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To examine CTL activities, splenocytes (1.0 × 106/mL) were harvested and then primed in vitro for 1 h in the presence of IL-2 (10 ng/mL). Those cells were incubated with mature DCs (1 × 105/mL) at a ratio of 10:1 in 96-well plates for 3 days to obtain specific-CTL cells. The specific CTLs (effector cells, 5 × 104/mL) were collected and mixed with B16 cells (target cells, 5 × 103/mL) into 96-well plates to give effector/target cells a ratio of 10:1. The cells were treated with various concentrations of AHG for 44 h. The CTL cell cytotoxicity was measured by MTT assay. The activity of CTLs was calculated by the following formula: CTL cell activity (%) = (ODT − (ODS − ODE))/ODT × 100%, where ODT is optical density value of target cells control, ODS is optical density value of test samples and ODE is optical density value of control effector cells. | study | 100.0 |
The cells were seeded in 96-well plates and cultured as mentioned above. Then, 20 μL MTT (5 mg/mL) was added to each well. The plates were incubated for 4 h. DMSO solution (150 μL) was added to resolve the colored material, and the absorbance of each well was measured at 570 nm on a microplate reader (BioRad 680, Hercules, CA, USA). | study | 99.94 |
The mice were randomly divided into six groups with 8 animals for each. One group was selected as NC group, which was treated with physiological saline, while the other five groups of mice were subjected to immunosuppression by administration of CY (100 mg/kg) intraperitoneally for 3 days. Then mice in AHG groups were intraperitoneally injected AHG at the dose of 1, 5 or 10 mg/kg body weight, respectively, once a day for the next 7 consecutive days, while mice in positive control group were treated with 1 mg/kg body weight LNT. Other mice and model control (MC) group were only given physiological saline at the same intervals. The animals were sacrificed by decapitation at 24 h after the last administration and heart, liver, kidney and spleen tissues were excised rapidly for further analysis. | study | 100.0 |
The splenocytes prepared as described above were adjusted to a final density of 5 × 106/mL, and stimulated with or without Con A (5 μg/mL) and LPS (10 μg/mL), respectively. The cells were incubated in 96 well plates at 37 °C in an incubator with 5% CO2 for 48 h. Lymphocyte proliferation was assessed by the absorbance at 570 nm through following MTT assay. | study | 99.94 |
The cytokines (IL-2, TNF-α, IFN-γ and IL-4) of the collected supernatants from splenocytes culture medium was determined using ELISA kits according to the manual of the manufacturer. The concentrations of IL-2, TNF-α, IFN-γ and IL-4 were elucidated by the absorbance values at 450 nm. | study | 99.94 |
Splenocytes (1 × 106/mL) were seeded in the 24-well plate, and incubated with the fluorescent calcium prober Fluo-3/AM at 37 °C for 45 min. Post incubation, the cells obtained by centrifugation were washed twice with PBS. Fluorescent signals were then detected by flow cytometry. | study | 99.94 |
Before three days of finishing administration, all the mice were immunized intraperitoneally with 0.2 mL of 5% (v/v) SRBC. On 9th day, after measuring the left footpad thickness, the mice were again challenged with 20 μL of 20% (v/v) SRBC in another paw. The increase footpad thickness was considered as foot swelling (in mm) after 24 h. | study | 99.94 |
The homogenate of liver, heart or kidney was prepared in 0.1 g/mL wet weight of ice-cold physiological saline. The samples were centrifuged at 2000 g at 4 °C for 10 min, and the supernatants were collected for the measurement of protein, T-AOC, MAD, CAT, SOD and GSH-Px. These antioxidant parameters were determined by commercially available assay kits in accordance with the manufacturer protocols. | study | 99.94 |
All results were expressed as means ± standard deviation (SD). Statistical significance of different group was evaluated by the use of one-way analysis of variance (ANOVA) using IBM SPSS statistics version 19.0 (IBM SPSS Inc., Chicago, IL, USA). P values less than 0.05 were considered statistically significant. | study | 96.2 |
The current study demonstrated that holothurian glycosaminoglycan (AHG) could successfully improve immunoregulation activity in vitro, and possesses the capacity to restore CY induced immunosuppression in vivo. Based on these findings and the data accumulated, it was suggested that AHG has huge potential to be an effective immunomodulatory agent and adjuvant in clinical treatment of all cancer patients. Further investigation should be conducted to understand the overall intracellular process associated with the AHG-induced immune response for the development of a novel marine medicine. | study | 99.94 |
Specifications TableTableSubject areaBiologyMore specific subject areaVeterinary microbiology, anaerobes, Clostridium perfringens, Clostridium difficileType of dataTables, figures and textHow data was acquiredAnalysis of clinical data and characteristics of bacterial isolatesData formatFiltered and analyzedExperimental factorsDogs and cats attended in veterinary clinics, and the Clostridium perfringens and C. difficile isolates obtained from their fecesExperimental featuresAnalysis of general data about participating clinics, and the demographic and clinical features of recruited animals; genetic and phenotypic profiling of isolatesData source locationUniversidad Complutense de Madrid, Madrid, SpainData accessibilityData is provided with this article | other | 99.56 |
Value of the data•First detailed analysis of the prevalence of Clostridium perfringens and Clostridium difficile shedding by small animals (dogs and cats) in the Madrid region (Spain).•Detailed phenotypic and genetic data of recovered isolates is provided, which may be useful for comparison in future epidemiological surveys.•Given the recent emergence of antibiotic-resistant strains of C. difficle, information on the antimicrobial susceptibility profiles of the isolates obtained in this survey may be particularly valuable. | study | 99.94 |
The data shown in Section 1.1 of this article provide detailed information on the veterinary clinics that participated in a recent survey of the prevalence of fecal shedding of Clostridium perfringens and C. difficile by dogs and cats which was carried out in the Madrid region (Spain) . Furthermore, the demographic and clinical features of recruited animals are detailed in Section 1.2, and Section 1.3 provides extensive data on the genetic and phenotypic characteristics of recovered bacterial isolates. | study | 99.94 |
An overview of the 17 veterinary clinics that participated in the study (hereafter referred to as clinics A to Q) is provided in Table 1. Two clinics (L and P) did not return a questionnaire of general data about their centre (see Section 2) and in two other cases (clinics H and K) the returned questionnaire was incomplete. Participating clinics were scattered within the Madrid region (14 were located in the capital city, two in other municipalities within the metropolitan area and one in a rural location) and varied widely in their year of opening (from 1981 to 2014), number of cases attended per week (x±S.D.=37.6±18.7 and 16.5±11 for dogs and cats, respectively), number of fecal cultures requested per week (1.6±2.4 and 0.9±1.8), and other parameters (Table 1). These clinics also differed in the antibiotics used for the treatment of diarrhea, but 12 of them (80% for which pharmacological data were available) reported the use of metronidazole for the treatment of these conditions. Only three clinics (20%; F, J and O) acknowledged frequent request of microbiological culturing for anaerobes, and five clinics (33.3%; D, F, H, N and O) reported occasional suspicion of C. difficile and/or C. perfringens involvement in severe cases of diarrhea.Table 1Overview of the characteristics of the veterinary clinics that participated in the study.aTable 1ClinicOpening yearNo. cases per weekbNo. diarrea cases per weekbNo. fecal samples per weekbNo. fecal cultures per weekbA201461 (55 D, 6 C)2.5 (2 D, 0.5 C)1 (1 D)0.5 (0.5 D)B199482 (50 D, 32 C)2.5 (2 D, 0.5 C)3.5 (3 D, 0.5 C)0C200911 (10 D, 1 C)5.5 (5 D, 0.5 C)1 (1 D)0D201440 (22 D, 18 C)8 (7 D, 1 C)7 (5 D, 2 C)0E199340 (20 D, 20 C)2 (2 D)4 (2 D, 2 C)4 (2 D, 2 C)F1981100 (70 D, 30 C)14 (10 D, 4 C)2.3 (2 D, 0.3 C)0.31 (0.3 D, 0.01 C)G198427.5 (16 D, 11.5 C)11.5 (8 D, 3.5 C)1.5 (1 D, 0.5 C)1.5 (1 D, 0.5 C)H200255 (35 D, 20 C)5 (4 D, 1 C)NANAI199270 (60 D, 10 C)8 (7 D, 1 C)1.5 (1 D, 0.5 C)0J200490 (48 D, 42 C)12 (8 D, 4 C)16 (10 D, 6 C)14 (8 D, 6 C)K1985NANA10 (6 D, 4 C)10 (6 D, 4 C)LNANANANANAM200147.5 (35 D, 12.5 C)4.5 (4 D, 0.5 C)4.5 (4 D, 0.5 C)3 (2.5 D, 0.5 C)N199935 (25 D, 10 C)4 (3 D, 1 C)4 (3 D, 1 C)1 (1 D)O201026 (20 D, 6 C)3 (2.5 D, 0.5 C)0.5 (0.5 D)0.5 (0.5 D)PNANANANANAQ200572 (60 D, 12 C)20 (20 D)2 (2 D)0Totalc54.1±25.3 (37.6±18.7 D, 16.5±11 C)7.3±5.1 (6±4.6 D, 1.3±1.4 C)4.2±4.1 (3±2.5 D, 1.2±1.7 C)2.5±4.1 (1.6±2.4 D, 0.9±1.8 C)aSee also Fig. 1.bD, dogs; C, cats. All figures refer to the last 12-month period.cx±S.D. | study | 99.94 |
The demographic characteristics of recruited animals are summarized in Table 2 and Fig. 1, Fig. 2. A total of 142 animals, including 105 dogs and 37 cats (73.9% and 26.1% of total, respectively; Fig. 1A) of diverse breeds (Fig. 2), were recruited for the study. The male/female ratio of animals varied widely among clinics, with the overall values for dogs and cats being similar (56.2%, 43.8% and 56.8%, 43.2%, respectively; Fig. 1C). The age distribution of sampled animals also showed ample variation among clinics, but the overall values were similar for the dog and cat subpopulations: 20%, 45.7%, 32.4% of dogs and 10.8%, 54.1%, 32.4% of cats had <1 year, 1–6 years and ≥7 years, respectively (Table 2).Fig. 1Characteristics of the animal populations recruited for this study. A) Bar plot showing the distribution of recruited animals per species: dog, green bars; cats, orange bars. For each participating clinic (A to Q), the overall number of animals is indicated between parentheses. B) Bar plot of the overall age distribution of dogs (n=105; green bars) and cats (n=37; orange bars) recruited for the study. C) Pie charts of the overall sex distribution of dogs (n=105) and cats (n=37) included in the study. Blue and pink sectors represent male (M) and female (F) subpopulations, respectively. D) Bar plot of the antimicrobial treatments administered to recruited dogs and cats (green and orange bars, respectively) ≤30 days before sampling. E) Bar plot of recent pharmacological treatments administered to recruited dogs and cats (green and orange bars, respectively) ≤30 days before sampling. Abbreviations: ACE, angiotensin-converting-enzyme; NSAIDs, nonsteroidal anti-inflammatory drugs; PPIs, proton pump inhibitors. In panels D and E, pharmacological treatment data were not available for a total of 48 animals (32 dogs and 16 cats).Fig. 1Fig. 2Bar plots showing the breed distribution of recruited dogs (n=105, left panel) and cats (n=37, right panel). *Other dog breeds (n): Boxer (3), Setter (3), Shih Tzu (3), American Staffordshire Terrier (2), Belgian Shepherd (2), Border Collie (2), Chinese Shar-Pei (2), Cocker Spaniel (2), Golden Retriever (2), Labrador Retriever (2), Pug (2), Shiba Inu (2), Andalusian Hound (1), Chihuahua (1), Dalmatian (1), Galician Palleiro (1), Hound (1), Mastiff (1), Miniature Pinscher (1), Pit bull (1), Poodle (1), Water dog (1).Fig. 2Table 2Overview of the animals that were recruited for this studya.Table 2ClinicNbDogsCatsnSex ratiocAge distributionDays since last episode of diarrheanSex ratiocAge distributionDays since last episode of diarrhea<1 yr1–6 yr≥7 yr0≤30>30<1 yr1–6 yr≥7 yr0≤30>30A662:133.3%66.7%0%0%66.7%33.3%0–––––––B15121:525%25%50%25%0%75%32:10%33.3%66.7%0%0%100%C843:150%50%0%0%25%75%41:10%25%75%50%0%50%D321:10%100%0%0%0%100%10:10%0%100%0%0%100%E432:10%66.7%33.3%0%66.7%33.3%10:10%0%100%100%0%0%F1252:30%20%80%0%40%60%74:314.3%85.7%0%0%0%100%G171511:420%40%40%13.3%26.7%60%20:10%100%0%0%0%100%H554:1100%0%0%20%20%60%0–––––––I654:120%60%20%40%20%40%11:00%0%100%0%100%0%J*832:10%66.7%0%33.3%66.7%0%53:220%60%0%40%60%0%K221:00%50%50%0%0%100%0–––––––L*220:10%0%50%50%0%50%0–––––––M27224:74.5%59.1%36.4%0%13.6%86.4%53:220%60%20%0%0%100%N*987:112.5%37.5%50%12.5%0%37.5%11:00%0%100%0%0%100%O1141:125%75%0%0%25%75%75:214.3%57.1%28.6%0%0%100%P110:1100%0%0%100%0%0%0–––––––Q665:116.7%50%33.3%33.3%33.3%33.3%0–––––––Total*14210559:4620%45.7%32.4%13.3%21.9%61%3721:1610.8%54.1%32.4%13.5%10.8%75.7%aSee also Fig. 1. Asterisks indicate that there were some recruited animals with missing data and thus the sum of percentages can be <100%.bTotal number of animals that were recruited (i.e. dogs and cats).cNumber of males: number of females. | study | 99.94 |
Characteristics of the animal populations recruited for this study. A) Bar plot showing the distribution of recruited animals per species: dog, green bars; cats, orange bars. For each participating clinic (A to Q), the overall number of animals is indicated between parentheses. B) Bar plot of the overall age distribution of dogs (n=105; green bars) and cats (n=37; orange bars) recruited for the study. C) Pie charts of the overall sex distribution of dogs (n=105) and cats (n=37) included in the study. Blue and pink sectors represent male (M) and female (F) subpopulations, respectively. D) Bar plot of the antimicrobial treatments administered to recruited dogs and cats (green and orange bars, respectively) ≤30 days before sampling. E) Bar plot of recent pharmacological treatments administered to recruited dogs and cats (green and orange bars, respectively) ≤30 days before sampling. Abbreviations: ACE, angiotensin-converting-enzyme; NSAIDs, nonsteroidal anti-inflammatory drugs; PPIs, proton pump inhibitors. In panels D and E, pharmacological treatment data were not available for a total of 48 animals (32 dogs and 16 cats). | study | 100.0 |
Bar plots showing the breed distribution of recruited dogs (n=105, left panel) and cats (n=37, right panel). *Other dog breeds (n): Boxer (3), Setter (3), Shih Tzu (3), American Staffordshire Terrier (2), Belgian Shepherd (2), Border Collie (2), Chinese Shar-Pei (2), Cocker Spaniel (2), Golden Retriever (2), Labrador Retriever (2), Pug (2), Shiba Inu (2), Andalusian Hound (1), Chihuahua (1), Dalmatian (1), Galician Palleiro (1), Hound (1), Mastiff (1), Miniature Pinscher (1), Pit bull (1), Poodle (1), Water dog (1). | study | 85.56 |
The overall proportion of dogs and cats with diarrhea on the sampling date were very similar (13.3% and 13.5%, respectively), and in both cases most animals had not suffered any episode of diarrhea within the preceding 30 days (61% and 75.7%, respectively) (Table 2). Only 24.7% (18/73) of dogs and 14.3% (3/21) of cats for which medication data was available were under antibiotic treatment on the sampling date or within the previous 30 days, with metronidazole and amoxicillin ranking first and second, respectively (Fig. 1D). Other pharmacological treatments of sampled animals are shown in Fig. 1E. | study | 99.94 |
The demographic data and clinical features of animals yielding positive fecal cultures for C. perfringens and/or C. difficile are detailed in Table 3 (see also Álvarez-Pérez et al. ).Table 3Signalment and clinical data of animals yielding positive fecal cultures for Clostridium perfringens and/or C. difficile, and characteristics of recovered isolates.Table 3ClinicAnimalaSpecies, breedbAge (yr.)bSexDiagnosisOther medical conditions of relevanceDiarrhoeacAntibiotic treatment(s)dGenotype (antimicrobial resistances) ofC. perfringensisolateseGenotype (antimicrobial resistances) ofC. difficileisolatesfAA/02Dog, Mixed-breed5FRoutine analysisNoneNoNonep063, p064, p065–BB/02Dog, Whippet13FRoutine analysisLymphomaNoNonep067–B/06Dog, Whippet7FRoutine analysisNoneNoNonep027, p028 [2 isolates]–B/07Dog, Schnauzer9FRoutine analysisNoneNoNonep011, p012, p023–B/08Dog, Schnauzer9FRoutine analysisNoneNoNonep002, p047, p051RT154/d05 (PEN), RT154/d06 (PEN), RT154/d07 (PEN)B/11Dog, Mixed Setter<1FRecent history of Isospora infectionNoneYesMetronidazolep035, p036 [2 isolates]–B/12Dog, Mixed Miniature Pinscher6FRoutine analysisNoneNoNonep037–B/13Cat, European shorthair9FRoutine analysisNoneNoNonep003 (MTZ), p006, p031–B/14Cat, European shorthair1MRoutine analysisNoneNoNonep069, p072–DD/02Dog, Hound1FRoutine analysisNoneNoNonep087 (MTZ), p088 (LZD), p095–EE/03Dog, Yorkshire Terrier11MGastroenteritis with vomitingHeart failureYes (5 d)Amoxicillin-clavulanic acidNART?/d13 (CLI/ERY/PEN) [2 isolates], RT?/d14 (CLI/ERY/PEN)FF/09Cat, Persian6MRoutine analysisNoneNoNonep004, p005–GG/01Dog, Labrador12MRoutine analysisNoneYes (7 d)Doxycyclinep106–Follow-up analysisNoneNoNonep041, p092, p097–G/05Dog, Boxer<1MRoutine analysisFood allergyNoNonep030 (MTZ), p050, p096–G/06Cat, Persian2FRoutine analysisNoneNoNonep026–HH/03Dog, Mastiff<1FRoutine analysisNoneNoNonep078 (PEN), p102 (IPM, PEN), p103–H/05Dog, Pit bull<1MRoutine analysisNoneNoNonep008, p034, p079 (IPM, LZD, PEN)–II/02Cat, European shorthair14MDigestive diseaseNoneYes (3 d)Nonep054, p055–I/04Dog, Mixed-breed1HDigestive diseaseNoneYes (0 d)Nonep082, p099–Follow-up analysisNoneNoNonep052 (LZD)–JJ/01Dog, Schnauzer?FRoutine analysisFood allergyYes (10 d)Nonep053, p058 (ERY, LVX)–J/03Dog, Golden Retriever4MAcute enteritisNoneYes (0 d)Nonep013, p017, p018–J/07Dog,?1MRoutine analysisNoneYes (8 d)Nonep101–KK/02Dog, Poodle10MRoutine analysisHeart murmurNoNonep089–MM/04Dog, Giant Schnauzer11FRoutine analysisNoneYes (30 d)Nonep090[2 isolates], p091–Follow-up analysisNoneYes (5 d)Nonep043 (TET), p076 (TET), p077–M/06Dog, Brittany6FRoutine analysisObesityNoNonep060, p061, p066–Follow-up analysisObesityNoNonep068, p070 (TET), p104–M/07Dog, Brittany3FEntetitisRecent Toxocara canis infection, obesityYes (25 d)Nonep081–Follow-up analysisObesityNoNonep033, p042 (TET), p056–M/08Dog, Mixed Hound3FRoutine analysisNoneNoNonep040, p062, p080–M/13Cat, European shorthair4FRoutine analysisNoneNoNonep025, p029, p071–M/14Dog, Mixed-breed8FRoutine analysisRecent removal of mammary tumorNoAmoxicillinp016, p020, p021RT106/d01 (PEN), RT106/d03 (PEN), RT106/d15 (PEN)Follow-up analysisNoneNoNonep014[2 isolates], p015–M/23Dog, Yorkshire Terrier12FRoutine analysisFrequent pseudo-pregnancies, benign mammary tumorsNoNonep032–M/25Dog, German Shepherd5FRoutine analysisObesityNoNonep001 (TET), p057–Follow-up analysisObesityNoNonep083 (TET), p084 (LZD, TET), p085 (LZD. TET)–M/26Dog, Mixed-breed2MRoutine analysisSeizure disorderNoNonep038, p039–Follow-up analysisSeizure disorderNoNonep086 (MTZ), p093, p100–NN/01Dog, Mixed Shih Tzu1MRoutine analysisNoneNoNonep044, p045, p046–N/12&14gDog, Shih Tzu12MRoutine analysisNoneYes (0 d)Nonep019, p024–N/19&20gDog, French Bulldog7MRoutine analysisNoneNoNone–RT009-like/d04, RT009-like/d08 (PEN), RT009-like/d09 (PEN), RT009-like/d10 (PEN), RT009-like/d11 (PEN), RT009-like/d12OO/04Cat, European shorthair4MRoutine analysisNoneNoNonep094–O/11Dog, Border Collie2MRoutine analysisNoneYes (7 d)Nonep007, p009, p010–QQ/03Dog, Maltese7MRoutine analysisFrequent gastrointestinal problems, heart diseaseNoNonep073 (MTZ), p074, p075 (LZD)RT106/d01 (PEN) [2 isolates], RT106/d02 (PEN)Q/04Dog, Mixed Labrador2MAllergic colitisFrequent gastrointestinal problemsYes (0 d)Sulfadiazine/trimethoprimp059–Follow-up analysisNoneNoNonep022, p098–Q/05Dog, Dachshund14MRoutine analysisNoneNoNonep048, p049–aRecruited animals were designated by a capital letter (corresponding to the clinic of origin) followed by a virgule and consecutive numbers.b?: Missing data.cNo: no diarrhea episodes were reported within the previous 30 days. For positive responses, the number of days since the last episode of diarrhea is indicated between parentheses.dAntibiotic treatment(s) administered within the previous 30 days. For an overview of other pharmacological treatments see Fig. 1E.eAFLP genotypes were arbitrarily designated by a lower case ‘p’ followed by a number (see Table 4). In vitro resistance to benzylpenicillin (PEN), erythromycin (ERY), imipenem (IPM), levofloxacin (LVX), linezolid (LZD), metronidazole (MTZ) and/or tetracycline (TET) is indicated between parentheses. The number of isolates belonging to each strain type (when different to one) is shown between square brackets. Dashes mean that C. perfringens was not isolated from the corresponding animal. NA: not analyzed (a single fecal swab was available for some animals, and this was used for C. difficile testing).fIn this case, ribotype (RT) and AFLP fingerprinting information is included. AFLP genotypes were arbitrarily designated by a lower case ‘d’ followed by a number (see Table 5). In vitro resistance to benzylpenicillin (PEN), clindamycin (CLI) and/or erythromycin (ERY) is indicated between parentheses. Additionally, all C. difficile isolates displayed resistance to levofloxacin and imipenem. The number of isolates belonging to each strain type (when different to one) is shown between square brackets. RT?: unknown ribotype. Dashes mean that C. difficile was not isolated from the corresponding animal.gThese animals were sampled twice during the study period. | study | 99.94 |
AFLP genotypes were arbitrarily designated by a lower case ‘p’ followed by a number (see Table 4). In vitro resistance to benzylpenicillin (PEN), erythromycin (ERY), imipenem (IPM), levofloxacin (LVX), linezolid (LZD), metronidazole (MTZ) and/or tetracycline (TET) is indicated between parentheses. The number of isolates belonging to each strain type (when different to one) is shown between square brackets. Dashes mean that C. perfringens was not isolated from the corresponding animal. NA: not analyzed (a single fecal swab was available for some animals, and this was used for C. difficile testing). | study | 100.0 |
In this case, ribotype (RT) and AFLP fingerprinting information is included. AFLP genotypes were arbitrarily designated by a lower case ‘d’ followed by a number (see Table 5). In vitro resistance to benzylpenicillin (PEN), clindamycin (CLI) and/or erythromycin (ERY) is indicated between parentheses. Additionally, all C. difficile isolates displayed resistance to levofloxacin and imipenem. The number of isolates belonging to each strain type (when different to one) is shown between square brackets. RT?: unknown ribotype. Dashes mean that C. difficile was not isolated from the corresponding animal. | study | 100.0 |
Table 3 includes an overview of the genetic and phenotypic characteristics of the bacterial isolates obtained from recruited animals. Additionally, the toxinotypes, PCR ribotypes (only for C. difficile isolates), amplified fragment length polymorphism (AFLP) genotypes and antimicrobial susceptibility profiles of C. perfringens and C. difficile isolates are detailed in Table 4, Table 5, respectively.Table 4Characteristics of the Clostridium perfringens isolates obtained in the study.Table 4IsolateaToxinotypebAFLP genotypecAntibiotic susceptibility (MIC, µg/ml)dAMCCLIERYIPMLVXLZDMTZPENRIFTECTETTGCVANA/02P1A (cpe, cpb2*)p0630.1250.2520.0640.54160.50.0080.064811A/02P2A (cpb2*)p0640.0320.0320.0640.0640.250.12580.1250.0040.03240.1250.5A/02P3A (cpe)p0650.0320.0320.0320.0640.250.12580.0640.0020.0320.1250.0640.5B/02P1Ap0670.0320.06420.0640.254160.0640.008<0.0160.1250.0640.5B/06P1A (cpb2*)p0270.0640.06420.0640.254160.0320.004<0.01640.250.5B/06P2A (cpb2*)p0280.0320.12520.50.254160.1250.0040.03240.50.5B/06P3A (cpb2*)p028<0.0160.520.50.254160.0640.0080.03240.50.5B/07P1A (cpe)p0110.0320.12510.0320.254160.0320.004<0.01620.1250.25B/07P2A (cpe)p0120.0320.12520.0640.254160.0640.0080.03240.1250.25B/07P3A (cpe)p023<0.0160.12520.0640.252160.1250.0080.03240.1250.25B/08P1A (cpb2*)p0470.0320.06420.1250.254160.0640.004<0.01640.1250.5B/08P2A (cpb2*)p0020.0320.2520.0640.252160.0640.0080.03220.50.5B/08P3A (cpb2*)p0510.0160.12520.1250.254160.0640.0080.03240.250.5B/11P1A (cpb2*)p0350.1250.01610.0640.251160.1250.004<0.01640.1250.5B/11P2A (cpb2*)p0360.0160.2520.0640.25280.1250.0040.01640.50.5B/11P3A (cpb2*)p0360.0320.2520.0080.52160.1250.0040.03240.50.5B/12P1Ap0370.0320.0160.1250.0640.25180.0320.004<0.01610.1250.5B/13P1A (cpb2*)p0030.0640.06420.0320.5432 (R)0.0640.0080.03220.0640.5B/13P2Ap0060.0320.06420.250.54160.1250.0040.06480.1250.5B/13P3Ap0310.0640.2520.0640.254160.1250.008<0.01680.1250.5B/14P1Ap0690.0640.12520.0640.52160.250.0080.03240.250.5B/14P2Ap0720.032220.0320.25<0.01680.0640.004<0.01640.250.5D/02P1E (cpe)p0870.064<0.01610.50.25232 (R)0.0640.0020.03240.0640.5D/02P2E (cpe)p0880.0640.06440.1250.258 (R)80.1250.0040.03240.251D/02P3E (cpe)p0950.0320.03210.0640.51160.1250.0040.03220.250.5F/09P2Ap0040.1250.0640.50.250.250.580.250.0040.0320.1250.0640.5F/09P3Ap0050.0640.064210.25480.250.0080.06440.0641G/01P1Ap1050.0640.0640.0640.0640.51160.0640.004<0.01610.1250.5G2/01P1†A (cpb2*)p0920.0640.06440.1250.25480.1250.0080.06440.250.5G2/01P2†A (cpb2*)p0970.064<0.0160.0640.1250.254160.1250.0080.01640.0640.5G2/01P3†A (cpb2*)p0410.0320.12520.0640.25480.1250.0040.01680.250.5G/05P1A (cpb2*)p0300.1250.06420.0640.25432 (R)0.1250.0080.03280.1250.5G/05P2A (cpb2*)p0500.0640.2510.0640.25280.50.004<0.01640.250.5G/05P3Ap096<0.0160.12510.0320.5440.1250.004<0.01640.1250.5G/06P1Ap0260.1250.12520.0640.54160.1250.0080.03280.251H/03P1Ap10280.1252≥32 (R)0.254168 (R)0.0040.03280.1250.5H/03P2Ap07840.032210.254168 (R)0.0040.03240.1250.5H/03P3Ap1030.0320.06410.1252280.0640.0040.06480.50.5H/05P1Ap07980.1252≥32 (R)0.258 (R)168 (R)0.0040.03280.50.5H/05P2Ap0080.0320.12510.0640.25140.0640.0020.01640.1250.5H/05P3Ap034<0.0160.06420.1250.254160.0320.0080.03240.1250.5I/02P1Ap0540.0640.06420.1250.252160.1250.0040.01640.1250.5I/02P2Ap0550.032<0.01640.0640.50.25160.064<0.002<0.01640.0640.5I/04P1A (cpe)P0990.1250.0320.250.1250.251160.1250.0040.0320.1250.0320.5I/04P2Ap0820.0320.2520.0320.250.5160.125<0.002<0.01640.1250.5I2/04P1†A (cpe)p0520.0320.12520.0640.258 (R)160.1250.0080.03240.250.5J/01P1A (cpb2*)p0530.0640.12520.0640.54160.1250.0080.03280.51J/01P2A (cpb2*)p0580.0640.2532 (R)0.125≥32 (R)2160.1250.0080.03240.1250.5J/03P1Ap0180.0640.03220.0640.54160.0640.004<0.0160.1250.0640.5J/03P2A (cpb2*)p0130.1250.0320.1250.0640.254160.064<0.002210.0322J/03P3A (cpb2*)p0170.0160.0320.1250.0320.252160.0640.004<0.01620.0320.5J/07P1A (cpb2)p1010.016<0.0160.1250.0640.251160.0320.0080.0320.1250.0320.5K/02P1Ap0890.0320.12520.50.254160.1250.0040.03240.1250.5M/04P1A (cpe)p0900.0640.12520.0640.254160.1250.0080.06480.50.5M/04P2A (cpe)p0900.0320.125210.54160.0640.0040.06440.50.5M/04P3A (cpe)p0910.032<0.016<0.0160.50.254160.0640.0160.06440.1250.5M2/04P1†A (cpb2*)p0430.0320.03220.250.254160.0320.0040.01616 (R)10.5M2/04P2†A (cpb2*)p0760.0320.06420.50.25480.0640.0080.01616 (R)0.50.5M2/04P3†A (cpb2*)p0770.0320.0320.0640.0640.5280.0640.0040.01640.250.5M/06P1A (cpb2*)p0660.1250.06420.0640.254160.50.0080.064821M/06P2A (cpb2*)p0600.0320.06410.0640.252160.1250.0040.03240.250.5M/06P3A (cpe, cpb2*)p0610.0320.0320.50.1250.254160.1250.0040.03240.1250.5M2/06P1†Ap0680.0640.06420.50.54160.250.0080.03280.1250.5M2/06P2†Ap0700.0160.12520.0320.254160.1250.0040.03216 (R)0.50.5M2/06P3†Ap1040.0640.0320.250.1250.25480.1250.0080.01640.50.5M/07P1Ap0810.250.03220.1250.254810.0080.03240.50.5M2/07P1†A (cpb2*)p042<0.0160.03220.0640.254160.1250.0080.01616 (R)10.5M2/07P2†Ap0560.0320.06440.1250.25480.1250.0040.03240.250.5M2/07P3†Ap0330.0320.06410.1250.5480.250.0080.03280.250.5M/08P1A (cpb2*)p0620.0640.520.0640.254160.25<0.0020.032820.5M/08P2Ap0400.0320.0640.250.0640.254160.1250.0080.01610.0640.5M/08P3Ap0800.032<0.0160.0320.0640.5180.064<0.0020.03240.1250.5M/13P1A (cpb2*)p0290.1250.2520.0640.254160.0640.0040.032841M/13P2A (cpb2*)p0250.0320.0320.0320.0320.250.12580.064<0.0020.03240.0320.5M/13P3A (cpe)p0710.032120.1250.5280.0640.0020.03240.1250.5M/14P1A (cpe)p0160.0160.06420.0640.25480.0640.0040.0160.1250.0640.5M/14P2A (cpe)p0200.1250.520.0640.25240.1250.004<0.01620.0320.5M/14P3A (cpe)p0210.0320.12520.0320.25280.0640.0080.01640.1250.5M2/14P1†A (cpb2*)p014<0.0160.03220.1250.25180.064<0.0020.01640.1250.5M2/14P2†A (cpb2*)p0150.0640.0320.0320.0640.252160.0640.0040.01640.50.5M2/14P3†A (cpb2*)p0140.0640.03220.1250.252160.0640.0040.01640.50.5M/23P1A (cpb2)p0320.0640.2520.0160.252160.250.0080.03240.1251M/25P1Ap0010.125240.0640.54160.250.0080.12516 (R)21M/25P2Ap0570.0320.01620.0640.25280.0320.0020.06480.1250.5M2/25P1†Ap0830.0320.12520.0640.5480.1250.0040.06416 (R)10.5M2/25P2†Ap0840.0320.12520.1250.258 (R)80.1250.0080.03216 (R)20.5M2/25P3†Ap0850.0320.12520.1250.258 (R)80.1250.0040.03216 (R)10.5M/26P1Ap0390.1250.12540.0640.254160.250.0040.03280.251M/26P2Ap0380.032<0.01610.0640.25280.0640.0020.01680.0640.5M2/26P1†Ap1000.0320.032210.254160.1250.0040.03240.250.5M2/26P2†Ap0860.0160.06420.1250.5432 (R)0.1250.0080.03220.1250.5M2/26P3†A (cpb2*)p0930.0160.06420.50.5480.0640.0040.03240.250.5N/01P1A (cpb2*)p0440.1250.12520.0640.254160.0640.004<0.01680.251N/01P2A (cpb2*)p0450.0320.06420.0640.54160.250.0040.06480.50.5N/01P3A (cpb2*)p0460.0320.12540.0640.5180.1250.004<0.01680.250.5N/12P1A (cpe)p0240.0640.06420.1250.254160.0640.0080.03240.50.5N/14P1Ap0190.0320.0320.250.1250.25280.0320.004<0.01640.1250.5O/04P1Ap0940.1250.06420.250.2541610.0080.03240.1250.5O/11P1A (cpb2*)p0070.064<0.0160.0640.0640.25480.125<0.0020.01640.1250.5O/11P2Ap0090.0320.06410.0320.25180.0320.0040.03280.1250.5O/11P3Ap0100.0320.06420.0640.25280.0640.0040.03280.1250.5Q/03P1Ap0730.1250.01620.0640.25432 (R)0.0640.004<0.01640.1250.5Q/03P2Ap0750.0640.03220.1250.258 (R)80.250.0080.03240.1250.5Q/03P3Ap0740.0320.12520.1250.254160.1250.0080.03240.250.5Q/04P2Ap0590.0320.12520.1250.254160.1250.0040.01640.1250.5Q2/04P1†A (cpb2*)p022<0.0160.12520.0640.254160.1250.0040.01640.250.5Q2/04P3†Ap0980.0320.01640.0640.254160.1250.0040.03220.1250.5Q/05P1A (cpb2*)p0480.016<0.0160.250.0640.51160.016<0.002<0.01610.1250.5Q/05P2A (cpb2*)p049<0.0160.12520.0640.52160.250.0040.01640.1250.5aIsolates whose names only differ in the last number were retrieved from the same animal (e.g. H/05P1, H/05P2 and H/05P3; see Table 3). Daggers (and the number 2 after the clinic's code) indicate isolates that were obtained in the follow-up analysis.bcpe: possession of the gene encoding for enterotoxin. cpb2: possession of the gene encoding for β2 toxin, with asterisks indicating atypical forms of the gene (as determined by PCR amplification).cAccording to the UPGMA dendrogram shown in Fig. 1 of Álvarez-Pérez et al. .dMinimum inhibitory concentration (MIC). AMC, amoxicillin/clavulanic acid; CLI, clindamycin; ERY, erythromycin; IPM, imipenem; LVX, levofloxacin; LZD, linezolid; MTZ, metronidazole; PEN, benzylpenicillin; RIF, rifampicin; TEC, teicoplanin; TET, tetracycline; TGC, tigecycline; VAN, vancomycin. R: MIC value above the breakpoint for in vitro resistance.Table 5Characteristics of the Clostridium difficile isolates obtained in the study.Table 5IsolateaPCR ribotypebAFLP genotypecAntibiotic susceptibility (MIC, µg/ml)dAMCCLIERYIPMLVXLZDMTZPENRIFTECTETTGCVANB/08D1RT154d0610.1250.25≥32 (R)≥32 (R)0.250.064≥32 (R)<0.0020.0640.0320.0160.25B/08D2RT154d070.250.0640.25≥32 (R)≥32 (R)0.250.0322 (R)<0.0020.0320.0320.0320.125B/08D3RT154d050.1250.0320.064≥32 (R)≥32 (R)0.1250.03232 (R)<0.0020.064<0.016<0.0160.125E/03D1RT?d130.5≥256 (R)≥256 (R)≥32 (R)≥32 (R)10.032≥32 (R)<0.0020.0640.5<0.0160.125E/03D2RT?d130.25≥256 (R)≥256 (R)≥32 (R)≥32 (R)0.50.064≥32 (R)0.0040.03210.0320.125E/03D3RT?d140.5≥256 (R)≥256 (R)≥32 (R)≥32 (R)10.125≥32 (R)<0.0020.0320.50.0320.125M/14D1RT106d150.50.0640.125≥32 (R)≥32 (R)0.250.064≥32 (R)<0.0020.032<0.016<0.0160.125M/14D2RT106d010.250.251≥32 (R)≥32 (R)0.1250.0644 (R)0.0040.032<0.016<0.0160.25M/14D3RT106d030.50.1250.25≥32 (R)≥32 (R)0.50.1252 (R)<0.0020.0320.032<0.0160.125N/19D1RT009-liked080.50.0640.25≥32 (R)≥32 (R)0.50.0324 (R)<0.0020.0640.032<0.0160.25N/19D2RT009-liked040.064<0.0160.016≥32 (R)≥32 (R)0.064<0.0161<0.0020.032<0.016<0.0160.125N/19D3RT009-liked090.25<0.0160.064≥32 (R)≥32 (R)0.125<0.0164 (R)<0.0020.0320.032<0.0160.125N/20D1RT009-liked100.250.0640.125≥32 (R)≥32 (R)0.250.032≥32 (R)<0.0020.0320.032<0.0160.25N/20D2RT009-liked1110.0160.032≥32 (R)≥32 (R)0.250.064≥32 (R)<0.0020.032<0.0160.0320.125N/20D3RT009-liked1210.1250.25≥32 (R)≥32 (R)0.1250.0641<0.0020.064<0.016<0.0160.125Q/03D1RT106d010.250.0640.064≥32 (R)≥32 (R)0.1250.032≥32 (R)<0.0020.032<0.016<0.0160.125Q/03D2RT106d020.250.0640.25≥32 (R)≥32 (R)0.1250.0322 (R)<0.0020.0640.0320.0320.25Q/03D3RT106d010.250.1250.125≥32 (R)≥32 (R)0.250.0322 (R)<0.0020.0640.064<0.0160.25aIsolates were retrieved from the same animal (B/08D1, B/08D2 and B/08D3; see Table 3).bToxin profiles: RT009-like, A-B-CDT- (but with a positive PCR result for tcdB); RT106, A+B+CDT-; RT154, A+B+CDT-; RT? (unknown ribotype), A-B-CDT-.cAccording to the UPGMA dendrogram shown in Fig. 2 of Álvarez-Pérez et al. .dMinimum inhibitory concentration (MIC). AMC, amoxicillin/clavulanic acid; CLI, clindamycin; ERY, erythromycin; IPM, imipenem; LVX, levofloxacin; LZD, linezolid; MTZ, metronidazole; PEN, benzylpenicillin; RIF, rifampicin; TEC, teicoplanin; TET, tetracycline; TGC, tigecycline; VAN, vancomycin. R: MIC value above the breakpoint for in vitro resistance. | study | 100.0 |
Minimum inhibitory concentration (MIC). AMC, amoxicillin/clavulanic acid; CLI, clindamycin; ERY, erythromycin; IPM, imipenem; LVX, levofloxacin; LZD, linezolid; MTZ, metronidazole; PEN, benzylpenicillin; RIF, rifampicin; TEC, teicoplanin; TET, tetracycline; TGC, tigecycline; VAN, vancomycin. R: MIC value above the breakpoint for in vitro resistance. | other | 99.94 |
Minimum inhibitory concentration (MIC). AMC, amoxicillin/clavulanic acid; CLI, clindamycin; ERY, erythromycin; IPM, imipenem; LVX, levofloxacin; LZD, linezolid; MTZ, metronidazole; PEN, benzylpenicillin; RIF, rifampicin; TEC, teicoplanin; TET, tetracycline; TGC, tigecycline; VAN, vancomycin. R: MIC value above the breakpoint for in vitro resistance. | other | 99.94 |
Our survey was performed during one week (from November 24 to December 1, 2015) in a total of 17 primary care veterinary clinics from the Madrid region (Spain). The staff of participating clinics received training for data and sample collection, and email and telephonic support was available throughout the duration of the study. Veterinarians of participating centers were asked to select two swab samples of all feces shed by dogs and cats at their clinic, regardless of the age, origin and clinical condition of the animals, and to send those samples to a central reference laboratory at the Faculty of Veterinary Medicine of Complutense University of Madrid. Additionally, the staff of each participating clinic had to complete a questionnaire of general data about the centre and a second questionnaire for each pair of fecal swabs obtained requesting data on the sample (collection date, consistency of feces and presence of blood) and the animal of origin (species, breed, sex, age, clinical status and episodes of diarrhea and medication(s) within the previous 30 days). An informed consent and agreement to participate in the study was obtained from the owners of each animal before enrolment. Animals were always handled by experienced veterinary practitioners in strict accordance with good animal practice and the Spanish legislation. | study | 100.0 |
The owners of animals yielding a positive culture for C. difficile and/or C. perfringens were invited to participate in a follow-up survey performed four months after the first study (in March 2016). In this case, fecal swab samples and clinical information of animals was obtained as explained above. | study | 99.94 |
The microbiology procedures used for C. perfringens and C. difficile isolation from fecal samples, and the methods used for toxin profiling, PCR ribotyping, AFLP subtyping and in vitro antimicrobial susceptibility testing of recovered isolates are detailed in our previous publication . | study | 99.9 |
RIG-I-like receptors, Toll-like receptors (TLRs), Nod-like receptors, and DNA sensors are germline-encoded pattern-recognition receptors (PRRs) that serve as the first line of defense against invading pathogens, including bacteria and viruses [1, 2]. PRRs possess the abilities to recognize pathogen-associated molecular patterns (PAMPS) and subsequent to the detection of PAMPs, PRRs trigger the activation of common type I interferon (IFN), NF-κB, inflammasome, and autophagy signaling pathways [1–5]. The consequent activation of these pathways induces the expression of cytokines and chemokines that facilitate the host pathogen defense and thus initiate the adaptive immune response. Among the PRRs, two RNA helicases such as Retinoic acid-inducible gene 1 (RIG-I, DDX58) and melanoma differentiation-associated protein 5 (MDA5), belonging to RIG-I-like receptor family, are considered to be the most important cytosolic viral RNA sensors . Although both RIG-I and MDA5 can respond to West Nile virus and dengue virus , they also recognize specific sets of viruses . While MDA5 primarily senses positive-strand viruses from the Picornaviridae family, such as Encephalomyocarditis virus (EMCV), RIG-I recognizes RNA viruses, including Sendai virus (SeV), vesicular stomatitis virus (VSV), and influenza virus [7, 9, 10]. Further studies and structural analysis of RIG-I and MDA5 showed that RIG-I recognizes the 5´-triphosphate group (5´-ppp) and blunt end of short (low molecular weight) RNAs with high affinity [11–14]. In contrast, MDA5 recognizes the internal duplex structure of long (high molecular weight) double strand (ds)RNAs with a weaker affinity [1, 15], thus resulting in the aggregation of MDA5 and its interaction with MAVS (VISA, IPS-1, Cardif) to trigger downstream type I IFN signaling cascade [16–19]. Of note, previous observation demonstrates that higher-order structure RNA comprising of both dsRNA and single strand RNA from EMCV-infected cells contain MDA5-stimulating activity , suggesting that other protein factors are involved in the selection and recognition of EMCV viral RNA. To this end, such potential candidate may be LGP2 (Laboratory of Genetics and Physiology 2), a cytoplasmic DExH helicase that shares the domain structure of RIG-I and MDA-5 with the exception of the CARD domains . Paradoxically, LGP2 has been reported to exert both positive and negative effects on RIG-I and MDA5 regulation in different cell types in response to different viruses [15, 21–24], thus questioning its’ specificity to MDA5-mediated viral RNA selection and recognition. Based on these premises, the identification of protein factors that are involved in the selection and recognition of MDA5 for viral RNA is warranted. | study | 99.44 |
In this study, our investigation directed towards the identification of factors required for recognition of MDA5 for viral mRNA revealed that the RNA helicase, DHX29 functions as a dsRNA co-sensor and exclusively interacts with MDA5 for enhanced RNA recognition in response to high molecular weight polyinosinic-polycytidylic acid [HMW-Poly(I:C)] treatment or EMCV infection in immune cells. In this respect, it is worth-mentioning that RNA helicases play critical roles in multiple biological processes, including protein translation initiation and stress granule formation [25–27]. In addition to the potential involvement in innate immune recognition and antiviral immunity [28, 29], several RNA helicases have also been identified as key factors in protein translation initiation [25, 30]. Recently, the RNA helicase DHX29 has been reported to be required for the formation and scanning of 43S translation initiation complexes on mRNAs with 5´-structured untranslated regions (UTRs) [31–33]. More importantly, mRNAs with internal ribosomal entry sites (IRES) occur in many Picornaviridae family viruses, including EMCV, and require DHX29 during translation initiation [34, 35]. Despite the important role of DHX29 in translation initiation of mRNAs with 5´ structured UTRs, particularly those from picornaviruses, its’ role in innate immune response to virus remains largely unknown. Interestingly, our mechanistic studies identify a previously unrecognized role of DHX29 in MDA5-mediated EMCV-specific type I IFN signaling and provide insights on the molecular mechanism by which DHX29 recognizes dsRNA and specifically interacts with MDA5 to render enhanced antiviral immunity. | study | 100.0 |
Despite the identification of many positive and negative regulators that control RIG-I function [36, 37], the knowledge on the functional regulation of MDA5 is scanty. In order to identify proteins involved in MDA5 regulation, we performed a functional screening of a cDNA expression sub-library for their ability to inhibit or enhance type I IFN signaling [38–42]. For this purpose, human embryonic kidney (HEK) 293T cells (293T cells) were cotransfected with the IFN-β promoter luciferase reporter, Renilla luciferase internal control (pRL-TK), and cDNA expression plasmids followed by stimulation with intracellular HMW Poly(I:C), low molecular weight (LMW) Poly(I:C), or intracellular DNA duplex Poly(dA:dT). Among them, DHX29 was identified as a positive regulator of intracellular HMW Poly(I:C)-induced IFN-β-Luc reporter activity as well as interferon-stimulated response element (ISRE)-luciferase (Luc) reporter activity, which only requires interferon regulatory factor 3 (IRF3) activation (Fig 1A). In contrast, DHX29 failed to increase ISRE-Luc and IFN-β-Luc activities in TLR3-expressing 293T cells (293T-TLR3) stimulated with LMW Poly(I:C) or 293T cells stimulated with Poly(dA:dT) or intracellular LMW Poly(I:C) (Fig 1B–1D). Consistent with these results, the ectopic expression of DHX29 markedly increased IFN-β transcription as well as TBK1 and IRF3 phosphorylation in response to intracellular HMW Poly(I:C), but not to intracellular LMW Poly(I:C) or Poly(dA:dT) treatment (Fig 1E and 1F). Similar increase in IFIT1 (encodes ISG56), IFIT2 (encodes ISG54), and CCL5 mRNA expression, was observed in response to intracellular HMW Poly(I:C) in DHX29 overexpressed cells (S1A–S1C Fig). Taken together, these results strongly suggest that DHX29 specifically enhances MDA5, but not TLR3 or RIG-I, mediated type I IFN signaling pathway. | study | 100.0 |
(A) 293T cells and (B) 293T-TLR3 cells were cotransfected with IFN-β or ISRE promoter luciferase reporter (100 ng), Renilla luciferase internal control (pRL-TK), and empty vector or increasing concentrations of pcDNA3.1-HA-DHX29 (0, 50, and 100 ng). Transfected 293T and 293T-TLR3 cells were stimulated with intracellular (IC) HMW Poly(I:C) (5 μg/ml) and LMW Poly(I:C) (10 μg/ml), respectively. Fold changes in IFN-β-luciferase (Luc) and ISRE-Luc activities were determined. (C) IFN-β-Luc and ISRE-Luc activities in 293T cells stimulated with Poly(dA:dT) (5 μg/ml) (D) IFN-β-Luc and ISRE-Luc activities in 293T cells stimulated with IC LMW Poly(I:C) (5 μg/ml). (E and F) Real-time PCR of IFN-β (IFNB) mRNA (E) and immunoblot analysis (F) of TBK1 and IRF3 phosphorylation in empty vector- and pcDNA3.1-Flag-DHX29 (100 ng)-transfected 293T cells treated with IC HMW Poly(I:C), IC LMW Poly(I:C), or Poly(dA:dT). (G) Real-time PCR analysis of DHX29 mRNA expression in different human tissues by two different primer sets (Primer set #1 and Primer set #2). (H) Cell lysates (30 ug of total protein) of human lung cell lines MRC5, A549, H441, monocyte, dendritic cell (DC), T cell, B cell, mouse MEF, CD4-T cells, CD19-positive B cells, BMDM, BMDC, peritoneal macrophage (pMac), RAW 264.7, neutrophil were loaded on SDS-PAGE for western blotting analysis using anti-DHX29 and anti-β-actin antibodies. Data from (A-E) are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant. | study | 100.0 |
DHX29 is expressed in human tissues and immune cells, especially high in prostate, spleen, mammary gland, testis, kidney and thymus using two sets of primers (#1 and #2) (Fig 1G). In addition to high expression of DHX29 in human lung cells , we showed that DHX29 protein could be detected in human peripheral blood mononuclear cells (PBMCs), monocyte, B cells, T cells, dendritic cells as well as in mouse T cells, B cells, bone marrow derived dendritic cells (BMDCs), peritoneal macrophages, bone marrow derived macrophages (BMDMs) and neutrophils (Fig 1H). Thus, our systematic investigation indicates that DHX29 is ubiquitously expressed in all tissues and cell types tested from human and mice, albeit with different expression levels, suggesting that DHX29 might have cell-type specific function in response to viral infection. | study | 100.0 |
We first assessed the effect of HMW Poly(I:C) stimulation on DHX29 expression or induction, and found that DHX29 expression was not affected by HMW Poly(I:C) or EMCV stimulation (S2A and S2B Fig). We next performed knockdown (KD) experiments and observed that three different DHX29-specific lentivirus short hairpin RNA (shRNA) constructs could effectively knockdown endogenous DHX29 expression (S2C Fig). Furthermore, changes in cell viability were not observed for 3 days following transfection of shRNA targeting DHX29 (S2D Fig). | study | 100.0 |
DHX29 KD markedly reduced TBK1 and IRF3 phosphorylation in response to intracellular HMW Poly(I:C) treatment, but not in response to LMW Poly(I:C) or Poly(dA:dT), in 293T cells (Fig 2A). Consistent with these results, DHX29 KD greatly reduced ISRE-Luc and IFN-β-Luc activities in 293T cells stimulated with intracellular HMW Poly(I:C), which could be rescued by ectopic expression of HA-DHX29 (Fig 2B). However, DHX29 KD had little or no effect on ISRE-Luc and IFN-β-Luc activities in 293T-TLR3 cells stimulated with LMW Poly(I:C) or 293T cells stimulated with intracellular LMW Poly(I:C) or Poly(dA:dT) (S2E–S2G Fig). Furthermore, we found that the expression of several endogenous genes, including IFN-β, IFIT1 and IFIT2, were reduced in cells transduced with DHX29-specific shRNAs in response to intracellular HMW Poly(I:C), but not intracellular LMW Poly(I:C) or poly(dAdT) (Fig 2C and S2H–S2J Fig), suggesting specific effects of DHX29 on HMW Poly(I:C)-induced type I IFN signaling. | study | 100.0 |
(A) Immunoblot analysis of TBK1 and IRF3 phosphorylation in scrambled (Scr) shRNA- and DHX29 shRNA-transfected 293T cells stimulated with intracellular (IC) HMW Poly(I:C), IC LMW Poly(I:C), or Poly(dA:dT). (B) ISRE- and IFN-β-luciferase (Luc) activities in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with IC HMW Poly(I:C). ISRE-Luc activity was also determined in 293T cells cotransfected with HA-DHX29 (200 ng) and Scr shRNA or DHX29 shRNA. IFN-β-Luc and ISRE-Luc activities were normalized to the Renilla luciferase internal control and presented as the fold increase relative to unstimulated control cells. (C) Real-time PCR analysis of IFNB mRNA expression in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with IC HMW Poly(I:C), IC LMW Poly(I:C), or Poly(dA:dT). (D-F) PBMCs (D) and THP-1 (E) and RAW264.7 (F) cells were transfected with Scr siRNA or DHX29 siRNA and stimulated with LMW Poly(I:C), HMW Poly(I:C) or Poly(dA:dT) in lyo/vec form (Vec). IFNB, IFIT1, and IFIT2 mRNA expression levels and IFN-β production were determined using real-time PCR analysis and ELISA, respectively. Data from (B-F) are representative of three independent experiments and plotted as the mean ± s.d. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. | study | 100.0 |
To further confirm the role of DHX29 in the type I IFN signaling pathway, the effect of DHX29 KD on the expression of type I IFN-stimulated genes and IFN-β production was determined in human and mouse immune cells. Human THP-1 cells, human peripheral blood mononuclear cells (PBMCs), and mouse RAW cells were transfected with DHX29-specific small interfering RNA (siRNA). High efficiency siRNA-mediated KD of DHX29 was observed in all three types of cells (S3A Fig). Compared with the scrambled siRNA control, DHX29 KD markedly reduced IFN-β, IFIT1, IFIT2, and CCL5 mRNA expression and IFN-β production in PBMCs, THP-1 and RAW cells after treatment with LMW Poly(I:C) or HMW Poly(I:C) in lyo/vec form (Vec), but not to Poly(dA:dT) (Fig 2D–2F and S3B–S3E Fig). Taken together, our results suggest that DHX29 plays an important role in type I IFN signaling, and this function is conserved between human and mouse immune cells. | study | 100.0 |
To further substantiate the biological role of DHX29 in type I IFN signaling in both human and mouse primary cells, we performed DHX29 knockout (KO) experiments in human peripheral blood mononuclear cells (PBMCs), mouse bone marrow-derived dendritic cells (BMDCs) and peritoneal macrophages using a lentiviral-based Cas9-P2A-puromycin fusion CRISPR (clustered regularly interspaced short palindromic repeats). Of note, both human and murine DHX29-sgRNA/Cas9 lentiCRISPR could efficiently knock out DHX29 in puromycin-selected bulk PBMCs, BMDCs, and peritoneal macrophages, as determined by western blot and a surveyor assay (Fig 3A–3C and S4A–S4D Fig). DHX29 KO markedly reduced TBK1 and IRF3 phosphorylation and IFN-β production in response to HMW Poly(I:C) in lyo/vec form (Vec) and EMCV, but did not affect IRF3 phosphorylation and IFN-β production in response to LMW Poly(I:C) Vec, Poly(dA:dT) or VSV in human PBMCs (Fig 3A and S4E Fig). Similar results were observed with DHX29 KO by DHX29 sgRNA/Cas9 lentiCRISPR in murine BMDCs and peritoneal macrophages (Fig 3B and 3C and S4F and S4G Fig). Thus, these results suggest that DHX29 KO markedly reduced type I IFN signaling in response to EMCV, but not VSV in immune cells, in contrast to what was observed in human airway epithelial cells . It should be noted that all our KO experiments were performed on day 3–4 post transduction, since further long-term culture (after day 4) of DHX29 KO cells caused cell death (S4H Fig), which is consistent with previous observations [31–33, 44], showing that DHX29 is essentially required for protein translation, cell proliferation and survival. Collectively, our results gathered by deploying DHX29 shRNA KD and DHX29 KO approaches in human and murine primary immune cells, clearly suggest that DHX29 is required for type I IFN signaling in response to HMW Poly(I:C) treatment and EMCV infection. | study | 100.0 |
(A) Human PBMCs, (B) murine BMDCs, and (C) murine peritoneal macrophages (pMAC) were transduced with control or mixed DHX29 sgRNA/Cas9 lentiCRISPR viruses. Transduced cells were selected by puromycin treatment for 48 h to eliminate untransduced cells. Puromycin-resistant cells were cultured with fresh medium, left untreated or stimulated with HMW Poly(I:C) or Poly(dA:dT) in lyo/vec form (Vec) or infected with EMCV or VSV at an MOI of 10. Phosphorylation of IRF3 and IFN-β protein were determined by western blot and ELISA. | study | 100.0 |
To further investigate the role of DHX29 in antiviral immune response and clarify the discrepancy between our data and those using human lung airway epithelial cells on the specificity of antiviral immunity against EMCV and VSV, we examined whether the extent of DHX29 expression can modulate EMCV and VSV infection. The mRNA level and titer of EMCV, but not VSV, were significantly reduced in DHX29 stably transfected (mDHX29) mouse embryonic fibroblasts (MEFs), when compared with empty vector-transduced MEFs (Fig 4A). Conversely, mRNA and titer of EMCV, but not VSV, were dramatically increased in DHX29 siRNA-transfected MEFs in comparison to scrambled siRNA-transfected MEFs (Fig 4B). Our results suggest that the expression level of DHX29 can specifically modulate the infection ability of EMCV. | study | 100.0 |
(A) DHX29 stably transfected (mDHX29) MEFs were infected with EMCV (multiplicity of infection [MOI] 5) or VSV (MOI 0.5). EMCV and VSV mRNA levels by real-time PCR analysis and virus titers were determined. Virus titers were expressed as plaque-forming units (pfu)/ml. (B) EMCV and VSV mRNA levels and titers were determined in scrambled (Scr) siRNA- and DHX29 siRNA-transfected MEFs 24 h post-infection with EMCV (MOI 5) or VSV (MOI 1). (C) MDA5-knockout (MDA5-/-) and WT MEFs and (D) RIG-I-knockout (RIG-I-/-) and WT MEFS were transfected with DHX29 siRNA or Scr siRNA and then infected with EMCV or VSV at the indicated MOIs. IFN-β production was determined by ELISA. Data from (A-D) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant. | study | 100.0 |
Because EMCV infection is known to activate MDA5-mediated type I IFN signaling, we further determined whether MDA5 is required for DHX29-mediated antiviral immunity. Our investigation with MDA5 wild-type (WT) and deficient MEFs demonstrated the lack of IFN-β secretion in MDA5-deficient MEFs in response to EMCV regardless of DHX29 status (Fig 4C). In contrast, DHX29 KD in WT MEFs markedly decreased IFN-β release after EMCV infection, when compared with scrambled shRNA-transduced MEFs (Fig 4C). By contrast, we did not observe any difference in IFN-β production in DHX29 KD or MDA5-deficient MEFs in response to VSV infection (Fig 4C). Overall, our single and double knock down studies strongly indicate that DHX29-mediated antiviral response to EMCV is dependent upon MDA5. Subsequently, we pursued to determine whether RIG-I is required for DHX29-mediated antiviral immunity. We performed similar experiments with WT and RIG-I-deficient MEFs, and found no difference in IFN-β production between scrambled siRNA transfected WT and scrambled siRNA-transfected RIG-I-deficient MEFs or between DHX29-specific siRNA-transfected WT and DHX29-specific siRNA transfected RIG-I-deficient MEFs in response to EMCV infection (Fig 4D). However, we observed marked reduction in IFN-β production in both DHX29-specific siRNA-transfected (WT and RIG-I-deficient) MEFs, compared with scrambled siRNA transfected (WT and RIG-I-deficient) MEFs after EMCV infection (Fig 4D), suggesting that DHX29 KD, but not RIG-I deficiency, is responsible for the impairment of EMCV-induced IFN-β production. In contrast, there was no appreciable change in IFN-β production in response to VSV infection between DHX29 siRNA- and scrambled siRNA-transfected WT MEFs (Fig 4D). As expected, RIG-I-deficient MEFs failed to produce IFN-β after VSV infection regardless of DHX29 status (Fig 4D). Taken together, these results clearly suggest that both DHX29 and MDA5 are specifically required for EMCV-, but not VSV-, induced antiviral immune response in MEFs. | study | 100.0 |
We next sought to determine whether DHX29 physically interacts with RNA sensors or adaptor molecules involved in the type I IFN signaling pathway. Accordingly, co-immunoprecipitation (CO-IP) assays were performed using 293T cells transfected with HA-DHX29 and Flag-RIG-I, Flag-MAVS, or Flag-MDA5. Both human and mouse DHX29 interacted with MDA5, but not with RIG-I or MAVS (Fig 5A and S5A Fig). After demonstrating the specific interaction between DXH29 and MDA5, we investigated the effect of increasing amount of DHX29 expressing vector on the activation of MDA5. | study | 100.0 |
(A) Whole cell lysates (WCL) obtained from 293T cells cotransfected with HA-DHX29 and Flag-RIG-I, Flag-MDA5, or Flag-MAVS plasmids were immunoprecipitated with anti-HA beads. The immunoprecipitated product was immunoblotted with anti-Flag antibody. (B) Luciferase activity of 293T cells transfected with an ISRE (upper panel) or NF-ĸB (bottom panel) luciferase reporter, together with vector for RIG-I, MDA5, MAVS, TBK1, along with empty vector (no wedge) or with increasing amounts (wedge) of expression vector for DHX29. (C and D) Coimmunoprecipitation assays were performed on WCL obtained from THP-1 cells infected with HMW Poly(I:C) (C) or EMCV (MOI 10) (D) at the indicated time points. Immunoprecipitation with anti-DHX29 antibody and immunoblotting with anti-MDA5, phosphorylated (p)-IRF3, and total IRF3 antibodies were performed. (E) DHX29 stably transfected (mDHX29) MEFs were stimulated with HMW Poly(I:C) at the indicated time points. WCL were immunoprecipitated with anti-Flag beads and immunoblotted with anti-MDA5, p-IRF3, and total IRF3 antibodies. (F) Purified HA-DHX29 and Flag-MDA5 were co-incubated with or without HMW Poly(I:C) for 4 h. Pull-down experiments were performed with anti-Flag beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (G) Confocal imaging analysis of the colocalization of DHX29 (red) and MDA5 (green) with or without HMW Poly(I:C) or EMCV stimulation for 5 h. Scale bar = 10 μm. (H) Time-course fluorescent microscopy analysis of the colocalization of DHX29 (red) and MDA (green) stimulated by HMW Poly(I:C) at indicated time points were recorded. Scale bar = 10 μm. (I) MDA5 deletion constructs were generated from full-length MDA5 (FL-MDA5) (top panel). WCL obtained from 293T cells cotransfected with HA-DHX29 and FL-MDA5, N-MDA5, M-MDA5, or C-MDA5 were immunoprecipitated with anti-Flag beads. The immunoprecipitated product was immunoblotted with anti-HA and anti-Flag antibodies. Data from (B) are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant. | study | 100.0 |
293T cells were co-transfected with expression vectors encoding proteins RIG-I, MDA5, MAVS and TBK1 together with increasing amounts of expression vector that encodes DHX29 protein along with the ISRE or NF-ĸB luciferase reporter. We found that DHX29 specifically enhanced activation of the luciferase reporters induced by MDA5, but not others (Fig 5B). To further determine whether the endogenous interaction between DHX29 and MDA5 occur under physiological conditions, we treated THP-1 or mDHX29-expressing MEFs with intracellular HMW Poly(I:C) or EMCV infection. Immunoprecipitation and immunoblot analysis revealed no interaction between endogenous DHX29 and MDA5 in THP-1 cells stimulated with intracellular LMW Poly(I:C) (S5F Fig). The endogenous DHX29-MDA5 interaction was only observed following intracellular HMW Poly(I:C) treatment or EMCV infection (Fig 5C–5E). Consistently, DHX29 was detected in a complex with MDA5 in the presence of HMW Poly(I:C) by in vitro pull-down experiments using purified DHX29 and MDA5 proteins (Fig 5F). Colocalization of DHX29 and MDA5 after HMW Poly(I:C) or EMCV stimulation was detected by confocal imaging (Fig 5G). Specifically, the interaction of DHX29 with MDA5 was observed starting from 150 min after HMW Poly(I:C) stimulation analyzed by time-recording fluorescent microscopy (Fig 5H). | study | 100.0 |
Interestingly, under stringent condition in the presence of our lysis buffer containing 1% Triton X-100, DHX29 did not interact with LGP2 or RIG-I, even after various kinds of stimulations. But in much lesser stringent condition such as in the presence of cell lysis buffer containing 0.1% NP-40 which was used in a recent report , we detected some weak association (S5G and S5H Fig). These results suggest that DHX29 specifically interacts with MDA5, but not with RIG-I or LGP2 protein, in a stringent condition which is optimal for determining the association in CO-IP assay. We further assessed the relative contribution of LGP2 and DHX29 to MDA5-mediated type I IFN signaling by knocking down or overexpression approaches. We found that KD of DHX29 markedly reduced IFN-β-luc activity induced by HMW Poly(I:C), which could be partially restored when LGP2 was overexpressed. Similarly, LGP2 KD strikingly reduced IFN-β-luc activity induced by HMW Poly(I:C), which could be partially restored when DHX29 was overexpressed (S5I Fig). Ectopic expression of both LGP2 and DHX29 further enhanced IFN-β-luc activity. Conversely, KD of LGP2 and DHX29 further markedly reduced IFN-β-luc activity (S5I Fig). These results suggest that LGP2 and DHX29 have additive and independent effects on MDA5-mediated type I IFN signaling pathway. Therefore, DHX29 enhances MDA5-mediated type I IFN signaling, independent of LGP2. | study | 100.0 |
Additional experimental evidence document that the phosphorylation level of IRF3 induced by TBK1 is not affected by DHX29 in both WT and MAVS knockout cell (S5B Fig), All together the results obtained from luciferase reporter assay and interaction data in RIG-I knockout cell showed the similar pattern as observed in WT cell (Fig 5A and 5B, S5C and S5D Fig). Cumulatively, all these data suggest that DHX29 specifically functions in concert with MDA5 and acts upstream of MAVS. To determine whether the interaction between DHX29 and MDA5 is dependent upon ligand stimulation, we treated DHX29- and MDA5-expressing 293T cells with intracellular HMW Poly(I:C) and found that the interaction between DHX29 and MDA5 was induced following intracellular HMW Poly(I:C) treatment (S5E Fig). Furthermore, DHX29 KD decreased the MDA5-MAVS interaction in response to HMW Poly(I:C) stimulation (S5J Fig). Henceforth, our mechanistic studies bolster us to suggest that DHX29 specifically binds to MDA5 and promotes the MDA5-MAVS interaction to enhance type I IFN signaling. To identify the interacting domains between MDA5 and DHX29, we generated different deletion constructs of MDA5, and tested for their abilities to interact with DHX29 (Fig 5I). DHX29 interacted with full-length MDA5 and the CARD domain-containing N-terminal fragment (N-MDA5), but not with M-MDA5 or MC-MDA5 (Fig 5I). | study | 100.0 |
To determine the specific role of DHX29 protein domains in the type I IFN pathway, four different DHX29 constructs (DHX29a [1–530 aa], DHX29b [1–830 aa], DHX29c [530–1369 aa], and DHX29d [830–1369 aa]) were generated and tested for their abilities to interact with MDA5 (Fig 6A). DHX29a and DHX29b, like full-length (FL) DHX29, were capable of binding to MDA5, whereas DHX29c and DHX29d failed to do so (Fig 6A), suggesting that the N-terminus (1–530 aa) of DHX29 is essential for DHX29 to interact with MDA5. In Fig 5I, DHX29 was shown to interact with N-MDA5. To confirm DHX29 interacts with MDA5 through N-DHX29 (DHX29a) and N-MDA5, we immunoprecipitated DHX29a with FL-MDA5 or deletions of MDA5, and found that DHX29a interacted only with FL- or N-MDA5 (S6A Fig). We next tested each of the four DHX29 constructs for their abilities to enhance type I IFN signaling using the ISRE-Luc reporter. The DHX29a and DHX29b constructs did not increase but rather inhibited intracellular HMW Poly(I:C)- or MDA5-induced ISRE-Luc activity, whereas DHX29c and DHX29d had no effect on the ISRE-luc activity compared with the vector control (Fig 6B and S6B Fig), suggesting that full-length DHX29 is required for its ability to enhance MDA5-mediated type I IFN signaling. | study | 100.0 |
(A) Schematic of DHX29 truncated forms (top panel). 293T cells were transfected with Flag-MDA5 and full-length HA-DHX29 (FL-DHX29), HA-DHX29a, HA-DHX29b, HA-DHX29c, or HA-DHX29d plasmids. Immunoprecipitation with anti-HA beads and immunoblotting with anti-HA and anti-Flag antibodies were performed (bottom panel). (B) 293T cells were cotransfected with ISRE-luciferase (Luc) and FL-DHX29, empty vector (vec), DHX29a, DHX29b, DHX29c, or DHX29d plasmids and stimulated with IC HMW Poly(I:C). ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold increase relative to stimulated FL-DHX29 control cells. (C) Cell lysates were prepared from 293T cells transfected with full-length HA-DHX29 or HA-DHX29a or HA-DHX29c alone or in combination with Flag-MDA5, and then incubated with biotin-labeled HMW Poly(I:C) for 4 h. Immunoprecipitation was performed with anti-biotin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (D) Purified HA-DHX29 in the presence or absence of Flag-MDA5 was incubated with biotin-labeled LMW Poly(I:C) or HMW Poly(I:C) for 4 h. Pull-down experiments were performed with NeutrAvidin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (E) Purified Flag-MDA5 in the presence or absence of BSA (control), FL-DHX29, DHX29a, DHX29b, DHX29c, or DHX29d protein was incubated with HMW Poly(I:C) for 4 h. Pull-down experiments were performed with anti-biotin beads, followed by immunoblotting with anti-Flag or anti-HA antibody. (F) Schematic of DHX29 mutations in walker A (WAM-DHX29) and walker B (WBM-DHX29) sites. Purified Flag-MDA5 in the presence or absence of BSA (control), HA-DHX29, HA-WAM-DHX29, HA-WBM-DHX29 protein was incubated with HMW Poly(I:C) for 4 h. Immunoprecipitation with NeutrAvidin beads and immunoblotting with anti-Flag or anti-HA antibody were performed. (G) 293T cells co-transfected with ISRE-Luc and BSA (control), HA-DHX29, WAM-DHX29, or WBM-DHX29 were stimulated with IC HMW Poly(I:C). ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold decrease relative to stimulated FL-DHX29 control cells. Data from (B and G) are representative of three independent experiments and plotted as the mean ± s.d. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. NS, not significant. | study | 100.0 |
To identify the molecular mechanisms by which DHX29 enhances MDA5-mediated type I IFN signaling, we tested whether DHX29 has the ability to bind to HMW Poly(I:C). To test this possibility, we performed a pull-down experiment with NeutrAvidin-beads from cell lysates of 293T cells cotransfected with biotin-labeled HMW Poly(I:C) along with MDA5, DHX29 fragments (FL, a or c), or MDA5 in combination with DHX29 fragments. We found that unlike DHX29a, FL-DHX29, DHX29c and MDA5 alone or in combination could be immunoprecipitated with anti-biotin beads (Fig 6C), suggesting that DHX29 functions as an RNA co-sensor to recognize HMW Poly(I:C). Furthermore, we performed similar experiments using LMW Poly(I:C) and found that only a small amount of DHX29 could be immunoprecipitated with anti-biotin beads (S6C Fig), suggesting that DHX29 preferentially binds to HMW Poly(I:C). | study | 100.0 |
It has been known that MDA5 has a poor binding affinity for HMW Poly(I:C) and viral dsRNA [1, 15]. Therefore, we reasoned that DHX29 may enhance type I IFN signaling by recognizing the HMW Poly(I:C) and binding to MDA5. To test this possibility, we determined the ability of purified MDA5 and DHX29 to bind to HMW Poly(I:C). Indeed, we found that MDA5 interacted weakly to biotin-labeled HMW Poly(I:C), whereas DHX29 exhibited a strong HMW Poly(I:C)-binding ability, but not LMW Poly(I:C) (Fig 6D), suggesting that DHX29 is an RNA co-sensor. Importantly, co-incubation of MDA5 with DHX29 markedly enhanced the ability of MDA5 to bind to HMW Poly(I:C) (Fig 6D). To determine the DHX29 domain(s) required to enhance the HMW Poly(I:C)-binding capacity of MDA5, pull-down experiments were performed using purified full-length and truncated DHX29 proteins. Full-length DHX29, but not its truncated forms, enhanced the ability of MDA5 to bind to HMW Poly(I:C) (Fig 6E). Interestingly, full-length DHX29 and truncated DHX29c, but not DHX29a, DHX29b, or DHX29d, were pulled down with biotin-labeled HMW Poly(I:C) (Fig 6E), suggesting that DHX29c (containing the DEXD and helicase domains) has a strong HMW Poly(I:C)-binding ability. Taken together, these results suggest that DHX29 interacts with MDA5 through its N-terminus and binds to HMW Poly(I:C) through its DEXD and helicase domains, thus bringing MDA5 and HMW Poly(I:C) together and enhancing their interaction. These results may explain why full-length DHX29 is required to enhance the HMW Poly(I:C)-binding capacity of MDA5. | study | 100.0 |
Because DHX29 contains two ATP binding motifs (Walker A and B) in the DEXD motif-containing domain , we next sought to determine whether these ATP binding motifs are necessary for DHX29 to sense HMW Poly(I:C) by introducing mutations in the Walker A and B sites of DHX29 (WAM-DHX29 and WBM-DHX29). Unlike full-length DHX29, WAM-DHX29 and WBM-DHX29 failed to bind to HMW Poly(I:C) or to increase the HMW Poly(I:C)-binding ability of MDA5 (Fig 6F). Consistent with these observations, we showed that like DHX29a and DHX29b, WAM-DHX29 and WBM-DHX29 did not increase but rather inhibited MDA5-mediated ISRE-Luc activity, as compared to full-length DHX29 (Fig 6G). The inhibitory effects of WAM-DHX29, WBM-DHX29, DHX29a, and DHX29b on HMW Poly(I:C)-induced ISRE-Luc activity were increased with increasing amounts of gene expression (S6D Fig), thus exerting a dominant negative effect on DHX29 function as an RNA sensor. Taken together, these results indicate that DHX29 functions as a sensor to recognize HMW Poly(I:C) and to interact with MDA5. The N-terminal domain of DHX29 is required for MDA5 binding, whereas the DEXD (Walker A and B)-containing domain is required for RNA binding. Thus, both the N-terminal and DEXD/helicase domains of DHX29 are required for its function as an RNA co-sensor to promote MDA5-mediated type I IFN signaling. | study | 100.0 |
Dephosphorylation of MDA5 and RIG-I is a key step of their activation [37, 45, 46]. To test whether DHX29 is required for MDA5 activation, we used wildtype, non-interacting (DHX29c) and inactive (WAM-DHX29 and WBM-DHX29) form of DHX29 to test the phosphorylation level of MDA5 and RIG-I. However, we did not detect any significant change in phosphorylation of RIG-I and MDA5 with different forms of DHX29 in the presence or the absence of stimulation (S6E and S6F Fig). | study | 100.0 |
Several recent studies show that MDA5 forms filaments upon binding to dsRNA, which is critical for the activation of the IFN-β signaling pathway [47, 48]. To determine whether DHX29 promotes the aggregation of MDA5 filaments along dsRNA, we performed electrophoretic mobility shift assays (EMSAs). Although DHX29 and MDA5 protein alone could bind to dsRNA oligonucleotides, dsRNA-binding activity in both mature (higher band) and intermediate forms (lower band) was enhanced when DHX29 and MDA5 were combined (Fig 7A). To further confirm the binding enhancement, we utilized atomic force microscopy (AFM) to image the RNA-protein complex. DHX29 was able to bind to Poly(I:C) but did not form filaments (Fig 7B). However, DHX29 promoted the aggregation of MDA5 filaments on Poly(I:C) dsRNA (Fig 7B). Because the mica plate flattens at the molecular level, the height (vertical distance) attribution could be used to represent the diameter of the RING structure of the protein-RNA complex (Fig 7C). We obtained 10 individual “section mode” analyses of the RNA-protein complexes. Compared with the 2 nm diameter of dsRNA alone (standard height for double strand HMW Poly[I:C]), the diameter of the dsRNA-MDA5-DHX29 complex was around 4 nm, and the diameter of both the dsRNA-MDA5 and dsRNA-DHX29 complexes was around 3 nm (Fig 7C). These results suggest that DHX29 interacts with dsRNA and MDA5 to form the aggregation of DHX29-MDA5 along dsRNA, by which DHX29 promotes MDA5 activation. | study | 100.0 |
(A) Electrophoretic mobility shift assay of purified MDA5 incubated with 24 bp RNA for 40 min on ice in the presence or absence of DHX29 protein. (B) AFM images of purified MDA5 protein in the presence and absence of DHX29 protein. (C) Section mode analysis was done to determine the height of the RNA-protein complexes. a, RNA alone (control); b1 and b2, RNA-DHX29; c, RNA-MDA5; and d, MDA5 plus DHX29. Data are plotted as the mean ± s.d. of ten independent site measurements. *P < 0.05, **P <0.01, ***P <0.001 vs. the corresponding control. (D) A proposed working model to illustrate the critical role of DHX29 in the control and activation of MDA5-mediated RNA recognition and type I IFN signaling. | study | 100.0 |
RIG-I and MDA5 are key RNA sensors that recognize different sets of viruses. MDA5 primarily senses positive-strand viruses from the Picornaviridae family, such as EMCV, whereas RIG-I recognizes SeV, VSV, and influenza virus [7, 9, 10]. Although it is not clear how RIG-I and MDA5 specifically respond to different viruses, it is likely that differences in viral RNA ligand specificity (5´ ppp end of short RNA for RIG-I vs. internal duplex structure of long dsRNA for MDA5) and RNA binding affinity (high affinity for RIG-I vs. low affinity for MDA5) may contribute to the distinct viral recognition of MDA5 and RIG-I in the innate immune response. Recently, several RNA helicases have been implicated in viral immunity and protein translation . For example, DDX1, DDX3, DDX5, DDX6, DDX24, DDX56, DDX60, DHX9, and DHX30 are implicated with human immunodeficiency virus, hepatitis B virus, hepatitis C virus, herpes simplex virus, VSV replication and innate immune response [26, 49, 50]. In particular, DDX1, DDX21, and DHX36 form a complex with adapter protein, TIR-domain-containing adapter-inducing interferon-β (TRIF) to recognize dsRNA from influenza virus and reovirus . DDX41 can recognize intracellular DNA and activate STING-mediated type I IFN signaling . However, potential RNA helicase(s) involved in the recognition of EMCV remain to be identified. In this study, we have provided compelling evidence that the RNA helicase DHX29 functions as an RNA co-sensor along with MDA5, and plays a critical role in innate immune signaling and immunity in response to HMW Poly(I:C) treatment and EMCV infection, but definitely not to LMW Poly(I:C) treatment or VSV infection. However, in contrary to our observation, a previous study reported that DHX29 is expressed solely in lung airway cells, but not in human PBMCs (such as B cells and T cells), primary monocytes, THP-1 cells and 293T cells, and thus does not play a role in type I IFN signaling in immune cells . Moreover, previous study suggested that DHX29 interacts with RIG-I and functions as a co-receptor for sensing poly I:C, poly dAdT:dAdT and VSV infection . However, the interaction between DHX29 and MDA5 was never explored in their study . | study | 99.94 |
To demonstrate the importance of DHX29 expression, several studies have shown that DHX29 is essentially required for protein translation, cell proliferation and survival [31–33, 44]. Consistent with these results, we show that KD or KO of DHX29 leads to cell death after 5 days in cell culture. More importantly, we demonstrate that both DHX29 and MDA5 function as RNA co-sensors and are required for the dsRNA-binding and type I IFN signaling in response to HMW poly I:C and EMCV, but not VSV infection. Our data on the specific recognition of EMCV by DHX29 are quite concordant with several recent studies showing the absolute requirement of DHX29 for the formation and scanning of 43S translation initiation complexes on mRNAs with 5´-structured UTRs, such as those from EMCV [31–33]. The translation of such mRNAs requires DHX29 in 43S translation initiation complexes to unwind structured mRNA and identify the correct initial start codon [34, 35]. These studies also suggest that DHX29 can recognize mRNAs with 5´-structured UTRs present in many Picornaviridae family viruses, including EMCV. Our results obtained with shRNA-mediated KD as well as CRISPR/Cas9-mediated KO of DHX29 in various cell types, further substantially demonstrate the specificity and requirement of DHX29 as an RNA co-sensor in the innate immune response to HMW Poly(I:C) treatment and EMCV infection. Moreover, the presence of DXH29 in both human and mouse cells supports for its functional conservation between human and mouse. Hence, our results unequivocally establish that DHX29 is expressed in all types of immune cells tested, including PBMC, monocyte, B cells, T cells, macrophages and BMDC, and is essentially required for cell proliferation and survival; Importantly, our results have clearly demonstrated that DHX29 functions as an MDA5-specific RNA sensor in response to HMW Poly(I:C) and EMCV, but not to LMW Poly(I:C) or VSV, in immune cells tested. Further evidence shows that DHX29 alone cannot trigger type I IFN signaling in immune cells; it must interact with MDA5, but not RIG-I, to activate the type I IFN signaling pathway in various immune cell types, including THP-1, human PBMCs, murine macrophages and DCs. Of note, DHX29 does not interact with MDA5 in uninfected or untreated cells, but rather is induced after HMW Poly(I:C) treatment or EMCV infection. DHX29 does not interact with LGP2 or RIG-I even after viral infection, suggesting that the N-terminus of DHX29 specifically binds to the N-terminal (CARD-containing) domain of MDA5. However, unlike full-length DHX29, the truncated N-terminus of DHX29 (without the C-terminus) inhibits MDA5-mediated type I IFN signaling, suggesting that full-length DHX29 is required for its ability to enhance type I IFN signaling. | study | 100.0 |
The role of LGP2 in antiviral immunity is controversial. Both positive and negative effects on RIG-I- and MDA5-mediated type I IFN signaling have been reported in response to different viruses [15, 21–24]. One study shows that LGP2 is not essential for the induction of innate immune response, but rather is required for controlling antigen-specific CD8-positive T cell survival in response to virus infection . However, several studies suggest that LGP2 works upstream of RIG-I and MDA5 to potentiate viral RNA-induced signaling and is essential for RIG-I- and MDA5-mediated type I IFN signaling and antiviral immunity [21, 54]. In particular, LGP2 recognizes L region of EMCV genome and promotes the type-I interferon signal in response to EMCV infection . Our results indicate that both LGP2 and DHX29 could markedly affect MDA5-mediated type I IFN response to HMW poly(I:C). Co-expression of LGP2 and DHX29 increases MDA5-mediated type I IFN signaling more than those obtained with either LGP2 or DHX29 expression alone. Ectopic expression of DHX29 can partially rescue HMW poly(I:C)-induced type I IFN signaling reduced by LGP2 KD. Thus, our study suggests that DHX29 positively regulate MDA5-mediated type I IFN signaling independent of LGP2. Consistent with this notion, we show that DHX29 does not interact with LGP2. Thus, our results identify a previously unrecognized role of DHX29 in MDA5-mediated type I IFN signaling and anti-EMCV immunity. Another MDA5 activator PACT was reported to promote MDA5 signaling by promoting MDA5 oligomerization , it will be of interest to investigate the relationship between DHX29 and PACT in the near future studies. | study | 100.0 |
To understand the molecular mechanisms by which DHX29 enhances MDA5-mediated type I IFN signaling, we show that DHX29 has the ability to bind to HMW Poly(I:C) with a strong binding affinity compared with the low HMW Poly(I:C)-binding affinity of MDA5. This is in agreement with previous studies showing that MDA5 has a poor binding affinity for HMW Poly(I:C) and viral dsRNA [1, 15]. Coexpression of DHX29 and MDA5 markedly increases the binding capacity of MDA5 to HMW Poly(I:C), suggesting that DHX29 and MDA5 function as co-sensors for detecting EMCV infection. Further experiments demonstrate that the DEXD (containing Walker A and B ATP binding motifs) and helicase domains of DHX29 are responsible for HMW Poly(I:C) binding. Thus, it appears that DHX29 specifically interacts with MDA5 through its N-terminus and binds to HMW Poly(I:C) through its C-terminal DEXD and helicase domains, thus bringing MDA5 and HMW Poly(I:C) together. Indeed, our EMSA and AFM results show that the dsRNA-binding activity of DHX29 and MDA5 is much better than MDA5 alone. Overall, further analysis is needed to clarify whether the helicase activity of DHX29 is required for its function as co-sensor for MDA5-dsRNA. The potential role of DHX29 in the modulating of ATPase activity and oligomerization of MDA5 [57–59] requires further investigation, although our study strongly indicates that DHX29 bring HMW Poly(I:C) and MDA5 together for enhanced dsRNA binding capacity and consequent type I IFN signaling. Although DHX29 mainly functions as a core component of translation initiation complexes required for the translation of mRNA with 5´-structured UTR under normal physiological conditions, it may bring these structured RNAs in close proximity to MDA5 to promote MDA5-RNA binding and MDA5 aggregation along dsRNA after EMCV infection or HMW Poly(I:C) treatment, which in turn leads to the activation of MDA5 for consequent downstream type I IFN signaling. | study | 100.0 |
Based on the results presented here and previously published data, we propose a working model to illustrate the role of DHX29 in MDA5-mediated innate immune response to HMW Poly(I:C) treatment and EMCV infection (Fig 7D). During normal physiological conditions, DHX29 is mainly involved in its function in the protein translation. However, DHX29 may switch its function to sense and initiate MDA5-mediated type I IFN signaling after EMCV infection or HMW Poly(I:C) treatment. By contrast, the major function of LGP2 is to regulate both RIG-I and MDA5 mediated innate immune signaling in response to different viral infection. Overall, our findings identify DHX29 as an RNA co-sensor with MDA5 in innate immune response and provide molecular insights into the mechanisms by which DHX29 controls EMCV-specific and MDA5-mediated antiviral immunity. | study | 100.0 |
Buffy coats of blood from healthy donors (from the Gulf Coast Regional Blood Center, Houston, TX) were used for isolation of PBMCs (human peripheral blood mononuclear cells) by density-gradient centrifugation with Lymphoprep (Nycomed Pharm). All blood samples were anonymized. The use of PBMCs was in accordance with institutional guidelines on human cell research and the approved protocol (Protocol #IBC00000357) by the Institutional Review Board of Houston Methodist Research Institute. Animal experiments in this study were approved and carried out in accordance with protocol (Protocol #AUP-0115-0005) provided by the Institutional Animal Care and Use Committee (IACUC) at Houston Methodist Research Institute. IACUC uses the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals, which is based on the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. | other | 99.9 |
Human embryonic kidneys (HEK) 293T (2x105) cells (ATCC, CRL-3216) were seeded into 24-well plates the day before transfection. Cells were cotransfected with the IFN-β or ISRE promoter luciferase reporter and Renilla luciferase internal control (pRL-TK) using Lipofectamine 2000 (Invitrogen) as previously described . Cells were stimulated with DHX29, RIG-I, or MDA5 ligand 24 h post-transfection. Empty pcDNA3.1 HA/Flag/Myc tagged vector was used to ensure equal amounts of DNA among the wells. Luciferase activity was determined using the Dual-Luciferase Assay (Promega) with the Luminoskan Ascent Luminometer (Thermo Scientific) as previously described . Reporter gene activity was normalized to the internal control. | study | 100.0 |
Encephalomyocarditis Virus (EMCV; ATCC, VR-1762) and Vesicular stomatitis virus (VSV, Indiana; ATCC, VR-1415) were amplified in Vero cells (African green monkey kidney cell line; ATCC, CCL-81) and 293T cells, respectively, and stored at -80°C until use. After three cycles of freeze-thawing to release intracellular virus, the supernatant was collected by spinning and used in plaque-forming assays. | study | 99.94 |
We designed human and murine DHX29 sgRNA as previously described . Four hDHX29 and 4 mDHX29 sgRNAs were designed and cloned into the BsmB1 site of lentiCRISPR vector containing Cas9-P2A-puromycin as previously described and verified by sequencing analysis. The sgRNA-containing plasmids were transfected into 293T cells with pCMV-VSV-G plasmids and pΔ8.9. After two days, the virus-containing medium was subjected to ultracentrifugation (20000 x g at 4°C for 2 h) and frozen at -80°C. Murine Bone marrow-derived dendritic cells (BMDCs) were generated by flushing bone marrow cells from femurs and tibiae of mice and were cultured in RPMI media containing 10% FBS supplemented with GM-CSF and IL-4. Peritoneal macrophages (pMAC) were obtained by injecting mice with 4% (v/v) thioglycollate (Beckton Dickson), and peritoneal cavities were flushed after 3 days with RPMI media/2% FBS. Isolated PBMCs, BMDCs, and pMAC were transduced with 4 mixed DHX29-sgRNA-containing lentiCRISPR viruses, respectively. Transduced cells were selected in the presence of puromycin (Invivogen) for 48 h and subjected to ligand stimulation or viral infection. | study | 100.0 |
The immunofluorescence assays and confocal microscopy were conducted as described previously . 293T cells grown on glass-bottom dish (MatTek) in complete medium were co-transfected with fluorescently tagged expression vectors for DHX29 (DsRed2) and low concentration of MDA5 (GFP) for 12–16 hr. Cells were either left untreated or treated with LMW Poly(I:C) or EMCV for 5 h. Cells were live-recorded at every 50 min or fixed in 4% paraformaldehyde solution in PBS at room temperature for 15 min. The nucleus was then labeled with DAPI for 5 min in the dark and then followed by three washes in PBS. Samples were then visualized using Nikon Eclipse Ti-E microscope. All acquired images were analyzed and the correlation coefficient (r) of pixel intensity values was extracted by using the Nikon NIS-Elements AR package or the ImageJ (NIH) software. | study | 99.94 |
For in-cell pull-down assays, clarified whole cell lysates (WCL) transfected with plasmid constructs expressing HA- or Flag-tagged protein were preincubated with NeutrAvidin beads for 2 h (Pierce). Biotin-labeled Poly (I:C) was added, and WCL were incubated for an additional 4 h at 4°C. For cell-free pull-down assays, WCL transfected with HA- or Flag-tagged protein were incubated with anti-HA or anti-Flag beads (Sigma), and proteins were eluted from the beads using the corresponding peptide. The purified proteins were then added to the NeutrAvidin beads and coincubated with Poly(I:C) for 4 h. After incubation, protein complexes were washed 5 times, boiled in 4X loading buffer, and subjected to SDS-PAGE. | study | 100.0 |
24bp HPLC-grade RNA oligonucleotide 5´-GCGUCGUACGCUAG CGUACGACGC-3´ was purchased from Integrated DNA Technologies. Sample preparation was performed as previously described . Samples were subjected to 4% native PAGE and stained with gel-red (Biotium). For AFM, we followed previously described protocol with modification. Briefly, we mixed 10 ng HMW Poly(I:C) and 5 ng purified MDA5 protein in 10 μl of buffer and then incubated in the presence or absence of 10 ng DHX29 in buffer A (pH 7.4, 50 mM NaH2PO4, 50 mM glycine, 14 mM succinic acid, 10 mM MgCl2, 10 μM ZnCl2, 5 mM DTT and 2.5 mM ATP) for 40 min on ice. The mixtures were dropped onto the flat mica plate (molecular level) for 10 min and then washed 5 times with PBS to remove excess salt. AFM imaging and analysis were carried out using the Bruker MultiMode AFM with scanasyst, PeakForce QNM imaging modes, Peak Force Tapping technology, and NanoScope Analysis software. | study | 99.94 |
(A-C) Real-time PCR analysis of (A) IFIT1, (B) IFIT2, and (C) CCL5 mRNA expression in Flag-DHX29- and empty vector-transfected 293T cells stimulated with IC HMW Poly(I:C). Data are plotted as the mean ± s.d. Results of (A-C) are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to Fig 1 in the main text. | study | 100.0 |
(A) Immunoblot analysis showing DHX29 expression in THP-1 cells stimulated with intracellular (IC) HMW Poly(I:C) (A, upper) or EMCV (A, lower) at the indicated time points. β-actin was used as a loading control. (B) Real-time PCR analysis of IFNB and DHX29 mRNA levels in THP-1 cells stimulated with IC HMW Poly(I:C). (C) Real-time PCR analysis of the KD efficiency of DHX29-specific shRNAs. Scrambled (Scr) siRNA was used as a control. (D) 293T cells were cotransfected with HA-vector or HA-DHX29 and Scr shRNA or DHX29 shRNA. Cell viability was determined at various time points post-transfection using bromophenol blue. (E) ISRE-luciferase (Luc) (left panel) and IFN-β-Luc activities (right panel) in Scr shRNA- and DHX29 shRNA-transfected 293T-TLR3 cells stimulated exogenously with naked Poly(I:C). (F and G) ISRE-Luc (left panel) and IFN-β-Luc (right panel) activities in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with (F) IC LMW Poly(I:C) and (G) IC Poly(dA:dT). ISRE-Luc and IFN-β-Luc activities are expressed as the fold increase relative to the control. (H-J) Real-time PCR analysis of IFIT1, IFIT2, and CCL5 mRNA levels in Scr shRNA- and DHX29 shRNA-transfected 293T cells stimulated with IC HMW Poly(I:C). Data from (A-J) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to Fig 2 in the main text. | study | 100.0 |
(A) Real-time PCR analysis of the KD efficiency of DHX29 siRNA in human PBMCs, human THP-1 cells, and mouse RAW cells. (B, C) Real-time PCR analysis of IFIT2 and CCL5 mRNA levels in scrambled (Scr) siRNA- and DHX29 siRNA-transfected THP-1 cells stimulated with HMW Poly(I:C) or Poly(dA:dT). (D, E) Real-time PCR analysis of IFIT2 and CCL5 mRNA levels in Scr siRNA- and DHX29 siRNA-transfected RAW cells stimulated with HMW Poly(I:C) or Poly(dA:dT) lyo/vec. Data from (A-E) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to Fig 2 in the main text. | study | 100.0 |
(A, B) Western blot of DHX29-sgRNA-Cas9 LentiCRISPR transduced 293T cells (A) or MEFs (B) after puromycin selection and SURVEYOR assays. (C, D) SURVEYOR assay of DHX29-sgRNA-Cas9 LentiCRISPR mixture transduced human PBMC (C) or murine cell (BMDC, pMAC) (D) after puromycin selection. (E-G) Immunoblot analysis of p-TBK1 in control sgRNA- or DHX29 sgRNA-transduced PBMC (E), BMDC (F), or peritoneal macrophages (pMAC) (G), left untreated or stimulated by LMW Poly(I:C). VSV, Poly(dA:dT), EMCV or HMW Poly(I:C). (H) Morphology of Control-Cas9 or DHX29-sgRNA-Cas9 LentiCRISPR transduced pMAC at Day 9 post transduction. Scale bar = 10μm. Related to Fig 3 in the main text. | study | 100.0 |
(A) 293T cells were cotransfected with HA-mouse (m)DHX29 and Flag-mRIG-I, Flag-mMDA5, or Flag-mMAVS. Whole cell lysates (WCL) were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA or anti-Flag antibodies. (B) Scrambled or DHX29 siRNA transfected WT MEF and MAVS knockout MEF were stimulated with Flag-TBK1 overnight. The WCL were subjected to immunoblot with indicated antibodies. (C) Luciferase assay of RIG-I knockout 293T cell transfected with increase amount of DHX29, followed by stimulation of intracellular (IC) LMW Poly(I:C), HMW Poly(I:C), MDA5 or MAVS. (D) RIG-I Knockout 293T cells were cotransfected with HA-DHX29 and Flag-MAVS or Flag-MDA5. WCL were immunoprecipitated with anti-HA beads and immunoblotted with anti-HA or anti-Flag antibodies. Anti-RIG-I antibody is used to verify the RIG-I knockout cell in whole cell lysate. (E) HA-DHX29- and Flag-MDA5 (20 ng)-transfected 293T cells were stimulated with intracellular (IC) HMW Poly(I:C) at the indicated time points. WCL were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA, phosphorylated (p)-IRF3, and IRF3 antibodies. (F) WCL obtained from THP-1 cells stimulated with IC HMW Poly(I:C) at the indicated time points were immunoprecipitated with anti-DHX29 antibody and immunoblotted with MDA5, p-IRF3, and IRF3 antibodies. (G) 293T cells transfected with HA-DHX29 and Flag-RIG-I or Flag-MDA5 were infected with indicated kind of stimulation at 8hr. The cell lysate was immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA antibodies. (H) WCL obtained from 293T cells transfected with HA-DHX29 and Flag-LGP2 after 6hr EMCV treatment were immunoprecipitated with anti-Flag beads and immunoblotted with anti-HA and anti-Flag antibodies. (I) IFN-β- Luc activities in 293T cells transfected with indicated plasmids post HMW Poly(I:C) treatment were determined. (J) 293T cells expressing Flag-MAVS and Flag-MDA5 were transfected with DHX29 siRNA or scrambled (Scr) siRNA and then stimulated with IC HMW Poly(I:C). WCL were immunoprecipitated with anti-HA antibody and immunoblotted with anti-HA, anti-Flag, p-IRF3, and IRF3 antibodies. WCL were immunoprecipitated with anti-biotin beads and immunoblotted with anti-Flag and anti-HA antibodies. Data from (C, I) are plotted as the mean ± s.d. and are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Related to Fig 5 in the main text. | study | 100.0 |
(A) WCL obtained from 293T cells co-transfected with Flag-tagged full-length (FL)-MDA5, N-MDA5, M-MDA5, or C-MDA5 and HA-DHX29a were immunoprecipitated with anti-Flag beads. The immunoprecipitated product was immunoblotted with anti-HA and anti-Flag antibodies. (B) 293T cells were cotransfected with ISRE-luciferase (Luc) and FL-DHX29, empty vector (vec), DHX29a, DHX29b, DHX29c, or DHX29d plasmids and stimulated by overexpressing MDA5. ISRE-Luc activity was normalized to the Renilla luciferase internal control and presented as the fold increase relative to stimulated FL-DHX29 control cells. (C) 293T cells transfected with Flag-MAVS, HA-DHX29, Flag-MDA5, or Flag-MDA5 plus HA-DHX29 were incubated with biotin-labeled LMW Poly(I:C) for 4 h. (D) ISRE-luciferase (Luc) activity in 293T cells transfected with DHX29 and increasing concentrations (0, 100, and 200 ng per well) of WAM-DHX29, WBM-DHX29, DHX29a, or DHX29b and stimulated with intracellular (IC) HMW Poly(I:C). ISRE-Luc activity is expressed as the fold increase relative to the unstimulated control. (E, F) 20ng Flag-RIG-I (E) or Flag-MDA5 (F) transfected 293T cells were co-transfected with wildtype DHX29, DHX29c, WAM and WBM of DHX29, followed by stimulation for 6 hrs or not. The lysates were immunoprecipitated with anti-Flag beads and immunoblotted with indicated antibodies. Cal A was added 1hr before lysate collection. Data from (B, D) are plotted as the mean ± s.d. Results are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. IC Poly(I:C)-stimulated cells (two-tailed Student's t-test). Related to Fig 6 in the main text. | study | 100.0 |
Metal complexes with the tridentate ligand 2,6-bis[(1H-pyrazol-1-yl)methyl]pyridine are known to be catalysts of polyethylene polymerization (Singh et al., 2003 ▸; Watson et al., 1987 ▸; Son et al., 2014 ▸; Kim & Kang, 2015 ▸). 2,6-Bis[(1H-pyrazol-1-yl)methyl]pyridine was oxidized to pyridine-2,6-dicarboxylate (pdc) by metal nitrate (Unuigboje & Anyile, 2007 ▸). The pdc molecule has been recognized as a component of bacterial spores, and is also useful in a variety of processes as an enzyme inhibitor, plant preservative and food sanitizer (Cui et al., 2011 ▸). The pdc molecule has been selected as a primary dibasic tridentate ligand and a metal complex with pdc was reported to be a new chemical sensor (Mistri et al., 2013 ▸). Attention has been paid to the design of various N-donor ligands with special structural properties in order to investigate the specific stereochemical requirements of a particular metal-binding site (Mukherjee, 2000 ▸). Various substituted N-donor heterocyclic ligands such as imidazole and pyrazole have been selected as a second ligand, so that the structural and electronic effects on the biologically important Cu—N bond could be probed (Ang et al., 1991 ▸; Chen et al., 2011 ▸; Lin et al., 2009 ▸; Liu et al., 2005 ▸). As part of these continuing studies, the title complex has been synthesized and characterized by single crystal X-ray diffraction. | study | 98.75 |
The molecular structure of the title compound is shown in Fig. 1 ▸. The CuII atom is coordinated by three O atoms and two N atoms from tridentate pyridine-2,6-dicarboxylate (pdc), pyrazole and water ligands. The coordination geometry around the CuII atom is a distorted square pyramid as indicated by the τ value of 0.113 (Addison et al., 1984 ▸). The CuII atom lies in the center of the basal plane defined by two nitrogen atoms (N2 from pdc and N14 from pyrazole) and two oxygen atoms (O9 and O12 from pdc). The plane including the CuII atom is almost planar, with an r.m.s. deviation of 0.0847 Å from the corresponding least-squares plane defined by the five constituent atoms. The pyrazole ring is twisted by 66.61 (10)° from the basal plane. The apical Cu1—O19 bond length of 2.217 (2) Å is much longer than those of the basal Cu—O lengths [Cu1—O9 = 2.026 (2) Å and Cu1—O12 = 2.058 (2) Å]. | study | 100.0 |
In the crystal, O—H⋯O hydrogen bonds (O19—H19B⋯O21, O20—H20B⋯O13 and O20—H20A⋯O10iii; symmetry code as in Table 1 ▸) link the complex molecule to the non-coordinating water molecules (Fig. 1 ▸). Two crystallographically independent non-coordinating water molecules are also linked to each other by O—H⋯O hydrogen bonds (O21—H21A⋯O20iv and O21—H21B⋯O20v; Table 1 ▸). Adjacent complex molecules are connected by other O—H⋯O and N—H⋯O hydrogen bonds (N15—H15⋯O12i and O19—H19A⋯O9ii; Table 1 ▸). The above-mentioned intermolecular interactions stabilize and link the components into a two-dimensional network parallel to the ab plane (Fig. 2 ▸). | study | 100.0 |
A search of the Cambridge Structural Database (Version 5.37 with two updates, Groom et al., 2016 ▸) returned 1448 entries for crystal structures related to the name pyridine-2,6-dicarboxylato. Most of them are crystal structures of metal complexes. However, there are only four entries with a secondary ligand of a pyrazolyl derivative bonded to a transition metal, viz. a Cu complex (Lin et al., 2009 ▸; Wang et al., 2014 ▸) and Zn and Co complexes (Zhang et al., 2011 ▸). | review | 52.28 |
A solution of copper nitrate trihydrate (0.072 g, 0.3 mmol) in acetonitrile (5 ml) was added to a solution of 2,6-bis[(1H-pyrazol-1-yl)methyl]pyridine (0.072 g, 0.3 mmol) in acetonitrile (5 ml) in a high-pressure vessel. After sealing the high-pressure vessel, the resulting solution was stirred for three days at 403 K. The precipitate formed was removed by filtration, and the filtrate was washed with acetonitrile and dichloromethane to get a dark-green powder. Single crystals of the title compound were obtained from its aqueous solution by slow evaporation of the solvent at 333 K within five days. | study | 100.0 |
Crystal data, data collection and structure refinement details are summarized in Table 2 ▸. H atoms of the water molecules and the NH group were located in a difference-Fourier map and refined freely [refined distances; O—H = 0.70 (5)–0.91 (6) Å and N—H = 0.93 (4) Å]. Other H atoms were positioned geometrically and refined using a riding model, with C—H = 0.93 Å, and with U iso(H) = 1.2U eq(C). | study | 99.94 |
Today approximately 3000 species of stick insects, also known as walking sticks (Phasmatodea), are known. An interesting feature of Phasmatodea is that 10–25% of the known species are either obligatory or facultative parthenogenetic . Notably, walking sticks may present with low effective population sizes (e.g., Peruphasma schultei) . Additionally, even in tropical habitats, walking stick eggs can require several months to develop, while individual animals live on average 1–2 years . Theoretical work suggests that longer lifespans like this might be associated with higher cell turnover rates . In practice, these factors may lead either to more chromosome abnormalities per generation or a higher likelihood of fixing chromosome abnormalities even if they are slightly deleterious. | study | 99.44 |
Genome sizes of Phasmatodea are close to that of orthopteran grasshoppers , which arose ~40.5 million years ago . However, Phasmatodea are much older, and many subgroups evolved in this order in the last ~200 million years . Chromosome numbers in this group are highly variable, and polyploidy is well documented in parthenogenetic taxa. Interestingly, in this group of insects, large genome sizes up to 8 picogram per haploid genome have been observed. The diploid chromosome number in stick insects ranges from 21 (male) or 22 (female), found in several species, up to 88 in Carausius furcillatus . Also, polyploid species with structurally diploid complements have been reported . Thus, many unique features of genome architecture and inheritance patterns can be observed in Phasmatodea. However, cytogenetic studies on them are principally hampered by high chromosome numbers (polyploidy) or poor chromosome morphology, or both. | review | 86.0 |
Of special interest for basic research is how chromosomal evolution and regulation work during parthenogenetic and clonal reproduction are typical for many species of stick insects. In some species, parthenogenetic populations may be observed in parallel to sexual populations ; how this is regulated and if and how they interact is still not known. Additionally, it needs to be considered that parthenogenetic reproduction may lead to diversity and selection of the most adapted clones. This has been shown by the breeding of stick insects over generations: some cultures reproduced successfully and stayed phenotypically unchanged, while others underwent unexpected changes. For example, changes in normally red wing color to white or pink in Peruphasma schultei in captivity have been reported recently . In a parthenogenetic lineage, new genetic and karyotypic variants normally do not derive from the recombination of the existing genetic and karyotypic elements within the population. Considering the possibility of different egg maturation mechanisms, as observed in Clonopsis gallica, restricted recombination may be possible also here . Still, in most cases parthenogenetic reproduction can be suggested to diminish possible genetic diversity, reducing genotypic and phenotypic variability for natural selection. It should be expected that this kind of evolutionary scenario leads to genomes adapted to long parthenogenetic reproduction, and possibly an accumulation of harmful mutations. In this context, structural chromosome re-patterning, such as fissions, translocations, and polyploidy have been observed . Thus, one major adaptation of parthenogenetic strains is probably whole or segmental polyploidy or amplification of some genomic elements, or both. In alternate or intermixing parthenogenetic and sexual reproducing populations, sexual reproduction can address problems in correct chromosome pairing and recombination during meiosis. This could require special genome and karyotype organization, preventing multivalent formation and ensuring genetic balance. | study | 99.8 |
Even though only comparably scanty data are available on karyotypes and chromosome structure of stick insects, it is known that there are different sex chromosome systems in Phasmatodea. Most stick insects have, besides numerous autosomes, only one gonosome—i.e., one X chromosome. This X0-system is most likely the ancestral sex chromosome system (68 taxa, present in 36 of 46 studied genera). The second largest group of Phasmatodea species exhibits an XY sex chromosome system (13 species, eight genera) . Additionally, Didymuria violescens has males with both X0 and XY complements. Out of 95 studied bisexual species, nine were found with XX/XY, while in the others an XX/X0 sex chromosome system was observed . | study | 100.0 |
Another peculiar karyotypic feature of stick insects relates to the presence/absence and different numbers and sizes of cytological visible satellites, varying among co-generic and even within the same species . For two species from the genus Leptynia, Leptynia montana (2n = 38/37; XX/X0) , L. attenuate (2n = 36/36; XX/XY) (subspecies L. attenuata attenuate, L. attenuata iberica, L. attenuata algarvica) regions enriched for 45S ribosomal deoxyribonucleic acid (rDNA) and telomeric repeats (TTAGG)n were reported . | study | 99.94 |
Despite an increasing interest in Phasmatodea genetics, cytogenetic studies are not very frequent, compared to other model insects such as Drosphila. This may be due to problems in obtaining chromosomes, as laboratory cultures of stick insects often consist only of parthenogenetic females. Accordingly, karyotyping is not an easy task, as the most convenient and useful material for cytogenetic analysis in insects is male testes. Ovarioles can also successfully be used for chromosome preparation, but they contain only a few dividing cells—in our experience, only one of four laboratories used to prepare animal and/or specifically insect chromosomes, was able to obtain stick insect chromosomes for the present study. | study | 100.0 |
Chromosomes of Phasmatodea were up to now mainly studied by routine staining approaches (i.e., Giemsa). Even centromere banding staining (C-banding) of stick insect chromosomes was described only for a few species . Molecular cytogenetics, i.e., fluorescence in situ hybridization (FISH) in stick insects, was applied in a single study . Thus, here we intended to provide karyotypic characterization of the following five species from four families applying C-banding, and to characterize the distribution of 18S rDNA and telomeric repeats by FISH: | study | 99.94 |
Medauroidea extradentata (Vietnam) is a species first reported in 1907 ; it can be bisexual in captivity, but also parthenogenetic. Cytogenetics was previously done resulting in a karyotype of 2n = 38 ; later others reported a karyotype 37,X0 for male. | other | 99.9 |
Sipyloidea sipylus (Southeast Asia; also, due to humans, Madagascar) was first described in 1859 , and established lab strains are mainly parthenogenetic; in the wild, bisexual populations can also be found. The first description of its karyotype was published in 1967 . In females the chromosome number was determined initially to be 80 ± 2 and it was suggested that S. sipylus is tetraploid . Later, the chromosome number in S. sipylus was listed as 4n = 80 . | study | 99.56 |
Five species of stick insects were maintained and bred as laboratory cultures: Medauroidea extradentata (Phasmatidae) , Sungaya inexpectata (Heteropterygidae) , Sipyloidea sipylus (Diapheromeridae) , Phaenopharos khaoyaiensis (Diapheromeridae) , and Peruphasma schultei (Pseudophasmatidae) . Only P. schultei presented with a bisexual population in our setting. The other four species included exclusively parthenogenetic females. M. extradentata, S. inexpectata, S. sipylus, and P. khaoyaiensis were fed with rasp- and blackberry leaves, P. schultei with privet (Ligustrum) leaves. All of them were kept on their fodder plants up to the cytogenetic analyses. | study | 99.94 |
Thirty- to 40-day-old embryos from stick insect eggs were used for the preparation of metaphase chromosomes—initial preliminary work (our own unpublished data) indicated that eggs of this age were best suited for chromosome preparations. The embryos were dissected out of the eggs and placed in a 0.9% sodium citrate solution at room temperature. For 20–30 min they were fixed in a cold methanol:glacial acetic acid mixture (3:1). Fixed embryos were macerated in a drop of 60% acetic acid on slides and air-dried . The per-species available metaphases for the present study are listed in Table 1 (~2 metaphases per embryo; in M. extradentata the amount was doubled). | study | 100.0 |
Meiotic chromosome preparation was performed from testes of P. schultei according to standard protocols. Males were injected with 0.1 mL of 0.1% colchicine in double-distilled water for 1.5–2.0 h prior to fixation of testes in ethanol:glacial acetic acid (3:1) for 15 min, and then kept in 70% ethanol. Air-dried chromosome preparations were obtained by squashing testis follicles in 45% acetic acid and then freezing them on dry ice. | study | 99.94 |
The probe for (TTAGG)n telomeric DNA was generated by polymerase chain reaction (PCR) with the oligonucleotides (TTAGG)5 and (CCTAA)5 as primers as described . Labeling was achieved by additional PCR cycles replacing dTTP (=desoxythymidintriphosphat) by Tetramethyl-Rhodamine-5-dUTP (Tamra-5-dUTP). For detection of 18S rDNA a fragment of human 18S rDNA cloned in pHr13 165 was applied. The rDNA-probe was labeled with Alexa 488-5-dUTP by nick-translation according standard protocols . | study | 99.9 |
Two-color FISH was carried out as described , with small modifications. Briefly, chromosome preparations were denatured in 70% formamide/2x saline-sodium citrate (SSC) solution for 5 min at 75 °C, and dehydrated in standard series of precooled ethanol at −20 °C; the DNA probes in hybridization mixture were denatured separately for 5 min at 95 °C; then hybridization was carried out overnight in a humid chamber at 37 °C. After hybridization, slides were washed under low stringency conditions (3 times for 5 min each, in 50% formamide/2xSSC at 45 °C, 2xSSC at 45 °C and 0.2xSSC at 45 °C). Counterstaining was done by Vectashield Antifade Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories) applied directly under a coverslip that was then sealed with rubber cement. | other | 99.8 |
Microscopy and imaging were done at the Centre for Microscopy of Biological Objects (Institute of Cytology and Genetics, Novosibirsk, Russia) with an AxioImager.M1 (Zeiss, Jena, Germany) fluorescence microscope equipped with #49, #46HE, #43HE filter sets (Zeiss), ProgRes MF (JenaOptik, Jena, Germany) CCD camera. Software package ISIS5 version (MetaSystems, Altlussheim, Germany) was used for image acquisition and analysis. | other | 99.94 |
In four species, M. extradentata, S. inexpectata, S. sipylus, and P. khaoyaiensis chromosome analyses were performed only on embryos obtained from parthenogenetic females. Chromosome numbers were determined, patterns of C-banding described, and FISH with a labeled fragment of 18S rDNA and telomeric repeats was carried out. In P. schultei, additionally to chromosomes from embryos, meiotic chromosomes of two males were analyzed. | study | 99.94 |
The chromosome number published earlier for this species was 2n = 38 for female . We could confirm that the karyotype contained two pairs of metacentrics, one pair of submetacentric and 16 acrocentric chromosomes. C-banding of M. extradentata is shown in Figure 1a; in detail: in the pericentric regions of all chromosomes apart from the largest acrocentric chromosome, large- or medium-sized C-positive regions were observed. Intercalary C-positive regions were also discovered in two pairs of acrocentrics. In the majority of acrocentrics and in all metacentrics small dot-like C-positive regions were identified in terminal regions of long (=q)-arms. The most impressive C-banding pattern was discovered in a pair of large submetacentrics. Their short arms consisted of large proximal C-positive regions, flanked by a small C-negative region and the small dot-like C-positive region on the tip of the chromosome arm; an identical dot-like C-positive region was also present on the end of its long arm. | study | 100.0 |
Interestingly, FISH with the labeled fragment of 18S rDNA stained the whole short arm of the latter mentioned submetacentric chromosome (Figure 1b). FISH signals of the labeled 18S rDNA on these chromosomes were very strong and made it difficult to observe signals in other chromosomal regions (Figure 1b). Nevertheless, an additional small FISH signal could be observed near the termini of some other chromosomes. For detailed analysis, the level of the weak signal was artificially increased (Figure 1c). In this way, small clusters of 18S rDNA were detected in numerous sites. They varied in size and were colocalized or localized near to the clusters of telomeric repeats. Additionally, interstitial clusters of telomeric repeats (Interstitial Telomeric Sequences—ITSs) were present in many chromosomes. Usually they were located not far from clusters of telomeric repeats in the termini of long arms. In some chromosomes, they were close to the clusters of telomeric repeats in the termini of long arms and were almost fused with them (Figure 1b,c). In the future, the pattern of the here applied probes together with other repetitive DNA probes might be used to clearly identify individual chromosome pairs. An attempt to do this based on C-banded chromosomes is shown in Figure 2a. | study | 100.0 |
S. inexpectata had 44 chromosomes per metaphase plate including two pairs of large metacentric, and 20 pairs of acrocentric chromosomes of different sizes. Pericentric C-positive regions were present in all chromosomes. One arm of the largest metacentric chromosome pair was C-positive (Figure 3a). Two-color FISH showed that the C-positive region on this chromosome pair was enriched for 18S rDNA; telomeric repeats were also present in this region. The latter were dispersed along the heterochromatic region, but due to their small numbers they produced a FISH signal of lower intensity (Figure 3b–e). On other chromosomes, clusters of telomeric repeats were located at the chromosomal tips. Additionally, ITSs were observed in C-negative regions of some chromosomes (Figure 3c,e), and besides the classical clusters of telomeric repeats dispersed telomeric repeats were also observed in pericentric C-positive regions of some chromosomes (Figure 3c,e) of S. inexpectata. For the karyotype see Figure 2a. | study | 100.0 |
Karyotype of S. sipylus was earlier described and chromosome number determined as 4n = 80 published by More . Despite the selection of metaphase plates for chromosome analysis according to features suggesting their unbrokenness, the chromosome number in metaphase plates varied in this study from 53 to 71. The chromosome plates of analyzed embryos included two pairs of large metacentrics, remaining chromosomes were submetacentics, varying in size from medium to small. Besides pericentric C-positive regions, C-bands were present in telomeric position of some chromosomes. In different chromosomes, they were in short or long arms. In a few chromosomes, interstitial C-positive regions were also identified. One pair of the large metacentrics was characterized with a large C-positive region. It was separated by a pericentric C-band with a small C-negative region similar in size to the short arm of medium acrocentrics (Figure 4a). According to the data obtained here this species is not tetraploid (Figure 2b). | study | 100.0 |
In the majority of studied embryos, two-color FISH revealed that exclusively the large C-positive regions of the aforementioned chromosome pair were enriched for 18S rDNA (Figure 4b,c). Besides these chromosomes, only one embryo had an additional chromosome containing a small cluster of 18S rDNA (Figure 4c). | study | 100.0 |
For P. khaoyaiensis the chromosome number was 70 for the majority of analyzed cells(Figure 2c), thus, a female karyotype of 34,XX is suggested. In a minority of cells, only 68 chromosomes were detectable, which was most likely an artifact. The analyzed chromosome plates of P. khaoyaiensis included one large, one medium and one small pair of metacentric chromosomes, while the remaining chromosomes were acrocentrics or acrocentrics containing a small short arm. C-positive regions were localized in the pericentric regions of almost all chromosomes. Moreover, in pericentric regions, C-positive stretches were also detected in terminal and intercalary positions of some chromosomes. Among all analyzed embryos, one large chromosome including a large C-positive arm and a small C-negative arm was observed. According to C-banding pattern, no chromosome homologous to it was found (Figure 5a). Subsequent two-color FISH revealed that the large C-positive long arm was enriched for telomeric repeats and repeats homologous to the fragment of 18S rDNA. Furthermore, an erstwhile unidentifiable homologous chromosome was found (Figure 5b). In chromosome spreads of P. khaoyaiensis there were two chromosomes containing cluster of 18S rDNA. They showed the same C-negative arms similar in size, but one of them contained a small short arm, while the other contained a large C-positive arm, both of them enriched for 18S rDNA and telomeric repeats (Figure 5b). In Figure 5c a karyotype is presented; gonosomes were not identified. | study | 100.0 |
Subsets and Splits