Opisthorchis viverrini (O. viverrini) induces intrahepatic cholangiocarcinoma (ICC). Inflammatory cytokines such as interleukin (IL) and tumor necrosis factor (TNF) are substances in the immune system that promote inflammation and causes disease to progress. Genes that help express proinflammatory cytokines can affect an individual’s susceptibility to disease, especially in cancer-related chronic inflammation. This study aimed to investigate risk factors for ICC with a focus on opisthorchiasis and polymorphisms of proinflammatory cytokines (IL-1β and TNF-α).Chronic inflammation and repeated infection with O. viverrini-IgG antibody was assessed using enzyme linked immunosorbent assay. IL-1β and TNF-α polymorphisms were analyzed using a polymerase chain reaction with high resolution melting analysis. Associations between variables and ICC were assessed using conditional logistic regression.This study was a nested case-control study within a cohort study. 219 subjects who developed a primary ICC were identified and matched with two non-cancer controls from the same cohort based on sex and age at recruitment (±3 years). An TNF-α  and high infection intensity were significantly related to an increased risk of ICC (p < 0.05).Subjects with a high infection intensity had higher risk of ICC than those who had a low level . Subjects with all genotypes of IL-1β and TNF-α are not a risk of ICC, but an individual with O. viverrini infection has an effect on all genotypes of the TNF-α gene that might promote ICC. Primary prevention of ICC in high-risk areas is based on efforts to reduce O. viverrini infection.Polymorphisms of  Cancer in Thailand, Vol. VII, 2007–2009, showed that it is the most common cancer in men  and the third most common cancer in women (ASR = 30.9). In Khon Kaen, located in Northeast Thailand, the incidence of ICC is ASR 57.4 in men, and 23.1 in women per 100,000 :5′- AATTTCTCAGCCTCCTACTTC-3′ and [R]: 5′- GTTTGGTATCTGICCGTTTC-3′. The TNF-α G308A gene was amplified by using [F]: 5′- TAGGTTTTGAGGGGCATG -3′ and [R]: 5′- CTGGGICCCTGACTGATTT-3′ in a PCR reaction. Both genetic polymorphisms were performed in a LightCycler® 480 Real-Time PCR System with a final volume of 20 μl containing 10 μl of master mix, 4.4 μl of H2O, 3 mM of MgCl2, 0.3 μM of each primer and 200 ng of the DNA template. The data of high resolution melting analysis was analyzed using the LightCycler 480® Gene Scanning Software version 1.5 (Roche). Normalized melting curves and melting peaks of sequence variation were evaluated and compared with the wild-type sample. Different plots of melting peaks are illustrated in Fig. IL-1β C-511 T and Fig. TNF-α G-308A. Sequence variations were distinguished by the different shape of melting curves . To improve the genotyping quality and validation, 10% of random genotyping samples were confirmed by the PCR with restriction fragment length polymorphism techniques (PCR-RFLP).The amplification of O. viverrini infection intensity and proinflammatory cytokines polymorphisms (IL-1β and TNF-α) with the risk of developing ICC and odds ratios (ORs) were assessed and estimated at the 95% confidence and conditional logistic regression. The information about smoking, alcohol, and diet that was used for the adjusted ORs from the multivariate analysis in Table O. viverrini infection intensity by proinflammatory cytokines polymorphisms (IL-1β and TNF-α) were also analyzed. Results that showed a p-value < 0.05 were statistically significant from the native control.The link between Out of 23,584 participants, 219 (0.9%) subjects developed ICC. This was comprised of 92 females and 127 males and with a median age of 57 years  possessed a higher risk for ICC than those who did not (OD ≤0.24) . In comparison, participants with TT variant of IL-1β C-511 T polymorphisms had a decreased risk of ICC . Results of interactions between O. viverrini infection intensity and IL-1β and TNF-α polymorphisms on the risk of ICC have been shown in Table TNF-α in all genotypes  who had high infection intensity (IgG antibody > 0.24) had an increased risk of ICC and were statistically significant . There were no interactions between O. viverrini infection intensity and the polymorphisms of IL-1β and TNF-α that influenced the risk of ICC.Table O. viverrini infection intensity detected by the IgG antibody was a risk factor for ICC and was comparable to previously reported studies in Northeast Thailand – Khon Kaen, [O. viverrini infection with TNF-α codon 308 AA variant. Participants with only O. viverrini infection intensity had adjusted OR 2.1 (95% CI: 1.2–3.9), but participants who had both O. viverrini infection intensity together with AA variant of TNF-α had an increased OR of 2.8 (95% CI: 1.4–5.8).In this work, our results successfully demonstrated the risk factors for ICC. For example, on Kaen,  Nakhon Pon Kaen,  and Ubonon Kaen, . ResultsIL-1β C-511 T and TNF-α G308A polymorphisms found in the controls were consistent with other studies in Thailand [IL-1β codon 511 were 45.9% vs. 46.4% and 54.1% vs. 53.6%, respectively. In addition, the allele distribution of TNF-α G308A was also consistent as reported before [The genotype frequencies of Thailand , 24. PreIL-1β C-511 T and TNF-α G-308A polymorphisms with the risk of various cancers, especially in chronic hepatitis B virus infection-linked HCC [IL-1β C-511 T and TNF-α G308A polymorphisms on ICC risk until now, the study on cytokines expression related cancer has been reported which acts as a diagnostic marker for cancer. The detection of serum levels of cytokines (such as IL-6 or IL-10) may be linked to the process of carcinogenesis or poor prognosis [O. viverrini infection was evaluated. Eleven cytokine profiles , measured by flow cytometry, revealed that both pro-inflammatory and anti-inflammatory cytokines were increased in the O. viverrini-associated ICC compared to uninfected normal controls [Very limited information exists in Thailand that reported the associations of nked HCC , 24. Altrognosis –28. In ocontrols .The basis of diagnosis of ICC was rarely done by histology (6.9%), and it is possible that other cases of fatal liver disease showing features of biliary obstruction, with a low serum alpha-fetoprotein, were possibly included in the case group. Despite this, the overall results support our hypothesis and are a pivotal factor in formulating experiments for future projects.IL-1β and TNF-α. Polymorphisms of IL-1β C-511 T and TNF-α G-308A are not a risk of ICC but an individual with O. viverrini infection has effects on all genotypes of the TNF-α gene  that might promote ICC. Primary prevention of ICC in high-risk areas is based on efforts to reduce O. viverrini infection.There was a known association between the polymorphisms of


TNF‐α) prevented sarcopenia and prevented age‐related change in muscle fiber phenotype. Furthermore, TNF‐α ablation reduced the number of satellite cells in aging muscle and promoted muscle cell fusion in vivo and in vitro. Because CD68+ macrophages are important sources of TNF‐α and the number of CD68+ macrophages increases in aging muscle, we tested whether macrophage‐derived TNF‐α affects myogenesis. Media conditioned by TNF‐α‐null macrophages increased muscle cell fusion in vitro, compared to media conditioned by wild‐type macrophages. In addition, transplantation of bone marrow cells from wild‐type mice into TNF‐α‐null recipients increased satellite cell numbers and reduced numbers of centrally nucleated myofibers, indicating that myeloid cell‐secreted TNF‐α reduces muscle cell fusion. Transplanting bone marrow cells from wild‐type mice into TNF‐α‐null recipients also increased sarcopenia, although transplantation did not restore the age‐related change in muscle fiber phenotype. Collectively, we show that myeloid cell‐derived TNF‐α contributes to muscle aging by affecting sarcopenia and muscle cell fusion with aging muscle fibers. Our findings also show that TNF‐α that is intrinsic to muscle and TNF‐α secreted by immune cells work together to influence muscle aging.Sarcopenia is age‐related muscle wasting that lacks effective therapeutic interventions. We found that systemic ablation of tumor necrosis factor‐α ( We found that ablating TNF‐α resulted in reduced sarcopenia and produced hypernucleation of aging myofibers. Furthermore, our in vitro experiments showed that TNF‐α intrinsic to satellite cells and TNF‐α secreted by macrophages both influence myogenesis. We then specifically tested the importance of myeloid‐cell‐secreted TNF‐α in vivo by performing transplantation of wild‐type bone marrow cells (BMCs) into TNF‐α‐null mice and found that myeloid‐cell‐derived TNF‐α contributed to sarcopenia and reduced muscle cell fusion, establishing the role of myeloid‐cell‐derived TNF‐α in influencing muscle aging.In this study, we used a mouse model with systemic genetic ablation of 22.1TNF‐α affected age‐related changes in muscle. TNF‐α‐null mice had no detectable expression of TNF‐α in muscles at 12 or 24 months of age  and old (24 months old) wild‐type mice and showed that aging is associated with greater expression of TNF‐α  cultures from adult wild‐type and TNF‐α‐null mice. Muscle cells were treated with conditioned media for 2 days before induction of differentiation, followed by another 5 days of treatment with conditioned media after induction of differentiation. Muscle cells receiving conditioned media from BMDMs isolated from TNF‐α‐null mice had more nuclei per fiber than cultures receiving media conditioned by wild‐type BMDMs  Guide for the Care and Use of Laboratory Animals and were approved by the University of California, Los Angeles Institutional Animal Care and Use Committee. Following euthanasia, muscles were collected from wild‐type mice (C57 BL/6) and 4.2Frozen cross sections were cut from the midbelly of quadriceps muscles at a thickness of 10 μm. Hematoxylin‐stained sections were used to quantify muscle fiber CSA and central nucleation. Total nucleation was quantified in sections immunolabeled with antidystrophin and propidium iodide staining, as described in Supporting Information Appendix Other frozen sections were immunolabeled with rabbit anti‐sMHC, rat anti‐CD68, or mouse anti‐Pax7 and then used to quantify numbers of slow muscle fibers, macrophages, or satellite cells, respectively. Sections were also double‐labeled with anti‐CD68 and mouse anti‐TNF‐α to identify TNF‐α‐expressing macrophages, as described in Supporting Information Appendix 4.3Frozen muscles were homogenized and RNA was isolated, cDNA generated, and QPCR performed as described previously (Villalta et al., 4.4Primary myoblasts were isolated following a previous protocol (Wehling‐Henricks et al., 4.5Bone marrow cells were isolated following a previously described protocol (Wang et al., 4.6SEM. One‐way analysis of variance was used to test whether differences between three or more groups were significant at p < 0.05. Significant differences were identified using Tukey's post hoc test. Comparisons of two groups of values were analyzed using the unpaired, two‐tailed t test.Data are presented as mean ± None declared.All authors participated in experimental design, experimentation, data analysis, and preparation of the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


The major limitation of organ transplantation is the shortage of available organs. Xenotransplantation is considered to be an effective way to resolve the problem. Immune rejection is a major hurdle for the successful survival of pig xenografts in primate recipients. Cytokines play important roles in inflammation and many diseases including allotransplantation, however, their roles in xenotransplantation have been less well investigated.We assessed the role of several cytokines in xenotransplantation using an in vitro model of human antibody-mediated complement-dependent cytotoxicity (CDC). Porcine aortic endothelial cells (PAECs) and porcine iliac endothelial cells (PIECs) were selected as target cells. The complement regulators  and junction protein genes were assessed by real-time PCR, flow cytometry, or western-blotting assay. Flow cytometry assay was also used to evaluate C3 and C5b-9 deposition, as well as the extent of human IgM and IgG binding to PIECs. Gene silencing was used to reduce genes expression in PIECs. Gene overexpression was mediated by adenovirus or retrovirus.Recombinant human TNF-α increased the cytotoxicity of PAECs and PIECs in a human antibody-mediated CDC model. Unexpectedly, we found that the expression of complement regulators  increased in PIECs exposed to human TNF-α. Human TNF-α did not modify C3 or C5b-9 deposition on PIECs. The extent of human IgM and IgG binding to PIECs was not affected by human TNF-α. Human TNF-α decreased the expression of Occludin in PIECs. Gene silencing and overexpression assay suggested that Occludin was required for human TNF-α-mediated cytotoxicity of PIECs in this model. P38 gene silencing or inhibition of P38 signaling pathway with a specific inhibitor, SB203580, inhibited the reduction of Occludin expression induced by TNF-α, and suppressed TNF-α-augmented cytotoxicity of PIECs.Our data suggest that human TNF-α increases the cytotoxicity of porcine endothelial cells in a human antibody-mediated CDC model by downregulating P38-dependent Occludin expression. Pharmacologic blockade of TNF-α is likely to increase xenograft survival in pig-to-primate organ xenotransplantation.The online version of this article (10.1186/s12964-019-0386-7) contains supplementary material, which is available to authorized users. Organ transplantation is an effective therapy for patients with end-stage organ failure . HoweverIn pig-to-human xenotransplantation, porcine vascular endothelial cells (ECs) are the first defense. They interact with human immune cells, and are activated by cytokines or chemokines produced by the human immune cells . PorcineThe complement system consists of several tightly-regulated proteins that play important roles in the inflammatory response and in host defense , 9. ThreIn pig-to-primate organ transplantation, cytokines and chemokines are secreted by recipient cells . PreviouIn order to explore this topic, we chose eight human cytokines  and evaluated their effects in a human antibody-mediated CDC model. Porcine ECs were chosen as the target cells. We demonstrated that TNF-α promoted human antibody-mediated CDC of porcine ECs and was dependent on P38-mediated reduction of Occludin expression. This provides a possible explanation for the pathological role of TNF-α in xenotransplantation., IL-6, TNF-α, G-CSF, G-MCSF, IFNγ, and IL-4 were purchased from R&D Systems . FITC-conjugated goat anti-human IgM, IgG or isotype-matched antibodies were purchased from Invitrogen . FITC-conjugated goat anti-mouse IgG was obtained from Jackson ImmunoReasearch Laboratories . Anti-actin, anti-pJNK, anti-JNK, anti-pP38, anti-P38, anti-ERK and anti-Cleaved Caspase 3 were purchased from Cell Signaling Technology ; anti-pERK from Santa Cruz Biotechnology ; anti-CD46 from Bio-rad ; anti-CD55 from LifeSpan BioSciences ; anti-Claudin 2, anti-C3 (Alexa Flour® 488), anti-C3c (FITC) and anti-C5b-9 from Abcam ; anti-Occludin and anti-Zo 1 from Thermo Fisher Scientific ; SB203580, SP600125 and PD98059 from Selleck ; Cell Counting Kit-8 (CCK8) from Dojindo Laboratories ; and Neutral Red from Sangon Biotech .Recombinant human (rh) IL-2, IL-15, IL-82. Porcine iliac endothelial cells (PIECs) were purchased from the Type Culture Collection of the Chinese Academy of Sciences; the cells were cultured with RPMI-1640 containing 10% (vol/vol) FBS, 1% (vol/vol) P/S at 37 °C with 5% CO2. Porcine aortic endothelial cells (PAECs) were isolated from one Chinese Wuzhishan wild-type pig, and cultured as previously described ) for 30 min at 37 °C. After washing with PBS, 10% goat serum was used to prevent non-specific binding. Then the cells were incubated with FITC-conjugated goat anti-human IgM or IgG antibody for 30 min at 4 °C in the dark. Secondary antibody only, without serum, acted as a control.Human IgM and IgG binding using flow cytometry was performed as previously described , 18. In 6) were incubated with 20% human serum for 30 min at 37 °C. Then the cells were incubated with anti-CD46, anti-CD55, or C5b-9 antibody for 1 h at 4 °C in the dark. Isotype antibody acted as a control. After washing, the cells were incubated with FITC-conjugated goat anti-mouse IgG for 30 min at 4 °C in the dark.For CD46 and CD55 expression, and C5b-9 deposition, PIECs (1 × 106) were incubated with 20% human serum for 30 min at 37 °C. Then the cells were incubated with anti-C3 (Alexa Flour® 488) or anti-C3c (FITC) antibody for 1 h at 4 °C in the dark. Corresponding isotype antibodies acted as controls.For C3 deposition, PIECs . The degree of antibody binding was assessed by geometric mean fluorescence intensity (Gmean).3VO4, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail . The protein concentration was assessed by BCA Protein Assay Kit (Thermo Fisher) according to the instructions. Every protein sample (20 μg) was separated by 10% SDS-PAGE. After the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes , the PVDF membranes were blocked with 5% skim milk at room temperature for 1 h and then were incubated with primary antibody overnight at 4 °C. After incubation with secondary antibody for 1 h at room temperature, the blots were visualized with enhanced chemiluminescence (ECL) detection reagents (Millipore).The procedure of western-blotting analysis has been previously reported . In brieCells were grown on 3 μm pore Transwell filters (Corning) until confluent. After the cells were washed twice with PBS, 100 μg/ml fluorescein isothiocyanate (FITC)-conjugated 40 kDa dextran (Sigma) was added to the apical compartment for 30 min. The fluorescence of basolateral medium was measured using a spectrofluorometer (Thermo Fisher) at an excitation wavelength of 492 nm and detection wavelength of 520 nm.p values < 0.05 were considered significant.Experimental data are presented as the mean ± SEM using Prism software (GraphPad software). Statistical significance between the groups was calculated by using a two-tailed Student’s t test (Microsoft Office Excel software). Previously, we found that recombinant human angiopoietin-1 (rhAng-1) and rhAng-2 protected PIECs from human antibody-mediated CDC . Human IPIECs were treated with recombinant human TNF-α, IL-2, IL-15, IL-8, IL-6, G-CSF, G-MCSF, IFNγ, IL-4 or medium for 48 h, and then exposed to human serum (heat-inactivated serum as a control) to induce antibody-mediated CDC. As expected, we found that human TNF-α increased the cytotoxicity of PIECs in the human antibody-mediated CDC model. Cytotoxicity (by CCK8) increased from 43 to 66%  and found that rhTNF-α also promoted the cytotoxicity of PAECs Fig. d. AdditiSince TNF-α could induce cell apoptosis in several cells directly, we wanted to know whether TNF-α caused PIEC apoptosis. We found that TNF-α alone did not induce cleaved caspase 3 in PIECs exposed to TNF-α for 48 h  suppress the activation of complement and subsequently reduce CDC , 21. We Next, we asked whether TNF-α affected complement activation. We assessed C3 (with anti-C3 and anti-C3c antibodies) and C5b-9 deposition after the PIECs were incubated with human serum and found that TNF-α did not affect C3 or C5b-9 deposition on PIECs  but not heat-inactivatd human serum minus IgG, IgA and IgM (HIHS[−]) Fig. d-e. HoweTogether, these data demonstrate that complement regulators, complement activation and antibody binding are not the primary factors in the TNF-α promotion of cytotoxicity of PIECs.A previous report suggested that IL-4 increased the expression of the junction gene Claudin 5 to mediate the protection of porcine ECs from human antibody-mediated CDC . We wantBecause upregulation of Claudin 5 suppresses human antibody-mediated CDC, we asked whether downregulation of Zo 1, Claudin 2, or Occludin promoted human antibody-mediated CDC. PIECs were infected with lentivirus encoding the corresponding siRNA to knockdown Zo 1, Claudin 2, or Occludin. The mRNA and protein levels of Zo 1, Claudin 2, or Occludin were significantly reduced in corresponding infected PIECs  in PIECs  were activated in PAECs by human TNF-α . We alsoSurprisingly, we found that inhibition of P38 with SB203580 obviously suppressed TNF-α-augmented cytotoxicity of PIECs exposed to TNF-α for 24 h or 48 h with 10% serum or 20% serum Fig. d. The cyAccording to the above findings, we predicted that TNF-α-decreased Occludin expression was dependent on P38. To prove our hypothesis, we inhibited P38 signaling pathway with P38 inhibitor or P38-specific siRNA oligos in PIECs exposed to TNF-α, and checked Occludin expression with real-time PCR and western-blotting assay. The mRNA level of Occludin was significantly increased in the SB203580 + TNF-α group compared to the DMSO+TNF-α group Fig. a. We obtOccludin is an important tight junction protein which plays a critical role in maintaining barrier function –26. MoreCollectively, TNF-α and Occludin knockdown increased the permeability of PIECs, though this might not be the primary reason for the TNF-α-augmented cytotoxicity of PIECs.Pro-inflammatory cytokines play important roles in immune regulation. Many diseases and allotransplantation are associated with cytokine dysregulation –33. HowePreviously, we also found that human (and porcine) TNF-α induced the expression of pro-inflammatory genes and a pro-coagulation gene (tissue factor) in porcine (or human) ECs , 34. BasBlockade of TNF-α in xenotransplantation has been reported by several groups –38. NeutComplement regulators  are important negative regulators that restrain the over-activation of complement . In the Dalmasso et al. reported that IL-4 protected porcine ECs from human antibody-mediated CDC partially through upregulating the junction protein, Claudin 5, that strengthened the barrier function of porcine ECs . We foun2+ ions, is thought to be the prime reason for MAC-induced cell death [2+ ions or other ions by interaction with MAC; ii) Cell ion pumps put excess ions out of the cell, and Occludin might promote this through interacting with ion pumps; iii) Since ion pumps put the ions out of the cell, which requires energy, Occludin may be involved in energy metabolism. These hypotheses need to be investigated in the future.Collectively, our data demonstrated a novel function of Occludin which protected ECs from human antibody-mediated CDC. Our findings suggested that TNF-α promoted the killing of PIECs through regulating the expression of Occludin. We found that antibody binding, complement activation, MAC formation, or barrier disruption was not the major reason for TNF-α’s enhancement of cytotoxicity of PIECs. Based on these observations, we predicted that Occludin-mediated protection of the cells from human antibody-mediated CDC was likely associated with protection of the cells from MAC-mediated cytolysis. The inward leakage of water and ions, particularly a large influx of Call death . OccludiSeveral papers have reported that TNF-α regulated the expression of Occludin –29, 40. In our setting, TNF-α decreased the expression of Occludin at mRNA and protein levels. However, how TNF-α-mediated the reduction of Occludin protein expression is an interesting scientific question. MAPKs  are intracellular signaling pathways that play a pivotal role in many complex cellular programs, such as cell proliferation, differentiation, development, and survival , 42. Her2-induced Occludin loss was also reversed by SB202190 (a specific P38 inhibitor) in rats [Here, we found that P38 siganling pathway was essential for TNF-α-mediated Occludin reduction. Tai et al. reported that amyloid-β induced Occludin down-regulation dependent on P38 activation in human brain ECs . CdCl2-i in rats . HoweverBesides the transcription level, P38 might regulate the protein level of Occludin directly. Several papers reported that Occludin served as a substrate for matrix metalloproteinases 9 (MMP9) , 46. TNFIn the present study, we found that retrovirus-mediated ectopic expression of Occludin in PIECs protected the PIECs from human antibody-mediated CDC. The data suggested that in vivo gene transfer might be a promising way to protect porcine cells, tissues, or organs from the complement-mediated injury. However, we need more evidence before we overexpress Occludin in animal models of xenotransplantation. We are also investigating whether Occludin affects other important aspects of xenotransplantation, such as coagulation and inflammation. If the data suggest that Occludin indeed mediates the protection of xenografts, Occludin-transgenic pigs may need to be put on the agenda. Retroviruses are not suitable for clinical application, and are known to cause oncogenesis . Use of In conclusion, in the present study, we found that human TNF-α promoted the cytotoxicity of PIECs by decreasing the expression of Occludin. The P38 signaling pathway was essential for TNF-α to mediate the reduction of Occludin expression and to increase human antibody-mediated CDC. Our study provides a possible explanation for the pathological role of TNF-α in xenotransplantation. In pig-to-human xenotransplantation, we suggest that blockade of TNF-α would probably decrease xenograft rejection and increase graft survival.Additional file 1:Table S1. Listing of primers and primer sequences for real-time PCR. (DOC 47 kb)Additional file 2:Figure S1. TNF-α did not cause PIEC death. (A) PIECs were treated with or without rhTNF-α (20 ng/ml) for 48 h, rhTNF-α (20 ng/ml) + cycloheximide  or rhTNF-α (20 ng/ml) + CHX (Low: 2 μg/ml) for 2.5 h as positive controls. Lysates were analyzed by western-blotting with antibodies against Cleaved Caspase 3 and actin. (B) PIECs were treated with rhTNF-α (20 ng/ml) or medium as a negative control (NC) for 0, 24 h, 48 h, 72 h, or 96 h. The cell number was assessed with CCK8. (C) PIECs were treated with recombinant human IL-2 (20 ng/ml), IL-15 (100 ng/ml), IL-8 (100 ng/ml), IL-6 (20 ng/ml), G-CSF (100 ng/ml), G-MCSF (50 ng/ml), IFNγ (50 ng/ml), IL-4 (20 ng/ml), or medium as a negative control (NC) for 48 h. The cell number was assessed with CCK8. Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05 by Student’s t test. L.E. = long exposure, S.E. = short exposure. (DOC 147 kb)Additional file 3:Figure S2. Occludin did not affect expression of other junction genes in PIECs. (A) PIECs were infected with control virus (EV) or retrovirus encoding Occludin. After 4 days, total RNA was collected and the mRNA levels of indicated genes were measured by RT-PCR. (B-C) PIECs were infected with control lentivirus or with lentivirus expressing Occludin- (B) or Claudin 2- (C) specific siRNA. After 4 days, total RNA was collected and the mRNA levels of indicated genes were measured by RT-PCR. Data are representative of at least three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01 by Student’s t test. (DOC 287 kb)Additional file 4:Figure S3. Loss of P38 in PIECs was mediated by P38-specific siRNA oligos. (A-B) PIECs were transfected with control siRNA oligo or with P38-specific siRNA oligos. After 48 h, total RNA was collected and the mRNA levels of P38 were measured by RT-PCR (A), and lysates were analyzed by western-blotting (right side) with antibodies against P38, or actin (B). Data are representative of at least three independent experiments (mean ± SEM). ***p < 0.001 by Student’s t test. (DOC 98 kb)


When faced with potential predators, animals instinctively decide whether there is a threat they should escape from, and also when, how, and where to take evasive action. While escape is often viewed in classical ethology as an action that is released upon presentation of specific stimuli, successful and adaptive escape behaviour relies on integrating information from sensory systems, stored knowledge, and internal states. From a neuroscience perspective, escape is an incredibly rich model that provides opportunities for investigating processes such as perceptual and value-based decision-making, or action selection, in an ethological setting. We review recent research from laboratory and field studies that explore, at the behavioural and mechanistic levels, how elements from multiple information streams are integrated to generate flexible escape behaviour. Escape behaviours are not only simple stimulus-reactions but are under cognitive control, allowing the study of processes such as decision-making and action selection in tractable organisms in ethological settings.Successful escape relies on integrating multiple external and internal variables, such as for computing flight trajectories towards shelter, and implementing trade-offs by choosing between actions that satisfy competing motivations.Some neural mechanisms of escape are innate and conserved across species, but are subject to control and modification by multiple systems, including the neocortex, which allow experience to be flexibly incorporated into escape behaviour.New tools to quantify behaviour while recording neural activity enable analysis of ethologically-relevant behaviours in complex environments, and will advance our understanding of the neural basis of natural behaviours. Escape  and a value component (is it threatening or not?). Animals must detect, identify, and evaluate threats based on sensory features such as shape, size, speed, or smell. For most animal species there are stimuli that innately carry negative valence, which is essential for surviving and escaping from predators without needing to rely on prior exposure to learn that they are threatening. This can be implemented through the evolution of dedicated channels with specialised sensory detectors, which activate defensive circuits and lead to stereotyped behaviour. For example, the rodent olfactory system has a circuit specialised for the detection of chemical substances emitted by other species, innately threatening stimuli but no adverse outcome occurs, presumably to minimise costs associated with unnecessary escape responses Although reflex-like stimulus–response couplings are important for survival, the evaluation of threats should be flexible enough to adapt to dynamic contingencies, and be modulated by expectations arising from experience and prior beliefs. For example, prey should adjust their behaviour based on the frequency and outcome of past encounters with predators, and adapt to the current level of predatory risk. In theoretical terms this is equivalent to a prior – at high predation risk, the threshold for detecting or reacting to threats should be lowered, and raised for low predation risk. Such flexible adaptation has been demonstrated in laboratory and field studies, where animals quickly learn to suppress escape responses if they are repeatedly presented with escape vigour in subsequent encounters Across species, experience with threats can also lead to heightened sensitivity to future stimuli. Many animals respond to repeated approaches from predatory threats by increasing the probability of escape and Habituation and sensitisation are two opposite adaptation strategies, and whether an individual responds to repeated threat encounters with one or the other is likely to depend on factors such as the initial perceived threat-level of the stimulus and the frequency of encounter, and in fact both can occur in the same animal on different timescales In addition to updating innate processes for classifying sensory stimuli as threats, surviving encounters with predators should also lead to learning new associations between features of the sensory space that might predict future encounters In mammals, the circuits for encoding predator cues and predator context-specific memories are not as well understood as those that process simple aversive stimuli such as foot shocks. Overall, they seem to involve the same core associative-learning circuit elements of the basolateral amygdala, hippocampus, and distributed cortical regions, but with the additional integration of threat-instructive signals from networks such as the medial hypothalamic defensive system In addition to modulation from past experience, threat detection is also affected by the current state of the environment, and foraging animals often display threat-assessment behaviours It is interesting to consider that key areas for threat detection and expression of defensive behaviours, such as the SC and amygdala, are also involved in the control of spatial attention and saliency maps After a sensory stimulus has been detected and valued as being a threat that requires immediate action, the escape circuit should in principle be engaged as soon as possible, and evolution has shaped neural circuits to ensure that this can be achieved. The Mauthner cell is perhaps the best-studied example – it receives direct synaptic input from the cranial nerve VIII carrying information from hair cells, and commands the initiation of fast C-start escape responses by projecting across the midline to activate contralateral motoneurons, allowing millisecond-long latencies between threat detection and escape start Box 1+ neurons, can recapitulate the effects of hunger on defence Hunger has a powerful and well demonstrated effect on behavioural choices and learning Alt-text: Box 1Initiating escape can be the result of a decision-making process that requires integrating dynamic evidence about the threat, and decisions take time. Therefore, the escape reaction time should in part reflect the time over which decision-making processes occur, such as evidence accumulation. It is important to note, however, that even in this case reaction time does not necessarily reflect exclusively the time it takes to decide to escape: animals may have made a commitment to escape, but not initiated the action, such as during ambush encounters with a striking predator, where an animal waits to take evasive action until it is too late for the predator to adjust its trajectory. In addition, economic models of escape, supported by experimental evidence from field studies, suggest that animals escape only when the costs of remaining  are higher than the costs of fleeing  Overall, the decision to initiate escape is not simply a threat but detection process, it is a computation that can integrate multiple external and internal variables, and currently we know very little about how the brain implements this process.Escape is only one of several possible actions in the defensive repertoire of most animals, and, for each encounter with a threat, the defensive action with the highest likelihood of success should in principle be chosen. The optimal choice depends on multiple factors about both the environment and the properties of the threat, which should be taken into account when selecting escape or alternative actions. A canonical defence alternative to escape is freezing behaviour, which has the main goal of avoiding detection Another factor known to control escape selection is the presence and value of a desirable resource. For example, when crayfish are close to a food source, they prefer to freeze in response to fast-expanding looming stimuli instead of escaping with a tail-flip In contrast to invertebrates, the neural implementation of defensive action selection in mammals is poorly understood. For learned threats in laboratory animals exposed to fear conditioning protocols, the central amygdala (CeA) is a critical node in controlling freezing behaviour through projections to the ventrolateral PAG (vlPAG) Box 2For the predator, successful predation includes at least five timepoints: detection, classification, approach, subjugation, and consumption (i) As prey behavioural responses are guided by the predator's current and expected actions, understanding the predator’s behaviour can help to infer and interpret the goals, strategies, order, and kinematics of prey behaviour (ii) Many prey act as predators themselves, and evidence suggests that the computations for detection, sensorimotor transformations, and movements that underlie predation and evasion are sufficiently similar that both can be carried out by overlapping neural structures. For foraging animals, sensory cues that are very similar can arise from prey, predators, or harmless agents, and misidentification can carry considerable fitness costs Interestingly, despite their opposing goals, some of the movements required for predation and evasion are so similar Alt-text: Box 2Once the escape action has been selected, a key consideration is where to escape to. One of the simplest actions is to increase the distance from the threat by moving away from it. Escape responses in fish again provide an excellent example of such evasive action, where C-start escapes bend the fish with the head pointing away from the stimulus, followed by a second phase of swimming away. However, the success of the escape action will increase if it is sufficiently flexible to adapt to the properties of the threat, such as different predator strategies , and to The most basic information to extract from the threat is its type and location, which can dictate the direction of escape not only for simple behaviours but also for more complex actions  seem to encode a variable correlated with threat-stimulus saliency, which activates the dPAG network to initiate escape once dmSC activity exceeds a threshold A crucial determinant of escape success is taking into account the spatial features of the local environment, such as routes to shelter and the presence of obstacles, which have been shown to modulate escape responses across phyla. In fish and frogs, the onset of C-start responses is sensitive to stationary obstacles: in open water the initial C-start angle is a function of the angle of the approaching threat, but when these animals are close to a wall their escape trajectories cannot be predicted from the threat-stimulus approach angle, and are instead biased away from the wall, even if the animal needs to turn towards the approaching threat Many animals, including fish, lizards, and rodents, escape towards a known refuge Box 3Congregating with conspecifics can be an advantageous antipredation strategy. As the number of potential prey increases, the chance that a given individual will be predated decreases through dilution Foraging in a group can enhance threat detection in several ways. First, many animal species emit dedicated alarm signals to warn conspecifics of threatening situations. Various modalities of signals have been described, including chemical alarm signals In addition to threat detection, being in a group setting also influences escape execution, which becomes extremely dynamic due to the additional navigation constraints imposed by the presence of nearby animals also trying to escape. Fish schools, which are thought to be primarily an antipredator adaptation, provide an excellent example of collective escape behaviour. Schools of sand eels can execute diverse coordinated escape actions such as split, join, or hourglass formation Alt-text: Box 3In addition, the local social environment can also modulate escape behaviour . Schoolide novo on the spot. This raises interesting parallels with heuristics-based decision-making, which might rely on similar processes, and thus investigation of escape mechanisms might shed light on this important component of behaviour. Although the neurophysiological mechanisms of threat detection and escape initiation have been studied in detail in some species, we know very little about how cognitive control of escape is implemented at the mechanistic level (see Outstanding Questions). As new software tools for rigorous behavioural quantification in freely behaving animals become available Outstanding QuestionsHow, and where in the brain, is the choice between escape and other defensive actions computed? Prey can exhibit multiple defensive behaviours that are flexibly selected as a function of environment and threat contingencies, but which neural circuits implement this action selection? Are models of action selection developed for learned actions valid for instinctive behaviours, or are there specialised ‘low-level’ modules for computing instinctive choice of defence actions? Do the basal ganglia play a role in escape behaviour? To what extent do the apparent defensive strategies rely on deliberative processes?How does the mammalian brain coordinate complex escapes? Fleeing to shelter may require navigation through complex environments and negotiation of obstacles or multiple route options, but where are these computations made? Can they be independently implemented by subcortical structures, or do they require coordination with cortical circuits? Where are variables that matter for escaping successfully encoded in the brain, and where are they integrated into escape decisions? Learning of these variables, such as shelter location, can be an extremely fast process, but how is this implemented at the neuronal and synaptic level?How are experience-dependent changes in escape behaviour achieved at a neuronal level? Are they implemented as ‘top-down’ cortical control over subcortical areas, or are the midbrain circuits underlying innate behaviours themselves plastic? Are changes such as threat habituation long-lasting or even permanent? How are prior beliefs and expectations about threat and escape encoded and updated, and how do they influence the core escape-circuit modules?Although escape behaviour may appear to be simple, there is overwhelming evidence at the behavioural level that much more is involved than simple feedforward sensorimotor transformations. For systems neuroscience, escape behaviour provides a powerful ethological paradigm for studying the neural basis of cognitive processes such as perceptual and value-based decision-making, or goal-directed actions. While escape is often perceived as a simple stimulus-reaction, the lack of apparent explicit deliberation should not be taken as an indication of a simple computational process. Escape might need to be implemented under strong timing constraints and favour short reaction times, but even very fast escape responses can integrate multiple variables such as spatial constraints of the environment and economic trade-offs. The difference with other actions might be that the results of the computations relevant for successful escape are cached and ready to use (pre-computed cognitive constructs) instead of being computed


Human noroviruses (HuNoVs) are the leading cause of viral foodborne outbreaks worldwide. To date, no available methods can be routinely used to detect infectious HuNoVs in foodstuffs. HuNoVs recognize Histo-Blood Group Antigens (HBGAs) through the binding pocket (BP) of capsid protein VP1, which promotes infection in the host cell. In this context, the suitability of human HBGA-binding assays to evaluate the BP integrity of HuNoVs was studied on GII.4 virus-like particles (VLPs) and GII.4 HuNoVs during natural ageing at 20 °C and heat treatments. Our results demonstrate that this approach may reduce the over-estimation of potential infectious HuNoVs resulting from solely using the genome detection, even though some limitations have been identified. The specificity of HBGA-binding to the BP is clearly dependent on the HGBA type (as previously evidenced) and the ionic strength of the media without disturbing such interactions. This study also provides new arguments regarding the ability of VLPs to mimic HuNoV behavior during inactivation treatments. The BP stability of VLPs was at least 4.3 fold lower than that of HuNoVs at 20 °C, whereas capsids of both particles were disrupted at 72 °C. Thus, VLPs are relevant surrogates of HuNoVs for inactivation treatments inducing significant changes in the capsid structure. HuNoVs, which belong to the family Caliciviridae, are mainly genogroup I (GI) and II (GII) NoVs which are subdivided into several genotypes3. Genogroup II, genotype 4 (GII.4) NoV is the most prevalent, and has been causing for about 70% of outbreaks since 20024. Infected individuals release high concentrations of HuNoVs in their feces, ranging from 106 to 109 genome copies (gc)/gram of stools over a 4- to 8-week period5. The infectious dose for HuNoVs has not been clearly defined yet. For Norwalk virus (NV), the 50% human infectious dose (HID50) was estimated between 18 and 2,800 genomic equivalents for secretor individuals and by considering the viral aggregation7. Thus, high viral shedding in the environment combined with a relatively low HID50 contributes to promote HuNoV transmission to the human population. HuNoVs are transmitted through the fecal-oral route, either directly by close contact with infected people or indirectly through consumption of contaminated water and food8.Human noroviruses (HuNoVs), responsible for acute gastroenteritis, are the leading cause of viral foodborne outbreaks in Europe and USA each year9.HuNoVs are non-enveloped viruses (about 40 nm in diameter) with a capsid of icosahedral symmetry, and a single-stranded positive-sense RNA  containing three open reading frames (ORFs). ORF1 encodes non-structural proteins while ORF2 and ORF3 encode for the major (VP1) and the minor (VP2) capsid proteins, respectively. The capsid is composed of 180 copies of VP1 assembled into 90 dimers and few copies of VP2. VP1 is organized into two domains, the shell domain (S) and the protruding domain (P) which is subdivided into P1 and P2 subdomains12. The food vehicles most commonly involved in HuNoV foodborne outbreaks are raw and minimally processed foods . Even though no regulation exists in Europe for the management of HuNoVs, their detection in foodstuffs put on the market is on the rise for the last few years. This trend is related to the publication of the ISO 15216-1 standard in 2013, allowing the genome detection of HuNoVs in vulnerable foodstuffs13. Based on representative sampling, the prevalence of HuNoV genomes ranges from 9.0% to 71.6% in shellfish and from 5.0% to 28.2% in fresh produce14. Although its interest is unquestionable for viral outbreak investigations, the routine use of this ISO standard13 for surveillance in foodstuffs has limitations due to its inability to discriminate infectious from non-infectious particles when HuNoV genomes are detected15. This drawback is related to the fact that HuNoVs cannot be routinely cultivated in laboratories, despite recent advances for the in vitro replication of few strains of HuNoVs using human intestinal enteroids16. From our point of view, it is important to underline that even if the cellular models of HuNoVs were mastered by control laboratories over the coming years, this approach would not be suitable for routine food testing owing to its complexity, high cost, and time-consumption.The occurrence of HuNoV foodborne outbreaks underlines that these viruses can remain infectious for a substantial time in the environmenti) enable better management of the viral hazard and (ii) improve the effectiveness of risk management strategies in industries. To solve this critical issue, numerous strategies have been implemented over the past two decades. Firstly, a number of cultivable enteric viruses were used as HuNoV surrogates to assess the virucidal effect of disinfection treatments applied in food and water industries. Among HuNoV surrogates, feline calicivirus, murine NoV (MNV), Tulane virus (TV), and F-specific RNA bacteriophages (FRNAPH) are commonly used18. VLPs of HuNoVs are also used as well20. They are morphologically and immunologically similar to the native virions22.In this context, it becomes necessary to develop a suitable molecular method for discriminating infectious from non-infectious HuNoVs in food and water in order to (i) the detection of the whole genome23, (ii) the use of enzymatic pre-treatments or intercalating dyes before genome detection25, and (iii) the detection of oxidative damages on the capsid26. The use of receptor binding to assess the capsid integrity is another promising approach29. Several studies have demonstrated that Histo-Blood Group Antigens (HBGAs) display a role of receptors or co-factors for HuNoVs which promote the infection in host cells33. HBGAs are complex carbohydrates linked to glycolipids or glycoproteins found on the red blood cells and mucosal epithelial cells. They can also be present as free antigens in biological fluids such as saliva34. The biosynthesis of HBGAs is governed by the ABO and the fucosyl-transferase FUT2 and FUT3 gene families35. The expression of active or non-active FUT2 defines the secretor or non-secretor status, respectively35.Secondly, several methods have been proposed to discriminate infectious from non-infectious HuNoVs such as The aim of this study was to investigate the ability of GII.4 VLPs to mimic the HuNoV behavior and the suitability of the HBGA-binding assays to avoid the over-estimation of potential infectious HuNoV number given by genome detection during natural ageing at 20 °C over time and heat treatments  by using the most prevalent HuNoV genotype worldwide (i.e. GII.4). First, the capsid integrity of GII.4 VLPs was studied during these inactivation treatments using receptor-binding enzyme linked immunosorbent assays (ELISA) and transmission electronic microscopy (TEM). The influence of the ionic strength of the medium on VLP binding to HBGAs was explored to assess the VLP stability and the nature of interactions between VLPs and HBGAs. Then, the HBGA-binding capacity of intact GII.4 HuNoVs extracted from human stools was evaluated using HBGA-binding followed by RNA amplification by quantitative RT-PCR (RT-qPCR) under the same conditions as those used with GII.4 VLPs. Finally, we compared the representativeness of using VLPs as HuNoV surrogates for a same genotype during inactivation treatments. We also discussed the benefits of using HBGA-binding assays to assess the capsid integrity and the suitability of this strategy to indicate the infectivity of HuNoVs after inactivation treatments.−) , A Lewis-positive (ALe+) , BLe− , BLe+ , and OLe+ . OLe− type was not found in the tested saliva samples. This distribution is consistent with the prevalence of 20% of non-secretor individuals in the Caucasian population37.HBGA types were determined for the 27 human saliva samples by ELISA and were distributed as follows: non-secretor phenotypes , A Lewis-negative (ALe450) values obtained under the same conditions, VLPs recognized all HBGAs from secretor individuals . Conversely, no VLP binding was observed for the five non-secretor saliva samples (positive/negative (P/N) ratio < 2). These results are consistent with published data37. Nevertheless, variable relative affinities were highlighted regarding interactions between VLPs and saliva samples from secretor individuals. This could be explained by the amount of HBGAs in each saliva37. The extent of VLP binding was particularly high for one secretor saliva corresponding to OLe+ type (OD450 = 0.625 at 1 µg/mL of GII.4 VLPs). Consequently, this sole saliva sample was used for all experiments performed in this study to evaluate the effect of natural ageing at 20 °C and heat treatments  on the NoV binding capacity.The binding profile of GII.4 VLPs to all saliva samples was performed using HBGA-binding ELISA  which becomes limiting. In tested conditions for natural ageing and heat treatments (10.0 µg/mL of GII.4 VLPs), the first log10 reduction of HBGA-binding does not have a substantial impact on the OD450 values. Conversely, the first 2 log10 reductions lead to a decrease of OD450 values until the LOD. So, we considered that 2 log10 reductions (T99) were obtained when OD450 values were equal to the LOD.The sensitivity and the linearity of the HBGA-binding ELISA method was investigated using OLe+ saliva is shown in Fig. + saliva was added as a negative control to check the specificity of VLP-HBGA interactions. For all trials, no VLP binding was observed after sodium periodate treatment (OD450 < 0.200) due to the oxidative cleavage of saccharide rings42. According to the OD450 values, a progressive reduction in VLP binding to OLe+ saliva was observed over time until reaching a complete loss of binding capacity after 63 and 70 days under low (10 mM PBS) and high (150 mM PBS) ionic strength conditions, respectively (P/N ratio < 2). From these days, the LOD was reached, consequently, 2 log10 reductions of the OD450 values were obtained. Under the high ionic strength condition, no significant difference was observed after 70 days between untreated and sodium periodate-treated samples . Under the low ionic strength condition, after 63 days, differences between untreated and sodium periodate-treated saliva samples appeared at the limit of significance . Based on the GII.4 VLP binding profiles, natural ageing at 20 °C affects gradually the BP integrity. However, changes in the magnitude of the binding profile were highlighted as a function of the ionic strength. For the first 63 days, the relative affinity of the VLP binding was significantly higher at high ionic strength compared to low ionic strength . Between 70 and 98 days, the VLP binding was low and statistically different between the two ionic strength conditions . After 105 days, no statistically significant difference was observed between the two ionic strength conditions .The effect of natural ageing at 20 °C over 105 days under two ionic strength conditions (10 mM and 150 mM PBS solutions) on GII.4 VLP binding to OLe+ saliva, the ODt/OD0 ratio was determined in 10 mM and 150 mM PBS solutions . For the others points, significant differences appeared . The ionic strength of the medium seems to influence the magnitude of the binding profile but without disturbing the specificity of VLP-HBGA interactions. Consequently, the binding profiles of GII.4 VLPs to OLe+ saliva after inactivation by heating were only performed under the high ionic strength condition.To better evaluate the influence of the ionic strength on GII.4 VLP binding to OLeons Fig. . Similar10 reductions in GII.4 VLP binding to OLe+ saliva after exposure to heat treatments  for 30 min were represented by the log (ODt/OD0) at 450 nm . The behavior of VLP binding was not statistically different for these two heat treatments . Because of the non-linear relationship between OD450 values and GII.4 VLP concentration, it is only possible to consider that after 30 min at 50 °C and 60 °C, the reduction of the VLP binding to OLe+ saliva is lower than 1 log10. Conversely, a significant reduction in the VLP binding was observed at 72 °C for the first 3 min until reaching a complete loss of the OD450nm detection after 10 min . After 2 min at 72 °C, 2 log10 reductions were achieved because OD450 values were equal to the LOD. According to the log10 reduction profiles, temperatures below 60 °C for 30 min affect slightly the BP integrity whereas 72 °C can be considered as a critical temperature which disturbs the BP from the early stage of treatment.The log43. According to these data, TEM results are consistent with the log10 reduction profiles of the VLP binding after heat treatments.TEM observations were performed to evaluate the influence of heat treatments on the overall structure of GII.4 VLPs   suggesting the persistence of potentially intact capsids because the genomes were not accessible to RNase A. Based on linear regression, the T90 was estimated at 294 days. During the whole treatment, a fraction of purified HuNoVs (mainly ranging between the LOD and the limit of quantification (LOQ) of the method) were able to bind to OLe+ saliva treated with sodium periodate, suggesting non-specific interactions between capsids and denatured HBGAs. Concerning GII.4 HuNoV binding to untreated OLe+ saliva, no significant variation was observed for the first 154 days with genome detection rates ranging from 4.50 to 5.20 log10 . A 1.2 log10 reduction in the binding capacity was shown between day 154 and day 294, achieving almost the LOQ determined at 1,000 gc/mL . Based on linear regression over the whole time period, the T90 was estimated at 166 days.The decay curves of the GII.4 HuNoV genomes treated with ribonuclease A (RNase A) and the binding capacity of viral particles to OLe10 reductions in GII.4 HuNoV binding to OLe+ saliva after exposure to heat treatments  for 30 min were determined using the log (C/C0) . Conversely, a statistically significant reduction was shown at 72 °C after 2 min until a complete loss of the genome detection after 3 min . As described for VLPs, temperatures below 60 °C for 30 min affect slightly the BP, whereas 72 °C seems to impair the BP integrity. Interestingly, the behavior of viral capsids of GII.4 VLPs and HuNoVs seems to be similar after heat treatments in the tested conditions  was used to compare the loss of GII.4 VLP and HuNoV binding to OLe+ saliva after natural ageing at 20 °C and heating at 72 °C  and the first time point when the LOD was reached (0.1 µg/mL). For HuNoVs, T99 values were derived from the slope of the linear regression analysis. After natural ageing at 20 °C, the T99 value of HuNoVs (333 days) is at least 4.3 fold higher than that of VLPs (ranging between 63 and 77 days) under the low ionic strength condition, demonstrating a different behavior of GII.4 VLPs and HuNoVs. Considering the similarity of the T99 values of VLPs at 10 mM and 150 mM PBS solutions, this result reinforces the fact that the ionic strength of the medium does not disturb the specificity of VLP-HBGA interactions. After heating at 72 °C, the T99 values were similar for GII.4 HuNoVs and VLPs and estimated at 0.002 days and ranging between 0.001 and 0.002 days, respectively.Because of the absence of linear relationship between OD13 in 2013 because of its inability to provide information about HuNoV infectivity when their genome is detected in vulnerable foodstuffs in prospective approaches such as surveys. Among all those proposed, the strategy based on the receptor/co-factor (HBGAs) binding to the BP to assess the capsid integrity of HuNoVs appeared promising41. The first objective of this study was to evaluate the ability of VLPs to mimic the behavior of HuNoVs for a same genotype (i.e. GII.4) during inactivation treatments. The second objective was to highlight the benefits/limitations of HuNoV binding to human HBGAs to potentially discriminate infectious from non-infectious particles or to prove the capsid integrity.For the past two decades, many studies have tried to develop the most reliable strategy to distinguish infectious from non-infectious HuNoVs in foodstuffs and in the environment with the aim to improve the management of HuNoV hazard but also to better assess the effectiveness of inactivation treatments. These efforts have increased even more since the publication of the ISO 15216-1 standard44. Such interactions are strongly affected by the ionic strength of the medium. The results indicate that VLPs are only weakly affected by the ionic strength range of 10 to 150 mM at pH 7.4 over a long period of time at 20 °C, as observed for other complete viruses46. In the same way, the capsids of GII.4 VLPs and HuNoVs are both disrupted after heating at 72 °C, as it was observed for GII.4 VLPs at 68 °C by Moore et al.41. Ausar et al.19 have also shown that the native quaternary structure of NV is maintained until 60 °C but irreversible capsid disruptions occur above 65 °C. Structural changes in the capsid proteins and/or loss of binding affinity to host cells could occur above a critical temperature, as demonstrated for MS2 phage at 72 °C47.Our study provides new arguments to discuss whether VLPs might be suitable models to represent the behavior of HuNoVs. GII.4 VLPs are only self-assemblies of VP1 that also involve electrostatic or hydrophobic interactions in the environment48. Further research should be conducted to determine the critical contact time in the 60–72 °C temperature range.Depending on the contact time, a critical temperature range of 60 °C to 72 °C leading to the disruption of the capsid may be proposed for VLPs as well as for HuNoVs. Surprisingly, the exact same critical temperature range has also been suggested for MNV43. But the main critical difference may be the stability of VLPs at 20 °C, which is at least 4.3 fold lower than that of HuNoVs. As highlighted by the T99 values, the reduction in VLP binding to human HBGAs was substantially more rapid than for HuNoVs under the same conditions. To our knowledge, no study has described the effect of natural ageing at 20 °C on the BP integrity of HuNoVs. Some differences may also exist at higher temperatures (above 50 °C) but the duration of treatment was not sufficient to draw any conclusions. Our results are consistent with the literature data regarding the effect of heat on GII.4 VLPs28. Hence, lesser stability of VLPs can be noted, at least in the BP region. It may be related to the absence of the viral genome or the VP2 protein which may play a role on HuNoV stability49. Dika et al.50 have shown that MS2 VLPs, which lack the genome and the maturation protein, have a capsid that expresses a different charge from that of the native particle, thus suggesting a different capsid organization. To summarize, GII.4 VLPs may be suitable for mimicking the behavior of HuNoVs with the exception of particle size and stability of BP below 72 °C. Thus, VLPs can be considered as relevant surrogates of HuNoVs when inactivation treatments induce significant changes in the capsid integrity, as described for some disinfectants51 and high-pressure processing29.On the other hand, our study also underlines major behavioral differences between GII.4 VLPs and HuNoVs. One difference is the size of the particles because part of VLPs have a smaller size (15 nm) than that expected for HuNoVs of about 40 nm18. HBGA-binding to the BP provides evidence of the presence of HuNoV capsids as it was confirmed by the lack of action of RNase A on the viral genome, suggesting persistence of potentially intact capsids. Conversely, this approach has several limitations that should be highlighted. The first point concerns methodological aspects. The specificity of HuNoV-HBGA interactions is dependent not only on the HBGA type (as previously demonstrated39), but also on the ionic strength of the media even though it does not disturb these interactions. The impact of the ionic strength is critical when comparing two different environmental media. Different interactions may be explained by the global charge of the viral particle. Considering the isoelectric point of GII.4 VLPs estimated to be between pH 5 and 644, the global charge of the particles at pH 7.4 is negative under the tested conditions. By increasing the ionic strength, the electric double layer of viral particles could be compressed leading to a decrease in repulsive electrostatic interactions and an increase in hydrophobic interactions between VLPs and HBGAs. Dika et al.52 pointed out this complex interplay by showing the adhesion features of different viral surrogates (i.e. FRNAPH) to abiotic surfaces as a function of ionic strength.Bearing in mind the aforementioned limitations of VLPs, it is possible to discuss the relevance of HBGA-binding assays to reduce the over-estimation of the number of potential infectious HuNoVs given by the genome detection method. Substantial advantages of HBGA-binding approach can be suggested. Interactions between HuNoVs and HBGAs are very specific for numerous genotypes of HuNoVs. Although their role is not clearly defined in the host-cell infection process, HuNoV-HBGA interactions could be correlated with infectivity in inactivation assays90 estimated at 166 days) compared to the whole encapsidated HuNoVs at 20 °C (T90 estimated at 294 days). This result indicates that at least two kinds of HuNoV particles are present, both having a capsid (as shown by the resistance to RNase A) and a genome but only one being capable of binding to HBGAs. Even though there is no evidence that the latter are really infectious, the use of HBGA-binding assays may reduce the over-estimation resulting from solely using the genome detection method. Our results are consistent with a previous study describing the persistence of GII HuNoV genomes in drinking water at 21 °C since the T90 was estimated at 298 days using a non-linear model10. Seitz et al.53 have also shown that the NV remained infectious in groundwater at room temperature in the dark for 61 days and the capsid remained intact for at least 427 days. Finally, HuNoV capsids may specifically bind to HBGAs when inactivated particles have a structural integrity of the capsid but an altered genome, or an intact genome with capsid damage(s) located in surrounding regions of the BP.The second point is related to a more rapid reduction in the number of HuNoVs able to bind to human HBGAs . The working suspensions were kept at 4 °C before use.GII.4 VLPs  were kindly provided by the National Reference Center for Gastroenteritis Viruses . Production and purification of GII.4 VLPs were performed using the baculovirus-expressed VP1 system, as described previously55 and kindly provided by the NRCgev . The purification of GII.4 HuNoVs was performed using a method described previously56, with significant modifications. Briefly, 1 g of fecal suspensions of GII.4 HuNoVs was added to 10 mL of 150 mM PBS solution at pH 7.4. Samples were clarified by addition of 1/3 (v/v) of chloroform, vortexed for 60 sec and centrifuged at 2,500 × g for 5 min. Supernatant was submitted to a second clarification step as described previously. The clarified samples were purified by dialysis using Float-A-Lyzer G2 with Biotech Cellulose Ester membranes . The membranes were dialyzed in a tank containing 10 L of 10 mM PBS at 4 °C overnight with gentle stirring. Dialysate samples were recovered and filtered using a 0.22 µm cellulose acetate membrane. After each step, the loss of HuNoV genomes was controlled. The viral genomes from 100 µL of purified HuNoV suspensions were extracted directly using the NucliSENS® easyMAG® kit . The HuNoV genomes were detected using an RNA UltraSense One-Step quantitative RT-PCR system , according to the ISO 15216-1 standard recommendations13. Cq values were determined using CFX Maestro software . Quantification of HuNoV gc was performed using a standard curve of plasmids with a concentration range of 5 to 105 gc/reaction mixture. Until use, the purified HuNoV suspensions were stored at 4 °C in the dark at a final concentration of 2.106 gc/mL.GII.4 HuNoVs from human stools were genotyped37, and informed consent was obtained from all donors. All experiments using saliva were performed in accordance with relevant guidelines and regulations. After harvesting, saliva samples were boiled at 100 °C for 5 min and then centrifuged at 10,000 × g for 5 min. Supernatants were stored at −20 °C until use. The presence of A, B, O blood group antigens and Lewis antigens in saliva samples at a dilution of 1:1,000 in 150 mM PBS was determined by ELISA, as described previously22.Saliva samples from 27 healthy adult volunteers were collected in 50 mL sterile polypropylene tubes. The saliva samples used in this study were described previously following approval by the Nantes University Hospital Review Board (study number BRD02/2-P)6 gc/mL of purified GII.4 HuNoVs in 10 mM PBS solution were exposed to natural ageing at 20 °C. This treatment, corresponding to the application of circadian cycles with a 24 h rotation of natural light and obscurity between October 2018 and July 2019, was evaluated over 105 days and 294 days for VLPs and HuNoVs, respectively. To that end, 20 separate 1 mL aliquots of VLPs and HuNoVs were placed in 2 mL Protein LoBind tubes. These aliquots were derived from a unique stock solution of 20 mL of VLPs and HuNoVs. For each sample, four and two analyses were performed for VLPs and HuNoVs, respectively.One mL of 10 µg/mL purified GII.4 VLPs in 10 mM and 150 mM PBS solutions and 1 mL of 2.105 gc/mL of purified GII.4 HuNoVs in 150 mM PBS solution were separately exposed at 50 °C, 60 °C, and 72 °C for 30 min. Before adding 100 µL of 100 µg/mL GII.4 VLPs or 2.106 gc/mL of GII.4 HuNoVs in borosilicate 4.9 glass tubes , 900 µL of 150 mM PBS solution was pre-warmed in a glycol bath GR150 S5  to minimize the effect of the temperature rise. After heating, treated samples were immediately placed on ice to stop the reaction. The temperature was monitored in the glycol bath but also in a control tube containing 1 mL of 150 mM PBS solution using temperature probes  coupled to a data logger . Experiments were performed in triplicate for VLPs and HuNoVs under the same conditions.One mL of 10 µg/mL purified GII.4 VLPs and 1 mL of 2.1022, with minor adjustments. Briefly, 100 µL of supernatant of saliva samples of known HBGA types were coated in 96-well microtiter plates at a dilution of 1:1,000 in 150 mM PBS at 4 °C overnight. Negative controls were systematically added using saliva-coated wells treated with sodium periodate as described previously42, with slight modifications. After blocking with 200 µL of 5% non-fat dried milk at 37 °C for 2 h, 100 µL of native and treated GII.4 VLPs diluted in PBS solutions was incubated at 37 °C for 1 h. The VLP binding to the saliva samples was detected with anti-GII.4 VLP MAbs kindly provided by the NRCgev  at a 1:10,000 dilution in 150 mM PBS for 1 h at 37 °C, followed by the addition of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  at a dilution of 1:30,000 in 150 mM PBS for 1 h at 37 °C. Between each step, the wells were washed three times with 200 µL of 150 mM PBS containing 0.05% Tween 20 using a microplate washer . The enzyme activity was detected using the TMB kit . After incubation for 15 min at room temperature, the reaction was stopped by adding 100 µL of 1 M sulfuric acid. The OD values of the enzyme activity were read at 450 nm with an enzyme immunoassay spectrum reader . All samples were analyzed in duplicate. Inconsistent results were rejected if the gap between OD450 value duplicates was above 0.1. To give confidence, the P/N ratio was calculated by dividing the mean OD450 values of the sample by the mean OD450 values of the negative control. When P/N ratio was above 2, results were considered positive. When P/N ratio was below 2, results were considered negative57.HBGA-binding ELISA was performed using the method described previously+ saliva sample and various contents of GII.4 VLPs (ranging from 0.05 and 10.0 µg/mL) diluted in 10 mM and 150 mM PBS solutions. The experiments were performed in four replicates.The sensitivity and the linearity of the HBGA-binding ELISA method were determined using the supernatant of OLe56. Briefly, supernatant of OLe+ saliva sample was coated in 96-well microtiter plates as described above. Negative controls were also added using the same saliva sample treated with sodium periodate. After blocking with non-fat dried milk, 100 µL of native and treated GII.4 HuNoV suspensions, previously treated by RNase A58, was incubated at 37 °C for 1 h. Between each step, the wells were washed three times as described above. The bound capsid proteins of HuNoVs were lysed directly in the wells using 150 µL of a buffer containing 5 M of guanidium thiocyanate for 10 min at room temperature. For each condition , 10 wells were pooled on the same 96-well microtiter plate to analyze 1 mL of purified HuNoVs. The viral genomes were purified and detected as described previously. The HuNoV genomes were also detected after RNase A treatment. The LOD was determined at 200 gc/mL and the LOQ was determined at 1,000 gc/mL.This method was performed using a combination of approaches described previouslyGII.4 VLPs exposed to heating were observed by TEM after negative staining with phosphotungstic acid. Briefly, 10 µL of heated GII.4 VLPs were absorbed to carbon coated copper grids by incubating at room temperature for 3 min. The excess was eliminated and the copper grids were negatively stained with 2% phosphotungstic acid by incubating at room temperature for 1 min. The stained copper grids were air dried for 3 min and then observed by TEM.p > 0.05), parametric tests were applied. A paired or unpaired sample Student’s t-test was performed for dependent or independent data following a normal distribution, respectively. Non-parametric tests were used for data sets having a non-normal distribution (p < 0.05). For dependent or independent data with non-normal distribution, a Wilcoxon signed-rank test or a Mann-Whitney U test was applied. For all tests, the significance level was set to 0.05.All statistical analyses were performed using R statistical software (Rx64 v.3.5.3). The Shapiro-Wilk test was performed to check normality of the data with alpha level of 0.05. If data set followed a normal distribution (supplementary info


Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.The traditional Chinese herb  It is considered as the final common stage of numerous acute and chronic diseases, such as myocardial infarction, myocarditis, hypertension and diabetes. Despite advances in pharmacological or non‐pharmacological therapies, heart failure continues to be a global pandemic, directly affecting at least 26 million people globally annually, and its prevalence is increasing.Tumour necrosis factor‐alpha (TNF‐α) is a typical pro‐inflammatory cytokine that has a pivotal role in the pathological processes of heart failure.TNF‐α–induced myocyte apoptosis involves complicated mechanisms.Lonicerae Japonicae Flos, as a traditional medicinal herb known as ‘Jin Yin Hua', is used to treat various diseases including acute fever, headache and acute dysentery.Lonicerae Japonicae Flos, which was used in traditional Chinese formula, had significant benefits in the treatment of heart failure in clinical as well as animal research.Lonicerae Japonicae Flos may be associated with its major active ingredient chlorogenic acid . Earlier studies indicated that CGA has anti‐inflammatory,Therefore, we investigated the effect of CGA on cardiovascular disease in a mouse model of TAC induced heart failure. The results of in vivo experiments show that CGA has cardioprotective effects and inhibited the high expression of TNF‐α in a heart failure mouse model. We used human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to explore whether CGA might have cardioprotective effects against the TNF‐α–induced apoptosis of myocardial cells and elucidate the underlying mechanism(s). Taken together, our findings demonstrated that CGA effectively alleviated TNF‐α overexpression induced injury in a TAC heart failure mouse model and protected hiPSC‐CMs from TNF‐α–induced apoptosis. Moreover, NF‐κB/p65 and JNK signals participated in the inhibitory effects of CGA on cardiomyocyte apoptosis.2All animal experiments were performed in accordance with the ‘National Institute of guiding principles of the care and use of experimental animals' by the China Physiological Society. This study was approved by the Animal Research Ethics Committee of the Beijing University of Chinese Medicine (BUCM‐4‐2018060445‐2049). Male C57BL/6N mice (SCXK(Jing)2016‐0006) were provided by the Beijing Vital River Laboratory Animal Technology Co. Ltd.  and raised in clean conditions at a temperature of 22 ± 1°C with 55 ± 5% humidity and a 12 hours light/dark cycle. After 1 week of adaptation, 27 C57BL/6N mice were randomly divided into four groups: (a) control group(n = 6); (b) sham + double distilled water (DDW)group (n = 6); (c) TAC + DDW group(n = 6): the TAC‐induced mice heart failure model was performed as previously described,2.1Echocardiography was performed four weeks after the TAC operation using a Vevo 2100 ultrasound . The centre‐frequency of the corresponding probe (MS‐400) was 30 MHz. Mouse chest hair was shaved and they were anaesthetized with isoflurane. Then the mice were put in a supine position. Two dimensional views of the left parasternal short axis and left ventricle in the long axis were assessed. In these views, 10 cardiac cycles were noted at every measured point. The bisecting, fractional shortening (FS) and ejection fraction (EF) were calculated though left ventricle (LV) and motion (m)‐mode measurements.2.2The heart tissues of mice were fixed by 4% paraformaldehyde and dehydrated with different grades of ethanol. Then the heart tissues were embedded in paraffin and cut into 3‐μm sections. Tissue sections were deparaffinized by xylene, rehydrated via different grades of ethanol and stained with haematoxylin and eosin. Then digital images were observed under a microscope .2.32O2 was added to the deparaffinized cardiac tissue sections for 20 minutes to reduce endogenous peroxidase activity. The sections were heated in a microwave in retrieval solution for 15 minutes to retrieve antigens. The slides were subsequently incubated in 10% goat serum for 2 hours at room temperature to block non‐specific binding. Then the slides were incubated with TNF‐α primary antibody  at 4°C overnight. The next day, the slides were incubated with secondary antibody  for 30 minutes at room temperature. Finally, they were visualized with 3,3′‐diaminobenzidine tetrahydrochloride (DAB) staining. Three slices from each group were randomly selected and semi‐quantitative image analysis using ImageJ software .Paraffin‐embedded cardiac tissue sections from different groups were deparaffinized by xylene and then rehydrated in different grades of ethanol. Then 3% H2.42. The hiPSCs were assessed for pluripotency  were cultured in PSCeasy hESCs/iPSCs medium . The cells were passaged every 3‐4 days at 80% confluence by ethylenediaminetetraacetic acid (EDTA) (HyClone) at 37°C and 5% COy Figure A.Cardiac differentiation of hiPSCs was performed as previously reportedhiPSC‐CMs were dissociated by CardioEasy CM dissociation enzyme set . Briefly, 1 mL of collagen‐based enzyme I was added to each 6‐well plate for 30 minutes at 37°C, and then 1 mL of trypsin‐based enzyme 2 was added for another 10‐20 minutes. Digested hiPSC‐CMs were seeded onto 6‐well plates by 5% KnockOut™ Serum Replacement (KSR) .For all trials in our study, hiPSC‐CMs were seeded at 80%‐90% density according to our experimental protocol. hiPSC‐CMs were randomly divided into four experimental groups: (a) control group: cardiomyocytes continuously cultured by 5% KSR; (b) TNF‐α group: cardiomyocytes treated with 40 ng/mL TNF‐α  for 24 hours; (c) 0.1 μmol/L CGA treatment group: cardiomyocytes pre‐incubated with 0.1 μmol/L CGA  for 12 hours and then co‐incubated with CGA and TNF‐α for 24 hours; and (d) 1 μmol/L CGA: cardiomyocytes pre‐incubated with 1 µmol/L CGA for 12 hours and then co‐incubated with CGA and TNF‐α for 24 hours. All drugs were dissolved in pre‐warmed 5% KSR and then were added directly to hiPSC‐CMs. In the control groups, equivalent volumes of culture medium were added.2.5Cells were grown on coverslips and then fixed with 4% paraformaldehyde for 30 minutes. Cells were blocked with 5% goat serum and 5% bovine serum albumin (BSA) at room temperature, and then incubated with primary antibodies: rabbit polyclonal anti‐OCT4 , mouse monoclonal anti‐TRA‐1‐81 , mouse monoclonal anticardiac troponin T (cTnT)  and rabbit polyclonal anti‐α‐actinin  at 4°C overnight. After washing with PBS, the slices were incubated with secondary antibodies: chicken antirabbit IgG Alexa Fluor 594 , chicken antimouse IgG Alexa Fluor 488 , goat antirabbit IgG Alexa Fluor 488  and goat antimouse IgG Alexa Fluor 594  at room temperature for 1 hour. Then the slices were rinsed in PBS three times and mounted with DNA‐specific 4′,6‐diamidino‐2‐phenylindole . Fluorescence was evaluated by a confocal laser scanning microscope .2.6Cell viability was examined by a Cell Counting Kit‐8 (CCK‐8)  to detect the cellular dehydrogenase activity of living cells. Briefly, different groups of hiPSC‐CMs in 96‐well gelatin‐coated plates received 10 μL CCK‐8 solution. Then the hiPSC‐CMs were incubated for 1‐4 hours at 37°C. An automatic microplate reader  was used to evaluate the absorbance of each sample at 450 nm.2.75 cells/mL were resuspended in 1 × Binding Buffer and then incubated with 5 μL FITC Annexin V and 5 μL PI for 15 minutes at RT (25°C) in the dark. The hiPSC‐CMs were incubated with 400 μL of 1 × Binding Buffer and analysed by flow cytometry (BD:LSRFortessa).The proportions of apoptotic hiPSC‐CMs in different treatment groups, including control group, TNF‐α group, CGA treatment group, and QNZ  , SB2032.8®, M8650). hiPSC‐CMs were cultured on 96‐well collagen‐coated plates. Different groups of hiPSC‐CMs were washed with cold PBS and incubated with 100 μL JC‐1 work solution for 30 minutes at 37°C in the dark. The image was then scanned using a high‐content analyzer , using the MetaXpress high‐content analysis system  to set the cell parameters and automatically calculate the average fluorescence intensity of the cells.Changes in the mitochondrial membrane potential of different groups of hiPSC‐CMs were evaluated by JC‐1 staining , Bcl2 , caspase‐3 , cleaved caspase‐3 , p65NF‐κB , p‐p65NF‐κB p38 , p‐p38 , ERK1/2 , p‐ERK1/2 (Phospho‐p44/42 MAPK (Erk1/2)XP®rabbit mAb, 4370), JNK  and p‐JNK  band densities were normalized to GAPDH . The data were analysed by ImageJ software.After pre‐treatment with 0.1 μmol/L as well as 1 μmol/L CGA for 12 hours, followed by treatment with 40 ng/mL TNF‐α for 24 hours, the nuclear proteins and all cell lysates of hiPSC‐CMs were extracted using cell protein extraction kits. hiPSC‐CMs were treated with a protease and phosphatase inhibitor cocktail, and then a Bicinchoninic acid disodium (BCA) protein assay was performed to evaluate the protein concentrations  according to the manufacturer's instructions. All samples were subsequently adjusted to the same volume by 2 × 4% sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 minutes. Then equal amounts of protein were resolved by electrophoresis on 10% SDS‐PAGE. The fractionated proteins of all samples were transferred to a polyvinylidene fluoride (PVDF) (MerckMillipore NO:IPVH00010) membrane by electrophoresis for 90 minutes at 300 mA. The membranes were blocked with 5% non‐fat milk powder for 1 hour and incubated with the appropriate concentration of primary antibodies (1:1000) at 4°C overnight. The membranes were cut in pieces according to the band size before incubation with primary antibodies. The membranes were subsequently incubated with an appropriate concentration of secondary antibodies  for 1 hour at room temperature in the dark. The protein bands in the membrane were visualized by a UVP Bio Imaging System. GAPDH proteins were evaluated by the same procedure. Stripping Buffer （CWBIO,Stripping Buffer,CW0056M）was used for the recycle of deproteinized membrane in our experiment. Steps are as follows: Wash membrane with TBST for three times, 10 minutes each. Immerse it into appropriate volume of WB Stripping Buffer, incubate at room temperature for 30 minutes and shake slowly. Take out membrane with tweezer. Elute with TBST once and wash for 5 minutes. Seal with 5% defatted milk powder and incubate with the appropriate concentration of next primary antibodies (1:1000) at 4°C overnight. Finally, the reported data for Bax  at 24 hours and their viability was evaluated by CCK‐8 assay. The results indicated that TNF‐α treatment led to a dose‐dependent reduction in cell viability. Specifically, the viability of hiPSC‐CMs was significantly decreased to approximately 40% after 24 hours and TNF‐α treatment at concentrations up to 40 ng/mL  of the inner mitochondrial membrane, leading to mitochondrial membrane depolarization as well as pro‐apoptotic substance release.3.3To explore the effect of CGA on TNF‐α–induced hiPSC‐CMs apoptosis, we assessed whether CGA has cytotoxic effects on hiPSC‐CMs. No changes in cell viability were observed for hiPSC‐CMs after CGA treatment for 12 hours, even at the maximal dose of CGA Figure A. MoreovCaspase‐3, activated through the proteolytic processing of procaspase‐3 into 12 and 17 kDa subunits, which are early markers of apoptosis, in turn activates the mitochondrial apoptotic pathway. In addition, the apoptotic process includes a variety of regulatory genes, of which, the Bcl2 protein family serves as a crucial regulatory factor in the mitochondrial apoptotic pathway and is comprised of death inhibitors  and death activators . These regulate the release of (pro)apoptotic intermembrane proteins. To further characterize the anti‐apoptotic effect of CGA, the protein expressions of Bcl2, Bax, caspase‐3 and cleaved caspase‐3 were determined by western blot. CGA pre‐treatment significantly suppressed the TNF‐α–induced upregulation of caspase‐3 and cleaved caspase‐3 protein expressions Figure A‐C. Furt3.4NF‐κB plays a vital role in cell apoptosis when exposed to different stimuli, such as TNF‐α. NF‐κB transcription can ultimately result in cell apoptosis. To further investigate whether the NF‐κB signalling pathway regulates the anti‐apoptotic effects of CGA, we treated hiPSC‐CMs with an NF‐κB inhibitor (QNZ) for 4 hours before TNF‐α treatment. Annexin V/PI double staining assays showed that QNZ inhibited TNF‐α–induced hiPSC‐CMs apoptosis Figure A,B. Thes3.5MAPKs, including p38MAPK, ERK1/2 and JNK, are related to the regulation of many cellular events, such as cell proliferation, survival and apoptosis, which are specifically activated by TNF‐α. To explore the molecular mechanism by which the MAPK pathway is involved in the effect of CGA on TNF‐α–induced apoptosis, we separately treated hiPSC‐CMs with a p38MAPK (SB203580) inhibitor, ERK1/2 inhibitor LY3214996) and JNK inhibitor (SP600125) for 4 hours before TNF‐α treatment. Annexin V/PI double staining assays revealed that TNF‐α–induced apoptosis was reversed by the JNK inhibitor (SP600125) but not by the ERK1/2 inhibitor (LY3214996) or p38MAPK (SB203580) inhibitor (Figure  and JNK 4Lonicerae Japonicae Flos, has numerous pharmacological effects, including anti‐inflammatory and anti‐apoptotic effects. In the current study, evidence from our in vivo study demonstrated that CGA has cardioprotective effects and mitigated TNF‐α–induced toxicity in a TAC heart failure mouse model. In the present in vitro study, we further showed that CGA attenuated TNF‐α–induced apoptosis in hiPSC‐CMs via multiple mechanisms. CGA inhibited TNF‐α–induced hiPSC‐CMs apoptosis by controlling the mitochondrial apoptotic pathway. In addition, CGA specifically regulated the activation of NF‐κB/p65 and JNK signals to exert anti‐apoptotic effects.The vital role of TNF‐α in the pathogenesis of heart failure has been widely accepted, and TNF‐α might also provide useful prognostic information as a biomarker for heart failure.High levels of inflammasome TNF‐α might disrupt mitochondrial membrane potential and provoke opening of the mPTP in pathological conditions.−/−mice were resistant to TNF‐α–induced cardiomyopathy and had enhanced cardiac function after myocardial infarction.NF‐κB is a transcription factor that regulates a variety of genes that participate in several crucial physiological processes such as survival, inflammation or immune responses.MAPKs are protein serine/threonine kinases comprising three major signalling pathways, namely ERK1/2, JNK/SAPK and p38MAPK, which participate in cell proliferation, differentiation and apoptosis.In conclusion, this study provides initial evidence that CGA effectively regulates the mitochondrial apoptotic pathway and markedly inhibits the activation of NF‐κB and JNK. These effects might have vital roles in the mechanism of CGA‐mediated myocardial protection Figure . If thesThe authors have no conflict of interest to declare.Lei Tian, Cong‐Ping Su, Qing Wang, Fu‐Jian Wu and Rui Bai contributed to the study design, data collection, study execution and preparation of the manuscript; Shu‐Zhen Guo, Feng Lan, Wen‐Jing Lu and Wei Wang were responsible for conception, design, and financial support; and Hui‐Min Zhang and Jin‐Ying Liu contributed to data analysis. All authors read and approved the final manuscript.The data that support the findings of this study are available from the corresponding author upon reasonable request. Click here for additional data file.


Psittacine beak and feather disease virus (PBFDV) has been reported in many countries, such as Australia, Poland, the United States, South Africa, etc. In this study, the complete genome of a PBFDV isolate was determined and characterized from budgerigars in China. Psittacine beak and feather disease virus (PBFDV) has been reported in many countries, such as Australia, Poland, the United States, South Africa, etc. In this study, the complete genome of a PBFDV isolate was determined and characterized from budgerigars in China. Circovirus in the family Circoviridae. The genome of PBFDV is a 2-kb circular single-stranded DNA molecule possessing two major open reading frames (ORFs) encoding a replication-associated protein (Rep) and the capsid protein (Cap) (Psittacine beak and feather disease virus (PBFDV) is a nonenveloped virus with a diameter of 14 to 16 nm and belongs to the genus in (Cap) , 4.PBFDV is distributed worldwide with a wide range of hosts, including more than 60 species of psittacine birds . PBFDV h6–The genome of the PBFDV-GD isolate was found to be 2,000 bp long, with 52% GC content, and contains two ORFs which encode Rep (289 amino acids [aa]) and Cap (237 aa). Our data showed that PBFDV-GD shares about 77% to 90% similarity with the PBFDV genomes deposited in GenBank from Australia, South Africa, and New Zealand, suggesting that the PBFDV circulating in China is different from those PBFDV strains found in Australia, South Africa, and New Zealand.Given the extensive international bird trade, PBFDV has spread worldwide and poses a threat to psittacine birds. The PBFDV strain identified in the diseased budgerigars indicates that a different lineage of PBFDV is circulating in China. Although PBFDV was identified in the diseased budgerigars, it remains to be confirmed whether PBFDV was the sole cause of the observed disease. Further active surveillance is needed to determine the prevalence of PBFDV in China.MK120438.The complete genome sequence reported here was submitted to GenBank under the accession number