Background: Patients with Crohn’s disease (CD) experience severely reduced quality of life, particularly those who do not respond to conventional therapies. Antitumor necrosis factor (TNF)α is commonly used as first-line therapy; however, many patients remain unresponsive to this treatment, and the identification of response predictors could facilitate the improvement of therapeutic strategies.Methods: We screened Gene Expression Omnibus (GEO) microarray cohorts with different anti-TNFα responses in patients with CD (discovery cohort) and explored the hub genes. The finding was confirmed in independent validation cohorts, and multiple algorithms and in vitro cellular models were performed to further validate the core predictor.Results: We screened four discovery datasets. Differentially expressed genes between anti-TNFα responders and nonresponders were confirmed in each cohort. Gene ontology enrichment revealed that innate immunity was involved in the anti-TNFα response in patients with CD. Prediction analysis of microarrays provided the minimum misclassification of genes, and the constructed network containing the hub genes supported the core status of TLR2. Furthermore, GSEA also supports TLR2 as the core predictor. The top hub genes were then validated in the validation cohort . Furthermore, ROC analyses demonstrated the significant predictive value of TLR2 (AUC: 0.829), TREM1 (AUC: 0.844), and CXCR1 (AUC: 0.841). Moreover, TLR2 expression in monocytes affected the immune–epithelial inflammatory response and epithelial barrier during lipopolysaccharide-induced inflammation (p < 0.05).Conclusion: Bioinformatics and experimental research identified TLR2, TREM1, CXCR1, FPR1, and FPR2 as promising candidates for predicting the anti-TNFα response in patients with Crohn’s disease and especially TLR2 as a core predictor. Inflammatory bowel diseases (IBDs), including ulcerative colitis (UC) and Crohn’s disease (CD), are chronic intestinal inflammatory disorders. Treatment strategies for CD focus on maintaining remission and preventing recurrence. Commonly used medications to treat CD include mesalazine, locally active steroids (such as budesonide), systemic steroids, thiopurines (such as azathioprine and mercaptopurine), methotrexate, and biological therapies  . In partAmong these therapies, anti-TNFα antibodies have been used for more than 26 years and are considered the most reliable biological therapy for the treatment of CD . Loss ofAccordingly, in this study, we aimed to identify novel predictors of anti-TNFα primary nonresponse in CD using independent bioinformatics analyses of multiple cohorts and experimental validation in cell models.http://www.ncbi.nlm.nih.gov/geo/) database for Crohn’s disease data with the following inclusion criteria: 1) key words“(Crohn OR CD OR IBD OR inflammatory bowel disease) AND ”; 2) Homo sapiens; 3) expression profiling by array OR high throughput sequencing; 4) submitted date <01/10/2022; and 5) datasets or series. After that, we reviewed every data under the following exclusive criteria: 1) recruited Crohn patients<6; 2) no Crohn’s anti-TNFα therapy record; 3) no anti-TNF response record; 4) responder or nonresponders <3; and 5) no clear endoscopy evaluations. For discovery cohorts, we tried to choose homogeneous data. For example, all the anti-TNFα therapy response outcomes were based on the endoscopy measure after therapy. Since adequate discovery data of candidates were lacking, we chose mixed Crohn’s disease majority’s data without a clear subtype for validation cohort’s selection in a slightly relaxed range.We searched the Gene Expression Omnibus  were further analyzed using Gene Ontology (GO) enrichment in the Metascape database (https://metascape.org) (https://string-db.org/)  . The comape.org) . Furtherdb.org/) . To idendb.org/) .We further performed prediction analysis of microarrays (PAM), a type of classification based on nearest centroids. The PAM R package provides an accurate predictor that may outperform much more complicated methods . Owing tThe top differentially expressed genes were further assessed in the discovery cohort, and the top five differentially expressed genes were further validated in the discovery cohort of responders and nonresponders with IBDs . Receiver operating characteristic (ROC) curve analysis was then performed to evaluate the predictive values of these top genes in response to anti-TNFα therapy in patients with CD.https://singlecell.broadinstitute.org/single_cell) held by the Broad Institute of MIT and Harvard. The Crohn’s disease single-cell data include 27 volunteers’ 201,883 single-cell transcriptomes. Then we obtained the cell types’ tSNE (t-Distributed Stochastic Neighbor Embedding) map and TLR2 expression tSNE map on the interactive visualization web tools.We explored Crohn’s disease data entitled “PREDICT 2021 paper: CD” on the single-cell portal  or infliximab , and Cell Counting Kit-8 (CCK8) assays  were performed to determine the appropriate concentration to use in subsequent experiments. THP1 cells were then transfected with a Toll-like receptor (TLR) 2 overexpression vector or TLR2 small interfering RNA (siRNA) using Lipo 2000 . Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were further performed to validate the overexpression and knockdown of TLR2 in THP1 cells . The TLR2 inhibitor C29  was added to block TLR2 overexpression in the coculture system. Caco2 cells were treated with 100 ng/ml LPS for 24 h and then with 100 μg/ml infliximab for another 24 h in the lower chambers of 6-well Transwell plates. The cells were then cocultured with TLR2-overexpressing THP1 cells cultured with 50 μM C29. Total RNA from the Caco2 cells was obtained after 24 h of coculture, and epithelial inflammation and tight junctions  were assessed using qPCR. RT-qPCR and Western blotting were carried out as described previously  and colonic epithelial cells  to validate the bioinformatics results. Caco2 and THP1 cells were obtained from the Cancer Research Institute of Central South University, China. THP1 and Caco2 cells were cultured in RPMI-1640 or MEN medium  supplemented with 10% fetal bovine serum  at 37°C in an atmosphere containing 5% COeviously . All prip values less than 0.05 were considered significant.Statistical analysis of bioinformatics data was performed using the R package. One-way analysis of variance (ANOVA) was used to compare multiple randomized sets of data. Unpaired t-tests were used to compare double randomized data. Results with A total of 30 data were included in the selection of further analysis. After carefully checking every data by the exclusion criteria, 25 data were excluded. Due to the limited eligible data for analysis, we choose total Crohn’s disease data  as discovery cohorts, which will help in revealing a reliable differential network between responders and nonresponders. For the validation cohort, we choose another mixed IBD data (GSE159034) dominated by Crohn’s disease patients without clear subtype information. The detailed information on included data is listed in p < 0.05 (CXCR) 1, CXCR2, TLR2, and triggering receptor expressed on myeloid cells (TREM1) were common differentially expressed genes. Furthermore, GO enrichment , and FPR2 were the top five differential genes.From the analysis of differentially expressed genes between responders and nonresponders, due to the insufficient significant differential genes under the adj p-value, we identified 4690, 3422, 1020, and 2811 differentially expressed genes in GSE16879, GSE52746, GSE107865, and GSE111761 datasets, respectively, under p < 0.05 . Some ofp < 0.05  by a Venp < 0.05 . C-X-C mrichment  of all drichment . STRING richment , includirichment . The degrichment  revealedMMP) 1, MMP3, regulator of G protein signaling 2, and alpha-2-macroglobulin, in our predictive model , representing one of the top three enrichment gene sets  . Unlike ene sets . Importagene set . Howeverp < 0.05 or 0.001). Thus, we hypothesized that TLR2 in immune cells may have biological effects on the colonic response to infliximab. Moreover, after tSNE reanalysis of TLR2 expression and cell types in public single-cell sequencing data of Crohn’s disease patients. Data from the Single Cell Portal support the major contributor of TLR2 expression in colonic tissues is the mononuclear phagocyte system, and lead to our THP-1 selection in the coculture validation : 0.829, p = 0.001, 95% confidence interval (CI): 0.680–0.979; TREM1. AUC: 0.844, p < 0.001, 95% CI: 0.716–0.873; CXCR1, AUC: 0.841, p < 0.001, 95% CI: 0.708–0.974; FPR1, AUC: 0.894, p < 0.001, 95% CI: 0.778–1.0; FPR2, AUC: 0.824, p < 0.001, 95% CI: 0.678–0.969].To validate and evaluate these newly built predictors’ value, we first displayed the basic expression profiles of top differential genes using a scatter plot for GSE16879 . The expr groups . ROC anaTLR2 expression on the colonic response to anti-TNFα therapy ] and improves the reduced epithelial permeability  in the LPS-induced model. Furthermore, the overexpression of TLR2 in THP1 cells amplified colonic epithelial inflammation, as measured by IL-1β, IL-6, and MCP1 (p < 0.05 or 0.001), and reduced the expression of tight junction proteins, such as occludin and ZO-1, even in the context of infliximab rescue. The TLR2 inhibitor C29 alleviated TLR2 overexpression-induced amplification of epithelial inflammation and impaired permeability during the infliximab therapy. By contrast, TLR2 knockdown showed an effective response to infliximab rescue after the LPS induced inflammation. (p < 0.05 or 0.001).A coculture system of THP1 and Caco2 cells was established to elucidate the effects of  therapy . Efficieng siRNA , and we blotting . CCK8 asblotting . ImportaSaccharomyces cerevisiae antibodies, fecal calprotectin, fecal lactoferrin, genetic polymorphisms, and prior anti-TNF therapy), and immune–epithelial biomarkers   . Mucosald S100A8 . Furtherd S100A8 . Neverthsponders . In a stsponders . In anotsponders . Althougsponders . TherefoTLR4, TLR2, TLR1, TLR8, CCR1, CD86, CCL4, HCK, and FCGR2A) were identified, and the pathway enrichment highlighted the interaction between the TLR pathway and FcγR signaling. Genes such as TLR4, TLR8, and CCL4 have also shown predictive value in nonresponsive intestinal tissue  are associated with the primary nonresponse to anti-TNFα therapy in patients with CD  are associated with the primary response  in pediatric patients with IBD have been shown to be promising markers for predicting the anti-TNF therapy response. Furthermore, TLR2 variants  have been shown to be associated with the response to anti-TNF treatment in patients with psoriasis (TNFα rs1799724 and TLR9 rs187084)  , we obse therapy . TLR2 isoutcomes . TLR2 cafunction . Higher esponses  and is lTLR2 expression in coculture experiments, and our reanalysis of single-cell data of Crohn’s disease supports that the mononuclear phagocyte system is the major contributor of TLR2 expression in the colon and leads to the THP-1 selection in the coculture validation. In our coculture system, higher TLR2 expression in monocytes amplified the inflammatory response and caused a worse response to infliximab treatment. Moreover, higher levels of inflammatory cytokines and reduced tight junction protein expression were associated with nonresponse. We also found that the TLR2 inhibitor C29 rescued the nonresponse outcome and therefore linked TLR2 expression to the response to anti-TNFα treatment. Similarly, higher colonic TLR2 expression results in poor survival outcomes in patients with colorectal cancer (TLR2. Nevertheless, the baseline disease status in responders and nonresponders with CD is different. Responders have a higher Pediatric Crohn’s Disease Activity Index than nonresponders prior to therapy, indicating more severe inflammation and elevated TLR2 in responders (TLR2-knockout mice exhibit more severe colitis than wild-type mice (TLR2 expression may result in poor outcomes, whereas low TLR2 expression may provide some health benefits; this phenomenon may enable the identification of the optimal cutoff value for the application of TLR2 as a biomarker.In this study, GSEA identified the monocyte-associated pathway as a top enriched pathway, and TLR2 is highly related to the activation of monocytes and the inflammatory reaction . At the l cancer . Howeversponders . Additioype mice . High TLTREM1 gene in patients with CD. Nonresponse to anti-TNFα therapy in patients with CD is associated with higher blood TREM1 levels (TREM1 levels predict better anti-TNFα response in patients with IBD (TREM1. However, another study indicated that TREM1 was upregulated in nonresponders but that low blood TREM1 levels may predict poor anti-TNFα outcomes (TREM1 cutoff value should be determined in additional studies (TLR2 expression in CD should be further evaluated in a larger prospective cohort. In addition, there are several factors supporting TLR2 as a priority predictor of anti-TNFα response. First, we found TLR2 and TREM1 as core differential genes, but TLR2 is more robust after the PAM results’ interaction. Second, TLR2 is proved to be a more robust predictor than TREM1 among the discovery cohort. Third, previous ideal IBD anti-TNFα predictors like TREM1 are also questioned as not a robust predictor of clinical or endoscopic outcomes following adalimumab treatment in patients with UC or CD (TLR2 will provide a candidate of predictors in the Crohn’s disease anti-TNFα therapy. We support the subgroup predictors’ classification in the management of IBD. However, we will not be surprised to see the future applicability in other autoimmune diseases like rheumatoid arthritis because predictors may well indicate the TNFα’s origin and release dynamics.Interestingly, such discrepancies have also been observed in studies of the 1 levels , whereaswith IBD . These foutcomes . Howeveroutcomes . A publi studies . We alsoUC or CD .. Up to TLR2 is a promising predictor for the response to anti-TNFα therapy in patients with CD. Future research should focus on determining the optimal cutoff value for TLR2 expression in a larger cohort of patients with CD.However, there were some limitations to this study as well, such as the number of patients included in the study was small. Because of the coronavirus disease 2019 pandemic and the limited number of patients taking anti-TNFα therapy, frequent loss to follow-up, and the relatively high cost of biologics in China , we failTLR2, TREM1, CXCR1, FPR1, and FPR2 as promising candidates for predicting response to anti-TNFα therapy in patients with CD. Our findings provided evidence that TLR2 could be a potential predictor for the anti-TNFα nonresponse in patients with CD, which could facilitate the establishment of novel approaches to alleviate disease burden.In conclusion, bioinformatics analysis and experimental validation showed that innate immunity played critical roles in the response of patients with CD to anti-TNFα therapy. Moreover, we identified


Dry-off is a typical management practice, but research on the effects of dry-off on feeding behavior in high-yielding cows is limited. The present study investigated the effects of 2 diet energy densities: lactation diet  versus a lactation diet diluted with 30% straw , both offered ad libitum, and 2 daily milking frequencies (2× vs. 1×) during the 7 d before dry-off day (d 0), and the effects of an injection of either a dopamine agonist  or saline (SAL) following the last milking on d 0 , on automatically monitored feeding behavior and rumination time in 119 clinically healthy, loose-housed, pregnant, lactating Holstein cows during the week before and after d 0. From d 0, all cows were fed the same dry-cow diet ad libitum. Data were analyzed in R using mixed-effects models. Over the days before d 0, REDU cows spent 30% more time feeding at a 50% lower feeding rate, visited both assigned and unassigned feed bins more frequently, and spent more time ruminating than NORM cows. No clear behavioral effects of reduced milking frequency were found. Within 24 h following injection, CAB cows spent approximately 40% less time feeding at a lower feeding rate, visited their feed bin 28% less often, and spent 40% less time ruminating than SAL cows, irrespective of treatment before dry-off. The current study demonstrates that reducing diet energy density for 1 wk before dry-off led to clear behavioral changes in high-yielding cows. Administering CAB after the last milking induced decreased feeding behavior lasting approximately 24 h, indicating collateral effects other than reduced prolactin secretion. In most commercial dairy farms, milk production is artificially stopped approximately 2 mo before the expected calving date in a process called “drying-off” . The dryNEB) before dry-off  in accordance with the Danish Ministry of Environment and Food Act No. 474 . The experimental work was conducted according to Good Clinical Practice Guideline VICH GL19 , and theAMS; DeLaval AB).The experiment was described in detail by NORM; REDU; Between d −14 and −8, cows were fed a partially mixed normal lactation diet NORM;  in automCAB) or saline (SAL). After dry-off, all experimental cows were fed a dry-cow diet ad libitum . A visit to a feed bin was defined from the moment the cow put her head inside the feed bin while placing her collar past the feed bin gate and opening the gate until she removed her head from inside the feed bin area. At each visit to the assigned feed bin, visit duration  and amount of feed consumed  were recorded. The DMI of each feed was calculated by multiplying the as-fed intake from each feed bin visit by the respective DM percentage . SubsequRumination time was recorded from d −14 to 6 using the sensor Heatime Ruminact (SCR Engineers Ltd.) attached to the left side of the neck collar of each experimental cow as validated by glmmTMB v.1.0.2.1; emmeans v.1.5.0; P-value < 0.1 are presented and discussed herein.Data were analyzed in R v.4.0.3 . The bas21 = 0.4, P = 0.544) or in minutes per kilogram of DM . Furthermore, absolute rumination time was analyzed at the 2-h level for 48 h from 1000 h on d −1 to 0959 h on d 1. The analysis was performed with a Gaussian linear mixed-effects model including sensor type, type of injection, 2-h interval, and 2-way interaction between type of injection and 2-h interval as fixed effects. Batch and cow were included as random effects. The average baseline over d −6, −4, and −3 was included as covariate. The model contained a continuous-time autoregressive covariance structure of order 1 to account for repeated measures of each cow over 2-h intervals.Feeding time and feeding rate were analyzed with Gaussian linear mixed-effects models. Frequency of visits and frequency of visit attempts were analyzed with negative bionomial generalized linear mixed-effects models with a log-link function. The fit of the visit attempts model was not initially confirmed. Therefore, the 4-way interaction was removed. Afterward, the fit of the reduced model was reassessed and not rejected. Rumination time data were analyzed with Gaussian linear mixed-effects modeling as described above, including sensor type  as fixed effect. No effect of sensor type was detected on rumination time data, in either minutes/day , had a 50% lower feeding rate , visited their feed bin approximately 30% more often , and attempted to visit unassigned feed bins more than twice as often  compared with NORM cows. Additionally, REDU cows spent, on average, more time ruminating than NORM cows, both in absolute terms  and per DMI .For variables recorded in the baseline period, no differences among treatments were observed. Over d −6, −4, −3 and −1, REDU cows spent more time feeding , displayed lower feeding rate on d 0 , and visited their feed bin approximately 28% less often on d 0  than SAL cows. Additionally, on d 0, CAB cows spent over 40% less time ruminating than SAL cows , with an average nadir of 7 min/2-h interval at 3 to 5 h after injection . On d 1 and 5, 1×-CAB cows (45 ± 2.1 and 48 ± 2.0 min/kg of DM) had the longest rumination time per DMI, followed by 2×-CAB cows (44 ± 2.1 and 41 ± 2.1 min/kg of DM), 2×-SAL cows (42 ± 2.1 and 41 ± 2.1 min/kg of DM), and 1×-SAL cows .Cows administered CAB spent less time feeding on d 0 (122 ± 6.0 vs. 197 ± 5.9 min/d) and 1 (188 ± 6.0 vs. 203 ± 5.9 min/d; χThe current study demonstrated that typical dry-off management practices, especially reducing diet energy concentration through straw dilution of the lactation diet for a week before last milking, can induce changes in feeding behavior. The increased feeding time and decreased feeding rate in cows fed the REDU diet before dry-off was consistent with previous studies examining the effects of low energy diet providing during dry-off e.g., . FurtherAt the dry-off day, cows that were abruptly dried off yielded around 25 kg/d of milk, whereas cows gradually dried off by either feeding REDU diet or being milked 1× yielded approximately 30% less, and cows gradually dried off by feeding the REDU diet and being milked 1× yielded approximately 45% less . HoweverThe administration of CAB to inhibit prolactin secretion collaterally induced a marked reduction in DMI  as well We encourage future studies examining the effects of these dry-off treatments on cows' feeding behavior to include extra feeding behavior measures, which could not be recorded in the present study. For instance, knowledge about the particle size distribution of feed left in the feed bins would have allowed for investigation of feed sorting, which positively correlates with feeding time and negatively correlates with feeding rate . AdditioThe current study demonstrated that diluting the lactation diet with straw in the week before dry-off, leading to changes in physical characteristics of the diet and reduction of diet energy concentration, clearly induced changes in feeding behavior in high-yielding cows. No clear feeding behavioral effects of daily milking frequency were seen during the experimental period. Cabergoline induced decreased feeding behavior lasting approximately 24 h following administration, indicating collateral effects other than reduced prolactin secretion.


The purpose of this study was to identify natural molecules able to alleviate ER stress and inhibit cardiomyocyte cell death through SIRT1 activation. Several phenolic compounds, abundant in vegetables, fruits, cereals, wine, and tea, were reported to stimulate the deacetylase activity of SIRT1. Here, we evaluated the cardioprotective effect of ten of these phenolic compounds against severe ER stress using cardiomyoblast cells and mice. Among the molecules tested, we showed that ferulic acid, pterostilbene, and tyrosol significantly protect cardiomyocytes and mice heart from cardiac alterations induced by severe ER stress. By studying the mechanisms involved, we showed that the activation of the PERK/eIF2α/ATF4/CHOP pathway of the UPR was reduced by ferulic acid, pterostilbene, and tyrosol under ER stress conditions, leading to a reduction in cardiomyocyte apoptosis. The protection afforded by these phenolic compounds was not directly related to their antioxidant activity but rather to their ability to increase SIRT1-mediated deacetylation of eIF2α. Taken together, our results suggest that ferulic acid, pterostilbene, and tyrosol are promising molecules to activate SIRT1 to protect the heart from the adverse effects of ER stress.Disturbances in Endoplasmic Reticulum (ER) homeostasis induce ER stress, which has been involved in the development and progression of various heart diseases, including arrhythmias, cardiac hypertrophy, ischemic heart diseases, dilated cardiomyopathy, and heart failure. A mild-to-moderate ER stress is considered beneficial and adaptative for heart functioning by engaging the pro-survival unfolded protein response (UPR) to restore normal ER function. By contrast, a severe or prolonged ER stress is detrimental by promoting cardiomyocyte apoptosis through hyperactivation of the UPR pathways. Previously, we have demonstrated that the NAD XBP1, ATF4), and the increase in the expression of genes encoding ER chaperones, proteins involved in the ER-associated protein degradation pathway (ERAD), and proteins of the autophagy pathway. However, when the ER is under chronic or excessive stress and the UPR fails to restore ER homeostasis, apoptosis is triggered to eliminate damaged cells. This can be correlated with the pathogenesis of a wide range of human diseases [The rough endoplasmic reticulum (ER) is a central organelle involved in the synthesis, folding, and quality control of transmembrane and secretory proteins. The impairment of ER homeostasis, caused by physiological or pathological conditions such as calcium homeostasis perturbations or oxidative stress, results in the accumulation of unfolded and misfolded proteins inside the ER lumen, a condition known as ER stress . With thdiseases .Numerous animal models and increasing human studies have shown that ER stress is involved in the development and progression of a large number of heart diseases including ischemic heart diseases, arrhythmias, cardiac hypertrophy, and heart failure . Current+-dependent deacetylase Sirtuin 1 (SIRT1) has been shown to be cardioprotective in response to a number of cardiac insults including ischemia, pressure overload, cardiac hypertrophy, or aging [+ axis and to protect cells from drug-induced cytotoxicity [As a stress sensor, the NADor aging . The proor aging . Recentlor aging , and proor aging . Therefotoxicity ,13. Amontoxicity . Subsequtoxicity ,18,19,20toxicity ,22,23.The current study was aimed at identifying bioactive natural molecules with the ability to alleviate ER stress and inhibit cardiomyocyte cell death. The ten phenolic compounds resveratrol, berberine, butein, catechin, ferulic acid, isoliquiritigenin, malvidin, piceatannol, pterostilbene, and tyrosol were demonstrated to protect the heart from various stresses and are independently suggested to activate SIRT1, although this is seen primarily in non-cardiovascular cell models  17,20,2,26,27,28To eliminate the potential toxic effects of the ten selected phenolic compounds , we firsTo evaluate the effect of the phenolic compounds on ER-stress-induced cell death in cardiomyocytes, adult rat ventricular myocytes (ARVM) were pre-treated or not with the different molecules for 1 h, then severe ER stress was induced with the well-known ER stressor tunicamycin . The percentage of dead cells was measured by fluorescence microscopy using FDA viability assay. As expected, after 24 h of treatment with TN, the percentage of dead cells significantly increased compared to the control . The phyTo investigate the effects of ferulic acid, pterostilbene, and tyrosol on cardiac function in the context of severe ER stress in vivo, adult male C57BL6/J mice were injected for 5 days with one of the phenolic compounds . ER stress was induced on the 3rd day by TN injection , and the cardiac function was evaluated by echocardiography on day 5, 2 h after the last phytochemical injection A,B. MiceTo further investigate the molecular mechanisms involved in the cardioprotective effect of ferulic acid, pterostilbene, and tyrosol, the cardiac H9c2 cell line was used. H9c2 cells were pre-treated for 1 h with or without ferulic acid (5 µM), pterostilbene (0.5 µM), or tyrosol (1 µM) before induction of severe ER stress by TN. After 48 h, cell death was analyzed by flow cytometry using FDA assay. As expected, TN increased H9c2 cell death (47.8 ± 3.8%) in comparison with the controls (11.8 ± 2%) A–C. Feru2O2 were analyzed in H9c2 cells by flow cytometry using MitoSOX™ Red and H2DFCDA probes, respectively. After 24 h of treatment, TN did not significantly modify the percentage of cells with an elevated content of mitochondrial superoxide anions (2O2 (2O2 (250 µM), a well-known generator of reactive oxygen species. As shown in 2O2 increased both ROS production  every day during 5 days. At day 3, mice were given i.p. injection of tunicamycin (TN) 2 mg/kg body weight or the equivalent volume of vehicle (Dextrose 150 mM) to induce severe ER stress as previously reported . EchocarTransthoracic echocardiography was performed using a 15 MHz transducer under 2% isoflurane gas anesthesia. Two-dimensional-guided (2D) M-mode echocardiography was used to determine the left ventricular chamber volume at systole and diastole and contractile parameters such as ejection fraction (EF).2/95% air. Severe ER stress was induced by TN treatment at 10 µg/mL . EX-527 Sirtuin-1 inhibitor was used at 5 µM . Its IC50 value was determined using the in vitro Fluor de Lys deacetylation assay and a purified human SIRT1 range from 38 to 150 nM. Berberine, Catechin, Ferulic Acid, Malvidin, Tyrosol, and Resveratrol were purchased from Merck-Sigma . Butein, Isoliquiritigenin, Piceatannol, and Pterostilbene were purchased from Cayman . All phenolic phytochemicals were diluted in DMSO.The H9c2 rat cardiomyoblast cell line was purchased from ATCC . Cells were cultured in DMEM medium with 1 mM pyruvate and 4.5 g/L glucose  and supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin , and 10% FBS  at 37 °C under 5% CO2−. Once in the mitochondria, MitoSOX™ Red reagent is oxidized by superoxide anions and exhibits red fluorescence detected by flow cytometry. CM-H2DCFHDA was used to measure cellular H2O2 levels. After 24 h of treatment, cells were centrifuged, washed with PBS, and incubated with 2 μM of MitoSOX™ Red for 10 min or with 1 µM CM-H2DCFHDA for 15 min at 37 °C and then analyzed by flow cytometry. A FC500 flow cytometer  was used for the analyses.Cell viability was assessed by using a fluorescein diacetate  fluorescent probe. After 48 h of treatment, cells were trypsinized and incubated for 10 min at 37 °C with 0.2 µg/mL FDA and analyzed by flow cytometry. Cell size was assessed based on FSC and SSC patterns. Plasma membrane integrity was estimated by adding 10 µg/mL of propidium iodide (PI)  just before flow cytometric analysis. DNA fragmentation (Sub G1 population) was analyzed by measuring DNA content. Briefly, after 48 h of treatment, cells were trypsinized and fixed in 70% ethanol during 24 h at −20 °C. Cells were then incubated overnight at 4 °C in dark with 50 µg/mL PI and 250 µg/mL RNAse A  and analyzed by flow cytometry. MitoSOX™ Red was used to measure the relative levels of the mitochondrial superoxide anion O2+-free Ringer solution. All animal experimental procedures were approved by the animal ethics committee of Paris-Saclay University, authorized by the French government (Authorization Number: B9201901), and complied with directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Hearts were then perfused retrogradely through the ascending aorta according to the Langendorff method and washed for 5 min at a constant flow of 6 mL/min with Ca2+-free Ringer solution at 37 °C with oxygen. Enzymatic dissociation was performed by perfusing during 45 min at a constant flow of 4 mL/min with Ringer solution with 1 mg/mL collagenase A  and 20 µM of free Ca2+ adjusted following 300 µM EGTA at 37 °C with oxygen. Ventricles were then separated from atria, finely chopped, and gently agitated to dissociate individual cells. Cell suspension was then filtered and the cells were allowed to settle down. The supernatant was discarded to eliminate dead cells and debris and resuspended during 5 min in Ca2+-free Ringer solution containing a progressively increasing calcium concentration. Cells were plated into dishes coated with laminin on DMEM medium with 4.5 g/L glucose and 1 mM pyruvate supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin. After 2 h of plating, the medium was changed and cardiomyocytes were treated for 24 h.Adult Wistar male rats (250–300 g) were anesthetized by intraperitoneal injection of Dolethal (150 mg/kg), and the beating hearts were rapidly collected after dissection and put in a cold CaTo analyze cell death, adult rat ventricular myocytes (ARVM) were stained with 0.2 µg/mL of FDA  for 10 min. FDA-negative cardiomyocytes were considered as dead cells. More than 1000 cells were counted for each condition on a Leica fluorescence microscope , and results were expressed as a percentage of FDA-negative cells.®  according to the manufacturer’s instructions. Total RNA (1 µg) was reverse transcribed using a BioRad iScript reverse transcription kit. Real-time PCR was performed using the SYBR® green method on the CFX96 TouchTM Real-Time PCR Detection system  from 2.5 ng cDNA. mRNA levels for all target genes were normalized to Ywhaz and RPL32. The PCR primers were obtained from Eurofins and are listed in RNA was isolated from cultured cells treated during 6 h with or without TN and phytochemicals, using TRI ReagentCells were lysed in a RIPA lysis buffer  supplemented with cocktails of proteases , phosphatases , and deacetylase inhibitors  for 30 min at 4 °C. Proteins (25 µg) were separated by 4–20% Tris-Glycine gel electrophoresis and transferred to PVDF membranes (Fisher scientific). Membranes were incubated overnight at 4 °C with the following antibodies: anti-GRP78, anti-GRP94, and anti-eIF2α from Cell Signaling Technology , anti-CHOP from Genetex (GTX11419), anti-Phospho-eIF2α from Invitrogen (44728G), and anti-Actin from Santa Cruz (sc-47778). Proteins were detected on an iBright FL1000 Imager  by using the ECL method according to the manufacturer’s instructions . To determine the working range of each antibody, different protein quantities, antibody concentrations, and times of exposure were tested. To calculate the relative density (RD), ImageJ software was used, and the intensity of each protein was normalized to actin. The data obtained were then expressed as the ratio of the intensity of the protein in treated cells to that of the corresponding protein in untreated cells.4 cells per well, and were transfected with 8 pmol of either non-targeting control siRNA or SIRT1 siRNA according to the manufacturer’s instructions. 24 h after transfection, the medium was removed, and cells were pre-treated or not for 1 h with ferulic acid, pterostilbene, or tyrosol and then incubated for 48 h with TN before analysis.SIRT1 siRNA (sc-108043) and non-targeting control siRNA were purchased from Santa Cruz (sc-37007). H9c2 cells were plated in 24-well plates overnight at 35 × 10To analyze the acetylation level of eIF2α, H9c2 cells were lysed in a denaturing IP buffer  supplemented with a cocktail of inhibitors of proteases, phosphatases, and deacetylases. Lysates were heated at 90 °C for 5 min and then diluted in a non-denaturing buffer to trap SDS , supplemented with protease, phosphatase, and deacetylase inhibitor cocktails. eIF2α was immunoprecipitated using an anti-eIF2α antibody coated on G magnetic beads . Immunoprecipitated proteins were run on an SDS-PAGE gel and were revealed with anti-eIF2α or anti-acetylated lysine antibodies .p-value was * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control or siCTL; # p < 0.05, ## p < 0.01, ### p < 0.001 versus TN or siCTL + TN; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 versus phytochemical + TN; § p < 0.05, §§ p < 0.01, §§§ p < 0.001 versus siSIRT1; £ p < 0.05, ££ p < 0.01, £££ p < 0.001 versus siCTL + phytochemical + TN; ¤ p < 0.05, ¤¤ p < 0.01, ¤¤¤ p < 0.001 versus H2O2.All data were presented as means ± S.E.M. Data were analyzed using Sigma Stat . One-way ANOVA was used to assess differences among groups, followed by Newman–Keuls post hoc tests. Differences between groups were considered significant if the Interestingly, the three phenolic compounds identified in our study as inhibitors of ER-stress-induced cardiac cell death are widely available in our diet. Indeed, ferulic acid is the most abundant polyphenol found in cereals and some fruits , pterost


Escherichia coli (STEC) is one of the leading causes of foodborne illnesses in North America and can lead to severe symptoms, with increased fatality risk for young children. While E. coli O157:H7 remains the dominant STEC serotype associated with foodborne outbreaks, there has been an increasing number of non-O157 STEC outbreaks in recent years. For the food industry, lytic bacteriophages offer an organic, self-limiting alternative to pathogen reduction—one that could replace or reduce the use of chemical and physical food processing methods. From EHEC-enriched sewage, we isolated a novel bacteriophage, vB_EcoM-4HA13 (4HA13). Phenotypic characterizations revealed 4HA13 to possess a myoviral morphotype, with a high specificity to non-motile O111 serotype, and a long latent period (90 min). Through genomic analyses, this 52,401-bp dsDNA phage was found to contain 81 CDS, but no detectable presence of antibiotic resistance, integrase, or virulence genes. A BLASTn search for each of the identified 81 CDS yielded homologues with low levels of similarity. Comparison of RNA polymerase and terminase large subunit amino acid sequences led to the proposal and acceptance of a new bacteriophage family, Chaseviridae, with 4HA13 representing a new species and genus. The discovery of this phage has broadened our current knowledge of bacteriophage diversity.Shiga toxin-producing  Escherichia coli strain, regardless of their microorganism of study. This bacterium’s speed of replication and robustness of growth are just two of the many traits that have made E. coli the darling of the molecular biology world. Ironically, these same traits are partly why their pathogenic brethren remains a continuous source of consternation for safe food production. It is unlikely an over-exaggeration that every microbiology laboratory has at least one E. coli groups, strains capable of producing the Shiga toxin (STEC) are of the most concern to food safety. While cases with less virulent E. coli are usually self-limiting, resolving within a matter of days, patients afflicted with STEC can exhibit bloody diarrhea, and 5–10% of cases can be complicated by hemolytic uremic syndrome, with possibility of long-term kidney damage and death [Of the six pathogenic nd death ,3,4. Natnd death ,7.E. coli-driven foodborne illnesses. However, non-O157 cases have been increasing, largely due to improved laboratory testing and reporting inclusion [From 2015 to 2020, there were a combined 27 national outbreaks investigated by federal agencies in Canada and the US, leading to product recalls and financial loss ,9. Withinclusion . Non-O15nclusion ,12. As rnclusion .TM—the only phage-based treatment for STEC available commercially. Despite sharing a target host, there can be great diversity between phage candidates. In an effort to better understand Escherichia phage diversity, Korf et al. isolated and compared 50 E. coli phages [E. coli phages isolated from sewage [E. coli phages [E. coli phage groups: T-even (subfamilies Tevenvirinae and Vequintavirinae)-, T1 (family Drexlerviridae)-, T5 (family Demerecviridae subfamily Markadamsvirinae) T3-, and T7 (family Autographiviridae subfamily Studiervirinae)-related phages. It was found that recognition of host receptors and response to different bacterial immunity systems were associated with the genomic features of the tested phages and determined their host range profiles. Hence, continual isolation and characterization of phages is necessary not only to increase the robustness of phage banks for food safety applications, but also to further our current understanding of phage diversity and provide critical information for the ongoing efforts for a genome-based taxonomical system [As their natural predators, lytic bacteriophages (phages) are good biocontrol candidates for pathogenic microbes. Applications in food safety can either be as a standalone alternative or as part of a hurdle technology to chemical and physical food processing. New STEC phages continue to be isolated and tested in bacterial challenge studies in different food matrices and abiotic surfaces as reviewed by Pinto et al. and Wang et al. ,15. O157i phages . While pm sewage . Howeveri phages . The stul system .Escherichia phage vB_EcoM-4HA13. While kinetic studies suggested this phage to be a less-than-desirable candidate for biocontrol applications, its high specificity can be used to develop a detection assay for E. coli O111 strains. Furthermore, comparative genomic analyses showed this phage to be representative of a novel species and genus, and instigated the proposal of a new phage family, Chaseviridae [In this study, we isolated and characterized the eviridae .E. coli strains were used in this study (v/v) glycerol at −80 °C until further use.Twenty-two is study . The strg for 10 min, the supernatant was filtered using a 0.45 µm cellulose acetate syringe filter . The filtrate was spotted, in 10 µL serial dilutions, against each of the enrichment strains on TSA plates with a semi-solid top layer containing 4 mL of tryptic soy broth with 0.5% (w/v) agar, 200 µL of overnight bacterial culture, and 2 mM CaCl2. The plates were incubated at 25 °C for 18–24 h. Prospective plaques were picked and diffused in 500 µL of CM buffer  gelatin) for a minimum of 3 h at room temperature, and re-spotted against the same bacterial strain. This purification process was repeated three times before propagation.Raw primary sludge was obtained from the Guelph Wastewater Treatment Plant in Guelph, Ontario, Canada. Fifteen milliliters of the sewage sample were added to an equivalent volume of 2X TSB enriched with a 500 µL cocktail of non-O157 STEC strains (HA2018015 (O111:NM), HA2018016 (O26:H11), HA2018017 (O103:H2), HA2018018 (O145:NM), and HA2018019 (O121:H19)), incubated aerobically at 25 °C with 180 rpm for 18–20 h. After centrifugation at 10,000× 2 were combined in a 20:10:1 (v/v) ratio, and incubated at 25 °C for 15 min to allow for phage adsorption. Ten milliliters of TSB with 0.5% (w/v) agar and 300 µL of the phage-bacteria-CaCl2 mixture were added to large 150 mm TSA plates. After solidification, the plates were incubated at 25 °C for 18–24 h. Ten milliliters of CM buffer were added to each plate and left for diffusion for at least 3 h at room temperature prior to collection and centrifugation at 10,000× g for 10 min. The pooled supernatant was filtered using a 0.45 µm cellulose acetate bottle filtration system (VWR Canada) and titred prior to downstream applications.4HA13 was propagated using an optimized solid agar approach. Phage lysate, overnight bacterial culture, and 2 M CaClE. coli strains using a microtitre plate was carried out by diluting phage lysate and bacteria with TSB supplemented with 2 mM CaCl2 to a final concentration of 107 particles/mL. Equal volumes of the diluted phage and bacteria were added to a 384-well flat-bottom microtitre plate , in triplicates, for an input ratio (phage/bacteria) of 1. Three sets of controls, in triplicates, were included: (1) blank (TSB with 2 mM CaCl2), (2) sterility (diluted phage and TSB with 2 mM CaCl2), and (3) growth (diluted bacteria and TSB with 2 mM CaCl2). The optical density (OD600) of each well was read every 30 min for 24 h using a microplate reader , with continuous orbital shaking (180 cpm) at 25 °C. Sample readings were standardized using the average of the blank controls and analyzed using the Phage–Host Interaction Data Analyzer (PHIDA), which automatically assigns a phage–host interaction designation based on the length of bacteria growth delay and the end-point optical density [Turbidimetric phage host range determination against 22  density .E. coli strains on TSA plates with a semi-solid top layer. The plates were incubated at 25 °C for 18–24 h and the resultant plaque counts were compared with the reference titre of the phage on its propagation host.EOP was determined by spotting 10 µL serial dilutions of the phage lysate against 22 −6 to 100. The assay was performed, in duplicates, in TSB at 25 °C for a duration of 16 h. Optical density values were read by a microplate reader (Agilent Technologies). Determination of MV50 and virulence index were determined as described in [Phage virulence was quantified using the methodology established by Storms et al. scaled down to a total volume of 100 µL and an adjusted input ratio range of 10ribed in .The one-step growth curve and adsorption assay were performed in triplicates, and in accordance with Kropinski with modifications ,24. Freeg) at 4 °C for 1 h. The pellet was washed with 1 mM HEPES twice before resuspension in 30 µL of 1 mM HEPES and allowed to diffuse at 4 °C overnight. Phage particles were affixed to carbonized copper grids (150 square mesh) and excess liquid was removed with filter paper. The grids were then stained with 2% (w/v) uranyl-acetate before imaging with a Tecnai F20  transmission electron microscope coupled with a Gatan 4k CCD camera. Phage dimensions were measured using ImageJ and average values were obtained from a minimum of 10 images [A milliliter of phage lysate was pelleted in a tabletop centrifuge at max speed 25,000× ,000× g aGenomic DNA was extracted using a PureLink Viral DNA/RNA Mini Kit (Thermo Scientific) per the manufacturer’s protocol. All kit reagents, except for the carrier tRNA, were scaled up 5–10-fold to accommodate the lower phage titre. DNA was eluted in molecular-grade water and quantified with the Qubit dsDNA BR assay kit (Thermo Scientific) and the Qubit 2.0 fluorometer (Thermo Scientific). Its quality was assessed with a Nanodrop spectrophotometer (Thermo Scientific) prior to library preparation and sequencing at the Ottawa Research and Development Centre. DNA was normalized to 200 ng and mechanically sheared using Covaris M220 . An NGS library was prepared using the NexSeq AmpFREE Low DNA Library Kit , and pooled with other libraries before sequenced with the MiSeq sequencer  using 2 × 300 bp MiSeq Reagent Kit v3 (600-cycles). All kits were used per their manufacturers’ protocol.Escherichia phage Mangalitsa (GenBank Accession: MN045229) using progressiveMauve [The genome was de novo assembled using the DNASTAR Lasergene suite version 16  using a base of 200,000 reads. Contigs were polished and re-assembled to produce a draft genome, which was then re-aligned to a homologue, iveMauve .Genome annotation was performed using a combination of RAST, CPT PAP Structural Workflow v2021.02, and CPT PAP Functional Workflow v2021.01 ,28. IdenPhage termini of 4HA13 was determined using PhageTerm 1.0.11 . The preA pairwise similarities heat map of 4HA13 and its top 49 BLASTn hits from NCBI were generated by VIRIDIC ,37,38. E8–109 PFU/mL) was treated with 10 µg/mL of DNase I and RNase A  and incubated at room temperature with gentle shaking for 1 h. After centrifugation at 7000× g at 4 °C for 18 h, the pellets were resuspended in 5–10 mL SM buffer  at 4 °C for 3–4 h. The collected SM buffer was centrifuged at 12,000× g at 4 °C for 10 min and the supernatant was transferred to a fresh conical tube. Cesium chloride was gradually added to the supernatant, in four portions, until the final density of the mixture reached 0.817 g/mL. The phage-CsCl mixture was centrifuged in OptiSeal tubes  at 155,000× g at 4 °C for 24 h . The resultant visible band was extracted using a 20 g needle and syringe (BD Canada) and added to a fresh preparation of CsCl in SM buffer. The centrifugation and extraction process was repeated prior to an overnight dialysis using 3500 MWCO Slide-A-Lyzer dialysis cassettes (Thermo Scientific) against 1% (w/v) ammonium bicarbonate at 4 °C. Dialysis was repeated.A liter of filtered phage lysate  for 3 min at the flow rate of 5 mL/min using Solvent A (0.1% FA) at 300 µL/min, and separated on a NanoEase m/z HSS C18 T3 analytical column  at the flow rate of 300 nL/min for 90 min. A linear gradient from 2% to 30% of Solvent B  at the duration of 65 min was used for peptide elution, followed by flushing with 85% solvent B for 15 min and re-equilibrating the column with solvent A for 10 min. An MS survey scan was acquired with a high resolution of 120,000 at the mass region of m/z 350 to 1800, and MS/MS measurements were performed on multi-charged ions of 2+ to 7+ using low-energy collision-induced dissociation at data-dependent acquisition mode. Dynamic exclusion was set to 30 s. Proteins from 4HA13 were reduced with 10 mM dithiothreitol (DTT), alkylated with 55 mM iodoacetamide, dialyzed against 10 mM ammonium bicarbonate, and dried using a CentriVap centrifugal concentrator . The tryptic digestion of proteins was performed using sequencing-grade trypsin  in 25 mM ammonium bicarbonate at a 1:100 ratio of trypsin-to-protein substrates. The resulting peptides were dried and reconstituted with 0.2% formic acid (FA), and identified using an online ACQUITY ultra performance liquid chromatography (UPLC) M-class  coupled with an Orbitrap Fusion mass spectrometer (MS) (Thermo Scientific). The peptides were trapped by a NanoEase p value < 0.05, and the identified MS/MS spectra were verified manually.The raw LC MS/MS data were searched against the 4HA13 protein sequences using the Mascot Server version 2.6.0 . The seaBased on TEM micrographs, vB_EcoM-4HA13 has a myoviral morphotype with a 53.2 ± 2.6 nm wide isometric capsid . Its taiE. coli strains tested, 4HA13 demonstrated a host specificity for non-motile O111 (O111:NM); in liquid assay, detection was delayed for 13 h .At input ratios of 10one hour . Higher E. coli O111:NM [p < 0.001). The finding of residual 4HA13 phage particles that had a slower adsorption rate supports previously reported biphasic adsorption kinetics and population heterogeneity in other phages [Compared with some of the other recently published STEC phages, 4HA13 adsorbs poorly to its host,  O111:NM ,42,43,44r phages ,46,47,48r phages . The lowr phages ,42,43,444HA13 has a linear dsDNA genome length of 52,401 bp with a GC content of 42.8%  of 50–53%, well below the proposed threshold for a new genus (70%) [4HA13 has little sequence similarity to known phages. Its closest nucleotide homologues, Proteus phage Myduc and Escherichia phage vB_EcoM_SA92KD, but does not belong to any of the subclades. Compared with Erwinia phage Faunus, it has an intergenomic similarity of 50.8% and complete alignment of 70% of both genomes. Due to the low similarities between the homologues, we constructed a phylogenetic tree using PATRIC global protein families (PGFams) of the 24 VIRIDC-identified relatives and it placed 4HA13 in a clade with Erwinia phage Faunus .Further comparative proteomics work carried out by Anany et al. identified 25 shared genes between 4HA13 and its homologues, which confirmed the five aforementioned genes  . Their pm/z 449.7904 and m/z 1128.0223 displayed a set of the N-terminal and C-terminal fragments, in which the mass increment of 42.010 Da of the only internal Lys residues  indicated the modification by acetylation. The reversible acetylation enables lysine residues in the proteins that are resistant to trypsin digestion. It plays an important role in the epigenetic regulation and the phage cell signaling [LC MS/MS analyses of the 4HA13 proteins followed by a Mascot data search identified 15 proteins with ≥50% sequence coverage including major capsid, head, tail structural, and non-structural components of the phage . Two comignaling ,54.Escherichia phage vB_EcoM-4HA13, which was highly specific for the non-motile E. coli O111 strain. Non-O157 STEC continues to be a threat to food safety, increasing its reported incidence rate to surpass that of O157. Bacteriophages have several advantages over current chemical and physical microbial reduction approaches—their specificity, environmental friendliness, and little impact on the food’s organoleptic properties . This stE. coli O111 strains after including more O111 strains in its host range study [Although 4HA13 is free of detectable integrase, antimicrobial resistance, or virulence-encoding genes, its narrow host range and poor adsorption kinetics do not bode well for its candidacy as a biocontrol agent. An ideal phage candidate for microbial reduction applications should have a wide enough host range to effectively reduce the pathogen, but narrow enough not to impact beneficial members of the bacterial community. It should also have rapid and effective adsorption, a short latent period, and a large burst size. The reason is twofold: (1) ease of high-titred preparation, and (2) fast and high reduction of the pathogen. Additional guidelines and considerations for an ideal phage candidate have been postulated in several works ,57,58,59ge study .Chaseviridae, and 4HA13 as the paradigm for the new genus Sabourvirus [Based on comparative genomics, 4HA13 is suggested to be a novel species representing a new genus. Additional work carried out by Anany et al. on 4HA13 and its VIRIDIC clade relatives led to the proposal of a new phage family, ourvirus . This is


Dietary adsorbents, such as bentonite clay, have been used to reduce AFs exposure in animal husbandry and carry over to edible tissues and products. To investigate the efficacy of adding bentonite clay to animal diets in reducing the concentration of AFB1, AFB2, AFG1, AFG2, and the metabolites AFM1 and AFM2 in animal-derived foods , chicken and cattle plasma and cattle ruminal fluid, a sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed. High-throughput sample preparation procedures were optimized, allowing the analysis of 96 samples per analytical batch and consisted of a liquid extraction using 1% formic acid in acetonitrile, followed by a further clean-up using QuEChERS (muscle tissue), QuEChERS in combination with Oasis® Ostro (liver tissue), Oasis® Ostro , and Oasis® PRiME HLB . The different procedures were validated in accordance with European guidelines. As a proof-of-concept, the final methods were used to successfully determine AFs concentrations in chicken and cattle samples collected during feeding trials for efficacy and safety evaluation of mycotoxin detoxifiers to protect against AFs as well as their carry-over to animal products.Aflatoxins (AFs) frequently contaminate food and animal feeds, especially in (sub) tropical countries. If animals consume contaminated feeds, AFs (mainly aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and their major metabolites aflatoxin M1 (AFM1) and M2 (AFM2)) can be transferred to edible tissues and products, such as eggs, liver and muscle tissue and milk, which ultimately can reach the human food chain. Currently, the European Union has established a maximum level for AFM1 in milk (0.05 µg kg Aspergillus fungi, which frequently contaminate agricultural crops, especially in developing countries with warm climates, poor food quality, safety control, production technologies, and bad crop storage conditions + m/z 331.1). In a blank chicken plasma sample, no peaks were present at the elution zone of the analytes of interest  ; 8 min , AFB1-spinterest B and in Fluorescence detectors are often used for the analysis of AFs in different matrices due to the native fluorescence properties of these components and because of the high sensitivity and specificity of these types of detectors. However, AFB2 and AFG2 are naturally strongly fluorescent molecules, while AFB1 and AFG1 need pre- or post-column derivatization to improve fluorescent properties and, as a consequence, method sensitivity ,13.+ ions were selected as precursor ions since it was observed by Chen et al. that fragments of [M + NH4]+ and [M + Na]+ precursor ions were not stable t stable . The twoThe results of the method validation experiments for chicken plasma are shown in −1 (chicken plasma) and LOQ to 10 ng mL(g)−1 (other matrices)  and goodness-of-fit coefficients (g) fulfilled the acceptance criteria and were ≥0.99 and ≤20%, respectively. Other authors reported calibration curves in the same range for human plasma [−1 in human breast milk [−1 for AFM1 in cow milk [−1 [−1 [−1 for AFB1 and AFB2, 0.1–100 µg kg−1 for AFG1, AFG2 and AFM1 [Matrix-matched calibrator samples were used to construct calibration curves in order to compensate for matrix effects. Calibration curves were linear and covered a concentration range of LOQ to 200 ng mLces) see . A weighn plasma ,53, pig n plasma ,21,27, an plasma . Calibraast milk  and 0.02 [−1 [−1  were rep−1) and at two or three different concentration levels −1, medium, 5 ng mL(g)−1 and high, 50 ng mL(g)−1; the latter concentration level was only evaluated in chicken plasma). The acceptance criteria were fulfilled for all compounds at the specified levels. The between-run precision and accuracy were evaluated at the same concentration levels, and the results also fell within the predefined ranges (see The within-run accuracy and precision were tested at the LOQ level (0.025–0.50 ng mL(g)−1 in chicken and cattle plasma and cattle milk; 0.10–0.50 ng mL−1 in cattle ruminal fluid; 0.05–0.25 µg kg−1 in chicken muscle; 0.05–0.50 µg kg−1 in chicken liver; 0.025–0.50 µg kg−1 in chicken eggs, depending on the aflatoxin. The calculated LOD values ranged between 0.002 and 0.060 ng mL−1 (chicken and cattle plasma); 0.002 and 0.038 ng mL−1 (cattle milk); 0.012 and 0.132 ng mL−1 ; 0.006 and 0.040 µg kg−1 (chicken tissues); 0.002 and 0.097 µg kg−1 (chicken eggs). These LOQ values fell in the same range or even lower than those mentioned by other authors: 0.01–2.0 ng mL−1 in rat plasma [−1 for AFs in human plasma or serum [−1 for AFB1 and AFM1 in pig and chicken plasma [−1 for AFB1, AFB2, AFG1 and AFG2 in chicken liver [−1 in swine liver [−1 in rat tissues [−1) [−1), implying the suitability of the current method for analysis of AFs in food at trace levels [LOQ values ranged between 0.025–0.50 ng mLt plasma ,27; 0.1–or serum ,53; 1 ngn plasma ,22; 2 µgen liver ; 0.12–0. tissues ,27. The ues [−1) ,37,54. Mues [−1) ,37,54, i−1 for AFM1 in milk. Therefore, LOQ and LOD values for AFs in milk should be as low as possible. The LOQ values for AFs in milk obtained with the presented method ranged between 0.025 and 0.05 µg kg−1, which is sufficiently low to detect these contaminants at the maximum allowed level by the EU (except for AFM2). LOQ values for AFs reported by other authors ranged between 0.08–0.16 ng mL−1 in human breast milk [−1 in cow milk [The EU legislation has set a maximum allowed level of 0.05 µg kgast milk ; 0.02–0.cow milk ,13,28,44−1 [−1 in various animal-derived food matrices [LOQ values in pork meat ranged between 0.1–0.2 µg kg−1  and betwmatrices .The obtained LOQ and LOD values in all matrices were sufficiently low, and hence it was possible to quantify the analytes of interest accurately in samples that were taken from laying hens, broiler chickens, and cattle as a part of toxicokinetic and in vivo efficacy studies with mycotoxin detoxifying agents ,56,57. In the reported procedure, no carry-over was observed for AFG2 and AFM1. For AFB1, AFB2, and AFG1, limited carry-over  was observed in the first solvent sample that was analyzed after the highest calibrator sample and was further reduced to ≤0.03% in the second solvent sample. In the third solvent sample, no peaks of AFB1, AFB2, and AFG1 could be detected. Although the impact of this carry-over on method accuracy and precision is negligible, it is advised to analyze at least one solvent sample after the highest calibrator and/or QC samples.Method selectivity was evaluated by comparing extracted blank samples with spiked samples. Within 1 min of the elution zone of each analyte, no interfering peaks (S/N > 3) were detected, ensuring proper quantification in the different matrices.The stability of AFs in sample extracts that were stored in the autosampler (8 °C) during at least nine days (cattle plasma) and 43 days (chicken plasma) shows that long analytical batches can be run, which makes the current method suitable for toxicokinetic and/or monitoring studies. Results of stability experiments further showed that the mean measured AFs concentrations fell within the accuracy acceptance criteria  after thThe stability of AFs under different conditions was also shown by other authors: AFs in stock solutions were stable for at least 12 months in plastic tubes stored at −20 °C and for at least two months in working solutions stored at 4 °C ; AFB1 waThe obtained results indicated there were no problems with stability during the routine analysis of samples.13C17-AFB1, 13C17-AFG1, and 13C17-AFM1). Moreover, matrix-matched calibration curves were prepared in the present study to reduce further the influences of matrix effects on the UHPLC-MS/MS instrument. Matrix-matched calibration was also used by other authors [LC-MS/MS is a very specific and selective analytical technique, but it has been demonstrated that co-eluting matrix interferences may have an impact on ionization efficiency ,39,59. T authors ,35,36 anE) between 70 to 110% are generally accepted when analyzing food [E were obtained for cattle plasma (57.5–80.0%), cattle ruminal fluid (61.4–75.1%), chicken plasma (66.1–73.5%), and chicken muscle (114–142.5%). RE’s were rather low for cattle milk (24.7–47.8%), chicken liver (28.5–39.3%), and chicken eggs (7.5–23.9%)  see . Results−1 feed); or from cows  fed either a control diet or a diet contaminated with AFB1  alone or in combination with a selected mycotoxin binder (see −1 feed) is shown in To prove the applicability of the methods, incurred biological samples were analyzed that were collected from chickens  fed with a diet containing a high AFs concentration  and major phase-I hydroxylation metabolites (AFM1 and AFM2) were included, as these were identified by Jurišić et al.  as approAnother metabolite, the AFB1-lysine adduct, which is considered an important biomarker of chronic exposure in serum or plasma, was not included in the current analysis methods because albumin/lysin adducts would have precipitated during the first step of the sample preparation procedure (consisting of liquid extraction with 1% FA in ACN) . Helix pomatia was performed during the sample preparation procedure since glucuronidation and/or sulfation have not been reported as main phase-II detoxification pathways for AFB1. Phase-II biotransformation reactions of AFB1 consist mainly of glutathione conjugation. However, Vidal et al.  mentioneThe above results demonstrate that the developed methods can be applied not only for toxicokinetic studies with aflatoxins in chickens and cattle but also for residue determination, food safety monitoring, and efficacy testing of potential mycotoxin binders and/or modifiers.Methods were developed and in-house validated for the quantitative determination of the aflatoxins AFB1, AFB2, AFG1, and AFG2 and their hydroxylated metabolites (AFM1 and AFM2) in chicken plasma, edible tissues (muscle and liver) and eggs; and cattle plasma, ruminal fluid, and milk. The novelty of the methods consisted of the optimization of high-throughput sample preparation procedures for each matrix to allow the processing of ≥96 samples per day. The sample preparation procedures were rather generic, but special attention was paid to the removal of PLs. Hence, these procedures can also be tested for the extraction of other mycotoxins in animal biological matrices, which can be a future application of the presented methods. Furthermore, chromatographic analysis was performed within 10 min, which allowed the analysis of a large number of samples in a 24-h period.The different procedures were validated in accordance with European guidelines , and the results generally met the predefined acceptance criteria. As a proof-of-concept, the final methods were applied to successfully determine AFs concentrations in incurred chicken and cattle samples taken during a feeding trial to evaluate the efficacy and safety of mycotoxin detoxifying agents to protect against AFs as well as their carry-over to animal products.13C17-AFB1, 13C17-AFM1, and 13C17-AFG1, were purchased as 0.5 µg mL−1 solutions in ACN from Biopure . All analytical standards were stored at the temperature suggested by the manufacturer. The standards of AFB1, AFB2, AFG1, AFG2, AFM1, and AFM2 were obtained from Fermentek Ltd.  and had a certified purity of ≥ 98%. The internal standards (IS), 4), sodium chloride (NaCl)) and were obtained from VWR . Methanol (MeOH), ACN, and formic acid (FA) were of ULC-MS grade and were obtained from Biosolve . ULC-MS grade water was taken from a Milli-Q system . All other solvents and reagents had an analytical grade , Oasis® PRiME HLB 96-well plates (30 mg), 2 mL square 96-well collection plates and 96-well cap-mats with square plugs  were purchased from Waters .Oasis−1), AFB2 (1 mg mL−1), AFG1 (1 mg mL−1), AFG2 (1 mg mL−1), AFM1 (0.1 mg mL−1) and AFM2 (0.1 mg mL−1) were prepared in ACN. An individual working solution (WSind) with a concentration level of 100 µg mL−1 was prepared for AFB1, AFB2, AFG1, and AFG2 in ACN. Mixed working solutions of all aflatoxins (WSmix) at concentrations of 1000 ng mL−1, 100 ng mL−1, 10 ng mL−1, 1 ng mL−1, and 0.1 ng mL−1 were prepared by appropriate dilution of the SS and WSind in ACN. Standard stock solutions (SS) of AFB1 (1 mg mLIS_mix) of the ISs, containing 13C17-AFB1, 13C17-AFM1, and 13C17-AFG1 at a concentration level of 10 ng mL−1 was prepared in ACN. A mixed working solution  and eggs were obtained from broiler chickens or laying hens, and blank plasma, milk, and ruminal fluid samples were obtained from dairy cattle that received no AFs. These blank samples were used for the preparation of matrix-matched calibrator and quality control (QC) samples and were stored at ≤−15 °C.Incurred plasma, tissue (muscle and liver), and egg samples from chickens and plasma, milk, and ruminal fluid samples from cattle were analyzed.−1 feed for four weeks, are shown in Chicken plasma, tissues , and egg samples were taken as a part of an in vivo efficacy and safety study with a mycotoxin detoxifying agent to reduce the carry-over of AFs to chicken products. Four hundred laying chickens  were bought from a commercial farm. The trial was conducted for four weeks, and samples  were collected at the end of the trial. More details concerning the animal experiments will be presented in a forthcoming article by Ochieng et al. . The resDairy cattle plasma, ruminal fluid, and milk samples were taken as part of an in vivo efficacy and safety study with a mycotoxin detoxifier to reduce the carry-over of AFs to milk. Therefore, 24 animals (18 Borans and 6 Friesian-Boran crosses) in the early lactation (days in milk (mean ± SD) = 30.7 ± 5.7, body weight (mean ± SD) = 341.0 ± 33.8 kg) were used. The Borans were randomly divided into three groups of six animals each and assigned to one of three experiments, while the 6 crosses were only used for one experiment  . The BorMore details concerning the animal experiments in cattle were described by Kemboi et al. .IS_mix 10 ng mL−1) and 100 µL of ACN, followed by vortex mixing and equilibration for 5 min at room temperature. After the addition of three hundred (300) µL of 1% FA in ACN, samples were vortex mixed (15 s) and centrifugated . The supernatant was applied to an Oasis® Ostro 96-well plate (25 mg) and allowed to pass through the 96-well plate by vacuum application (15 mm Hg) for 5 min, and the filtrate was collected in a 96-well collector plate. After evaporation under a nitrogen stream (~40 °C), the dry residue was reconstituted in 200 µL of water/MeOH  and the 96-well collector plate was vortex mixed for 15 s. A mat cap was applied onto the 96-well collector plate, and a 5.0-µL aliquot of each sample was analyzed using the UHPLC-MS/MS instrument.To 100 µL of chicken or cattle plasma were added 25 µL of the mixed IS working solution , the samples were extracted on a vertical rotary apparatus for 10 min (80 rpm), followed by a centrifugation step for 10 min at 1200× g. One (1) mL of the supernatant was applied to an Oasis PRiME HLB 96-well plate (30 mg of sorbent/well), and the filtrate was collected into a 2-mL 96-well square collector plate. The sample was evaporated to dryness under a nitrogen stream (~60 °C) and re-dissolved in 250 µL of water/MeOH . A 5.0-µL aliquot was injected into the UHPLC-MS/MS instrument. To 1.0 mL of cattle milk or ruminal fluid was added 25 µL of the WSIS_mix 10 ng mL−1 was added, followed by vortex mixing and equilibration for 5 min at room temperature. Liquid extraction was performed by the addition of three mL of 1% FA in ACN, followed by vortex mixing (5 min), extraction on a rotary apparatus for 10 min (80 rpm), and vortex mixing (5 min). The sample was centrifuged for 10 min at 1200× g, after which the supernatant was transferred to an extraction tube containing 0.2 g of sodium chloride (NaCl) and 0.8 g of magnesium sulfate (MgSO4). Next, the sample was vortex mixed again for 1 min, followed by a centrifugation step of 10 min at 1200× g. The supernatant was pipetted into another tube and evaporated to dryness under a nitrogen stream (~40 °C). Two hundred and fifty (250) µL of 1% FA in ACN were added to the dry residue, followed by vortex mixing for 15 s. The sample was transferred to an Oasis Ostro 96-well plate (25 mg) and allowed to pass through the 96-well plate by the application of a vacuum (15 mm Hg) for 10 min. The eluate was collected in a square 96-well collector plate and diluted with 250 µL of water, followed by gentle mixing (1000 rpm). The 96-well plate was covered with a cap mat, and a 5.0-µL aliquot was injected onto the UHPLC-MS/MS instrument. To 1.0 g of chicken liver, 25 µL of the WSIS_mix 10 ng mL−1 was added, followed by vortex mixing and equilibration for 5 min at room temperature. Two (2) mL of water and 3 mL of 1% FA in ACN were added, each followed by a vortex mixing step of 2 min and 5 min, respectively, on a multi-tube vortex mixer. Extraction was performed for 15 min on a rotary apparatus (80 rpm), followed by a vortex mixing step (5 min) and centrifugation for 10 min at 1200× g. The supernatant was added to another extraction tube containing 0.2 g of NaCl and 0.8 g of MgSO4, followed by a 1-min vortex mixing step. After centrifugation (10 min at 1200× g), 500 µL of the supernatant was added to an autosampler vial and diluted by the addition of 200 µL of water, followed by vortex mixing (30 s). A 5.0-µL aliquot was analyzed using the UHPLC-MS/MS instrument. One (1.0) g of chicken muscle was transferred to a 15-mL extraction tube, and 25 µL of the WSIS_mix 10 ng mL−1 was added, followed by vortex mixing and equilibration for 5 min at room temperature. Liquid extraction was performed using 3 mL of 1% FA in ACN, followed by vortex mixing (5 min) and extraction on a rotary apparatus for 10 min (80 rpm). After centrifugation for 10 min at 1200× g, the supernatant was added to another tube and evaporated under a gentle nitrogen stream (40 °C). To the dry residue, 250 µL of 1% FA in ACN was added, followed by vortex mixing for 15 s. Thereafter the sample was applied onto an Oasis Ostro 96-well plate (25 mg). The sample was allowed to pass through the 96-well plate by vacuum application (15 mm Hg) for 10 min, and the eluate was collected in a 96-well 2-mL collector plate. After the addition of 250 µL of water to each well and gentle mixing , the 96-well collector plated was covered with a cap mat, and a 5.0-µL aliquot was analyzed using the UHPLC-MS/MS instrument.One (1.0) g egg was weighed in a 15-mL extraction tube, and 25 µL of the WSThe UHPLC system was an Acquity UPLC H-Class Quaternary Solvent Manager in combination with a Flow-Through-Needle Sample Manager with temperature controlled tray and column oven from Waters. An Acquity UPLC HSS T3 column  in combination with an Acquity HSS T3 1.8 μm Vanguard pre-column, both from Waters, was used for chromatographic separation. −1.The mobile phase A was water, while the mobile phase B was MeOH. The following gradient was used: 0–1.0 min , 3.0 min (linear gradient to 90% B), 3.0–7.0 min , 7.3 min (linear gradient to 80% A), 7.3–10.0 min . The flow rate was 0.3 mL minThe column oven and autosampler tray were kept at 40 °C and 8 °C, respectively. ® MS/MS system with an electrospray ionization (ESI) probe that operated in the positive ionization mode . The divert valve was programmed to send the UPLC effluent to the mass spectrometer between 3.0 and 6.0 min.The UHPLC column effluent was transferred to a Xevo TQ-S−1 of all aflatoxins and the ISs at a flow rate of 10 µL min−1 in combination with the mobile phase . Mass spectrometric parameters were determined by infusing working solutions of 100 ng mL−1, cone gas: 150 L h−1, nebulizer pressure: 6.9 bar, LM resolution 1 and 2: 2.8, HM resolution 1 and 2: 15, ion energy 1 and 2: 0.2 and 0.8, respectively, collision gas flow: 0.15 mL min−1. The following parameters were selected: capillary voltage: 3.2 kV, source offset: 50 V, source temperature: 150 °C, desolvation temperature: 600 °C, desolvation gas: 800 L hMS/MS data acquisition was performed in the multiple reaction monitoring (MRM) mode. The MRM transitions that were measured for all analytes are shown in E) and extraction recovery (RE). The UHPLC-MS/MS method was validated in-house for the analytes of interest  by analyzing spiked blank biological samples obtained from untreated chickens and cattle. The following parameters were evaluated as recommended by European guidelines and in accordance with the literature ,63,64,65−1), 1 g of blank chicken tissues (liver and muscle) and eggs, and 1 mL of blank cattle milk or ruminal fluid (concentration range: LOQ–10 ng mL−1 or µg kg−1) were used. Three different calibration curves were extracted on three different analysis days. The correlation coefficients (r) and goodness-of-fit coefficients (gof) were evaluated and had to comply with the acceptance criteria for r ≥ 0.99 and g ≤ 20%, respectively [0, 1/x1, 1/x2) was selected based on VICH GL49 guidelines [For the preparation of matrix-matched calibration curves, 100 µL of blank chicken and cattle plasma , six blank samples that were spiked at a low (LOQ), medium, and high concentration level were analyzed in the same run. For the evaluation of the between-run accuracy and precision (reproducibility), at least three blank samples spiked at the same concentration levels were analyzed on three different days. The results had to fulfill acceptance criteria for accuracy as described in VICH GL49. The relative standard deviation (RSD) was calculated and had to be below the RSDax value . −1 or µg kg−1 were analyzed on the same day [The LOQ was defined as the lowest concentration of the analyte for which the method was validated with an accuracy and precision that fell within the specified ranges. The LOQ was also set as the lowest point of the calibration curve. To determine the LOQ, six samples spiked at a concentration level between 0.025 and 0.50 ng mLsame day . The lowest concentration that could be recognized by the detector with a signal-to-noise (S/N) ratio of ≥ 3 was defined as the LOD. The LOD values were calculated using the mean S/N of the blank samples spiked at the LOQ level .The reconstitution solvent was injected after the highest calibration sample to evaluate the presence or absence of carry-over. If a peak was observed in the elution zone of an analyte or the IS, it had to be below 20% of the LOQ and below 5% for the IS .E) and matrix effects (ME) quantitatively, three sets of samples were prepared: set A consisted of standard solutions of the analytes of interest (concentration: 0.50 and 5.0 ng mL−1 or µg kg−1); the two other sets consisted of matrix-matched samples that were spiked with AFs after (set B) and before (set C) extraction at the same concentration levels as the set-A samples. The RE and ME (%) were calculated by dividing the peak areas of AFs in the respective samples, i.e., RE = C/B × 100 and ME = B/A × 100.To assess extraction recovery (Rstab_extr) in the matrix during storage at ≤−15 °C (QCstab_matrix) and during three freeze-thaw cycles (QCfreeze_thaw). For each stability experiment, blank samples were spiked with AFs at a concentration of 0.50 and 5.0 ng mL−1 or µg kg−1. The stability of AFs was evaluated in extracted samples  were extracted as specified above  were frozen and stored in the freezer at ≤−15 °C and thawed at room temperature. During each cycle, samples were frozen for at least 12 h before they were thawed [freeze_thaw samples were extracted and analyzed on the UHPLC-MS/MS instrument. Quantification was performed using a freshly prepared matrix-matched calibration curve.e thawed . After tStability in matrix. The QCstab_matrix samples were put in the freezer at ≤−15 °C immediately after preparation. After a certain storage period, the samples were thawed and analyzed using the above described sample preparation procedure and UHPLC-MS/MS method. Samples were quantified using a freshly prepared matrix-matched calibration curve.stab_extr, QCfreeze_thaw, and QCstab_matrix sample stability were considered acceptable if the mean concentration determined at the specified stability time point agreed with the spiked theoretical concentrations within the accuracy acceptance criteria [QCcriteria ,65.