each quintile representing 20% of the population . Statistical analysis We conducted data analysis using R version 4.2.0. To accommodate the complex survey design of NDHS 2022, we employed a weighted analysis approach using the “survey” package . Multicollinearity was assessed prior to analysis, with a Variance Inflation Factor (VIF) of less than 10 considered an acceptable threshold for multicollinearity. Categorical variables were expressed as frequencies, percentages, and 95% confidence interval (CI). To determine the relationship between independent variables and maternal health service utilization variables (including ≥ 4 ANC visits, ID, and PNC visit, combined coverage of ≥ 4 ANC visits and ID, and combined coverage of ≥ 4 ANC visits, ID and PNC visit within two days of delivery), we performed univariate and multivariable binary logistic regression analyses. The outcomes of the logistic regression analysis were reported as crude odds ratio (COR) and adjusted odds ratio (AOR), each accompanied by their respective 95% CI. Ethical consideration NDHS 2022 obtained ethical approval from the institutional review board of ICF International, United States of America (Reference number: 180657.0.001.NP.DHS.01) and the ethical review board of Nepal Health Research Council (Reference number: 494/2021). Upon approval of our request for access to data, we downloaded NDHS 2022 dataset from https://dhsprogram.com/data/available-datasets.cfm . Written informed consent was obtained in the original survey. In the case of minors, both the assent as well as consent were obtained before enrollment in the survey.
Two stage stratified sampling technique was used in NDHS 2022. Seven provinces were divided into urban and rural settings leading to a total of 14 sampling stratum. A strategy of implicit stratification with proportionate allocation was adopted at the lowest administrative levels that included shorting the sampling frame inside each stratum prior to sample selection, including administrative units from various levels, and using a probability-proportional-to-size strategy during the first sampling step. A total of 476 primary sampling units (PSUs) were chosen using a probability-proportional-to-size of the PSU of which 248 PSUs were from urban settings, while 228 were from rural settings. A household listing was carried out in the selected PSUs, yielding a sampling frame. Segmentation was done in sub-wards when the projected number of homes exceeded 300, and only one segment was selected for the survey. The likelihood of selection was related to the size of the segment. A total of 14,243 households were chosen, and among them, 13,833 were confirmed as being inhabited. From these inhabited households, successful interviews were conducted with 13,786, resulting in a response rate exceeding 99%. All females aged 15 to 49 who were either permanent inhabitants of the designated houses or visitors who had spent the night before in those households, were eligible to participate in the interviews. In this study, we have analyzed the data from 1891 women who had live births two years preceding the survey ( ).
Data collection for the survey was conducted between January 5 to June 22, 2022. The DHS Program’s model questionnaires were modified to address Nepal’s unique demographics and health problems in consultation with various stakeholders, including government departments, agencies, non-governmental organizations, and funders. After the English versions of the survey tools were completed, they were translated into Nepali, Maithili, and Bhojpuri, the three most common languages spoken in Nepal. The translated versions of the questionnaire (household, woman, and man) were then digitized to facilitate data collection using computer-assisted personal interviews (CAPI). Data for the NDHS 2022 was gathered by 19 teams. Each team comprised of a supervisor, one male interviewer, three female interviewers, and a biomarker specialist .
The dependent variables for this study are maternal health service utilization variables, which includes (i) ≥ 4 ANC visits, (ii) ID, and (iii) PNC visit for mother and newborn the first two days of delivery, (iv) combined coverage of ≥ 4 ANC visits and ID, and (v) Continuum of care coverage: combined coverage of ≥ 4 ANC visits, ID, and PNC visit for mother and newborn during the first two days of delivery. Additional information on variables can be obtained from the full report .
Independent variables include place of residence (urban, rural), province (Koshi, Madhesh, Bagmati, Gandaki, Lumbini, Karnali, Sudurpashchim), age at delivery ( < 20 years, 20-34 years and ≥ 35 years), ethnicity (Brahmin/ Chhetri, Dalit, Janajati, Madhesi, other), wealth quintile (poorest, poorer, middle, richer, richest), education (no education, basic, secondary or higher), distance to facility ( < 30 min, 30-59 min, 1-2 hours, and ≥ 2 hours), birth order (one, two, three or more), insurance coverage (no, yes), media exposure-for example newspapers, TV, and radio (no, yes) and internet use (no, yes). To calculate wealth quintiles, households were assigned scores based on the variety of consumer goods they own, such as televisions, bicycles, or cars, as well as housing characteristics like drinking water sources, toilet facilities, and flooring materials. These scores were derived using principal component analysis. National wealth quintiles were then determined by assigning each household a score, ranking individuals within the household population according to these scores, and dividing the population into five equal groups, with each quintile representing 20% of the population .
We conducted data analysis using R version 4.2.0. To accommodate the complex survey design of NDHS 2022, we employed a weighted analysis approach using the “survey” package . Multicollinearity was assessed prior to analysis, with a Variance Inflation Factor (VIF) of less than 10 considered an acceptable threshold for multicollinearity. Categorical variables were expressed as frequencies, percentages, and 95% confidence interval (CI). To determine the relationship between independent variables and maternal health service utilization variables (including ≥ 4 ANC visits, ID, and PNC visit, combined coverage of ≥ 4 ANC visits and ID, and combined coverage of ≥ 4 ANC visits, ID and PNC visit within two days of delivery), we performed univariate and multivariable binary logistic regression analyses. The outcomes of the logistic regression analysis were reported as crude odds ratio (COR) and adjusted odds ratio (AOR), each accompanied by their respective 95% CI.
NDHS 2022 obtained ethical approval from the institutional review board of ICF International, United States of America (Reference number: 180657.0.001.NP.DHS.01) and the ethical review board of Nepal Health Research Council (Reference number: 494/2021). Upon approval of our request for access to data, we downloaded NDHS 2022 dataset from https://dhsprogram.com/data/available-datasets.cfm . Written informed consent was obtained in the original survey. In the case of minors, both the assent as well as consent were obtained before enrollment in the survey.
Out of total participants, 17.37% (95% CI: 15.54, 19.36) of participants at the national level, 15.68% (95% CI: 13.28, 18.42) in urban settings, and 20.59% (95% CI: 18.12, 23.30) in rural settings had age at delivery < 20 years. The highest proportion of participants were Janajati, constituting 30.77% (95% CI: 27.29, 34.48) at the national level, 29.50% (95% CI: 25.10, 34.31) in urban settings, and 33.19% (95% CI: 27.72, 39.15) in rural settings. Regarding education, the proportion of participants having secondary or higher-level education was 47.49% (95% CI: 44.03, 50.97) at the national level, 50.67% (95% CI: 46.02, 55.30) in urban settings, and 41.45% (95% CI: 36.75, 46.31) in rural settings. Approximately 18.49% (95% CI: 15.91, 21.38) of participants at the national level, 17.55% (95% CI: 14.21, 21.47) in urban settings, and 20.28% (95% CI: 16.63, 24.49) in rural settings had no formal education. Additionally, 86.30% (95% CI: 83.55, 88.66) of participants at the national level, 90.67% (95% CI: 87.25, 93.24) in urban settings, and 78.00% (95% CI: 72.85, 82.41) in rural settings reached the nearest health facility within 30 minutes. At the national level, 11.3% (95% CI: 9.23, 13.77) of participants had insurance coverage, 45.76% (95% CI: 42.53, 49.02) had media exposure, and 54.04% (95% CI: 50.72, 57.33) used the internet ( ). Similarly, 79.73% (95% CI: 76.05, 82.98) in urban setting and 82.3% (95% CI: 78.67, 85.43) in rural settings had ≥ 4 ANC visits, 80.93% (95% CI: 77.32, 84.09) in urban setting and 76.39% (95% CI: 72.34, 80.00) in rural settings had ID, 62.92% (95% CI: 59.08, 66.59) in urban settings and 61.87% (95% CI: 57.60, 65.97) in rural settings had a PNC visit within two days of delivery, 67.98% (95% CI: 64.00, 71.72) in urban settings and 66.83% (95% CI: 62.36, 71.02) in rural settings had both ≥ ANC visits and ID, and 51.12% (95% CI: 47.29, 54.93) in urban settings and 50.8% (95% CI: 46.35, 55.25) in rural settings had ≥ 4 ANC visits, ID, and a PNC visit within two days of delivery ( ). Regarding mothers’ age at delivery, 45.88% (95% CI: 39.85, 52.04) of participants with age at delivery < 20 years, 52.48% (95% CI: 49.17, 55.77) of participants with age at delivery 20-34 years, and 43.8% (95% CI: 31.1, 57.37) of participants with age at delivery ≥ 35 years had completed ≥ 4 ANC visits, had ID, and had PNC visit within two days of delivery. Among ethnic groups, 58.19% (95% CI: 53.26, 62.97) completed ≥ 4 ANC visits, ID, and PNC visit within two days of delivery among Brahmin/Chhetri, while the proportion was 40.16% (95% CI: 34.07, 46.57) among Madheshi. Among provinces, the percentage of participants completing all three components of the continuum of care was highest in Gandaki at 60.05% (95% CI: 51.93, 67.66) and lowest in Madhesh at 35.95% (95% CI: 30.22, 42.11). While 70.14% (95% CI: 62.97, 76.44) of participants in the richest wealth quintile completed all components of the continuum of care, the proportion was 39.43% (95% CI: 34.85, 44.2) in the poorest wealth quintile. Similarly, there was variation in coverage by educational level, distance to facility, birth order, insurance coverage, media exposure, and internet use, as presented in . Compared to participants in Koshi province, participants in Sudurpashchim province had higher odds of completing ≥ 4 ANC visits (AOR: 2.55, 95% CI: 1.45, 4.49) and had borderline association with ID (AOR: 1.92, 95% CI: 1.03, 3.58). Madhesh province had lower odds of having ID (AOR: 0.43, 95% CI: 0.24, 0.75) and PNC visit (AOR: 0.57, 95% CI: 0.35, 0.93) within two days of delivery. Bagmati province had lower odds (AOR: 0.61, 95% CI: 0.38, 0.98) for completing PNC visit compared to Koshi province. Regarding urban/rural settings, participants in rural setting had marginally higher odds (AOR: 1.50, 95% CI: 1.10, 2.04) of completion of ≥ 4 ANC visits while variables like ID and PNC visit were not associated ( ). Compared to participants in poorest wealth quintile, participants in richest wealth quintile (AOR: 2.38, 95% CI: 1.09, 5.24) had higher odds of having ≥ 4 ANC visits. Similarly, compared to poorest wealth quintile, participants in the middle (AOR: 1.78, 95% CI:1.15, 2.75), richer (AOR: 2.22, 95% CI: 1.27, 3.88) and richest (AOR: 9.87, 95% CI: 3.58, 27.20) wealth quintile had higher odds of having ID. Participants who had secondary or higher education had higher odds of having ID with AOR of 1.90 (95% CI: 1.24, 2.91). Compared to participants who reside at locations with distance to health facility ≥ 2 hours, participants having distance to facility < 30 minutes (AOR: 2.17, 95% CI: 1.21, 3.91) and 1-2 hours (AOR: 2.69, 95% CI: 1.34, 5.39) had higher odds of completion of ≥ 4 ANC visits. Compared to participants with birth order one, participants with birth order three or more had lower odds of completing ≥ 4 ANC visits (AOR: 0.54, 95% CI: 0.35, 0.83), ID (AOR: 0.27, 95% CI: 0.18, 0.42) and PNC visit (AOR: 0.56, 95% CI: 0.41, 0.76). Among other variables, internet use was associated with completion of ≥ 4 ANC visits (AOR: 1.61, 95% CI: 1.14, 2.29) and having ID (AOR: 1.46, 95% CI: 1.07, 1.98) ( ). Residents of Sudurpashchim province had higher odds of having both ≥ 4 ANC visits and ID (AOR: 2.69, 95% CI: 1.64, 4.43) and completing all three components of care, i.e., ≥ ANC visits + ID + PNC visit (AOR: 1.65, 95% CI: 1.04, 2.61). Similarly, rural residents had marginally higher odds of completing continuum of care compared to urban residents (AOR: 1.33, 95% CI: 1.04, 1.70) ( ). Participants in poorer (AOR: 1.71, 95% CI: 1.23, 2.37), middle (AOR: 1.81, 95% CI: 1.22, 2.69), richer (AOR: 2.65, 95% CI: 1.75, 4.01), and richest (AOR: 5.86, 95% CI: 3.01, 11.40) quintile had higher odds of completing both ≥ 4 ANC visits and ID. Similarly, participants in poorer (AOR: 1.50, 95% CI: 1.07, 2.12), middle (AOR: 1.54, 95% CI: 1.06, 2.23), richer (AOR: 2.04, 95% CI: 1.41, 2.94), and richest (AOR: 2.98, 95% CI: 1.83, 4.83) quintile had higher odds of completing ≥ 4 ANC visits, ID and PNC visit within two days of delivery ( ). Odds of completing continuum of care was higher among those who reside 30-59 minutes of travel time to health facility (AOR: 2.24, 95% CI: 1.03, 4.86) compared to those who reside in location with ≥ 2 hours of travel time. Participants with birth order three or more had lower odds of completing ≥ 4 ANC visits and ID (AOR: 0.37, 95% CI: 0.25, 0.53) and completing all three continuum of care components (AOR: 0.50, 95% CI: 0.36, 0.69). Participants who used internet had higher odds of completing ≥ 4 ANC visits and ID (AOR: 1.62, 95% CI: 1.25, 2.11) and complete all three continuum of care components (AOR: 1.38, 95% CI: 1.09, 1.76) ( ).
Nepal is committed to achieve SDG target of reducing MMR to 70 per 100,000 live birth and NMR to 12 per 1,000 live births by 2030 . As in some other LMICs, considering the current MMR of 151 per 100,000 live births and NMR of 21 per 1000 live births, achieving SDG target seems challenging without accelerating the service utilization across continuum of care [ , , ]. Several policy documents in Nepal, such as Nepal Health Sector Strategic Plan 2023-2030 , SMNH Roadmap 2030 , and ANC and PNC Continuum of Care Guideline , and the NENAP 2016 , have underscored the importance of ensuring a continuum of care for MNH services for achieving maternal and newborn health related targets. These documents have also outlined corresponding strategies and interventions. Despite these efforts, the stagnant NMR and relatively high MMR indicate the need to further improve service utilization across continuum of care. The aim of this study was to assess the status and explore the factors associated with continuum of care for MNH. Among the total participants, 80.6% of participants had ≥ 4 ANC visits which was greater than the coverage level of 53.1% in 2011 , 70.8% in 2016 and 77.9% in 2019 . Similarly, ID rate in our study was 79.4% which was higher than the previous two surveys: 35% in 2011 and 57% in 2016 as per NDHS and 77.5% in 2019 as per Nepal Multiple Indicator Cluster Survey (NMICS) . The coverage of ≥ 4 ANC visits and ID rates in Nepal are more than some of Southeast Asian countries. For instance, in India, the coverage of ≥ 4 ANC visits was 57.9% in 2019-21, in Bangladesh, it was 45.8% in 2017-18, and in Pakistan, it stood at 52.8% . Nepal also has higher ID rate than Bangladesh, where the rate was 65% , and in Pakistan, where the rate was 66% . However, the ID in this study was lower than that in India where it was 89% . Regarding PNC, 62.56% of the participants had PNC visits for both mother and newborn. In the previous NDHS survey conducted in 2016, 57% of women reported having received a PNC in the first two days of delivery . Similarly, the PNC visit within two days of delivery was 69.4% in the MICS 2019 . The recent DHS survey conducted in Pakistan and Bangladesh have reported lower coverage of PNC compared to Nepal with 60% in Pakistan and 55% in Bangladesh . However, in India, PNC visit in the first two days of delivery was 82% which is higher than this study. Steady increase in coverage of ≥ 4 ANC visits, ID and PNC service could be the result of policy commitment Nepal has on MNH services in last few decades. With the overarching goal of enhancing MNH outcomes across the country and to overcome financial barrier in service utilization, Nepal initiated maternity incentive scheme in 2005. The scheme initially incentivized transport for facility-based deliveries in 25 districts with low Human Development Index which later evolved into the nationwide Aama Program in 2009, encompassing delivery care. Subsequent policy changes integrated ANC incentives in 2012. The program offers financial incentives to both women and health facilities, with reimbursement rates varying based on the type of service provided and the ecological belt . The Maternity incentive scheme serves as a motivating factor for expanding service coverage, benefiting both mothers and health facilities. Furthermore, the free newborn care program was launched which aimed to reduce financial barriers to accessing newborn care services. This incentive-driven approach might have also contributed to overall progress in service utilization. Although a good proportion of participants completed ≥ 4 ANC visits, the proportion steadily declined, with only 51% completing all three components of the continuum of care for MNH services. While Nepal government has made significant progress in improving MNH services like ANC visits, ID and PNC checkups, additional efforts are needed to ensure that the chain is maintained across continuum of care. Counselling during 1 st ANC visit for the follow up visits, and for ID could be useful strategy which is already reflected in policies of government of Nepal. Ensuring quality of care and gaining trust would require regular supervision and quality assurance activities at health facility level. Establishing inter-institutional linkages between federal, provincial and local government health facilities has the potential to strengthen referral system, enhance the management of complex deliveries and address specific MNH conditions. Such linkages could contribute to saving lives by improving care for both mother and newborn while also fostering greater trust in facilities. Encouraging and supporting Female Community Health Volunteers (FCHVs) in maintaining a list of pregnant women along with their contact information, improving the documentation of personal details in registers, and using mobile phone systems to send appointment reminders to mothers could also improve service utilization across continuum of care. There are provincial differences in coverage of ≥ 4 ANC visits, ID, PNC visit, ≥ ANC visits + ID and all three components of continuum of care considered in the study. Women residing in Sudurpashchim province had higher odds of completing ≥ 4 ANC visits, had borderline association with ID and had higher odds completing all three components of care, i.e., ≥ ANC visits + ID + PNC visit. Madhesh province had lower odds of having ID and PNC visit within two days of delivery. Variations based on administrative divisions and geographic area were also reported in multiple previous studies in different settings like India , Pakistan , and Ethiopia . Factors like variation in socio-economic and developmental status, service availability and readiness across facilities in respective region/provinces, quality of the service may be partially responsible for provincial differences in utilization of different components of continuum of care as well as specific service in isolation. Nepal had massive changes in health system with three tiers of governments being functional and local governments taking responsibility of basic health services including MNH services. The transfer of authority to local level governments provides flexibility to tailor interventions or strategies to local needs. This may have some impacts on differential service utilization and coverage status across the provinces, resulting from innovations tried at local levels. Differences based on provinces and geography could have been observed also because of differences in program implementation strategies, management efficiency and socio-cultural variation among provinces. Additional studies, preferably qualitative, are needed to gain detailed understanding of the reasons causing variation of the service utilization across provinces. For most services, our study shows disparity in coverage of service based on wealth quintile. Other studies conducted in Ethiopia , India , and Zambia found that the household wealth quintile was positively associated with ID. Similar to other studies conducted in Nepal , our study also identified that women of higher wealth quintile were more likely than women with lower wealth quintile to have ID. Similarly, PNC visit within two days of delivery was found lower in poorer wealth quintile in some of the previous studies conducted in Nepal and Ethiopia which align with our study. Having wealth or a higher socio-economic status often means having a higher disposable income, which increases the ability to bear both direct and indirect expenses related to service utilization. Additionally, it provides better access to information and a stronger social network, all of which are positively associated with increased service utilization. Women with high financial standing may have the freedom to decide on the use of household incomes. Persisting disparities in service utilization despite the availability of free maternal health services and incentives raise concern about the effectiveness of the program and point out the need to reevaluate the program. The expansion of community-based services and awareness campaigns, bolstering primary healthcare facilities, and implementation of other measures targeting the economically marginalized are of utmost importance. SMNH roadmap recommends developing a transition plan to revise financial incentives for delivery care and ANC visits with the objective of shifting away from universal incentives to targeting those who currently do not use services, such as the poorest or those in remote areas, while continuing incentives for women in regions with low ANC and ID rates . However, in-depth studies on the potential impact of changes in incentives program is needed before policy or programmatic changes. Our findings show significant differences in ID based on maternal education level in line with the other studies conducted in Nepal and other parts of the world [ , , , ]. This could be because education improves women’s understanding, facilitates their information access and enables a thorough understanding of advocacy messages communicated through media, the internet, and healthcare providers. Government of Nepal also has the School Health and Nursing Service Program, guided by the National Health Policy, 2019, which has been implemented in 1,011 government schools . Information on adolescent sexual and reproductive health are also covered in the school health program, which could also have served as source of information apart from the content covered in curriculum. Better possession of information about the need of services, where the services are available and that the services are available free of cost with travel incentives may have resulted in better coverage of ID among those who had secondary or higher-level education. However, no association was observed between the completion of ≥ 4 ANC visits, PNC, and the continuum of care with educational level in adjusted analysis in our study. This could be an area for further research, especially qualitative studies aimed at clearly identifying the factors that limit women’s access to these services despite having the necessary information. Our findings are based on further analysis from nationally representative data collected in globally standardized tool, taking into account the recent federal structure of Nepal. As the survey has used standardized definition for variables, and has used globally accepted survey methodology, findings are comparable with findings from other countries. However, as the survey was undertaken while COVID-19 pandemic was ongoing, there could be some over or underestimation in case of some variables.
Several factors influenced MNH service utilization and continuum of care, including wealth quintile, education level, place of residence, and internet use. Addressing these disparities is imperative to ensure that all groups of the population can benefit equally from improved MNH services. In addition, establishing inter-linkage between different services and strengthening referral mechanism across different facilities can accelerate progress toward MNH goals, reducing preventable deaths and ensuring the well-being of mothers and newborns. Nepal has recently transitioned to federal structure with 7 provincial and 753 local governments, having decision making authority. This could be an opportunity to test innovative strategies or interventions to increase coverage of continuum of care which can be subsequently scaled up throughout the country.
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Genetic risk factors associated with ocular perfusion pressure in primary open-angle glaucoma | c9daa55f-5580-4ce6-bfd3-9da13664365f | 11934579 | Pathologic Processes[mh] | Primary open-angle glaucoma (POAG), the leading cause of irreversible vision loss, is a progressive optic neuropathy characterised by degeneration of retinal ganglion cells and their axons; it affects approximately 65 million people worldwide . Alterations in posterior displacement of the lamina cribrosa with blockade of axoplasmic flow are putative features of POAG ; however, its precise pathogenesis remains to be elucidated owing to multiple risk factors involved in its pathophysiology [ , – ]. The proposed risk factors for POAG include ageing, elevated intraocular pressure (IOP), vascular factors, genetic factors, and ocular phenotypes, such as corneal hysteresis (CH), corneal resistance factor (CRF), and refractive error (RE) [ , , ]. There has been tremendous growth in our understanding of glaucoma genetics over the past decade, with over 125 genes identified as being associated with POAG and more than 100 novel single nucleotide polymorphisms (SNPs) being linked to IOP alone . However, except for IOP, genetic studies on POAG risk factors and their effects are lacking. This is crucial because glaucoma is primarily a complex polygenic disease. Several studies have suggested that insufficient ocular blood flow is a risk factor for POAG [ – ], consistent with the vascular theory of glaucoma [ – ]. Although autoregulation of blood vessels might compensate for reduced blood flow to a certain extent , several studies have shown that a lower mean ocular perfusion pressure (MOPP) may be a risk factor for POAG [ , , ]. Some studies have shown a significant association between MOPP and POAG [ , , ], and fluctuations in MOPP are significantly associated with POAG ; however, others have reported a non-significant or limited impact of MOPP on POAG risk . Furthermore, a statistically significant correlation does not indicate a causal relationship, and therefore, a prospective cohort study is necessary to improve our understanding of the effect of MOPP on POAG. Only a few studies have reported a causal association between MOPP and POAG . Investigating MOPP as a risk factor for POAG using genetic data could unveil novel findings for POAG pathology with respect to vascular theory . Mendelian randomisation (MR) is a genetic epidemiological technique that uses genetic variants associated with potential risk factors as instrumental variables (IVs) to assess the causal effects on disease outcomes . In contrast to observational studies, MR analysis uses genetic variants distributed independently in the population and supports causal interference on the effects of risk factors. Previous studies using MR [ – ] have shown that ocular risk factors, such as CH, CRF, IOP, and RE, have a genetic causal association with POAG. To date, no genome-wide association study (GWAS) or MR analysis has been conducted for MOPP. Considering MOPP as a risk factor, it might help unveil currently unknown mechanisms of POAG development. In the present study, we aimed to elucidate representative genetic features of MOPP and investigate whether MOPP is a causal factor for POAG, using MR analysis. Considering the results of a recent study that showed that deep ocular phenotypes such as CH, CRF, IOP, and RE to be associated with the genetic burden in POAG , we analysed these phenotypes using mediation and multivariable (MV) MR analysis. We adopted a two-stage approach, which included the following: (1) Discovery of genetic variants related to ocular risk factors of POAG using GWAS. Since large biobanks such as the UK Biobank (UKBB) do not provide summary information on MOPP, we needed to perform GWAS on primary data after obtaining study permission from UKBB (project ID: 96390). (2) Based on the first stage, identification of the causal association of MOPP with POAG using two-sample (TS) MR and MVMR. This study followed the Strengthening and Reporting of Genetic Association Studies (STREGA) guidelines, an extension of the STROBE statement, to ensure robust and transparent reporting of genetic association findings .
Study design and sample phenotype The study protocol was approved by the Institutional Review Board of Veterans Health Service Medical Centre (IRB No. 2022-03-034). The need for informed consent was waived owing to the retrospective nature of the study; the data were anonymised and de-identified. In addition, the UKBB committee approved the project (project ID: 96390) and material transfer agreement. Among the approximately 500,000 samples from UKBB, our analysis included 459,195 individuals of European ancestry, excluding those of other ethnicities. In this study, CH, CRF, IOP, RE, and MOPP were clinically defined as follows: (1) CH (data field id 5256 and 5264) and (2) CRF (data field id 5257 and 5265) were measured using a Reichert Ocular Response Analyzer (Reichert, Inc., Depew, NY) according to a predetermined protocol (available at http://biobank.ctsu.ox.ac.uk/crystal/refer.cgi?id¼100236 ) provided by UKBB. (3) IOP measurements were based on the corneal compensated IOP (data field id 5254 and 5262) and the maximum value in two eyes. (4) RE (data field id 7390 and 7389) was measured using a Tomey RC 5000 auto-refractor (Tomey GmbH, Germany). The mean spherical equivalent (MSE) was calculated using the following formula: spherical power + (0.5 × cylindrical power). The worse MSE values (across the left and right eyes) were used in our analysis. (5) MOPP was calculated from IOP using the corneal compensated IOP and the maximum value in two eyes ( [12pt]{minimal}
$$\:=\:[+\:(-)]-$$ , where SBP and DBP denote the systolic and diastolic blood pressure, respectively). UKBB provides two forms of IOP: the Goldmann applanation tonometer-measured IOP (GAT-IOP, data field id 5255 and 5263) and corneal compensated IOP (IOPcc, data field id 5254 and 5262). MOPP can be computed by selecting the average IOP (IOPmean) and highest IOP (IOPmax) of both eyes. Because an individual has two eyes, four types of IOP can be calculated: GAT-IOPmean, GAT-IOPmax, IOPcc_mean, and IOPcc_max; the same applies to MOPP. GWAS analysed four categories of IOP phenotypes, although no significant difference was observed between them owing to their high similarity in the pilot analysis. Because IOPcc is theoretically ideal , and IOPmax is better than IOPmean for characterising POAG and the systemic factors that demonstrate the features of IOP, this study defined IOPcc_max as the ‘IOP’, which was used for calculating the MOPP. Genotyping and imputation Genotyping was performed using the UKBB data, which identified 805,426 single-nucleotide polymorphisms in approximately 500,000 individuals . Sample and SNP quality controls were performed. Samples with sex inconsistencies or call rates of < 95% were excluded from the analysis. SNPs were excluded from the analysis if they had > 5% missingness, a minor allele frequency of < 0.05, or a deviation from the Hardy–Weinberg equilibrium with a P < 1.00 × 10 − 6 . After quality control, imputation was performed with IMPUTE4 using UK10K, 1000 Genomes Phase 3, and the Haplotype Reference Consortium panel as reference data . Therefore, 80,863,435 imputed SNPs were used in the analysis (Fig. a). Gene-based analysis of GWAS Genome-wide analysis of CH, CRF, IOP, RE, and MOPP were conducted using a linear model with PLINK . Age, sex, and 10 principal component scores were included as covariates. We performed a gene-based analysis using MAGMA (‘generalised gene-set analysis of GWAS data’) with our GWAS results as input data . Gene-level analysis was conducted using the SNP-wise mean model (default parameters) to compute P -values. European samples from 1000 Genomes Phase 3 were used as a reference panel to adjust for linkage disequilibrium (LD) between SNPs. The input SNPs were mapped to 19,230 protein-coding genes for CH, CRF, and RE and 19,273 protein-coding genes for IOP and MOPP. The significant genes were considered to be those with a false discovery rate (FDR)-adjusted P -value ( P FDR ) below 0.05, using the Benjamini–Hochberg procedure . Quantile–quantile (QQ), Manhattan, and colocalisation plots were generated using LocusFocus for the GWAS results . To eliminate the possible confounding effects of population stratification, we calculated the genomic inflation factor (GIF). A GIF value close to 1 indicates the absence of genomic inflation. Furthermore, to investigate the differential expression of significant genes in POAG, we utilised a dataset from the GEO database (accession no.: GSE27276 ) . Differential gene expression analysis was performed using the R package ‘limma’. Functional annotation and pathway enrichment analysis Functional annotation and pathway enrichment analysis were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool . The gene list was analysed for associations with diseases (DisGeNET) , functional annotations, and gene ontology (GO) terms, as well as enrichment in biological pathways. Specifically, the analysis included the biological process, cellular component, and molecular function categories for GO and pathway databases including KEGG, Reactome, and WikiPathways. An enrichment analysis was conducted using Fisher’s exact test, and the results were considered statistically significant when the adjusted P -value was less than 0.05. Heritability and genetic correlation (GC) Heritability of CH, CRF, IOP, RE, and MOPP and their GCs with POAG were estimated using GWAS summary statistics from bivariate LD score regression . The LD reference panel for analysis was based on European ancestry information obtained from the 1000 Genomes Project . Instrumental variable selection The instrumental variables for CH, CRF, IOP, RE, and MOPP were selected from SNPs that showed significant associations in GWAS using the UKBB datasets. The outcome dataset was the summary statistics of a genome-wide meta-analysis of 16,677 cases and 199,580 controls from European samples (Fig. b) . To avoid sample overlap, we used GWAS summary statistics from the European samples, excluding the UKBB subjects. To select appropriate IVs for the MR analysis, we utilized the ‘ld_clump’ function from the R package ‘ieugwasr’. This function was applied to perform LD pruning, where variants were excluded based on whether they exhibited strong LD with other variants. Specifically, variants within a 10,000 kb distance or with an LD (r²) greater than 0.001 were removed. Additionally, we excluded variants that were absent from the reference panel to ensure that only high-quality, accessible variants were included. Variants that were nominally associated with POAG ( P < 0.05) were also removed to minimize any potential bias in the MR analysis. After these quality control steps, we identified the genome-wide SNPs associated with CH, CRF, IOP, RE, and MOPP. Only the SNPs that passed these filtering criteria were selected as IVs for the MR analysis. These steps ensure that the final set of IVs used in the MR analysis are independent, non-pleiotropic, and genetically robust, providing reliable results for assessing causal relationships. F -statistics ( F ) indicated instrumental strength, and an F value larger than 10 suggested that the analysis was unlikely to be affected by weak instrument bias . To detect pleiotropic outlier SNPs, we used Cochran’s Q-test, Rucker’s Q’ statistic, and MR-PRESSO test . Additionally, the violation extent of the ‘NO Measurement Error’ (NOME) assumption was quantified using I 2 statistics, where I 2 values above 90 indicated less estimate dilution in the MR analysis . Univariable TSMR bidirectional analysis Univariable TSMR was used to test the causal inferences between CH, CRF, IOP, RE, MOPP, and POAG. We performed traditional and robust MR analysis using different models. First, we used inverse variance-weighted (IVW) analysis to provide unbiased estimates when all IV assumptions were satisfied . We conducted a leave-one-out sensitivity analysis to evaluate the robustness of our results and to detect any potential influence of individual genetic variants on the overall estimates. By iteratively excluding each SNP, we assessed whether the exclusion of any variant substantially altered the causal estimates, which would indicate any potential heterogeneity or pleiotropy. The MR-Egger method was employed to account for pleiotropic effects and estimate the causal effect . In addition, we applied three extensions to the robust MR methods to relax the assumptions by considering multiple IVs and allowing some to be invalid: (1) robust regression, (2) penalised weights, and (3) penalised robust regression . We conducted reverse MR analysis by switching the roles of the exposure and outcome. MR analysis was performed using the ‘TwoSampleMR’ and ‘MendelianRandomization’ packages in R software. The significance threshold for MR was set at 0.01 (0.05/5), considering Bonferroni correction. In this study, we primarily used the robust and penalized robust MR methods, which focus on selecting the most valid instrumental variables (IVs) and excluding unreliable ones, ensuring more significant and reliable estimates. These methods are particularly effective in minimizing bias from invalid IVs, making them suitable for our analysis. Additionally, weighted median and mode-based MR methods were also applied as complementary approaches to perform further sensitivity analysis. These methods allow for more flexibility in the inclusion of IVs, offering alternative estimates under different assumptions regarding pleiotropy. While these methods are robust to certain violations, such as the presence of invalid IVs, they may be less powerful in the presence of heterogeneity or pleiotropy, which could lead to non-significant results. In our study, the results from the weighted median and mode-based methods did not reach statistical significance, possibly due to the inclusion of less robust IVs or the impact of pleiotropy. MV TSMR analysis MVMR extends univariable (UV) MR by including all phenotypes in the same model and jointly estimating their causal effects on POAG risk. The IVs selected for each phenotype in the UVMR analysis were directly carried over to the MVMR models without modification. Specifically, the same set of SNPs identified as IVs in the UVMR for each individual phenotype was used in the MVMR analysis. Through factor analysis, we selected the variables to include in the MVMR models (Model 1 = CH, IOP, and RE; Model 2 = CH, MOPP, and RE). While UVMR allows us to examine the causal effect of each phenotype individually, MVMR allows for the simultaneous assessment of multiple phenotypes, accounting for potential correlations among the IVs related to each phenotype. This correlation between IVs is explicitly incorporated into the MVMR analysis to ensure the robustness and accuracy of the results. To infer causal effects using MVMR, the analysis was performed using the ‘MVMR’ package in R software using the MV-IVW and MVMR-Egger methods. Mediation analysis using structural equation models To estimate the direct and indirect effects using the structural equation modelling (SEM) framework, we assumed a mediation analysis model corresponding to the directed acyclic graph (DAG) in Fig. . To reinforce the theoretical foundation of the proposed pathways, additional relevant studies were systematically reviewed and incorporated. Specifically, the study by Wong et al. was included to support the “IOP/MOPP→RE→POAG” pathway . Furthermore, the study by Deol et al. highlighting the physiological interplay in the “IOP/MOPP→CH→POAG” pathway was also reviewed and integrated . These studies were selected based on their relevance to the proposed mechanisms and their contributions to understanding the physiological processes involved in POAG. We evaluated the mediation effects of CH or RE on the causal pathway from IOP/MOPP to POAG using the ‘sem’ function from the R package ‘lavvan’.
The study protocol was approved by the Institutional Review Board of Veterans Health Service Medical Centre (IRB No. 2022-03-034). The need for informed consent was waived owing to the retrospective nature of the study; the data were anonymised and de-identified. In addition, the UKBB committee approved the project (project ID: 96390) and material transfer agreement. Among the approximately 500,000 samples from UKBB, our analysis included 459,195 individuals of European ancestry, excluding those of other ethnicities. In this study, CH, CRF, IOP, RE, and MOPP were clinically defined as follows: (1) CH (data field id 5256 and 5264) and (2) CRF (data field id 5257 and 5265) were measured using a Reichert Ocular Response Analyzer (Reichert, Inc., Depew, NY) according to a predetermined protocol (available at http://biobank.ctsu.ox.ac.uk/crystal/refer.cgi?id¼100236 ) provided by UKBB. (3) IOP measurements were based on the corneal compensated IOP (data field id 5254 and 5262) and the maximum value in two eyes. (4) RE (data field id 7390 and 7389) was measured using a Tomey RC 5000 auto-refractor (Tomey GmbH, Germany). The mean spherical equivalent (MSE) was calculated using the following formula: spherical power + (0.5 × cylindrical power). The worse MSE values (across the left and right eyes) were used in our analysis. (5) MOPP was calculated from IOP using the corneal compensated IOP and the maximum value in two eyes ( [12pt]{minimal}
$$\:=\:[+\:(-)]-$$ , where SBP and DBP denote the systolic and diastolic blood pressure, respectively). UKBB provides two forms of IOP: the Goldmann applanation tonometer-measured IOP (GAT-IOP, data field id 5255 and 5263) and corneal compensated IOP (IOPcc, data field id 5254 and 5262). MOPP can be computed by selecting the average IOP (IOPmean) and highest IOP (IOPmax) of both eyes. Because an individual has two eyes, four types of IOP can be calculated: GAT-IOPmean, GAT-IOPmax, IOPcc_mean, and IOPcc_max; the same applies to MOPP. GWAS analysed four categories of IOP phenotypes, although no significant difference was observed between them owing to their high similarity in the pilot analysis. Because IOPcc is theoretically ideal , and IOPmax is better than IOPmean for characterising POAG and the systemic factors that demonstrate the features of IOP, this study defined IOPcc_max as the ‘IOP’, which was used for calculating the MOPP.
Genotyping was performed using the UKBB data, which identified 805,426 single-nucleotide polymorphisms in approximately 500,000 individuals . Sample and SNP quality controls were performed. Samples with sex inconsistencies or call rates of < 95% were excluded from the analysis. SNPs were excluded from the analysis if they had > 5% missingness, a minor allele frequency of < 0.05, or a deviation from the Hardy–Weinberg equilibrium with a P < 1.00 × 10 − 6 . After quality control, imputation was performed with IMPUTE4 using UK10K, 1000 Genomes Phase 3, and the Haplotype Reference Consortium panel as reference data . Therefore, 80,863,435 imputed SNPs were used in the analysis (Fig. a).
Genome-wide analysis of CH, CRF, IOP, RE, and MOPP were conducted using a linear model with PLINK . Age, sex, and 10 principal component scores were included as covariates. We performed a gene-based analysis using MAGMA (‘generalised gene-set analysis of GWAS data’) with our GWAS results as input data . Gene-level analysis was conducted using the SNP-wise mean model (default parameters) to compute P -values. European samples from 1000 Genomes Phase 3 were used as a reference panel to adjust for linkage disequilibrium (LD) between SNPs. The input SNPs were mapped to 19,230 protein-coding genes for CH, CRF, and RE and 19,273 protein-coding genes for IOP and MOPP. The significant genes were considered to be those with a false discovery rate (FDR)-adjusted P -value ( P FDR ) below 0.05, using the Benjamini–Hochberg procedure . Quantile–quantile (QQ), Manhattan, and colocalisation plots were generated using LocusFocus for the GWAS results . To eliminate the possible confounding effects of population stratification, we calculated the genomic inflation factor (GIF). A GIF value close to 1 indicates the absence of genomic inflation. Furthermore, to investigate the differential expression of significant genes in POAG, we utilised a dataset from the GEO database (accession no.: GSE27276 ) . Differential gene expression analysis was performed using the R package ‘limma’.
Functional annotation and pathway enrichment analysis were performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool . The gene list was analysed for associations with diseases (DisGeNET) , functional annotations, and gene ontology (GO) terms, as well as enrichment in biological pathways. Specifically, the analysis included the biological process, cellular component, and molecular function categories for GO and pathway databases including KEGG, Reactome, and WikiPathways. An enrichment analysis was conducted using Fisher’s exact test, and the results were considered statistically significant when the adjusted P -value was less than 0.05.
Heritability of CH, CRF, IOP, RE, and MOPP and their GCs with POAG were estimated using GWAS summary statistics from bivariate LD score regression . The LD reference panel for analysis was based on European ancestry information obtained from the 1000 Genomes Project .
The instrumental variables for CH, CRF, IOP, RE, and MOPP were selected from SNPs that showed significant associations in GWAS using the UKBB datasets. The outcome dataset was the summary statistics of a genome-wide meta-analysis of 16,677 cases and 199,580 controls from European samples (Fig. b) . To avoid sample overlap, we used GWAS summary statistics from the European samples, excluding the UKBB subjects. To select appropriate IVs for the MR analysis, we utilized the ‘ld_clump’ function from the R package ‘ieugwasr’. This function was applied to perform LD pruning, where variants were excluded based on whether they exhibited strong LD with other variants. Specifically, variants within a 10,000 kb distance or with an LD (r²) greater than 0.001 were removed. Additionally, we excluded variants that were absent from the reference panel to ensure that only high-quality, accessible variants were included. Variants that were nominally associated with POAG ( P < 0.05) were also removed to minimize any potential bias in the MR analysis. After these quality control steps, we identified the genome-wide SNPs associated with CH, CRF, IOP, RE, and MOPP. Only the SNPs that passed these filtering criteria were selected as IVs for the MR analysis. These steps ensure that the final set of IVs used in the MR analysis are independent, non-pleiotropic, and genetically robust, providing reliable results for assessing causal relationships. F -statistics ( F ) indicated instrumental strength, and an F value larger than 10 suggested that the analysis was unlikely to be affected by weak instrument bias . To detect pleiotropic outlier SNPs, we used Cochran’s Q-test, Rucker’s Q’ statistic, and MR-PRESSO test . Additionally, the violation extent of the ‘NO Measurement Error’ (NOME) assumption was quantified using I 2 statistics, where I 2 values above 90 indicated less estimate dilution in the MR analysis .
Univariable TSMR was used to test the causal inferences between CH, CRF, IOP, RE, MOPP, and POAG. We performed traditional and robust MR analysis using different models. First, we used inverse variance-weighted (IVW) analysis to provide unbiased estimates when all IV assumptions were satisfied . We conducted a leave-one-out sensitivity analysis to evaluate the robustness of our results and to detect any potential influence of individual genetic variants on the overall estimates. By iteratively excluding each SNP, we assessed whether the exclusion of any variant substantially altered the causal estimates, which would indicate any potential heterogeneity or pleiotropy. The MR-Egger method was employed to account for pleiotropic effects and estimate the causal effect . In addition, we applied three extensions to the robust MR methods to relax the assumptions by considering multiple IVs and allowing some to be invalid: (1) robust regression, (2) penalised weights, and (3) penalised robust regression . We conducted reverse MR analysis by switching the roles of the exposure and outcome. MR analysis was performed using the ‘TwoSampleMR’ and ‘MendelianRandomization’ packages in R software. The significance threshold for MR was set at 0.01 (0.05/5), considering Bonferroni correction. In this study, we primarily used the robust and penalized robust MR methods, which focus on selecting the most valid instrumental variables (IVs) and excluding unreliable ones, ensuring more significant and reliable estimates. These methods are particularly effective in minimizing bias from invalid IVs, making them suitable for our analysis. Additionally, weighted median and mode-based MR methods were also applied as complementary approaches to perform further sensitivity analysis. These methods allow for more flexibility in the inclusion of IVs, offering alternative estimates under different assumptions regarding pleiotropy. While these methods are robust to certain violations, such as the presence of invalid IVs, they may be less powerful in the presence of heterogeneity or pleiotropy, which could lead to non-significant results. In our study, the results from the weighted median and mode-based methods did not reach statistical significance, possibly due to the inclusion of less robust IVs or the impact of pleiotropy.
MVMR extends univariable (UV) MR by including all phenotypes in the same model and jointly estimating their causal effects on POAG risk. The IVs selected for each phenotype in the UVMR analysis were directly carried over to the MVMR models without modification. Specifically, the same set of SNPs identified as IVs in the UVMR for each individual phenotype was used in the MVMR analysis. Through factor analysis, we selected the variables to include in the MVMR models (Model 1 = CH, IOP, and RE; Model 2 = CH, MOPP, and RE). While UVMR allows us to examine the causal effect of each phenotype individually, MVMR allows for the simultaneous assessment of multiple phenotypes, accounting for potential correlations among the IVs related to each phenotype. This correlation between IVs is explicitly incorporated into the MVMR analysis to ensure the robustness and accuracy of the results. To infer causal effects using MVMR, the analysis was performed using the ‘MVMR’ package in R software using the MV-IVW and MVMR-Egger methods.
To estimate the direct and indirect effects using the structural equation modelling (SEM) framework, we assumed a mediation analysis model corresponding to the directed acyclic graph (DAG) in Fig. . To reinforce the theoretical foundation of the proposed pathways, additional relevant studies were systematically reviewed and incorporated. Specifically, the study by Wong et al. was included to support the “IOP/MOPP→RE→POAG” pathway . Furthermore, the study by Deol et al. highlighting the physiological interplay in the “IOP/MOPP→CH→POAG” pathway was also reviewed and integrated . These studies were selected based on their relevance to the proposed mechanisms and their contributions to understanding the physiological processes involved in POAG. We evaluated the mediation effects of CH or RE on the causal pathway from IOP/MOPP to POAG using the ‘sem’ function from the R package ‘lavvan’.
Clinical characteristics of the study population Sample data from 459,195 European individuals from UKBB, and, of them, 98,661 samples with ocular phenotypes were used for the analysis, and the phenotypic characteristics are summarised in Fig. ; Table . The proportion of male participants was 45.7%, and the mean age of the population was 56.76 years. The mean CH, CRF, IOP, MOPP, and RE were 11.41 mmHg, 11.39 mmHg, 17.36 mmHg, 50.19 mmHg, and − 0.64 dioptre, respectively. GWAS analysis We conducted GWAS using 97,338 samples for CH, CRF, and RE and 96,743 samples for IOP and MOPP with 80,863,435 imputed SNPs. The GIF was 1.107 for CH, 1.018 for CRF, 1.154 for IOP, 1.136 for RE, and 1.162 for MOPP, suggesting no evidence of inflation in the GWAS results. The Manhattan and QQ plots for the gene-based analysis of GWAS allowed visualisation of the distribution of several association signals across the genome and enabled the identification of significant loci associated with the traits of interest [Supplementary Fig. (a)–(e)]. LD clumping was conducted with significant SNPs, whereas SNPs with LD with other variants or those absent from the European LD reference panel were excluded from the results. Among the significant genes for each phenotype, we identified candidate marker genes based on differential expression for the retinal ganglion cell type (Table ). We found a total of 14 marker genes [ MAGI1 ( P FDR = 1.14 × 10 − 3 ) and ZFHX3 ( P FDR = 4.62 × 10 − 2 ) for CH; RBMS3 ( P FDR = 1.55 × 10 − 2 ), MAGI1 ( P FDR = 3.98 × 10 − 3 ), and TRHDE ( P FDR = 4.02 × 10 − 2 ) for CRF; TUBB3 ( P FDR = 3.18 × 10 − 2 ) and POU6F2 ( P FDR = 2.08 × 10 − 2 ) for IOP; RBMS1 ( P FDR = 2.36 × 10 − 2 ), ZMAT4 ( P FDR = 2.99 × 10 − 5 ), GRIA4 ( P FDR = 2.62 × 10 − 13 ), LRFN5 ( P FDR = 2.20 × 10 − 6 ), RBFOX1 ( P FDR = 4.70 × 10 − 3 ), and GRIN2A ( P FDR = 2.18 × 10 − 3 ) for RE; CEP85L ( P FDR = 1.53 × 10 − 3 ) and RBPMS (P FDR = 8.13 × 10 − 5 ) for MOPP]. Of them, POU6F2 have been reported as a significant marker associated with IOP in the previous study . The co-localisation figures from LocusFocus for each gene are shown in Supplementary Fig. [in the artery (aorta and coronary), heart (left ventricle and atrial appendage) and whole blood tissue]. Furthermore, the decreased expression of MAGI1 ( P = 4.33 × 10 − 3 ), ZFHX3 ( P = 2.29 × 10 − 2 ), TRHDE ( P = 6.64 × 10 − 2 ), ZMAT4 ( P = 1.06 × 10 − 2 ), LRFN5 ( P = 3.29 × 10 − 2 ), RBFOX1 ( P = 3.33 × 10 − 2 ), GRIN2A ( P = 3.36 × 10 − 2 ), and RBPMS ( P = 9.57 × 10 − 3 ) was associated with POAG in the GEO dataset ( GSE27276 ) (Table ). The functinal annotation and pathway analysis revealed significant enrichment of several biological pathways and molecular functions in neurobiology (Supplementary Table ). Heritability and GC The estimated heritability for CH, CRF, IOP, RE, and MOPP was 7.4%, 9.5%, 6.9%, 21.0%, and 12.1%, respectively, without inflation in the GWAS results (Table ). We identified strong shared genetics for CRF, IOP, RE, and MOPP with POAG (GC = − 0.264, P = 4.58 × 10 − 7 for CRF; GC = − 0.665, P = 8.03 × 10 − 25 for IOP; GC = 0.130, P = 3.20 × 10 − 3 for RE; GC = 0.325, P = 3.08 × 10 − 16 for MOPP). No genetic association was observed between CH and POAG (GC = 0.033, P = 0.552) (Table ). Univariable TSMR bidirectional analysis A total of 44, 47, 10, 85, and 8 genome-wide SNPs were associated with CH, CRF, IOP, RE, and MOPP, respectively, and were identified as IVs for MR analysis (Supplementary Table ). The leave-one-out analysis revealed that no single genetic variant had a disproportionately large effect on the overall estimates, supporting the robustness of our findings and the absence of significant heterogeneity or pleiotropy. The results of this analysis are presented in Supplementary Fig. . For reverse MR, 11, 17, 5, 9, and 9 genome-wide SNPs associated with POAG were used for CH, CRF, IOP, RE, and MOPP, respectively (Supplementary Table ). All IVs for bidirectional MR were entirely unrelated to the outcomes ( P > 0.05). We found no evidence of weak instrument bias ( F > 10), no violation of the NOME assumption ( I 2 > 90), and no heterogeneity or outlier pleiotropy ( P of Q-test, Q’-test, and MR-PRESSO > 0.05) (Supplementary Table ). The correlation matrix of instruments for all variables (Fig. ) illustrates the relationships among the genetic instruments, which is essential for evaluating the assumption of instrument independence in the subsequent MVMR analysis. Based on the analysis from the robust and penalised robust MR methods (IVW and MR-Egger), which downweighs outliers, we observed that CH [odds ratio (OR) = 0.998 with P < 0.001 for all robust methods], CRF (OR = 0.998 with P < 0.001 for all robust methods), and MOPP (OR = 0.998 with P < 0.001 for all robust methods) were inversely associated with POAG, whereas IOP (OR = 1.002 with P = 0.009 for robust and penalised robust IVW methods) was positively associated with POAG (Fig. and Supplementary Tables – ). However, the association was null when non-robust IVW and MR-Egger methods were used. Notably, in the reverse MR-Egger analysis, presence of POAG had significant association with MOPP (OR = 4.066 with P = 0.001 for MR-Egger and penalised MR-Egger; OR = 3.971 with P < 0.001 for robust MR-Egger and penalised robust MR-Egger) (Fig. and Supplementary Table ). The intercepts of various MR-Egger methods suggested the presence of horizontal pleiotropy, suggesting that some IVs may influence MOPP through pathways other than POAG. Violation of MR assumption, as is likely in this case, can lead to biased or unreliable estimates. Thus, the findings concerning the impact of POAG on MOPP should be interpreted with caution. MV MR analysis Since the correlation matrix reveals a strong positive correlation between CH and CRF, a negative correlation between CH and IOP, and another negative correlation between IOP and MOPP (Fig. ). Next, to control for pleiotropic effects, based on the results of factor analysis, we performed an MVMR analysis including CH, IOP, and RE jointly using Model 1, and CH, MOPP and RE jointly using Model 2 (Table ). In the univariable MR analysis, CH was significant, whereas RE was not; however, in the MVMR analysis, CH ( Ps > 0.05) was no longer significant and RE (OR = 0.934 with P < 0.001 in MV-IVW; OR = 0.929 with P < 0.001 in MV-MR-Egger) was significant in Model 1 (Fig. and Supplementary Table ). In Model 2, which included MOPP, the intercept of the MVMR-Egger analysis was non-zero, indicating heterogeneity among the IVs. The results in Model 2 revealed that CH (OR = 1.008 with P = 0.503) was not significant, whereas RE (OR = 0.945 with P < 0.001) was, and MOPP (OR = 0.944 with P < 0.001) remained significant (Fig. and Supplementary Table ). Mediation analysis using structural equation models Based on the MVMR results assessing the direct effect, it can be inferred that CH, RE, and MOPP has an indirect effect on POAG. The mediation analysis using SEM in the context of a DAG model [Fig. , DAG (A)-2 and Table ] for MOPP, CH, and POAG showed that the mediation effect of MOPP through CH on POAG (a*b) was significant (Estimate = -0.001, P < 0.001), indicating a small significant mediation (Table ). In addition, the mediation analysis [Fig. , DAG (B)-2] for MOPP, RE, and POAG demonstrated that both the direct effect of MOPP on POAG (f) and the indirect effect of MOPP through RE on POAG (d*e) were significant (Estimate = -0.002, P < 0.001 for both).
Sample data from 459,195 European individuals from UKBB, and, of them, 98,661 samples with ocular phenotypes were used for the analysis, and the phenotypic characteristics are summarised in Fig. ; Table . The proportion of male participants was 45.7%, and the mean age of the population was 56.76 years. The mean CH, CRF, IOP, MOPP, and RE were 11.41 mmHg, 11.39 mmHg, 17.36 mmHg, 50.19 mmHg, and − 0.64 dioptre, respectively.
We conducted GWAS using 97,338 samples for CH, CRF, and RE and 96,743 samples for IOP and MOPP with 80,863,435 imputed SNPs. The GIF was 1.107 for CH, 1.018 for CRF, 1.154 for IOP, 1.136 for RE, and 1.162 for MOPP, suggesting no evidence of inflation in the GWAS results. The Manhattan and QQ plots for the gene-based analysis of GWAS allowed visualisation of the distribution of several association signals across the genome and enabled the identification of significant loci associated with the traits of interest [Supplementary Fig. (a)–(e)]. LD clumping was conducted with significant SNPs, whereas SNPs with LD with other variants or those absent from the European LD reference panel were excluded from the results. Among the significant genes for each phenotype, we identified candidate marker genes based on differential expression for the retinal ganglion cell type (Table ). We found a total of 14 marker genes [ MAGI1 ( P FDR = 1.14 × 10 − 3 ) and ZFHX3 ( P FDR = 4.62 × 10 − 2 ) for CH; RBMS3 ( P FDR = 1.55 × 10 − 2 ), MAGI1 ( P FDR = 3.98 × 10 − 3 ), and TRHDE ( P FDR = 4.02 × 10 − 2 ) for CRF; TUBB3 ( P FDR = 3.18 × 10 − 2 ) and POU6F2 ( P FDR = 2.08 × 10 − 2 ) for IOP; RBMS1 ( P FDR = 2.36 × 10 − 2 ), ZMAT4 ( P FDR = 2.99 × 10 − 5 ), GRIA4 ( P FDR = 2.62 × 10 − 13 ), LRFN5 ( P FDR = 2.20 × 10 − 6 ), RBFOX1 ( P FDR = 4.70 × 10 − 3 ), and GRIN2A ( P FDR = 2.18 × 10 − 3 ) for RE; CEP85L ( P FDR = 1.53 × 10 − 3 ) and RBPMS (P FDR = 8.13 × 10 − 5 ) for MOPP]. Of them, POU6F2 have been reported as a significant marker associated with IOP in the previous study . The co-localisation figures from LocusFocus for each gene are shown in Supplementary Fig. [in the artery (aorta and coronary), heart (left ventricle and atrial appendage) and whole blood tissue]. Furthermore, the decreased expression of MAGI1 ( P = 4.33 × 10 − 3 ), ZFHX3 ( P = 2.29 × 10 − 2 ), TRHDE ( P = 6.64 × 10 − 2 ), ZMAT4 ( P = 1.06 × 10 − 2 ), LRFN5 ( P = 3.29 × 10 − 2 ), RBFOX1 ( P = 3.33 × 10 − 2 ), GRIN2A ( P = 3.36 × 10 − 2 ), and RBPMS ( P = 9.57 × 10 − 3 ) was associated with POAG in the GEO dataset ( GSE27276 ) (Table ). The functinal annotation and pathway analysis revealed significant enrichment of several biological pathways and molecular functions in neurobiology (Supplementary Table ).
The estimated heritability for CH, CRF, IOP, RE, and MOPP was 7.4%, 9.5%, 6.9%, 21.0%, and 12.1%, respectively, without inflation in the GWAS results (Table ). We identified strong shared genetics for CRF, IOP, RE, and MOPP with POAG (GC = − 0.264, P = 4.58 × 10 − 7 for CRF; GC = − 0.665, P = 8.03 × 10 − 25 for IOP; GC = 0.130, P = 3.20 × 10 − 3 for RE; GC = 0.325, P = 3.08 × 10 − 16 for MOPP). No genetic association was observed between CH and POAG (GC = 0.033, P = 0.552) (Table ).
A total of 44, 47, 10, 85, and 8 genome-wide SNPs were associated with CH, CRF, IOP, RE, and MOPP, respectively, and were identified as IVs for MR analysis (Supplementary Table ). The leave-one-out analysis revealed that no single genetic variant had a disproportionately large effect on the overall estimates, supporting the robustness of our findings and the absence of significant heterogeneity or pleiotropy. The results of this analysis are presented in Supplementary Fig. . For reverse MR, 11, 17, 5, 9, and 9 genome-wide SNPs associated with POAG were used for CH, CRF, IOP, RE, and MOPP, respectively (Supplementary Table ). All IVs for bidirectional MR were entirely unrelated to the outcomes ( P > 0.05). We found no evidence of weak instrument bias ( F > 10), no violation of the NOME assumption ( I 2 > 90), and no heterogeneity or outlier pleiotropy ( P of Q-test, Q’-test, and MR-PRESSO > 0.05) (Supplementary Table ). The correlation matrix of instruments for all variables (Fig. ) illustrates the relationships among the genetic instruments, which is essential for evaluating the assumption of instrument independence in the subsequent MVMR analysis. Based on the analysis from the robust and penalised robust MR methods (IVW and MR-Egger), which downweighs outliers, we observed that CH [odds ratio (OR) = 0.998 with P < 0.001 for all robust methods], CRF (OR = 0.998 with P < 0.001 for all robust methods), and MOPP (OR = 0.998 with P < 0.001 for all robust methods) were inversely associated with POAG, whereas IOP (OR = 1.002 with P = 0.009 for robust and penalised robust IVW methods) was positively associated with POAG (Fig. and Supplementary Tables – ). However, the association was null when non-robust IVW and MR-Egger methods were used. Notably, in the reverse MR-Egger analysis, presence of POAG had significant association with MOPP (OR = 4.066 with P = 0.001 for MR-Egger and penalised MR-Egger; OR = 3.971 with P < 0.001 for robust MR-Egger and penalised robust MR-Egger) (Fig. and Supplementary Table ). The intercepts of various MR-Egger methods suggested the presence of horizontal pleiotropy, suggesting that some IVs may influence MOPP through pathways other than POAG. Violation of MR assumption, as is likely in this case, can lead to biased or unreliable estimates. Thus, the findings concerning the impact of POAG on MOPP should be interpreted with caution.
Since the correlation matrix reveals a strong positive correlation between CH and CRF, a negative correlation between CH and IOP, and another negative correlation between IOP and MOPP (Fig. ). Next, to control for pleiotropic effects, based on the results of factor analysis, we performed an MVMR analysis including CH, IOP, and RE jointly using Model 1, and CH, MOPP and RE jointly using Model 2 (Table ). In the univariable MR analysis, CH was significant, whereas RE was not; however, in the MVMR analysis, CH ( Ps > 0.05) was no longer significant and RE (OR = 0.934 with P < 0.001 in MV-IVW; OR = 0.929 with P < 0.001 in MV-MR-Egger) was significant in Model 1 (Fig. and Supplementary Table ). In Model 2, which included MOPP, the intercept of the MVMR-Egger analysis was non-zero, indicating heterogeneity among the IVs. The results in Model 2 revealed that CH (OR = 1.008 with P = 0.503) was not significant, whereas RE (OR = 0.945 with P < 0.001) was, and MOPP (OR = 0.944 with P < 0.001) remained significant (Fig. and Supplementary Table ).
Based on the MVMR results assessing the direct effect, it can be inferred that CH, RE, and MOPP has an indirect effect on POAG. The mediation analysis using SEM in the context of a DAG model [Fig. , DAG (A)-2 and Table ] for MOPP, CH, and POAG showed that the mediation effect of MOPP through CH on POAG (a*b) was significant (Estimate = -0.001, P < 0.001), indicating a small significant mediation (Table ). In addition, the mediation analysis [Fig. , DAG (B)-2] for MOPP, RE, and POAG demonstrated that both the direct effect of MOPP on POAG (f) and the indirect effect of MOPP through RE on POAG (d*e) were significant (Estimate = -0.002, P < 0.001 for both).
In this study, we identified 14 genes related to known risk factors for POAG, including CH, CRF, IOP, RE, and MOPP, through gene-based analysis of GWAS data. After adjusting for all ocular phenotypes using MVMR, we observed a direct effect of RE, IOP, and MOPP on POAG. Importantly, when considering the comprehensive results of UVMR, MVMR, and mediation analysis, it consistently demonstrated that lower MOPP has direct as well as indirect effects on POAG. A previous MR study reported no causal association or a limited effect of blood pressure on POAG . However, our study provides evidence of a consistent and potential association between MOPP and other ocular phenotypes in patients with POAG. The GWAS results for MOPP and ocular phenotypes are significant, with new features to be declared. Three loci associated with MOPP were also associated with blood pressure [rs7559141 (intergenic GYPC and TEX51 ), rs12509595 (intergenic PRDM8 and FGF5 ), rs2681485 (intronic ATP2B1 )]. NPR3 The SNP (rs9292468, intergenic NPR3 and LINC02120 ) for MOPP is related to the natriuretic peptide receptor, which is a secondary signal in the regulation of IOP with a potential link to POAG . In addition, one gene expression study using human tissues from African Americans and Caucasian Americans showed differently expressed NPR3 in the optic nerve head astrocyte transcriptome . NPR3 plays an important role in the clearance of natriuretic peptides that modulate blood pressure , which might be linked to MOPP and POAG pathogenesis. The SNP, rs4657477 ( TMCO1 ) is a well-known POAG locus related to IOP elevation [ – ], and rs28892906 ( AFAP1 ) is a well-known locus related to POAG and is independently associated with the IOP and the cup-disc ratio , which were found in GWAS. However, these SNPs were not used as IVs due to their potential violation of MR assumptions as confounders. Enrichment analysis of MOPP-related genes showed that CEP85L and RBPMS were significantly expressed in retinal ganglion cells in glaucoma. CEP85L (centrosomal protein 85 like) is a protein-coding gene related to lissencephaly and plays an essential role in neuronal cell migration. Despite the paucity of glaucoma research on this gene, CEP85L has been linked to cardiovascular GWAS for blood pressure . RBPMS (RNA binding protein) is a protein-coding gene related to prostate stromal sarcoma and retinal ischaemia via pathway exercise-induced circadian regulation. RBPMS is a significant marker for retinal ganglion cells in glaucoma models [ – ]. The discovery of a relationship between these modifications in RBPMS and MOPP is notable. A previous epidemiological study showed that lower systolic, diastolic, and mean perfusion pressures are associated with a higher prevalence of POAG , consistent with the results of cross-sectional studies [ , , ]. Forward MR results demonstrated that a lower MOPP can compromise blood flow to the optic nerve head. This ischemic environment may contribute to optic nerve damage, increasing the risk of POAG. This finding aligns with prior evidence that vascular dysregulation and insufficient ocular perfusion play a significant role in glaucoma progression. However, reverse MR results showed that POAG was positively associated with MOPP. Several points should be considered for understanding these results. The significant MR-Egger intercept (Supplementary Tables − ) highlights the complexity of the relationship between POAG and MOPP, potentially involving shared genetic pathways or confounding factors that cannot be fully accounted for in this analysis. While the MR-Egger slope suggests an inverse association, the limitations of the method and the presence of horizontal pleiotropy call for cautious interpretation of this result. In addition, in this study, the subjects were not strictly controlled in order to study the overall trend of the real-world cohort, MOPP may have increased due to the use of IOP-lowering medication for glaucoma treatment. It is worthwhile to contrast the findings of this investigation with those of recent studies on the causal relationship between myopia (RE in our study) and POAG [ – ]. A previous study demonstrated that the causal relation between myopia and POAG is mediated by IOP , whereas in our study, the causal pathway from MOPP to POAG demonstrated a mediation effect of CH, RE, or both CH and RE. In addition, a study on POAG polygenic risk scores showed that ocular phenotypes, including IOP, CH, and RE, predict the risk stratification of POAG , consistent with our findings. The originality of our study lies in the fact that it included MOPP in the model to investigate the role of vascular theory among the risk factors for POAG. A key strength of our study is its utilisation of a large dataset from UKBB and the analysis of a new clinical dataset, which revealed genetic loci related to ocular phenotypes as risk factors for POAG. Functional annotation and the pathway enrichment analysis revealed the candidate mechanisms related to POAG via identified loci. In addition, through MVMR and mediation analysis, we meticulously analysed the relationships between POAG and ocular phenotypes. However, this study has several limitations. First, our study population is limited to individuals with European ancestry; therefore, concluding the relationship between MOPP and POAG in other ethnic groups is challenging owing to the limited ethnic diversity of the study population. This limitation can be overcome by conducting replication studies in other ethnic groups, such as East Asian and African populations. Second, it should be understood that the selection of IOP, which prioritized higher values in both eyes, may lead to exaggerated GWAS results. Additionally, the selection of IOP measurement using IOPcc instead of GAT-IOP should also be considered, as it may lead to some differences in clinical settings. Therefore, the findings should be critically evaluated. Third, an aberration in the causal linkage of MOPP to POAG was observed in the reverse MR analysis. However, these results are attributed to the high likelihood of accompanying IOP abnormalities and POAG as opposed to causal effects. Consequently, care must be taken when interpreting the results of reverse MR. Fourth, for the TSMR analysis, we tried to minimise data overlap within UKBB as much as possible. However, approximately 5% of the UKBB dataset does exhibit overlap . Fifith, the discrepancy observed between the UVMR and MVMR analysis, where the protective effect of CF on POAG is seen in UVMR but reversed in MVMR, may be attributed to the differences in the structures of the models. The UVMR analysis evaluates the effect of a single exposure without accounting for potential confounders, whereas MVMR includes multiple exposures and covariates, which could alter the observed relationships. Despite a relatively low correlation between CH and MOPP, which suggests minimal multicollinearity, the inclusion of additional covariates in MVMR may influence the direction of the effect. Further analyses are required to explore the independent effects of CH and MOPP on POAG and to evaluate how confounders interact with these exposures. Finally, if the subjects had any conditions or were using medications that could affect IOP or blood pressure, adjustments might have been necessary; however, no such interventions were made in this study. Since some previous studies applied corrections while others did not , we aimed to minimize potentially arbitrary adjustments and analysed the data as they were collected using real-world data for MOPP. Given that the analysis was conducted without applying an operational definition, it can be inferred that, in cases of glaucoma treatment, the use of IOP-lowering drugs effectively led to an increase in MOPP. Hence, further studies with corrections including those for specific ocular conditions may be necessary.
We successfully determined 14 genetic loci related to CH, CRF, IOP, RE, and MOPP, indicating a potential association with POAG. In addition, a significant genetic causal link was observed between MOPP and POAG. Finally, we found that a lower MOPP has direct and indirect causal effects on POAG. Our findings suggest that, in addition to IOP, MOPP may serve as a potential causal factor of POAG, providing valuable insights into the pathophysiology and vascular theory of POAG.
Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 2
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Land use and soil characteristics affect soil organisms differently from above-ground assemblages | 8984c4a5-96b1-45d7-8a83-8ba31dd13883 | 9673366 | Microbiology[mh] | Terrestrial biodiversity continues to decline globally in the face of increasing human impacts , with land-use change and intensification the biggest driver of recent biodiversity loss . Species extinction rates are estimated to be around 10–1000 times higher than the background rate , with 1 million plant and animal species threatened with extinction and the Living Planet Index (which reflects trends in vertebrate population size) declined by 69% between 1970 and 2022 . However, these assessments and indicators focus on data-rich taxa, especially vertebrates, and so may not reflect broader biodiversity patterns . Organisms that live in the soil and leaf litter (henceforth, soil biodiversity) are particularly poorly represented in indicators and assessments of the global state of nature . This is despite the fact that they comprise 23% of described living species, support ecosystem services, such as nutrient cycling, soil formation and water quality , valued at $2.1 trillion per year worldwide and form the second largest carbon pool on Earth . This poor representation partly reflects data limitations: taxonomic discovery is less complete for many groups of soil species than those above ground, their distributions are less well known, and their assemblage structure is less often quantified . Additionally, because soil biodiversity samples are often not identified to the species level and because soil-dwelling species may be more taxonomically inclusive (‘lumped’) than above-ground species , estimates of diversity may not be comparable with those for better-known taxa. Although soil and above-ground communities are linked mechanistically , they often show different patterns of diversity . Soil characteristics can also affect biodiversity within both soil and above-ground assemblages. Soils are a fundamental determinant of plant communities , with soil biota being linked to them directly through symbiosis and herbivory, and indirectly via decomposition and nutrient cycling , but global patterns of soil fauna biomass may not follow plant biomass . We analyse biodiversity data from 19,651 above-ground and 7155 soil assemblages (comprising vertebrates, invertebrates, plants, and fungi) (Table ) in different land uses worldwide (Fig. and Table ), alongside global datasets of soil characteristics . Because soil assemblage data are often less taxonomically precise than data from above-ground assemblages, the response variable we model is the summed abundance of all taxa sampled. This measure is very much less sensitive to change than more information-rich measures that incorporate species identity , but it has the advantage that significant differences between models cannot be artefacts of differences in taxonomic precision. The Soil Biodiversity Observation Network (SoilBON) have proposed population abundance as an Essential Biodiversity Variable . To accommodate heterogeneity due to the wide range of sampling methods and macroecological gradients in the dataset we used mixed-effects models to test three main hypotheses (expanded on in Additional file : Table S1): (1) Because some land uses imply very different levels of perturbation to soil versus above-ground microenvironments, and because soil organisms are less mobile and more sensitive to microclimate change , we expect differences in how assemblages from these two settings respond to land use. For example, using land to rear livestock may impact soil structure much less than above-ground habitat structure, while soil organisms may take longer than above-ground taxa to recolonise sites recovering from physical soil disturbance [ – ]. (2) Because physical properties of soil, such as pH and soil texture, themselves mediate the impacts of land use microenvironments, we expect these properties to influence assemblage responses to land use. For instance, moisture retention by clay-rich soils may mitigate the warming and drying effects of agriculture. (3) Although above-ground and soil assemblages are linked mechanistically, we do not expect soil properties to shape their assemblage-level responses to land use in the same way. To offset the different geographic biases in soil versus above-ground assemblages we ran a weighted model. Weights were calculated by dividing the number of soil sites by the number of above-ground sites within each biome. In addition to the models required to test the three hypotheses, we also constructed a set of simpler models with single or additive terms to fully characterise which terms in the full model contributed the most explanatory power. We also undertook two sensitivity analyses. Because the biome-weighting is not commonplace (despite the ubiquity of geographic biases in biodiversity databases ), our first sensitivity analysis repeated the modelling without it. The second sensitivity analysis addresses the point that soil and above-ground assemblage data sets obviously have very different compositions in terms of which major taxonomic groups are well represented. While different impacts of land use on soil and above-ground assemblages would still be important even if they simply reflected such taxonomic differences, we also ran the same models using only the data for invertebrate taxa.
Estimated effects of land use differed markedly between soil and above-ground biota (Table ); relative to primary vegetation, soil assemblages had lower abundance than above-ground assemblages in secondary vegetation and (especially) plantation forest and cropland, but higher abundance in pasture (Fig. ). Soil properties, especially bulk density, affected how soil fauna abundance responds to land use (Fig. , Table ). These effects were not consistent among land uses; for example, abundance correlated positively with bulk density among cropland and plantation sites but not among pasture sites (Fig. ). Soil properties also mediated the responses of above-ground assemblages to land use, in ways that differed from how they shaped the responses of soil assemblages (Table ). Like the soil biota, effects on above-ground biodiversity were not consistent among land uses, e.g., the positive correlation between above-ground organism abundance and organic carbon was more pronounced in cropland and pasture than in other land uses (Fig. ). As expected with such heterogeneous data, most of the explained variation was attributed to random effects; but interactions increased the explanatory power of the fixed effects by nearly half, from 14% to 20% (Additional file : Fig. S1). The unweighted model found broadly similar patterns between above-ground and soil assemblage responses to land use but had lower explanatory power (see Additional file ). Compared to the pattern shown in Fig. , analysis of the invertebrate-only data found a bigger difference in the effects of plantations on soil versus above-ground assemblages, but a negligible difference in how cropland affected them (see Additional file ).
Land use—the recent main driver of biodiversity loss worldwide —affects soil assemblages differently from those above ground. As hypothesised, cropland—where tillage, pesticides and fertilisers disturb soil biodiversity —reduces abundance even more in the soil than above ground. In contrast, pasture—with relatively little physical disturbance of the soil and often increased nutrient input —shows the opposite pattern. Clear-felling and replanting with different tree species has been previously found to have the strongest negative impact on biodiversity , and we also find a strong negative effect of plantation on biodiversity. The much lower relative abundance in soil than above-ground assemblages (Fig. ) may be explained by the acidified soil and recalcitrant leaf litter typical of conifer plantations (the dominant type in the soil assemblage data), together with drier soils in plantations that have a reduced under-story . Soil organism abundance has not recovered in secondary vegetation as much as above-ground abundance (Fig. ), in keeping with our hypothesis that soil biota recovers more slowly to disturbance than above-ground biodiversity. As well as affecting the overall abundance of soil organisms, soil properties also mediated how land use affected soil assemblages (left-hand column of Fig. ). Perhaps more surprisingly, soil properties also affected how above-ground assemblages responded to land use, in ways that differed from their effect on the responses of soil assemblages (right-hand column of Fig. , Table ). Above-ground abundance generally increased with organic carbon, which is as expected given the latter’s close link with plant productivity . Abundance was generally higher in more clay-rich soils, which typically have more nutrients and retain water better . These effects of soil properties will include both direct, and indirect effects medicated by biotic interactions, but we were unable to separate these as few studies collected data on above-ground and soil biota concurrently. The greater impact of cropland and plantation forestry on soil biota than above-ground assemblages shown by our models is a cause for serious concern. To feed the growing population, scenarios include the world’s croplands increasing in area, being managed more intensively, or both . The rapid recent expansion of plantation forests may accelerate further if they receive subsidies for carbon sequestration, despite their impacts on biodiversity . Pathways to sustainable development must avoid the diminution of soil assemblages that would undermine the long-term provision of soil ecosystem services . This highlights the likely importance of soil biodiversity for the ecological intensification of agriculture and of considering soil biodiversity explicitly in formulating conservation policy . Our division of assemblages into soil and above-ground was based on how they were sampled rather than on ecosystem ecology. Many organisms sampled by above-ground methods spend part of their life cycle in the soil (e.g., many flies, bees and beetles), or even have much of their biomass underground (e.g., most plants). The soil assemblages have a very different taxonomic composition from those above-ground (Table ) and different taxonomic groups are expected to respond differently to land use and soil properties [ , , ]. An example of this can be seen in the invertebrate-only results (see Supplemental Information), here both above-ground and soil invertebrates are equally impacted in cropland, but above-ground invertebrate abundance is greater than soil invertebrates in plantation sites. Further work with models incorporating functional traits robust to coarse taxonomic resolution would be valuable. Better documentation of the taxonomic and functional diversity of soil fauna would also help overcome some of these limitations, so we echo calls for better soil biodiversity information systems . Additionally, except for fungi, micro-organisms are unrepresented in both soil and above-ground datasets—assemblage data from metabarcoding and metagenomic approaches will enable the use of more information-rich biodiversity measures. Above-ground and soil taxa may be active and sampled at very different spatial scales, and soil property data with a spatial resolution of 250 m used here may not accurately reflect that experienced by the biota. Site-specific soil property data were available for some studies used in this analysis but were too insufficient or inconsistent to incorporate. Better standardisation of soil biodiversity surveys with a minimum level of environmental measurements collected would be a valuable contribution to the field as would explicit tests of spatial and temporal heterogeneity . Likewise, models that consider other drivers, such as climate change, alongside land use will also improve understanding. Correlative models such as ours are sufficient for developing indicators and models for monitoring and combating biodiversity loss but there is also a need for an improved understanding of the mechanisms linking land use, soil properties, and biodiversity responses . Future analysis of this dataset using structural equation models (SEMs) could be used towards this, to disentangle the direct and indirect effects of soil properties and land use on communities. However, the limitations of our data and models do not detract from the central implication that soil and above-ground assemblages respond differently to land use: inferences drawn from what lives above ground cannot safely be extended to the soil biota.
We show that soil biodiversity does not respond the same way to land use and soil properties as above-ground assemblages. The most widely used indicators of biodiversity, e.g. the Red List Index and the Living Planet Index , include few or no soil taxa . This means that current indicators, models, and frameworks for monitoring and combating biodiversity loss may be insufficient to safeguard the soil biodiversity needed to underpin ecosystem function.
Biodiversity data In the absence of a well-developed catalogue of global soil biodiversity , we initially searched within the PREDICTS database for soil assemblage data, defining soil assemblages as those sampled within the soil; at the soil surface, or in the leaf litter. The database is a global compilation of studies that have each compared non-cultivated species assemblages at multiple sites facing different land-use and related pressures . To the 59 studies (from 38 source publications with 1356 sites and 1570 taxa) of soil assemblage data previously in the PREDICTS database, we added 46 further studies (from 25 sources with 2726 sites and 3857 taxa (Tables and ). Above-ground assemblage data came from the other 509 studies (from 422 source publications, with 20,634 sites and 22,721 taxa) in the PREDICTS database at that time (October 2016). The fraction of taxa resolved to species level was over twice as high in the above-ground assemblages as in the soil assemblages (58% versus 28%). Given this, plus the likelihood that species-level taxa are more inclusive in soil than above-ground organisms (i.e., the latter tend to be subdivided more finely when species are demarcated ), measures that use compositional information (such as diversity indices, or even numbers of species) cannot be compared between soil and above-ground assemblages. We therefore used the summed abundance across all sampled taxa—which is unaffected by taxonomic precision—as the site-level response variable. Whenever sampling effort varied among sites within a study, any abundance data sensitive to it (i.e., metrics not already reported as numbers per unit time, distance, area, or volume) were divided by sampling effort. Finally, abundance values were rescaled within each study to have a maximum value of 1, reducing among-study heterogeneity and thereby aid model convergence. Explanatory variables Using the information in the original papers, each site was classified into one of six categories of land use—primary vegetation, secondary vegetation, plantation forest, cropland, pasture or urban—and either low, medium, or high use intensity (see Additional file and for full definitions). Most combinations of land use and use intensity (henceforth, LUI) had large enough sample sizes in both the above-ground and soil subsets, but even after targeted literature searching to augment the database’s holdings of urban data, there were insufficient sites for robust comparison of above- and below-ground in urban land use sites. The above-ground subset comprised primarily arthropods, plants, and vertebrates whereas the soil biodiversity subset was mostly arthropods (Table ). Nine soil properties widely reported to influence soil biodiversity (Additional file : Table S1) were obtained from the SoilGrids250m database using ESRI ArcGIS 10.3 . Values were not available for 49 sites, which were therefore removed from the analysis. We averaged the values from depths 0, 5, 15 and 30 cm as no biodiversity data sources sampled at depths greater than 30 cm. The soil properties were expected to be collinear so, before model construction began, generalised variance inflation factors (GVIFs) were calculated . Among the soil texture properties, the percentage of clay had the lowest GVIF so was chosen in preference to percentages of silt or sand. Successively dropping the variable with the highest GVIF until all remaining GVIFs were low enough to suggest collinearity was not a major issue (all GVIF < 1.5), led to all soil moisture properties being dropped, while pH, bulk density, organic carbon, and clay percentage were retained. Biome weighting To offset the geographic bias in soil versus above-ground assemblages (Fig. ), we applied weights in the models. Weights were calculated by dividing the number of soil sites by the number of above-ground sites within each biome (Additional file : Table S2). Weights were calculated separately for the invertebrate-only subset (not shown). Statistical analysis All analyses were carried out in R 3.5.1 . Because total abundance contained non-integers even before rescaling, it was log(x + 1) transformed before modelling with Gaussian errors. The studies in the dataset vary widely in many aspects of sampling. We therefore fitted mixed-effects models (as implemented in lme4 version 1.1.18.1 with bobyqa numerical optimisation) to reduce heterogeneity caused by among-study differences in sampling methodology and macroecological gradients such as latitude. Our previously listed hypotheses were tested by comparing a maximally complex model with three simpler models (see Additional file for model structures) that lacked the hypothesised effects. The full model included six main fixed effects—land-use type and intensity (LUI), above-ground or soil assemblage (habitat layer), and the four soil properties (rescaled to the range 0-1 to aid fitting)—plus each soil property’s interaction with land-use type and habitat layer, the interaction between land-use type and habitat layer, and the three-way interactions of each soil property with land-use type and habitat layer. The random-effects structure was chosen by comparing the Akaike’s Information Criterion (AIC) of models having the full set of fixed effects plus, as random intercepts, (a) spatial block nested within study identity, (b) spatial block identity, or (c) study identity ; models with random slopes did not converge. The optimal random-effects structure was then retained for all models. To ascertain the influence of use intensity on the full model was compared to one with only land use. To test whether above-ground and soil assemblages respond differently to land use, the full model was compared to one in which habitat layer could not interact with other explanatory variables. The importance of soil properties in shaping assemblage responses to land use was tested by comparing the full model to one in which soil properties were included as main effects but could not interact with LUI. Whether effects of soil properties differ for above-ground and soil assemblages was tested by comparing the full model to one in which neither the soil properties nor their interactions with land use could interact with habitat layer. In addition to the models required to test the three hypotheses, we also constructed a set of simpler models with single or additive terms to fully characterise which terms in the full model contributed most explanatory power. The variance explained by fixed effects alone (marginal R 2 glmm ) and fixed and random effects combined (conditional R 2 glmm ) were calculated using the MuMIn package as measures of explanatory power. The random effects and spatial blocks intended to accommodate the heterogeneity among studies are expected to explain much more of the variance than the fixed effects. Consequently, when comparing models in terms of the explanatory power of their fixed effects, we compare their marginal R 2 glmm / (1 - conditional R 2 glmm ). We repeated the analyses without the weighting procedure used to compensate for the different geographic biases of above-ground and soil assemblage data (results in Additional file ).
In the absence of a well-developed catalogue of global soil biodiversity , we initially searched within the PREDICTS database for soil assemblage data, defining soil assemblages as those sampled within the soil; at the soil surface, or in the leaf litter. The database is a global compilation of studies that have each compared non-cultivated species assemblages at multiple sites facing different land-use and related pressures . To the 59 studies (from 38 source publications with 1356 sites and 1570 taxa) of soil assemblage data previously in the PREDICTS database, we added 46 further studies (from 25 sources with 2726 sites and 3857 taxa (Tables and ). Above-ground assemblage data came from the other 509 studies (from 422 source publications, with 20,634 sites and 22,721 taxa) in the PREDICTS database at that time (October 2016). The fraction of taxa resolved to species level was over twice as high in the above-ground assemblages as in the soil assemblages (58% versus 28%). Given this, plus the likelihood that species-level taxa are more inclusive in soil than above-ground organisms (i.e., the latter tend to be subdivided more finely when species are demarcated ), measures that use compositional information (such as diversity indices, or even numbers of species) cannot be compared between soil and above-ground assemblages. We therefore used the summed abundance across all sampled taxa—which is unaffected by taxonomic precision—as the site-level response variable. Whenever sampling effort varied among sites within a study, any abundance data sensitive to it (i.e., metrics not already reported as numbers per unit time, distance, area, or volume) were divided by sampling effort. Finally, abundance values were rescaled within each study to have a maximum value of 1, reducing among-study heterogeneity and thereby aid model convergence.
Using the information in the original papers, each site was classified into one of six categories of land use—primary vegetation, secondary vegetation, plantation forest, cropland, pasture or urban—and either low, medium, or high use intensity (see Additional file and for full definitions). Most combinations of land use and use intensity (henceforth, LUI) had large enough sample sizes in both the above-ground and soil subsets, but even after targeted literature searching to augment the database’s holdings of urban data, there were insufficient sites for robust comparison of above- and below-ground in urban land use sites. The above-ground subset comprised primarily arthropods, plants, and vertebrates whereas the soil biodiversity subset was mostly arthropods (Table ). Nine soil properties widely reported to influence soil biodiversity (Additional file : Table S1) were obtained from the SoilGrids250m database using ESRI ArcGIS 10.3 . Values were not available for 49 sites, which were therefore removed from the analysis. We averaged the values from depths 0, 5, 15 and 30 cm as no biodiversity data sources sampled at depths greater than 30 cm. The soil properties were expected to be collinear so, before model construction began, generalised variance inflation factors (GVIFs) were calculated . Among the soil texture properties, the percentage of clay had the lowest GVIF so was chosen in preference to percentages of silt or sand. Successively dropping the variable with the highest GVIF until all remaining GVIFs were low enough to suggest collinearity was not a major issue (all GVIF < 1.5), led to all soil moisture properties being dropped, while pH, bulk density, organic carbon, and clay percentage were retained.
To offset the geographic bias in soil versus above-ground assemblages (Fig. ), we applied weights in the models. Weights were calculated by dividing the number of soil sites by the number of above-ground sites within each biome (Additional file : Table S2). Weights were calculated separately for the invertebrate-only subset (not shown).
All analyses were carried out in R 3.5.1 . Because total abundance contained non-integers even before rescaling, it was log(x + 1) transformed before modelling with Gaussian errors. The studies in the dataset vary widely in many aspects of sampling. We therefore fitted mixed-effects models (as implemented in lme4 version 1.1.18.1 with bobyqa numerical optimisation) to reduce heterogeneity caused by among-study differences in sampling methodology and macroecological gradients such as latitude. Our previously listed hypotheses were tested by comparing a maximally complex model with three simpler models (see Additional file for model structures) that lacked the hypothesised effects. The full model included six main fixed effects—land-use type and intensity (LUI), above-ground or soil assemblage (habitat layer), and the four soil properties (rescaled to the range 0-1 to aid fitting)—plus each soil property’s interaction with land-use type and habitat layer, the interaction between land-use type and habitat layer, and the three-way interactions of each soil property with land-use type and habitat layer. The random-effects structure was chosen by comparing the Akaike’s Information Criterion (AIC) of models having the full set of fixed effects plus, as random intercepts, (a) spatial block nested within study identity, (b) spatial block identity, or (c) study identity ; models with random slopes did not converge. The optimal random-effects structure was then retained for all models. To ascertain the influence of use intensity on the full model was compared to one with only land use. To test whether above-ground and soil assemblages respond differently to land use, the full model was compared to one in which habitat layer could not interact with other explanatory variables. The importance of soil properties in shaping assemblage responses to land use was tested by comparing the full model to one in which soil properties were included as main effects but could not interact with LUI. Whether effects of soil properties differ for above-ground and soil assemblages was tested by comparing the full model to one in which neither the soil properties nor their interactions with land use could interact with habitat layer. In addition to the models required to test the three hypotheses, we also constructed a set of simpler models with single or additive terms to fully characterise which terms in the full model contributed most explanatory power. The variance explained by fixed effects alone (marginal R 2 glmm ) and fixed and random effects combined (conditional R 2 glmm ) were calculated using the MuMIn package as measures of explanatory power. The random effects and spatial blocks intended to accommodate the heterogeneity among studies are expected to explain much more of the variance than the fixed effects. Consequently, when comparing models in terms of the explanatory power of their fixed effects, we compare their marginal R 2 glmm / (1 - conditional R 2 glmm ). We repeated the analyses without the weighting procedure used to compensate for the different geographic biases of above-ground and soil assemblage data (results in Additional file ).
Additional file 1. Contains Tables S1-S4 and Figures S1-S6, providing further information on data sources, model structures and results of sensitivity analyses.
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The behavioral signature of stepwise learning strategy in male rats and its neural correlate in the basal forebrain | 5fa52c7c-85cf-4b6f-bac7-acb4c0c92f92 | 10362048 | Physiology[mh] | Associative learning is essential for survival and allows animals and humans to predict future reward based on environmental stimuli , or their own actions – . Understanding the algorithmic principles of associative learning has been a central question in psychology and neuroscience – , and has broad implications in machine learning and artificial intelligence – . While the learning of stimulus-reward and action-reward associations have been historically studied under the separate labels of Pavlovian , and instrumental , conditioning, most learning scenarios require the synergistic contribution from both types of learning strategies. For example, when a new reward-predicting stimulus is introduced to the environment, the Pavlovian strategy might not be sufficient because oftentimes the reward would not be delivered unless animals take specific actions. In experimental settings, such actions could be a lever press, or a saccade toward a target, or multiple licks before the reward is delivered. In these scenarios, reward is obtained only when sensory stimuli and animals’ actions occur in a specific order as a behavioral sequence , – . Compared to the wealth of knowledge about Pavlovian and instrumental conditioning, little is understood about how animals learn behavioral sequences that contain both stimuli and actions. Converging views from theoretical studies support the idea that reward-predicting behavioral sequences can be efficiently learned using a strategy that we will refer to as stepwise learning: learning starts from the event closest to the reward, while earlier events are learned in later steps. This learning strategy was initially proposed by Skinner and more recently elaborated into formal learning models , . Similar learning dynamics are also predicted by reinforcement learning algorithms, in which states that are closer to the final reward are learned first . The stepwise learning strategy has also been successfully used in various animal training scenarios to incrementally chain single behaviors into long sequences over multiple training steps . The goal of the current study is to test whether animals use the stepwise learning strategy to learn reward-predicting behavioral sequences that contain both stimuli and actions. We seek to identify the behavioral and neural signatures of this learning process that can delineate the discrete steps of learning. A major challenge in understanding this type of learning is that behavioral sequences are controlled not only by the experimenter but also by the animal, which is free to take various actions. We reason that, at the beginning of learning, animals’ actions would be less constrained and therefore would generate a large repertoire of behavioral sequences that may or may not lead to the rewarding outcome. As the stepwise learning process unfolds, the repertoire of behavioral sequences should become increasingly selective as well as more frequently rewarded. Therefore, the behavioral signature of the stepwise learning strategy may reside in how the entire repertoire of behavioral sequences become sequentially refined during the learning process. In the current study, we identified such a behavioral signature, which corresponded to the discrete steps in the stepwise learning process. In order to validate the behavioral signature for the stepwise learning strategy, we focused on a special subset of noncholinergic neurons in the basal forebrain (BF), which are referred to as BF bursting neurons – . Previous studies have found that BF bursting neurons convey a reward-prediction error signal , , , , and show highly robust phasic bursting responses to reward-predicting sensory stimuli irrespective of their sensory modalities – . Moreover, such responses only emerge after reward-based associative learning , and are tightly coupled with behavioral performance and promotes faster decision speeds – . These observations suggest that increased BF bursting neuron activities toward a behavioral event reflects that the event has been learned as a reward predictor. By observing the temporal evolution of BF bursting neuron activities throughout the learning process in the current study, we predict that BF responses should mirror the stepwise learning process: BF activity should first emerge toward the last behavioral event closest to the reward, and subsequently develop toward the earlier behavioral events. Such behavioral and neurophysiological findings will provide important insights on how behavioral sequences are learned. A model for learning behavioral sequences using the stepwise learning strategy To gain intuition about the stepwise learning strategy, we first considered a toy example in which a three-element sequence A-B-C predicted reward (Fig. ). This sequence can be learned using the stepwise strategy in three discrete steps, starting from the event closest to the reward and sequentially incorporating earlier events (Fig. ). As more behavioral events are learned as reward predictors in each step, only behavioral sequences that contain all the learned events would predict reward and therefore preferentially executed, while incompatible sequences that do not contain all the learned events would not predict reward and therefore be eliminated from the behavioral repertoire. As a result, the discrete steps of learning would correspond to the stepwise elimination of non-rewarded behavioral sequences that share subsets of behavioral elements (Fig. ). We hypothesized that this sequential refinement of reward-seeking behaviors might provide a behavioral signature of the stepwise learning strategy. Sequential refinement of reward-seeking behaviors during new learning To test whether animals indeed use the stepwise strategy to learn behavioral sequences that contain both sensory stimuli and their own actions, we trained adult Long-Evans rats in an auditory discrimination task. Rats entered the fixation port to initiate each trial, where they encountered three trial types (S left ; S right ; catch) with equal probabilities that respectively indicated sucrose water reward in the left or right port, or no reward in the case of catch trials (no stimulus) (Fig. ). During the initial auditory discrimination phase, S left and S right were two distinct sound stimuli. After reaching asymptotic performance, rats entered the new learning phase (first new learning session denoted as the D 0 session), in which the S right sound stimulus was switched to a novel light stimulus that minimized sensory generalization from past experience (Fig. and Table ). During the new learning phase, rats maintained stable levels of performance toward the previously learned S left sound stimulus (Fig. ) (94.3 ± 4.8% correct, 109 ± 20 trials per session, mean ± std). Within the first three sessions of the new learning phase, all rats ( N = 7) began responding correctly in the new light trials (the first such session denoted as the D 1 session) and maintained stable levels of >90% correct response rates afterwards (Fig. and Table ). To understand how the repertoire of behavioral sequences evolved during learning, we examined all possible behavioral sequences that the animal might experience (Fig. ). This approach allowed us to identify non-rewarded behavioral sequences that were not associated with specific trial types but were, nonetheless, highly relevant for the learning process. We identified three types of behavioral sequences whose frequencies consistently increased during the new learning phase. These three types of behavioral sequences included the rewarded licking behavior in the new light trials (light licks), as well as two types of non-rewarded behavioral sequences: catch licks and no-fixation licks (Fig. ). Catch licks refers to licking responses toward the right reward port in catch trials when no sensory stimulus was presented. No-fixation licks refers to the situation in which rats directly licked at the right reward port without first entering the center fixation port. All three behavioral sequences shared the common feature of licking the right reward port (lick-right). Both no-fixation licks and catch licks were largely absent before the new learning phase, and emerged and peaked during early sessions of new learning, before subsequently diminished in later sessions (Fig. ). No-fixation licks occurred most frequently at the D 1 session, while catch licks peaked in the same session ( N = 1/7) or later in most animals ( N = 6/7). We will denote the peak of catch licks as the D 2 session in each animal. The consistent temporal order of the D 1 and D 2 sessions within each animal allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The temporal dynamics of the three types of rightward licks during new learning (Fig. ) showed that non-rewarded behaviors were sequentially eliminated while rewarded behaviors were preserved. This temporal dynamics resembled the pattern of sequential refinement of behavioral sequences predicted by the stepwise learning strategy (Fig. ), and likely corresponded to the discrete steps in the underlying learning process. To further test whether such patterns of sequential refinements represent a general feature of behavioral sequence learning, we trained a separate cohort of animals and observed similar behavioral signatures regardless of the sensory modality of the stimulus or the laterality of the new learning side (Fig. ). These observations support the idea that animals do use the stepwise strategy to learn behavioral sequences. In the following analyses, we tested additional predictions of the stepwise learning strategy at both behavioral and neurophysiological levels. Initial learning was characterized by the rapid emergence of reward-seeking behaviors and corresponding increases in BF activities To validate the behavioral observations and understand the underlying neural dynamics, we recorded BF neuronal activity throughout the learning process (Fig. ) and used the consistent S left sound as the control stimulus to identify stable populations of BF bursting neurons (Figs. and ). A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. ). The population response of BF bursting neurons were highly consistent across animals (Fig. ), and remained remarkably stable in S left trials throughout the learning process (Fig. ). The inclusion of S left trials therefore allowed us to record from stable and representative populations of BF bursting neurons throughout the learning process, and to investigate how their activities dynamically evolved in other trial types during learning at single trial resolution (Fig. ). We first applied this approach to understand the behavioral and neural dynamics in the D 1 session because all three types of rightward licks emerged in this session (Fig. , middle panel). Detailed analysis of behavioral responses from a representative session (Fig. ) revealed that the three types of rightward licking behaviors emerged abruptly after a transition point (see Methods for definition). Rightward licking behaviors were mostly absent before the transition point, and rapidly switched to almost 100% licking after the transition point. This pattern of abrupt transition was consistently observed across all animals (Fig. ), while the behavioral performance and BF activities in S left sound trials remained relatively stable (Fig. ). At the neuronal level, there was a corresponding increase in the activity of BF bursting neurons that rapidly emerged after the transition in rightward reward-seeking behaviors (Figs. e and ). This increase in BF activity was most prominent in the epoch before the trial outcome as animals approached the reward port (Fig. ), but this activity was not consistently aligned with intervening behavioral events (Fig. ). In contrast, in trials before the transition point, BF bursting neurons did not show similar activity increases in the corresponding time window after exiting the fixation port (Figs. e and ). We will refer to the maximum BF activity in this window as the BF evaluation response (see Methods for definition) because it reflected animals’ internal evaluation when no sensory stimuli were presented during this epoch and because it was not consistently associated with intervening behavioral events. The emergence and subsequent elimination of non-rewarded behaviors were mirrored by changes in BF activity The stepwise learning model predicted that, as animals learned about the lick-right action as the first reward predictor, animals would initially engage in all three types of rightward licking behaviors because they all contained this reward predictor (Fig. ). We therefore examined the respective prevalence of the three types of rightward licking behaviors after the transition point in the D 1 session (Fig. ). All three types of rightward licking behaviors emerged immediately after the transition point. Moreover, after about 60 trials, no-fixation licks began to decline and occurred less frequently than light licks and catch licks (Fig. ). At the neuronal level, the corresponding prediction was that the lick-right action would be associated with increases in BF activities during the first learning step, which should be similarly present in all three types of rightward licking behaviors. Indeed, BF evaluation responses quickly increased after the transition point in all three types of rightward licking behaviors (Fig. ). The amplitudes of BF evaluation responses were similar between the three types of rightward licking behaviors within the first 60 trials after the transition. Subsequently, the BF evaluation response in no-fixation licks declined in the next 60 trials, relative to the other two types of rightward licking behaviors (Fig. ). These results support that the first step of the stepwise learning process corresponded to the first 60 trials after the transition point in the D 1 session. All three types of rightward licking behaviors were present during this step of learning. BF activity also increased to similar levels in all three types of behavioral sequences whenever animals approached and licked the right reward port, regardless of whether they had exited from the center fixation port. These results further suggest that the second step of learning started at roughly 60 trials after the transition, at which point animals learned about the second reward predictor: exiting the fixation port (Fig. ). As a result, the no-fixation lick sequence was no longer compatible with the learned reward predictors, which resulted in diminished BF evaluation responses and the elimination of this behavior from the behavioral repertoire. The other two types of rightward licks, light licks and catch licks, remained compatible and maintained high levels of BF evaluation responses. BF neurons did not respond to the new light stimulus during initial learning A further prediction of the stepwise learning model was that, at both the first and the second steps of learning, animals had not learned to use the new light stimulus as a reward predictor, and therefore the new light stimulus would not elicit responses in BF bursting neurons during these steps (Fig. ). We tested this prediction by comparing BF activities between light and catch trials in the D 1 session (Fig. ). Indeed, BF activities in light trials were highly similar to those in catch trials (in the absence of the light stimulus) in the epochs before exiting the fixation port. This was true regardless of whether animals subsequently licked at the right reward port. This observation confirmed the prediction that the new light stimulus did not activate BF bursting neurons in the D 1 session, despite the near-perfect behavioral performance in light trials after the behavior transition point. On the other hand, in the epoch after exiting the fixation port, there were similar increases in BF activity when animals licked at the right reward port in both light and catch trials (Fig. ). This increase in BF activity corresponded to the BF evaluation response described earlier (Fig. ), which reliably distinguished between lick and no lick trials, but not between light and catch trials (Fig. ). These observations support the idea that light and catch trials were treated as the same in the D 1 session, and that the light stimulus has not been learned as a reward predictor at this stage of learning. BF responses to light onset emerged later when the light stimulus was used to guide reward-seeking behavior When did the third step of the stepwise learning process take place? Since the third step was when animals learned to use the light stimulus as a reward predictor to guide reward-seeking behaviors, it should correspond to the time point when the behavioral performance in light and catch trials began to diverge. We noted that the behavioral pattern in light and catch trials were highly similar prior to the D 2 session (Fig. ), and the similarity was best illustrated in the fine behavioral and neuronal dynamics within the D 1 session (Fig. ). In contrast, during the D 2 session, the behavioral pattern in light and catch trials began to show a small but significant difference (Fig. ). This pattern suggests that the D 2 session might be when the third step of learning took place. The corresponding prediction at the neuronal level was that BF bursting neurons should begin to respond to the light stimulus in the D 2 session. We tested this prediction by comparing BF activities between light and catch lick trials in the D 2 session, and found significant differences in all epochs, including the presence of a phasic response to the light onset (Fig. ). This observation supports the idea that BF responses to the new light emerged in the D 2 session. To better understand how BF responses to the new light emerged in the D2 session, we further compared BF responses in light lick and catch lick trials between (1) late trials in the D 2 −1 session; (2) early trials in the D 2 session; (3) late trials in the D 2 session (Fig. ). At the end of the D 2 −1 session, BF neurons did not show response to the new light (paired t -test between light lick and catch lick trials, p = 0.51, N = 6). In early D 2 trials, BF phasic responses to the new light were clearly visible in 4 animals. Comparison between late trials in the D 2 −1 session and the early D 2 trials showed a trend toward increasing BF responses (Fig. , significant at p < 0.05 level; Fig. , paired t -test, p = 0.065, N = 6). Moreover, during the D 2 session, BF responses to the new light increased in all animals between early and late trials (Fig. , paired t -test, p = 0.003, N = 7). This increase took place mostly during the sustained phase of the BF response that was better aligned with fixation port exit than with stimulus onset (Fig. ). Together, these observations suggest that BF responses to the new light developed partly offline between the D 2 −1 and D 2 session, and partly strengthened during the D 2 session. Comparing the temporal dynamics of behavioral and BF responses further suggests that the emergence of BF responses to the new light preceded the elimination of catch licks during the third step of learning (Fig. ). Stronger BF responses to the new light reflected better learning and faster decisions After the third step of learning took place in the D 2 session, did the learning about the new light plateau or did the learning continue to progress? At the neuronal level, BF phasic responses to the new light continued to grow stronger after the D 2 session (Fig. ). At first glance, the continual increases in BF responses to the new light stimulus did not match with the hit rates in light trials, which had already plateaued in the D 2 session (Fig. ). However, as we have shown earlier (Figs. and ), hit rates in light trials could be a poor index of learning about the new light stimulus because light licks in the early learning sessions were not driven by the light stimulus but by later events in the behavioral sequence (exiting fixation and lick-right) as reward predictors. Those behavioral events enabled rightward licking responses in the absence of the light stimulus (catch licks and no-fixation licks). Instead of the hit rate in light trials, a better behavioral index for the learning about the light stimulus would be the difference in the levels of behavioral performance between light and catch trials. This behavioral index accounted for the contributions from the later events in the behavioral sequence that were shared between light and catch licks, and isolated the contribution of the light stimulus to the rightward licking behavior. We found that this index of light learning was strongly correlated with the amplitude of BF phasic responses to the light stimulus in individual sessions (Fig. ). This observation therefore supports the idea that the learning about the light stimulus continued to grow stronger after the D 2 session. Another dimension of the learning about the light stimulus was the change in animals’ decision speeds, measured by reaction times (RTs). Previous studies have shown that stronger BF bursting responses are quantitatively coupled with, and causally lead to, faster RTs , . Such observations predicted that there would be corresponding decreases in RTs toward the light stimulus after the D 2 session. In support of this prediction, we found that stronger phasic bursting responses to light onset were coupled with faster RTs in individual sessions (Fig. ). Together, these observations support the idea that the learning about the new light was reflected by the amplitude of BF phasic response to the light stimulus. In addition, BF activities not only reflected the learning about the light stimulus, but also predicted reward-seeking behaviors in the absence of the light stimulus. For example, in catch trials, stronger BF activities after exiting the fixation port predicted rightward licking behavior and discriminated lick trials from no lick trials (Fig. ). Moreover, increased BF activities before the start of licking predicted longer durations of licking in catch lick trials when no reward was delivered (Fig. ). These observations provided additional support for the idea that the activity of BF bursting neurons promoted reward-seeking behaviors. Reward expectations negatively modulated BF responses to trial outcomes A final validation of the learning dynamics came from how BF responses to the trial outcome were modulated by reward expectations and BF activities in earlier epochs. Previous studies have found that the response of BF bursting neurons to the reward was negatively modulated by reward expectation , , , . Such properties would predict that, in the current experiment, BF responses to the reward should decrease throughout the stepwise learning process. Indeed, BF responses to the right reward decreased over trials in the D 1 session (Figs. e2 and ), and continued to decrease over subsequent sessions (Fig. ). These results support that animals learned to better predict the rewarding outcome across different steps of the learning process. We further investigated whether the response of BF bursting neurons to trial outcomes were negatively correlated with BF responses in earlier epochs. Such a negative correlation is a hallmark feature of reward prediction error encoding and has been previously reported in BF bursting neurons , . Indeed, at the per session level, we found that the amplitude of BF responses to the reward in light trials was strongly and negatively correlated with the amplitude of BF phasic bursting response to the light onset (Fig. ). Moreover, in pre-D 2 sessions where BF responses to the new light stimulus had yet to develop, we found that BF responses to the trial outcome were negatively correlated with BF evaluation responses in the same trial (Fig. ). This negative correlation at the single trial level was observed not only when the reward was delivered (light licks) but also when the reward was absent (no-fixation licks and catch licks) (Fig. b, ). The fact that these patterns were observed in catch licks and non-fixation licks supports that animals were expecting to receive reward in those trials, and the extent of reward expectation was similarly reflected in BF evaluation responses and negatively modulated BF responses to the trial outcome. Together, these observations support that BF bursting neurons encoded reward prediction error, and that BF activities in earlier epochs (stimulus onset or evaluation window) reflected animals’ reward expectations. To gain intuition about the stepwise learning strategy, we first considered a toy example in which a three-element sequence A-B-C predicted reward (Fig. ). This sequence can be learned using the stepwise strategy in three discrete steps, starting from the event closest to the reward and sequentially incorporating earlier events (Fig. ). As more behavioral events are learned as reward predictors in each step, only behavioral sequences that contain all the learned events would predict reward and therefore preferentially executed, while incompatible sequences that do not contain all the learned events would not predict reward and therefore be eliminated from the behavioral repertoire. As a result, the discrete steps of learning would correspond to the stepwise elimination of non-rewarded behavioral sequences that share subsets of behavioral elements (Fig. ). We hypothesized that this sequential refinement of reward-seeking behaviors might provide a behavioral signature of the stepwise learning strategy. To test whether animals indeed use the stepwise strategy to learn behavioral sequences that contain both sensory stimuli and their own actions, we trained adult Long-Evans rats in an auditory discrimination task. Rats entered the fixation port to initiate each trial, where they encountered three trial types (S left ; S right ; catch) with equal probabilities that respectively indicated sucrose water reward in the left or right port, or no reward in the case of catch trials (no stimulus) (Fig. ). During the initial auditory discrimination phase, S left and S right were two distinct sound stimuli. After reaching asymptotic performance, rats entered the new learning phase (first new learning session denoted as the D 0 session), in which the S right sound stimulus was switched to a novel light stimulus that minimized sensory generalization from past experience (Fig. and Table ). During the new learning phase, rats maintained stable levels of performance toward the previously learned S left sound stimulus (Fig. ) (94.3 ± 4.8% correct, 109 ± 20 trials per session, mean ± std). Within the first three sessions of the new learning phase, all rats ( N = 7) began responding correctly in the new light trials (the first such session denoted as the D 1 session) and maintained stable levels of >90% correct response rates afterwards (Fig. and Table ). To understand how the repertoire of behavioral sequences evolved during learning, we examined all possible behavioral sequences that the animal might experience (Fig. ). This approach allowed us to identify non-rewarded behavioral sequences that were not associated with specific trial types but were, nonetheless, highly relevant for the learning process. We identified three types of behavioral sequences whose frequencies consistently increased during the new learning phase. These three types of behavioral sequences included the rewarded licking behavior in the new light trials (light licks), as well as two types of non-rewarded behavioral sequences: catch licks and no-fixation licks (Fig. ). Catch licks refers to licking responses toward the right reward port in catch trials when no sensory stimulus was presented. No-fixation licks refers to the situation in which rats directly licked at the right reward port without first entering the center fixation port. All three behavioral sequences shared the common feature of licking the right reward port (lick-right). Both no-fixation licks and catch licks were largely absent before the new learning phase, and emerged and peaked during early sessions of new learning, before subsequently diminished in later sessions (Fig. ). No-fixation licks occurred most frequently at the D 1 session, while catch licks peaked in the same session ( N = 1/7) or later in most animals ( N = 6/7). We will denote the peak of catch licks as the D 2 session in each animal. The consistent temporal order of the D 1 and D 2 sessions within each animal allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The temporal dynamics of the three types of rightward licks during new learning (Fig. ) showed that non-rewarded behaviors were sequentially eliminated while rewarded behaviors were preserved. This temporal dynamics resembled the pattern of sequential refinement of behavioral sequences predicted by the stepwise learning strategy (Fig. ), and likely corresponded to the discrete steps in the underlying learning process. To further test whether such patterns of sequential refinements represent a general feature of behavioral sequence learning, we trained a separate cohort of animals and observed similar behavioral signatures regardless of the sensory modality of the stimulus or the laterality of the new learning side (Fig. ). These observations support the idea that animals do use the stepwise strategy to learn behavioral sequences. In the following analyses, we tested additional predictions of the stepwise learning strategy at both behavioral and neurophysiological levels. To validate the behavioral observations and understand the underlying neural dynamics, we recorded BF neuronal activity throughout the learning process (Fig. ) and used the consistent S left sound as the control stimulus to identify stable populations of BF bursting neurons (Figs. and ). A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. ). The population response of BF bursting neurons were highly consistent across animals (Fig. ), and remained remarkably stable in S left trials throughout the learning process (Fig. ). The inclusion of S left trials therefore allowed us to record from stable and representative populations of BF bursting neurons throughout the learning process, and to investigate how their activities dynamically evolved in other trial types during learning at single trial resolution (Fig. ). We first applied this approach to understand the behavioral and neural dynamics in the D 1 session because all three types of rightward licks emerged in this session (Fig. , middle panel). Detailed analysis of behavioral responses from a representative session (Fig. ) revealed that the three types of rightward licking behaviors emerged abruptly after a transition point (see Methods for definition). Rightward licking behaviors were mostly absent before the transition point, and rapidly switched to almost 100% licking after the transition point. This pattern of abrupt transition was consistently observed across all animals (Fig. ), while the behavioral performance and BF activities in S left sound trials remained relatively stable (Fig. ). At the neuronal level, there was a corresponding increase in the activity of BF bursting neurons that rapidly emerged after the transition in rightward reward-seeking behaviors (Figs. e and ). This increase in BF activity was most prominent in the epoch before the trial outcome as animals approached the reward port (Fig. ), but this activity was not consistently aligned with intervening behavioral events (Fig. ). In contrast, in trials before the transition point, BF bursting neurons did not show similar activity increases in the corresponding time window after exiting the fixation port (Figs. e and ). We will refer to the maximum BF activity in this window as the BF evaluation response (see Methods for definition) because it reflected animals’ internal evaluation when no sensory stimuli were presented during this epoch and because it was not consistently associated with intervening behavioral events. The stepwise learning model predicted that, as animals learned about the lick-right action as the first reward predictor, animals would initially engage in all three types of rightward licking behaviors because they all contained this reward predictor (Fig. ). We therefore examined the respective prevalence of the three types of rightward licking behaviors after the transition point in the D 1 session (Fig. ). All three types of rightward licking behaviors emerged immediately after the transition point. Moreover, after about 60 trials, no-fixation licks began to decline and occurred less frequently than light licks and catch licks (Fig. ). At the neuronal level, the corresponding prediction was that the lick-right action would be associated with increases in BF activities during the first learning step, which should be similarly present in all three types of rightward licking behaviors. Indeed, BF evaluation responses quickly increased after the transition point in all three types of rightward licking behaviors (Fig. ). The amplitudes of BF evaluation responses were similar between the three types of rightward licking behaviors within the first 60 trials after the transition. Subsequently, the BF evaluation response in no-fixation licks declined in the next 60 trials, relative to the other two types of rightward licking behaviors (Fig. ). These results support that the first step of the stepwise learning process corresponded to the first 60 trials after the transition point in the D 1 session. All three types of rightward licking behaviors were present during this step of learning. BF activity also increased to similar levels in all three types of behavioral sequences whenever animals approached and licked the right reward port, regardless of whether they had exited from the center fixation port. These results further suggest that the second step of learning started at roughly 60 trials after the transition, at which point animals learned about the second reward predictor: exiting the fixation port (Fig. ). As a result, the no-fixation lick sequence was no longer compatible with the learned reward predictors, which resulted in diminished BF evaluation responses and the elimination of this behavior from the behavioral repertoire. The other two types of rightward licks, light licks and catch licks, remained compatible and maintained high levels of BF evaluation responses. A further prediction of the stepwise learning model was that, at both the first and the second steps of learning, animals had not learned to use the new light stimulus as a reward predictor, and therefore the new light stimulus would not elicit responses in BF bursting neurons during these steps (Fig. ). We tested this prediction by comparing BF activities between light and catch trials in the D 1 session (Fig. ). Indeed, BF activities in light trials were highly similar to those in catch trials (in the absence of the light stimulus) in the epochs before exiting the fixation port. This was true regardless of whether animals subsequently licked at the right reward port. This observation confirmed the prediction that the new light stimulus did not activate BF bursting neurons in the D 1 session, despite the near-perfect behavioral performance in light trials after the behavior transition point. On the other hand, in the epoch after exiting the fixation port, there were similar increases in BF activity when animals licked at the right reward port in both light and catch trials (Fig. ). This increase in BF activity corresponded to the BF evaluation response described earlier (Fig. ), which reliably distinguished between lick and no lick trials, but not between light and catch trials (Fig. ). These observations support the idea that light and catch trials were treated as the same in the D 1 session, and that the light stimulus has not been learned as a reward predictor at this stage of learning. When did the third step of the stepwise learning process take place? Since the third step was when animals learned to use the light stimulus as a reward predictor to guide reward-seeking behaviors, it should correspond to the time point when the behavioral performance in light and catch trials began to diverge. We noted that the behavioral pattern in light and catch trials were highly similar prior to the D 2 session (Fig. ), and the similarity was best illustrated in the fine behavioral and neuronal dynamics within the D 1 session (Fig. ). In contrast, during the D 2 session, the behavioral pattern in light and catch trials began to show a small but significant difference (Fig. ). This pattern suggests that the D 2 session might be when the third step of learning took place. The corresponding prediction at the neuronal level was that BF bursting neurons should begin to respond to the light stimulus in the D 2 session. We tested this prediction by comparing BF activities between light and catch lick trials in the D 2 session, and found significant differences in all epochs, including the presence of a phasic response to the light onset (Fig. ). This observation supports the idea that BF responses to the new light emerged in the D 2 session. To better understand how BF responses to the new light emerged in the D2 session, we further compared BF responses in light lick and catch lick trials between (1) late trials in the D 2 −1 session; (2) early trials in the D 2 session; (3) late trials in the D 2 session (Fig. ). At the end of the D 2 −1 session, BF neurons did not show response to the new light (paired t -test between light lick and catch lick trials, p = 0.51, N = 6). In early D 2 trials, BF phasic responses to the new light were clearly visible in 4 animals. Comparison between late trials in the D 2 −1 session and the early D 2 trials showed a trend toward increasing BF responses (Fig. , significant at p < 0.05 level; Fig. , paired t -test, p = 0.065, N = 6). Moreover, during the D 2 session, BF responses to the new light increased in all animals between early and late trials (Fig. , paired t -test, p = 0.003, N = 7). This increase took place mostly during the sustained phase of the BF response that was better aligned with fixation port exit than with stimulus onset (Fig. ). Together, these observations suggest that BF responses to the new light developed partly offline between the D 2 −1 and D 2 session, and partly strengthened during the D 2 session. Comparing the temporal dynamics of behavioral and BF responses further suggests that the emergence of BF responses to the new light preceded the elimination of catch licks during the third step of learning (Fig. ). After the third step of learning took place in the D 2 session, did the learning about the new light plateau or did the learning continue to progress? At the neuronal level, BF phasic responses to the new light continued to grow stronger after the D 2 session (Fig. ). At first glance, the continual increases in BF responses to the new light stimulus did not match with the hit rates in light trials, which had already plateaued in the D 2 session (Fig. ). However, as we have shown earlier (Figs. and ), hit rates in light trials could be a poor index of learning about the new light stimulus because light licks in the early learning sessions were not driven by the light stimulus but by later events in the behavioral sequence (exiting fixation and lick-right) as reward predictors. Those behavioral events enabled rightward licking responses in the absence of the light stimulus (catch licks and no-fixation licks). Instead of the hit rate in light trials, a better behavioral index for the learning about the light stimulus would be the difference in the levels of behavioral performance between light and catch trials. This behavioral index accounted for the contributions from the later events in the behavioral sequence that were shared between light and catch licks, and isolated the contribution of the light stimulus to the rightward licking behavior. We found that this index of light learning was strongly correlated with the amplitude of BF phasic responses to the light stimulus in individual sessions (Fig. ). This observation therefore supports the idea that the learning about the light stimulus continued to grow stronger after the D 2 session. Another dimension of the learning about the light stimulus was the change in animals’ decision speeds, measured by reaction times (RTs). Previous studies have shown that stronger BF bursting responses are quantitatively coupled with, and causally lead to, faster RTs , . Such observations predicted that there would be corresponding decreases in RTs toward the light stimulus after the D 2 session. In support of this prediction, we found that stronger phasic bursting responses to light onset were coupled with faster RTs in individual sessions (Fig. ). Together, these observations support the idea that the learning about the new light was reflected by the amplitude of BF phasic response to the light stimulus. In addition, BF activities not only reflected the learning about the light stimulus, but also predicted reward-seeking behaviors in the absence of the light stimulus. For example, in catch trials, stronger BF activities after exiting the fixation port predicted rightward licking behavior and discriminated lick trials from no lick trials (Fig. ). Moreover, increased BF activities before the start of licking predicted longer durations of licking in catch lick trials when no reward was delivered (Fig. ). These observations provided additional support for the idea that the activity of BF bursting neurons promoted reward-seeking behaviors. A final validation of the learning dynamics came from how BF responses to the trial outcome were modulated by reward expectations and BF activities in earlier epochs. Previous studies have found that the response of BF bursting neurons to the reward was negatively modulated by reward expectation , , , . Such properties would predict that, in the current experiment, BF responses to the reward should decrease throughout the stepwise learning process. Indeed, BF responses to the right reward decreased over trials in the D 1 session (Figs. e2 and ), and continued to decrease over subsequent sessions (Fig. ). These results support that animals learned to better predict the rewarding outcome across different steps of the learning process. We further investigated whether the response of BF bursting neurons to trial outcomes were negatively correlated with BF responses in earlier epochs. Such a negative correlation is a hallmark feature of reward prediction error encoding and has been previously reported in BF bursting neurons , . Indeed, at the per session level, we found that the amplitude of BF responses to the reward in light trials was strongly and negatively correlated with the amplitude of BF phasic bursting response to the light onset (Fig. ). Moreover, in pre-D 2 sessions where BF responses to the new light stimulus had yet to develop, we found that BF responses to the trial outcome were negatively correlated with BF evaluation responses in the same trial (Fig. ). This negative correlation at the single trial level was observed not only when the reward was delivered (light licks) but also when the reward was absent (no-fixation licks and catch licks) (Fig. b, ). The fact that these patterns were observed in catch licks and non-fixation licks supports that animals were expecting to receive reward in those trials, and the extent of reward expectation was similarly reflected in BF evaluation responses and negatively modulated BF responses to the trial outcome. Together, these observations support that BF bursting neurons encoded reward prediction error, and that BF activities in earlier epochs (stimulus onset or evaluation window) reflected animals’ reward expectations. Results from the current study support that animals used a stepwise strategy to learn behavioral sequences that contain both sensory stimuli and their own actions. Behavioral events were learned sequentially, starting from the event closest to the reward and sequentially expanded to earlier events (Fig. ). The behavioral signature of this stepwise learning process was the sequential refinement of rightward licking behaviors, in which non-rewarded licking behaviors (no-fixation licks and catch licks) were sequentially eliminated while the rewarded behavior (light licks) was preserved (Fig. ). Learning about each behavioral event as a new reward predictor was accompanied by the emergence of BF responses toward that event, which conveyed animals’ reward prediction toward that event. Increased BF activities first emerged in the epoch before animals entered the reward port (Figs. e and ), while responses to the earlier event (light stimulus) developed later (Figs. and ). The evolution of BF activities mirrored the behavioral response patterns, which was initially increased in all three types of rightward licks (Figs. e and ) and subsequently decreased in non-rewarded licks as those behaviors were eliminated (Figs. b and ). Throughout the learning process, the activity of BF bursting neurons encoded reward prediction error signals (Fig. ) and their increased activities consistently predicted reward-seeking behaviors and faster reaction times (Fig. ). These results therefore identified the behavioral and neurophysiological signatures of the stepwise learning strategy when animals learned behavioral sequences. The current study focused on the learning of behavioral sequences that contain both sensory stimuli and actions, which reflect behavioral contexts that are commonly encountered both in experimental and natural settings. The learning dynamics that we described in this study cannot be easily accounted for using either Pavlovian , or instrumental , conditioning alone. In this regard, stepwise learning provides a new framework to understand the learning of behavioral sequences. Theoretically, long behavioral sequences are difficult to learn because the number of sequence permutations grows exponentially as a function of the sequence length. However, modeling studies have suggested that such learning can be greatly accelerated using the stepwise strategy, which reduces the number of sequence permutations to a linear function of the sequence length . The current study provides behavioral and neurophysiological evidence to support that animals indeed adopt the stepwise learning strategy to learn behavioral sequences. At each step of learning, animals explored the subset of behavioral sequences that shared common sequence elements learned in previous steps (Figs. and ). Such explorations allowed animals to distinguish those sequences, which were initially indistinguishable at the beginning of that learning step, and selectively eliminate subsets of non-rewarded sequences. From this perspective, the stepwise learning strategy offers an intuitive explanation of why animals committed certain types of ‘errors’ (non-rewarded licks), and suggests that those behaviors in fact represented genuine reward-seeking efforts at earlier stages of learning. By learning the associative relationship one step at a time, the stepwise learning strategy likely minimized the cognitive burden of the animal during the learning process and enabled the efficient learning of complex sequences. Our results suggest that stepwise learning is likely a general strategy for learning behavioral sequences because its behavioral and neural signatures were also observed in other learning settings. In a separate cohort of animals, we showed that the behavioral signatures of sequential refinements were similarly observed when the sensory modalities of the stimulus were reversed, and regardless of the laterality of the new learning side (Fig. ). Moreover, even during the learning of the most simple behavioral sequence containing only two elements (stimulus-action), similar behavioral and neural signatures were also observed (Fig. ). It is important to note that, while these results support that stepwise learning is a widely-used strategy for learning behavioral sequences, they do not preclude other possible learning strategies. In particular, if animals had previously encountered similar behavioral events, such experiences could be generalized to the new learning context, and allow animals to use a forward chaining strategy to learn behavioral sequences, instead of the backward chaining order in the stepwise learning strategy . For example, if animals had previously learned that light stimuli could predict reward, they could generalize that experience to the current learning task and view the new light stimulus as a potential reward predictor. Such generalizations would lead animals to engage in reward-seeking behaviors in light trials and quickly discover the light-reward association, bypassing the learning of behavioral events later in the sequence. Such strategies perhaps underlie the accelerated learning dynamics that we observed in one animal (animal #7, Fig. ), which also featured the strongest BF responses to the light stimulus during the initial encounter of the new light (Fig. ). Our results revealed that the true temporal dynamics about the learning of the new stimulus can be very different from the dynamics of behavioral performance levels in the new stimulus trial. We found that, despite the near-perfect behavioral performance in light trials that began after the transition point in the D 1 session (Figs. and ), the learning of the new light stimulus did not begin until the D 2 session (Figs. and ). Moreover, the learning of the new light stimulus continued to grow after the D 2 session despite the plateaued behavioral performance in light trials (Figs. and ). During early stages of learning, while the light stimulus was clearly perceptible and had been consistently paired with reward over many trials, the reward-seeking behavior in light trials was not driven by the light stimulus but by later behavioral events in the sequence (fixation port exit and lick-right). The critical factors that allowed us to reach this conclusion were the analysis of non-rewarded licking responses and the inclusion of catch trials in our task design. If not for these factors, we would incorrectly conclude that the learning about the new light stimulus occurred much earlier. The discrepancy between behavioral performance in light trials and the learning about the light stimulus highlights the potential mismatch in which salient physical events (such as the light stimulus) are not always automatically used by animals to predict reward, especially during the early stages of learning. The light stimulus was only incorporated as a reward predictor in later stages of the learning process, despite its continued presence from the beginning of the new learning. This observation indicates that, even in the absence of changes in reward contingencies in the environment, the learning process can lead to the addition of new reward predictors. Such structural revisions of the internal reward prediction model pose a fundamental challenge to theories and models of learning that assume a static set of reward predictors. Using models with incorrect reward predictors will lead to incorrect interpretations, regardless of how well the model fits the behavioral performance. The current study extends our understanding about the roles of BF bursting neurons in the encoding of reward prediction error. Previous studies have demonstrated that BF responses to rewards are negatively modulated by reward expectation , , and support the idea that BF neurons encode a reward prediction error signal , . The current study further extends this idea and shows that BF bursting neurons similarly encode reward prediction error in the context of new learning, and such encoding is robust even at the single trial level (Fig. ). This robust encoding of reward prediction error by BF bursting neurons quantitatively conveys the amount of reward prediction associated with each behavioral event at each learning step. As a result, the stepwise learning process was mirrored by the activity of BF bursting neurons, which provides a neural correlate of the stepwise learning process that we were able to track throughout the learning process in single trials. Given that reward prediction error information is also conveyed by other neuromodulatory systems, including midbrain dopaminergic neurons , , and BF cholinergic neurons – , BF bursting neurons are likely part of a broader network with partly correlated activities. Whether the other systems also provide similar neural correlates of stepwise learning remains to be determined. Previous studies have also established that BF bursting neurons serve as a bidirectional gain modulation mechanism for reward-seeking behaviors, where increased BF activities promote faster reaction times , while the inhibition of BF activities leads to rapid behavioral stopping . Manipulations of BF activities using electrical stimulation further suggest that BF bursting neurons likely play a causal role that can modulate decision speed when their activities are increased or inhibited . The current study extends this idea to the context of new learning, by showing that increased BF activities were tightly coupled with reward-seeking behaviors at multiple levels throughout the learning process (Figs. – ), and quantitatively predicted faster reaction times (Fig. ) and longer licking durations (Fig. ). The engagement in reward-seeking behaviors was particularly important in the context of learning behavioral sequences, because such explorations were essential for discovering the relationship between earlier events in the behavioral sequence and the rewarding outcome. Taken together, BF bursting neurons may serve the role of transforming the encoding of reward-prediction error into promoting reward-seeking behaviors during the new learning process. Finally, several studies have suggested that BF bursting neurons are likely a subset of GABAergic neurons , , , but their specific cellular marker(s) remains to be determined. Such marker information will be needed to conduct selective manipulation experiments that specifically target BF bursting neurons to directly test their causal role in the learning process. Ethics statement All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals and approved by the National Institute on Aging (NIA) Animal Care and Use Committee and by the Institutional Animal Care and Use Committee at the National Yang Ming Chiao Tung University, Taiwan (NYCU). Subjects Seven male Long-Evans rats (Charles River, NC), aged 3–6 months and weighing 300–400 g were used for the recording experiment. Rats were housed in a 12/12 day/night cycle and were provided with 10–12 dry pellets per day and unrestricted access to water. Rats were trained in daily sessions lasting 60–90 min. A separate cohort of eight male Long Evans rats (National Laboratory Animal Center, Taiwan) were used for behavioral testing (Fig. ) and an additional recording experiment (Fig. ). During training and recording procedures, these rats were water restricted to their 85–90% weight and were trained in a daily 60-min session. Water-restricted rats received 15 min water access at the end of each training day with free access on weekends. Apparatus Plexiglass operant chambers (11″ L × 8 ¼″ W × 13″ H), custom-built by Med Associates Inc. (St. Albans, VT), were contained in sound-attenuating cubicles (ENV-018MD) each with an exhaust fan that helped mask external noise. Each chamber’s front panel was equipped with an illuminated nose-poke port (ENV-114M) located in the center (horizontal axis) as the fixation port, which was equipped with an infrared (IR) sensor to detect the entry of the animals’ snout into the port. On each side of the center nose-poke port there were two reward ports (CT-ENV-251L-P). Two IR sensors were positioned to detect reward-port entry and sipper-tube licking, respectively. Sucrose solution (13.3%) was used as reward and delivered through the sipper tubes located in the reward ports. Reward delivery was controlled by solenoid valves (Parker Hannifin Corp #003-0111-900, Hollis, NH) and calibrated to provide 10 µl of solution per drop. Each chamber was equipped with a ceiling-mounted speaker (ENV-224BM) to deliver auditory stimuli, and a stimulus light (ENV-221) positioned above the center fixation port to serve as the new light stimulus. For the additional behavioral testing (Fig. ), one stimulus light each was added above the left and the right reward ports to serve as the sensory cue in the visual discrimination experiment, and water was used as the reward. Behavioral training protocols were controlled by Med-PC software (Versions IV & V, Med Associates Inc.), which stored all event timestamps at 1 or 2 ms resolution and also sent out TTL signals to the neurophysiology recording systems. Behavioral training procedures Rats were trained in operant chambers that were dimly lit. Rats were first trained in an auditory or visual discrimination task. See Table and Fig. for details of the stimuli used for each animal. Trials were separated by an unsignaled inter-trial interval (ITI) lasting 4–6 s. Fixation or licks during the ITI reset the ITI timer. After the ITI, the center fixation port was illuminated, which was turned off only when rats poked the fixation port. Rats were required to maintain fixation in the center nose-poke port for a variable amount of foreperiod. Four different foreperiods (0.35, 0.5, 0.65, and 0.8 s) were used, pseudorandomly across trials. After the foreperiod, one of three conditions was randomly presented with equal probabilities: a S right stimulus indicated reward on the right port; a different S left stimulus indicated reward on the left port; or the absence of stimulus (catch trial) indicated no reward. An internal timestamp was recorded in catch trials to mark the onset of the would-be stimulus. Early fixation port exit before the end of the foreperiod led to the re-illumination of the center fixation port. Licking in the correct port within a 3 s window after stimulus onset led to three drops of reward, delivered starting at the 3rd lick. The delivery of reward at the 3rd lick created an expectation for trial outcome at this time point, which was dissociated in time from the initiation of licking (1st lick). We will therefore refer to the time point of the 3rd lick also as the trial outcome event. Trials with licking responses ended after 1 s following the last lick, while trials without licking responses ended after the 3 s response window. The ending of each trial started the ITI timer. After reaching asymptotic behavioral performance in the auditory or the visual discrimination task, a new learning phase was introduced by replacing either the S right or S left stimuli by a novel sensory stimulus in a different sensory modality, while all other aspects of the task remained the same. In the group that were initially trained with auditory discrimination, the S right sound stimulus was replaced by the central light above the fixation port to indicate reward on the right port (Table ). In the group there were initially trained with visual discrimination, either the S right or S left light stimuli was replaced by a 6 kHz sound (70 dB) played from a speaker above the center fixation port (Fig. ). Stereotaxic surgery and electrode Surgery was performed under isoflurane anesthesia similar to our earlier study . Multiple skull screws were inserted to anchor the implant, with one screw over the cerebellum serving as the common electrical reference and a separate screw over the opposite cerebellum hemisphere serving as the electrical ground. Craniotomies were opened to target bilateral BF (AP –0.6 mm, ML ± 2.25 mm relative to Bregma) . The electrode contained two bundles of 16 polyimide-insulated tungsten wires (38 µm diameter; California Fine Wire, CA), each bundle ensheathed in a 28-gauge stainless steel cannula and controlled by a precision microdrive. The impedance of individual wire was ~ 0.1 MΩ measured at 1 kHz (niPOD, NeuroNexusTech, MI or Open Ephys Acquisition Board). During surgery, the cannulae were lowered to DV 6.5 mm below cortical surface using a micropositioner (Model 2662, David Kopf Instrument or Robot Stereotaxic, Neurostar GmbH) at a speed of 2–50 µm/s. After reaching target depth, the electrode and screws were covered with dental cement (Hygenic Denture Resin), and electrodes further advanced to 7.5 mm below the cortical surface. Rats received ibuprofen and topical antibiotics after surgery for pain relief and prevention of infection, and were allowed one week to recover with ad libitum food and water. Cannulae and electrode tip locations were verified with cresyl violet staining of histological sections at the end of the experiment. All electrodes were found at expected positions between AP [–0.2, –1.2] mm, ML [1.5, 3] mm, relative to Bregma, and DV [7.5, 8.5] mm relative to cortical surface (Fig. ). Data acquisition and spike sorting Electrical signals were referenced to a common skull screw placed over the cerebellum. Electrical signals were filtered (0.3 Hz to 7.5 kHz) and amplified using Cereplex M digital headstages and recorded using a Neural Signal Processor (Blackrock Microsystems, UT). Single unit activity was further filtered (250 Hz to 5 kHz) and recorded at 30 kHz. Spike waveforms were sorted offline to identify single units using the KlustaKwik sorting algorithm followed by a custom Python GUI (version 2.7) for manual curation. Only single units with clear separation from the noise cluster and with minimal (<0.1%) spike collisions (spikes with less than 1.5 ms interspike interval) were used for further analyses, consistent with previous studies of BF bursting neurons – . Additional cross-correlation analysis was used to remove duplicate units recorded simultaneously across multiple electrodes – . Recording during the new learning phase After surgery, BF neuronal activity was monitored while rats were re-trained in the auditory discrimination task to asymptotic performance level. During this re-training phase, BF electrode depths were adjusted slightly (by advancing electrodes at 125 µm increment) until a stable population of BF single units can be recorded. At this point, the new learning phase with the light as the new S right stimulus was introduced and rats were trained and recorded daily with BF electrodes remained at the same depth. This approach allowed us to monitor the activity of a large population of BF neurons and follow its temporal evolution across sessions. Data analysis Data were analyzed using custom Matlab (R2018b, MATLAB The MathWorks Inc., Natick, MA) scripts. Define different behavioral response types Licking responses were defined for stimulus (S left and S right ) and catch trials if rats licked at least three times in the reward port within the 3 s window after stimulus onset (or the corresponding timestamp for the would-be stimulus in catch trials). Licking responses to the correct reward port were rewarded with three drops of water, delivered starting at the 3rd lick (referred to as trial outcome event). During the new learning phase, licking responses in the new stimulus and catch trials were predominantly to the reward port associated with the new stimulus. No-fixation licks corresponded to licking responses to the reward port associated with the new stimulus that were not preceded by poking the center fixation port. Specifically, no-fixation licks were defined based on three criteria: (1) rats made at least three consecutive licks in the reward port; (2) the interval between the last exit from the fixation port and the first lick must be greater than 2 s; (3) the interval between the last exit from the reward port and the subsequent first lick in the same reward port must be greater than 1 s. These duration thresholds were determined based on the empirical licking patterns across animals. In the analyses of learning dynamics in the D 1 session (Figs. and ), no-fixation licks were treated as rightward licking trials, even though such behaviors were self-initiated and not imposed by the task design. Reaction time (RT) in light trials in a session (Fig. ) was defined as the median of the interval between the onset of the light stimulus and the exit from the fixation port in light lick trials. Lick duration in catch trials in a session (Fig. ) was defined as the median of the interval between the first and the last lick in catch lick trials. Define the D 0 , D 1 and D 2 learning landmarks During the new learning phase, three sessions (D 0 , D 1 , D 2 ) were identified in individual animals as landmarks that demarcated distinct stages of new learning (Fig. ). The D 0 session was defined as the very first session the new light stimulus was introduced. The D 1 session was defined as the first session when animals began to respond correctly in the new stimulus trials and obtained reward in the associated reward port in at least three trials. The D 2 session was defined as the session in which catch licks occurred most frequently. The D 1 and D 2 landmarks allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The specific timing of the three landmark sessions in each animal are provided in Table (also see Fig. ). One animal (ID#7) with accelerated learning dynamics, in which D 1 and D 2 occurred in the same session, was excluded from analyses of D 1 neural dynamics (Figs. and ) to ensure that neural activities associated with D 2 did not confound the neural dynamics in the D 1 session. The BF neuronal activity in this animal was included in the analysis of D 2 neural dynamics (Fig. ) and showed the strongest phasic response to the light onset among all animals, consistent with its accelerated learning dynamics. Identification of the behavioral transition point in the D 1 session The behavioral transition points in D 1 sessions (Figs. and ) were identified based on behavioral response patterns in three trial types combined: light trials, catch trials and no-fixation licks. The behavioral response pattern in each trial was coded as either 1 or 0 based on whether animals licked in the right reward port in that trial. The behavioral transition point was defined as the point with the largest difference in licking responses between the 20 trials before and the 20 trials after that point. In 5/7 animals, the first trial after the behavioral transition was a rewarded light lick trial. In the other two animals, the transition point was adjusted to the closest light lick trial by 2 or 4 trials, respectively. Identification of BF bursting neurons BF bursting neurons were defined as BF single units whose average firing rates during the [0.05, 0.2]s window after stimulus onset increased by more than 2 spikes/s in the S left sound trials compared to the corresponding window in catch trials (Fig. ). This contrast between sound trials and catch trials was necessary because it removed the nonstationary baseline before stimulus onset and allowed us to ask whether BF neurons truly responded to the sound stimulus. In addition, BF bursting neurons should have baseline firing rates (during the [−1, 0]s window relative to the trial start signal) less than 10 spikes/s. A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. and Table ). Since BF electrodes remained at the same depth throughout the recording sessions, the same BF single units might be recorded in multiple sessions. The large number of BF bursting neurons recorded in each session allowed us to treat them as a representative sample of all BF bursting neurons, whose responses to the S left sound were highly stable throughout the learning process (Fig. ). This strategy ensured that we were following functionally the same neuronal ensemble and could track how BF bursting neurons acquired responses to the new light during learning, regardless of whether the identities of these BF neurons were exactly the same in each session. One session with only one BF bursting neuron was excluded from the analysis of BF population activities. Population BF responses to behavioral events The spike timestamps of all BF bursting neurons in a single session were pooled together to approximate the population activity of all BF bursting neurons. Population peri-stimulus time histograms (PSTHs) were calculated with 10-ms bins, and normalized by the number of BF bursting neurons in a session. To properly assess whether BF bursting neurons responded to the onset of the new light stimulus (Figs. – ), it was important to disambiguate such stimulus-onset responses from the increased BF activities after fixation port exit (Fig. ). To achieve this goal, PSTHs to the stimulus onset were calculated based only on spikes that occurred before fixation port exit in individual trials, resulting in different interval lengths (between stimulus onset to fixation port exit) across trials. Accordingly, the calculation of the mean PSTH across all trials in a session was adjusted for the different number of trials at different interval lengths. The mean PSTHs were further truncated at the median interval length of that session to reduce noisy estimates of PSTHs at long interval lengths due to lower number of trials. When PSTHs were averaged across animals, the averaged PSTHs were further truncated at the mean of median interval latencies across animals. This truncation procedure resulted in the uneven lengths of PSTHs across individual animals (Figs. , , S and S ) and across sessions (Fig. ). This procedure was also applied to calculating the BF responses before fixation port exit to include only spikes that occurred after stimulus onset (Figs. , and S ), and for calculating BF responses during licking in catch lick trials (Fig. ). The time windows used to quantify average BF activity in different epochs were indicated in respective figures, and corresponded to the following: [0.05, 0.2]s after S left sound onset; [0.1, 0.3]s after S right light stimulus onset; [0.1, 0.3]s after the timestamp for the would-be stimulus in catch trials; [0.05, 0.35]s after the 3rd lick for outcome responses; [−0.3, 0]s and [0, 0.3]s relative to the fixation port exit. The epoch for calculating evaluation response is described below. Evaluation response (Figs. e, b, a and b, ) refers to the increased BF activity after exiting the fixation port and before the trial outcome (3rd lick). The evaluation response reflected animals’ internal evaluation because no additional sensory stimuli were presented during this epoch and this activity was not consistently aligned with intervening behavioral events (Fig. ). Specifically, the evaluation response was calculated in individual trials and defined as the maximum firing rate of any 500 ms window during the evaluation epoch, which corresponded to the interval between [fix-out, outcome], with additional adjustments according to trial types. In light lick and catch lick trials, the evaluation epoch was defined as [fix-out, outcome] in each trial. The epoch durations in light lick and catch lick trials within each session were used as the reference point for other trial types as described next. In no-fixation licks, in which the fix-out event was absent, the duration of evaluation epoch was set as the 95th percentile of the evaluation epoch durations in light lick and catch lick trials, and the epoch should begin at least 0.5 s before reward port entry. In light no lick and catch no lick trials, in which the 3rd lick event was absent, the duration of the evaluation epoch was set as the median of the evaluation epoch durations in light lick and catch lick trials. These adjustments in the definition of evaluation epochs, as well as its calculation of maximum firing rate within the epoch, took into consideration the behavioral variability across trial types, learning stages and individual animals. To evaluate the dynamic changes of BF activities around the transition point in the D 1 session (Figs. e and ), single trial evaluation and outcome responses were smoothed using moving median over 10 trials. The smoothed trends were aligned at the transition point and then averaged across all animals. Only trials with smoothed trend data from at least 4 animals were plotted in the group average (Fig. ). Statistics Statistical comparisons were conducted using the Statistics and Machine Learning Toolbox (version 11.3) in MATLAB (R2018a) ( https://www.mathworks.com/ ). Two-sided paired t -test (ttest.m) was used to compare behavioral and neural activity differences between two groups (Figs. d, a, c2, and ). Repeated measures analysis of variance (ranova.m) was used for comparisons involving more than 2 groups, by specifying the appropriate within-subject models (Figs. a3, b3, and ). Comparisons of PSTHs between two groups (Figs. a, b, c1, d, and ) was conducted for each 100 ms sliding window (10 ms step) using two-sided paired t -test. Significance level was set at p < 0.01 for three consecutive bins. Pearson correlation (corrcoef.m) was used to determine the relationship between neuronal activities and/or behavior (Figs. b, c, a, b2, and ). Receiver operating characteristic (ROC) and area under curve (AUC) analysis To determine whether the activity of BF bursting neurons differentiated between trial types within each D 1 session (Fig. ), we compared BF activity for each 100 ms sliding window (10 ms step) using the AUC measure of ROC analysis (auc.m by Alois Schloegl). At each sliding window, BF population activity was calculated for each light and catch trial, and distributions of BF activities were compared between light vs catch trials or between lick vs no lick trials. Significance level was set at p < 0.001 using 10,000 trial-shuffled random permutations (two-sided). To determine whether BF activity differentiated between lick and no lick trials within the same trial type (light or catch trials) (Fig. ), we compared BF activity in the [0, 500] ms window after exiting the fixation port. For each session and each trial type, lick and no lick trials must each constitute at least 10% of that trial type to be included in the analysis. Catch trials from all sessions were included in this analysis. Only light trials before the D 2 session (pre-D 2 ) were included in this analysis because BF responses to the onset of the light stimulus had not developed in those sessions. Significance level was set at p < 0.05 using 1000 trial-shuffled random permutations (two-sided). Reporting summary Further information on research design is available in the linked to this article. All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals and approved by the National Institute on Aging (NIA) Animal Care and Use Committee and by the Institutional Animal Care and Use Committee at the National Yang Ming Chiao Tung University, Taiwan (NYCU). Seven male Long-Evans rats (Charles River, NC), aged 3–6 months and weighing 300–400 g were used for the recording experiment. Rats were housed in a 12/12 day/night cycle and were provided with 10–12 dry pellets per day and unrestricted access to water. Rats were trained in daily sessions lasting 60–90 min. A separate cohort of eight male Long Evans rats (National Laboratory Animal Center, Taiwan) were used for behavioral testing (Fig. ) and an additional recording experiment (Fig. ). During training and recording procedures, these rats were water restricted to their 85–90% weight and were trained in a daily 60-min session. Water-restricted rats received 15 min water access at the end of each training day with free access on weekends. Plexiglass operant chambers (11″ L × 8 ¼″ W × 13″ H), custom-built by Med Associates Inc. (St. Albans, VT), were contained in sound-attenuating cubicles (ENV-018MD) each with an exhaust fan that helped mask external noise. Each chamber’s front panel was equipped with an illuminated nose-poke port (ENV-114M) located in the center (horizontal axis) as the fixation port, which was equipped with an infrared (IR) sensor to detect the entry of the animals’ snout into the port. On each side of the center nose-poke port there were two reward ports (CT-ENV-251L-P). Two IR sensors were positioned to detect reward-port entry and sipper-tube licking, respectively. Sucrose solution (13.3%) was used as reward and delivered through the sipper tubes located in the reward ports. Reward delivery was controlled by solenoid valves (Parker Hannifin Corp #003-0111-900, Hollis, NH) and calibrated to provide 10 µl of solution per drop. Each chamber was equipped with a ceiling-mounted speaker (ENV-224BM) to deliver auditory stimuli, and a stimulus light (ENV-221) positioned above the center fixation port to serve as the new light stimulus. For the additional behavioral testing (Fig. ), one stimulus light each was added above the left and the right reward ports to serve as the sensory cue in the visual discrimination experiment, and water was used as the reward. Behavioral training protocols were controlled by Med-PC software (Versions IV & V, Med Associates Inc.), which stored all event timestamps at 1 or 2 ms resolution and also sent out TTL signals to the neurophysiology recording systems. Rats were trained in operant chambers that were dimly lit. Rats were first trained in an auditory or visual discrimination task. See Table and Fig. for details of the stimuli used for each animal. Trials were separated by an unsignaled inter-trial interval (ITI) lasting 4–6 s. Fixation or licks during the ITI reset the ITI timer. After the ITI, the center fixation port was illuminated, which was turned off only when rats poked the fixation port. Rats were required to maintain fixation in the center nose-poke port for a variable amount of foreperiod. Four different foreperiods (0.35, 0.5, 0.65, and 0.8 s) were used, pseudorandomly across trials. After the foreperiod, one of three conditions was randomly presented with equal probabilities: a S right stimulus indicated reward on the right port; a different S left stimulus indicated reward on the left port; or the absence of stimulus (catch trial) indicated no reward. An internal timestamp was recorded in catch trials to mark the onset of the would-be stimulus. Early fixation port exit before the end of the foreperiod led to the re-illumination of the center fixation port. Licking in the correct port within a 3 s window after stimulus onset led to three drops of reward, delivered starting at the 3rd lick. The delivery of reward at the 3rd lick created an expectation for trial outcome at this time point, which was dissociated in time from the initiation of licking (1st lick). We will therefore refer to the time point of the 3rd lick also as the trial outcome event. Trials with licking responses ended after 1 s following the last lick, while trials without licking responses ended after the 3 s response window. The ending of each trial started the ITI timer. After reaching asymptotic behavioral performance in the auditory or the visual discrimination task, a new learning phase was introduced by replacing either the S right or S left stimuli by a novel sensory stimulus in a different sensory modality, while all other aspects of the task remained the same. In the group that were initially trained with auditory discrimination, the S right sound stimulus was replaced by the central light above the fixation port to indicate reward on the right port (Table ). In the group there were initially trained with visual discrimination, either the S right or S left light stimuli was replaced by a 6 kHz sound (70 dB) played from a speaker above the center fixation port (Fig. ). Surgery was performed under isoflurane anesthesia similar to our earlier study . Multiple skull screws were inserted to anchor the implant, with one screw over the cerebellum serving as the common electrical reference and a separate screw over the opposite cerebellum hemisphere serving as the electrical ground. Craniotomies were opened to target bilateral BF (AP –0.6 mm, ML ± 2.25 mm relative to Bregma) . The electrode contained two bundles of 16 polyimide-insulated tungsten wires (38 µm diameter; California Fine Wire, CA), each bundle ensheathed in a 28-gauge stainless steel cannula and controlled by a precision microdrive. The impedance of individual wire was ~ 0.1 MΩ measured at 1 kHz (niPOD, NeuroNexusTech, MI or Open Ephys Acquisition Board). During surgery, the cannulae were lowered to DV 6.5 mm below cortical surface using a micropositioner (Model 2662, David Kopf Instrument or Robot Stereotaxic, Neurostar GmbH) at a speed of 2–50 µm/s. After reaching target depth, the electrode and screws were covered with dental cement (Hygenic Denture Resin), and electrodes further advanced to 7.5 mm below the cortical surface. Rats received ibuprofen and topical antibiotics after surgery for pain relief and prevention of infection, and were allowed one week to recover with ad libitum food and water. Cannulae and electrode tip locations were verified with cresyl violet staining of histological sections at the end of the experiment. All electrodes were found at expected positions between AP [–0.2, –1.2] mm, ML [1.5, 3] mm, relative to Bregma, and DV [7.5, 8.5] mm relative to cortical surface (Fig. ). Electrical signals were referenced to a common skull screw placed over the cerebellum. Electrical signals were filtered (0.3 Hz to 7.5 kHz) and amplified using Cereplex M digital headstages and recorded using a Neural Signal Processor (Blackrock Microsystems, UT). Single unit activity was further filtered (250 Hz to 5 kHz) and recorded at 30 kHz. Spike waveforms were sorted offline to identify single units using the KlustaKwik sorting algorithm followed by a custom Python GUI (version 2.7) for manual curation. Only single units with clear separation from the noise cluster and with minimal (<0.1%) spike collisions (spikes with less than 1.5 ms interspike interval) were used for further analyses, consistent with previous studies of BF bursting neurons – . Additional cross-correlation analysis was used to remove duplicate units recorded simultaneously across multiple electrodes – . After surgery, BF neuronal activity was monitored while rats were re-trained in the auditory discrimination task to asymptotic performance level. During this re-training phase, BF electrode depths were adjusted slightly (by advancing electrodes at 125 µm increment) until a stable population of BF single units can be recorded. At this point, the new learning phase with the light as the new S right stimulus was introduced and rats were trained and recorded daily with BF electrodes remained at the same depth. This approach allowed us to monitor the activity of a large population of BF neurons and follow its temporal evolution across sessions. Data were analyzed using custom Matlab (R2018b, MATLAB The MathWorks Inc., Natick, MA) scripts. Define different behavioral response types Licking responses were defined for stimulus (S left and S right ) and catch trials if rats licked at least three times in the reward port within the 3 s window after stimulus onset (or the corresponding timestamp for the would-be stimulus in catch trials). Licking responses to the correct reward port were rewarded with three drops of water, delivered starting at the 3rd lick (referred to as trial outcome event). During the new learning phase, licking responses in the new stimulus and catch trials were predominantly to the reward port associated with the new stimulus. No-fixation licks corresponded to licking responses to the reward port associated with the new stimulus that were not preceded by poking the center fixation port. Specifically, no-fixation licks were defined based on three criteria: (1) rats made at least three consecutive licks in the reward port; (2) the interval between the last exit from the fixation port and the first lick must be greater than 2 s; (3) the interval between the last exit from the reward port and the subsequent first lick in the same reward port must be greater than 1 s. These duration thresholds were determined based on the empirical licking patterns across animals. In the analyses of learning dynamics in the D 1 session (Figs. and ), no-fixation licks were treated as rightward licking trials, even though such behaviors were self-initiated and not imposed by the task design. Reaction time (RT) in light trials in a session (Fig. ) was defined as the median of the interval between the onset of the light stimulus and the exit from the fixation port in light lick trials. Lick duration in catch trials in a session (Fig. ) was defined as the median of the interval between the first and the last lick in catch lick trials. Define the D 0 , D 1 and D 2 learning landmarks During the new learning phase, three sessions (D 0 , D 1 , D 2 ) were identified in individual animals as landmarks that demarcated distinct stages of new learning (Fig. ). The D 0 session was defined as the very first session the new light stimulus was introduced. The D 1 session was defined as the first session when animals began to respond correctly in the new stimulus trials and obtained reward in the associated reward port in at least three trials. The D 2 session was defined as the session in which catch licks occurred most frequently. The D 1 and D 2 landmarks allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The specific timing of the three landmark sessions in each animal are provided in Table (also see Fig. ). One animal (ID#7) with accelerated learning dynamics, in which D 1 and D 2 occurred in the same session, was excluded from analyses of D 1 neural dynamics (Figs. and ) to ensure that neural activities associated with D 2 did not confound the neural dynamics in the D 1 session. The BF neuronal activity in this animal was included in the analysis of D 2 neural dynamics (Fig. ) and showed the strongest phasic response to the light onset among all animals, consistent with its accelerated learning dynamics. Identification of the behavioral transition point in the D 1 session The behavioral transition points in D 1 sessions (Figs. and ) were identified based on behavioral response patterns in three trial types combined: light trials, catch trials and no-fixation licks. The behavioral response pattern in each trial was coded as either 1 or 0 based on whether animals licked in the right reward port in that trial. The behavioral transition point was defined as the point with the largest difference in licking responses between the 20 trials before and the 20 trials after that point. In 5/7 animals, the first trial after the behavioral transition was a rewarded light lick trial. In the other two animals, the transition point was adjusted to the closest light lick trial by 2 or 4 trials, respectively. Identification of BF bursting neurons BF bursting neurons were defined as BF single units whose average firing rates during the [0.05, 0.2]s window after stimulus onset increased by more than 2 spikes/s in the S left sound trials compared to the corresponding window in catch trials (Fig. ). This contrast between sound trials and catch trials was necessary because it removed the nonstationary baseline before stimulus onset and allowed us to ask whether BF neurons truly responded to the sound stimulus. In addition, BF bursting neurons should have baseline firing rates (during the [−1, 0]s window relative to the trial start signal) less than 10 spikes/s. A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. and Table ). Since BF electrodes remained at the same depth throughout the recording sessions, the same BF single units might be recorded in multiple sessions. The large number of BF bursting neurons recorded in each session allowed us to treat them as a representative sample of all BF bursting neurons, whose responses to the S left sound were highly stable throughout the learning process (Fig. ). This strategy ensured that we were following functionally the same neuronal ensemble and could track how BF bursting neurons acquired responses to the new light during learning, regardless of whether the identities of these BF neurons were exactly the same in each session. One session with only one BF bursting neuron was excluded from the analysis of BF population activities. Population BF responses to behavioral events The spike timestamps of all BF bursting neurons in a single session were pooled together to approximate the population activity of all BF bursting neurons. Population peri-stimulus time histograms (PSTHs) were calculated with 10-ms bins, and normalized by the number of BF bursting neurons in a session. To properly assess whether BF bursting neurons responded to the onset of the new light stimulus (Figs. – ), it was important to disambiguate such stimulus-onset responses from the increased BF activities after fixation port exit (Fig. ). To achieve this goal, PSTHs to the stimulus onset were calculated based only on spikes that occurred before fixation port exit in individual trials, resulting in different interval lengths (between stimulus onset to fixation port exit) across trials. Accordingly, the calculation of the mean PSTH across all trials in a session was adjusted for the different number of trials at different interval lengths. The mean PSTHs were further truncated at the median interval length of that session to reduce noisy estimates of PSTHs at long interval lengths due to lower number of trials. When PSTHs were averaged across animals, the averaged PSTHs were further truncated at the mean of median interval latencies across animals. This truncation procedure resulted in the uneven lengths of PSTHs across individual animals (Figs. , , S and S ) and across sessions (Fig. ). This procedure was also applied to calculating the BF responses before fixation port exit to include only spikes that occurred after stimulus onset (Figs. , and S ), and for calculating BF responses during licking in catch lick trials (Fig. ). The time windows used to quantify average BF activity in different epochs were indicated in respective figures, and corresponded to the following: [0.05, 0.2]s after S left sound onset; [0.1, 0.3]s after S right light stimulus onset; [0.1, 0.3]s after the timestamp for the would-be stimulus in catch trials; [0.05, 0.35]s after the 3rd lick for outcome responses; [−0.3, 0]s and [0, 0.3]s relative to the fixation port exit. The epoch for calculating evaluation response is described below. Evaluation response (Figs. e, b, a and b, ) refers to the increased BF activity after exiting the fixation port and before the trial outcome (3rd lick). The evaluation response reflected animals’ internal evaluation because no additional sensory stimuli were presented during this epoch and this activity was not consistently aligned with intervening behavioral events (Fig. ). Specifically, the evaluation response was calculated in individual trials and defined as the maximum firing rate of any 500 ms window during the evaluation epoch, which corresponded to the interval between [fix-out, outcome], with additional adjustments according to trial types. In light lick and catch lick trials, the evaluation epoch was defined as [fix-out, outcome] in each trial. The epoch durations in light lick and catch lick trials within each session were used as the reference point for other trial types as described next. In no-fixation licks, in which the fix-out event was absent, the duration of evaluation epoch was set as the 95th percentile of the evaluation epoch durations in light lick and catch lick trials, and the epoch should begin at least 0.5 s before reward port entry. In light no lick and catch no lick trials, in which the 3rd lick event was absent, the duration of the evaluation epoch was set as the median of the evaluation epoch durations in light lick and catch lick trials. These adjustments in the definition of evaluation epochs, as well as its calculation of maximum firing rate within the epoch, took into consideration the behavioral variability across trial types, learning stages and individual animals. To evaluate the dynamic changes of BF activities around the transition point in the D 1 session (Figs. e and ), single trial evaluation and outcome responses were smoothed using moving median over 10 trials. The smoothed trends were aligned at the transition point and then averaged across all animals. Only trials with smoothed trend data from at least 4 animals were plotted in the group average (Fig. ). Statistics Statistical comparisons were conducted using the Statistics and Machine Learning Toolbox (version 11.3) in MATLAB (R2018a) ( https://www.mathworks.com/ ). Two-sided paired t -test (ttest.m) was used to compare behavioral and neural activity differences between two groups (Figs. d, a, c2, and ). Repeated measures analysis of variance (ranova.m) was used for comparisons involving more than 2 groups, by specifying the appropriate within-subject models (Figs. a3, b3, and ). Comparisons of PSTHs between two groups (Figs. a, b, c1, d, and ) was conducted for each 100 ms sliding window (10 ms step) using two-sided paired t -test. Significance level was set at p < 0.01 for three consecutive bins. Pearson correlation (corrcoef.m) was used to determine the relationship between neuronal activities and/or behavior (Figs. b, c, a, b2, and ). Receiver operating characteristic (ROC) and area under curve (AUC) analysis To determine whether the activity of BF bursting neurons differentiated between trial types within each D 1 session (Fig. ), we compared BF activity for each 100 ms sliding window (10 ms step) using the AUC measure of ROC analysis (auc.m by Alois Schloegl). At each sliding window, BF population activity was calculated for each light and catch trial, and distributions of BF activities were compared between light vs catch trials or between lick vs no lick trials. Significance level was set at p < 0.001 using 10,000 trial-shuffled random permutations (two-sided). To determine whether BF activity differentiated between lick and no lick trials within the same trial type (light or catch trials) (Fig. ), we compared BF activity in the [0, 500] ms window after exiting the fixation port. For each session and each trial type, lick and no lick trials must each constitute at least 10% of that trial type to be included in the analysis. Catch trials from all sessions were included in this analysis. Only light trials before the D 2 session (pre-D 2 ) were included in this analysis because BF responses to the onset of the light stimulus had not developed in those sessions. Significance level was set at p < 0.05 using 1000 trial-shuffled random permutations (two-sided). Licking responses were defined for stimulus (S left and S right ) and catch trials if rats licked at least three times in the reward port within the 3 s window after stimulus onset (or the corresponding timestamp for the would-be stimulus in catch trials). Licking responses to the correct reward port were rewarded with three drops of water, delivered starting at the 3rd lick (referred to as trial outcome event). During the new learning phase, licking responses in the new stimulus and catch trials were predominantly to the reward port associated with the new stimulus. No-fixation licks corresponded to licking responses to the reward port associated with the new stimulus that were not preceded by poking the center fixation port. Specifically, no-fixation licks were defined based on three criteria: (1) rats made at least three consecutive licks in the reward port; (2) the interval between the last exit from the fixation port and the first lick must be greater than 2 s; (3) the interval between the last exit from the reward port and the subsequent first lick in the same reward port must be greater than 1 s. These duration thresholds were determined based on the empirical licking patterns across animals. In the analyses of learning dynamics in the D 1 session (Figs. and ), no-fixation licks were treated as rightward licking trials, even though such behaviors were self-initiated and not imposed by the task design. Reaction time (RT) in light trials in a session (Fig. ) was defined as the median of the interval between the onset of the light stimulus and the exit from the fixation port in light lick trials. Lick duration in catch trials in a session (Fig. ) was defined as the median of the interval between the first and the last lick in catch lick trials. 0 , D 1 and D 2 learning landmarks During the new learning phase, three sessions (D 0 , D 1 , D 2 ) were identified in individual animals as landmarks that demarcated distinct stages of new learning (Fig. ). The D 0 session was defined as the very first session the new light stimulus was introduced. The D 1 session was defined as the first session when animals began to respond correctly in the new stimulus trials and obtained reward in the associated reward port in at least three trials. The D 2 session was defined as the session in which catch licks occurred most frequently. The D 1 and D 2 landmarks allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The specific timing of the three landmark sessions in each animal are provided in Table (also see Fig. ). One animal (ID#7) with accelerated learning dynamics, in which D 1 and D 2 occurred in the same session, was excluded from analyses of D 1 neural dynamics (Figs. and ) to ensure that neural activities associated with D 2 did not confound the neural dynamics in the D 1 session. The BF neuronal activity in this animal was included in the analysis of D 2 neural dynamics (Fig. ) and showed the strongest phasic response to the light onset among all animals, consistent with its accelerated learning dynamics. 1 session The behavioral transition points in D 1 sessions (Figs. and ) were identified based on behavioral response patterns in three trial types combined: light trials, catch trials and no-fixation licks. The behavioral response pattern in each trial was coded as either 1 or 0 based on whether animals licked in the right reward port in that trial. The behavioral transition point was defined as the point with the largest difference in licking responses between the 20 trials before and the 20 trials after that point. In 5/7 animals, the first trial after the behavioral transition was a rewarded light lick trial. In the other two animals, the transition point was adjusted to the closest light lick trial by 2 or 4 trials, respectively. BF bursting neurons were defined as BF single units whose average firing rates during the [0.05, 0.2]s window after stimulus onset increased by more than 2 spikes/s in the S left sound trials compared to the corresponding window in catch trials (Fig. ). This contrast between sound trials and catch trials was necessary because it removed the nonstationary baseline before stimulus onset and allowed us to ask whether BF neurons truly responded to the sound stimulus. In addition, BF bursting neurons should have baseline firing rates (during the [−1, 0]s window relative to the trial start signal) less than 10 spikes/s. A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. and Table ). Since BF electrodes remained at the same depth throughout the recording sessions, the same BF single units might be recorded in multiple sessions. The large number of BF bursting neurons recorded in each session allowed us to treat them as a representative sample of all BF bursting neurons, whose responses to the S left sound were highly stable throughout the learning process (Fig. ). This strategy ensured that we were following functionally the same neuronal ensemble and could track how BF bursting neurons acquired responses to the new light during learning, regardless of whether the identities of these BF neurons were exactly the same in each session. One session with only one BF bursting neuron was excluded from the analysis of BF population activities. The spike timestamps of all BF bursting neurons in a single session were pooled together to approximate the population activity of all BF bursting neurons. Population peri-stimulus time histograms (PSTHs) were calculated with 10-ms bins, and normalized by the number of BF bursting neurons in a session. To properly assess whether BF bursting neurons responded to the onset of the new light stimulus (Figs. – ), it was important to disambiguate such stimulus-onset responses from the increased BF activities after fixation port exit (Fig. ). To achieve this goal, PSTHs to the stimulus onset were calculated based only on spikes that occurred before fixation port exit in individual trials, resulting in different interval lengths (between stimulus onset to fixation port exit) across trials. Accordingly, the calculation of the mean PSTH across all trials in a session was adjusted for the different number of trials at different interval lengths. The mean PSTHs were further truncated at the median interval length of that session to reduce noisy estimates of PSTHs at long interval lengths due to lower number of trials. When PSTHs were averaged across animals, the averaged PSTHs were further truncated at the mean of median interval latencies across animals. This truncation procedure resulted in the uneven lengths of PSTHs across individual animals (Figs. , , S and S ) and across sessions (Fig. ). This procedure was also applied to calculating the BF responses before fixation port exit to include only spikes that occurred after stimulus onset (Figs. , and S ), and for calculating BF responses during licking in catch lick trials (Fig. ). The time windows used to quantify average BF activity in different epochs were indicated in respective figures, and corresponded to the following: [0.05, 0.2]s after S left sound onset; [0.1, 0.3]s after S right light stimulus onset; [0.1, 0.3]s after the timestamp for the would-be stimulus in catch trials; [0.05, 0.35]s after the 3rd lick for outcome responses; [−0.3, 0]s and [0, 0.3]s relative to the fixation port exit. The epoch for calculating evaluation response is described below. Evaluation response (Figs. e, b, a and b, ) refers to the increased BF activity after exiting the fixation port and before the trial outcome (3rd lick). The evaluation response reflected animals’ internal evaluation because no additional sensory stimuli were presented during this epoch and this activity was not consistently aligned with intervening behavioral events (Fig. ). Specifically, the evaluation response was calculated in individual trials and defined as the maximum firing rate of any 500 ms window during the evaluation epoch, which corresponded to the interval between [fix-out, outcome], with additional adjustments according to trial types. In light lick and catch lick trials, the evaluation epoch was defined as [fix-out, outcome] in each trial. The epoch durations in light lick and catch lick trials within each session were used as the reference point for other trial types as described next. In no-fixation licks, in which the fix-out event was absent, the duration of evaluation epoch was set as the 95th percentile of the evaluation epoch durations in light lick and catch lick trials, and the epoch should begin at least 0.5 s before reward port entry. In light no lick and catch no lick trials, in which the 3rd lick event was absent, the duration of the evaluation epoch was set as the median of the evaluation epoch durations in light lick and catch lick trials. These adjustments in the definition of evaluation epochs, as well as its calculation of maximum firing rate within the epoch, took into consideration the behavioral variability across trial types, learning stages and individual animals. To evaluate the dynamic changes of BF activities around the transition point in the D 1 session (Figs. e and ), single trial evaluation and outcome responses were smoothed using moving median over 10 trials. The smoothed trends were aligned at the transition point and then averaged across all animals. Only trials with smoothed trend data from at least 4 animals were plotted in the group average (Fig. ). Statistical comparisons were conducted using the Statistics and Machine Learning Toolbox (version 11.3) in MATLAB (R2018a) ( https://www.mathworks.com/ ). Two-sided paired t -test (ttest.m) was used to compare behavioral and neural activity differences between two groups (Figs. d, a, c2, and ). Repeated measures analysis of variance (ranova.m) was used for comparisons involving more than 2 groups, by specifying the appropriate within-subject models (Figs. a3, b3, and ). Comparisons of PSTHs between two groups (Figs. a, b, c1, d, and ) was conducted for each 100 ms sliding window (10 ms step) using two-sided paired t -test. Significance level was set at p < 0.01 for three consecutive bins. Pearson correlation (corrcoef.m) was used to determine the relationship between neuronal activities and/or behavior (Figs. b, c, a, b2, and ). To determine whether the activity of BF bursting neurons differentiated between trial types within each D 1 session (Fig. ), we compared BF activity for each 100 ms sliding window (10 ms step) using the AUC measure of ROC analysis (auc.m by Alois Schloegl). At each sliding window, BF population activity was calculated for each light and catch trial, and distributions of BF activities were compared between light vs catch trials or between lick vs no lick trials. Significance level was set at p < 0.001 using 10,000 trial-shuffled random permutations (two-sided). To determine whether BF activity differentiated between lick and no lick trials within the same trial type (light or catch trials) (Fig. ), we compared BF activity in the [0, 500] ms window after exiting the fixation port. For each session and each trial type, lick and no lick trials must each constitute at least 10% of that trial type to be included in the analysis. Catch trials from all sessions were included in this analysis. Only light trials before the D 2 session (pre-D 2 ) were included in this analysis because BF responses to the onset of the light stimulus had not developed in those sessions. Significance level was set at p < 0.05 using 1000 trial-shuffled random permutations (two-sided). Further information on research design is available in the linked to this article. Supplementary Information Peer Review File Reporting Summary |
Food profiling goes green: Sustainable analysis strategies for food authentication | 6d4971b4-e198-414b-b38b-04068dea7243 | 11865702 | Biochemistry[mh] | INTRODUCTION In 2015, the United Nations (UN) adopted 17 sustainable development goals (SDGs) with the 2030 Agenda. These also include goals that directly or indirectly affect our food, primarily SDG 2 (zero hunger), but also SDG 3 (good health and well‐being), SDG 6 (clean water and sanitation), SDG 12 (responsible consumption and production), SDG 13 (climate action), SDG 14 (life below water), as well as SDG 15 (life on land) (United Nations, 2015. The 17 goals ( https://sdgs.un.org/goals , accessed on April 28th, 2024). In order to meet these goals with regard to our nutrition, objective analytical methods are required that can monitor compliance with these goals. At the same time, it must be ensured that the analytical methods themselves are sustainable. However, it must of course also be taken into account that reliable and usable analysis results are achieved . Typical parameters that can be easily checked analytically include the nutritional values of foods or the detection of residues, contaminants, and additives, provided these are known. In such cases, “classic” targeted analyses can be carried out, which are specifically aimed at detecting a few analytes. However, analyzing unknown compounds is more difficult, as it is not possible to search for them specifically. Such cases occur, especially when illegal substances are used. Therefore, it may be necessary to use non‐targeted methods that are not only aimed at detecting a few molecules or elements but are suitable for screening approaches . Other challenging questions, for which non‐targeted approaches are appropriate, concern the verification of geographical origin. This can be important to drawing conclusions about the transport routes taken, the regionality, and the CO 2 balance. In addition, non‐targeted methods may be of interest to verify organic farming methods (organic vs. conventional), to detect the use of genetic engineering techniques, and to analytically confirm botanical or zoological identities, for example, to ensure species protection. Moreover, analytical testing of food freshness is relevant to estimate the shelf life and, if necessary, extend it through suitable storage conditions, thus reducing the proportion of spoiled food that ultimately has to be thrown away . In addition to ensuring sustainability aspects, such issues have also become important for individual consumers, who are increasingly paying attention to such “soft” factors that can influence their purchasing decisions when buying food. As the production of sustainably produced food is often more expensive and associated with certain quality parameters, consumers also accept higher purchase prices. This, in turn, can lead to food fraud in order to achieve higher economic profits . For the often difficult scientific questions described above, non‐targeted omics strategies (genomics, proteomics, metabolomics, isotopolomics, and metallomics; see also Section ) are particularly suitable to initially identify markers or for detecting unknown compounds in the first place. Such approaches require the use of high‐resolution analytical equipment, which is usually associated with a high consumption of chemicals and energy. However, following a non‐targeted approach, it is often possible to develop targeted methods for the most suitable markers and thus reduce the analytical effort and become more sustainable . Nevertheless, for particularly challenging questions, it may also be necessary to combine different omics methods to achieve a higher molecular and elemental resolution (food profiling/panomics approach). However, this strategy also requires more resources, so a cost‐benefit analysis may be necessary . 1.1 A few words about relevant definitions Various terms have now been defined around the field of “Green Chemistry.” The most important of these are listed in Table . The two terms “Green Chemistry” and “Sustainable Chemistry” are frequently used interchangeably. However, a closer look at the literature shows that there are numerous definitions . The concept of “Green Chemistry” originates from Paul Anastas and John Warner in 1998, who formulated “The 12 Principles of Green Chemistry.” Their objectives were to minimize chemical waste as well as harmful influences on the environment and to increase the efficiency of chemical syntheses. A very commonly used definition in this context is: Green chemistry is the design of chemical products and processes that reduce or eliminate the use and generation of hazardous substances . Since the term “green” is associated with other definitions, especially in German‐speaking countries, such as the rejection of genetic engineering and nuclear energy, and also occurs in the name of a German party (Bündnis 90/Die Grünen), the term “Sustainable Chemistry” is preferred by organizations such as the International Union of Pure and Applied Chemistry, the Organization for Economic Co‐operation and Development (OECD) as well as the German Chemical Society. The OECD defined “Sustainable Chemistry” as a scientific concept that seeks to improve the efficiency with which natural resources are used to meet human needs for chemical products and services (OECD, 2024). Sustainable chemistry. https://www.oecd.org/chemicalsafety/risk‐management/sustainable‐chemistry/ , accessed on April 28th, 2024) . Despite the similarity of these definitions, some experts emphasized that there are differences. Thus, Anastas formulated in 2016: If sustainability is the goal, green chemistry will show the way ! . The term “Green Chemistry” initially referred primarily to synthetic processes in the chemical industry. Around 2000, the term “Green Analytical Chemistry” emerged, focusing on the establishment of environmentally friendly analytical methods and sample preparation procedures. Analogous to the concept of “Green Chemistry”, Gałuszka et al. published “The 12 Principles of Green Analytical Chemistry” in 2013, including four key goals: (i) elimination or reduction of chemical substances; (ii) minimization of energy consumption; (iii) proper management of waste; and (iv) increasing safety for the operator . A few words about relevant definitions Various terms have now been defined around the field of “Green Chemistry.” The most important of these are listed in Table . The two terms “Green Chemistry” and “Sustainable Chemistry” are frequently used interchangeably. However, a closer look at the literature shows that there are numerous definitions . The concept of “Green Chemistry” originates from Paul Anastas and John Warner in 1998, who formulated “The 12 Principles of Green Chemistry.” Their objectives were to minimize chemical waste as well as harmful influences on the environment and to increase the efficiency of chemical syntheses. A very commonly used definition in this context is: Green chemistry is the design of chemical products and processes that reduce or eliminate the use and generation of hazardous substances . Since the term “green” is associated with other definitions, especially in German‐speaking countries, such as the rejection of genetic engineering and nuclear energy, and also occurs in the name of a German party (Bündnis 90/Die Grünen), the term “Sustainable Chemistry” is preferred by organizations such as the International Union of Pure and Applied Chemistry, the Organization for Economic Co‐operation and Development (OECD) as well as the German Chemical Society. The OECD defined “Sustainable Chemistry” as a scientific concept that seeks to improve the efficiency with which natural resources are used to meet human needs for chemical products and services (OECD, 2024). Sustainable chemistry. https://www.oecd.org/chemicalsafety/risk‐management/sustainable‐chemistry/ , accessed on April 28th, 2024) . Despite the similarity of these definitions, some experts emphasized that there are differences. Thus, Anastas formulated in 2016: If sustainability is the goal, green chemistry will show the way ! . The term “Green Chemistry” initially referred primarily to synthetic processes in the chemical industry. Around 2000, the term “Green Analytical Chemistry” emerged, focusing on the establishment of environmentally friendly analytical methods and sample preparation procedures. Analogous to the concept of “Green Chemistry”, Gałuszka et al. published “The 12 Principles of Green Analytical Chemistry” in 2013, including four key goals: (i) elimination or reduction of chemical substances; (ii) minimization of energy consumption; (iii) proper management of waste; and (iv) increasing safety for the operator . GREEN SAMPLE COLLECTION AND STORAGE When implementing non‐targeted strategies, it is often a major challenge to obtain a sufficiently large number of authentic samples, transport them, and store them properly so that chemical or physical changes are as minimal as possible. The challenge is particularly relevant in metabolomics and proteomics approaches, whereas in isotopolomics, metallomics, or genomics methods, such factors can be somewhat neglected. It is also crucial that these steps are standardized and carried out under realistic market conditions. On the one hand, this is necessary for the subsequent implementation of the methods in routine analysis, which is the main objective, and on the other hand, to be able to supplement the model with additional reference samples at any time . As part of a study to determine the origin of asparagus, we traveled several 1000 km with a refrigerated van in order to meet these scientific requirements . In another project to identify truffle species, a truffle trader was a partner in our consortium. He had direct access to the local markets and was also able to import the goods directly from these harvesting countries. In most cases, the reference material was sent directly to our research center, which was again sustainable as it helped to avoid additional transport . In both studies, samples were measured using various omics technologies. This approach aimed to extract the maximum information content from the samples and to evaluate the suitability of different technologies for addressing various questions and food matrices. These two examples underline that collaborations between scientific institutes and industrial partners are invaluable to improving the ecology of analytical methods. This approach is also followed by numerous countries and their monitoring authorities, including the EU, which is actively establishing internationally cooperating reference centers to ensure the authenticity and integrity of the food chain (Regulation (EU) 2017/625). Another approach is to procure samples through importers, but then the authenticity of the samples cannot usually be sufficiently proven, so it is advisable to analyze a significantly larger number of samples to identify possible false declarations. Alternatively, checking the samples with additional analytical strategies can help, but this again increases the analysis effort with a negative impact on sustainability. For instance, we employed this approach in a study aimed at identifying marker compounds using a metabolomics‐based method to detect marjoram admixtures in oregano. The samples for this study were obtained with the help of importers. During method development, it was noticed that some of the oregano samples had greater similarities with the marjoram samples. DNA analyses proved that the supposedly pure oregano samples contained large amounts of marjoram. This example underlines the critical importance of using suitable reference materials . Depending on the sample matrix and analysis strategy, refrigeration or freezing may be necessary for sample preservation. For optimal preservation, flash freezing with liquid nitrogen followed by storage at temperatures as low as −80°C in freezers or even down to −196°C in liquid nitrogen is often recommended. The use of freezers with a temperature of 86°C is quite widespread and does not necessitate regular refilling of nitrogen, which simplifies maintenance . However, their power consumption is notably high. For instance, the annual energy consumption of a −86°C freezer (capacity 740 L, ∼10.5 kW h/day) is equivalent to the energy consumption of a family of two to three people (∼9.5–14 kW h/day). (Eppendorf, 2020. CryoCube. CryoCube‐Operation manual. https://www.eppendorf.com/product‐media/doc/en/230895/Freezers_Operating‐manual_CryoCube‐F740hi‐hiw.pdf , accessed on April 28th, 2024); DeStatis, 2023. Stromverbrauch. https://www.destatis.de/DE/Themen/Gesellschaft‐Umwelt/Umwelt/UGR/private‐haushalte/Tabellen/stromverbrauch‐haushalte.html , accessed on April 28th, 2024). To keep the cooling efficiency of freezers as high as possible, measures such as regular cleaning of the filters and removing ice from the doors can help. The choice of location for the freezer is also relevant. A place directly by a window, where the freezer may be warmed by the sun, is less suitable than a well‐ventilated room in which waste heat is dissipated to the outside. Using organization systems and clear sample labels can make sample collection easier and reduce the amount of time doors are open. It is also advisable to store samples in containers of minimal size to minimize dead volumes and maximize efficient use of the required space. Moreover, careful consideration should be given to the required amount of material and the potential need for aliquoting, as the composition of samples can be impacted by thawing and freezing cycles, potentially affecting the analysis outcomes . Additionally, it is worth considering the feasibility of using higher temperatures and/or storing the samples under inert gases. Alternatively, drying samples can further improve storage stability. For example, in a study involving raw and freeze‐dried milk samples, it was demonstrated that freeze‐drying resulted in only minor changes in the metabolome. Furthermore, the freeze‐dried samples remained stable when stored at both −20 and +4°C for several months, with no significant alterations in the metabolome observed. However, larger differences in the freeze‐dried samples were detected when stored at room temperature . It can be assumed that the stability is highly dependent on the food matrix and may need to be evaluated accordingly. Drying samples also has an analytical advantage, as the analytes are concentrated and the comparability of the samples is increased . Nevertheless, it must also be taken into account that energy must first be used. The required energy expenditure can be at least partially reduced by a possible reduction in the sample quantity. Furthermore, it is worth contemplating whether dry foods typically not subjected to freezing, like tea, spices, or cookies, should be stored directly at room temperature. Such and further considerations are also the focus of the “Freezer Challenge,” an initiative by non‐profit organizations striving to maximize the efficiency of cooling capacity utilization (The Freezer Challenge, 2024. https://www.freezerchallenge.org/ , accessed on April 28th, 2024). In order to minimize the effort of sampling, as well as the transport and storage capacities, there are also extraction processes, rapid tests, lab‐on‐the‐chip systems, and portable devices that can be used directly on site for selected questions. However, these processes and detection systems are usually developed primarily for targeted analyses or only have a limited resolution, which reduces the area of application accordingly. Nevertheless, they offer promising potential for selected scientific questions. Alternatively, it is possible to send the laboratory to the samples. For example, specially equipped container laboratories could be used here, similar to those used during the Covid pandemic (Universität Hamburg, Container Lab. https://www.csmc.uni‐hamburg.de/research/artefact‐lab/artefact‐lab/container.html , accessed on June 29th, 2024). GREEN SAMPLE PREPARATION Sample preparation is a crucial step in performing analytical methods, as it ensures that the food is converted into a measurable form. During sample preparation, undesirable components of the matrix are removed and, if possible, the relevant analytes or analyte groups are concentrated. Due to the high influence of sample preparation on the sustainability of an analytical process, López‐Lorente et al. published in 2022 “The Ten Principles of Green Sample Preparation.” Many of the principles established are redundant with the principles of “Green Chemistry” and “Green Analytical Chemistry” since all approaches are based on the same goal . 3.1 Headspace technologies A very effective way to make sample preparation “green,” that is, as environmentally friendly as possible, is to avoid it completely, or at least to work solvent‐free. In this regard, gas chromatography (GC) analyses and the implementation of static headspace experiments are particularly suitable. However, this approach is only applicable to volatile compounds and may lack sensitivity. There are various strategies to achieve enrichment of analytes in the vapor space. This includes the addition of saturated salt solutions (e.g., with NaCl) to reduce the solubility of organic compounds, increase their vapor pressure (salting‐out effect), or make adjustments to the pH value . Alternatively, a vacuum can also be applied to increase the concentration of analytes in the vapor space and accelerate the enrichment process (vacuum‐assisted techniques) . Furthermore, analytes can be driven off using a gas stream. Such techniques are often combined with sorbents (see Section 3.2) to concentrate the analytes first and then thermally desorb the volatile compounds after a certain time (dynamic headspace analyses). If the gas flow bubbles through the sample matrix, this is referred to as the “purge and trap” technique . 3.2 Sorbent‐based microextraction techniques What sorbent‐based techniques have in common is that they use sorbents to which analytes are first adsorbed or absorbed. This leads to an increased concentration of the analytes, whereby often only small amounts of sample quantities are required. Furthermore, analytes can be simply washed, and interfering matrix components can be removed. Compared to classical approaches such as liquid–liquid extraction (LLE) or solid–liquid extraction, which involve high amounts of solvents, the need for solvents can be significantly reduced (Figure ). There is now a wide range of sorbents available to address diverse analytical challenges. These include normal‐phase and reversed phase (RP)‐based sorbents, ion exchangers, molecularly imprinted polymers, aptamers, ionic liquids (ILs), carbon‐based nanomaterials, and many others. Some sorbents exhibit high selectivity, making them ideal for targeted analyses, whereas others are suitable for non‐targeted methods. Additionally, combining different sorbents can expand the range of extractable analytes . The use of sorbents in the form of solid‐phase extraction (SPE) cartridges or columns and solid‐phase microextraction (SPME) techniques is very common . In headspace SPME, a coated quartz glass fiber with the sorbent is introduced into the headspace of the sample via a syringe‐like holder. Alternatively, the fiber can also be immersed directly into the sample. Selective‐access membrane coatings can be utilized to protect the fiber and remove interference (membrane extraction SPME) . In addition, SPME procedures can also be coupled with liquid chromatography (LC) applications. However, the utilization of SPME techniques in conjunction with LC applications is currently of minor importance, and the scope of studies is correspondingly limited. This is mainly because the online integration of SPME into LC systems is quite challenging. For this reason, SPE‐based studies are more likely to be carried out on LC systems, whereas these are not suitable for GC applications due to the solvents . A further development of SPME is SPME Arrow, which was introduced by Restek Corporation. Instead of a fiber, a solid stainless‐steel core is used here, which tapers toward the front and can be coated in various ways. Notably, this design is not only more robust but also offers a larger surface area, enabling the extraction of a greater amount of analytes . The high‐capacity sorptive extraction alternative from Markes International, which has a thicker stainless steel rod, also offers a larger surface area . Additionally, thin‐film SPME membranes, available from Gerstel GmbH, offer further options with comparatively high capacities and fast extraction kinetics . In‐tube SPME applications use a tube or capillary containing the sorbent instead of a fiber. The technique is very suitable for online coupling in both GC and LC systems, in particular in nanoLC systems. In‐tube SPME can also be coupled directly to suitable instruments, such as mass spectrometers. This approach further reduces required solvent volumes and enables high‐throughput analyses to be conducted . A miniaturized version of SPE is microextraction by packed sorbent (MEPS). Similar to SPE, MEPS facilitates extraction, pre‐concentration, and clean‐up with small sample volumes, but in the µL range rather than the mL range . A further advancement of MEPS is the µSPEed variant, marketed by the Australian company ePrep. The µSPEed version features a one‐way check valve, enabling a unidirectional flow path through the sorbent. This ensures that solvents flow only in one direction. Consequently, analytes with poor sorbent binding are prevented from being flushed out. Moreover, this configuration enhances pressure stability and facilitates the use of sorbent particles with diameters <3 µm, resulting in an increased surface area. In contrast, traditional MEPS approaches employ particle sizes of 50–60 µm . Sorbents can also be added directly to a sample solution, known as dispersive SPE (DSPE). After mixing the sorbent and the sample solution, during which the analytes are adsorbed, the sorbent is separated by centrifugation. Alternatively, magnetic sorbents can also be employed (magnetic dispersive SPE), which can be separated using a magnetic field. Consequently, this approach is a modification of the classic DSPE . A notably eco‐friendly variation of DSPE is µSPE, which utilizes a reduced amount of sorbent. The sorbent is then immobilized, for example, in pipette tips (pipette‐tip micro‐SPE) or in spin columns. With the pipette version, analytes can be bound or eluted by repeatedly drawing up and draining the sample solution or small volumes of solvent using a pipette . Alternatively, the sorbent can also be freely movable in the pipette tip between two filters, allowing for more intensive mixing (disposable pipette extraction) . In addition, a suitable immersive method for liquid samples is stir bar sorptive extraction (SBSE), for which a stir bar is used that is coated with the sorbent. The stir bar is placed in the sample solution and stirred for some time so that the analytes can be absorbed by the sorbent. Compared to SPME, higher enrichment factors are achieved with SBSE . An aptamer‐based application was recently presented to detect allergenic milk proteins from the food matrix using SBSE and matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MS) . Alternatively, the stir bar can also be placed in the headspace of a liquid or solid sample (headspace sorptive extraction) . In fabric phase sorptive extraction, a flexible fabric on which the sorbent has been bound is immersed in a sample solution and stirred for some time so that the analytes can be absorbed . Dry, undissolved samples can also be triturated directly with a sorbent. This process is also known as matrix solid‐phase dispersion. The mixture can then be packed into a pre‐fritted empty cartridge . 3.3 Liquid‐based microextraction techniques Instead of LLE, liquid‐phase microextraction (LPME) methods also offer a more environmentally friendly alternative, as they often reduce the use of organic solvents to below 100 µL (Figure ). There are several approaches to implementing LPME, including single‐drop microextraction (SDME), which is based on using a single drop (0.5–30 µL) of an organic solvent at the end of a microsyringe needle for extraction . In the direct immersion variant, utilized for extracting nonvolatile analytes, the extraction drop is immersed in an aqueous sample solution. The drop can also be positioned in the headspace of a sample to collect volatile and semi‐volatile analytes . In the hollow fiber LPME (HF‐LPME) approach, an organic solvent is immobilized inside a porous hydrophobic HF, which serves as a supported liquid membrane. The same solvent is also present in the lumen of this fiber and serves as an acceptor phase. The fiber is immersed in an aqueous sample solution (donor phase), allowing analytes to migrate into the organic phase (two‐phase system). Alternatively, the fiber can contain an organic solvent serving as a supported liquid membrane to immobilize an aqueous solution, which serves as an acceptor phase (three‐phase system) . The advantage of using a fiber over SDME lies in the better stabilization of the solvent and the barrier effect of the fiber against particles and macromolecules, thereby preventing interference. To reduce the time required and improve efficiency, further enhancements have been developed. These include the use of electrical potentials (electro‐membrane microextraction) to accelerate the migration of charged compounds, or carrier‐mediated three‐phase HF‐LPME, where the formation of ion‐pair complexes supports the migration. Two‐phase HF‐LPME is particularly suitable for extracting nonpolar compounds, whereas three‐phase HF‐LPME can be used to extract ionizable compounds and metals . Through targeted and non‐targeted studies of human milk using GC–MS analyses, a direct comparison between an HF‐LPME approach and a conventional LLE method revealed improved detection of metabolites in terms of enrichment factors, sample throughput, and metabolite coverage . In dispersive liquid–liquid microextraction, a mixture of a water‐immiscible solvent (extractant) and a water‐miscible solvent (disperser) is added to an aqueous sample solution. After injecting a small amount of this mixture into the sample solution, a dispersion is formed in which the organic immiscible solvent exists in small droplets, thus providing a large contact surface for the extraction process. The solvents are then separated from each other using centrifugation . Cloud point extraction, involving the addition of surfactants to the sample solution, is another method suitable for concentrating specific analytes. By heating the solution above the “cloud point,” micelles are formed, encapsulating the target compounds. The micelles can then be separated from the solvent via centrifugation. The surfactant‐rich phase obtained can be directly used for analysis or subjected to back‐extraction . Efforts are underway to make this process more environmentally friendly. For example, extraction can be conducted at room temperature to save energy, or different surfactants can be used to increase extraction efficiency . 3.4 Methods for increasing extraction efficiency and cell disruption To improve the extraction efficiency of the different approaches and minimize chemical usage, extraction times, and energy consumption, additional physical and enzymatic processes can be employed. This includes the use of ultrasonic waves to induce cavitation bubbles, whose implosion generates local turbulence, facilitating the mixing of analytes and solvents. Moreover, ultrasonic waves can disrupt cell structures, which is not possible with simple mixing . Alternatively, cell structures can be broken up relatively easily using ball milling techniques . Enzymes can also be used, as they catalyze reactions in water, and the process can therefore be classified as environmentally friendly . Microwaves are often used to initially excite water molecules in the sample or the extractant, releasing heat into the environment, which in turn disrupts cell structures and accelerates extraction. Microwaves are sometimes used under high pressure in closed vessels, which further increases the reaction kinetics. When combined with nitric acid and hydrogen peroxide, microwave digestions can prepare samples for metal analysis by converting organic components into CO 2 and water under these conditions. This method not only saves time but also reduces the use of chemicals and energy consumption . Recently, it has been demonstrated that even low concentrations, such as 20%, of nitric acid are effective in converting food into an extract suitable for inductively coupled plasma (ICP)‐MS analysis by microwave extraction . Accelerated solvent extraction or pressurized fluid extraction also works under increased pressure and temperature. The sample is placed in stainless steel tubes, which have frits at the ends and through which various solvents are passed to extract the analytes. Both organic solvents and water (known as pressurized hot water extraction or subcritical water extraction) can be used as extraction solvents. In particular, the suitability of these solvents makes this method more sustainable than traditional approaches such as Soxhlet extraction. In addition, this procedure is faster and requires small amounts of solvent . Pulsed electric field (PEF) is a nonthermal extraction method in which short, high‐intensity pulses are applied to the sample material positioned between two electrodes. This leads to transient electroporation of the cell membranes, allowing intramolecular compounds to pass into the extractant . In contrast, high‐voltage electrical discharges (HVED) apply a high voltage to two electrodes that are immersed in the sample solution, forming a plasma channel. This generates shock waves, turbulence, UV waves, and radicals, ultimately leading to the disruption of cell structures. Samples extracted with HVED have higher antioxidant capacities compared to those treated with ultrasonic waves and LLEs, indicating the method's gentleness . However, the use of PEF and HVED methods to prepare samples for food analysis has so far been carried out to a limited extent, as these methods are currently predominantly used for extracting bioactive substances such as phenols in the industrial sector . Another alternative could be cold plasma‐assisted extraction, in which gases such as argon, helium, or nitrogen are used to create a plasma that is directed at the sample and breaks up cell structures. This approach has also been used primarily for extractions on an industrial scale and is considered to be very environmentally friendly. However, there are currently only a few studies available, and, to our knowledge, no comparative study has been carried out in the context of food analysis on a laboratory scale, so further research steps must first be undertaken in order to be able to assess whether this method is also suitable for cell disruption in this context . 3.5 Green solvents In the best case, environmentally compatible solvents possess high selectivity, solvent capability, low toxicity, low environmental impact, and are biodegradable or recyclable. They can also be produced cost‐effectively from renewable raw materials. In addition, solvents with low vapor pressure and high boiling points are preferred to reduce environmental pollution, but the latter can also be detrimental to the analytical processes if the solvent has to be removed later . The “greenest” solvent is water. Ethanol is also a comparatively green solvent. However, both have the disadvantage that they are polar solvents, which means that they cannot be used to extract nonpolar compounds, which is why numerous alternatives have been developed . ILs are salts that have a melting point below 100°C. They are thermally and chemically very stable, with high viscosity, low vapor pressure, and low flammability. Imidazolium, phosphonium, ammonium, and pyrrolidinium are often used as cations, whereas chloride, dicyanamide, and tetrafluoroborate are common anions. The properties of ILs can be adjusted very selectively, depending on the cations and anions used, which is why they are also referred to as “designer solvents.” They are classified as “green” due to their low vapor pressure and low flammability. However, they are often highly toxic, and their synthesis is energy‐intensive, requiring fossil raw materials. In addition, achieving biodegradability with ILs is also challenging. Magnetic ILs contain paramagnetic metal ions that can be easily separated from large sample volumes . Deep eutectic solvents (DESs) and natural DESs (NADESs) are created by mixing hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). The formation of hydrogen bonds results in a lower melting point than that of the starting components. Although DESs use synthetic substances, NADESs are derived from natural sources. Most of the starting materials used are biodegradable. Choline chloride is often used as an HBA for the production of DESs or NADESs. Suitable HBDs include organic acids as well as sugars or polyols. Compared to ILs, production is much easier, and the compounds are mostly nontoxic . Supramolecular DESs contain macromolecules such as cyclodextrins and can lead to better extraction efficiencies compared to DESs . Alternatively, extraction can also be carried out with solvents or gases under supercritical conditions. CO 2 is used particularly frequently. As subcritical CO 2 has low polarity, various modifiers are added in this process. The approach is considered particularly environmentally friendly as CO 2 can be reused, sourced from industrial processes, and is the second most environmentally friendly solvent after water . In recent years, switchable hydrophilic solvents (SHSs) have gained popularity. SHSs offer the ability to reversibly change physical properties, such as switching from a hydrophobic to a hydrophilic state. Amine‐based SHSs are protonated in the presence of CO 2 and form amine carbonate salts (Figure ). Expulsion of CO 2 through heating or using a stream of knitted material converts them back into their hydrophobic form. Fatty acids are also suitable as SHSs and can be converted into hydrophilic salts with sodium hydroxide, reverting to hydrophobic fatty acids when the pH shifts to the acidic range . Headspace technologies A very effective way to make sample preparation “green,” that is, as environmentally friendly as possible, is to avoid it completely, or at least to work solvent‐free. In this regard, gas chromatography (GC) analyses and the implementation of static headspace experiments are particularly suitable. However, this approach is only applicable to volatile compounds and may lack sensitivity. There are various strategies to achieve enrichment of analytes in the vapor space. This includes the addition of saturated salt solutions (e.g., with NaCl) to reduce the solubility of organic compounds, increase their vapor pressure (salting‐out effect), or make adjustments to the pH value . Alternatively, a vacuum can also be applied to increase the concentration of analytes in the vapor space and accelerate the enrichment process (vacuum‐assisted techniques) . Furthermore, analytes can be driven off using a gas stream. Such techniques are often combined with sorbents (see Section 3.2) to concentrate the analytes first and then thermally desorb the volatile compounds after a certain time (dynamic headspace analyses). If the gas flow bubbles through the sample matrix, this is referred to as the “purge and trap” technique . Sorbent‐based microextraction techniques What sorbent‐based techniques have in common is that they use sorbents to which analytes are first adsorbed or absorbed. This leads to an increased concentration of the analytes, whereby often only small amounts of sample quantities are required. Furthermore, analytes can be simply washed, and interfering matrix components can be removed. Compared to classical approaches such as liquid–liquid extraction (LLE) or solid–liquid extraction, which involve high amounts of solvents, the need for solvents can be significantly reduced (Figure ). There is now a wide range of sorbents available to address diverse analytical challenges. These include normal‐phase and reversed phase (RP)‐based sorbents, ion exchangers, molecularly imprinted polymers, aptamers, ionic liquids (ILs), carbon‐based nanomaterials, and many others. Some sorbents exhibit high selectivity, making them ideal for targeted analyses, whereas others are suitable for non‐targeted methods. Additionally, combining different sorbents can expand the range of extractable analytes . The use of sorbents in the form of solid‐phase extraction (SPE) cartridges or columns and solid‐phase microextraction (SPME) techniques is very common . In headspace SPME, a coated quartz glass fiber with the sorbent is introduced into the headspace of the sample via a syringe‐like holder. Alternatively, the fiber can also be immersed directly into the sample. Selective‐access membrane coatings can be utilized to protect the fiber and remove interference (membrane extraction SPME) . In addition, SPME procedures can also be coupled with liquid chromatography (LC) applications. However, the utilization of SPME techniques in conjunction with LC applications is currently of minor importance, and the scope of studies is correspondingly limited. This is mainly because the online integration of SPME into LC systems is quite challenging. For this reason, SPE‐based studies are more likely to be carried out on LC systems, whereas these are not suitable for GC applications due to the solvents . A further development of SPME is SPME Arrow, which was introduced by Restek Corporation. Instead of a fiber, a solid stainless‐steel core is used here, which tapers toward the front and can be coated in various ways. Notably, this design is not only more robust but also offers a larger surface area, enabling the extraction of a greater amount of analytes . The high‐capacity sorptive extraction alternative from Markes International, which has a thicker stainless steel rod, also offers a larger surface area . Additionally, thin‐film SPME membranes, available from Gerstel GmbH, offer further options with comparatively high capacities and fast extraction kinetics . In‐tube SPME applications use a tube or capillary containing the sorbent instead of a fiber. The technique is very suitable for online coupling in both GC and LC systems, in particular in nanoLC systems. In‐tube SPME can also be coupled directly to suitable instruments, such as mass spectrometers. This approach further reduces required solvent volumes and enables high‐throughput analyses to be conducted . A miniaturized version of SPE is microextraction by packed sorbent (MEPS). Similar to SPE, MEPS facilitates extraction, pre‐concentration, and clean‐up with small sample volumes, but in the µL range rather than the mL range . A further advancement of MEPS is the µSPEed variant, marketed by the Australian company ePrep. The µSPEed version features a one‐way check valve, enabling a unidirectional flow path through the sorbent. This ensures that solvents flow only in one direction. Consequently, analytes with poor sorbent binding are prevented from being flushed out. Moreover, this configuration enhances pressure stability and facilitates the use of sorbent particles with diameters <3 µm, resulting in an increased surface area. In contrast, traditional MEPS approaches employ particle sizes of 50–60 µm . Sorbents can also be added directly to a sample solution, known as dispersive SPE (DSPE). After mixing the sorbent and the sample solution, during which the analytes are adsorbed, the sorbent is separated by centrifugation. Alternatively, magnetic sorbents can also be employed (magnetic dispersive SPE), which can be separated using a magnetic field. Consequently, this approach is a modification of the classic DSPE . A notably eco‐friendly variation of DSPE is µSPE, which utilizes a reduced amount of sorbent. The sorbent is then immobilized, for example, in pipette tips (pipette‐tip micro‐SPE) or in spin columns. With the pipette version, analytes can be bound or eluted by repeatedly drawing up and draining the sample solution or small volumes of solvent using a pipette . Alternatively, the sorbent can also be freely movable in the pipette tip between two filters, allowing for more intensive mixing (disposable pipette extraction) . In addition, a suitable immersive method for liquid samples is stir bar sorptive extraction (SBSE), for which a stir bar is used that is coated with the sorbent. The stir bar is placed in the sample solution and stirred for some time so that the analytes can be absorbed by the sorbent. Compared to SPME, higher enrichment factors are achieved with SBSE . An aptamer‐based application was recently presented to detect allergenic milk proteins from the food matrix using SBSE and matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometry (MS) . Alternatively, the stir bar can also be placed in the headspace of a liquid or solid sample (headspace sorptive extraction) . In fabric phase sorptive extraction, a flexible fabric on which the sorbent has been bound is immersed in a sample solution and stirred for some time so that the analytes can be absorbed . Dry, undissolved samples can also be triturated directly with a sorbent. This process is also known as matrix solid‐phase dispersion. The mixture can then be packed into a pre‐fritted empty cartridge . Liquid‐based microextraction techniques Instead of LLE, liquid‐phase microextraction (LPME) methods also offer a more environmentally friendly alternative, as they often reduce the use of organic solvents to below 100 µL (Figure ). There are several approaches to implementing LPME, including single‐drop microextraction (SDME), which is based on using a single drop (0.5–30 µL) of an organic solvent at the end of a microsyringe needle for extraction . In the direct immersion variant, utilized for extracting nonvolatile analytes, the extraction drop is immersed in an aqueous sample solution. The drop can also be positioned in the headspace of a sample to collect volatile and semi‐volatile analytes . In the hollow fiber LPME (HF‐LPME) approach, an organic solvent is immobilized inside a porous hydrophobic HF, which serves as a supported liquid membrane. The same solvent is also present in the lumen of this fiber and serves as an acceptor phase. The fiber is immersed in an aqueous sample solution (donor phase), allowing analytes to migrate into the organic phase (two‐phase system). Alternatively, the fiber can contain an organic solvent serving as a supported liquid membrane to immobilize an aqueous solution, which serves as an acceptor phase (three‐phase system) . The advantage of using a fiber over SDME lies in the better stabilization of the solvent and the barrier effect of the fiber against particles and macromolecules, thereby preventing interference. To reduce the time required and improve efficiency, further enhancements have been developed. These include the use of electrical potentials (electro‐membrane microextraction) to accelerate the migration of charged compounds, or carrier‐mediated three‐phase HF‐LPME, where the formation of ion‐pair complexes supports the migration. Two‐phase HF‐LPME is particularly suitable for extracting nonpolar compounds, whereas three‐phase HF‐LPME can be used to extract ionizable compounds and metals . Through targeted and non‐targeted studies of human milk using GC–MS analyses, a direct comparison between an HF‐LPME approach and a conventional LLE method revealed improved detection of metabolites in terms of enrichment factors, sample throughput, and metabolite coverage . In dispersive liquid–liquid microextraction, a mixture of a water‐immiscible solvent (extractant) and a water‐miscible solvent (disperser) is added to an aqueous sample solution. After injecting a small amount of this mixture into the sample solution, a dispersion is formed in which the organic immiscible solvent exists in small droplets, thus providing a large contact surface for the extraction process. The solvents are then separated from each other using centrifugation . Cloud point extraction, involving the addition of surfactants to the sample solution, is another method suitable for concentrating specific analytes. By heating the solution above the “cloud point,” micelles are formed, encapsulating the target compounds. The micelles can then be separated from the solvent via centrifugation. The surfactant‐rich phase obtained can be directly used for analysis or subjected to back‐extraction . Efforts are underway to make this process more environmentally friendly. For example, extraction can be conducted at room temperature to save energy, or different surfactants can be used to increase extraction efficiency . Methods for increasing extraction efficiency and cell disruption To improve the extraction efficiency of the different approaches and minimize chemical usage, extraction times, and energy consumption, additional physical and enzymatic processes can be employed. This includes the use of ultrasonic waves to induce cavitation bubbles, whose implosion generates local turbulence, facilitating the mixing of analytes and solvents. Moreover, ultrasonic waves can disrupt cell structures, which is not possible with simple mixing . Alternatively, cell structures can be broken up relatively easily using ball milling techniques . Enzymes can also be used, as they catalyze reactions in water, and the process can therefore be classified as environmentally friendly . Microwaves are often used to initially excite water molecules in the sample or the extractant, releasing heat into the environment, which in turn disrupts cell structures and accelerates extraction. Microwaves are sometimes used under high pressure in closed vessels, which further increases the reaction kinetics. When combined with nitric acid and hydrogen peroxide, microwave digestions can prepare samples for metal analysis by converting organic components into CO 2 and water under these conditions. This method not only saves time but also reduces the use of chemicals and energy consumption . Recently, it has been demonstrated that even low concentrations, such as 20%, of nitric acid are effective in converting food into an extract suitable for inductively coupled plasma (ICP)‐MS analysis by microwave extraction . Accelerated solvent extraction or pressurized fluid extraction also works under increased pressure and temperature. The sample is placed in stainless steel tubes, which have frits at the ends and through which various solvents are passed to extract the analytes. Both organic solvents and water (known as pressurized hot water extraction or subcritical water extraction) can be used as extraction solvents. In particular, the suitability of these solvents makes this method more sustainable than traditional approaches such as Soxhlet extraction. In addition, this procedure is faster and requires small amounts of solvent . Pulsed electric field (PEF) is a nonthermal extraction method in which short, high‐intensity pulses are applied to the sample material positioned between two electrodes. This leads to transient electroporation of the cell membranes, allowing intramolecular compounds to pass into the extractant . In contrast, high‐voltage electrical discharges (HVED) apply a high voltage to two electrodes that are immersed in the sample solution, forming a plasma channel. This generates shock waves, turbulence, UV waves, and radicals, ultimately leading to the disruption of cell structures. Samples extracted with HVED have higher antioxidant capacities compared to those treated with ultrasonic waves and LLEs, indicating the method's gentleness . However, the use of PEF and HVED methods to prepare samples for food analysis has so far been carried out to a limited extent, as these methods are currently predominantly used for extracting bioactive substances such as phenols in the industrial sector . Another alternative could be cold plasma‐assisted extraction, in which gases such as argon, helium, or nitrogen are used to create a plasma that is directed at the sample and breaks up cell structures. This approach has also been used primarily for extractions on an industrial scale and is considered to be very environmentally friendly. However, there are currently only a few studies available, and, to our knowledge, no comparative study has been carried out in the context of food analysis on a laboratory scale, so further research steps must first be undertaken in order to be able to assess whether this method is also suitable for cell disruption in this context . Green solvents In the best case, environmentally compatible solvents possess high selectivity, solvent capability, low toxicity, low environmental impact, and are biodegradable or recyclable. They can also be produced cost‐effectively from renewable raw materials. In addition, solvents with low vapor pressure and high boiling points are preferred to reduce environmental pollution, but the latter can also be detrimental to the analytical processes if the solvent has to be removed later . The “greenest” solvent is water. Ethanol is also a comparatively green solvent. However, both have the disadvantage that they are polar solvents, which means that they cannot be used to extract nonpolar compounds, which is why numerous alternatives have been developed . ILs are salts that have a melting point below 100°C. They are thermally and chemically very stable, with high viscosity, low vapor pressure, and low flammability. Imidazolium, phosphonium, ammonium, and pyrrolidinium are often used as cations, whereas chloride, dicyanamide, and tetrafluoroborate are common anions. The properties of ILs can be adjusted very selectively, depending on the cations and anions used, which is why they are also referred to as “designer solvents.” They are classified as “green” due to their low vapor pressure and low flammability. However, they are often highly toxic, and their synthesis is energy‐intensive, requiring fossil raw materials. In addition, achieving biodegradability with ILs is also challenging. Magnetic ILs contain paramagnetic metal ions that can be easily separated from large sample volumes . Deep eutectic solvents (DESs) and natural DESs (NADESs) are created by mixing hydrogen bond acceptors (HBAs) and hydrogen bond donors (HBDs). The formation of hydrogen bonds results in a lower melting point than that of the starting components. Although DESs use synthetic substances, NADESs are derived from natural sources. Most of the starting materials used are biodegradable. Choline chloride is often used as an HBA for the production of DESs or NADESs. Suitable HBDs include organic acids as well as sugars or polyols. Compared to ILs, production is much easier, and the compounds are mostly nontoxic . Supramolecular DESs contain macromolecules such as cyclodextrins and can lead to better extraction efficiencies compared to DESs . Alternatively, extraction can also be carried out with solvents or gases under supercritical conditions. CO 2 is used particularly frequently. As subcritical CO 2 has low polarity, various modifiers are added in this process. The approach is considered particularly environmentally friendly as CO 2 can be reused, sourced from industrial processes, and is the second most environmentally friendly solvent after water . In recent years, switchable hydrophilic solvents (SHSs) have gained popularity. SHSs offer the ability to reversibly change physical properties, such as switching from a hydrophobic to a hydrophilic state. Amine‐based SHSs are protonated in the presence of CO 2 and form amine carbonate salts (Figure ). Expulsion of CO 2 through heating or using a stream of knitted material converts them back into their hydrophobic form. Fatty acids are also suitable as SHSs and can be converted into hydrophilic salts with sodium hydroxide, reverting to hydrophobic fatty acids when the pH shifts to the acidic range . GREEN INSTRUMENTAL OMICS ANALYSES FOR FOOD As already mentioned in the introduction to this publication, the various analytical strategies for examining food are often divided into targeted and non‐targeted analyses. In addition, the focus of the various analyses is on different cellular levels, which are of varying suitability for answering the various questions in this context. An overview of the various approaches that are relevant in this regard and in the following chapters is shown in Table . 4.1 DNA Genomics analyses are often used for taxonomic differentiation of varieties and species, as well as for the identification of microorganisms. Despite their importance, to the best of our knowledge, there are no publications explicitly addressing aspects of “Green Chemistry” in this context. However, the implementation of genomic analyses has undergone significant advancements in recent years, particularly through the development of new next‐generation sequencing methods (NGS). These methods allow a high degree of parallelization and contribute to a more environmentally friendly analysis by reducing the amount of chemicals required compared to traditional Sanger sequencing. Third and fourth‐generation NGS sequencers no longer require DNA amplification, further reducing chemical consumption. Additionally, devices such as the MinION sequencer by Oxford Nanopore Technologies have become significantly smaller, making their production more sustainable . DNA fingerprinting analyses do not require the DNA sequence to be determined, but fragment lengths are analyzed by first amplifying the fragments using polymerase chain reaction (PCR) and then detecting them. Numerous options are available for such procedures, such as restriction fragment length polymorphism analysis, amplified fragment length polymorphism PCR, or randomly amplified polymorphic DNA PCR. The fragments are detected using gel electrophoresis after the DNA molecules have been stained with a dye . For many years, staining was carried out with ethidium bromide, which, however, has a very high toxicity. Numerous alternatives have now been developed to protect laboratory staff and the environment. A comparative overview was recently published and can be found in the literature . Capillary electrophoresis can also be used as an alternative to detection on agarose gels. These methods are not only faster and offer better efficiency as well as resolution, but they also enable automation and require significantly smaller sample quantities, so that fewer chemicals are needed . Alternatively, DNA microarrays can also be used for detection, which support a high degree of parallelization and also require comparatively small amounts of chemicals and sample volumes . Furthermore, lateral flow assays provide a very simple and environmentally friendly option for detecting certain DNA sequences. Although these methods are not considered omics applications per se, they are suitable as downstream methods and for long‐term sustainable implementation in routine processes . A more environmentally friendly alternative to classical PCR are multiplex PCR approaches in which several sequences are amplified simultaneously, thus increasing the degree of parallelization . Although classical PCR amplifications require energy to reach the required temperatures for each step, a loop‐mediated isothermal amplification method has the potential to operate in the best case at room temperature but uses a DNA polymerase with strand displacement activity. Instead of a thermophilic DNA polymerase, which is commonly used, other strategies are based on a DNA polymerase I fragment from the extremophile bacterium Deinococcus radiodurans . 4.2 Peptides and metabolites High‐resolution mass spectrometers or nuclear magnetic resonance (NMR) spectroscopy instruments are generally used for metabolomics and proteomics‐based studies. In MS analyses, the analytes often pre‐separated chromatographically to enhance analytical coverage and achieve better sensitivity. Both energy consumption and the use of chemicals for chromatographic separation can be reduced by shortening analysis times. Additionally, miniaturization processes are increasingly in demand in this field. NanoLC devices are frequently employed alongside (U)HPLC instruments, particularly in proteomics studies. These devices significantly reduce the amount of solvent required for chromatographic separation and can also increase sensitivities. Further advantages arise in the chromatographic resolution of peaks . Similar effects, although not to the same extent, can also be attained in (U)HPLC applications. The use of smaller core–shell particles in smaller columns also improves the resolution of these instruments, leading to corresponding savings in analysis times and solvents. This improvement is accompanied by increased back pressure, which can be easily managed by UHPLC instruments. Alternatively, monolithic columns are suitable for higher flow rates and generate low back pressure, thereby shortening the analysis time . The choice of solvent also has a significant impact on the sustainability of LC applications. Depending on the stationary phase, water is typically used along with organic solvents such as methanol or acetonitrile. More environmentally friendly alternatives include ethanol, isopropanol, acetone, ILs, ethyl acetate, ethyl lactate, and propylene carbonate. When selecting suitable solvents, it is important to consider not only the properties of the stationary phase but also which detector will be used to detect the analytes following the separation. Some solvents have cut‐offs at certain wavelengths when working with UV detectors or are not compatible with ionization sources when using a mass analyzer, as they can lead to ion suppression or are not sufficiently volatile. This consideration also applies to green micellar LC approaches, where aqueous solutions with surfactants above the critical micelle concentration are used. Consequently, analytes are detected with UV or fluorescence detectors . Separation using supercritical CO 2 or water offers another alternative but requires more complex equipment. In particular, supercritical fluid chromatography (SFC) approaches with CO 2 as eluent have become increasingly popular in recent years. This is primarily due to the improved commercial availability of the equipment, especially with the introduction of hybrid SFC/UHPLC instruments. Furthermore, a wide variety of chromatographic phases are now accessible. Additionally, couplings with mass spectrometers via ESI or APCI sources are feasible, allowing for extensive non‐targeted analyses . A comparative study between RP chromatography and SFC, conducted for the detection of pesticides in food matrices, indicates superior chromatographic separation and ion source efficiency in SFC measurements . However, studies conducted in the context of non‐targeted authenticity analyses of food using SFC are currently rare. Nonetheless, there are examples of the authentication of oils . SFC separations have been predominantly used for detecting small organic molecules, with rare applications in proteomics analyses. However, it is basically possible to separate peptides using SFC approaches . Similar to LC applications, sustainable GC analyses benefit from the use of smaller instruments and column dimensions, as well as faster separation gradients, to reduce gas consumption while achieving good separation and low detection limits of the analytes. In this context, micro‐GC applications are of great importance. To our knowledge, these smaller devices have not yet been used for authentication issues. Nevertheless, there could be great potential in the development of environmentally friendly methods . Other sustainable aspects of GC applications relate to the selection of carrier gas. As a rule, helium, nitrogen, or hydrogen are used, with helium being the most prevalent choice. However, the availability of helium on Earth is finite. Due to its lightness compared to air, helium rises in the Earth's atmosphere and gradually escapes into space. Although there are methods for collecting and recycling helium, they are still relatively expensive and are therefore currently only used on a limited basis . Hydrogen offers a more environmentally friendly alternative, but the risk of explosion must be taken into account. However, analytically very good results can be achieved with hydrogen. In contrast to nitrogen, which produces the worst chromatographic results and is not suitable for GC–MS combinations because it is too easily ionized . Further savings options when carrying out green GC analyses enable low‐pressure conditions in conjunction with MS analyzers, which accelerate the analyses while maintaining separation performance as the optimal linear carrier gas velocity increases. In addition, heating devices with low thermal mass technology enable faster heating and cooling of the column, resulting in further savings in energy consumption . Recently, high‐resolution MS analyzers have often been coupled with ion mobility cells. Parasta et al. recently highlighted the environmental benefits of GC‐IMS couplings compared to GC–MS couplings, particularly since IM measurements do not require a vacuum. Additionally, the carrier gas can be reused. Meanwhile, several studies have been published in the field of food authentication using GC‐IM instruments. A comprehensive overview can be found in the cited review paper . As already mentioned, avoiding the production of a sample extract is the most environmentally friendly option. In this regard, the use of mass spectrometers in particular offers a wide range of options through coupling with ambient ion techniques or specific sampling techniques. These include, for example, direct analysis in real time, desorption electrospray ionization, laser ablation with electrospray ionization, probe ESI, rapid evaporative ionization MS, atmospheric solids analysis probe, paper spray MS, or open port sampling interface. What all of these techniques have in common is that no chromatographic separation is required, although some may utilize organic solvents or gases, albeit in minimal quantities. Additionally, they can only detect a rather small number of analytes, as the detectable mass range is often limited to analytes with a lower mass to charge ration ( m/z ) (<2000) . In addition, there are numerous efforts to miniaturize mass spectrometric devices. Promising results have already been achieved in this regard. However, currently the published studies in this area are quite limited, but there could still be great potential for future applications . Depending on how NMR analyses are conducted, they may also incorporate green elements, particularly considering that, according to a literature study, many metabolomics experiments require only a mixture of 10% deuterated water and 90% regular water to produce suitable extracts . One disadvantage, however, is the high demand for liquid helium to cool NMR magnets. Nevertheless, this aspect is taken into account in the development of new devices. Bruker Biospin has succeeded in significantly reducing the helium consumption of NMR systems and has recently started offering systems for the recovery of helium that enable savings of up to 95 % (Bruker BioSpin, 2023). Bruker Advances NMR Magnet Portfolio, Innovates in Industrial Solutions. https://www.bruker.com/en/news‐and‐events/news/2023/bruker‐advances‐nmr‐magnet‐portfolio.html , accessed on April 28th, 2024). Alternatively, in recent years, small low‐field benchtop NMR instruments have also proven to be suitable for tracing the authenticity of food, even if the information content is reduced. These devices have field strengths of 40–100 MHz and only require a power supply . Further green modifications of MS and NMR‐based approaches can also be made if the often non‐targeted analyses are converted into simpler targeted analyses. Less high‐resolution, smaller devices, and faster methods can be used for this step . In addition to NMR and MS instruments, vibrational techniques are also suitable for detecting signals from metabolites or peptides/proteins and using them for authentication. Although the resolution of such devices is comparatively low and specific molecules cannot be identified, authenticity analyses can still be conducted with them. The main advantage is that, in many cases, the samples can be measured directly, and the devices only require electricity. Miniaturized handheld versions are also available so that measurements can be carried out on‐site and there is no need to transport or store the sample. Both IR and Raman methods are used. For IR analyses, it is often necessary to first lyophilize the samples, as the water bands can overlay a lot of information . However, this does not necessarily have to be the case, as was recently shown using almonds as an example . The water content is generally not relevant for Raman analyses. With spatially offset Raman spectroscopy, food packaging no longer even needs to be opened, as this technology can be used to measure through many materials . 4.3 Elements The analysis of metals and stable isotope ratios can also be used for authentication. In this context, ICP‐MS instruments are particularly important, as they enable a high degree of parallelization with regard to metal analyses. The ionization of the elements takes place in an argon plasma at temperatures of up to 10 000°C. Argon is present in the air in high quantities and can be obtained as a byproduct of ammonia synthesis, making this process sustainable . Even with ICP‐MS analyses, it is possible to bypass sample extraction and instead carry out sample ablation with lasers or electrothermal vaporization . X‐ray fluorescence (XRF) spectroscopy and laser‐induced breakdown spectroscopy also do not require sample extraction, which makes these methods very sustainable. Even though they are not as sensitive and resolving as ICP‐MS instruments, they have still proven to be useful for authentication . However, XRF spectroscopy measurements involve the use of X‐rays, which could potentially pose a danger to technical personnel. Nevertheless, these devices are very safe because the emitters are weak and shielded. XRF spectroscopy analyses are also classified as environmentally friendly methods due to the factors mentioned . Isotope ratio MS focuses on the analysis of 2 H/ 1 H, 13 C/ 12 C, 15 N/ 14 N, 18 O/ 16 O, and 34 S/ 32 S. The isotope ratios of hydrogen and oxygen can be used, in particular, to determine geographical origins. However, the ratio of 13 C/ 12 C can be used to distinguish C3, C4 organisms as well as plants that exhibit crassulacean acid metabolism. Furthermore, the ratios of nitrogen and sulfur isotopes primarily provide information about the use of fertilizers and soil conditions. MS analyzers are also used to determine these ratios. Ionization occurs using EI sources after the ions have been converted into gases. To achieve this, the samples are burned either without or with oxygen, depending on which isotope ratios are to be determined. This is followed by gas chromatographic separation, usually with helium as carrier gas, and mass spectrometric detection. Stable isotope analyses benefit from the fact that there is no need to extract the samples; only combustion is required, which involves generating high temperatures of up to 1800°C. Nevertheless, this method is relatively environmentally friendly because it requires few reagents and generates little waste . DNA Genomics analyses are often used for taxonomic differentiation of varieties and species, as well as for the identification of microorganisms. Despite their importance, to the best of our knowledge, there are no publications explicitly addressing aspects of “Green Chemistry” in this context. However, the implementation of genomic analyses has undergone significant advancements in recent years, particularly through the development of new next‐generation sequencing methods (NGS). These methods allow a high degree of parallelization and contribute to a more environmentally friendly analysis by reducing the amount of chemicals required compared to traditional Sanger sequencing. Third and fourth‐generation NGS sequencers no longer require DNA amplification, further reducing chemical consumption. Additionally, devices such as the MinION sequencer by Oxford Nanopore Technologies have become significantly smaller, making their production more sustainable . DNA fingerprinting analyses do not require the DNA sequence to be determined, but fragment lengths are analyzed by first amplifying the fragments using polymerase chain reaction (PCR) and then detecting them. Numerous options are available for such procedures, such as restriction fragment length polymorphism analysis, amplified fragment length polymorphism PCR, or randomly amplified polymorphic DNA PCR. The fragments are detected using gel electrophoresis after the DNA molecules have been stained with a dye . For many years, staining was carried out with ethidium bromide, which, however, has a very high toxicity. Numerous alternatives have now been developed to protect laboratory staff and the environment. A comparative overview was recently published and can be found in the literature . Capillary electrophoresis can also be used as an alternative to detection on agarose gels. These methods are not only faster and offer better efficiency as well as resolution, but they also enable automation and require significantly smaller sample quantities, so that fewer chemicals are needed . Alternatively, DNA microarrays can also be used for detection, which support a high degree of parallelization and also require comparatively small amounts of chemicals and sample volumes . Furthermore, lateral flow assays provide a very simple and environmentally friendly option for detecting certain DNA sequences. Although these methods are not considered omics applications per se, they are suitable as downstream methods and for long‐term sustainable implementation in routine processes . A more environmentally friendly alternative to classical PCR are multiplex PCR approaches in which several sequences are amplified simultaneously, thus increasing the degree of parallelization . Although classical PCR amplifications require energy to reach the required temperatures for each step, a loop‐mediated isothermal amplification method has the potential to operate in the best case at room temperature but uses a DNA polymerase with strand displacement activity. Instead of a thermophilic DNA polymerase, which is commonly used, other strategies are based on a DNA polymerase I fragment from the extremophile bacterium Deinococcus radiodurans . Peptides and metabolites High‐resolution mass spectrometers or nuclear magnetic resonance (NMR) spectroscopy instruments are generally used for metabolomics and proteomics‐based studies. In MS analyses, the analytes often pre‐separated chromatographically to enhance analytical coverage and achieve better sensitivity. Both energy consumption and the use of chemicals for chromatographic separation can be reduced by shortening analysis times. Additionally, miniaturization processes are increasingly in demand in this field. NanoLC devices are frequently employed alongside (U)HPLC instruments, particularly in proteomics studies. These devices significantly reduce the amount of solvent required for chromatographic separation and can also increase sensitivities. Further advantages arise in the chromatographic resolution of peaks . Similar effects, although not to the same extent, can also be attained in (U)HPLC applications. The use of smaller core–shell particles in smaller columns also improves the resolution of these instruments, leading to corresponding savings in analysis times and solvents. This improvement is accompanied by increased back pressure, which can be easily managed by UHPLC instruments. Alternatively, monolithic columns are suitable for higher flow rates and generate low back pressure, thereby shortening the analysis time . The choice of solvent also has a significant impact on the sustainability of LC applications. Depending on the stationary phase, water is typically used along with organic solvents such as methanol or acetonitrile. More environmentally friendly alternatives include ethanol, isopropanol, acetone, ILs, ethyl acetate, ethyl lactate, and propylene carbonate. When selecting suitable solvents, it is important to consider not only the properties of the stationary phase but also which detector will be used to detect the analytes following the separation. Some solvents have cut‐offs at certain wavelengths when working with UV detectors or are not compatible with ionization sources when using a mass analyzer, as they can lead to ion suppression or are not sufficiently volatile. This consideration also applies to green micellar LC approaches, where aqueous solutions with surfactants above the critical micelle concentration are used. Consequently, analytes are detected with UV or fluorescence detectors . Separation using supercritical CO 2 or water offers another alternative but requires more complex equipment. In particular, supercritical fluid chromatography (SFC) approaches with CO 2 as eluent have become increasingly popular in recent years. This is primarily due to the improved commercial availability of the equipment, especially with the introduction of hybrid SFC/UHPLC instruments. Furthermore, a wide variety of chromatographic phases are now accessible. Additionally, couplings with mass spectrometers via ESI or APCI sources are feasible, allowing for extensive non‐targeted analyses . A comparative study between RP chromatography and SFC, conducted for the detection of pesticides in food matrices, indicates superior chromatographic separation and ion source efficiency in SFC measurements . However, studies conducted in the context of non‐targeted authenticity analyses of food using SFC are currently rare. Nonetheless, there are examples of the authentication of oils . SFC separations have been predominantly used for detecting small organic molecules, with rare applications in proteomics analyses. However, it is basically possible to separate peptides using SFC approaches . Similar to LC applications, sustainable GC analyses benefit from the use of smaller instruments and column dimensions, as well as faster separation gradients, to reduce gas consumption while achieving good separation and low detection limits of the analytes. In this context, micro‐GC applications are of great importance. To our knowledge, these smaller devices have not yet been used for authentication issues. Nevertheless, there could be great potential in the development of environmentally friendly methods . Other sustainable aspects of GC applications relate to the selection of carrier gas. As a rule, helium, nitrogen, or hydrogen are used, with helium being the most prevalent choice. However, the availability of helium on Earth is finite. Due to its lightness compared to air, helium rises in the Earth's atmosphere and gradually escapes into space. Although there are methods for collecting and recycling helium, they are still relatively expensive and are therefore currently only used on a limited basis . Hydrogen offers a more environmentally friendly alternative, but the risk of explosion must be taken into account. However, analytically very good results can be achieved with hydrogen. In contrast to nitrogen, which produces the worst chromatographic results and is not suitable for GC–MS combinations because it is too easily ionized . Further savings options when carrying out green GC analyses enable low‐pressure conditions in conjunction with MS analyzers, which accelerate the analyses while maintaining separation performance as the optimal linear carrier gas velocity increases. In addition, heating devices with low thermal mass technology enable faster heating and cooling of the column, resulting in further savings in energy consumption . Recently, high‐resolution MS analyzers have often been coupled with ion mobility cells. Parasta et al. recently highlighted the environmental benefits of GC‐IMS couplings compared to GC–MS couplings, particularly since IM measurements do not require a vacuum. Additionally, the carrier gas can be reused. Meanwhile, several studies have been published in the field of food authentication using GC‐IM instruments. A comprehensive overview can be found in the cited review paper . As already mentioned, avoiding the production of a sample extract is the most environmentally friendly option. In this regard, the use of mass spectrometers in particular offers a wide range of options through coupling with ambient ion techniques or specific sampling techniques. These include, for example, direct analysis in real time, desorption electrospray ionization, laser ablation with electrospray ionization, probe ESI, rapid evaporative ionization MS, atmospheric solids analysis probe, paper spray MS, or open port sampling interface. What all of these techniques have in common is that no chromatographic separation is required, although some may utilize organic solvents or gases, albeit in minimal quantities. Additionally, they can only detect a rather small number of analytes, as the detectable mass range is often limited to analytes with a lower mass to charge ration ( m/z ) (<2000) . In addition, there are numerous efforts to miniaturize mass spectrometric devices. Promising results have already been achieved in this regard. However, currently the published studies in this area are quite limited, but there could still be great potential for future applications . Depending on how NMR analyses are conducted, they may also incorporate green elements, particularly considering that, according to a literature study, many metabolomics experiments require only a mixture of 10% deuterated water and 90% regular water to produce suitable extracts . One disadvantage, however, is the high demand for liquid helium to cool NMR magnets. Nevertheless, this aspect is taken into account in the development of new devices. Bruker Biospin has succeeded in significantly reducing the helium consumption of NMR systems and has recently started offering systems for the recovery of helium that enable savings of up to 95 % (Bruker BioSpin, 2023). Bruker Advances NMR Magnet Portfolio, Innovates in Industrial Solutions. https://www.bruker.com/en/news‐and‐events/news/2023/bruker‐advances‐nmr‐magnet‐portfolio.html , accessed on April 28th, 2024). Alternatively, in recent years, small low‐field benchtop NMR instruments have also proven to be suitable for tracing the authenticity of food, even if the information content is reduced. These devices have field strengths of 40–100 MHz and only require a power supply . Further green modifications of MS and NMR‐based approaches can also be made if the often non‐targeted analyses are converted into simpler targeted analyses. Less high‐resolution, smaller devices, and faster methods can be used for this step . In addition to NMR and MS instruments, vibrational techniques are also suitable for detecting signals from metabolites or peptides/proteins and using them for authentication. Although the resolution of such devices is comparatively low and specific molecules cannot be identified, authenticity analyses can still be conducted with them. The main advantage is that, in many cases, the samples can be measured directly, and the devices only require electricity. Miniaturized handheld versions are also available so that measurements can be carried out on‐site and there is no need to transport or store the sample. Both IR and Raman methods are used. For IR analyses, it is often necessary to first lyophilize the samples, as the water bands can overlay a lot of information . However, this does not necessarily have to be the case, as was recently shown using almonds as an example . The water content is generally not relevant for Raman analyses. With spatially offset Raman spectroscopy, food packaging no longer even needs to be opened, as this technology can be used to measure through many materials . Elements The analysis of metals and stable isotope ratios can also be used for authentication. In this context, ICP‐MS instruments are particularly important, as they enable a high degree of parallelization with regard to metal analyses. The ionization of the elements takes place in an argon plasma at temperatures of up to 10 000°C. Argon is present in the air in high quantities and can be obtained as a byproduct of ammonia synthesis, making this process sustainable . Even with ICP‐MS analyses, it is possible to bypass sample extraction and instead carry out sample ablation with lasers or electrothermal vaporization . X‐ray fluorescence (XRF) spectroscopy and laser‐induced breakdown spectroscopy also do not require sample extraction, which makes these methods very sustainable. Even though they are not as sensitive and resolving as ICP‐MS instruments, they have still proven to be useful for authentication . However, XRF spectroscopy measurements involve the use of X‐rays, which could potentially pose a danger to technical personnel. Nevertheless, these devices are very safe because the emitters are weak and shielded. XRF spectroscopy analyses are also classified as environmentally friendly methods due to the factors mentioned . Isotope ratio MS focuses on the analysis of 2 H/ 1 H, 13 C/ 12 C, 15 N/ 14 N, 18 O/ 16 O, and 34 S/ 32 S. The isotope ratios of hydrogen and oxygen can be used, in particular, to determine geographical origins. However, the ratio of 13 C/ 12 C can be used to distinguish C3, C4 organisms as well as plants that exhibit crassulacean acid metabolism. Furthermore, the ratios of nitrogen and sulfur isotopes primarily provide information about the use of fertilizers and soil conditions. MS analyzers are also used to determine these ratios. Ionization occurs using EI sources after the ions have been converted into gases. To achieve this, the samples are burned either without or with oxygen, depending on which isotope ratios are to be determined. This is followed by gas chromatographic separation, usually with helium as carrier gas, and mass spectrometric detection. Stable isotope analyses benefit from the fact that there is no need to extract the samples; only combustion is required, which involves generating high temperatures of up to 1800°C. Nevertheless, this method is relatively environmentally friendly because it requires few reagents and generates little waste . CONCLUSIONS In recent years, significant progress has been made to enhance the sustainability of analytical processes in omics analysis, particularly in the assessment of food authenticity and the careful use of existing raw materials. However, in many cases, a compromise must be found between environmental friendliness and analytical performance. This balancing act is not always easy and certainly requires a lot of additional knowledge. Nevertheless, it can be anticipated that future advancements in this field will increasingly emphasize both aspects, striving to reconcile them accordingly. The aspects explained show that this process has already taken place many times. Nonetheless, there is undoubtedly more work to be done, and there remains an urgent need to sustain these efforts and foster the necessary awareness for the widespread adoption of “Green Analytical Chemistry” approaches. The authors declare no conflicts of interest. |
Utility of Temporal Bone Computed Tomography in Pediatric Emergency Medicine | 06faf412-73df-45ca-9899-698205199392 | 8967447 | Pediatrics[mh] | Up to seven million children in the United States undergo computed tomography (CT) annually, which has been raised as a public health concern due to radiation exposure and increased lifetime cancer risk. , For this reason, multiple algorithms have been developed to reduce the radiation dose associated with CT in the pediatric population. , The “as low as reasonably achievable” (ALARA) concept addresses methods for reducing the amount of radiation in a child while maintaining reliability of the diagnostic modality. The ALARA recommendations include developing weight-based protocols, considering alternative non-radiation modalities, and discouraging repeat CT imaging. Furthermore, young children may require sedation for imaging, adding additional risks, time, and cost. Ultimately, the best method of harm reduction is to avoid performing CT that will not inform or alter clinical decision-making. Temporal bone CT is used in the pediatric population to identify acute middle- and inner-ear pathologies, often in the setting of infectious or trauma evaluation. , Due to the complex bony anatomy, CT of the temporal bone requires a slice thickness of <1.0 millimeter and a high signal-to-noise ratio to minimize artifact for optimal visualization, thus requiring a higher radiation dose compared to routine head CT. Reducing the radiation dose of temporal bone CT below literature-derived protocols while maintaining accurate detection of findings of middle- and inner-ear structures is an area of active research. The high resolution of temporal bone CT aids in surgical planning by identifying infectious destruction of bone or the precise location of a temporal bone fracture that may otherwise be missed on lower-resolution imaging protocols. In the setting of temporal bone fracture, operative intervention with facial nerve decompression is indicated for patients with immediate facial nerve paresis and progressive decline in electroneuronography (ENoG) functioning to less than 10% of the normal side. Operative intervention is often indicated for complicated mastoiditis, with extracranial and intracranial sequelae encountered in 13–38% of mastoiditis cases. , However, the current criteria for diagnosing complicated mastoiditis are diverse, and there is a lack of consensus regarding the strategies for diagnosis and the role of CT in the pediatric population. The primary objective of our study was to characterize the use of temporal bone CT in the acute emergency setting and investigate the clinical utility of this imaging modality in diagnosis and management of acute infectious and traumatic pathology of the temporal bone in the pediatric population. Our goal was to identify patient characteristics and common indications for temporal bone CT at our institution and to determine the subsequent clinical and/or operative management for these patients. Recognizing the appropriate scenarios to order temporal bone CT may prevent unnecessary radiation exposure in children presenting with such pathologies. Population Health Research Capsule What do we already know about this issue? High-resolution computed tomography (CT) may be used in pediatric infectious and traumatic temporal bone etiologies, but radiation dose is a public health concern. What was the research question? What is the clinical utility of temporal bone CT in pediatric acute infectious and traumatic pathologies? What was the major finding of the study? Temporal bone CT is beneficial for acute mastoiditis, but its utility in trauma evaluation may be limited. How does this improve population health? We identify areas for potential reduction in the use of temporal bone CT, which may limit unnecessary radiation exposure in the pediatric population.
Following institutional review board approval at Penn State Hershey Medical Center, we conducted a retrospective review of pediatric emergency department (ED) visits at our institution between January 1, 2012 – December 31, 2016, for all pediatric patients who underwent CT temporal bone imaging over the specified time period. Patients with a primary International Classification of Disease s, 9 th or 10 th revision, diagnosis consistent with otitis externa, otitis media, mastoiditis, head trauma, temporal bone fracture, or otalgia were included to limit our study to the use of CT temporal bone imaging in the acute evaluation of infectious and traumatic etiologies. We collected data regarding patients’ presenting signs/symptoms and admission type (inpatient vs emergency), indications for CT temporal bone imaging, radiologic findings, and operative procedures performed within 30 days of CT imaging. For patients with suspected mastoiditis, we compared the operative and non-operative groups. The chart abstractors were not blinded to the study hypothesis. Statistical significance was determined by Fisher’s exact test with α = 0.05 implemented via the “stats” package in R v. 3.2.2 (The R Foundation for Statistical Computing, Vienna, Austria).
Within our patient cohort there were 96 temporal bone CTs. Most studies (N = 67; 70%) were associated with an inpatient admission, while the remaining patients were discharged from the ED (N = 29; 30%). The most common indications for imaging were evaluation of acute mastoiditis (N = 53; 55%) or trauma (N = 39; 41%). The otolaryngology service was consulted for 68 patients, representing 79% of our patient cohort. Otalgia, otorrhea, and post-auricular swelling were the most common presenting symptoms among patients with concern for mastoiditis. Mastoid tenderness, auricular proptosis, tympanic membrane opacification, and otorrhea were the most commons signs. Of the 53 patients with concern for mastoiditis, five had CT head performed prior to CT temporal bone, and otolaryngology was consulted for 66% of all infectious patients (N = 35) . There was a total of 27 otologic procedures among the cohort, which included myringotomy and tympanostomy tube insertion and mastoidectomy, with or without abscess incision and drainage. To determine the utility of temporal bone CT for patients with infectious concerns, we compared patient-reported symptoms, physical examination signs, and radiographic findings between patients who required operative intervention and those who did not . Presentation of altered mental status was significantly more prevalent in the operative group (N = 5) compared to the non-operative group (N = 0; P = 0.02). Also, proptosis ( P = 0.05) and post-auricular fluctuance ( P = 0.02) were more frequent among the operative group. Radiographic findings of complicated mastoiditis (ie, post-auricular abscess, Bezold’s abscess, sigmoid sinus thrombosis, intracranial abscess) were reported among 10 patients in the operative group, and no patients in the non-operative group ( P <0.01). No other clinical or radiologic findings were statistically associated with operative intervention; however, there were two patients in the operative group with facial nerve paralysis, which is a clear indication for operative management in this setting of infection. As expected, otolaryngology was consulted for all patients in the operative group (N = 27) compared to 31% (N = 8) of the non-operative group. Of the trauma patients (N = 39), the most common presenting otologic signs and symptoms included hemotympanum, bloody otorrhea, hearing loss, otalgia, mastoid tenderness, and otorrhea . Seventy-four percent of trauma patients who had CT temporal bone also had CT head (N =29), and 85% had otolaryngology consults (N = 33). The most common final diagnoses among trauma patients based on radiographic results were temporal bone fracture and mastoid effusion without radiographic evidence of fracture . Among those who had temporal bone CT in the setting of trauma, two patients had operative intervention, both for facial nerve decompression.
Temporal bone CT provides detailed anatomic information regarding the middle ear and temporal bone at the expense of a relatively high radiation dose, which is particularly undesirable in the pediatric population. To improve quality of care, clinicians should carefully weigh the risk of radiation exposure to the potential benefit of CT temporal bone imaging. Our study demonstrates that temporal bone CT is useful for surgical planning in the setting of complicated mastoiditis; however, mastoiditis remains primarily a clinical diagnosis that often does not require high-resolution imaging. – Traumatic fractures can often be presumed in the setting of air cell opacification on head CT without the need to visualize the fracture with a high-resolution image. Moreover, surgical intervention is typically not required in the absence of complete facial nerve paralysis. , Therefore, temporal bone CT should not be a routine study in the workup of these patients, especially in the pediatric population. Based on our findings, we have proposed approaches to obtaining temporal bone CT imaging between the specialties of emergency medicine and otolaryngology to maximize usefulness among pediatric patients with acute infectious concerns , as well as those with temporal bone fractures . In the setting of temporal bone fractures due to head trauma, it is possible that the patient will be sedated, intubated, or unable to follow commands. In such cases, assessment of facial paralysis may not be feasible and should be deferred until the patient is awake, and trauma evaluation should then proceed as indicated. However, it is important to note that if a patient is subsequently found to have a complete paralysis, the injury is treated as an immediate complete paralysis as opposed to delayed paralysis, and surgical intervention should be pursued. , In our pediatric ED population, nearly half of patients who underwent CT to evaluate for mastoiditis required operative intervention. This indicates that half of patients who received temporal bone CT did not require surgery, potentially exposing these children to unnecessary ionizing radiation. While it is possible that patients may require a temporal bone CT for admission for medical management of suspected mastoiditis, our data does highlight the importance of identifying clinical findings that raise suspicion for surgical intervention (ie, mental status changes, proptosis, fluctuance) when deciding whether temporal bone CT is necessary. The literature suggests that CT may be beneficial in confirming the diagnosis of mastoiditis in patients who do not present with a clear clinical picture. Future studies should examine scenarios in which CT can be avoided altogether in patients with acute infection and when head CT alone or other lower-radiation temporal bone CT techniques may be sufficient, thereby limiting radiation exposure if surgical intervention is unlikely. Additionally, recognizing the clinical signs and symptoms associated with acute infection of the temporal bone can help guide initial medical management in the emergency setting for situations when surgical intervention may be delayed or unnecessary. The literature suggests that uncomplicated mastoiditis, meaning no neurologic deficits or sepsis, should be managed with intravenous antibiotics, and CT temporal bone should only be obtained if deterioration or lack of clinical improvement is observed. , Of note, no patients in our cohort had sepsis, which would have allowed us to further assess the utility of CT temporal bone specifically among patients with this acute concern. Operative intervention should be considered as a reasonable next step following medical management in such cases of disease progression or lack of improvement. In the setting of trauma evaluation, children are often exposed to significant radiation due to extensive radiologic workup. If initial findings on head CT suggest temporal bone fracture, a temporal bone CT may, in practice, be obtained to better visualize the fracture. Studies consistently indicate that facial nerve decompression is recommended in temporal bone fracture if the patient has complete facial nerve paralysis and a loss of greater than 90% function on ENoG. , In such scenarios, temporal bone CT is useful in delineating the anatomical course of the facial nerve and locating the exact site of injury, guiding the decision on surgical approach (ie, trans-mastoid vs middle cranial fossa). Yet only 6% (N = 2) of trauma patients who underwent temporal bone CT in our study ultimately required facial nerve decompression. Even patients with incomplete facial nerve paralysis on initial presentation generally do well with expectant management. Surgical decompression for incomplete facial paralysis has a Grade D aggregate evidence level in the Otolaryngology – Head and Neck Surgery Clinical Practice Guidelines due to the lack of definitive benefits of surgery in this setting. Otherwise, temporal bone fractures are only monitored for hearing outcomes, which includes a delayed audiogram performed 3–6 weeks after injury to allow enough time for resolution of hemotympanum to accurately assess for ossicular discontinuity. Therefore, if there is no concern for complete facial nerve paralysis, a head CT alone obtained during routine trauma workup may be sufficient, since additional imaging is unlikely to change this management strategy and would increase radiation exposure. Following consultation with otolaryngology, consideration may be given for obtaining a temporal bone CT if there is concern for cerebrospinal fluid leak or otic capsule-violating fracture; however, even in these scenarios expectant management is often sufficient. , It is worth noting that significant trauma is required to cause a temporal bone fracture, and many patients with such a finding have other more serious injuries taking precedence. Our algorithm presupposes that no urgent/emergent neurosurgical pathology in or around the temporal bone is present, such as epidural hematoma or carotid canal injury, which may require additional imaging or other expedient management. Previous studies have demonstrated that the radiologic finding of mastoid air cell opacification is non-specific, rarely clinically significant, and found incidentally in the pediatric population at rates of 14% and 21% with CT and magnetic resonance imaging (MRI), respectively. , Additionally, Polat et al reported that only 17% of patients with mastoid opacification on MRI were found to have clinical infectious otologic disease. Therefore, despite the benefit of no radiation exposure with MRI, it is not necessarily superior to CT in ruling out this incidental finding. Furthermore, additional evaluation based on this finding can result in unnecessary treatment and expenditure of healthcare resources. , Our study demonstrates that there were 68 total otolaryngology referrals for CT temporal bone findings among the patient cohort. We found that 94% of trauma patients and 23% of infectious patients for whom otolaryngology was consulted ultimately did not require operative intervention. Therefore, CT head findings should be closely correlated with clinical examination to reduce unnecessary temporal bone CT, and further imaging should be based on a coordinated approach between services when specialty consultation is requested. This descriptive study has identified trends for certain inefficiency and redundancy at our institution. With this data, we have developed algorithms to streamline the decision process in ordering temporal bone CT in the acute setting to foster shared decision-making between clinicians and specialties to ultimately reduce radiation exposure among the pediatric population and improve quality of patient care.
There are limitations to the findings of our study. First, this was a retrospective review performed at a single institution with a small sample size, and thus some of our conclusions may not be generalizable to other populations as practice patterns may vary by institution. Additionally, the chart abstractors were not blinded to the study hypothesis. While the study demonstrates an opportunity to reduce the decision to perform a CT, our study did not evaluate whether that decision was made by the ED, trauma surgery, or the consulting service. However, we hope that by highlighting this opportunity it will foster more collaboration between services in providing multidisciplinary care.
Our study demonstrates that temporal bone CT can be beneficial in guiding diagnosis and management of acute infectious pathology in the pediatric emergency setting. Clinical examination findings such as mental status changes, proptosis, and fluctuance should guide decision-making surrounding the utility of temporal bone CT for mastoiditis. There may be circumstances in which imaging could be avoided to reduce radiation exposure. In traumatic pathology of the temporal bone, CT did not lead to operative intervention in patients without a clinically apparent facial nerve paralysis. We propose approaches to addressing CT imaging among pediatric patients with acute infectious concerns as well as those with temporal bone fractures, which emphasize using clinical findings likely to lead to operative intervention. By identifying scenarios in which imaging may be unnecessary, we hope to continue public health efforts to reduce ionizing radiation among the pediatric population.
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Comparison of medium- and long-term total knee arthroplasty follow-up with or without tourniquet | bc71a3b1-edf3-467e-9462-746d06ce0118 | 11866867 | Surgical Procedures, Operative[mh] | Recently, revision surgery has shown an increasing trend due to the increased demand for total knee arthroplasty (TKA) and expansion of surgical indications. According to the American Academy of Orthopaedic Surgeons, the number of TKA revision cases by 2030 will increase to six times that of 2005 . Aseptic loosening is a major indication for knee revision surgery, accounting for 28.7% of cases usually occurring at the bone-cement interface . Osteotomy surface and implant stability are affected by many factors. The depth of bone cement penetration into the cancellous bone is crucial for a successful initial TKA. It is related to bone cement thickness, placement time, penetration conditions, and many other factors . In theory, the bone cement penetration depth determines the implant’s tensile strength in the vertical direction and the shear strength in the horizontal direction. Bone cement covering 3–4 mm between the tibial implant and the tibial trabecula is best to avoid surrounding osteolysis and prosthesis loosening . Cancellous bones contain an interstitial space, and when the prosthesis is installed, properly pressing the unsolidified bone cement can better penetrate the bone trabeculae interstitial space. In contrast, blood seepage and fat drops on the osteotomy surface blocks bone cement infiltration and reduce its viscosity. Relevant studies have shown that bleeding and lipid droplets on the osteotomy surface reduce bone cement viscosity by approximately 50%, decreasing the thickness of the bone cement penetrating the cancellous bone , which may affect the service life of prostheses. In order to ensure the bone cement can penetrate smoothly. Tourniquet is used in traditional TKA because it can effectively reduce bleeding from the osteotomy surface, provide a dry osteotomy surface, facilitate bone cement occlusion, and improve prosthesis placement quality. Besides, It can ensures a clear surgical field, reduces frequent hemostatic operations, makes the operation smoother, shortens the operation time, and reduces infection risk . The short-term outcome of knee arthroplasty was evaluated based on the prosthesis stability and the knee joint range of motion (ROM). Long-term outcomes were primarily evaluated through survival analysis with revision surgery as the endpoint .With the popularity of the enhanced recovery after surgery (ERAS) concept in perioperative TKA , many researchers still do not advocate using tourniquets during surgery. Using intraoperative tourniquets increases invisible postoperative blood loss and ischemia–reperfusion injury risk, aggravates swelling of the affected limb, and is not conducive to early postoperative functional exercise. Notably, many researchers have studied whether using tourniquets affects the stability and survival period of knee prostheses. However, no consensus has been reached . Certain scholars believe that using tourniquets reduces the service life of knee prostheses. In contrast, others have an opposite conclusion, believing that using tourniquets does not affect the survival period of prostheses. Currently, there are no long-term follow-up studies to determine whether intraoperative tourniquet use affects the useful life of prostheses. This study’s objectives were: i) to compare the differences in knee function and quality of life between the two groups during follow-up. ii) to compare whether the use of a tourniquet affected bone cement penetration and the probability of generating radioactive rays by imaging measurements, and iii) to compare whether the use of a tourniquet affects the survival of knee prostheses during long-term follow-up.
Inclusion and exclusion criteria The medical ethics committee approved this study, and all participants provided informed consent. The inclusion criteria were: (1) patients with degenerative osteoarthritis of the knee who underwent knee arthroplasty for the first time, (2) Age 60–75 years old, body mass index < 35 kg/m 2 , and (3) all operations had been performed by the same senior physician with the same surgical approach. The exclusion criteria were: (1) patients with bilateral primary knee arthroplasty, (2) serious knee deformity, internal and external inversion > 20°, and (3) patients requiring secondary revision due to periprosthetic fractures. Study design and participants We retrospectively analyzed 254 patients with knee arthritis treated in our hospital from January 1, 2014, to June 1, 2015, among them, 217 patients met the inclusion criteria, 20 met the exclusion criteria, and a total of 197 patients were screened. Among the above 197 patients who met the inclusion and exclusion criteria, 8 patients who died due to extra-articular diseases, and 23 patients without follow-up information. Finally, 166 patients were included in this study. Our hospital’s surgical concept changed in 2014, and the patients were divided into two groups according to whether tourniquets were used during the operation. There were 80 cases in the tourniquet (T) group, including 31 males and 49 females, with an average age of 68.07 ± 6.75 years, and 86 cases in the non-tourniquet (NT) group, including 35 males and 51 females, with an average age of 68.41 ± 6.52 years. Before surgery, we evaluated the severity of knee arthritis by radiology using the Kellgren and Lawrence scores. Table shows the general information of the patients in the two groups. Surgical technique The patient was placed in the supine position and the surgical field was routinely disinfected and covered with a cloth. In group T, a tourniquet (pressure 260 mmHg) was applied throughout the entire process, whereas no tourniquet was applied in Group NT. All patients were administered 1 g tranexamic acid intravenously before surgery. A longitudinal incision was made in the anterior median, starting at the proximal side of the patella (5–10 cm) and ending at the tibial tubercle. A deep incision was made along the medial side of the patella to expose the joint cavity. According to certain operating procedures, the femoral end osteotomy was performed first, followed by the posterior tibial end osteotomy; the lower limb vertical line was restored, and bone cement was fully mixed when required. Then, the tibia and femur prostheses were implanted, excess bone cement was removed after it had solidified, and a polyethylene liner was installed and rinsed with a washing gun. The tourniquet in the T group was loosened for careful electrocoagulation. In contrast, the NT group had already performed inadvertent electrocoagulation at the bleeding site intraoperatively. Indwelling drainage tube was placed in both groups. And then, all patients were subsequently sutured layer-by-layer, bandaged with gauze, cotton pads, and elastic bandages. The two groups had similar postoperative treatment methods, including swelling reduction, pain relief, anti-inflammatory therapy, thrombosis prevention, and other routine treatments. Patients were instructed to exercise the ankle pump and leg lift functions under guidance on the first-day post-surgery. On the second postoperative day, the wound dressing was changed, the wound drainage tube was removed, the dressing was bandaged, and the patient was instructed to perform knee joint and flexion function exercises, and walked with the help of a walker and then gradually transitioned to normal walking without walker. Observation assessment We compared all patient’s perioperative data, including operation time, incision length, intraoperative blood loss, total blood loss, and length of hospital stay. Complications, such as wound infection, poor wound healing, and deep venous thrombosis (DVT) of the lower limbs, were recorded during follow-up. The hip knee ankle angle (HKA) and medial proximal tibial angle (MPTA) were measured before and after surgery. We measured the depth of bone cement penetration in each area of the postoperative radiographic prosthesis (Fig. ). We identified the radiolucent line between the osteotomy surface and the prosthesis using radiography at the last follow-up. Preoperative and postoperative HSS and knee range of motion (ROM) were recorded in both groups during follow-up. Data analyses Data were measured independently and averaged from three uninformed individuals. Statistical analysis was performed using the statistical package for social sciences 26.0, and measurement data were presented as (x̅ ± s). Normally distributed data was compared between both groups using an independent sample t -test, while counting data was compared using the χ 2 test or Fisher’s exact test. The grade data between both groups were compared using the Mann–Whitney U test. Statistical significance was set at P < 0.05.
The medical ethics committee approved this study, and all participants provided informed consent. The inclusion criteria were: (1) patients with degenerative osteoarthritis of the knee who underwent knee arthroplasty for the first time, (2) Age 60–75 years old, body mass index < 35 kg/m 2 , and (3) all operations had been performed by the same senior physician with the same surgical approach. The exclusion criteria were: (1) patients with bilateral primary knee arthroplasty, (2) serious knee deformity, internal and external inversion > 20°, and (3) patients requiring secondary revision due to periprosthetic fractures.
We retrospectively analyzed 254 patients with knee arthritis treated in our hospital from January 1, 2014, to June 1, 2015, among them, 217 patients met the inclusion criteria, 20 met the exclusion criteria, and a total of 197 patients were screened. Among the above 197 patients who met the inclusion and exclusion criteria, 8 patients who died due to extra-articular diseases, and 23 patients without follow-up information. Finally, 166 patients were included in this study. Our hospital’s surgical concept changed in 2014, and the patients were divided into two groups according to whether tourniquets were used during the operation. There were 80 cases in the tourniquet (T) group, including 31 males and 49 females, with an average age of 68.07 ± 6.75 years, and 86 cases in the non-tourniquet (NT) group, including 35 males and 51 females, with an average age of 68.41 ± 6.52 years. Before surgery, we evaluated the severity of knee arthritis by radiology using the Kellgren and Lawrence scores. Table shows the general information of the patients in the two groups.
The patient was placed in the supine position and the surgical field was routinely disinfected and covered with a cloth. In group T, a tourniquet (pressure 260 mmHg) was applied throughout the entire process, whereas no tourniquet was applied in Group NT. All patients were administered 1 g tranexamic acid intravenously before surgery. A longitudinal incision was made in the anterior median, starting at the proximal side of the patella (5–10 cm) and ending at the tibial tubercle. A deep incision was made along the medial side of the patella to expose the joint cavity. According to certain operating procedures, the femoral end osteotomy was performed first, followed by the posterior tibial end osteotomy; the lower limb vertical line was restored, and bone cement was fully mixed when required. Then, the tibia and femur prostheses were implanted, excess bone cement was removed after it had solidified, and a polyethylene liner was installed and rinsed with a washing gun. The tourniquet in the T group was loosened for careful electrocoagulation. In contrast, the NT group had already performed inadvertent electrocoagulation at the bleeding site intraoperatively. Indwelling drainage tube was placed in both groups. And then, all patients were subsequently sutured layer-by-layer, bandaged with gauze, cotton pads, and elastic bandages. The two groups had similar postoperative treatment methods, including swelling reduction, pain relief, anti-inflammatory therapy, thrombosis prevention, and other routine treatments. Patients were instructed to exercise the ankle pump and leg lift functions under guidance on the first-day post-surgery. On the second postoperative day, the wound dressing was changed, the wound drainage tube was removed, the dressing was bandaged, and the patient was instructed to perform knee joint and flexion function exercises, and walked with the help of a walker and then gradually transitioned to normal walking without walker.
We compared all patient’s perioperative data, including operation time, incision length, intraoperative blood loss, total blood loss, and length of hospital stay. Complications, such as wound infection, poor wound healing, and deep venous thrombosis (DVT) of the lower limbs, were recorded during follow-up. The hip knee ankle angle (HKA) and medial proximal tibial angle (MPTA) were measured before and after surgery. We measured the depth of bone cement penetration in each area of the postoperative radiographic prosthesis (Fig. ). We identified the radiolucent line between the osteotomy surface and the prosthesis using radiography at the last follow-up. Preoperative and postoperative HSS and knee range of motion (ROM) were recorded in both groups during follow-up.
Data were measured independently and averaged from three uninformed individuals. Statistical analysis was performed using the statistical package for social sciences 26.0, and measurement data were presented as (x̅ ± s). Normally distributed data was compared between both groups using an independent sample t -test, while counting data was compared using the χ 2 test or Fisher’s exact test. The grade data between both groups were compared using the Mann–Whitney U test. Statistical significance was set at P < 0.05.
General results and complications Compared with the T group, the amount of intraoperative blood loss increased significantly( P < 0.05), but the total blood loss did not differ significantly ( P > 0.05). In addition, There was no significant difference of the length of hospital stays and the operative time between two groups.There were no blood transfusions in the T group and NTgroup, and both groups did not differ significantly ( P = 1.000). No complications, such as DVT or pulmonary embolism, occurred in either group (Table ). Follow-up results All patients were followed up completely. With regard to cumulative incidence of revision surgery, reoperations and complications, each group had one case of aseptic loosening requiring revision surgery(1.25% vs 1.16%), and no cases of infection revision was reported in either group. The remaining patients did not have implant displacement, loosening, or bone-cement fractures. A total of 3 patients in group T(3.75%) and 4 patients in group NT(4.65%) had poor incision healing. All of these patients experienced delayed incision healing after antibiotic therapy, daily dressing changes, and local physical therapy. Postoperative imaging data indicated that HKA and MPTA differed slightly between both groups, but the difference was not statistically significant between the two groups. The pain in both groups was significantly improved compared with that before surgery, and no patients with no improvement or even aggravation of symptoms. No significant differences were observed in the preoperative and postoperative HSS scores or knee ROM between the two groups (Table ). At the last follow-up, we determined excellent and good outcomes based on the knee joint scores of both groups. In the T group, 75 cases were excellent, three were good, one was medium, and one was poor; the excellent and good rates were 97.5%. In the NT group, 81 cases were excellent, two were good, and three were medium; the excellent and good rates were 96.51%. See Table for further details. Cement penetration The depth of cement penetration measured at different points in different areas for both patient groups differed slightly, but the overall difference was not large. We observed statistically significant differences between the zone femur 3A observed in the lateral view (1.717 ± 0.7901 vs 1.985 ± 0.759, P = 0.027) and the average observation zone of the femur (1.646 ± 0.395 vs 1.788 ± 0.372, P = 0.018). However, they did not indicate that tourniquet use affected the infiltration of bone cement into the osteotomy surface. We speculate that this may have something to do with measurement errors. No significant difference was observed in the mean penetration depth of the bone cement between both groups ( P > 0.05) (Table ). Radiolucent lines The radiolucent lines (RLL) occurred mostly in zones tibial 1 and 2 (anteroposterior view) 、zones tibial 1 and 2 (lateral views) and in zones femur 1 and 2. In the lateral view between both groups, zone tibial 2 (35% vs. 15.12%, P = 0.003) and zone femur 1 (31.25% vs. 15.12%, P = 0.014) differed significantly, whereas there were no statistically significant differences in other areas ( P > 0.05). There was no statistically significant difference in the probability of progressive RLLs ( P > 0.05) (Table ).
Compared with the T group, the amount of intraoperative blood loss increased significantly( P < 0.05), but the total blood loss did not differ significantly ( P > 0.05). In addition, There was no significant difference of the length of hospital stays and the operative time between two groups.There were no blood transfusions in the T group and NTgroup, and both groups did not differ significantly ( P = 1.000). No complications, such as DVT or pulmonary embolism, occurred in either group (Table ).
All patients were followed up completely. With regard to cumulative incidence of revision surgery, reoperations and complications, each group had one case of aseptic loosening requiring revision surgery(1.25% vs 1.16%), and no cases of infection revision was reported in either group. The remaining patients did not have implant displacement, loosening, or bone-cement fractures. A total of 3 patients in group T(3.75%) and 4 patients in group NT(4.65%) had poor incision healing. All of these patients experienced delayed incision healing after antibiotic therapy, daily dressing changes, and local physical therapy. Postoperative imaging data indicated that HKA and MPTA differed slightly between both groups, but the difference was not statistically significant between the two groups. The pain in both groups was significantly improved compared with that before surgery, and no patients with no improvement or even aggravation of symptoms. No significant differences were observed in the preoperative and postoperative HSS scores or knee ROM between the two groups (Table ). At the last follow-up, we determined excellent and good outcomes based on the knee joint scores of both groups. In the T group, 75 cases were excellent, three were good, one was medium, and one was poor; the excellent and good rates were 97.5%. In the NT group, 81 cases were excellent, two were good, and three were medium; the excellent and good rates were 96.51%. See Table for further details.
The depth of cement penetration measured at different points in different areas for both patient groups differed slightly, but the overall difference was not large. We observed statistically significant differences between the zone femur 3A observed in the lateral view (1.717 ± 0.7901 vs 1.985 ± 0.759, P = 0.027) and the average observation zone of the femur (1.646 ± 0.395 vs 1.788 ± 0.372, P = 0.018). However, they did not indicate that tourniquet use affected the infiltration of bone cement into the osteotomy surface. We speculate that this may have something to do with measurement errors. No significant difference was observed in the mean penetration depth of the bone cement between both groups ( P > 0.05) (Table ).
The radiolucent lines (RLL) occurred mostly in zones tibial 1 and 2 (anteroposterior view) 、zones tibial 1 and 2 (lateral views) and in zones femur 1 and 2. In the lateral view between both groups, zone tibial 2 (35% vs. 15.12%, P = 0.003) and zone femur 1 (31.25% vs. 15.12%, P = 0.014) differed significantly, whereas there were no statistically significant differences in other areas ( P > 0.05). There was no statistically significant difference in the probability of progressive RLLs ( P > 0.05) (Table ).
Our study found that the use of tourniquet technique in TKA was similar to that of intraoperative tourniquet use in terms of prosthesis survival, reoperation, complications, knee function, and health status. With the popularity of ERAS, the use of tourniquets has decreased. According to relevant studies, the proportion of tourniquets used for knee arthroplasty has decreased from 90% in 2011 to 70% in 2014 . The tourniquet has certain benefits, but greater pressure causes compression injury to the thigh muscle, reduces the strength of the quadriceps muscle, aggravates postoperative pain, and delays postoperative rehabilitation . Regarding perioperative blood management of TKA, long-term tourniquet compression will lead to increased microvascular permeability, anoxia of vascular wall cells, slow blood flow, hypercoagulability aggravation, and increased probability of DVT . In addition, it is not conducive to the maintenance of intraoperative blood pressure, and the location of vascular injury prone to ischemia–reperfusion injury, cannot be detected immediately . Notably, some scholars have proposed that tourniquets reduce the amount of intraoperative overt blood loss but increase the amount of postoperative recessive blood loss. Zan et al. concluded that recessive blood loss after TKA exceeds 50% of the total blood loss , and is not dominant if the role of tourniquets in blood management is weighed using the total blood loss . Schnettler et al. showed that the total blood loss with tourniquet was 1215.34 ± 370.31 ml, while the total blood loss without tourniquet was 1007.22 ± 385.32 ml in their study, which was similar to our study results . In the NT group, the total blood loss was 1073.401 ± 226.452 ml. The total blood loss in the T group was 1054.275 ± 215.209 ml, and the difference was not statistically significant, indicating that the use of tourniquet in TKA did not affect the total blood loss.This was also confirmed by this study’s findings. Using tourniquets reduced the amount of intraoperative blood loss; however, the total amount did not differ significantly. As the operator and assistant become more skilled and the hemostatic operation becomes more mature, the amount of blood loss during the operation can be reduced and damage to the blood vessels and nerves can be avoided. Administering tranexamic acid intravenous drip before surgery has also been proven useful in numerous studies ; therefore, the use value of the tourniquet should be considered. Aseptic prosthesis loosening can be diagnosed by implant displacement, bone cement fracture, and continuous radio-clear bands with widths > 2 mm at the bone-cement interface . Currently, there is a lack of relevant literature confirming whether intraoperative tourniquets affect the medium- and long-term survival rates of prostheses. This is the first retrospective study with medium- and long-term follow-ups. TKA outcomes were evaluated primarily using Kaplan–Meier survival analysis with revision surgery as the endpoint. In contrast, short-term evaluation was based on the reconstructed lower limb contour and prosthesis placement quality . Joint arthroplasty can effectively reconstruct the vertical lines of the lower limbs. Theoretically, the deeper the bone cement penetrates, the better the prosthesis placement quality. Notably, some scholars have investigated whether tourniquets affect the penetration of bone cement into the bone trabeculae, but no consensus has been reached. We also discovered that some researchers' data did not truly reflect the depth of bone cement penetration in the knee prosthesis, and they measured the thickness of pure bone cement. When the bone-cement layer is too thick, stress occlusion is produced, and the local bone becomes loose, which is unconducive for long-term stability . Miller et al. discovered that a bone cement thickness of 3 mm achieved long-term stability of the prosthesis, whereas a thickness of < 1 mm affected the long-term survival rate of the prosthesis . Hegde et al. pointed tourniquet use improves cement penetration ; However, Andrade et al. showed that the use of tourniquets did not affect the penetration of bone cement . In this study, the anteroposterior and lateral radiographs of the knee after TKA showed that the depth of the bone cement penetration on the osteotomy surface was slightly lower in the NT group than in the T group. However, the difference was not statistically significant ( P > 0.05). RLL production is related to many factors, such as bone cement technology, bone cement type, type of prosthesis, and X-ray differences. Furthermore, we observed RLL occurrence at different points and found some differences, most of which were concentrated on the platform’s contact surface, where prosthesis loosening was most likely to occur. There were differences at some observation points, but they were not statistically significant ( P > 0.05). In addition, the revision rate between the two groups did not differ significantly ( P > 0.05), indicating that tourniquet use was not directly related to the prosthetic revision rate. Ozkunt et al. discovered that the tourniquet did not affect the stability of the prosthesis within 2 years post-TKA . Touzopoulos et al. tracked the occurrence of clear bands under a tibial prosthesis 3 years post-TKA, but there was no statistical difference in the stability of the prosthesis . Hoffmann et al. conducted a prospective study with an average of 5.3 years and concluded that using tourniquets in TKA had no significant differences in prosthesis survival, reoperation, complications, knee function, and health status . Xu conducted a 5–8-year retrospective study and discovered that using tourniquets increased the probability of RLL occurrence, but the revision rate did not differ significantly . In this study, measuring the depth of bone-cement penetration in different areas around the prosthesis and the occurrence of RLL during the medium- and long-term follow-ups, with an average of 8.8 years and the longest 10.25 years showed that tourniquet use did not affect the revision rate of both groups. This study’s limitation is that the optimal time for tourniquet use (full, half, or only bone cement use) and pressure values were not included in the data. In addition, radiography was not performed by the same physician, and the radiography quality seriously affected bone cement measurement. The posterior inclination of the tibial plateau and the standard body position at the time of radiography affected the measurement of bone cement in different areas. In summary, tourniquet use during TKA did not affect the medium- or long-term stability of knee prostheses. Improving perioperative bleeding control programs can greatly reduce the amount of intraoperative bleeding, tourniquet function can be completely replaced and tourniquet-related complications can be avoided.
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Epstein–Barr virus-associated smooth muscle tumor with multiple lesions in a patient with AIDS | 4178a60f-fd05-409f-8bbc-ee5c378f8e24 | 11784901 | Anatomy[mh] | We would like to express our sincere appreciation to Professor Zili Lv from the Pathology Department of the First Affiliated Hospital of Guangxi Medical University for her pathological support. This study was supported by the Guangxi Natural Science Foundation project (2024GXNSFAA010051 and 2024GXNSFAA010139). Contribution statement: concept and design – X.-M.Z., F.X., and B.-M.L. Acquisition, analysis, or interpretation of data – F.X. and X.-M.Z. Drafting manuscript: all authors. Conflicts of interest There are no conflicts of interest. There are no conflicts of interest. Supplemental Digital Content |
A dataset of single-cell transcriptomic atlas of Bama pig and potential marker genes across seven tissues | 20b8a028-634b-41eb-84e9-145ede99c4ad | 11899051 | Digestive System[mh] | The continual advancements and breakthroughs in transcriptomic technology [ – ] have allowed researchers to conduct in-depth studies at the transcriptomic level in Sus scrofa (pig or swine). The main aim is to discover intrinsic information, such as gene expression regulation mechanisms , immune responses , functional genes , and metabolic pathways . For example, one study completed transcriptome gene annotation across multiple species, including pigs . A highly integrated resource of transcriptomic features was provided by detailed analysis of the swine transcriptomic landscape, laying a solid foundation for research at the transcriptomic level in pigs . The rapid development of swine transcriptomic technology has significantly aided research on disease and improvements in breeding . Although traditional transcriptomic methods, such as bulk RNA sequencing, have significantly advanced the understanding of gene expression, they fail to capture cellular heterogeneity at the single-cell level . Single-cell RNA sequencing technologies have recently emerged as vital tools for investigating cellular heterogeneity and for single-cell research . Recently, a comprehensive account of the immense potential and challenges of single-cell technologies was provided . The feasibility of conducting systematic analyses and constructing comprehensive atlases at the single-cell, single-nucleus level even in tissues where endothelial cells constitute a minority has been demonstrated . Notably, during the global outbreak of African swine fever in 2018, virus-regulated signaling pathways were discovered via single-cell technologies, providing crucial scientific evidence for disease control strategies . Research on porcine single cells will profoundly impact the understanding of biological complexity and contribute to solving pressing issues such as disease . The Bama pig is a small pig breed that bears physiological, anatomical, nutritional, metabolic, and disease-related similarities to humans, making it extensively used in human disease research . Concurrently, extensive research has been conducted on Yucatan miniature pigs, particularly in cardiac , peripheral blood , colonic , and kidney pig tissues, via single-cell sequencing. However, the application of single-cell sequencing technology in transcriptomic studies of Bama pig remains relatively limited. Specifically, research into systematically defining unique marker genes in the visceral tissues of Bama pig is limited.
Animals and sample collection The research utilized an adult female purebred Chinese Bama pig (2 years old, 45 kg) provided by Hengshu Bio-Technology Co., Ltd., Yibin, Sichuan, China. The pig was maintained in a stably controlled laboratory environment, with the room temperature set at 25–28 °C and the humidity at 70%. The feed energy level required for animal maintenance was determined on the basis of the NRC (2012) and the Chinese Fatty Growing and Finishing Pig Feeding Standards (2004). After 12 h of fasting, the pig was placed in a restraint bag, and Zoletil ® 50 anesthetic was injected into the well-developed neck muscles using a 14-gauge needle at a dose of 5 mg/kg. Subsequently, the pig was slaughtered, and samples from seven organs or tissues, namely, the liver, spleen, lung, kidney, psoas major muscle (PMM), subcutaneous adipose tissue of the back (SAT), and greater omentum (GOM), were collected. Tissue preparation for single-cell (scRNA-seq) libraries (liver, spleen, lung, kidney): Tissues intended for single-cell RNA sequencing library generation were obtained from a local slaughterhouse. Freshly collected tissues were immediately placed on ice and processed within 30 min. Each type of tissue was dissociated and digested separately. To ensure effective digestion and maintain cell viability, after tissue dissociation, the cell suspensions were passed through a cell strainer to remove debris and lyse red blood cells. Cell viability for each tissue type was assessed at the core facility of Novogene Co., Ltd. (Beijing, China) via a flow cytometer (Acea Bioscience, Inc., US), which employs Hoechst 33,342 (Invitrogen, Cat# H3570) and propidium iodide (Thermo Fisher Scientific, Cat# P3566), with a viability threshold of over 80% required for subsequent sequencing analyses. Liver Fresh livers were sampled from five distinct anatomical regions: the left lateral lobe (LLL), left medial lobe (LML), right medial lobe (RML), right lateral lobe (RLL), and quadrate lobe (QL). Each region provided 1 g of tissue, which was washed twice with cold PBS. The tissues were mixed, chopped into small pieces, and then transferred to a 50 mL tube, to which 20 mL of digestion solution was added. This mixture contained 0.5 mg/mL collagenase type II (Gibco, Cat#17101015), 1.25 mg/mL protease (Sigma, Cat#P5147–100MG), and 7.5 µg/mL DNase I (Sigma‒Aldrich, Cat#D4527–10KU) in cold HBSS. The digestion was carried out at 37 °C for 15 min, with gentle shaking every 5 min. The reaction was terminated in cold MACS buffer containing 0.25% BSA (Sigma‒Aldrich, Cat# 10735096001) and 2 mM EDTA, and the sample was filtered through a 100 μm cell strainer (Sigma‒Aldrich, Cat# CLS431752-50EA), followed by flow cytometric cell staining. Spleen Fresh spleens were sampled from two anatomical sides, the visceral and parietal sides, with 1 g of tissue taken from each, and washed twice with cold PBS. The tissues were mixed, chopped into small pieces, and transferred to a 50 mL tube, where 10 mL of digestion solution was added. This mixture contained 20 mg/mL collagenase type IV (Gibco, Cat# 17104019), 1 U/mL dispase II (Gibco, Cat# 17105041), and 7.5 µg/mL DNase I in 10 mL of HBSS. The digestion was carried out at 37 °C for 15 min, with gentle shaking every 5 min. The reaction was terminated in cold MACS buffer containing 0.25% BSA and 2 mM EDTA, and the sample was sequentially filtered through 100 μm and 40 μm cell strainers, followed by flow cytometric cell staining. Lung Fresh lungs were sampled from seven different areas: the left apex, left medial, left main, right apex, right medial, accessory, and right main lobes. Each area provided 0.5 g of tissue, which was subsequently washed twice with cold HBSS. The tissues were chopped into small pieces and transferred to a 50 mL Falcon tube, to which 20 mL of digestion solution was added, containing 1 mg/mL collagenase type II, 2.5 mg/mL collagenase type IV, and 7.5 µg/mL DNase I. The digestion was conducted at 37 °C for 30 min, with gentle shaking every 5 min, and terminated in MACS buffer. The sample was diluted in cold HBSS and filtered through 100 μm and 40 μm cell strainers, followed by debris removal and flow cytometric cell staining. Kidney Fresh kidneys were sampled from four areas: the upper pole, lower pole, cortex, and medulla. Each area provided 1 g of tissue, which was subsequently washed twice with cold PBS. The tissues were chopped into small pieces and transferred to a 50 mL Falcon tube, to which 20 mL of digestion solution containing 1 mg/mL collagenase type II (Gibco, Cat# 17101015), 2 mg/mL collagenase type IV (Gibco, Cat# 17104019), 1 U/mL dispase II (Gibco, Cat# 17105041), and 7.5 µg/mL DNase I (Sigma‒Aldrich, Cat# D4527–10KU) was added. The digestion was conducted at 37 °C for 20 min, with gentle shaking every 5 min. The reaction was terminated in 20 mL of cold MACS buffer containing 0.25% BSA (Sigma‒Aldrich, Cat# 10735096001) and 2 mM EDTA, and the sample was filtered through 100 μm and 40 μm cell strainers, followed by flow cytometric cell staining. Sample collection for single-nucleus sequencing (snRNA-seq) The tissues used for single-nucleus sampling included visceral adipose (greater omentum fat, GOM), subcutaneous fat (SAT), and psoas major muscle (PMM) from a Bama pig, which were meticulously dissected while adhering to ethical guidelines. The collected tissues were washed with cold PBS, immediately frozen in liquid nitrogen, and stored at −80 °C until use. For nuclear extraction, the tissues were thawed, cut into small pieces, and transferred to homogenization buffer containing 20 mM Tris pH 8.0, 500 mM sucrose, 0.1% NP-40, 0.2 U/mL RNase inhibitor, 1% BSA, and 0.1 mM DTT. The tissues were homogenized via a pestle 15 times and filtered through a 40 μm strainer. The samples were then centrifuged at 4 °C for 10 min at 500×g, and the supernatant was carefully discarded. The pellet (nuclei) was resuspended in PBS containing 1% BSA and 20 U/µL RNase inhibitor and prepared for subsequent snRNA-seq library construction. 10× genomics library preparation and sequencing The samples were subsequently transported on dry ice to Novogene Co., Ltd. (Beijing, China). where the cDNA libraries were constructed and sequenced. The 10× Genomics Chromium single-cell 3′ gene expression solution was used. Among the seven data samples collected under the designated conditions, four datasets were from single-cell sequencing, and the other three datasets were from single-nucleus sequencing. All the experimental procedures were conducted in accordance with the manufacturer’s protocol ( www.10xgenomics.com/support/single-cell-gene-expression ). The quality control criteria for the preparation of single-cell suspension samples were a cell viability > 80%, a cell concentration of 700–1200 cells/µL, and a cell diameter of 5–30 μm. The cell nuclei were extracted from the PMM, SAT, and GOM samples at the time of sample preparation (10× Chromium Nuclei Isolation Kit) because of their excessively large cell diameters. Qualified cell and cell nuclei suspensions were loaded onto 10× Genomics Single-Cell 3.0 Chips. During this step, the cells were partitioned into gel beads-in-emulsion (GEMs) along with gel beads coated with 10× barcode oligonucleotides (including the 14-bp index and 10-bp UMIs (unique molecular identifiers)). After generating the GEMs, the samples were transferred into PCR tubes, and reverse transcription was performed via a T100 Thermal Cycler (Bio-Rad). cDNAs with both barcodes were amplified, and libraries were constructed via a single-cell 3′ Reagent Kit (v3) for each sample. The resulting libraries were sequenced on an Illumina NovaSeq 6000 System in PE150 mode. Cell demultiplexing and gene counting The raw sequencing data were used directly for sequence quality control and gene quantification via CellRanger (v7.1.0, 10× Genomics, using default parameters). The reference genome assembly was downloaded from Ensembl in FASTA format (Sscrofa11.1, GCA_000003025.6) together with the gene annotation GTF file (release 109). The cell metadata, which include barcodes.tsv, features.tsv, and gene expression matrix (*.mtx) files, were automatically generated via CellRanger. The initial data are available in additional files (Supplementary Table ). Quality control of cells and genes We used R software (version 4.2.3, https://www.r-project.org/ ) and the Seurat R package (version 4.4.0, https://satijalab.org/seurat/ ) for the downstream analyses. Initial mitochondrial quantification was conducted, and data quality control was performed according to the following criteria: genes expressed in < 10 cells were excluded; genes with exceedingly low or high overall expression (nFeature_RNA < 200, nFeature_RNA > 5000) were filtered out; and in single-nucleus sequencing tissues (SAT, GOM, and PMM), cells with mitochondrial gene percentages greater than 10% were eliminated. In single-cell tissues (lung, kidney, and spleen), cells with mitochondrial gene percentages greater than 30% were eliminated. In single-cell tissues (liver), cells with mitochondrial gene percentages greater than 50% were eliminated. The R package DoubletFinder (github.com/chris-mcginnis-ucsf/DoubletFinder) was subsequently used to remove doublet cells (DoubletRate = 0.075). Data normalization and cell clustering For the refined dataset, gene expression count information for each sample was normalized via the `NormalizeData` function, followed by feature selection of 3,000 variable genes via the `FindVariableFeatures` function with the “vst” method. The data were then scaled via the `ScaleData` function . Principal component analysis (PCA) and UMAP projection were executed through the `RunPCA` and `RunUMAP` functions, respectively. For different tissues, we used either 30 (single-nucleus datasets) or 20 (single-cell datasets) principal components as determined by the inflection point of the elbow plot for each tissue. Cell clusters were identified via the `FindClusters` function and visualized via UMAP. For the accurate determination of tissue-specific resolution sizes for each dataset, clustree software was employed. Specifically, the resolutions were set as follows: 0.8 for the liver, 0.2 for the spleen, 0.2 for the lung, 0.1 for the kidney, 0.5 for the PMM, 0.5 for the SAT, and 0.2 for the GOM. Differentially expressed genes and annotation To identify differentially expressed genes (DEGs) across different cell clusters, we employed the FindAllMarkers function from the Seurat package, utilizing the Wilcoxon rank sum test. This function was configured to detect only genes positively marking clusters, with a minimum percentage of expressing cells (min.pct) set at 25% and a log fold change (logfc.threshold) threshold of 0.25. We subsequently filtered these DEGs to include only those with an adjusted p value less than 0.05. After all the DEGs were identified, the cell types were manually annotated by referencing tissue-specific marker genes that have been documented in previously published studies [ , , – ]. These known marker genes are listed in additional files (Supplementary Table ), while the most typical marker genes used in our annotations are displayed in additional files ( ). Predicting marker genes On the basis of the differentially expressed genes (DEGs) identified, we annotated cell types using known marker genes. To uncover more potential marker genes for each cell type across the seven tissues, we further refined the criteria for gene selection: the gene’s pct1 value must exceed 0.7; avg_log2FC must be greater than 0.5; and the gene must exhibit pronounced expression specificity, using the percentage of the gene’s relative expression as one of the standards, such as a gene accounting for more than 70% of its total expression in a specific cell type. These criteria increase their potential as cell type-specific markers. Data integration We utilized the ‘merge’ function to integrate the postquality control single-cell datasets (liver, spleen, lung, and kidney) and single-nucleus datasets (PMM, SAT, and GOM) independently. We used Harmony , a method that relies on multidimensional scaling techniques, for the separate integration of single-cell and single-nucleus datasets. Harmony eliminates batch effects caused by technical and biological variations by harmonizing high-dimensional similarities between cells. Thus, Harmony addresses not only technical factors such as library preparation but also biological variations between cell subpopulations .
The research utilized an adult female purebred Chinese Bama pig (2 years old, 45 kg) provided by Hengshu Bio-Technology Co., Ltd., Yibin, Sichuan, China. The pig was maintained in a stably controlled laboratory environment, with the room temperature set at 25–28 °C and the humidity at 70%. The feed energy level required for animal maintenance was determined on the basis of the NRC (2012) and the Chinese Fatty Growing and Finishing Pig Feeding Standards (2004). After 12 h of fasting, the pig was placed in a restraint bag, and Zoletil ® 50 anesthetic was injected into the well-developed neck muscles using a 14-gauge needle at a dose of 5 mg/kg. Subsequently, the pig was slaughtered, and samples from seven organs or tissues, namely, the liver, spleen, lung, kidney, psoas major muscle (PMM), subcutaneous adipose tissue of the back (SAT), and greater omentum (GOM), were collected. Tissue preparation for single-cell (scRNA-seq) libraries (liver, spleen, lung, kidney): Tissues intended for single-cell RNA sequencing library generation were obtained from a local slaughterhouse. Freshly collected tissues were immediately placed on ice and processed within 30 min. Each type of tissue was dissociated and digested separately. To ensure effective digestion and maintain cell viability, after tissue dissociation, the cell suspensions were passed through a cell strainer to remove debris and lyse red blood cells. Cell viability for each tissue type was assessed at the core facility of Novogene Co., Ltd. (Beijing, China) via a flow cytometer (Acea Bioscience, Inc., US), which employs Hoechst 33,342 (Invitrogen, Cat# H3570) and propidium iodide (Thermo Fisher Scientific, Cat# P3566), with a viability threshold of over 80% required for subsequent sequencing analyses. Liver Fresh livers were sampled from five distinct anatomical regions: the left lateral lobe (LLL), left medial lobe (LML), right medial lobe (RML), right lateral lobe (RLL), and quadrate lobe (QL). Each region provided 1 g of tissue, which was washed twice with cold PBS. The tissues were mixed, chopped into small pieces, and then transferred to a 50 mL tube, to which 20 mL of digestion solution was added. This mixture contained 0.5 mg/mL collagenase type II (Gibco, Cat#17101015), 1.25 mg/mL protease (Sigma, Cat#P5147–100MG), and 7.5 µg/mL DNase I (Sigma‒Aldrich, Cat#D4527–10KU) in cold HBSS. The digestion was carried out at 37 °C for 15 min, with gentle shaking every 5 min. The reaction was terminated in cold MACS buffer containing 0.25% BSA (Sigma‒Aldrich, Cat# 10735096001) and 2 mM EDTA, and the sample was filtered through a 100 μm cell strainer (Sigma‒Aldrich, Cat# CLS431752-50EA), followed by flow cytometric cell staining. Spleen Fresh spleens were sampled from two anatomical sides, the visceral and parietal sides, with 1 g of tissue taken from each, and washed twice with cold PBS. The tissues were mixed, chopped into small pieces, and transferred to a 50 mL tube, where 10 mL of digestion solution was added. This mixture contained 20 mg/mL collagenase type IV (Gibco, Cat# 17104019), 1 U/mL dispase II (Gibco, Cat# 17105041), and 7.5 µg/mL DNase I in 10 mL of HBSS. The digestion was carried out at 37 °C for 15 min, with gentle shaking every 5 min. The reaction was terminated in cold MACS buffer containing 0.25% BSA and 2 mM EDTA, and the sample was sequentially filtered through 100 μm and 40 μm cell strainers, followed by flow cytometric cell staining. Lung Fresh lungs were sampled from seven different areas: the left apex, left medial, left main, right apex, right medial, accessory, and right main lobes. Each area provided 0.5 g of tissue, which was subsequently washed twice with cold HBSS. The tissues were chopped into small pieces and transferred to a 50 mL Falcon tube, to which 20 mL of digestion solution was added, containing 1 mg/mL collagenase type II, 2.5 mg/mL collagenase type IV, and 7.5 µg/mL DNase I. The digestion was conducted at 37 °C for 30 min, with gentle shaking every 5 min, and terminated in MACS buffer. The sample was diluted in cold HBSS and filtered through 100 μm and 40 μm cell strainers, followed by debris removal and flow cytometric cell staining. Kidney Fresh kidneys were sampled from four areas: the upper pole, lower pole, cortex, and medulla. Each area provided 1 g of tissue, which was subsequently washed twice with cold PBS. The tissues were chopped into small pieces and transferred to a 50 mL Falcon tube, to which 20 mL of digestion solution containing 1 mg/mL collagenase type II (Gibco, Cat# 17101015), 2 mg/mL collagenase type IV (Gibco, Cat# 17104019), 1 U/mL dispase II (Gibco, Cat# 17105041), and 7.5 µg/mL DNase I (Sigma‒Aldrich, Cat# D4527–10KU) was added. The digestion was conducted at 37 °C for 20 min, with gentle shaking every 5 min. The reaction was terminated in 20 mL of cold MACS buffer containing 0.25% BSA (Sigma‒Aldrich, Cat# 10735096001) and 2 mM EDTA, and the sample was filtered through 100 μm and 40 μm cell strainers, followed by flow cytometric cell staining. Sample collection for single-nucleus sequencing (snRNA-seq) The tissues used for single-nucleus sampling included visceral adipose (greater omentum fat, GOM), subcutaneous fat (SAT), and psoas major muscle (PMM) from a Bama pig, which were meticulously dissected while adhering to ethical guidelines. The collected tissues were washed with cold PBS, immediately frozen in liquid nitrogen, and stored at −80 °C until use. For nuclear extraction, the tissues were thawed, cut into small pieces, and transferred to homogenization buffer containing 20 mM Tris pH 8.0, 500 mM sucrose, 0.1% NP-40, 0.2 U/mL RNase inhibitor, 1% BSA, and 0.1 mM DTT. The tissues were homogenized via a pestle 15 times and filtered through a 40 μm strainer. The samples were then centrifuged at 4 °C for 10 min at 500×g, and the supernatant was carefully discarded. The pellet (nuclei) was resuspended in PBS containing 1% BSA and 20 U/µL RNase inhibitor and prepared for subsequent snRNA-seq library construction.
Fresh livers were sampled from five distinct anatomical regions: the left lateral lobe (LLL), left medial lobe (LML), right medial lobe (RML), right lateral lobe (RLL), and quadrate lobe (QL). Each region provided 1 g of tissue, which was washed twice with cold PBS. The tissues were mixed, chopped into small pieces, and then transferred to a 50 mL tube, to which 20 mL of digestion solution was added. This mixture contained 0.5 mg/mL collagenase type II (Gibco, Cat#17101015), 1.25 mg/mL protease (Sigma, Cat#P5147–100MG), and 7.5 µg/mL DNase I (Sigma‒Aldrich, Cat#D4527–10KU) in cold HBSS. The digestion was carried out at 37 °C for 15 min, with gentle shaking every 5 min. The reaction was terminated in cold MACS buffer containing 0.25% BSA (Sigma‒Aldrich, Cat# 10735096001) and 2 mM EDTA, and the sample was filtered through a 100 μm cell strainer (Sigma‒Aldrich, Cat# CLS431752-50EA), followed by flow cytometric cell staining.
Fresh spleens were sampled from two anatomical sides, the visceral and parietal sides, with 1 g of tissue taken from each, and washed twice with cold PBS. The tissues were mixed, chopped into small pieces, and transferred to a 50 mL tube, where 10 mL of digestion solution was added. This mixture contained 20 mg/mL collagenase type IV (Gibco, Cat# 17104019), 1 U/mL dispase II (Gibco, Cat# 17105041), and 7.5 µg/mL DNase I in 10 mL of HBSS. The digestion was carried out at 37 °C for 15 min, with gentle shaking every 5 min. The reaction was terminated in cold MACS buffer containing 0.25% BSA and 2 mM EDTA, and the sample was sequentially filtered through 100 μm and 40 μm cell strainers, followed by flow cytometric cell staining.
Fresh lungs were sampled from seven different areas: the left apex, left medial, left main, right apex, right medial, accessory, and right main lobes. Each area provided 0.5 g of tissue, which was subsequently washed twice with cold HBSS. The tissues were chopped into small pieces and transferred to a 50 mL Falcon tube, to which 20 mL of digestion solution was added, containing 1 mg/mL collagenase type II, 2.5 mg/mL collagenase type IV, and 7.5 µg/mL DNase I. The digestion was conducted at 37 °C for 30 min, with gentle shaking every 5 min, and terminated in MACS buffer. The sample was diluted in cold HBSS and filtered through 100 μm and 40 μm cell strainers, followed by debris removal and flow cytometric cell staining.
Fresh kidneys were sampled from four areas: the upper pole, lower pole, cortex, and medulla. Each area provided 1 g of tissue, which was subsequently washed twice with cold PBS. The tissues were chopped into small pieces and transferred to a 50 mL Falcon tube, to which 20 mL of digestion solution containing 1 mg/mL collagenase type II (Gibco, Cat# 17101015), 2 mg/mL collagenase type IV (Gibco, Cat# 17104019), 1 U/mL dispase II (Gibco, Cat# 17105041), and 7.5 µg/mL DNase I (Sigma‒Aldrich, Cat# D4527–10KU) was added. The digestion was conducted at 37 °C for 20 min, with gentle shaking every 5 min. The reaction was terminated in 20 mL of cold MACS buffer containing 0.25% BSA (Sigma‒Aldrich, Cat# 10735096001) and 2 mM EDTA, and the sample was filtered through 100 μm and 40 μm cell strainers, followed by flow cytometric cell staining.
The tissues used for single-nucleus sampling included visceral adipose (greater omentum fat, GOM), subcutaneous fat (SAT), and psoas major muscle (PMM) from a Bama pig, which were meticulously dissected while adhering to ethical guidelines. The collected tissues were washed with cold PBS, immediately frozen in liquid nitrogen, and stored at −80 °C until use. For nuclear extraction, the tissues were thawed, cut into small pieces, and transferred to homogenization buffer containing 20 mM Tris pH 8.0, 500 mM sucrose, 0.1% NP-40, 0.2 U/mL RNase inhibitor, 1% BSA, and 0.1 mM DTT. The tissues were homogenized via a pestle 15 times and filtered through a 40 μm strainer. The samples were then centrifuged at 4 °C for 10 min at 500×g, and the supernatant was carefully discarded. The pellet (nuclei) was resuspended in PBS containing 1% BSA and 20 U/µL RNase inhibitor and prepared for subsequent snRNA-seq library construction.
The samples were subsequently transported on dry ice to Novogene Co., Ltd. (Beijing, China). where the cDNA libraries were constructed and sequenced. The 10× Genomics Chromium single-cell 3′ gene expression solution was used. Among the seven data samples collected under the designated conditions, four datasets were from single-cell sequencing, and the other three datasets were from single-nucleus sequencing. All the experimental procedures were conducted in accordance with the manufacturer’s protocol ( www.10xgenomics.com/support/single-cell-gene-expression ). The quality control criteria for the preparation of single-cell suspension samples were a cell viability > 80%, a cell concentration of 700–1200 cells/µL, and a cell diameter of 5–30 μm. The cell nuclei were extracted from the PMM, SAT, and GOM samples at the time of sample preparation (10× Chromium Nuclei Isolation Kit) because of their excessively large cell diameters. Qualified cell and cell nuclei suspensions were loaded onto 10× Genomics Single-Cell 3.0 Chips. During this step, the cells were partitioned into gel beads-in-emulsion (GEMs) along with gel beads coated with 10× barcode oligonucleotides (including the 14-bp index and 10-bp UMIs (unique molecular identifiers)). After generating the GEMs, the samples were transferred into PCR tubes, and reverse transcription was performed via a T100 Thermal Cycler (Bio-Rad). cDNAs with both barcodes were amplified, and libraries were constructed via a single-cell 3′ Reagent Kit (v3) for each sample. The resulting libraries were sequenced on an Illumina NovaSeq 6000 System in PE150 mode.
The raw sequencing data were used directly for sequence quality control and gene quantification via CellRanger (v7.1.0, 10× Genomics, using default parameters). The reference genome assembly was downloaded from Ensembl in FASTA format (Sscrofa11.1, GCA_000003025.6) together with the gene annotation GTF file (release 109). The cell metadata, which include barcodes.tsv, features.tsv, and gene expression matrix (*.mtx) files, were automatically generated via CellRanger. The initial data are available in additional files (Supplementary Table ).
We used R software (version 4.2.3, https://www.r-project.org/ ) and the Seurat R package (version 4.4.0, https://satijalab.org/seurat/ ) for the downstream analyses. Initial mitochondrial quantification was conducted, and data quality control was performed according to the following criteria: genes expressed in < 10 cells were excluded; genes with exceedingly low or high overall expression (nFeature_RNA < 200, nFeature_RNA > 5000) were filtered out; and in single-nucleus sequencing tissues (SAT, GOM, and PMM), cells with mitochondrial gene percentages greater than 10% were eliminated. In single-cell tissues (lung, kidney, and spleen), cells with mitochondrial gene percentages greater than 30% were eliminated. In single-cell tissues (liver), cells with mitochondrial gene percentages greater than 50% were eliminated. The R package DoubletFinder (github.com/chris-mcginnis-ucsf/DoubletFinder) was subsequently used to remove doublet cells (DoubletRate = 0.075).
For the refined dataset, gene expression count information for each sample was normalized via the `NormalizeData` function, followed by feature selection of 3,000 variable genes via the `FindVariableFeatures` function with the “vst” method. The data were then scaled via the `ScaleData` function . Principal component analysis (PCA) and UMAP projection were executed through the `RunPCA` and `RunUMAP` functions, respectively. For different tissues, we used either 30 (single-nucleus datasets) or 20 (single-cell datasets) principal components as determined by the inflection point of the elbow plot for each tissue. Cell clusters were identified via the `FindClusters` function and visualized via UMAP. For the accurate determination of tissue-specific resolution sizes for each dataset, clustree software was employed. Specifically, the resolutions were set as follows: 0.8 for the liver, 0.2 for the spleen, 0.2 for the lung, 0.1 for the kidney, 0.5 for the PMM, 0.5 for the SAT, and 0.2 for the GOM.
To identify differentially expressed genes (DEGs) across different cell clusters, we employed the FindAllMarkers function from the Seurat package, utilizing the Wilcoxon rank sum test. This function was configured to detect only genes positively marking clusters, with a minimum percentage of expressing cells (min.pct) set at 25% and a log fold change (logfc.threshold) threshold of 0.25. We subsequently filtered these DEGs to include only those with an adjusted p value less than 0.05. After all the DEGs were identified, the cell types were manually annotated by referencing tissue-specific marker genes that have been documented in previously published studies [ , , – ]. These known marker genes are listed in additional files (Supplementary Table ), while the most typical marker genes used in our annotations are displayed in additional files ( ).
On the basis of the differentially expressed genes (DEGs) identified, we annotated cell types using known marker genes. To uncover more potential marker genes for each cell type across the seven tissues, we further refined the criteria for gene selection: the gene’s pct1 value must exceed 0.7; avg_log2FC must be greater than 0.5; and the gene must exhibit pronounced expression specificity, using the percentage of the gene’s relative expression as one of the standards, such as a gene accounting for more than 70% of its total expression in a specific cell type. These criteria increase their potential as cell type-specific markers.
We utilized the ‘merge’ function to integrate the postquality control single-cell datasets (liver, spleen, lung, and kidney) and single-nucleus datasets (PMM, SAT, and GOM) independently. We used Harmony , a method that relies on multidimensional scaling techniques, for the separate integration of single-cell and single-nucleus datasets. Harmony eliminates batch effects caused by technical and biological variations by harmonizing high-dimensional similarities between cells. Thus, Harmony addresses not only technical factors such as library preparation but also biological variations between cell subpopulations .
Experimental workflow and initial data quality We collected samples from seven different tissues and organs of Bama pig, including the liver, spleen, lung, kidney, psoas major muscle, subcutaneous back fat, and omental fat. These tissue samples were immediately refrigerated upon collection and underwent standardized cellular dissociation and digestion processes to ensure cell viability. The samples were subsequently sent to the laboratory for single-cell library construction and high-throughput sequencing. Using the 10x Genomics platform and CellRanger software for data processing, we achieved detailed cell typing and annotation for both whole and individual tissues. Batch effects were corrected via the Harmony algorithm during integrated analysis, ensuring data consistency and reliability (Fig. a). The proportion of reads uniquely mapped to the genome was above 58%, with the average number of reads per cell ranging from 56,339 to 184,315. Analysis via CellRanger revealed that the median gene expression per cell ranged from 1,000 to 2,696. The average sequencing saturation for the Bama pig samples was 77.10%, demonstrating high data utility. Furthermore, CellRanger analysis indicated that the number of viable cells typically met or exceeded the anticipated capture of 3,000 cells (Fig. b). The detailed raw data, such as the Q30 values for UMI sequences across all seven tissues exceeding 90%, are reported in additional files (Supplementary Table ). Overall, these data highlight the high-quality sequencing depth and excellent coverage of the Bama pig reference genome, providing a solid basis for further analyses. The raw single-nucleus sequencing data (SAT, GOM, and PMM) represent RNA found only within cell nuclei. RNA from mitochondrial (percent.mt) genes is generally absent in nucleus-only sequencing data; hence, the percentage of mt is typically lower in snRNA-seq data. Moreover, since single-nucleus sequencing captures only nascent RNA in nuclei, the quantity of RNA (nCount_RNA) is also expected to be lower than that of scRNA-seq methods, with correspondingly lower numbers of genes and reads (nFeature_RNA) per cell sequenced (Fig. c). Tissue-specific cell type classification To independently verify the reliability of each dataset and identify tissue-specific cell types or subtypes, we conducted separate analyses through cell annotation and classification for each dataset. After CellRanger processed the raw data output, we used Seurat to construct single-cell matrices and then performed quality control on each dataset. As previously mentioned, the percentage of mitochondrial genes is used as a proxy for cell death, but mitochondrial gene RNA is generally absent in nuclear data; therefore, the percent.mt is lower in single-nucleus sequencing data. Notably, Hepatocytes may have very high mitochondrial content ; hence, a threshold of 50% was set (liver, percent.mt < 50) to optimally preserve hepatocytes and remove dead or dying cells. In single-cell microfluidics processes, droplets containing two or more cells can occur, which are then mistakenly identified as single cells. To further remove cells with abnormal gene expression, we employed DoubletFinder , eliminating doublets. Finally, each cluster’s identity was assigned on the basis of established cell-specific marker gene expression. As expected, each tissue exhibited specific cell types; subcutaneous back fat (SAT) and omental fat (GOM) uniquely featured “fibro-/adipogenic progenitors” and “adipocytes”. The psoas major muscle (PMM) showed skeletal muscle fiber cells, “I myofiber”, “II myofiber 2B”, and “II myofiber 2X”. The spleen contains “dendritic cells”, which are crucial for initiating immune responses, particularly in antigen presentation . The lung features “alveolar type 1 cells”, which cover the surfaces of the alveoli for gas exchange, and “alveolar type 2 cells”, which are responsible for secreting surfactants to prevent alveolar collapse . The kidney displays “proximal tubule cells”, which are crucial for filtering waste from the blood and absorbing water and nutrients . Liver tissue includes “Hepatocytes”, which are associated with the metabolism of proteins, carbohydrates, and fats , and specialized immune cells in the liver, “Kupffer cells” (Fig. ). Integrative analysis results of the ScRNA-seq data After individually assessing and confirming the high quality of each tissue dataset, which featured a diverse array of cell types and tissue-specific cells, we found that integrative analysis was feasible. Batch effects between different datasets can compromise the authenticity of comparisons, even when the same sequencing technologies are used. To standardize comparisons, we merged the postquality control scRNA-seq samples from the liver, spleen, lung, and kidney and conducted dimensionality reduction clustering (resolution = 0.8). This approach effectively mitigated batch effects, yielding a consolidated dataset of 39,406 cells and 24,686 genes for comprehensive annotation and comparison. Typically, cells with more total RNA molecules also exhibit more diverse gene detection. The correlation coefficient between nCount_RNA (total RNA molecules in cells) and nFeature_RNA (number of distinct genes detected per cell) increased from 0.83 to 0.9 after quality control (Fig. a), indicating a significant increase in the quality of the subsequent datasets. After correction for batch effects, the integration of similar cell types across different tissues was significantly enhanced (Fig. b). In this integrated analysis, using a curated set of cell-specific marker genes (Fig. e), we annotated 12 cell types from 33 cell clusters (Fig. c). In addition to identifying most cell types previously detected in individual analyses (Fig. d), we also discovered four established marker genes common across tissues, namely, CRTAM , GNLY , IL7R , and KLRB , for identifying T cells in all four tissues. In terms of cell abundance, the spleen, which is predominantly composed of immune cells, represented 96.74%, whereas the kidney was characterized mainly by tubule cells. The liver and lung presented a more complex assortment of cell types. Integrative analysis results of the SnRNA-seq data Similarly, to compare snRNA-seq data under the same standard, snRNA-seq samples (SAT, GOM, and PMM) were merged and subjected to dimensionality reduction clustering (resolution = 0.4), resulting in a total of 20,090 nuclei and 22,991 genes after batch effect removal. The correlation coefficient between nCount_RNA (total RNA molecules in nuclei) and nFeature_RNA (number of distinct genes detected per nucleus) increased from 0.9 to 0.94 after quality control (Fig. a). Compared with the scRNA-seq dataset, the snRNA-seq dataset presented more pronounced batch effects (Fig. b), and the cells became more distinctly separated, with similar cell types clustering more closely after batch effect correction. In the snRNA-seq dataset, 12 cell types were annotated from 21 cell clusters via a curated set of cell-specific marker genes (Fig. e, c). In terms of cell abundance, each tissue presented unique cell types with high cell type abundance (Fig. d). Overall, the data integration highlights the reliability of the data, not only providing a comparative abundance of cell types under a unified standard but also serving as a reference for future data integration and other research endeavors. Potential marker genes In single-cell transcriptomics, high-quality data are often closely associated with gene quality. To confirm the high quality of our data, we used a verification procedure that involved the careful selection of 0–5 potential marker genes for each single-cell type to assess their predictive power. Using the filtering criteria in our methodology, we found that the genes summarized in our I additional files. (Supplementary Table ) clearly exhibited strong marker gene representation. Remarkably, our results show robust concordance between the functions of the predicted annotated genes and the original annotations of the cell types, confirming a high level of correspondence. Together, the results highlight the effectiveness of these genes as marker genes, which aligns perfectly with our expectations regardless of their specificity or expression levels. In summary, within a rigorous quality control framework, including meticulous batch effect removal, the final set of high-quality genes and cells provides additional potential marker genes for a thorough exploration of distinct cell types in miniature pig (Bama pig), offering resources for studying the heterogeneity between different tissues or organs at the transcriptomic level. One limitation of this study is the lack of replicates. Although we have collected rich single-cell transcriptomic data from seven different tissues and organs, and ensured high data quality through strict quality control steps, the absence of replicates means that our results may be influenced by individual variations or sample processing inconsistencies. Additionally, the study is based solely on single-cell transcriptomic data from Bama pigs, so it would be beneficial to integrate and compare data from other similar or different pig breeds to further validate and expand the identified marker genes and cell types. Furthermore, while we have identified several potential marker genes, these still require experimental validation to confirm their biological significance across different tissues and cell types. Therefore, future studies should consider incorporating replicates, integrating data from different pig breeds or experimental conditions to enhance the generalizability and robustness of the findings, and further validating the potential marker genes.
We collected samples from seven different tissues and organs of Bama pig, including the liver, spleen, lung, kidney, psoas major muscle, subcutaneous back fat, and omental fat. These tissue samples were immediately refrigerated upon collection and underwent standardized cellular dissociation and digestion processes to ensure cell viability. The samples were subsequently sent to the laboratory for single-cell library construction and high-throughput sequencing. Using the 10x Genomics platform and CellRanger software for data processing, we achieved detailed cell typing and annotation for both whole and individual tissues. Batch effects were corrected via the Harmony algorithm during integrated analysis, ensuring data consistency and reliability (Fig. a). The proportion of reads uniquely mapped to the genome was above 58%, with the average number of reads per cell ranging from 56,339 to 184,315. Analysis via CellRanger revealed that the median gene expression per cell ranged from 1,000 to 2,696. The average sequencing saturation for the Bama pig samples was 77.10%, demonstrating high data utility. Furthermore, CellRanger analysis indicated that the number of viable cells typically met or exceeded the anticipated capture of 3,000 cells (Fig. b). The detailed raw data, such as the Q30 values for UMI sequences across all seven tissues exceeding 90%, are reported in additional files (Supplementary Table ). Overall, these data highlight the high-quality sequencing depth and excellent coverage of the Bama pig reference genome, providing a solid basis for further analyses. The raw single-nucleus sequencing data (SAT, GOM, and PMM) represent RNA found only within cell nuclei. RNA from mitochondrial (percent.mt) genes is generally absent in nucleus-only sequencing data; hence, the percentage of mt is typically lower in snRNA-seq data. Moreover, since single-nucleus sequencing captures only nascent RNA in nuclei, the quantity of RNA (nCount_RNA) is also expected to be lower than that of scRNA-seq methods, with correspondingly lower numbers of genes and reads (nFeature_RNA) per cell sequenced (Fig. c).
To independently verify the reliability of each dataset and identify tissue-specific cell types or subtypes, we conducted separate analyses through cell annotation and classification for each dataset. After CellRanger processed the raw data output, we used Seurat to construct single-cell matrices and then performed quality control on each dataset. As previously mentioned, the percentage of mitochondrial genes is used as a proxy for cell death, but mitochondrial gene RNA is generally absent in nuclear data; therefore, the percent.mt is lower in single-nucleus sequencing data. Notably, Hepatocytes may have very high mitochondrial content ; hence, a threshold of 50% was set (liver, percent.mt < 50) to optimally preserve hepatocytes and remove dead or dying cells. In single-cell microfluidics processes, droplets containing two or more cells can occur, which are then mistakenly identified as single cells. To further remove cells with abnormal gene expression, we employed DoubletFinder , eliminating doublets. Finally, each cluster’s identity was assigned on the basis of established cell-specific marker gene expression. As expected, each tissue exhibited specific cell types; subcutaneous back fat (SAT) and omental fat (GOM) uniquely featured “fibro-/adipogenic progenitors” and “adipocytes”. The psoas major muscle (PMM) showed skeletal muscle fiber cells, “I myofiber”, “II myofiber 2B”, and “II myofiber 2X”. The spleen contains “dendritic cells”, which are crucial for initiating immune responses, particularly in antigen presentation . The lung features “alveolar type 1 cells”, which cover the surfaces of the alveoli for gas exchange, and “alveolar type 2 cells”, which are responsible for secreting surfactants to prevent alveolar collapse . The kidney displays “proximal tubule cells”, which are crucial for filtering waste from the blood and absorbing water and nutrients . Liver tissue includes “Hepatocytes”, which are associated with the metabolism of proteins, carbohydrates, and fats , and specialized immune cells in the liver, “Kupffer cells” (Fig. ).
After individually assessing and confirming the high quality of each tissue dataset, which featured a diverse array of cell types and tissue-specific cells, we found that integrative analysis was feasible. Batch effects between different datasets can compromise the authenticity of comparisons, even when the same sequencing technologies are used. To standardize comparisons, we merged the postquality control scRNA-seq samples from the liver, spleen, lung, and kidney and conducted dimensionality reduction clustering (resolution = 0.8). This approach effectively mitigated batch effects, yielding a consolidated dataset of 39,406 cells and 24,686 genes for comprehensive annotation and comparison. Typically, cells with more total RNA molecules also exhibit more diverse gene detection. The correlation coefficient between nCount_RNA (total RNA molecules in cells) and nFeature_RNA (number of distinct genes detected per cell) increased from 0.83 to 0.9 after quality control (Fig. a), indicating a significant increase in the quality of the subsequent datasets. After correction for batch effects, the integration of similar cell types across different tissues was significantly enhanced (Fig. b). In this integrated analysis, using a curated set of cell-specific marker genes (Fig. e), we annotated 12 cell types from 33 cell clusters (Fig. c). In addition to identifying most cell types previously detected in individual analyses (Fig. d), we also discovered four established marker genes common across tissues, namely, CRTAM , GNLY , IL7R , and KLRB , for identifying T cells in all four tissues. In terms of cell abundance, the spleen, which is predominantly composed of immune cells, represented 96.74%, whereas the kidney was characterized mainly by tubule cells. The liver and lung presented a more complex assortment of cell types.
Similarly, to compare snRNA-seq data under the same standard, snRNA-seq samples (SAT, GOM, and PMM) were merged and subjected to dimensionality reduction clustering (resolution = 0.4), resulting in a total of 20,090 nuclei and 22,991 genes after batch effect removal. The correlation coefficient between nCount_RNA (total RNA molecules in nuclei) and nFeature_RNA (number of distinct genes detected per nucleus) increased from 0.9 to 0.94 after quality control (Fig. a). Compared with the scRNA-seq dataset, the snRNA-seq dataset presented more pronounced batch effects (Fig. b), and the cells became more distinctly separated, with similar cell types clustering more closely after batch effect correction. In the snRNA-seq dataset, 12 cell types were annotated from 21 cell clusters via a curated set of cell-specific marker genes (Fig. e, c). In terms of cell abundance, each tissue presented unique cell types with high cell type abundance (Fig. d). Overall, the data integration highlights the reliability of the data, not only providing a comparative abundance of cell types under a unified standard but also serving as a reference for future data integration and other research endeavors.
In single-cell transcriptomics, high-quality data are often closely associated with gene quality. To confirm the high quality of our data, we used a verification procedure that involved the careful selection of 0–5 potential marker genes for each single-cell type to assess their predictive power. Using the filtering criteria in our methodology, we found that the genes summarized in our I additional files. (Supplementary Table ) clearly exhibited strong marker gene representation. Remarkably, our results show robust concordance between the functions of the predicted annotated genes and the original annotations of the cell types, confirming a high level of correspondence. Together, the results highlight the effectiveness of these genes as marker genes, which aligns perfectly with our expectations regardless of their specificity or expression levels. In summary, within a rigorous quality control framework, including meticulous batch effect removal, the final set of high-quality genes and cells provides additional potential marker genes for a thorough exploration of distinct cell types in miniature pig (Bama pig), offering resources for studying the heterogeneity between different tissues or organs at the transcriptomic level. One limitation of this study is the lack of replicates. Although we have collected rich single-cell transcriptomic data from seven different tissues and organs, and ensured high data quality through strict quality control steps, the absence of replicates means that our results may be influenced by individual variations or sample processing inconsistencies. Additionally, the study is based solely on single-cell transcriptomic data from Bama pigs, so it would be beneficial to integrate and compare data from other similar or different pig breeds to further validate and expand the identified marker genes and cell types. Furthermore, while we have identified several potential marker genes, these still require experimental validation to confirm their biological significance across different tissues and cell types. Therefore, future studies should consider incorporating replicates, integrating data from different pig breeds or experimental conditions to enhance the generalizability and robustness of the findings, and further validating the potential marker genes.
In the present study, we gathered data from seven distinct tissues and organs of a Bama pig. This high-quality dataset comprises 72,710 cells and serves as a valuable resource that offers a comprehensive single-cell reference for miniature pigs. This resource will not only aid in exploring the broad heterogeneity across different pig tissues and organs but also become a valuable resource and reference for identifying potential biomarkers unique to Bama pig.
Supplementary Material 1: Supplementary Figures. Comparative Bubble Plots of Top Marker Genes Across Seven Tissues (Kidney, Liver, Lung, Spleen, PMM, SAT, and GOM) Highlighting the Most Signi cant Markers for Each Cell Type Based on Normalized Gene Expression. Supplementary Material 2: Supplementary Table 1. CellRanger metrics and quality control cells for single-cell and single-nucleus datasets. Supplementary Table 2. Known marker genes for seven anatomical sites of Bama pigs with references from 17 studies. Supplementary Table 3. Detailed potential marker genes of distinct cell populations in each anatomical tissue, based on individual dataset analyses.
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Endoscopic Minimally Invasive Approach Versus Median Sternotomy for Multiple-Valve Surgery: A Propensity-Matched Analysis | 54a7f700-a893-49bd-b9ae-3a516b8be40d | 11782361 | Surgical Procedures, Operative[mh] |
Minimally invasive techniques in cardiac surgery constitute the state of art in the field of heart surgery offering an alternative to traditional surgery through a total sternotomy and can be applied in a wide spectrum of different procedures including coronary revascularization surgery, valve surgery, aortic surgery, as well as surgery of congenital malformations and anatomical abnormalities . Minimally invasive valve surgery (MIVS) has better postoperative outcomes than the traditional median sternotomy (MS) for single-valve disease in terms of cosmesis, respiratory functions, blood product use, and a lower overall cost . MIVS is now used safely in multiple centers around the world for isolated aortic valve , mitral valve , and tricuspid valve surgeries . Moreover, minimally invasive approaches to multiple-valve surgery (MIMVS) have also been described as feasible and safe . However, limited comparative data assessing the safety and feasibility of the minimally invasive approach to multiple-valve surgeries compared with MS is available through the current literature . There are several techniques for MIVS, including among others right anterior mini-thoracotomy (RAMT), partial upper sternotomy, and robot-assisted surgery . This study aims to present our experience with endoscopic MIMVS via RAMT, evaluate its early outcomes compared with those of the traditional approach, and emphasize its advantages and disadvantages over MS.
Ethical Approval The study was approved by the respective institutional review boards (Medical Association of North Rhine number 82/2021, local ethics board of the University of Bonn number 169/20). Individual patient consent for the study was waived. Study Design The electronic records of all patients who underwent combined multiple-valve surgeries at two cardiac referral centers in Germany (Siegburg Heart Center and Bonn Heart Center) from March of 2017 to March of 2023 were retrospectively reviewed for this study. Multiple-valve surgery was defined as any surgery involving more than one valve including combined mitral and tricuspid valve procedures. All perioperative data were prospectively collected from the clinic’s internal databases, archived patient files and examination results from referring clinics or practices, and electronically filed and catalogued in the electronic patient record database during the patient’s hospital stay; thus no missing data was encountered. The choice of a minimally invasive access rather than a median sternotomy was based on an individual assessment of the patient’s characteristics, associated comorbidities, and anatomical conditions. The patient’s informed preference and the surgeon’s experience were also considered. Prior to the surgery, computed tomography angiography of the aorta and arterial vascular system was performed, and the possibility of a minimally invasive approach was excluded in cases of severe aortic and pelvic artery calcifications, severe anatomical abnormalities, e.g., pronounced severe sternum excavatum, or strong adhesions of the lungs. Patients with extensive endocarditis were also excluded. All patients undergoing multiple-valve surgery were otherwise selected for a minimally invasive procedure. A total of 317 consecutive patients undergoing surgery for double or triple valve disease were included. Procedures with concomitant coronary artery bypass grafting and ascending aortic surgery were excluded. However, concomitant procedures such as left atrial appendage (LAA) ligature/clipping, atrial septal defect (ASD) or ventricular septal defect (VSD) closure, and cryomaze were included in the study. The patients were divided into MIMVS group, patients who underwent valve surgery via an endoscopic minimally invasive approach, and MS group for the valve surgeries via median sternotomy. Preoperative baseline characteristics of the patients between the two groups were then investigated and analyzed. Operative Techniques All patients were placed on the operating table in the supine position. Intubation was then performed following the induction of general anesthesia, using a single-lumen endotracheal tube. To monitor the heart and valve functions perioperatively, a transesophageal echocardiography (TEE) was used. Endoscopic MIMVS group Early in our experience with minimally invasive surgery, cannulation of the femoral vessels for cardiopulmonary bypass (CPB) in the endoscopic group was done surgically. Since January 2019, however, peripheral cannulation was achieved percutaneously while femoral artery closure was performed with the use of the MANTA vascular closure device (Essential Medical Inc., Malvern, Pennsylvania, PA, USA). Detailed steps of both surgical and percutaneous femoral cannulations were previously published . In the minimally invasive group, a single RAMT was used for surgical access. For surgeries involving the aortic valve, it consists of a small incision 3–4 cm long at the level of the second or third intercostal space, placed 2 cm to the right of the sternal border without dislocating or resecting the ribs or the cartilage junction. RAMT was otherwise achieved using either a small incision at the level of the fourth intercostal space or using a periareolar incision (nipple-cut). The pleural space was accessed laterally following a careful dissection of the intercostal tissue preserving the right internal thoracic vessels. Exposure of the surgical field was then aided with a soft tissue retractor (Valve Gate ® Soft Tissue Protector, Geister, Germany). Pericardiotomy followed above the phrenic nerve while a three-dimensional (3D) camera port (Aesculap Einstein Vision, Tuttlingen, Germany) is placed through a 10-mm incision in the intercostal space above the main incision. A small 5-mm incision for the Chitwood clamp (Scanlan International, Inc, St Paul, MN, USA) was then performed laterally to the camera port incision. CO 2 was then infused through the 3D camera port. A long cardioplegia catheter (Medtronic DLP 9F, Ref 10012) was placed in the ascending aorta and secured with a polypropylene purse string suture. The left ventricular vent was inserted in the right upper pulmonary vein after a polypropylene purse string suture. After aortic clamping and administration of crystalloid cardioplegia (Custodiol; Koehler Chemi, Alsbach-Haenlien, Germany), normothermic CPB was achieved. Depending on the procedure required, the concerned valves were then operated on, and in all cases, the aortotomy and/or the atrial incisions were closed in two layers. After the heart was de-aired, ventricular pacing wires were placed prior to declamping the aorta and the patient was then gradually weaned from CPB. Protamine/heparin was administered in a 1:1 ratio, and TEE was used to assess valvular function. Decannulation was then achieved by either slowly removing the arterial and venous cannulas from the groin followed by soft tissue and skin closure with resorbable sutures in case of surgical femoral access, or with the use of the MANTA system in case of a percutaneous approach. A pleural drain was placed through the clamp incision, the ribs were then secured, and the wound was finally closed in layers. MS group In the MS group, a standard median sternotomy was performed. In aortic valve surgery, CPB was initiated after a direct cannulation of the ascending aorta and the right atrium. Double cannulation of the superior and inferior vena cava was used when the mitral and/or tricuspid valves were involved. Antegrade Calafiore cardioplegia was administered using an aortic vent catheter, and then directly into the coronary ostia in case of aortic regurgitation. The remaining surgical steps were performed in standard fashion. Concomitant procedures When a concomitant left atrial cryoablation was performed, the Complete Left-Atrial Cox-Maze III Lesion Set was used with three lesion lines: superior pulmonary vein–inferior pulmonary vein, and superior pulmonary vein–LAA, inferior pulmonary vein–LAA. Atrial septal defect repair was done with either a simple PFO stitch or using a patch implantation. Variables The primary outcome was operation-related mortality, which was defined as inhospital mortality occurring within 30 postoperative days. Secondary endpoints were inhospital mortality of any cause and inhospital morbidity, which was defined as the occurrence of stroke, delirium, acute kidney injury, new-onset atrial fibrillation, atrioventricular block (AVB) requiring a temporary pacemaker (PM), low cardiac output syndrome (LCOS), number of procedure-related wound revision, and respiratory insufficiency requiring invasive management, namely prolonged intubation (≥ 24 h) and tracheostomy. Other outcome variables included were CPB time and aortic cross clamping (AoX) time for the intraoperative parameters, as well as nights spent in the intensive care unit (ICU), total postoperative hospital stay, number of re-exploration for intrathoracic bleeding, number of packed red blood cells (pRBC) transfused per patient, number of patients requiring transfusions, and patient discharge destination, namely whether a patient required rehabilitation (discharged to a rehabilitation center or another clinic) or were discharged home directly. Statistical Analysis Continuous variables were represented as mean ± standard deviation (SD) or as median (interquartile range) depending on the normality of their distribution. The latter was analyzed using inspection of the QQ plot and the Shapiro–Wilk normality test. The interquartile range (IQR) was expressed as an interval. Categorical variables were reported as counts and their respective percentages N . Prior to propensity matching, continuous outcome variables were compared with the non-parametric Mann–Whitney test. Pearson’s χ 2 test and Fisher’s exact test were used for comparing categorical outcomes between the two study groups where appropriate, depending on whether the minimum expected assumption was met. In all cases, a two-tailed p value of less than 0.05 was considered statistically significant. In order to take into account possible treatment bias due to confounding factors, propensity score matching was performed. The propensity score for each patient was calculated by logistic regression with adjustment for 13 key baseline variables: age, female sex, body mass index (BMI), New York Heart Association classification (NYHA) ≥ 3, hypertension, diabetes, endocarditis, nonelective surgery, heart failure, Euroscore II, three concomitant valve operation, previous heart surgery, peripheral artery disease, and coronary artery disease. Patients were matched using a 1 to 1 nearest neighbor propensity score matching without replacement with a caliper of 0.12. Standardized mean difference (SMD) was used instead of p values to assess the balance between the two groups . After matching, all SMD values for the covariates were below 0.1. A love plot of absolute standardized mean differences is shown in Fig. and a jitter plot of the propensity scores in Fig. . Comparisons between the two matched groups were carried out using either a paired t test or the Wilcoxon signed rank sum test for continuous variables, depending on the normality of their distribution, and binary categorical variables were compared using McNemar’s mid- p test while the Bhapkar test was used for categorical variables with more than two outcomes. All statistical analyses were performed using the R statistical software (R Foundation for Statistical Computing, Vienna, Austria). The MatchIt package was used for propensity score matching .
The study was approved by the respective institutional review boards (Medical Association of North Rhine number 82/2021, local ethics board of the University of Bonn number 169/20). Individual patient consent for the study was waived.
The electronic records of all patients who underwent combined multiple-valve surgeries at two cardiac referral centers in Germany (Siegburg Heart Center and Bonn Heart Center) from March of 2017 to March of 2023 were retrospectively reviewed for this study. Multiple-valve surgery was defined as any surgery involving more than one valve including combined mitral and tricuspid valve procedures. All perioperative data were prospectively collected from the clinic’s internal databases, archived patient files and examination results from referring clinics or practices, and electronically filed and catalogued in the electronic patient record database during the patient’s hospital stay; thus no missing data was encountered. The choice of a minimally invasive access rather than a median sternotomy was based on an individual assessment of the patient’s characteristics, associated comorbidities, and anatomical conditions. The patient’s informed preference and the surgeon’s experience were also considered. Prior to the surgery, computed tomography angiography of the aorta and arterial vascular system was performed, and the possibility of a minimally invasive approach was excluded in cases of severe aortic and pelvic artery calcifications, severe anatomical abnormalities, e.g., pronounced severe sternum excavatum, or strong adhesions of the lungs. Patients with extensive endocarditis were also excluded. All patients undergoing multiple-valve surgery were otherwise selected for a minimally invasive procedure. A total of 317 consecutive patients undergoing surgery for double or triple valve disease were included. Procedures with concomitant coronary artery bypass grafting and ascending aortic surgery were excluded. However, concomitant procedures such as left atrial appendage (LAA) ligature/clipping, atrial septal defect (ASD) or ventricular septal defect (VSD) closure, and cryomaze were included in the study. The patients were divided into MIMVS group, patients who underwent valve surgery via an endoscopic minimally invasive approach, and MS group for the valve surgeries via median sternotomy. Preoperative baseline characteristics of the patients between the two groups were then investigated and analyzed.
All patients were placed on the operating table in the supine position. Intubation was then performed following the induction of general anesthesia, using a single-lumen endotracheal tube. To monitor the heart and valve functions perioperatively, a transesophageal echocardiography (TEE) was used. Endoscopic MIMVS group Early in our experience with minimally invasive surgery, cannulation of the femoral vessels for cardiopulmonary bypass (CPB) in the endoscopic group was done surgically. Since January 2019, however, peripheral cannulation was achieved percutaneously while femoral artery closure was performed with the use of the MANTA vascular closure device (Essential Medical Inc., Malvern, Pennsylvania, PA, USA). Detailed steps of both surgical and percutaneous femoral cannulations were previously published . In the minimally invasive group, a single RAMT was used for surgical access. For surgeries involving the aortic valve, it consists of a small incision 3–4 cm long at the level of the second or third intercostal space, placed 2 cm to the right of the sternal border without dislocating or resecting the ribs or the cartilage junction. RAMT was otherwise achieved using either a small incision at the level of the fourth intercostal space or using a periareolar incision (nipple-cut). The pleural space was accessed laterally following a careful dissection of the intercostal tissue preserving the right internal thoracic vessels. Exposure of the surgical field was then aided with a soft tissue retractor (Valve Gate ® Soft Tissue Protector, Geister, Germany). Pericardiotomy followed above the phrenic nerve while a three-dimensional (3D) camera port (Aesculap Einstein Vision, Tuttlingen, Germany) is placed through a 10-mm incision in the intercostal space above the main incision. A small 5-mm incision for the Chitwood clamp (Scanlan International, Inc, St Paul, MN, USA) was then performed laterally to the camera port incision. CO 2 was then infused through the 3D camera port. A long cardioplegia catheter (Medtronic DLP 9F, Ref 10012) was placed in the ascending aorta and secured with a polypropylene purse string suture. The left ventricular vent was inserted in the right upper pulmonary vein after a polypropylene purse string suture. After aortic clamping and administration of crystalloid cardioplegia (Custodiol; Koehler Chemi, Alsbach-Haenlien, Germany), normothermic CPB was achieved. Depending on the procedure required, the concerned valves were then operated on, and in all cases, the aortotomy and/or the atrial incisions were closed in two layers. After the heart was de-aired, ventricular pacing wires were placed prior to declamping the aorta and the patient was then gradually weaned from CPB. Protamine/heparin was administered in a 1:1 ratio, and TEE was used to assess valvular function. Decannulation was then achieved by either slowly removing the arterial and venous cannulas from the groin followed by soft tissue and skin closure with resorbable sutures in case of surgical femoral access, or with the use of the MANTA system in case of a percutaneous approach. A pleural drain was placed through the clamp incision, the ribs were then secured, and the wound was finally closed in layers. MS group In the MS group, a standard median sternotomy was performed. In aortic valve surgery, CPB was initiated after a direct cannulation of the ascending aorta and the right atrium. Double cannulation of the superior and inferior vena cava was used when the mitral and/or tricuspid valves were involved. Antegrade Calafiore cardioplegia was administered using an aortic vent catheter, and then directly into the coronary ostia in case of aortic regurgitation. The remaining surgical steps were performed in standard fashion. Concomitant procedures When a concomitant left atrial cryoablation was performed, the Complete Left-Atrial Cox-Maze III Lesion Set was used with three lesion lines: superior pulmonary vein–inferior pulmonary vein, and superior pulmonary vein–LAA, inferior pulmonary vein–LAA. Atrial septal defect repair was done with either a simple PFO stitch or using a patch implantation.
The primary outcome was operation-related mortality, which was defined as inhospital mortality occurring within 30 postoperative days. Secondary endpoints were inhospital mortality of any cause and inhospital morbidity, which was defined as the occurrence of stroke, delirium, acute kidney injury, new-onset atrial fibrillation, atrioventricular block (AVB) requiring a temporary pacemaker (PM), low cardiac output syndrome (LCOS), number of procedure-related wound revision, and respiratory insufficiency requiring invasive management, namely prolonged intubation (≥ 24 h) and tracheostomy. Other outcome variables included were CPB time and aortic cross clamping (AoX) time for the intraoperative parameters, as well as nights spent in the intensive care unit (ICU), total postoperative hospital stay, number of re-exploration for intrathoracic bleeding, number of packed red blood cells (pRBC) transfused per patient, number of patients requiring transfusions, and patient discharge destination, namely whether a patient required rehabilitation (discharged to a rehabilitation center or another clinic) or were discharged home directly.
Continuous variables were represented as mean ± standard deviation (SD) or as median (interquartile range) depending on the normality of their distribution. The latter was analyzed using inspection of the QQ plot and the Shapiro–Wilk normality test. The interquartile range (IQR) was expressed as an interval. Categorical variables were reported as counts and their respective percentages N . Prior to propensity matching, continuous outcome variables were compared with the non-parametric Mann–Whitney test. Pearson’s χ 2 test and Fisher’s exact test were used for comparing categorical outcomes between the two study groups where appropriate, depending on whether the minimum expected assumption was met. In all cases, a two-tailed p value of less than 0.05 was considered statistically significant. In order to take into account possible treatment bias due to confounding factors, propensity score matching was performed. The propensity score for each patient was calculated by logistic regression with adjustment for 13 key baseline variables: age, female sex, body mass index (BMI), New York Heart Association classification (NYHA) ≥ 3, hypertension, diabetes, endocarditis, nonelective surgery, heart failure, Euroscore II, three concomitant valve operation, previous heart surgery, peripheral artery disease, and coronary artery disease. Patients were matched using a 1 to 1 nearest neighbor propensity score matching without replacement with a caliper of 0.12. Standardized mean difference (SMD) was used instead of p values to assess the balance between the two groups . After matching, all SMD values for the covariates were below 0.1. A love plot of absolute standardized mean differences is shown in Fig. and a jitter plot of the propensity scores in Fig. . Comparisons between the two matched groups were carried out using either a paired t test or the Wilcoxon signed rank sum test for continuous variables, depending on the normality of their distribution, and binary categorical variables were compared using McNemar’s mid- p test while the Bhapkar test was used for categorical variables with more than two outcomes. All statistical analyses were performed using the R statistical software (R Foundation for Statistical Computing, Vienna, Austria). The MatchIt package was used for propensity score matching .
Between March of 2017 and March of 2023, a total of 317 patients underwent multiple-valve surgeries, 123 (38.8%) were endoscopic MIMVS and 194 (61.2%) were performed via MS. Patient and Procedure Characteristics Prior to Propensity Score Matching Across both groups before matching, 167 (52.7%) patients were female, median age was 69 [61–75] years, and the median BMI was 26.12 [23.77–29.32] kg/m 2 . Fifty-three (16.7%) patients had undergone previous cardiac surgery and 86.1% of patients had a NYHA classification III or IV. The baseline characteristics of both groups prior to matching are summarized in Table . Within the endoscopic MIMVS group, 18 (14.6%) cases were non-elective, which was defined as cases that were not urgent or emergent. The most prevalent procedure was concomitant mitral valve and tricuspid valve repair, followed by mitral valve replacement associated with a tricuspid valve repair, with totals of 47 (38.2%) and 34 (27.6%), respectively. Associated LAA closure, cryoablation, ASD repair, and VSD repair were represented in 9 (7.3%), 18 (14.6%), 8 (6.5%), and 2 (1.6%) cases, respectively. One ASD repair was done using a patch. In the MS group, 49 (25.3%) patients received a non-elective surgery. Mitral valve and aortic valve replacement accounted for the most carried out operation, followed by concomitant aortic valve replacement and mitral valve repair, with totals of 75 (38.7%) and 33 (17%), respectively. Concomitant LAA closure, cryoablation, ASD repair, and VSD repair occurred with 8 (4.1%), 8 (4.1%), 10 (5.2%), and 4 (2.1%) patients, respectively. One ASD repair was done using a patch. Other procedural details as well as grafted valve types for both groups are detailed in Tables and . Patient and Procedure Characteristics of Matched Groups Balance between the two groups after propensity matching was assessed using absolute mean standardized differences. Patient characteristics of the overall matched population as well as both groups, MIMVS and MS, are summarized in Table . There was no significant imbalance between both groups except for the prevalence of atrial fibrillation and renal failure, which were present in 64.3% and 4.5% of patients in the MIMVS group, respectively, in contrast with 46.4% and 8% of patients of the MS group, respectively (SMD = 0.365 and 0.148 for atrial fibrillation and renal failure, respectively). Procedural details as well as grafted valve types for both groups are detailed in Tables and . Outcomes Prior to Propensity Score Matching Intraoperative and postoperative outcomes prior to propensity score matching are summarized in Table . Primary Endpoint: 30-Day Mortality Within the endoscopic MIMVS group, operation-related mortality, defined as inhospital mortality within 30 days postoperatively, occurred in 9 (7.3%) cases, which was non-significantly lower than in the median sternotomy group with 28 (14.4%) cases ( p = 0.055). Secondary Endpoints Inhospital mortality of any cause and at any time within the duration of hospitalization was also less prevalent in the endoscopic minimally invasive group than in the sternotomy group, with 11 (8.9%) versus 31 (16%) occurrences, respectively. This difference was, however, also statistically non-significant ( p = 0.07). Median CPB time and AoX time in the minimally invasive group were 106 [79–124.5] mins and 70 [53–80.5] mins, respectively, both longer than in the MS group but without statistical significance, where the respective durations were 100 [73–147] mins and 65 [48.25–95] mins ( p = 0.97 for CPB time and p = 0.81 for AoX time). Regarding postoperative inhospital morbidity, there were no statistically significant differences between the endoscopic MIMVS group and the MS group in the occurrences of stroke (2.4% vs 3.1%, respectively), delirium (4.9% vs 10.3%, respectively), acute kidney injury (5.7% vs 10.8%, respectively), new-onset atrial fibrillation (4.1% vs 4.6%, respectively), AVB requiring a temporary PM (0.8% vs 3.1%, respectively), LCOS (8.9% vs 7.2%, respectively), respiratory insufficiency (14.6% vs 12.4%, respectively), and wound revisions (3.3%). Median number of transfusions of pRBCs within the group of patients undergoing a mini-thoracotomy was 0 [0–3] transfusion, significantly lower than in the median sternotomy group, where the median was 1 [0–4], p = 0.014. The number of patients requiring transfusion of at least 1 unit of pRBCs was also statistically higher in the MS group, where 59.3% of patients received transfusions of pRBCs, versus 47.2% in the MIMVS group, with a p value of 0.035. Re-exploration for intrathoracic bleeding was only necessary in 1 (0.5%) patient from the MS group. ICU stay following the procedure was similar across both groups ( p = 0.75). Additionally, total hospital stay was also similar between the two groups, with a median stay of 12 [8–17.5] days in the MIMVS group, versus 13 [8.25–21] days for the MS group ( p = 0.36). Following hospitalization, patient discharge directly to their home was successful in more patients from the endoscopic minimally invasive group, with 53 (43.1%) patients versus 60 (30.9%). This difference was, however, not statistically significant ( p = 0.062). There was no incidence of repeat valve operations, paravalvular leak, or moderate to severe valve regurgitation across both groups. There was also no need for a conversion to sternotomy in all patients in the endoscopic MIMVS group. ECMO was required in 8.9% for the MIMVS group and 8.8% for the MS group, p = 0.56. All patients who required ECMO in both groups had an age above the average of the population of the study, as well as CPB times above the 75th percentile. Outcomes of Matched Groups Primary Endpoint: 30-Day Mortality Except for the median number of transfused pRBCs per patients (0 [0–3] units for MIMVS vs 1 [0–4.25] units for MS, p = 0.002), there were no statistically significant differences between the two groups after propensity score matching. Median CPB time in the MIMVS group was 105.5 [79.75–124] mins versus 98 [68.75–130.25] mins in the MS group ( p = 0.92). Similar results were also seen for aortic cross-clamping times (70 [53–80.25] mins vs 63.5 [46–90.25] mins in the MIMVS and MS groups, respectively, p = 0.76). Postoperative mortality within 30 days was still lower in the MIMVS group, albeit non-significantly, as was the overall inhospital mortality. All intraoperative and postoperative outcomes for the matched groups are summarized in Table .
Across both groups before matching, 167 (52.7%) patients were female, median age was 69 [61–75] years, and the median BMI was 26.12 [23.77–29.32] kg/m 2 . Fifty-three (16.7%) patients had undergone previous cardiac surgery and 86.1% of patients had a NYHA classification III or IV. The baseline characteristics of both groups prior to matching are summarized in Table . Within the endoscopic MIMVS group, 18 (14.6%) cases were non-elective, which was defined as cases that were not urgent or emergent. The most prevalent procedure was concomitant mitral valve and tricuspid valve repair, followed by mitral valve replacement associated with a tricuspid valve repair, with totals of 47 (38.2%) and 34 (27.6%), respectively. Associated LAA closure, cryoablation, ASD repair, and VSD repair were represented in 9 (7.3%), 18 (14.6%), 8 (6.5%), and 2 (1.6%) cases, respectively. One ASD repair was done using a patch. In the MS group, 49 (25.3%) patients received a non-elective surgery. Mitral valve and aortic valve replacement accounted for the most carried out operation, followed by concomitant aortic valve replacement and mitral valve repair, with totals of 75 (38.7%) and 33 (17%), respectively. Concomitant LAA closure, cryoablation, ASD repair, and VSD repair occurred with 8 (4.1%), 8 (4.1%), 10 (5.2%), and 4 (2.1%) patients, respectively. One ASD repair was done using a patch. Other procedural details as well as grafted valve types for both groups are detailed in Tables and .
Balance between the two groups after propensity matching was assessed using absolute mean standardized differences. Patient characteristics of the overall matched population as well as both groups, MIMVS and MS, are summarized in Table . There was no significant imbalance between both groups except for the prevalence of atrial fibrillation and renal failure, which were present in 64.3% and 4.5% of patients in the MIMVS group, respectively, in contrast with 46.4% and 8% of patients of the MS group, respectively (SMD = 0.365 and 0.148 for atrial fibrillation and renal failure, respectively). Procedural details as well as grafted valve types for both groups are detailed in Tables and .
Intraoperative and postoperative outcomes prior to propensity score matching are summarized in Table . Primary Endpoint: 30-Day Mortality Within the endoscopic MIMVS group, operation-related mortality, defined as inhospital mortality within 30 days postoperatively, occurred in 9 (7.3%) cases, which was non-significantly lower than in the median sternotomy group with 28 (14.4%) cases ( p = 0.055). Secondary Endpoints Inhospital mortality of any cause and at any time within the duration of hospitalization was also less prevalent in the endoscopic minimally invasive group than in the sternotomy group, with 11 (8.9%) versus 31 (16%) occurrences, respectively. This difference was, however, also statistically non-significant ( p = 0.07). Median CPB time and AoX time in the minimally invasive group were 106 [79–124.5] mins and 70 [53–80.5] mins, respectively, both longer than in the MS group but without statistical significance, where the respective durations were 100 [73–147] mins and 65 [48.25–95] mins ( p = 0.97 for CPB time and p = 0.81 for AoX time). Regarding postoperative inhospital morbidity, there were no statistically significant differences between the endoscopic MIMVS group and the MS group in the occurrences of stroke (2.4% vs 3.1%, respectively), delirium (4.9% vs 10.3%, respectively), acute kidney injury (5.7% vs 10.8%, respectively), new-onset atrial fibrillation (4.1% vs 4.6%, respectively), AVB requiring a temporary PM (0.8% vs 3.1%, respectively), LCOS (8.9% vs 7.2%, respectively), respiratory insufficiency (14.6% vs 12.4%, respectively), and wound revisions (3.3%). Median number of transfusions of pRBCs within the group of patients undergoing a mini-thoracotomy was 0 [0–3] transfusion, significantly lower than in the median sternotomy group, where the median was 1 [0–4], p = 0.014. The number of patients requiring transfusion of at least 1 unit of pRBCs was also statistically higher in the MS group, where 59.3% of patients received transfusions of pRBCs, versus 47.2% in the MIMVS group, with a p value of 0.035. Re-exploration for intrathoracic bleeding was only necessary in 1 (0.5%) patient from the MS group. ICU stay following the procedure was similar across both groups ( p = 0.75). Additionally, total hospital stay was also similar between the two groups, with a median stay of 12 [8–17.5] days in the MIMVS group, versus 13 [8.25–21] days for the MS group ( p = 0.36). Following hospitalization, patient discharge directly to their home was successful in more patients from the endoscopic minimally invasive group, with 53 (43.1%) patients versus 60 (30.9%). This difference was, however, not statistically significant ( p = 0.062). There was no incidence of repeat valve operations, paravalvular leak, or moderate to severe valve regurgitation across both groups. There was also no need for a conversion to sternotomy in all patients in the endoscopic MIMVS group. ECMO was required in 8.9% for the MIMVS group and 8.8% for the MS group, p = 0.56. All patients who required ECMO in both groups had an age above the average of the population of the study, as well as CPB times above the 75th percentile.
Within the endoscopic MIMVS group, operation-related mortality, defined as inhospital mortality within 30 days postoperatively, occurred in 9 (7.3%) cases, which was non-significantly lower than in the median sternotomy group with 28 (14.4%) cases ( p = 0.055).
Inhospital mortality of any cause and at any time within the duration of hospitalization was also less prevalent in the endoscopic minimally invasive group than in the sternotomy group, with 11 (8.9%) versus 31 (16%) occurrences, respectively. This difference was, however, also statistically non-significant ( p = 0.07). Median CPB time and AoX time in the minimally invasive group were 106 [79–124.5] mins and 70 [53–80.5] mins, respectively, both longer than in the MS group but without statistical significance, where the respective durations were 100 [73–147] mins and 65 [48.25–95] mins ( p = 0.97 for CPB time and p = 0.81 for AoX time). Regarding postoperative inhospital morbidity, there were no statistically significant differences between the endoscopic MIMVS group and the MS group in the occurrences of stroke (2.4% vs 3.1%, respectively), delirium (4.9% vs 10.3%, respectively), acute kidney injury (5.7% vs 10.8%, respectively), new-onset atrial fibrillation (4.1% vs 4.6%, respectively), AVB requiring a temporary PM (0.8% vs 3.1%, respectively), LCOS (8.9% vs 7.2%, respectively), respiratory insufficiency (14.6% vs 12.4%, respectively), and wound revisions (3.3%). Median number of transfusions of pRBCs within the group of patients undergoing a mini-thoracotomy was 0 [0–3] transfusion, significantly lower than in the median sternotomy group, where the median was 1 [0–4], p = 0.014. The number of patients requiring transfusion of at least 1 unit of pRBCs was also statistically higher in the MS group, where 59.3% of patients received transfusions of pRBCs, versus 47.2% in the MIMVS group, with a p value of 0.035. Re-exploration for intrathoracic bleeding was only necessary in 1 (0.5%) patient from the MS group. ICU stay following the procedure was similar across both groups ( p = 0.75). Additionally, total hospital stay was also similar between the two groups, with a median stay of 12 [8–17.5] days in the MIMVS group, versus 13 [8.25–21] days for the MS group ( p = 0.36). Following hospitalization, patient discharge directly to their home was successful in more patients from the endoscopic minimally invasive group, with 53 (43.1%) patients versus 60 (30.9%). This difference was, however, not statistically significant ( p = 0.062). There was no incidence of repeat valve operations, paravalvular leak, or moderate to severe valve regurgitation across both groups. There was also no need for a conversion to sternotomy in all patients in the endoscopic MIMVS group. ECMO was required in 8.9% for the MIMVS group and 8.8% for the MS group, p = 0.56. All patients who required ECMO in both groups had an age above the average of the population of the study, as well as CPB times above the 75th percentile.
Primary Endpoint: 30-Day Mortality Except for the median number of transfused pRBCs per patients (0 [0–3] units for MIMVS vs 1 [0–4.25] units for MS, p = 0.002), there were no statistically significant differences between the two groups after propensity score matching. Median CPB time in the MIMVS group was 105.5 [79.75–124] mins versus 98 [68.75–130.25] mins in the MS group ( p = 0.92). Similar results were also seen for aortic cross-clamping times (70 [53–80.25] mins vs 63.5 [46–90.25] mins in the MIMVS and MS groups, respectively, p = 0.76). Postoperative mortality within 30 days was still lower in the MIMVS group, albeit non-significantly, as was the overall inhospital mortality. All intraoperative and postoperative outcomes for the matched groups are summarized in Table .
Except for the median number of transfused pRBCs per patients (0 [0–3] units for MIMVS vs 1 [0–4.25] units for MS, p = 0.002), there were no statistically significant differences between the two groups after propensity score matching. Median CPB time in the MIMVS group was 105.5 [79.75–124] mins versus 98 [68.75–130.25] mins in the MS group ( p = 0.92). Similar results were also seen for aortic cross-clamping times (70 [53–80.25] mins vs 63.5 [46–90.25] mins in the MIMVS and MS groups, respectively, p = 0.76). Postoperative mortality within 30 days was still lower in the MIMVS group, albeit non-significantly, as was the overall inhospital mortality. All intraoperative and postoperative outcomes for the matched groups are summarized in Table .
Video-assisted RAMT has been shown to be a safe and feasible minimally invasive approach for multiple-valve surgeries . However, the current literature lacks comparative data assessing the feasibility and safety of a totally endoscopic access versus the more conventional median sternotomy RAMT is achieved through a small 3–5-cm incision, and the minimal invasiveness of this technique compared with a full sternotomy offers the advantage of a smaller operational scar and minimal postoperative pain, leading to a more favorable patient satisfaction . Re-exploration for Bleeding and Transfusion Requirements Aside from the better cosmesis, the smaller incision should also have a favorable effect on postoperative bleeding and blood product requirements , although these reports are mainly based on results from studies concerning single-valve minimally invasive surgeries. Blood transfusions have previously been linked to an increased risk of postoperative morbidity and mortality in patients undergoing heart surgeries, including a longer hospital stay and increased costs . When compared with full sternotomy, our study showed that endoscopic MIMVS was associated with a lower need for pRBC transfusions, and no difference in the need for a re-exploration surgery for bleeding. According to similar, albeit limited in number, comparative studies in the current literature, the favorable effect of MIMVS on transfusion requirements and re-exploration for bleeding is somewhat controversial. Atik et al. found similar transfusion rates and returns to the operation room for bleeding between the minimally invasive group and the median sternotomy group . In a study comparing the outcomes RAMT to re-sternotomy in mitral and tricuspid valve surgeries with previous sternotomies, Miura et al. also reported no difference in the number of transfusions between the two groups, as well as similar incidences of postoperative major complications, which included reoperation for bleeding . Indeed, another study comparing the outcomes of video-assisted RAMT for double valve surgeries to those of MS done by Qiao et al. showed a statistically significant lower amount of thoracic drainage in their study group comprising patients who underwent MIMVS, as well as no reoperation for bleeding across both groups, which would agree with the findings of our current study, although they did not report the number of required transfusions . Additionally, our findings concur with those of a propensity matched comparative study done by Zhao et al. . ICU and Hospital Lengths of Stay, and Rehabilitation Requirements Minimally invasive cardiac valve operations were previously shown to be associated with similar lengths of ICU stay and hospitalization, if not more favorable, compared with median sternotomy, as well as a decreased need for rehabilitation services following patient discharge . It has been proposed that this has the effect of lowering the overall costs of cardiac valve operations in association with a less traumatic surgical access and more cosmetic incisions . Our current series showed that patients who underwent endoscopic MIMVS had a comparable postoperative ICU stay as those in the MS group, as well as similar hospitalization duration. Within the current literature, similar comparative studies report comparable results regarding ICU length of stay and hospitalization duration . Atik et al. reported shorter ICU and hospital stay within the MIMVS group compared with the MS group overall, but there was no significant difference within the propensity matched patients, which would align with our findings. The same results were reported in a recent propensity matched retrospective study done by Paparella et al., which evaluated the outcomes of MIMVS compared with MS in concomitant mitral and tricuspid valve surgeries . In a retrospective study by Gumus et al. comparing RAMT and MS for multiple-valve surgery, both ICU and hospital stays were reported to be not significantly shorter in the MIMVS group . Similar results were also reported by Miura et al. . Data regarding rehabilitation requirements and cost-effectiveness of a minimally invasive approach has previously been reported for single-valve surgeries ; however, data is still lacking with regards to multiple-valve surgeries in comparison with median sternotomy. Respiratory Insufficiency and Invasive Respiratory Support Concerns for respiratory insufficiency following minimally invasive valve operations arise because they have been associated with longer operative times, as well as the risks associated with general anesthesia and mechanical ventilation . However, minimally invasive valve surgeries have an improved respiratory outcome for single valves . This can be in part be attributed to the preserved stability of the sternum and the improved pain score associated with the smaller surgical access . Our results show, however, that MIMVS was not associated with higher rates of respiratory insufficiency postoperatively, requiring invasive interventions or otherwise when compared with MS for multiple concomitant valve surgeries, both prior to and following propensity matching. These results are in line with other similar studies in the literature, which report similar rates of prolonged ventilation or respiratory insufficiency across both groups . Cardiopulmonary Bypass and Aortic Cross Clamping Time Prolonged AoX and CPB times are independent risk factors for postoperative morbidity, including acute kidney injury, as well as mortality in low- and high-risk patients undergoing minimally invasive heart surgeries . Furthermore, retrograde CPB presents an increased risk for stroke, delirium, and aortic dissection in contrast to anterograde CPB . Nonetheless, this current study showed similar rates of cerebrovascular events, delirium, and AKI across both groups, all of which resolved with either supportive treatment or spontaneously. This can be attributed to the fact that, even though the CPB and AoX times were slightly prolonged in the endoscopic MIMVS group without being statistically significant, median cross clamping time and IQR were still under 90 min. Reports by Al-Sarraf et al. indeed showed increased morbidity and mortality rates for AoX times of more than 90 min. While similar studies in the current literature have found an increase in CPB and AoX times for the minimally invasive groups, our findings did not show a statistically significant difference between MIMVS and MS for multiple-valve surgery . However, Atik et al. showed a decreased CPB time in the minimally invasive group. This can be explained by the different surgical techniques employed for the MIMVS, which consists of a right paramedian incision at the early stages of their study and later a partial upper sternotomy, instead of an endoscopic approach through right mini-thoracotomy . Mortality and Morbidity Mortality rates associated with minimally invasive valve surgeries are similar to those of conventional sternotomy . Furthermore, current studies comparing MIMVS specifically with MS also show similar mortality rates . Our findings are in agreement with these reports. Both early mortality, defined as mortality occurring within 30 days postoperatively, and overall mortality prior to patient discharge, were lower in the endoscopic MIMVS group than in the MS group in our study. However, the lower rate of 30-day mortality in the MIMVS group was not statistically significant for our sample, both before and after matching. This could be explained by the fact that the patients included in our study were operated on by a team that is skilled and experienced in the minimally invasive technique, while minimally invasive valve surgery has a notoriously steep learning curve. Additionally, our study was retrospective, and thus non-randomized, inherently subject to selection bias. Regarding other postoperative outcomes that could contribute to operative morbidity, our study showed similar incidences of LCOS, new onset atrial fibrillation, AVB requiring a PM, wound revisions, postoperative ECMO requirements, and coagulopathy, as well as pleural effusion and pneumothorax. These findings concur with the results of similar studies in the literature . However, Gumus et al. reported an increased incidence of delirium in the MIMVS group compared with the MS group , and Zhao et al. found an increased incidence of new-onset atrial fibrillation within patients in the MIMVS .
Aside from the better cosmesis, the smaller incision should also have a favorable effect on postoperative bleeding and blood product requirements , although these reports are mainly based on results from studies concerning single-valve minimally invasive surgeries. Blood transfusions have previously been linked to an increased risk of postoperative morbidity and mortality in patients undergoing heart surgeries, including a longer hospital stay and increased costs . When compared with full sternotomy, our study showed that endoscopic MIMVS was associated with a lower need for pRBC transfusions, and no difference in the need for a re-exploration surgery for bleeding. According to similar, albeit limited in number, comparative studies in the current literature, the favorable effect of MIMVS on transfusion requirements and re-exploration for bleeding is somewhat controversial. Atik et al. found similar transfusion rates and returns to the operation room for bleeding between the minimally invasive group and the median sternotomy group . In a study comparing the outcomes RAMT to re-sternotomy in mitral and tricuspid valve surgeries with previous sternotomies, Miura et al. also reported no difference in the number of transfusions between the two groups, as well as similar incidences of postoperative major complications, which included reoperation for bleeding . Indeed, another study comparing the outcomes of video-assisted RAMT for double valve surgeries to those of MS done by Qiao et al. showed a statistically significant lower amount of thoracic drainage in their study group comprising patients who underwent MIMVS, as well as no reoperation for bleeding across both groups, which would agree with the findings of our current study, although they did not report the number of required transfusions . Additionally, our findings concur with those of a propensity matched comparative study done by Zhao et al. .
Minimally invasive cardiac valve operations were previously shown to be associated with similar lengths of ICU stay and hospitalization, if not more favorable, compared with median sternotomy, as well as a decreased need for rehabilitation services following patient discharge . It has been proposed that this has the effect of lowering the overall costs of cardiac valve operations in association with a less traumatic surgical access and more cosmetic incisions . Our current series showed that patients who underwent endoscopic MIMVS had a comparable postoperative ICU stay as those in the MS group, as well as similar hospitalization duration. Within the current literature, similar comparative studies report comparable results regarding ICU length of stay and hospitalization duration . Atik et al. reported shorter ICU and hospital stay within the MIMVS group compared with the MS group overall, but there was no significant difference within the propensity matched patients, which would align with our findings. The same results were reported in a recent propensity matched retrospective study done by Paparella et al., which evaluated the outcomes of MIMVS compared with MS in concomitant mitral and tricuspid valve surgeries . In a retrospective study by Gumus et al. comparing RAMT and MS for multiple-valve surgery, both ICU and hospital stays were reported to be not significantly shorter in the MIMVS group . Similar results were also reported by Miura et al. . Data regarding rehabilitation requirements and cost-effectiveness of a minimally invasive approach has previously been reported for single-valve surgeries ; however, data is still lacking with regards to multiple-valve surgeries in comparison with median sternotomy.
Concerns for respiratory insufficiency following minimally invasive valve operations arise because they have been associated with longer operative times, as well as the risks associated with general anesthesia and mechanical ventilation . However, minimally invasive valve surgeries have an improved respiratory outcome for single valves . This can be in part be attributed to the preserved stability of the sternum and the improved pain score associated with the smaller surgical access . Our results show, however, that MIMVS was not associated with higher rates of respiratory insufficiency postoperatively, requiring invasive interventions or otherwise when compared with MS for multiple concomitant valve surgeries, both prior to and following propensity matching. These results are in line with other similar studies in the literature, which report similar rates of prolonged ventilation or respiratory insufficiency across both groups .
Prolonged AoX and CPB times are independent risk factors for postoperative morbidity, including acute kidney injury, as well as mortality in low- and high-risk patients undergoing minimally invasive heart surgeries . Furthermore, retrograde CPB presents an increased risk for stroke, delirium, and aortic dissection in contrast to anterograde CPB . Nonetheless, this current study showed similar rates of cerebrovascular events, delirium, and AKI across both groups, all of which resolved with either supportive treatment or spontaneously. This can be attributed to the fact that, even though the CPB and AoX times were slightly prolonged in the endoscopic MIMVS group without being statistically significant, median cross clamping time and IQR were still under 90 min. Reports by Al-Sarraf et al. indeed showed increased morbidity and mortality rates for AoX times of more than 90 min. While similar studies in the current literature have found an increase in CPB and AoX times for the minimally invasive groups, our findings did not show a statistically significant difference between MIMVS and MS for multiple-valve surgery . However, Atik et al. showed a decreased CPB time in the minimally invasive group. This can be explained by the different surgical techniques employed for the MIMVS, which consists of a right paramedian incision at the early stages of their study and later a partial upper sternotomy, instead of an endoscopic approach through right mini-thoracotomy .
Mortality rates associated with minimally invasive valve surgeries are similar to those of conventional sternotomy . Furthermore, current studies comparing MIMVS specifically with MS also show similar mortality rates . Our findings are in agreement with these reports. Both early mortality, defined as mortality occurring within 30 days postoperatively, and overall mortality prior to patient discharge, were lower in the endoscopic MIMVS group than in the MS group in our study. However, the lower rate of 30-day mortality in the MIMVS group was not statistically significant for our sample, both before and after matching. This could be explained by the fact that the patients included in our study were operated on by a team that is skilled and experienced in the minimally invasive technique, while minimally invasive valve surgery has a notoriously steep learning curve. Additionally, our study was retrospective, and thus non-randomized, inherently subject to selection bias. Regarding other postoperative outcomes that could contribute to operative morbidity, our study showed similar incidences of LCOS, new onset atrial fibrillation, AVB requiring a PM, wound revisions, postoperative ECMO requirements, and coagulopathy, as well as pleural effusion and pneumothorax. These findings concur with the results of similar studies in the literature . However, Gumus et al. reported an increased incidence of delirium in the MIMVS group compared with the MS group , and Zhao et al. found an increased incidence of new-onset atrial fibrillation within patients in the MIMVS .
This study has, however, several limitations, some of which are inherent to being a retrospective study, and thus non-randomized. Although propensity score matching was done to mitigate some of the treatment selection bias, the relatively small sample size limits the statistical power of our analyses. A larger multicenter prospective randomized trial is needed in the future. Furthermore, regarding the patients in the minimally invasive group, the MANTA vascular closure device was introduced mid-study in favor of an open surgical approach to femoral cannulation for cardiopulmonary bypass. The use of this percutaneous device at our institution was shown to have favorable outcomes with regards to morbidity and the length of stay in the ICU , when compared with the open surgical femoral access, and the inclusion of the patients who underwent a surgical femoral access for CPB in this study might have masked more favorable outcomes for the patients in the endoscopic MIMVS group, especially that within the patients who underwent a vascular closure through MANTA, there was no incidence of vascular closure device-related morbidity, including pseudoaneurysms, bleeding, or otherwise. Additionally, there was no follow-up as part of the study design. Finally, minimally invasive multiple-valve surgery is known to have a steep learning curve; however, the surgeons operating on the patients of this study are experienced in the totally endoscopic RAMT technique, which impacts further on the selection bias of our study, where patients selected for a median sternotomy were excluded from a minimally invasive approach as a result of contraindications and limitations that could not be taken into account when doing propensity matching. As a result of these inherent biases of our study, we cannot generalize the conclusions of our comparisons therein to a more general population. Although our present results reflect our experience with minimally invasive valve surgery at our institutions, performed by surgeons experienced in its techniques, the results of the comparisons with median sternotomy should be carefully considered.
Endoscopic concomitant multiple-valve surgery through RAMT is a feasible, safe, and effective approach. This study shows that this approach for MIMVS is associated with low transfusion requirements, short hospital stay, and a low overall inhospital mortality and morbidity in selected patients. It was also associated with comparable CPB time and aortic cross clamping duration with MS, without increase in associated morbidities such as acute kidney injury or neurological events.
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Educational readiness among health professionals in rheumatology: low awareness of EULAR offerings and unfamiliarity with the course content as major barriers—results of a EULAR-funded European survey | 192e9f65-564b-4495-8c64-ac0ff5577cb6 | 10230966 | Pediatrics[mh] | Health professionals in rheumatology (HPR) are a heterogeneous group of professionals with different roles, responsibilities and scopes of practice. Their training differs depending on the level of professional qualification and varying health systems across countries. Educational needs of HPR differ considerably across countries and professions. While health professionals in rheumatology (HPR) in Europe have a solid knowledge about the existence of the European Alliance of Associations for Rheumatology (EULAR), they have little awareness about its educational offerings: approximately 90% do not know the ‘Teach the Teacher Course’, up to 80% are not aware of travel bursaries. English as the primary language in educational offerings of EULAR is a significant obstacle for more than 50% of HPR whose mother tongue is not English. Even among HPR who, ‘felt comfortable taking a course in English’, would prefer educational offerings in the national language (52%). Higher educated HPR and HPR with more extended work experience are more likely to attend the annual EULAR congress, whereas HPR with less work experience are more likely to take up EULAR School of Rheumatology offerings. Better-educated HPRs provide better care. Increasing awareness and use of EULAR educational offerings can increase up-to-date knowledge of HPR, promote research and collaboration and, ultimately, contribute to better patient outcomes. Active involvement of national HPR organisations is needed to ensure the leading role of EULAR in educational opportunities in rheumatology. Millions of people worldwide lack access to high-quality healthcare because of shortages, the uneven geographical distribution of service provision and not sufficiently trained health professionals. Higher education of health professionals contributes to better patient outcomes; a study even demonstrated that a higher number of nurses with a bachelor’s degree (compared with a diploma or an associate degree) reduced hospital mortality rates. To ensure that health professionals continue their education and training over a more extended period of their working life and, thus, contribute to a high quality of healthcare, many countries decided to make continuing education mandatory. As a result, health professionals are increasingly required to participate in continuing education after completing their basic training. Health professionals in rheumatology (HPR) play a critical role in the care of people with rheumatic and musculoskeletal diseases (RMDs). HPR are from different professions and include nurses, occupational therapists, physical therapists, social workers, psychologists, pharmacists and others. The EULAR definition of HPR does not include registered medical practitioners. Basic training varies across countries, and harmonised postgraduate education could guarantee that patients with similar diseases receive similar quality of care in different countries. Vliet Vlieland et al conducted an educational needs survey in 2015, being the first inventory of the educational needs of HPR in Europe. However, after the EULAR School of Rheumatology (ESOR) was launched in 2017, the educational needs of HPR were not reassessed, and EULAR’s new educational offerings for HPR have not yet been evaluated. In addition, changes in the legislation in some countries, such as the mandatory registration (and reregistrations) of HPR in Austria, Croatia, Cyprus, Ireland, Serbia, the UK and others and the need for accreditation of postgraduate courses in some countries, may also have led to changes in educational requirements. Compared to 2015, national offerings may have also changed over time. Moreover, the COVID-19 pandemic has significantly changed some of the didactic preferences of postgraduate education. For example, it has made online access to high-level specialists, unbound by time and space, more widely accepted. This study aimed to (1) determine current HPR educational readiness, needs and preferences, (2) identify barriers to taking part in postgraduate education and (3) ask for feedback on the current offerings and activities of ESOR for HPR. Design and participants The Educational Subcommittee of the EULAR Committee of HPR, in collaboration with the EULAR Committee of Education and Training and the Paediatric Rheumatology European Society, developed an online survey. The questionnaire was adapted from the 2015 version by Vliet Vlieland et al and extended to include questions asking for feedback on current courses. The following main topics were covered in the survey: (1) characteristics of the respondents, current educational needs in terms of clinical practice and theoretical knowledge, RMDs that should be addressed in the courses, wishes and expectations regarding the organisation of EULAR/ESOR courses, (2) barriers for participation in the courses, familiarity and awareness of the educational offerings from EULAR/ESOR, and (3) feedback on the current educational offerings of EULAR/ESOR. The online survey was distributed using a free software programme ( www.soscisurvey.com ). It was tested in advance by the Educational Sub-Committee of the EULAR Committee of HPR, and the feedback was used to adapt the questionnaire. We intended to distribute the questionnaire in as many European countries as possible. For this reason, we translated the questionnaire into 24 languages (Czech, Croatian, Danish, Dutch, English, Estonian, Finnish, French, German, Greek, Hungarian, Italian, Norwegian, Polish, Portuguese, Romanian, Russian, Serbian, Slovak, Slovenian, Spanish, Swedish, Turkish and Ukrainian). With these languages, we could cover the 25 national member organisations of EULAR and, at the same time, the 20 most populous European countries. Each translation was peer debriefed by at least two native-speaking HPR. The English version of the questionnaire is found in the supplement . The entire questionnaire, with all translations and response options, can be requested by the corresponding author. 10.1136/rmdopen-2023-003120.supp1 Supplementary data The questionnaire was distributed at the end of June 2021 via networks of the national professional organisations, the EULAR HPR Newsletter, participants in previous EULAR HPR activities, national liaison persons, national HPR associations, universities and other educational institutions with a request to forward it to all health professionals in the field of rheumatology. A similar invitation/reminder to participate in the questionnaire was sent in September 2021. Reporting of the results followed the ‘Checklist for Reporting Results of Internet E-Surveys’ guideline. Data analysis The responses of all participants were recorded anonymously. Absolute frequencies and percentages were given for categorical data. Ordinal variables were reported with median and IQR, metric variables with mean and SD as well as median and IQR, both for the whole study population and subgroups (HPR in adult care (HPR-A) and HPR in paediatric care (HPR-P)). As a final step, we conducted subgroup analyses for the three most prominent professional groups (nurses, physiotherapists, occupational therapists) and the North, South, East and Central European regions. We used natural language processing and the Latent Dirichlet Allocation (LDA) to analyse the questionnaire’s free text fields (feedback on current EULAR/ESOR offerings). LDA is a technique used for unsupervised generative probabilistic topic modelling. It aims to extract the meanings of a predefined number of topics. Each topic is characterised by high-frequency words and words that are best suited to distinguish it from other topics. To perform LDA, we created a semantic space by downloading and translating all the answers into English. Then, we took the following steps: stemming the words, removing stop words, casting the text into lowercase only and removing punctuations, names and personal references. We then explored the frequency of words and correlations between the co-occurrence of words and extracted five topics based on LDA. The number of topics was determined by examining the heatmaps and the coherence of the words in the topics. Heatmaps are a popular graphical method for visualising high-dimensional data. In a heatmap, tables of data are coded as coloured cells. The matrix’s rows and columns are arranged, so patterns become highlighted. The dendrogram reflects the inter-relationships of the topics and shows similarities between responses. Regression analysis We used multiple logistic regression models to determine higher postgraduate educational readiness factors. We operationalised educational readiness by means of four variables: first, in terms of knowledge (Have you ever heard of EULAR/ESOR?) and second, in terms of action taken (Have you ever attended the annual EULAR Congress/ESOR offerings?). The independent variables were selected from participants’ personal data (age, gender, occupation, work with children/adults, the highest level of education completed, work experience with people with RMD and time spent working in clinical patient care/research/organisational roles). All variables were tested for multicollinearity prior to the regression analysis. All analyses were conducted using R ( http://www.r-project.org ). The Educational Subcommittee of the EULAR Committee of HPR, in collaboration with the EULAR Committee of Education and Training and the Paediatric Rheumatology European Society, developed an online survey. The questionnaire was adapted from the 2015 version by Vliet Vlieland et al and extended to include questions asking for feedback on current courses. The following main topics were covered in the survey: (1) characteristics of the respondents, current educational needs in terms of clinical practice and theoretical knowledge, RMDs that should be addressed in the courses, wishes and expectations regarding the organisation of EULAR/ESOR courses, (2) barriers for participation in the courses, familiarity and awareness of the educational offerings from EULAR/ESOR, and (3) feedback on the current educational offerings of EULAR/ESOR. The online survey was distributed using a free software programme ( www.soscisurvey.com ). It was tested in advance by the Educational Sub-Committee of the EULAR Committee of HPR, and the feedback was used to adapt the questionnaire. We intended to distribute the questionnaire in as many European countries as possible. For this reason, we translated the questionnaire into 24 languages (Czech, Croatian, Danish, Dutch, English, Estonian, Finnish, French, German, Greek, Hungarian, Italian, Norwegian, Polish, Portuguese, Romanian, Russian, Serbian, Slovak, Slovenian, Spanish, Swedish, Turkish and Ukrainian). With these languages, we could cover the 25 national member organisations of EULAR and, at the same time, the 20 most populous European countries. Each translation was peer debriefed by at least two native-speaking HPR. The English version of the questionnaire is found in the supplement . The entire questionnaire, with all translations and response options, can be requested by the corresponding author. 10.1136/rmdopen-2023-003120.supp1 Supplementary data The questionnaire was distributed at the end of June 2021 via networks of the national professional organisations, the EULAR HPR Newsletter, participants in previous EULAR HPR activities, national liaison persons, national HPR associations, universities and other educational institutions with a request to forward it to all health professionals in the field of rheumatology. A similar invitation/reminder to participate in the questionnaire was sent in September 2021. Reporting of the results followed the ‘Checklist for Reporting Results of Internet E-Surveys’ guideline. The responses of all participants were recorded anonymously. Absolute frequencies and percentages were given for categorical data. Ordinal variables were reported with median and IQR, metric variables with mean and SD as well as median and IQR, both for the whole study population and subgroups (HPR in adult care (HPR-A) and HPR in paediatric care (HPR-P)). As a final step, we conducted subgroup analyses for the three most prominent professional groups (nurses, physiotherapists, occupational therapists) and the North, South, East and Central European regions. We used natural language processing and the Latent Dirichlet Allocation (LDA) to analyse the questionnaire’s free text fields (feedback on current EULAR/ESOR offerings). LDA is a technique used for unsupervised generative probabilistic topic modelling. It aims to extract the meanings of a predefined number of topics. Each topic is characterised by high-frequency words and words that are best suited to distinguish it from other topics. To perform LDA, we created a semantic space by downloading and translating all the answers into English. Then, we took the following steps: stemming the words, removing stop words, casting the text into lowercase only and removing punctuations, names and personal references. We then explored the frequency of words and correlations between the co-occurrence of words and extracted five topics based on LDA. The number of topics was determined by examining the heatmaps and the coherence of the words in the topics. Heatmaps are a popular graphical method for visualising high-dimensional data. In a heatmap, tables of data are coded as coloured cells. The matrix’s rows and columns are arranged, so patterns become highlighted. The dendrogram reflects the inter-relationships of the topics and shows similarities between responses. We used multiple logistic regression models to determine higher postgraduate educational readiness factors. We operationalised educational readiness by means of four variables: first, in terms of knowledge (Have you ever heard of EULAR/ESOR?) and second, in terms of action taken (Have you ever attended the annual EULAR Congress/ESOR offerings?). The independent variables were selected from participants’ personal data (age, gender, occupation, work with children/adults, the highest level of education completed, work experience with people with RMD and time spent working in clinical patient care/research/organisational roles). All variables were tested for multicollinearity prior to the regression analysis. All analyses were conducted using R ( http://www.r-project.org ). Response rate In total, the questionnaire was viewed 3589 times, and 998 times it was started to be filled in. Of these, 667 HPR (66.8%) from 34 European countries completed the questionnaire ( and ). Participant characteristics The respondents were mainly women (n=762; 76.4%), and the mean age was 40.53 (±11.5) years. More than 50% of the participants had a bachelor’s degree or similar, 24.9% (n=245) had a master’s degree and 9.1% (n=90) had a PhD . Physiotherapists (n=350; 34.8%), nurses (n=248; 25.0%) and occupational therapists (n=189; 18.8%) were the most frequent professions who responded to the questionnaire. Of all responses, n=913 (91.5%) came from HPR working with adults, and n=85 (8.5%) from HPR in children’s/youth’s care. Educational readiness In terms of knowledge, familiarity with EULAR or ESOR was mainly associated with being a nurse, being older (in years), having more experience in the field of rheumatology, having a formally higher level of education and being more involved in research. We observed a similar pattern in participation at the annual EULAR congress. Nurses and HPR with higher levels of education and increased research activities were also associated with taking part in the congress. This result contrasts with attendance in ESOR courses. In particular, people with less work experience seem to feel more addressed by the offer than more experienced people (OR 0.91, CI 95% 0.81 to 0.99) . 10.1136/rmdopen-2023-003120.supp3 Supplementary data Educational needs: differences between health professionals in adult and paediatric care The highest-rated educational need in terms of clinical practice was professional development for paediatric HPR (3.91; on a scale of 1 to 5) and prevention for HPR in adult care (3.70). ‘Clinical characteristics’ were rated as the most important theoretical knowledge for both HPR-A and HPR-P (3.62 and 4.00, respectively). Practice organisation and management were rated lowest by HPR-A (3.08), and training in diagnostic assessments by HPR-P (2.78). According to the ratings provided by HPR-A and HPR-P, the RMDs that need to be covered in the courses received all high scores, ranging from 3.76 to 4.49 . Course organisation: live courses and prerecorded (online) lectures were preferred Live courses taking place face-to-face (n=463; 38.6%) and prerecorded online lectures accessible without time constraints (n=338; 28.2%) were preferred over other modalities. Almost half of the participants (n=377; 44.0%) considered a course duration of 1–2 days optimal, and two-thirds of HPR preferred the organisation of the courses by EULAR compared with national organisations. However, following the responses of the HPR, offerings should be available in national languages rather than in English . Even among HPR who said they ‘felt comfortable taking a course in English’ (n=419), more than half would still prefer courses in the national language (n=219; 52.3%). Barriers: lack of awareness of educational offerings and content Lack of awareness of the educational offerings (3.30; ±1.49) and lack of knowledge of the content of the offerings (3.51; ±1.50) were mentioned as the most important barriers to non-participation in EULAR/ESOR educational offerings (on a scale of 1–5). Likewise, the participants perceived the costs of ESOR courses (3.29; ±1.36) and the annual congress (3.46; ±1.34) as too high. Participants reported that despite their interest in EULAR’s educational offerings, they did not receive support in the form of time resources during work hours from their employers to attend the courses (3.32; ±1.39). The technical requirements for participation in an online course, such as the availability of computers, laptops or tablets, and an internet connection, were not considered obstacles to participation in the courses or classes . Feedback on the current offerings of ESOR for HPR Although almost two-thirds of HPR-A and half of HPR-P were familiar with EULAR, they reported little awareness or use of EULAR’s offerings for HPR. Three-quarters of HPR only had limited or no knowledge of travel bursaries, and 85% (HPR-A), respectively, 97% (HPR-P), were not aware of the Teach the Teacher Course. Sixty to 80% have never visited the EULAR/ESOR communication platforms, and 73.9% (HPR-A), respectively 90.7% (HPR-P) had never participated in the annual EULAR congress . Using LDA, we generated two topics from positive feedback and three from negative feedback ( and ). The comprehensive and up-to-date course content, along with the flexibility to attend classes without any time restrictions, were highly valued, resulting in overall positive responses ( online courses offer time flexibility and the possibility to complete the course at your own pace ). Barriers to participation in training were: uncertainty if there would be a profession-specific gain from participating in interdisciplinary courses ( The online course should have a clear benefit for the respective professional groups, even if it is a multidisciplinary course ), language limitations during the classes ( English is challenging in classes, especially when you want to ask questions ) and problems with the English language in the final exams ( English is challenging, especially in the exams ). 10.1136/rmdopen-2023-003120.supp2 Supplementary data Differences in educational preferences but not the delivery method As expected, our subgroup analysis revealed differences in educational preferences related to course content across professions and countries. However, we found surprisingly limited variation in the preferences for the delivery method of educational offerings. The most common selection for live/on-site courses lasting 1–2 days and being conducted in the national language remained consistent across professions and countries. The only tendency found (not significant, p=0.056) was that Eastern countries favoured discipline-specific content (37.1% to 34.3% multidisciplinary) compared with the other countries. Nurses were more likely to attend the EULAR Congress (36.3%) than other professional groups (PT 16.9%, OT 12.6%; OR 2.35 CI 95% 0.98 to 5.87, p=0.05). Detailed results of the subgroup analysis are found in appendices E-I and J-O. In total, the questionnaire was viewed 3589 times, and 998 times it was started to be filled in. Of these, 667 HPR (66.8%) from 34 European countries completed the questionnaire ( and ). The respondents were mainly women (n=762; 76.4%), and the mean age was 40.53 (±11.5) years. More than 50% of the participants had a bachelor’s degree or similar, 24.9% (n=245) had a master’s degree and 9.1% (n=90) had a PhD . Physiotherapists (n=350; 34.8%), nurses (n=248; 25.0%) and occupational therapists (n=189; 18.8%) were the most frequent professions who responded to the questionnaire. Of all responses, n=913 (91.5%) came from HPR working with adults, and n=85 (8.5%) from HPR in children’s/youth’s care. In terms of knowledge, familiarity with EULAR or ESOR was mainly associated with being a nurse, being older (in years), having more experience in the field of rheumatology, having a formally higher level of education and being more involved in research. We observed a similar pattern in participation at the annual EULAR congress. Nurses and HPR with higher levels of education and increased research activities were also associated with taking part in the congress. This result contrasts with attendance in ESOR courses. In particular, people with less work experience seem to feel more addressed by the offer than more experienced people (OR 0.91, CI 95% 0.81 to 0.99) . 10.1136/rmdopen-2023-003120.supp3 Supplementary data The highest-rated educational need in terms of clinical practice was professional development for paediatric HPR (3.91; on a scale of 1 to 5) and prevention for HPR in adult care (3.70). ‘Clinical characteristics’ were rated as the most important theoretical knowledge for both HPR-A and HPR-P (3.62 and 4.00, respectively). Practice organisation and management were rated lowest by HPR-A (3.08), and training in diagnostic assessments by HPR-P (2.78). According to the ratings provided by HPR-A and HPR-P, the RMDs that need to be covered in the courses received all high scores, ranging from 3.76 to 4.49 . Live courses taking place face-to-face (n=463; 38.6%) and prerecorded online lectures accessible without time constraints (n=338; 28.2%) were preferred over other modalities. Almost half of the participants (n=377; 44.0%) considered a course duration of 1–2 days optimal, and two-thirds of HPR preferred the organisation of the courses by EULAR compared with national organisations. However, following the responses of the HPR, offerings should be available in national languages rather than in English . Even among HPR who said they ‘felt comfortable taking a course in English’ (n=419), more than half would still prefer courses in the national language (n=219; 52.3%). Lack of awareness of the educational offerings (3.30; ±1.49) and lack of knowledge of the content of the offerings (3.51; ±1.50) were mentioned as the most important barriers to non-participation in EULAR/ESOR educational offerings (on a scale of 1–5). Likewise, the participants perceived the costs of ESOR courses (3.29; ±1.36) and the annual congress (3.46; ±1.34) as too high. Participants reported that despite their interest in EULAR’s educational offerings, they did not receive support in the form of time resources during work hours from their employers to attend the courses (3.32; ±1.39). The technical requirements for participation in an online course, such as the availability of computers, laptops or tablets, and an internet connection, were not considered obstacles to participation in the courses or classes . Although almost two-thirds of HPR-A and half of HPR-P were familiar with EULAR, they reported little awareness or use of EULAR’s offerings for HPR. Three-quarters of HPR only had limited or no knowledge of travel bursaries, and 85% (HPR-A), respectively, 97% (HPR-P), were not aware of the Teach the Teacher Course. Sixty to 80% have never visited the EULAR/ESOR communication platforms, and 73.9% (HPR-A), respectively 90.7% (HPR-P) had never participated in the annual EULAR congress . Using LDA, we generated two topics from positive feedback and three from negative feedback ( and ). The comprehensive and up-to-date course content, along with the flexibility to attend classes without any time restrictions, were highly valued, resulting in overall positive responses ( online courses offer time flexibility and the possibility to complete the course at your own pace ). Barriers to participation in training were: uncertainty if there would be a profession-specific gain from participating in interdisciplinary courses ( The online course should have a clear benefit for the respective professional groups, even if it is a multidisciplinary course ), language limitations during the classes ( English is challenging in classes, especially when you want to ask questions ) and problems with the English language in the final exams ( English is challenging, especially in the exams ). 10.1136/rmdopen-2023-003120.supp2 Supplementary data As expected, our subgroup analysis revealed differences in educational preferences related to course content across professions and countries. However, we found surprisingly limited variation in the preferences for the delivery method of educational offerings. The most common selection for live/on-site courses lasting 1–2 days and being conducted in the national language remained consistent across professions and countries. The only tendency found (not significant, p=0.056) was that Eastern countries favoured discipline-specific content (37.1% to 34.3% multidisciplinary) compared with the other countries. Nurses were more likely to attend the EULAR Congress (36.3%) than other professional groups (PT 16.9%, OT 12.6%; OR 2.35 CI 95% 0.98 to 5.87, p=0.05). Detailed results of the subgroup analysis are found in appendices E-I and J-O. This study aimed to identify the educational needs of HPR in paediatric and adult care. For the first time, we anonymously asked for feedback on the current educational offerings of EULAR/ESOR in 34 countries and described current barriers to the attendance of EULAR/ESOR offerings. It is already widely recognised that the successful implementation of postgraduate HPR education is of great importance to ‘increase the quantity, quality and relevance of health professionals, and in so doing strengthen the country health systems and improve population health outcomes’. Our study’s two most important findings were that (1) EULAR only succeeds in reaching a minority of HPRs in Europe and (2) that services do not appeal equally to the broad range and different educational levels of HPRs. Our study found that higher age, more professional experience, being a nurse, and a higher level of education contributed significantly to the knowledge of EULAR and ESOR and attendance at the annual EULAR congress. In terms of higher education level, we were able to show, for example, that having a PhD increases the likelihood of knowing about ESOR (OR 3.70) and whether one has ever attended the EULAR congress (OR 7.50). Accordingly, the EULAR congress is (more) attractive to older and formally more educated HPR. However, we observed a contrasting picture with the ESOR offerings. The courses were more likely to be attended by HPRs with less experience in the field of rheumatology (OR 0.91). Several strategies can be derived from our findings on how to achieve a better use of educational offerings among HPR in Europe. Our findings suggest that several strategies can be implemented to enhance the utilisation of educational offerings among HPR in Europe. For instance, educational providers could target HPR at earlier stages of their career or those with less formal education by reviewing their existing offerings to identify which groups of HPR are being addressed. This would enable providers to develop tailored educational programmes that meet the specific needs of these groups, thereby increasing their engagement and participation. To address the varying educational qualification levels of HPR in Europe, educational courses could be developed at different levels to lower the access barrier for early career HPR while also providing more formally educated and later career HPR with opportunities to continue their education at a higher level. This could be achieved by involving national organisations in disseminating educational offerings through their networks in national languages. Strengthening these organisations would increase the accessibility of educational opportunities to HPR in different regions of Europe. To minimise costs for EULAR while enhancing the utilisation of its course offerings in national countries, a ‘franchise’ model could be considered in addition to the teach-the-teacher courses. Under this model, EULAR would act as a ‘franchisor’ and permit national organisations to use its brand and course offerings. In exchange, national organisations would agree to pay a franchise fee to EULAR. Another important finding of our survey was that English as an educational and examination language is a major barrier for HPR, whose native language is not English. Between 40% and 55% of all respondents indicated they did not feel comfortable taking a course in English. This problem was particularly cited when HPR were under time pressure and had to ask questions or take an exam in English. Even among HPR who reported having sufficient English competence to feel comfortable taking a course in English, more than half would still prefer their national language. HPR from northern countries (Estonia, Denmark, Finland, Norway, Sweden) were more likely to consider themselves advanced or fluent in English than HPR from other countries; however, the majority of them (58.8%) still preferred their national language. Therefore, one feasible approach to increase attendance in EULAR courses could be to translate the content (and exams) or offer courses with subtitles in the national language. English as a language was also identified in the first Education Needs Survey as an important barrier to using EULAR offerings. Apparently, this has not changed in the last 7 years. One lesson learnt from the survey is that EULAR messages/evidence may need to be translated into other languages to facilitate their implementation in clinical care across Europe. A strength of our study was that we also analysed the free text entries (positive and negative feedback to the ESOR classes) with a natural language processing tool. We applied an unsupervised generative probabilistic method specifically designed for short text inputs, such as those typically found in questionnaires. The results of our project go beyond the interests of EULAR and ESOR. Although the findings are initially based on EULAR’s existing educational programme, they can also be useful for national education providers, for example, with regards to preferences in course content or delivery methods. We are aware that our study has certain limitations. Although we translated the questionnaire into 24 languages, we could not exceed the overall response rate of the 2015 survey. Our study had limited representation from northern and eastern European countries. One reason may have been the timing during the (summer) vacations and/or the COVID-19 pandemic, which limited overall opportunities for training and education and may have changed the priorities for HPR. The results of our survey show that the proportions of some professions were different across the countries. In Portugal and Denmark, for example, more nurses participated compared with other countries, such as Austria or Italy. Such differences could limit the generalisability of our results. In addition, we had some missing data because it was not made mandatory to answer all questions in the survey. We decided not to make the responses mandatory because we wanted to give HPR as much freedom as possible in responding to the questions. However, the missing data could, of course, distort the results. Due to the study design and the partly unequally distributed responses in terms of country and profession, the sample’s representativeness is not given, and the results should be generalised only with caution. HPR is often involved in supporting people with RMD in managing their disease. Their knowledge directly impacts people’s well-being and is critical to utilising other health resources. Therefore, ongoing education and training for the various healthcare professions are essential to ensure optimal care. Improving awareness of educational offers among national organisations and the challenge of reducing costs and language barriers are points of attention to promote future dissemination. EULAR and other international postgraduate training providers could use a ‘franchise’ model of their offerings tailored to local contexts. 10.1136/rmdopen-2023-003120.supp4 Supplementary data 10.1136/rmdopen-2023-003120.supp5 Supplementary data 10.1136/rmdopen-2023-003120.supp6 Supplementary data |
Evaluation of ready-to-use freezer stocks of a synthetic microbial community for maize root colonization | 96dc0a32-52bd-4783-a801-d3b95d37d9ab | 10783020 | Microbiology[mh] | Plant-associated microbiota play key roles in plant evolution, development, health, and stress resilience; studying these roles is critical for understanding the fundamental principles of plant biology and developing and applying biotechnology for sustainable agriculture ( ). Microorganisms improve plant host fitness by facilitating nutrient uptake ( ), providing defense against pathogens ( , ), and alleviating abiotic stress ( ). Dissecting and using the microbial mechanisms that benefit plants is becoming more important as increasing global population and climate change place greater demands on sustainable agricultural production ( ). Although the initial assembly of plant-associated microbiota is influenced by stochastic processes, mounting evidence suggests that healthy plants can select particular microorganisms for establishing beneficial communities that are complex and structured, yet reproducible ( ). Synthetic microbial communities (SynComs) that reduce the complexity of the plant-associated microbiota while maintaining key structures and functions allow for hypothesis-driven experiments with reproducible conditions ( , , ). Experiments using SynComs in gnotobiotic plant systems have described assembly patterns resulting from specific plant–microbe ( ) and microbe–microbe interactions ( , ), identified keystone species and assembly patterns ( , ), determined microbial niche specialization ( ), and led to the discovery of microbe-dependent heterosis in maize ( ). In short, SynComs are a powerful tool for unraveling complex plant−microbe and microbe–microbe interactions and defining the plant holobiont. Reproducible construction of even mildly complex SynComs requires significant time and labor. Recent evidence highlights the importance of reproducible SynCom construction, suggesting that the inoculation ratio of community members influences microbial community interactions ( ). Additionally, time- and labor-intensive construction of SynComs would become cost-prohibitive for SynComs developed as commercial products for widespread application in agriculture. In order to improve consistency between experiments and extend concepts and plant-beneficial mechanisms to agricultural production, new approaches to building SynComs are required that are less labor-intensive while providing consistent ratios of SynCom members. Here, we explore the stability and efficacy of fresh and frozen aliquots of a seven-member SynCom that has been developed as a simplified and representative SynCom for maize ( ). We used root colonization following inoculation on maize seeds to measure the stability and efficacy.
Preparation of fresh and frozen SynCom inocula The synthetic community used in this study contained the following bacterial species: Stenotrophomonas maltophilia AA1 (ZK5342), Brucella pituitosa AA2 (ZK5343), Curtobacterium pusillum AA3 (ZK5344), Enterobacter ludwigii AA4 (ZK5345), Chryseobacterium indologenes AA5 (ZK5346), Herbaspirillum robiniae AA6 (ZK5347), and Pseudomonas putida AA7 (ZK5348), as described in Niu et al. ( , ). Following those same studies, we used the “Sugar Bun” (Johnny’s Seeds, Cat. 267T) variety of maize as the plant host throughout this study. To determine the variability between different SynCom preparation events and the impact of freezing on the viability of the SynComs, we conducted two experiments in which four different SynCom master mixes were prepared and inoculated onto plants either directly (fresh) or after freezing. To determine if the duration of freezing had an impact on SynCom viability, we tested the mixes after 1 hour and after 1 week of freezing ( ). We constructed the synthetic community following the previously published protocol by Niu and Kolter ( ) with some modifications. We streaked freezer stocks of each species onto selective 0.1 x tryptic soy agar plates with species-specific antibiotics and then incubated the plates at 30°C for 2 days. Individual colonies from plates were inoculated into 5 mL of tryptic soy broth without dextrose (VWR) and shaken for 8 hours at 30°C. We transferred 0.1 mL of each species’ culture to a separate 250-mL flask with 125 mL of tryptic soy broth, and the flasks were shaken at 30°C for 14 to 16 hours. We centrifuged the cultures at 8,000 × g for 10 min at 4°C. Cell pellets were washed with 5 mL of phosphate-buffered saline (PBS) at pH 7.4 (Fisher Scientific), re-pelleted by centrifugation at 8,000 × g for 10 min at 4°C, and then re-suspended in 10 mL PBS. We combined species to build a SynCom master mix with S. maltophilia and P. putida at 10 7 cells/mL and all other species at 10 8 cells/mL. The concentrations were estimated from optical density (OD) readings calibrated to cells/mL using direct cell counting under a microscope. The SynCom master mix was divided into aliquots for plate counting, creating freezer stocks, and direct inoculation of the fresh microbial community onto sterilized maize seeds. We prepared fresh and frozen, ready-to-use stocks of the SynCom in the same exact way as follows: 1 mL of the master mix was added to cryotubes containing 0.7 mL of sterilized PBS:glycerol (1:1), yielding a final volume of 1.7 mL with 20% glycerol. The fresh stocks were never frozen. To use a stock, 1.7 mL of the stock was mixed with 8.3 mL PBS to yield a 10 mL inoculum with a final concentration of 10 6 –10 7 cells/mL. The resulting 10 mL of 10 6 –10 7 cells/mL SynCom mix (fresh or frozen) was added per liter of 0.5 × Murashige and Skoog medium (Caisson labs), yielding a final concentration of 10 4 –10 5 cells/mL of each bacterial species. This mix was then used for plant inoculation as described below. Four replicate SynCom master mixes were created for two experiments over the course of the study. In the first experiment (Experiment#1), a SynCom mix (master mix 1) was prepared and frozen at −80°C. One week later, another SynCom mix (master mix 2) was prepared and divided into a fresh and a frozen aliquot. The fresh aliquot from master mix 2 was immediately inoculated into surface-sterilized maize seeds (Fresh #1). At the same time, the frozen aliquot from master mix 1 was thawed and inoculated (Experiment 1: Frozen 7-day). The frozen aliquot of master mix 2 was frozen at −80°C for 1 hour; thereafter the mix was thawed and inoculated on the same day (Experiment 1: Frozen 1 h). In the second experiment (Experiment#2), two more SynCom mixes were prepared separately (master mix 3 and master mix 4). Master mix 3 was divided into three aliquots one to be inoculated fresh the same day (Fresh #2), after being frozen for 1 hour (Experiment 2: Frozen 1 h), and after being frozen for 1 week (Experiment 2: Frozen 7-day). Master mix 4 was prepared and inoculated fresh (Fresh #3) on the day that Frozen 7-day (Experiment 2) was inoculated into surface-sterilized maize seeds. Inoculation of surface-sterilized maize seeds and growth In a laminar flow hood, we surface-sterilized Sugar Bun seeds by submerging them in 70% (vol/vol) ethanol for 3 minutes, followed by submerging them in 2% (vol/vol) sodium hypochlorite solution for 3 minutes, and then washing them 10 times with sterilized deionized water. The final wash was plated onto 0.1X TSA plates to test for residual contamination after the sterilization procedure, as described by Niu et al. ( ). We inoculated 15 surface-sterilized seeds with the fresh SynCom inocula and 15 surface-sterilized seeds with the 1 hour frozen SynCom inocula, as previously described ( ). Surface-sterilized seeds were placed in sterile 7.5” × 15” Whirl-pak bags (Nabisco) filled with 150 mL of a calcined clay (“Pro’s Choice Rapid Dry”; Oil-Dri Corporation) and inoculated with 90 mL of 0.5 × Murashige and Skoog medium containing the SynCom. We sealed the bags with sterile AeraSeal breathable film (Excel Scientific, Inc.) to keep the system sterile while allowing for aeration. Bags were randomized and placed on a shelf with light-emitting diode (LED) growth lights (16 hours light, 8 hours dark, 23°C, ambient humidity). Emergence of seedlings was documented daily, and plants were harvested at 10 days post-emergence. Root harvest and absolute quantification of live microbial cells colonizing the roots Roots from 9 to 10 plants were harvested for the colonization assay according to Niu and Kolter ( ) with some modifications. Plants were removed from bags, and the roots were gently rinsed in deionized water. The whole primary root from each plant was harvested, and fresh weights were recorded (200–500 mg fresh weight). Roots were cut into small pieces using a sterile knife and vortexed in a 2-mL tube for 3 min in 1 mL of sterile PBS with six 3 mm sterilized glass beads to recover microbial cells from the root surface, as described in Niu and Kolter ( ). We serially diluted the cell suspension in PBS (10 −1 -10 −8 ) and plated the dilutions on species-specific selective media, as previously described ( ). After incubation for the required time for selective growth (16–60 hours at 30°C), the colonies were counted and counts normalized against root fresh weight. Viability of SynCom members following longer-term freezing To test the impact of freezing on the viability of each species in the SynCom mix, we prepared a SynCom master mix with 10 8 cells/mL for each species and prepared fresh and frozen, ready-to-use stocks of the SynCom from the master mix, as described previously. We measured the viability of each strain in fresh vials and vials from the same batch frozen for 1 hour, 7 days, and 4 months. To determine the viability, vials were diluted to 10 7 cells/mL in PBS and a dilution series (10 −1 -10 −8 ) was prepared for each mix. A total of 10 µL of each dilution in the series was plated in technical triplicates on selective plates according to Niu and Kolter ( ). Plates were incubated for 16–60 hours at 30°C, and the resulting colonies were counted. Statistical analysis To detect statistical differences between different SynCom master mixes, one-way ANOVA followed by Tukey’s HSD was performed on the data set using RStudio (1.4.1106) statistical software ( , ). Adjustment of P -values was done using the Benjamini–Hochberg method. For each experiment, log CFUs / g fresh weight of each species were tested separately against the type of the master mix used to inoculate the plants. Assumptions of normality were satisfied by looking at the data distribution along normal Q-Q plots. Assumptions of equal variance were tested using the Bartlett test.
The synthetic community used in this study contained the following bacterial species: Stenotrophomonas maltophilia AA1 (ZK5342), Brucella pituitosa AA2 (ZK5343), Curtobacterium pusillum AA3 (ZK5344), Enterobacter ludwigii AA4 (ZK5345), Chryseobacterium indologenes AA5 (ZK5346), Herbaspirillum robiniae AA6 (ZK5347), and Pseudomonas putida AA7 (ZK5348), as described in Niu et al. ( , ). Following those same studies, we used the “Sugar Bun” (Johnny’s Seeds, Cat. 267T) variety of maize as the plant host throughout this study. To determine the variability between different SynCom preparation events and the impact of freezing on the viability of the SynComs, we conducted two experiments in which four different SynCom master mixes were prepared and inoculated onto plants either directly (fresh) or after freezing. To determine if the duration of freezing had an impact on SynCom viability, we tested the mixes after 1 hour and after 1 week of freezing ( ). We constructed the synthetic community following the previously published protocol by Niu and Kolter ( ) with some modifications. We streaked freezer stocks of each species onto selective 0.1 x tryptic soy agar plates with species-specific antibiotics and then incubated the plates at 30°C for 2 days. Individual colonies from plates were inoculated into 5 mL of tryptic soy broth without dextrose (VWR) and shaken for 8 hours at 30°C. We transferred 0.1 mL of each species’ culture to a separate 250-mL flask with 125 mL of tryptic soy broth, and the flasks were shaken at 30°C for 14 to 16 hours. We centrifuged the cultures at 8,000 × g for 10 min at 4°C. Cell pellets were washed with 5 mL of phosphate-buffered saline (PBS) at pH 7.4 (Fisher Scientific), re-pelleted by centrifugation at 8,000 × g for 10 min at 4°C, and then re-suspended in 10 mL PBS. We combined species to build a SynCom master mix with S. maltophilia and P. putida at 10 7 cells/mL and all other species at 10 8 cells/mL. The concentrations were estimated from optical density (OD) readings calibrated to cells/mL using direct cell counting under a microscope. The SynCom master mix was divided into aliquots for plate counting, creating freezer stocks, and direct inoculation of the fresh microbial community onto sterilized maize seeds. We prepared fresh and frozen, ready-to-use stocks of the SynCom in the same exact way as follows: 1 mL of the master mix was added to cryotubes containing 0.7 mL of sterilized PBS:glycerol (1:1), yielding a final volume of 1.7 mL with 20% glycerol. The fresh stocks were never frozen. To use a stock, 1.7 mL of the stock was mixed with 8.3 mL PBS to yield a 10 mL inoculum with a final concentration of 10 6 –10 7 cells/mL. The resulting 10 mL of 10 6 –10 7 cells/mL SynCom mix (fresh or frozen) was added per liter of 0.5 × Murashige and Skoog medium (Caisson labs), yielding a final concentration of 10 4 –10 5 cells/mL of each bacterial species. This mix was then used for plant inoculation as described below. Four replicate SynCom master mixes were created for two experiments over the course of the study. In the first experiment (Experiment#1), a SynCom mix (master mix 1) was prepared and frozen at −80°C. One week later, another SynCom mix (master mix 2) was prepared and divided into a fresh and a frozen aliquot. The fresh aliquot from master mix 2 was immediately inoculated into surface-sterilized maize seeds (Fresh #1). At the same time, the frozen aliquot from master mix 1 was thawed and inoculated (Experiment 1: Frozen 7-day). The frozen aliquot of master mix 2 was frozen at −80°C for 1 hour; thereafter the mix was thawed and inoculated on the same day (Experiment 1: Frozen 1 h). In the second experiment (Experiment#2), two more SynCom mixes were prepared separately (master mix 3 and master mix 4). Master mix 3 was divided into three aliquots one to be inoculated fresh the same day (Fresh #2), after being frozen for 1 hour (Experiment 2: Frozen 1 h), and after being frozen for 1 week (Experiment 2: Frozen 7-day). Master mix 4 was prepared and inoculated fresh (Fresh #3) on the day that Frozen 7-day (Experiment 2) was inoculated into surface-sterilized maize seeds.
In a laminar flow hood, we surface-sterilized Sugar Bun seeds by submerging them in 70% (vol/vol) ethanol for 3 minutes, followed by submerging them in 2% (vol/vol) sodium hypochlorite solution for 3 minutes, and then washing them 10 times with sterilized deionized water. The final wash was plated onto 0.1X TSA plates to test for residual contamination after the sterilization procedure, as described by Niu et al. ( ). We inoculated 15 surface-sterilized seeds with the fresh SynCom inocula and 15 surface-sterilized seeds with the 1 hour frozen SynCom inocula, as previously described ( ). Surface-sterilized seeds were placed in sterile 7.5” × 15” Whirl-pak bags (Nabisco) filled with 150 mL of a calcined clay (“Pro’s Choice Rapid Dry”; Oil-Dri Corporation) and inoculated with 90 mL of 0.5 × Murashige and Skoog medium containing the SynCom. We sealed the bags with sterile AeraSeal breathable film (Excel Scientific, Inc.) to keep the system sterile while allowing for aeration. Bags were randomized and placed on a shelf with light-emitting diode (LED) growth lights (16 hours light, 8 hours dark, 23°C, ambient humidity). Emergence of seedlings was documented daily, and plants were harvested at 10 days post-emergence.
Roots from 9 to 10 plants were harvested for the colonization assay according to Niu and Kolter ( ) with some modifications. Plants were removed from bags, and the roots were gently rinsed in deionized water. The whole primary root from each plant was harvested, and fresh weights were recorded (200–500 mg fresh weight). Roots were cut into small pieces using a sterile knife and vortexed in a 2-mL tube for 3 min in 1 mL of sterile PBS with six 3 mm sterilized glass beads to recover microbial cells from the root surface, as described in Niu and Kolter ( ). We serially diluted the cell suspension in PBS (10 −1 -10 −8 ) and plated the dilutions on species-specific selective media, as previously described ( ). After incubation for the required time for selective growth (16–60 hours at 30°C), the colonies were counted and counts normalized against root fresh weight.
To test the impact of freezing on the viability of each species in the SynCom mix, we prepared a SynCom master mix with 10 8 cells/mL for each species and prepared fresh and frozen, ready-to-use stocks of the SynCom from the master mix, as described previously. We measured the viability of each strain in fresh vials and vials from the same batch frozen for 1 hour, 7 days, and 4 months. To determine the viability, vials were diluted to 10 7 cells/mL in PBS and a dilution series (10 −1 -10 −8 ) was prepared for each mix. A total of 10 µL of each dilution in the series was plated in technical triplicates on selective plates according to Niu and Kolter ( ). Plates were incubated for 16–60 hours at 30°C, and the resulting colonies were counted.
To detect statistical differences between different SynCom master mixes, one-way ANOVA followed by Tukey’s HSD was performed on the data set using RStudio (1.4.1106) statistical software ( , ). Adjustment of P -values was done using the Benjamini–Hochberg method. For each experiment, log CFUs / g fresh weight of each species were tested separately against the type of the master mix used to inoculate the plants. Assumptions of normality were satisfied by looking at the data distribution along normal Q-Q plots. Assumptions of equal variance were tested using the Bartlett test.
Four replicate SynCom master mixes were created at separate time points to test for differences in maize root colonization in terms of bacterial species colony-forming unit counts due to (1) duration of freezer storage or (2) SynCom preparation. Master mixes 1 and 2 had aliquots frozen for 1 hour or 7 days. Master mix 3 was used in its fresh form, frozen for 1 hour, or frozen for 7 days. Master mix 4 was used only in its fresh form in comparison to the 7 days frozen master mix 3 ( ). master mix 4 and master mix 3 were compared to determine the differences between different construction events and between fresh and frozen SynComs. Master mixes contained 10 7 cells/mL of Stenotrophomonas maltophilia and Pseudomonas putida and 10 8 cells/mL of Brucella pituitosa , Curtobacterium pusillum , Enterobacter ludwigii , Chryseobacterium indologenes , and Herbaspirillum robiniae . Variation of species abundances in roots from plants inoculated with fresh SynComs We inoculated sterilized seeds in calcined clay with Murashige and Skoog medium containing the SynCom bacteria and grew the plants under controlled conditions for 10 days post-emergence before we harvested roots for bacterial colony counting (see Materials and Methods). Comparing roots of plants inoculated with the same SynCom inoculum (e.g., master mix #2 frozen for 1 hour) showed that, for all inocula, the abundance (log CFUs/g of root fresh weight) of each SynCom species was highly variable between replicate plants. The standard deviations for species abundances in replicate plants ranged from a log-value of 0.18 ( E. ludwigii and C. pusillum ) to 2.93 ( H. robiniae ) ( ). Comparing roots of plants inoculated with different master mixes showed that the abundances varied considerably for some species ( ). All of the species had significantly higher abundances (CFUs/g of fresh root weight) in fresh mix 2 as compared to fresh mix 3 ( P < 0.05, ). When comparing fresh mixes 1 and 2 again, all species were found to have significantly higher abundances (CFUs/g of fresh root weight) in fresh mix 2 as compared to fresh mix 1 ( P < 0.05, ), with the exception of S. maltophilia ( ). None of the SynCom members were significantly different in terms of CFUs/g of fresh root weight between fresh mixes 1 and 3. Impact of freezing SynCom stocks on species abundances in roots Abundances of each SynCom species from plants inoculated with the frozen SynCom stocks compared with the corresponding fresh SynCom master mixes suggest that freezing had little impact on the ability of each species to colonize plant roots. Results from tests with two replicate master mix communities showed that only C. pusillum colonization was consistently altered by freezing in both replicate experiments ( ). For master mix 3, we found a significant difference in the colonization of all seven species when comparing the fresh (fresh #2) to the 7-day frozen SynCom ( ). Relative abundance of SynCom members on roots varies less than absolute abundance Despite variation in the absolute abundance of each community member (CFU/g of fresh root) using both frozen and fresh SynComs for seed inoculation ( ), the relative abundance of each SynCom member colonizing the root remained consistent across all of the SynComs, whether fresh or frozen ( ). The exception was C. pusillum, which showed significant differences (ANOVA followed by Tukey’s HSD, P < 0.05) between different mixes; however, these differences were not associated with a specific SynCom treatment. In other words, lower relative abundances for C. pusillum were observed in both fresh and frozen SynComs. The fact that relative abundances are more consistent between experiments suggests that overall community assembly is deterministic and reproducible between experiments. Additionally, the relative abundance values that we measured are consistent with those in the original characterization of the SynCom ( ). Impact of freezing SynCom stocks on species abundances in the inoculum We tested the impact of longer-term freezing on species culturability in the SynCom master mix by plating five aliquots of the same ready-to-use freshly prepared SynCom on selective media and after 1 hour, 7 days, and 4 months of storage at −80°C. The titer of each species in the SynCom frozen for different periods was compared with that in the fresh SynCom. Although there were significant differences in the viability of some of the strains due to freezing (viability of S. maltophilia and P. putida is slightly higher after 4 months of freezing, that of E. ludwigii is lower after 1 hour and 7 days, and that of H. robinae is lower at all freezing time points; ANOVA followed by Tukey’s HSD), the magnitude change in viability is negligible for 4 months of freezing ( ).
We inoculated sterilized seeds in calcined clay with Murashige and Skoog medium containing the SynCom bacteria and grew the plants under controlled conditions for 10 days post-emergence before we harvested roots for bacterial colony counting (see Materials and Methods). Comparing roots of plants inoculated with the same SynCom inoculum (e.g., master mix #2 frozen for 1 hour) showed that, for all inocula, the abundance (log CFUs/g of root fresh weight) of each SynCom species was highly variable between replicate plants. The standard deviations for species abundances in replicate plants ranged from a log-value of 0.18 ( E. ludwigii and C. pusillum ) to 2.93 ( H. robiniae ) ( ). Comparing roots of plants inoculated with different master mixes showed that the abundances varied considerably for some species ( ). All of the species had significantly higher abundances (CFUs/g of fresh root weight) in fresh mix 2 as compared to fresh mix 3 ( P < 0.05, ). When comparing fresh mixes 1 and 2 again, all species were found to have significantly higher abundances (CFUs/g of fresh root weight) in fresh mix 2 as compared to fresh mix 1 ( P < 0.05, ), with the exception of S. maltophilia ( ). None of the SynCom members were significantly different in terms of CFUs/g of fresh root weight between fresh mixes 1 and 3.
Abundances of each SynCom species from plants inoculated with the frozen SynCom stocks compared with the corresponding fresh SynCom master mixes suggest that freezing had little impact on the ability of each species to colonize plant roots. Results from tests with two replicate master mix communities showed that only C. pusillum colonization was consistently altered by freezing in both replicate experiments ( ). For master mix 3, we found a significant difference in the colonization of all seven species when comparing the fresh (fresh #2) to the 7-day frozen SynCom ( ).
Despite variation in the absolute abundance of each community member (CFU/g of fresh root) using both frozen and fresh SynComs for seed inoculation ( ), the relative abundance of each SynCom member colonizing the root remained consistent across all of the SynComs, whether fresh or frozen ( ). The exception was C. pusillum, which showed significant differences (ANOVA followed by Tukey’s HSD, P < 0.05) between different mixes; however, these differences were not associated with a specific SynCom treatment. In other words, lower relative abundances for C. pusillum were observed in both fresh and frozen SynComs. The fact that relative abundances are more consistent between experiments suggests that overall community assembly is deterministic and reproducible between experiments. Additionally, the relative abundance values that we measured are consistent with those in the original characterization of the SynCom ( ).
We tested the impact of longer-term freezing on species culturability in the SynCom master mix by plating five aliquots of the same ready-to-use freshly prepared SynCom on selective media and after 1 hour, 7 days, and 4 months of storage at −80°C. The titer of each species in the SynCom frozen for different periods was compared with that in the fresh SynCom. Although there were significant differences in the viability of some of the strains due to freezing (viability of S. maltophilia and P. putida is slightly higher after 4 months of freezing, that of E. ludwigii is lower after 1 hour and 7 days, and that of H. robinae is lower at all freezing time points; ANOVA followed by Tukey’s HSD), the magnitude change in viability is negligible for 4 months of freezing ( ).
Our goal with this study was to evaluate the potential of freezing aliquots of SynComs in lieu of constructing a new community for each experiment. Freezing aliquots of a SynCom master mix provides two benefits to SynCom–host studies. First, it reduces the amount of work required to culture individual members in order to construct the community each time an experiment is initiated, and second, our results suggest that the consistency of species relative abundances in frozen aliquots is equal to, if not better than, the consistency of re-culturing and constructing a fresh community for each experiment. Although the same protocol was used to prepare each replicated SynCom, the absolute abundances of the members of each SynCom harvested from maize roots significantly differed in each of the three communities. The variation in each member of the freshly constructed SynComs harvested from maize roots was quite high, ranging from standard deviation log-values of 0.18 ( E. ludwigii and C. pusillum ) to 2.93 ( H. robiniae ) ( ). Several SynCom studies report the log-scale variance in colonies on the root surface across replicates. For example, the original study of the SynComs tested here reports the range of variance for colonization of each of the seven community members ( ). Niu et al. reported that replicates collected at the same time point had a ± log variance ranging from 0.04 (SD for E. ludwigii ) to 1.78 (SD for P. putida ) ( ). These values are consistent with those of another study of a 12-member SynCom in maize that indicates colonization variance between a ± variance of 0.5–1.0 log ( ). Another study that explored the cryo-preservation of a 17-member SynCom also showed large differences in community composition and root colonization in Brachypodium ( ). Despite the high variance that we have presented here, and which was also reported in other studies, the variation in colonization is not reported in most published SynCom studies, even though it is potentially important in understanding the microbial colonization of plants. Finally, these data are consistent with those of other studies that show that the absolute abundance of each member of the community is often less important than the relative abundance or the ratio of community members ( ). Although considerable variations in the rhizosphere microbiome have been documented during vegetative growth ( ), we think that part of the significant differences observed in absolute species abundances is likely due to variation introduced during harvesting and plating for counting, rather than variability due to community construction or freezing ( ). In Experiment 1, all the root samples (fresh and frozen 1 hour and 7 days) were processed at the same time. In this experiment, only one out of 14 comparisons between frozen SynCom samples and fresh SynCom samples was significant. In Experiment 2, root samples inoculated with fresh mix #3 and 7-day frozen mix were harvested and processed at the same time, and none of the comparisons was significant, while all comparisons of the fresh mix #2, which was harvested earlier, to the 7-day frozen mix were significant ( ). Despite the high variability in absolute abundance of individual community members harvested from maize roots across each SynCom, the relative abundances of SynCom members were similar to each other and to the relative abundances reported previously ( ). This indicates that potential variation in microbial species ratios introduced by SynCom treatment did not impact overall colonization patterns of SynCom members on corn roots. However, since we did not measure ratios of SynCom members for each treatment by plating inocula prior to application to corn seeds, we do not know how much variation was present in ratios of SynCom member between treatments. The observed stability in relative ratios after colonization could thus either be due to limited variation of species ratios in the inoculum or deterministic establishment of specific relative ratios upon root colonization driven by interactions with the plant and/or other SynCom members independent of input ratios. The literature on inoculation of plant and animal hosts with SynComs supports both scenarios. For example, Carlström et al. ( ) showed that ratios of 62 SynCom members in the inoculum were not predictive of ultimate colonization patterns in the Arabidopsis phyllosphere, and some SynCom members consistently colonized to high relative abundances, while others consistently failed to colonize. The data from Carlström et al. indicate that at least in part colonization patterns are deterministic and independent of inoculum ratios. On the other hand, Venturelli et al. ( ) showed that ratios of SynCom members in the inoculum can impact the final community in a human gut microbiome SynCom. They demonstrated that after 72 hours of cultivation, 12% of the communities displayed legacy dependence on the initial ratio ( ). In summary, while in our study the relative colonization ratios of SynCom members on corn roots were similar across all treatments, it will be important to assess the impact of strongly shifted SynCom member ratios in the inoculum on root colonization patterns as such ratio shifts might be caused by longer-term storage of ready-to-use SynCom stocks. Our study has at least three limitations, which should be addressed in future work. First, we did not investigate how long ready-to-use stocks can be stored in the freezer before losing their ability to colonize on corn roots at reproducible relative abundances. Ideally, ready-to-use inocula allow for reproducible inoculation for at least 1 year or more to enable execution of multiple repeat experiments and potential sharing of ready-to-use stocks with other members of the scientific community for reproducibility across laboratories. Our tests showed that during 4 months of freezing, loss of viability was negligible, suggesting that stocks can be used even after much longer storage. Second, it has been previously shown that glycerol can impact plant growth ( ) and root development ( ) at similar concentrations to what was present in our final SynCom mixes applied to plants (~4.8 mM). We did not examine the persistence of glycerol from frozen stocks in this study or determine longer-term plant impacts. As the response to glycerol is plant species-specific ( ), this is something that would need to be resolved for each plant host. This highlights an important experimental design consideration, though in that it needs to be ensured that all plants would receive the same concentration of glycerol; for example, sterile control plants would need to receive the same concentration of glycerol as plants receiving the SynCom. Alternatively, future testing could be done to see if viability of the mix remains if glycerol is removed by centrifugation of the initial 10 mL mix prepared by dilution of the frozen aliquots. Third, often experiments with SynComs require constructing SynComs with different compositions such as, for example, the exclusion of suspected keystone species. For these types of experiments, it would still be beneficial to not have to grow all the community members de novo every time. Thus, mixing of SynComs from ready-to-use stocks of individual SynCom members or groups of SynCom members would be ideal. However, this has to our knowledge not been tested yet in any system and thus would require careful testing and validation. Finally, although we only studied the applicability of a frozen ready-to-use SynCom for this particular maize SynCom ( ), we have demonstrated the utility of frozen SynCom stocks in terms of reducing labor and improving inoculum consistency between experiments. The principle works, and the approach used here can be applied to validate frozen ready-to-use mixes for other SynComs.
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Editorial: Ethical considerations in electronic data in healthcare | 616afed9-ee2a-4701-ae54-cd7a6061d0d9 | 11287769 | Psychiatry[mh] | Electronic data has revolutionized the healthcare sector in the digital age, promising enhanced patient care, streamlined operations, and groundbreaking medical research. However, this transformation has complex ethical challenges that need careful consideration. The surge in electronic health records (EHRs), big data analytics, and telemedicine raises significant questions about privacy, consent, data ownership, and equity. Integrating these technologies into our healthcare systems is crucial to navigating these ethical dilemmas thoughtfully. This editorial explores the ethical considerations surrounding electronic data in healthcare, drawing insights from a series of articles that explore various facets of this multifaceted issue. These contributions collectively provide a comprehensive view of the challenges and propose pathways for ethically sound practices in managing electronic healthcare data.
One of the foremost ethical concerns is the protection of patient privacy in an era where data breaches and cyber-attacks are increasingly common. Carmichael et al. 's article, “ Personal Data Store Ecosystems in Health and Social Care ,” underscores the need for robust security measures to prevent unauthorized access to sensitive patient information. She highlights the tension between the accessibility of data for medical purposes and the imperative to protect patient confidentiality.
Informed consent is a cornerstone of ethical healthcare practices, but its application becomes complex with electronic data. Benevento et al. explore this Research Topic in their article, “ Measuring the willingness to share personal Health information: a systematic review .”
The question of data ownership is another critical ethical issue. In the article, “ Brave (in a) New World: An Ethical Perspective on Chatbots for Medical Advice ,” Erren et al. examine the legal and ethical implications of data ownership in the healthcare sector. They discuss the competing interests of patients, healthcare providers, and third-party companies, and advocate for policies that prioritize patient rights.
The digital divide presents a significant barrier to equitable healthcare. Adepoju et al. address this in their piece, “ Access to Technology, Internet Usage, and Online Health information-seeking behaviors in a racially diverse, lower-income population .” They highlight how disparities in digital access can exacerbate existing health inequalities, with marginalized communities often being the most disadvantaged. The authors advocate for policies and initiatives that promote digital literacy and provide equitable access to technology, ensuring that the benefits of electronic data in healthcare are shared broadly across all segments of society.
The utilization of big data in healthcare offers immense innovation potential, but it also poses significant ethical challenges. Pu et al. 's article, “ A Medical Big Data Access Control Model Based on Smart Contracts and Risk in the Blockchain Environment ,” investigates the ethical considerations of using large datasets for medical research and decision-making. He discusses the balance between the benefits of big data, such as improved patient outcomes and medical advancements, and the risks, including privacy violations and data misuse. Pu et al. emphasizes the need for ethical frameworks that guide the responsible use of big data while fostering innovation.
As we navigate the digital transformation of healthcare, it is imperative to address the ethical challenges associated with electronic data. Protecting patient privacy, ensuring informed consent, safeguarding against digital threats, promoting equity and access, and maintaining transparency and accountability are all critical components of ethical practice in this new landscape. The insights from the articles in this series highlight the complexities and propose thoughtful approaches to managing these Research Topic. The ethical considerations in healthcare data demand our attention and action. Together, these articles offer a roadmap for healthcare providers, policymakers, and technology developers to build a more ethical and inclusive healthcare system, where the promise of electronic data can be fully realized without compromising ethical standards.
1. “ Barriers and facilitators related to healthcare practitioner use of real-time prescription monitoring tools in Australia ” by Hoppe et al. : - Using an online survey, investigate the barriers and facilitators related to healthcare practitioners' use of real-time prescription monitoring (RTPM) tools in Australia. - Further research is needed to gain an understanding of healthcare practitioners' use of RTPM tools and how to minimize barriers and optimize use for the essential delivery of quality healthcare. 2. “ Measuring the willingness to share personal health information: a systematic review ” by Benevento et al. : - Analyze the determinants and describe the measurement of the willingness to disclose personal health information. - Systematic review of articles assessing willingness to share personal health information as a primary or secondary outcome. 3. “ Brave (in a) new world: an ethical perspective on chatbots for medical advice ” by Erren et al. : - Emphasizes the significant ethical challenges associated with the use of AI chatbots in medical contexts, such as privacy and confidentiality. - Discusses the necessity of regulating AI, particularly in the medical field, to avoid potential harms, and raises critical questions about who controls AI, how personal data is protected, and who is liable for the advice provided by AI. 4. “ Access to technology, internet usage, and online health information-seeking behaviors in a racially diverse, lower-income population ” by Adepoju et al. : - Examines access to technology, internet usage, and online health information-seeking behaviors, in a racially diverse, lower-income population using a survey. - Identifies the gap between technology adoption and effective use for health purposes, highlighting a critical area for improving public health efforts to leverage digital resources. - Revealed that higher income, higher education levels, and female gender were significantly associated with increased online health information-seeking behaviors. 5. “ Personal data store ecosystems in health and social care ” by Carmichael et al. : - Highlights the potential of personal data storage to transform health and social care through enhanced individual data control and usage. - Points out the significant challenges that need to be addressed for their successful adoption, such as Technical and Operational Hurdles, User Engagement, and Data Governance. 6. “ A Medical Big Data Access Control Model based on Smart Contracts and Risk in the Blockchain Environment ” by Pu et al. : - Proposes a smart contract and risk-based access control model (SCR-BAC) integrated with traditional risk-based access control and deploys risk-based access control policies in the form of smart contracts into the blockchain, thereby ensuring the protection of medical data. - Demonstrates that the access control model effectively curbs the access behavior of malicious doctors to a certain extent and imposes a limiting effect on the internal abuse and privacy leakage of medical big data. 7. “ Large language models in physical therapy: time to adapt and adept ” by Naqvi et al. : - Examines how large language models (LLMs) driven by deep ML can offer human-like performance but face challenges in accuracy due to vast data in Physical Therapy (PT) and rehabilitation practice. - Urges PTs to engage in learning and shaping AI models by highlighting the need for ethical use and human supervision to address potential biases. Through a comprehensive understanding and proactive management of these ethical issues, we can ensure that the digital revolution in healthcare is both transformative and just, benefiting all patients and society.
DM: Conceptualization, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing. MA-K: Conceptualization, Investigation, Methodology, Validation, Writing – review & editing.
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Improving the confidence level and surgical skills of undergraduate medical students by using pig-perineum simulation-based learning | fc24672c-31ff-4b9e-a0d2-fda71947569e | 11827413 | Surgical Procedures, Operative[mh] | Vaginal birth, especially in nulliparous women, is often associated with perineal tear and perineal lacerations. Sometimes, a perineal injury can extend deeper to the anal sphincter and rectal mucosa, known as an obstetric anal sphincter injury (OASI)Waldman . Perineal laceration, either occurring spontaneously or resulting from a surgical incision (episiotomy), is a common obstetric injury, occurring in nearly 90% of women with spontaneous vaginal delivery . Most lacerations heal without long term complications, but some lacerations can lead to prolonged pain, sexual dysfunction and embarrassment . Some lacerations need to be carefully identified and properly repaired at the time of delivery . According to the Medical Council of Thailand, medical students must be taught to perform normal labor, episiotomy and perineorrhaphy or repair of episiotomy wound after normal vaginal delivery. Medical graduates must be capable of effectively performing these procedures by themselves. However, perineal-repair surgical skills are challenging and need special techniques, not gained from general suturing practice. Unfortunately, the acquisition of those skills by medical students is usually hindered by time pressure and a long learning curve. Additionally, a recent survey reported that Thai medical graduates did not achieve the satisfactory confidence levels in performing normal vaginal delivery and episiotomy repair . Furthermore, perineal repair needs complex surgical skills, and the process may cause medical accidents, such as needle-stick injuries, especially in inexperienced trainees. A survey from Sri Lanka reported that 70% of medical students had experienced such events during suturing of episiotomy wounds . Therefore, medical schools need to provide programs that encourage medical students to practice these surgical skills. Additionally, performing tasks on live patients often cause anxiety and stress for new clinical students because of fear and lack of prior experience. Considering that this procedure involves a long learning curve, which means that much effort is needed and learning is initially difficult, the importance of training stands out. Students who receive simulation training participate more actively in the clinical environment during the course of clerkship. Simulation training is beneficial because it provides a safe environment for learning and practising obstetric skills with minimal risk, increases competency with maneuvers, and translates this competence into increased clinical participation and confidence without any harm to patients . Various teaching models in perineal repair for medical and midwifery students have been proposed, for example using a sponge model, a perineum-padsicle model, a porcine tongue simulator, and a beef tongue simulation model. These teaching modules are aimed at helping students, residents and other practitioners enhance their skills, so that they are able to effectively perform perineal repair, especially in cases of third- or fourth-degree lacerations (OASI). Previous studies on a porcine tongue simulator and a detailed beef tongue simulation model showed that repair workshop for residents could help improve their background knowledge and skills in repairing obstetric anal sphincter injury. In addition, a sponge model improved the repair skills of students regarding second-degree perineal laceration , as measured by the Objective Structured Assessment of Technical Skills (OSATS), and fourth-degree perineal laceration . We hypothesize that using real pig perineum for perineal repair practice better represents the human perineum compared to other parts of the animal body or artificial tissue. Accordingly, this preliminary study may provide a new simulation model that is theoretically superior to previously used models. Since it is essential for medical students to learn basic surgical techniques, as mentioned above, we developed a hands-on perineal repair workshop, using real pig perineum as a model, to teach medical students before they perform the procedure in real clinical practice. The objective of this study is to report the confidence levels of the medical students as well as their perspectives on the closeness to reality of pig-perineum as a simulator. A retrospective study of pig-perineum simulation-based learning (SBL) for medical students was conducted at the Department of Obstetrics and Gynecology, Faculty of Medicine, Chiang Mai University from June 2016 to June 2023, with ethical approval by the Institutional Review Board, Faculty of Medicine, Chiang Mai University (Research ID: OBG-2567-0283), in accordance with the ethical standards of the 1964 Declaration of Helsinki. Hands-on pig-perineum simulation-based learning workshop (Fig. ) This workshop was designed by the educational team of our department, and its implementation commenced in the 2015 academic year in the obstetric clerkship at the Faculty of Medicine, Chiang Mai University. Medical students participated in a pig-perineum repair workshop on identifying perineal laceration, knot-tying, and perineal laceration repair. The pig perineum used in the workshop was obtained from discarded pork products processed by Betagro Northern Agro Industry Company Limited. The workshop did not involve the use of live animals, nor were any animals commercially purchased or client-owned. These discarded materials, which would have otherwise been destroyed, were repurposed as educational tools. A half-day training program was conducted for all 5th- and 6th-year medical students who underwent obstetric clerkship rotation. The training program consisted of both lectures and practical training. Students had individual workstations. The first part of the training involved lectures on the following aspects: the anatomy of the perineum, episiotomy, accurate diagnosis of perineal tears, classification and identification of internal and external anal sphincter muscles, and methods of repairing second-degree perineal tears. We used real female-pig perineum tissue blocks as simulators, as shown in Fig. . Regarding anatomy, the porcine vaginal diameter is smaller than that of humans. We oriented the (pig) vagina to be an anal opening (simulator) and presumed the anal sphincter to be a perineal muscle (simulator), which was considered feasible and similar to that of humans. We adapted this simulator for the practice of proper suturing regarding second-degree perineal laceration. The second part, the hands-on clinical training, was divided into two steps: performing episiotomy on pig perineum and repairing second-degree perineal tears. Each participant was supervised by the trainers. Upon suturing completion, the medical students did a final assessment to confirm that the repair visually appeared to be accurate. Questionnaire survey Medical students’ confidence levels after attending the workshop were measured with a questionnaire (5-point Likert scale, with 1 being extremely unconfident and 5 being extremely confident). From the medical students’ perspectives, the pig-perineum effectively simulated the vaginal canal, anal canal, and anal sphincter, and the simulation training was helpful in instrument handling, knot typing and suturing of vaginal and perineal muscles. Moreover, the questionnaire also sought to evaluate their skills and satisfaction. The questionnaire was delivered immediately before and after the training session. The data obtained from the questionnaire were comprehensively reviewed after ethical approval. Statistical analysis Descriptive data were presented as frequencies and percentages, means and SD or medians and interquartile ranges, as appropriate. The Wilcoxon signed rank test was used to compare the pre- and post-simulation training self-rated confidence levels of perineal repair skills. All data were recorded in an electronic database and subsequently analyzed, using the Statistical Package for the Social Sciences (SPSS) software, version 26.0 IBM Corp, released 2019 (IBM SPSS Statistics for Windows, Version 26.0 Armonk, NY: IBM Corp). A p -value of less than 0.05 was defined as statistically significant. ) This workshop was designed by the educational team of our department, and its implementation commenced in the 2015 academic year in the obstetric clerkship at the Faculty of Medicine, Chiang Mai University. Medical students participated in a pig-perineum repair workshop on identifying perineal laceration, knot-tying, and perineal laceration repair. The pig perineum used in the workshop was obtained from discarded pork products processed by Betagro Northern Agro Industry Company Limited. The workshop did not involve the use of live animals, nor were any animals commercially purchased or client-owned. These discarded materials, which would have otherwise been destroyed, were repurposed as educational tools. A half-day training program was conducted for all 5th- and 6th-year medical students who underwent obstetric clerkship rotation. The training program consisted of both lectures and practical training. Students had individual workstations. The first part of the training involved lectures on the following aspects: the anatomy of the perineum, episiotomy, accurate diagnosis of perineal tears, classification and identification of internal and external anal sphincter muscles, and methods of repairing second-degree perineal tears. We used real female-pig perineum tissue blocks as simulators, as shown in Fig. . Regarding anatomy, the porcine vaginal diameter is smaller than that of humans. We oriented the (pig) vagina to be an anal opening (simulator) and presumed the anal sphincter to be a perineal muscle (simulator), which was considered feasible and similar to that of humans. We adapted this simulator for the practice of proper suturing regarding second-degree perineal laceration. The second part, the hands-on clinical training, was divided into two steps: performing episiotomy on pig perineum and repairing second-degree perineal tears. Each participant was supervised by the trainers. Upon suturing completion, the medical students did a final assessment to confirm that the repair visually appeared to be accurate. Medical students’ confidence levels after attending the workshop were measured with a questionnaire (5-point Likert scale, with 1 being extremely unconfident and 5 being extremely confident). From the medical students’ perspectives, the pig-perineum effectively simulated the vaginal canal, anal canal, and anal sphincter, and the simulation training was helpful in instrument handling, knot typing and suturing of vaginal and perineal muscles. Moreover, the questionnaire also sought to evaluate their skills and satisfaction. The questionnaire was delivered immediately before and after the training session. The data obtained from the questionnaire were comprehensively reviewed after ethical approval. Descriptive data were presented as frequencies and percentages, means and SD or medians and interquartile ranges, as appropriate. The Wilcoxon signed rank test was used to compare the pre- and post-simulation training self-rated confidence levels of perineal repair skills. All data were recorded in an electronic database and subsequently analyzed, using the Statistical Package for the Social Sciences (SPSS) software, version 26.0 IBM Corp, released 2019 (IBM SPSS Statistics for Windows, Version 26.0 Armonk, NY: IBM Corp). A p -value of less than 0.05 was defined as statistically significant. A total of 792 students participated in this pig-perineum simulation-based learning (SBL), including 475 (60%) 5th-year medical students and 317 (40%) 6th-year medical students. The survey included questions on confidence level, satisfaction, knowledge of perineal repair, and perspective on the closeness to reality of using pig perineum as a simulator for second-degree laceration repair. The mean confidence level among medical students before attending the workshop was 3.63 points. Notably, the lowest level of confidence was found among 5th year medical students (3.46 points). The assessment of the confidence level before and after training demonstrated a significant improvement of 10.6% in the mean score ( p -value < 0.001), as shown in Fig. . Both pre- and post-training scores were higher for 6th-year medical students, but greater improvement was seen in 5th-year medical students. Figure also displays the mean pre- and post-training confidence levels of the 5th- and 6th-year medical students. Most medical students either strongly agreed or agreed that the pig-perineum simulator was effective in replicating the anatomical structures of the vaginal canal, perineal muscle and anal sphincter. Moreover, they would be able to utilize pig perineum for practising knot-tying and suturing perineal lacerations, which can enhance surgical skills and boost confidence after training. Figure shows the details of their perspectives regarding the closeness to reality of pig perineum as a simulator. Furthermore, the medical students gave high ratings for their performance skills, such as evaluation degree of perineal laceration and repairing 2nd-, 3rd-, and 4th-degree perineal lacerations, after attending the workshop. They were satisfied (mean satisfaction score = 4.22) with the training and asserted that the simulation improved their knowledge and surgical skills, which can be applied in their clinical practice. Table shows the details of self-evaluation of the medical students regarding the hands-on pig-perineum SBL workshop for second-degree laceration suturing. Our study demonstrated that the medical students enrolled in hands-on pig-perineum SBL workshop showed an improvement of confidence level and satisfaction. Also, they had a positive attitude towards the pig-perineum simulator. A recent survey reported that Thai medical graduates did not achieve the satisfactory confidence levels in performing normal vaginal delivery and episiotomy repair . Additionally, tertiary medical schools are faced with a marked decrease in the number of vaginal deliveries all over the country. Thus, medical students currently have less experience than before, leading to even less confidence and competency in performing delivery and episiotomy repair. This emphasizes the necessity of providing more training opportunities for them. SBL offers significant advantages, as it provides a comfortable atmosphere for learners to practice without the fear of making mistakes, thus giving them hands-on experience in a safe environment . Therefore, SBL helps medical students to rapidly improve their surgical skills . As simulation becomes increasingly important in medical education, particularly for 3rd- and 4th-degree laceration repair, many training models have been used as simulators. Several studies have evaluated the effectiveness of teaching obstetric anal sphincter injury repair using practice models, and their results indicate improvement in resident skill sets after undergoing an educational workshop . Among the types of simulation, those involving animals are the closest to real life because the tissues are similar to those of humans. Pig-perineum has been used for teaching OASI repair, and this study advocates the realistic and practical features of pig-perineum simulator in perineal repair training. Pig-perineum SBL offers an attractive solution because it allows medical students to learn in a safe and controlled environment. For this reason, our obstetric and gynecological educational leadership team has made effort to achieve the use of pig-perineum simulators for perineal repair training. Students reported immediate increased comfort in assessing and repairing perineal lacerations, and they had high satisfaction levels and positive attitudes regarding this SBL. All the medical students stated that this simulation is valuable, and they would be able to utilize what they had learnt in clinical practice. To the best of our knowledge, this is the first study that not only showed an improvement of medical students’ confidence level after a pig-perineum SBL for second-degree perineal lacerations but also inspired them to enhance their surgical skills in knot-tying and perineal laceration repair. Although there might be a significant gap between confidence and real proficiency, this study demonstrates the benefits of pig-perineum SBL in obstetric clerkship. Therefore, we strongly believe that a pig-perineum SBL program with realistic hands-on training should be incorporated in the obstetric curriculum of medical students. The limitations of this study are as follows. Firstly, only the apprentices themselves evaluated the confidence levels and skills acquired in a single workshop, without assessment by validated tools. For future investigations, an evaluation, such as the Objective Structured Assessment of Technical Skills (OSATS), a theoretical test or the retention of knowledge, should be incorporated. Secondly, this study was based on self-reporting, which could lead to information bias. Lastly, the medical students were exposed to only one simulator intervention, without comparison with other training simulators. An alternative design should be allowed for comparison, to determine which simulator promotes efficiency and improves performance through skill acquisition better than the other. The strengths of this study include: (1) A relatively large sample size of the participants, probably enhancing the reliability of the conclusion. (2) The high similarity of the simulator to human perineum in actual practice, in terms of tissue consistency and anatomical structures and their complexities. This is different from several previous studies, which used artificial models or tissues from other organs for the animal models (such as pig or beef tongue). Based on such similarity, the use of the model to practice the identification of anatomical structures, knot-tying, suturing and repair theoretically helps medical students to rapidly develop their skills in actual practice on patients, with less complications and shortened learning curve. Pig-perineum simulation-based learning improves confidence levels in undergraduate medical students. Theoretically, this approach is possibly helpful in enhancing competency during actual clinical practice. Below is the link to the electronic supplementary material. Supplementary Material 1 |
Adeno-associated virus 2 CRISPR/Cas9-mediated targeting of hepatitis B virus in tree shrews | 62c841fa-c7fb-426c-8709-076497d6bc22 | 11909760 | Digestive System[mh] | Introduction Hepatitis B virus (HBV), a member of the family Hepadnaviridae , is an enveloped, circular, and partially double-stranded DNA virus that constitutes a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma ( ; ). HBV has eight well-known genotypes (A–H), with >8 % difference in the nucleotide sequence of the genome and a distinct geographic distribution ( ; ; ; ). Over 254 million people are chronically infected with HBV worldwide with 1.2 million new infections occurring annually. Current therapies with nucleoside/nucleotide analogs can suppress viral replication by attenuating the activity of viral reverse transcriptase ( ); however, they are unable to eliminate replicative HBV templates comprising covalently closed circular DNA (cccDNA) or integrated HBV DNA ( ). Moreover, integrated HBV DNA contributes to HBsAg production ( ) and is associated with carcinogenesis ( ; ). Therefore, the eradication of persistent HBV cccDNA or integrated HBV DNA from infected cells is crucial to achieve a complete cure for chronic HBV infection ( ; ), highlighting the need to explore alternative therapeutic approaches to eliminate this residual HBV DNA. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) is a genome editing tool that has shown potential as a therapeutic approach against viral infections ( ; ; ). Within this system, tools for delivering Cas9 and guide RNA (gRNA) are key factors influencing its efficiency. Adeno-associated virus (AAV), a nonpathogenic parvovirus, has become one of the most promising viral vectors for human gene therapy ( ). In particular, AAV2-based vectors may offer a flexible delivery method for CRISPR/Cas9 gene editing. We previously investigated the effects of AAV2 vector-mediated delivery of three gRNAs/Cas9 genes selected from 16 gRNAs ( ). These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting the highest efficacy, and being chosen for in vivo studies. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and reduced cccDNA levels in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced the effects of entecavir when used in combination, indicating a different mode of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA and cccDNA in liver tissues without significant cytotoxicity; next generation sequencing data showed no significant genomic mutations ( ). Although we confirmed the anti-HBV effect of AAV2/WJ11-Cas9 in vitro and in vivo , its efficacy needs to be further analyzed in immunocompetent animal models. Previously, chimpanzees have been used as natural infection models for HBV; however, high cost and ethical concerns restrict the experimental use of chimpanzees. Humanized chimeric mice ( ) are effective animal models of HBV infection ( ); however, characterization of the host immune response is not possible. To address this issue, we previously established a northern tree shrew ( Tupaia belangeri ; hereafter, “tupaia”) model ( ) and have used it to study HBV infection ( ; ; , ; ). HBV-F Mt (BCP/PC/2051), containing A1762T/G1764A, G1896A, and A2051C mutations, was observed in patients with HCC and could replicate more efficiently than HBV-F Wt ( ). We also investigated the immune response in the tupaia model ( ). In the present study, we aimed to understand the effect of AAV2/WJ11-Cas9 on HBV infection and host immune responses using the HBV-F Mt tupaia model.
Materials and methods 2.1 Ethics statement This study was conducted in strict accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science, and the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols for the tupaia experiment were approved by the regional ethics committee (VM13044). 2.2 Animals Northern tree shrews ( Tupaia belangeri ) were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences. The animals, aged 4 to 6 years (F1–F2), were born in the animal facilities of Kagoshima University, Japan. Nine tupaias were assigned to three groups: CRISPR/Cas9-treated, Mock-treated, and Normal ( n = 3). The Normal group consisted of tupaias that were not infected with HBV and were used for comparative histopathological and cytokine expression analyses. 2.3 Plasmids The AAVpro CRISPR/Cas9 Helper-Free System (AAV2) (Clontech Laboratories, Inc., Palo Alto, CA, USA) was used to construct the gRNA/Cas9 expression plasmids, and AAV2 particles were prepared as described previously ( ). The gRNA sequence of WJ-11 (positions 1859–1878) used in this study was ACTGTTCAAGCCTCCAAGCT. 2.4 Production and purification of AAV2 particles For the preparation of AAV2-Guide-it-Up and AAV2-Guide-it-Down-gRNA vectors (purchased from TaKaRa Bio Inc., Shiga, Japan), HEK293 cells were transiently transfected using the Xfect Transfection Reagent (Clontech Laboratories, Inc.) according to the manufacturer's instructions. Briefly, HEK293 cells were co-transfected with the pAAV-Guide-it-Up or pAAV-Guide-it-Down vector expression plasmid, an AAV2 ITR-containing plasmid carrying the Cas9/gRNA genes, a pRC2-mi342 plasmid expressing AAV2 Rep and Cap proteins, and pHelper plasmid expressing adenovirus helper proteins (Clontech Laboratories, Inc.). To accommodate the Streptococcus pyogenes Cas9, which is >4 kb in length, a dual AAV platform consisting of either the pAAV-Guide-it-Up or pAAV-Guide-it-Down vector was used. At 72 h post-transfection, cells were collected, and AAV2 particles were harvested using AAV extraction solutions A and B (Clontech Laboratories, Inc.) according to the manufacturer's instructions. The harvested AAV2 particles were stored at −80 °C for further use. SignaGen Laboratories prepared the AAV2 particles to a large scale (10 13 copies) for in vivo experiments. AAV2 vectors were purified using an AAVpro Purification Kit (all serotypes) (TaKaRa Bio Inc.) according to the manufacturer's instructions. After purification, the viral titer (viral genomic titer) was determined through real-time polymerase chain reaction (PCR) targeting the ITR domain, using an AAVpro Titration Kit (TaKaRa Bio Inc.), following the manufacturer's instructions. 2.5 Inoculation of AAV2/WJ11-Cas9 to HBV-infected tupaias To examine the anti-HBV effects of the HBV-specific AAV2/WJ11-Cas9 system in vivo , six adult tupaias infected with HBV genotype F mutant strain (GenBank LC831106.1) were used, with three tupaias used as non-treated controls (marked as 178, 191, and 386) and the others inoculated with AAV2/WJ11-Cas9 (marked as 323, 380, and 329). HBV genotype F (10 7 copies) was administered intravenously (iv) ( ) simultaneously with AAV2/WJ11 or mock treatment. A total of 5 × 10 12 vg copies of AAV2/WJ11-Cas9 or an equal volume of sterile Opti-MEM (non-treated control) were injected through the tail vein. Tupaia serum was collected on days −4, 1, 3, 5, 7, 10, and 14 post-inoculation. Serum HBV DNA titers, along with alanine aminotransferase (ALT) were measured at each indicated time point. Fourteen days post-inoculation, the tupaias were sacrificed under anesthesia after whole blood collection, and tissue samples were collected and stored at −80 °C for further characterization or placed in 10 % formaldehyde-PBS (WAKO) for histological examination (hematoxylin and eosin staining). Liver tissue was also applied for HBcAg detection, as described previously ( ). Intrahepatic HBV DNA and HBV cccDNA titers were determined from genomic DNA extracted from tupaia liver tissues using the phenol-chloroform extraction method. Serum ALT levels were determined using a Transnase Nissui kit (Nissui Pharmaceutical Co., Ltd.), and the data were standardized and are presented as IU/l. 2.6 HBV total DNA, cccDNA extraction and quantitation, and T7E1 assay Total DNA was isolated from tupaia liver tissues as previously described ( ). To purify HBV cccDNA, total DNA was treated with Plasmid-Safe ATP-Dependent DNase ( ) (Lucigen Corporation) at 37 °C for 60 min, followed by DNase inactivation at 70 °C for 30 min. The digested products were used as templates for real-time PCR to quantify HBV cccDNA, as described below. The T7E1 assay was performed as previously described ( ; ). The primers used were completely matched to HBV-Fmt sequence; 1st PCR sense (794) 5ʹ-CCGCTGTTACCAATTTTCTGTTATC-3ʹ, 1st PCR anti-sense (2051) 5ʹ-GAGGTGTGCAATGTTCTGGTGATT-3ʹ, 2nd PCR sense 5ʹ-(819) TGTGGGTATCCATTTAAATA-3ʹ, and 2nd PCR anti-sense (2027) 5ʹ-CTAAAGCATCCCGGTAGAGG-3ʹ. HBV DNA was quantified using real-time PCR as previously described ( ), with a detection limit of one copy of HBV DNA and HBV cccDNA per microgram of liver tissue. The primers and probes for the S gene included the forward primer HB-166-S21 (nucleotides (nts) 166–186): 5′-CAC ATC AGG ATT CCT AGG ACC-3′, the reverse primer HB-344-R20 (nts 344–325): 5′-AGG TTG GTG AGT GAT TGG AG-3′, and the TaqMan probe HB-242-S26FT (nts 242–267): 5′-CAG AGT CTA CAC TCG TGG ACT TC-3′. HBV cccDNA quantification was performed using qPCR with TaqMan chemistry, using the forward primer HBVcccDNA 1519-S25 (5′-ACG GGG CGC ACC TCT CTT TAC GCG G-3′), the reverse primer HBVcccDNA 1886-R25 (5′-CAA GGC ACA GCT TGG AGG CTT GAA C-3′), and the Taq-Man probe HBVcccDNA-1575 S26FT (5′6-FAM–CCG TGT GCA CTT CGC TTC ACC TCT GC-TAMRA-3′). Cycling was performed as 50 °C for 2 min, 95 °C for 10 min, and 53 cycles at 95 °C for 20 s and 65 °C for 1 min. PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad). Cytokines and TLR mRNAs were quantified via qRT-PCR using specific primers (Table S1), as previously reported ( ). 2.7 Measurement of HBsAg and HBcrAg HBsAg levels in the tupaia serum was measured using the two-step sandwich assay principle with a fully automated chemiluminescent enzyme immunoassay system (Lumipulse G 1200, Fujirebio, Tokyo, Japan), as described previously ( ). HBcrAg levels in tupaia serum were measured using a high-sensitivity HBcrAg assay, as described previously ( ). 2.8 Total RNA extraction from tupaia liver tissues and measurement of cytokines and TLR mRNA Total RNA was extracted from tupaia liver tissues using the RNeasy Plus Mini Kit (QIAGEN) following the manufacturer's instructions, and genomic DNA was removed using the gDNA Eliminator Spin Column. The concentration and purity of the extracted RNA were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Waltham, MA, USA). The samples were stored at −80 °C until needed for gene expression analysis. We analyzed the expression of TLR mRNAs (TLR1–9) in tupaia liver tissues using one-step qRT-PCR with Brilliant III Ultra-fast SYBR Green QRT-PCR Master Mix (Agilent Technologies, Santa Clara, CA, USA), following . The PCR process involved reverse transcription at 50 °C for 10 min, initial denaturation at 95 °C for 3 min, and 40 cycles at 95 °C for 5 s and 60 °C for 10 s. We also measured the expression of IL-6, TNF-α, IFN-γ, cGAS, and IFN-β mRNA using the same method, as described previously ( ; ). The gene-specific primer sequences are provided in Table S1. The gene copy number per microgram of liver RNA was calculated using a standard curve from pre-quantified gene copies. Each sample was measured in duplicate, and statistical analysis was performed to compare with uninfected controls. 2.9 ELISA and immunoblot assay Anti-HBc and anti-HBs antibodies in tupaia serum were detected via ELISA, as previously described ( ) with slight modifications. Purified HBc and HBs antigens (Beacle Co., Osaka, Japan) were diluted with a sodium carbonate buffer (pH 9.0) (5 μg/mL) and a 1 % Block Ace solution (KAC Co. Ltd., Japan) in phosphate-buffered saline (-) containing 0.05 % Tween 20. This solution was used to block and dilute the antibodies. Tupaia serum and anti-tupaia IgG rabbit serum (in-house) were diluted 10,000-fold for reaction. Anti-HBc rabbit polyclonal antibody (ab115992, Abcam Co.) and anti-HBs mouse monoclonal antibody (MoAb) (MA1-19263, ThermoFisher) were used as positive controls. Cas9 protein was detected in tupaia liver tissues via standard immunoblotting assay using an anti-Cas9 rabbit MoAb (#19,526, Cell Signaling Co.). Tissues were lysed in a lysis buffer (1 % SDS, 0.5 % NP40, 0.15 M NaCl, 10 mM Tris [pH 7.4], 5 mM EDTA, and 1 mM DTT). β actin was detected using an anti-β actin mouse MoAb (clone AC15, Merk Co.). Protein bands were visualized and quantified using a FUSION chemiluminescence imaging system (M&S Instruments, Inc.). 2.10 Statistical analysis Data are presented as the means ± SD. For statistical analysis, Student's t -test for two groups and one-way ANOVA and Dunnett's multiple comparisons were performed using GraphPad Prism software. Statistical significance was set at P < 0.05.
Ethics statement This study was conducted in strict accordance with the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science, and the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols for the tupaia experiment were approved by the regional ethics committee (VM13044).
Animals Northern tree shrews ( Tupaia belangeri ) were purchased from the Kunming Institute of Zoology, Chinese Academy of Sciences. The animals, aged 4 to 6 years (F1–F2), were born in the animal facilities of Kagoshima University, Japan. Nine tupaias were assigned to three groups: CRISPR/Cas9-treated, Mock-treated, and Normal ( n = 3). The Normal group consisted of tupaias that were not infected with HBV and were used for comparative histopathological and cytokine expression analyses.
Plasmids The AAVpro CRISPR/Cas9 Helper-Free System (AAV2) (Clontech Laboratories, Inc., Palo Alto, CA, USA) was used to construct the gRNA/Cas9 expression plasmids, and AAV2 particles were prepared as described previously ( ). The gRNA sequence of WJ-11 (positions 1859–1878) used in this study was ACTGTTCAAGCCTCCAAGCT.
Production and purification of AAV2 particles For the preparation of AAV2-Guide-it-Up and AAV2-Guide-it-Down-gRNA vectors (purchased from TaKaRa Bio Inc., Shiga, Japan), HEK293 cells were transiently transfected using the Xfect Transfection Reagent (Clontech Laboratories, Inc.) according to the manufacturer's instructions. Briefly, HEK293 cells were co-transfected with the pAAV-Guide-it-Up or pAAV-Guide-it-Down vector expression plasmid, an AAV2 ITR-containing plasmid carrying the Cas9/gRNA genes, a pRC2-mi342 plasmid expressing AAV2 Rep and Cap proteins, and pHelper plasmid expressing adenovirus helper proteins (Clontech Laboratories, Inc.). To accommodate the Streptococcus pyogenes Cas9, which is >4 kb in length, a dual AAV platform consisting of either the pAAV-Guide-it-Up or pAAV-Guide-it-Down vector was used. At 72 h post-transfection, cells were collected, and AAV2 particles were harvested using AAV extraction solutions A and B (Clontech Laboratories, Inc.) according to the manufacturer's instructions. The harvested AAV2 particles were stored at −80 °C for further use. SignaGen Laboratories prepared the AAV2 particles to a large scale (10 13 copies) for in vivo experiments. AAV2 vectors were purified using an AAVpro Purification Kit (all serotypes) (TaKaRa Bio Inc.) according to the manufacturer's instructions. After purification, the viral titer (viral genomic titer) was determined through real-time polymerase chain reaction (PCR) targeting the ITR domain, using an AAVpro Titration Kit (TaKaRa Bio Inc.), following the manufacturer's instructions.
Inoculation of AAV2/WJ11-Cas9 to HBV-infected tupaias To examine the anti-HBV effects of the HBV-specific AAV2/WJ11-Cas9 system in vivo , six adult tupaias infected with HBV genotype F mutant strain (GenBank LC831106.1) were used, with three tupaias used as non-treated controls (marked as 178, 191, and 386) and the others inoculated with AAV2/WJ11-Cas9 (marked as 323, 380, and 329). HBV genotype F (10 7 copies) was administered intravenously (iv) ( ) simultaneously with AAV2/WJ11 or mock treatment. A total of 5 × 10 12 vg copies of AAV2/WJ11-Cas9 or an equal volume of sterile Opti-MEM (non-treated control) were injected through the tail vein. Tupaia serum was collected on days −4, 1, 3, 5, 7, 10, and 14 post-inoculation. Serum HBV DNA titers, along with alanine aminotransferase (ALT) were measured at each indicated time point. Fourteen days post-inoculation, the tupaias were sacrificed under anesthesia after whole blood collection, and tissue samples were collected and stored at −80 °C for further characterization or placed in 10 % formaldehyde-PBS (WAKO) for histological examination (hematoxylin and eosin staining). Liver tissue was also applied for HBcAg detection, as described previously ( ). Intrahepatic HBV DNA and HBV cccDNA titers were determined from genomic DNA extracted from tupaia liver tissues using the phenol-chloroform extraction method. Serum ALT levels were determined using a Transnase Nissui kit (Nissui Pharmaceutical Co., Ltd.), and the data were standardized and are presented as IU/l.
HBV total DNA, cccDNA extraction and quantitation, and T7E1 assay Total DNA was isolated from tupaia liver tissues as previously described ( ). To purify HBV cccDNA, total DNA was treated with Plasmid-Safe ATP-Dependent DNase ( ) (Lucigen Corporation) at 37 °C for 60 min, followed by DNase inactivation at 70 °C for 30 min. The digested products were used as templates for real-time PCR to quantify HBV cccDNA, as described below. The T7E1 assay was performed as previously described ( ; ). The primers used were completely matched to HBV-Fmt sequence; 1st PCR sense (794) 5ʹ-CCGCTGTTACCAATTTTCTGTTATC-3ʹ, 1st PCR anti-sense (2051) 5ʹ-GAGGTGTGCAATGTTCTGGTGATT-3ʹ, 2nd PCR sense 5ʹ-(819) TGTGGGTATCCATTTAAATA-3ʹ, and 2nd PCR anti-sense (2027) 5ʹ-CTAAAGCATCCCGGTAGAGG-3ʹ. HBV DNA was quantified using real-time PCR as previously described ( ), with a detection limit of one copy of HBV DNA and HBV cccDNA per microgram of liver tissue. The primers and probes for the S gene included the forward primer HB-166-S21 (nucleotides (nts) 166–186): 5′-CAC ATC AGG ATT CCT AGG ACC-3′, the reverse primer HB-344-R20 (nts 344–325): 5′-AGG TTG GTG AGT GAT TGG AG-3′, and the TaqMan probe HB-242-S26FT (nts 242–267): 5′-CAG AGT CTA CAC TCG TGG ACT TC-3′. HBV cccDNA quantification was performed using qPCR with TaqMan chemistry, using the forward primer HBVcccDNA 1519-S25 (5′-ACG GGG CGC ACC TCT CTT TAC GCG G-3′), the reverse primer HBVcccDNA 1886-R25 (5′-CAA GGC ACA GCT TGG AGG CTT GAA C-3′), and the Taq-Man probe HBVcccDNA-1575 S26FT (5′6-FAM–CCG TGT GCA CTT CGC TTC ACC TCT GC-TAMRA-3′). Cycling was performed as 50 °C for 2 min, 95 °C for 10 min, and 53 cycles at 95 °C for 20 s and 65 °C for 1 min. PCR was performed using a CFX Connect Real-Time PCR Detection System (Bio-Rad). Cytokines and TLR mRNAs were quantified via qRT-PCR using specific primers (Table S1), as previously reported ( ).
Measurement of HBsAg and HBcrAg HBsAg levels in the tupaia serum was measured using the two-step sandwich assay principle with a fully automated chemiluminescent enzyme immunoassay system (Lumipulse G 1200, Fujirebio, Tokyo, Japan), as described previously ( ). HBcrAg levels in tupaia serum were measured using a high-sensitivity HBcrAg assay, as described previously ( ).
Total RNA extraction from tupaia liver tissues and measurement of cytokines and TLR mRNA Total RNA was extracted from tupaia liver tissues using the RNeasy Plus Mini Kit (QIAGEN) following the manufacturer's instructions, and genomic DNA was removed using the gDNA Eliminator Spin Column. The concentration and purity of the extracted RNA were measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Waltham, MA, USA). The samples were stored at −80 °C until needed for gene expression analysis. We analyzed the expression of TLR mRNAs (TLR1–9) in tupaia liver tissues using one-step qRT-PCR with Brilliant III Ultra-fast SYBR Green QRT-PCR Master Mix (Agilent Technologies, Santa Clara, CA, USA), following . The PCR process involved reverse transcription at 50 °C for 10 min, initial denaturation at 95 °C for 3 min, and 40 cycles at 95 °C for 5 s and 60 °C for 10 s. We also measured the expression of IL-6, TNF-α, IFN-γ, cGAS, and IFN-β mRNA using the same method, as described previously ( ; ). The gene-specific primer sequences are provided in Table S1. The gene copy number per microgram of liver RNA was calculated using a standard curve from pre-quantified gene copies. Each sample was measured in duplicate, and statistical analysis was performed to compare with uninfected controls.
ELISA and immunoblot assay Anti-HBc and anti-HBs antibodies in tupaia serum were detected via ELISA, as previously described ( ) with slight modifications. Purified HBc and HBs antigens (Beacle Co., Osaka, Japan) were diluted with a sodium carbonate buffer (pH 9.0) (5 μg/mL) and a 1 % Block Ace solution (KAC Co. Ltd., Japan) in phosphate-buffered saline (-) containing 0.05 % Tween 20. This solution was used to block and dilute the antibodies. Tupaia serum and anti-tupaia IgG rabbit serum (in-house) were diluted 10,000-fold for reaction. Anti-HBc rabbit polyclonal antibody (ab115992, Abcam Co.) and anti-HBs mouse monoclonal antibody (MoAb) (MA1-19263, ThermoFisher) were used as positive controls. Cas9 protein was detected in tupaia liver tissues via standard immunoblotting assay using an anti-Cas9 rabbit MoAb (#19,526, Cell Signaling Co.). Tissues were lysed in a lysis buffer (1 % SDS, 0.5 % NP40, 0.15 M NaCl, 10 mM Tris [pH 7.4], 5 mM EDTA, and 1 mM DTT). β actin was detected using an anti-β actin mouse MoAb (clone AC15, Merk Co.). Protein bands were visualized and quantified using a FUSION chemiluminescence imaging system (M&S Instruments, Inc.).
Statistical analysis Data are presented as the means ± SD. For statistical analysis, Student's t -test for two groups and one-way ANOVA and Dunnett's multiple comparisons were performed using GraphPad Prism software. Statistical significance was set at P < 0.05.
Results 3.1 Anti-HBV effects of the AAV2/WJ11-Cas9 system To examine the anti-HBV effects of the HBV-specific gRNA/Cas9 system in vivo in an immunocompetent HBV infection model, we injected AAV2/WJ11-Cas9 (5 × 10 12 copies/tupaia) into the tail vein of a tupaia ( ). Simultaneously, HBV genotype F-Mt (10 7 copies; entire nucleotide sequence is shown in GenBank LC831106.1) was injected intravenously as reported previously ( ). Blood samples were collected from tupaias on days −4, 1, 3, 5, 7, 10, and 14 days post HBV infection (dpi). We then measured HBV DNA and ALT levels in serum from mock-treated and AAV2/WJ11-Cas9-treated tupaias ( ). Significantly lower HBV DNA levels were observed in the serum of tupaia injected with AAV2/WJ11-Cas 9 than in mock-treated tupaia at 1, 7, 10, and 14 dpi ( A). Administration of AAV2/WJ11-Cas9 did not yield any significant changes in ALT levels, although the overall ALT values in the mock-treated group were significantly higher than those in the AAV2/WJ11-Cas9-treated group ( B). The peak of body weight in the mock-treated group was observed at 1 dpi (103 %), whereas the peak in the AAV2/WJ11-Cas9-treated group was observed at 3 dpi (106.3 %) ( C). Anti-HBs and anti-HBc antibodies could be detected in tupaia serum via ELISA at 14 dpi ( D). 3.2 Mock- and AAV2/WJ11-Cas9-treated tupaia liver tissues Pathological analysis was performed on the liver 14 dpi ( A–F). In mock-treated tupaias, 50 % to 60 % of the hepatocytes in the liver tissue exhibited balloon-like swelling due to hydropic degeneration ( A). The cytoplasm of some swollen hepatocytes was slightly eosinophilic and exhibited a ground-glass appearance ( B). Single-cell necrosis of the hepatocytes was not evident. Additionally, mild lymphocytic infiltration was observed in the interlobular connective tissues. In contrast, these abnormalities were absent in the livers of AAV2/WJ11-Cas9-treated tupaia ( C and D) or normal tupaia livers ( E and F). HBc antigen was detected in mock- ( G) and AAV2/WJ11-Cas9-treated tupaia liver tissues ( H), and a significant reduction of HBc antigen was observed in AAV2/WJ11-Cas9-treated tupaia liver tissues ( H). We next examined whether AAV2/WJ11-Cas9 could effectively suppress HBV DNA and cccDNA in the liver ( ). AAV2/WJ11-Cas9 treatment significantly suppressed HBV DNA expression compared to the mock control ( A). We also detected HBV cccDNA levels in liver genomic DNA, and a reduction was observed following AAV2/WJ11-Cas9 treatment ( B). Serum HBsAg and HBcrAg levels were measured at 14 dpi, and a significant reduction was observed following AAV2/WJ11-Cas9 treatment ( C and D). The presence of the AAV genome in liver tissue was confirmed through qPCR (5.4 × 10 4 copies/μg genome DNA; E). Cas9 protein expression was detected in the AAV2/WJ11-Cas9 treated group ( F); Cas9 protein was quantified and normalized to actin. The cleavage of the HBV genome by AAV2/WJ11-Cas9 was confirmed using the T7E1 assay ( ). HBV genome amplification via second-round PCR using the HBV-Fmt-819F and 2027R primers yielded the expected 1.2-kb fragment ( A, blue arrow head) from genomic DNA purified from the liver tissue of tupaia 329. We performed a T7E1 assay on the HBV genome (1.2 kb) fragment, as described previously ( ). The results showed digestion of AAV2/WJ11-Cas9-treated tupaia liver DNA, generating fragments of the expected size ( ca . 1.0 kb, A, orange arrowhead), which appeared after T7E1 assay ( A right). The amplified HBV genome (1.2 kb) was subcloned and sequenced ( B). Indel mutations in the HBV genome generated in target regions by AAV/WJ11-Cas9 are shown in B (i-1). 3.3 Molecular analysis of mock- and AAV2/WJ11-Cas9-treated tupaia liver tissues Given that AAV2/WJ11-Cas9-treated liver tissues showed significant recovery, we further characterized the modified host factors after AAV2/WJ11-Cas9 treatment. Consistent with the pathological analysis, the inflammatory cytokine IL-6 decreased to normal levels (Fig. S1). The level of TNF-α was relatively lower in AAV2/WJ11-Cas9-treated tupaia liver (Fig. S1). Additionally, the DNA sensor cyclic GMP-AMP (cGAMP) synthase, which suppresses HBV replication with STING ( ; ), was downregulated following AAV2/WJ11-Cas9 treatment. Conversely, IFN-β levels were increased after AAV2/WJ11-Cas9 treatment (Fig. S1). The level of IFN-g mRNA was highest in normal tupaia and decreased in mock-treated tupaia (Fig. S1). To further clarify the modifications of upstream sensors due to HBV infection and the subsequent recovery induced by AAV2/WJ11-Cas9, we examined the mRNA expression levels of TLR1–9 ( ). Following HBV-F infection, TLR2, TLR4, TLR6, TLR7, and TLR9 mRNAs were significantly upregulated and TLR1 and TLR4 were significantly downregulated in tupaia liver ( ). The modified mRNA expression levels of TLR1–9 induced by HBV-F infection returned to normal following AAV2/WJ11-Cas9 treatment, except for TLR5.
Anti-HBV effects of the AAV2/WJ11-Cas9 system To examine the anti-HBV effects of the HBV-specific gRNA/Cas9 system in vivo in an immunocompetent HBV infection model, we injected AAV2/WJ11-Cas9 (5 × 10 12 copies/tupaia) into the tail vein of a tupaia ( ). Simultaneously, HBV genotype F-Mt (10 7 copies; entire nucleotide sequence is shown in GenBank LC831106.1) was injected intravenously as reported previously ( ). Blood samples were collected from tupaias on days −4, 1, 3, 5, 7, 10, and 14 days post HBV infection (dpi). We then measured HBV DNA and ALT levels in serum from mock-treated and AAV2/WJ11-Cas9-treated tupaias ( ). Significantly lower HBV DNA levels were observed in the serum of tupaia injected with AAV2/WJ11-Cas 9 than in mock-treated tupaia at 1, 7, 10, and 14 dpi ( A). Administration of AAV2/WJ11-Cas9 did not yield any significant changes in ALT levels, although the overall ALT values in the mock-treated group were significantly higher than those in the AAV2/WJ11-Cas9-treated group ( B). The peak of body weight in the mock-treated group was observed at 1 dpi (103 %), whereas the peak in the AAV2/WJ11-Cas9-treated group was observed at 3 dpi (106.3 %) ( C). Anti-HBs and anti-HBc antibodies could be detected in tupaia serum via ELISA at 14 dpi ( D).
Mock- and AAV2/WJ11-Cas9-treated tupaia liver tissues Pathological analysis was performed on the liver 14 dpi ( A–F). In mock-treated tupaias, 50 % to 60 % of the hepatocytes in the liver tissue exhibited balloon-like swelling due to hydropic degeneration ( A). The cytoplasm of some swollen hepatocytes was slightly eosinophilic and exhibited a ground-glass appearance ( B). Single-cell necrosis of the hepatocytes was not evident. Additionally, mild lymphocytic infiltration was observed in the interlobular connective tissues. In contrast, these abnormalities were absent in the livers of AAV2/WJ11-Cas9-treated tupaia ( C and D) or normal tupaia livers ( E and F). HBc antigen was detected in mock- ( G) and AAV2/WJ11-Cas9-treated tupaia liver tissues ( H), and a significant reduction of HBc antigen was observed in AAV2/WJ11-Cas9-treated tupaia liver tissues ( H). We next examined whether AAV2/WJ11-Cas9 could effectively suppress HBV DNA and cccDNA in the liver ( ). AAV2/WJ11-Cas9 treatment significantly suppressed HBV DNA expression compared to the mock control ( A). We also detected HBV cccDNA levels in liver genomic DNA, and a reduction was observed following AAV2/WJ11-Cas9 treatment ( B). Serum HBsAg and HBcrAg levels were measured at 14 dpi, and a significant reduction was observed following AAV2/WJ11-Cas9 treatment ( C and D). The presence of the AAV genome in liver tissue was confirmed through qPCR (5.4 × 10 4 copies/μg genome DNA; E). Cas9 protein expression was detected in the AAV2/WJ11-Cas9 treated group ( F); Cas9 protein was quantified and normalized to actin. The cleavage of the HBV genome by AAV2/WJ11-Cas9 was confirmed using the T7E1 assay ( ). HBV genome amplification via second-round PCR using the HBV-Fmt-819F and 2027R primers yielded the expected 1.2-kb fragment ( A, blue arrow head) from genomic DNA purified from the liver tissue of tupaia 329. We performed a T7E1 assay on the HBV genome (1.2 kb) fragment, as described previously ( ). The results showed digestion of AAV2/WJ11-Cas9-treated tupaia liver DNA, generating fragments of the expected size ( ca . 1.0 kb, A, orange arrowhead), which appeared after T7E1 assay ( A right). The amplified HBV genome (1.2 kb) was subcloned and sequenced ( B). Indel mutations in the HBV genome generated in target regions by AAV/WJ11-Cas9 are shown in B (i-1).
Molecular analysis of mock- and AAV2/WJ11-Cas9-treated tupaia liver tissues Given that AAV2/WJ11-Cas9-treated liver tissues showed significant recovery, we further characterized the modified host factors after AAV2/WJ11-Cas9 treatment. Consistent with the pathological analysis, the inflammatory cytokine IL-6 decreased to normal levels (Fig. S1). The level of TNF-α was relatively lower in AAV2/WJ11-Cas9-treated tupaia liver (Fig. S1). Additionally, the DNA sensor cyclic GMP-AMP (cGAMP) synthase, which suppresses HBV replication with STING ( ; ), was downregulated following AAV2/WJ11-Cas9 treatment. Conversely, IFN-β levels were increased after AAV2/WJ11-Cas9 treatment (Fig. S1). The level of IFN-g mRNA was highest in normal tupaia and decreased in mock-treated tupaia (Fig. S1). To further clarify the modifications of upstream sensors due to HBV infection and the subsequent recovery induced by AAV2/WJ11-Cas9, we examined the mRNA expression levels of TLR1–9 ( ). Following HBV-F infection, TLR2, TLR4, TLR6, TLR7, and TLR9 mRNAs were significantly upregulated and TLR1 and TLR4 were significantly downregulated in tupaia liver ( ). The modified mRNA expression levels of TLR1–9 induced by HBV-F infection returned to normal following AAV2/WJ11-Cas9 treatment, except for TLR5.
Discussion The results of this study highlight the potential of WJ11/Cas9, delivered via AAV2 vectors, as a new therapeutic approach for suppressing HBV during the acute phase of infection in an immunocompetent model. Significant suppression of the viral load in serum HBV was observed in the AAV2/WJ11-Cas9-treated group at 1, 7, 10, and 14 dpi, compared with that in the mock-treated control group. Consistent with this observation, inflammation in liver tissues induced by HBV-F infection was significantly reduced following AAV2/WJ11-Cas9 treatment, including a reduction in hepatocyte necrosis. Among the inflammatory cytokines, IL-6 mRNA levels were significantly suppressed by AAV2/WJ11-Cas9. Elevated IL-6 mRNA levels are suggestive of more active hepatic necroinflammation and disease progression ( ; , ). Therefore, AAV2/WJ11-Cas9 may have therapeutic effects against HBV-induced hepatitis. Furthermore, suppression of HBV infection was associated with increased levels of IFN-β, consistent with the previous findings that HBV suppresses IFN-β in tupaia ( ). The suppression of IFN-β by HBV may primarily result from the downregulation of TLR3 which is a major regulator of IFN-β induction ( ). The cyclic GMP-AMP synthase pathway, a key DNA sensor that suppresses HBV replication through STING, is downregulated following AAV2/WJ11-Cas9 treatment, potentially compromising the immune response and allowing HBV to evade detection more effectively ( ). The expression of upstream TLRs was significantly modified by HBV-F but mostly returned to normal following AAV2/WJ11-Cas9 treatment, except for TLR5 . Upregulation of TLR2, TLR4, TLR6, TLR7, and TLR9 by HBV-F infection may contribute to the induction of inflammatory cytokine production ( ), which was downregulated by AAV2/WJ11-Cas9 treatment. TLR1 was significantly downregulated by HBV-F, which may reflect its role in suppressing inflammation ( ). TLR3 expression was also significantly suppressed by HBV-F, which likely contributed to the suppression of IFN-β induction, consistent with previous observations ( ). Thus, the expression of TLR1–9 , except for TLR5 , was shown to be closely linked with HBV-F in tupaia liver. In our previous work, we showed the suppressive effect of AAV2/WJ11-Cas9 on HBV genotype C ( ). In the present study, we show the suppressive effect of AAV2/WJ11-Cas9 on HBV-F. This effect may be attributed to the conserved nature of the gRNA sequence of WJ11 (nucleotide number 1859–1878) across HBV genotypes. The WJ11 gRNA sequence is perfectly conserved across HBV genotypes A–F, suggesting that AAV2/WJ11-Cas9 can be developed for pan-genotypic anti-HBV therapy. Although using the AAV2 vector alone as a negative control would have been more relevant to confirm the effect of AAV2/WJ11-Cas9 in targeting HBV, Cas9 expression and the detection of indel mutations in the HBV genome, as observed by the T7E1 assay, provide evidence of the direct effect of AAV2/WJ11-Cas9 on HBV genome disruption ( ). In a previous study, the efficiency of target-specific mutagenesis of HBV DNA in mouse livers was approximately 11.0 %, as measured by deep sequencing in a HBV hydrodynamic injection- and CRISPR/Cas9-treated mouse model ( ). Based on the results of this study, the mutagenesis rate was 3.8 %. Thus, increasing the amount of AAV2/WJ11-Cas9 may be necessary to achieve complete genome targeting in liver tissues during natural HBV infection. To further clarify the anti-HBV effect of AAV2/WJ11-Cas9, future studies should evaluate the effect of AAV vector alone on HBV DNA and cccDNA, although we already demonstrated a significantly higher effect of AAV2/WJ11-Cas9 compared to vector alone in HBV-infected humanized chimeric mice ( ). The results of this study indicated that the tupaia HBV infection model is a useful tool for characterizing host immune responses ( ; ; ). To evaluate AAV2/WJ11-Cas9, we used 5 × 10 12 copies for each animal, and transduction was confirmed via AAV detection in the liver tissues. Considering the body weight and liver size of tupaia (average body weight 150 g, average liver weight 10 g), it is estimated that at least 10 14–15 copies of AAV2/WJ11-Cas9 would be required for a human patient (body weight, 60 kg, liver 1–1.5 kg). This strongly suggests that improving delivery systems is essential to facilitate the application of CRISPR/Cas9 technology in human gene therapy. AAV vectors, particularly AAV2, are widely regarded as highly effective delivery vehicles for CRISPR-Cas9 components in vivo due to their high transduction efficiency and low immunogenicity. However, AAV8, a hepatotropic variant, could serve as a promising candidate for further optimization, contributing enhanced targeting specificity for liver cells and potentially improving the efficacy of gene editing in liver-associated diseases ( ; ; ). Our findings enhance our understanding of the immune response of immunocompetent HBV model animals in the acute phase of HBV infection, which could further advance translational medicine. However, this study had limitations that should be addressed. We focused on the acute phase of HBV infection at 14 dpi, which means that our results may not be fully generalizable to long-term or chronic treatment outcomes. Furthermore, the tupaia model, while valuable for HBV research, may not fully replicate human immune responses, thus limiting direct applicability. Certain markers, such as HBeAg or Anti-HBe antibody, were undetectable within the study timeframe, potentially missing important early immune responses. In addition, cytokine analysis was conducted solely at 14 dpi, potentially overlooking important immune response changes at later stages. These limitations underscore the importance of future research with an extended study duration, varied animal models, and broader immune response analyses to enhance the understanding and applicability of the findings. To our knowledge, this is the first study to evaluate the CRISPR/Cas9 system in an immunocompetent HBV infection model. Overall, our findings suggest that WJ11-Cas9 delivered using AAV2 vectors may represent a new therapeutic strategy for eliminating HBV infection. Additionally, this study provides valuable molecular insights into HBV-induced inflammation. However, chronic HBV infection remains a major global health problem due to the persistence of cccDNA and integrated HBV DNA replicative templates. Moreover, HBV mutations can occur during reverse transcription at an estimated rate of 1–3 × 10 −5 nucleotide substitutions per site per year, increasing the risk of resistance to nucleoside/nucleotide analog therapies. Consequently, alternative therapeutic approaches are required to treat effectively HBV infections. Genome editing enables the manipulation of target genes in various cell types using engineered nucleases. Compared with ZFNs and TALENS, the CRISPR/Cas9 system can be easily reprogrammed to cleave virtually any DNA sequence by redesigning native or engineered crRNAs. Although CRISPR/Cas9 has revolutionized gene editing, viral delivery in immunocompetent animals has not been thoroughly explored. Furthermore, evaluating CRISPR/Cas9 in an HBV-infected tupaia model could provide a reference for future therapeutic interventions in translational medicine. Accordingly, further studies are warranted to investigate the efficacy of the CRISPR/Cas9 therapeutic strategy for treating chronic HBV infection.
CRISPR/Cas9: Gene editing method using Cas9 enzymes that recognize guide RNA sequences ZFN (zinc finger nuclease): The first genome editing tool that recognizes FokI digestion site. TALEN: Transcription activator-like effector nuclease composed of TALE protein and FokI nuclease.
This work was supported by grants from the Japan Agency for Medical Research and Development ( K07S411687S , 23fk0310515h0002 , and 24fk0310515h0003 ) and the Ministry of Health, Labour, and Welfare of Japan .
Md Haroon Or Rashid: Writing – review & editing, Writing – original draft, Data curation. Mohammad Enamul Hoque Kayesh: Writing – review & editing, Writing – original draft, Validation, Data curation. Md Abul Hashem: Writing – review & editing, Data curation, Conceptualization. Tatsuro Hifumi: Writing – review & editing, Data curation. Shintaro Ogawa: Methodology, Data curation. Noriaki Miyoshi: Methodology, Data curation. Yasuhito Tanaka: Methodology, Data curation. Michinori Kohara: Writing – review & editing, Writing – original draft, Supervision, Data curation, Conceptualization. Kyoko Tsukiyama-Kohara: Writing – review & editing, Writing – original draft, Funding acquisition, Data curation, Conceptualization.
The authors declare no conflict of interest.
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Acute total intestinal volvulus caused by sclerosing mesenteritis: a case report and review of the literature | 0f35f6b2-2c24-4048-b462-89bc51e66150 | 11948711 | Digestive System[mh] | Acute total small intestine volvulus is a severe condition characterized by the near-complete rotation of the small intestine and its mesentery by over 180° around the mesenteric axis. This rotation is accompanied by the twisting of the mesenteric vessels, resulting in circulatory impairments of varying severity within the intestinal tract. In the wake of acute mesenteric ischemia, the affected intestinal segment rapidly progresses to necrosis, which can precipitate secondary septic shock. This life-threatening condition, if untreated, is inevitably fatal . Total small intestine volvulus in adults is an uncommon condition with an etiology that remains not fully understood. It is suspected to be associated with various factors, including small intestinal polyps, diverticula, adhesions, lipomas, uterine fibroids, and the aftermath of bariatric surgery, among other potential contributors [ – ]. A case report detailing an intestinal obstruction caused by a lipoma illustrates that mesenteric tumors can lead to mesenteric volvulus and small intestine volvulus as a result of the displacement of intestinal loops . Sclerosing mesenteritis is an uncommon idiopathic condition marked by chronic fat necrosis, inflammation, and fibrosis, predominantly involving the mesentery of the small intestine. Gross examination reveals diffuse thickening of the mesentery, with single or multiple confluent masses present at the mesenteric root, along with surface features of contraction, aggregation, and wrinkling. Patients typically exhibit a range of clinical symptoms, including abdominal pain, nausea, low-grade fever, weight loss, and intestinal dysfunction. On abdominal examination, half of the patients present with a distended abdomen, and palpation may reveal a poorly defined mass. While cases of mesenteric fat necrosis leading to intestinal obstruction have been documented, instances of sclerosing mesenteritis inducing total small bowel volvulus are exceptionally rare . The mesenteric mass resulting from sclerosing mesenteritis may alter the length, position, or fixation of the mesentery, thus providing an anatomical basis for intestinal volvulus. Such masses may compress or displace the intestinal tract, leading to the accumulation of intestinal contents or expansion, thereby increasing the weight of the intestinal tract. Additionally, the mass may restrict the normal movement of the intestinal tract, further elevating the risk of volvulus. When subjected to volvulus, it may compromise the primary vascular structures, including the superior mesenteric artery. This can rapidly result in necrosis of the entire small intestine. Within this article, we detail a case of the latter, offer a discussion on the findings, and provide a comprehensive review of the relevant literature.
Clinical presentation A 68-year-old Chinese female individual was hospitalized owing to severe, persistent, and colicky abdominal pain that occurred 6 h after engaging in physical labor. The intensity of the pain compelled her to adopt a knee-chest position. She reported a significant weight loss over the preceding 12 months. A total of 11 years prior, she had undergone a “left inguinal hernia repair” in the absence of any abdominal trauma. At the time of admission, she exhibited lower abdominal tenderness, yet without pronounced muscular rigidity. Imaging Abdominal computed tomography (CT) imaging (Fig. ) disclosed findings consistent with mesenteric volvulus accompanied by intestinal obstruction, suggesting a potential for intestinal necrosis. A high-density mass within the right mesentery raised the possibility of a tumor. Laboratory tests The patient had a white blood cell count of 19.9 × 10 9 /L with a neutrophil percentage of 86.7%. Her hemoglobin level was 130 g/L, blood lactate was 1.7 mmol/L, lactate dehydrogenase was 236.4 U/L, and creatine kinase was 77.5 U/L. Tumor markers included a carcinoembryonic antigen level of 0.89 ng/ml and a carbohydrate antigen 19–9 level of 6.2 U/ml. Diagnosis Her diagnosis was of intestinal volvulus with necrosis, without ruling out a mesenteric tumor. Surgical situation The intraoperative exploration exposed a substantial accumulation of turbid, purulent fluid within the abdominal cavity; the majority of the small intestine was purplish-black and grayish-white (Fig. ). After conversion to laparotomy, a firm mass with an irregular surface and limited mobility was identified at the base of the small intestine mesentery, resulting in a 1440° (four full turns) clockwise rotation of the mesenteric root (Fig. ). A segment of approximately 400 cm of the small intestine, extending from 5 cm above the duodenojejunal flexure to 3 cm below the ileocecal junction, was determined to be entirely necrotic. In an effort to manage the abdominal infection, a complete resection of the small bowel was carried out, along with the creation of a jejunostomy at the proximal jejunum. Postoperative pathology Postoperative pathology findings described a mesenteric mass measuring 4.0 cm × 3.5 cm × 3.0 cm. The mass was firm and hard, resisting sectioning (Fig. ). Microscopic examination revealed significant calcification, accompanied by a proliferation of fibrous tissue, chronic inflammatory cell infiltration, and areas of fat necrosis. The mesentery also showed signs of congestion, stasis, and hemorrhage. Three lymph nodes were sampled, and no tumor metastasis was detected (Fig. ). Due to the prompt intervention, the patient was spared from developing severe septic shock. However, the development of short bowel syndrome is now certain. The patient is projected to require long-term total parenteral nutrition or home parenteral nutrition, and may be a candidate for isolated intestinal transplant at an appropriate future date.
A 68-year-old Chinese female individual was hospitalized owing to severe, persistent, and colicky abdominal pain that occurred 6 h after engaging in physical labor. The intensity of the pain compelled her to adopt a knee-chest position. She reported a significant weight loss over the preceding 12 months. A total of 11 years prior, she had undergone a “left inguinal hernia repair” in the absence of any abdominal trauma. At the time of admission, she exhibited lower abdominal tenderness, yet without pronounced muscular rigidity.
Abdominal computed tomography (CT) imaging (Fig. ) disclosed findings consistent with mesenteric volvulus accompanied by intestinal obstruction, suggesting a potential for intestinal necrosis. A high-density mass within the right mesentery raised the possibility of a tumor.
The patient had a white blood cell count of 19.9 × 10 9 /L with a neutrophil percentage of 86.7%. Her hemoglobin level was 130 g/L, blood lactate was 1.7 mmol/L, lactate dehydrogenase was 236.4 U/L, and creatine kinase was 77.5 U/L. Tumor markers included a carcinoembryonic antigen level of 0.89 ng/ml and a carbohydrate antigen 19–9 level of 6.2 U/ml.
Her diagnosis was of intestinal volvulus with necrosis, without ruling out a mesenteric tumor.
The intraoperative exploration exposed a substantial accumulation of turbid, purulent fluid within the abdominal cavity; the majority of the small intestine was purplish-black and grayish-white (Fig. ). After conversion to laparotomy, a firm mass with an irregular surface and limited mobility was identified at the base of the small intestine mesentery, resulting in a 1440° (four full turns) clockwise rotation of the mesenteric root (Fig. ). A segment of approximately 400 cm of the small intestine, extending from 5 cm above the duodenojejunal flexure to 3 cm below the ileocecal junction, was determined to be entirely necrotic. In an effort to manage the abdominal infection, a complete resection of the small bowel was carried out, along with the creation of a jejunostomy at the proximal jejunum.
Postoperative pathology findings described a mesenteric mass measuring 4.0 cm × 3.5 cm × 3.0 cm. The mass was firm and hard, resisting sectioning (Fig. ). Microscopic examination revealed significant calcification, accompanied by a proliferation of fibrous tissue, chronic inflammatory cell infiltration, and areas of fat necrosis. The mesentery also showed signs of congestion, stasis, and hemorrhage. Three lymph nodes were sampled, and no tumor metastasis was detected (Fig. ). Due to the prompt intervention, the patient was spared from developing severe septic shock. However, the development of short bowel syndrome is now certain. The patient is projected to require long-term total parenteral nutrition or home parenteral nutrition, and may be a candidate for isolated intestinal transplant at an appropriate future date.
Sclerosing mesenteritis is an idiopathic, non-neoplastic condition distinguished by chronic fat necrosis, inflammation, and fibrosis. It was designated as mesenteric panniculitis by Ogden in the 1960s . This disease has been known by various names throughout medical history, such as retractile mesentitis, lipogranuloma of the mesentery, and isolated lipodystrophy, among others. As the lesion progresses, the extents of fat necrosis, fibrosis, and inflammation within it vary. Currently, these terms are regarded as synonymous, all referring to the same condition . Sclerosing mesenteritis occurs approximately twice as frequently in men as in women, with a predilection for adults over the age of 50 years, and it is less commonly seen in children. The etiology and pathogenesis of this condition remain elusive, with contributing factors including a history of abdominal surgery, abdominal trauma, autoimmune conditions, chronic ischemia, infections, and intra-abdominal tumors . The clinical presentation of sclerosing mesenteritis can include abdominal pain, weight loss, diarrhea, anorexia, and abdominal distension. Upon physical examination, abdominal tenderness and the presence of an abdominal mass may be noted. However, many patients are asymptomatic or have no apparent signs, with the condition often being discovered incidentally during imaging studies or surgery . In the case presented, the patient experienced weight loss 1 year prior to the onset of other symptoms. Diagnosing sclerosing mesenteritis primarily relies on abdominal computed tomography (CT), with the quintessential feature being involvement of the mesenteric root. The characteristic appearance includes a well-demarcated mesenteric fat mass with heterogeneous density, and “fat ring sign” can be observed, that is, an annular low-density ring that can be seen around the blood vessel wall. As the disease advances, CT images of advanced sclerosing mesenteritis typically reveal irregular masses with partial fusion of the mesenteric roots, which are denser than the preexisting soft tissue, indicative of the disease’s fibrotic nature, and may show internal calcification within the masses. Magnetic resonance imaging (MRI) is an equally effective diagnostic tool, which excels in displaying fat, soft tissue components, and the extent of vascular involvement. Fibrous tissue contains immobile protons and exhibits low signal in all sequences; T2-weighted imaging or fat suppression sequences are highly valuable in distinguishing benign terminal mesenteric inflammation from malignant tumors . In addition, MRI can also display whether major blood vessels and their branches are affected. As previously reported, mesenteric lipoma, mesenteric cystic lymphangioma, and sclerosing mesenteritis can cause intestinal volvulus and blood supply disorder, and it is difficult to distinguish them only from clinical manifestations . Imaging examination is an important basis for differential diagnosis. For example, lymphoma is usually accompanied by retroperitoneal lymph node infiltration; after chemotherapy, owing to fibrosis and mesenteric adhesion, it can show high-density mesenteric fog, but there will be no “fat ring sign.” Lipoma can show a homogeneous tumor of adipose tissue without fibrosis ; Metastasis often fused into a mass when it is implanted and spread in abdominal cavity and involves mesentery. There is no fat-density focus in the tumor and no “pseudocapsule sign” will appear. Currently, there is no consensus on the treatment of sclerosing mesenteritis. According to expert opinions, asymptomatic patients may be managed with observation alone, without immediate treatment. For those with symptoms such as abdominal pain or compression from an abdominal mass, the preferred initial pharmacological approach is a combination of tamoxifen and prednisone. Surgical intervention is typically reserved for patients who present with mechanical intestinal obstruction and do not respond to conservative medical management . When intestinal volvulus and circulatory disturbances occur, early disruption of the intestinal mucosal barrier may ensue, which can then cause sepsis owing to the displacement of intestinal microbiota. Therefore, sufficient antibiotics must be given to resist infection. Owing to intestinal obstruction, a large amount of fluid is lost, which can easily lead to water electrolyte imbalance and acid–base imbalance. Proper correction should be given before surgery. Surgery should remove as many necrotic and potentially inactive intestinal tubes as possible. The main causes of death for patients are infection and gastrointestinal stress ulcer bleeding. After surgery, attention should be paid to protecting the gastrointestinal mucosa and preventing multiple organ failure. Patients in this case have a poor prognosis. Generally, sclerosing mesenteritis is considered a benign condition, and the majority of cases remain stable over time. To our knowledge, this represents the first case report of volvulus resulting from sclerosing mesenteritis. Although exceedingly rare in clinical practice, sclerosing mesenteritis can result in severe volvulus of the mesenteric root, leading to ischemic necrosis of the entire small intestine. Consequently, sclerosing mesenteritis should be considered in the differential diagnosis when encountering cases of small intestine volvulus associated with abdominal masses. Early detection and prompt, decisive surgery are paramount for both saving lives and maximizing intestinal preservation.
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