H. pylori infection/exposure and inflammatory bowel disease prevalence/incidence, however, there are no reports of individual patients who developed a "non-transient” ulcerative colitis (UC) following H. pylori eradication.Epidemiological studies suggest an inverse association between H. pylori eradication and a 3-year follow-up of the progression to steroid-dependent colitis complicated with enteropathic arthritis and final containment of the disease with golimumab. In our patient, H. pylori eradication was associated with the development of pancolitis that evolved into clinically, endoscopically, and pathohistologically confirmed UC.We report a case of a 72-year-old female with an elderly-onset UC developed upon The case of our patient provides a unique clinical context for a growing body of literature suggesting molecular mechanisms involved in the interaction of genes, environment, and microbiota to be of critical importance in the etiopathogenesis of UC, and thus, provides a valuable set of complementary translational information for preclinical and epidemiological research on the topic. Helicobacter pylori (H. pylori) is an important human pathogen with 50–90% prevalence depending on the stage of economic development , toxin B [4.55 ng/ml]) and O&P exam were negative. Fecal calprotectin (FCP) was 113.9 mg/kg. The patient was prescribed a light dietary regimen, mesalazine . On the 10th day, a follow-up colonoscopy revealed persistent pancolitis and the patient reported diffuse pain in the lower hemiabdomen, hematochezia, and diarrhea. Successful eradication of H. pylori was confirmed with a stool antigen test. The patient was encouraged to remain adherent to the dietary regimen and therapy.Five days in eradication, the patient developed bloody diarrhea but remained highly motivated to continue with the regimen despite the adverse drug reactions (ADRs). Upon successful completion of eradication, colonoscopy was done due to persistent bloody diarrhea. Edematous mucosa of the entire colon and internal hemorrhoids (grade I/II) were revealed and pathohistology was in concordance with pancolitis Fig. C. C.diffC.difficile, and stool O&P remained unremarkable, and FCP was 130.8 mg/kg. Otilonium bromide and meal replacement were added to the therapy. The patient stopped smoking.After 2 months, the patient reported a reduction in symptom severity in the period of therapeutic adherence, however, diarrhea persisted despite therapy. In a period of noncompliance, she noticed traces of blood in the stool that disappeared upon a continuation of the recommended therapy. Bacteriological, C.difficile, O&P, and H. pylori remained unremarkable. FCP increased to 1162 mg/kg and c-reactive protein (CRP) was 10.5 mg/L. Colonoscopy revealed proctosigmoiditis, 1.5 cm flat cecal polyp, and several individual diverticula . HLA-typization indicated no relevant TPMT mutations and azathioprine was included in therapy.Three months later, the patient reported the development of painful shin nodules, pain in her wrists, and increased night sweats. The reported symptoms were considered a sign of enteropathic arthritis and methylprednisolone was initiated. Budesonide was added to therapy and celecoxib, denosumab, and vitamin D were initiated due to osteoporosis. Laboratory findings revealed FCP 986 mg/kg; CRP 6,9 mg/l; rheumatoid factor 50.4 IU/ml; IgG 26,7 g/l; pANCA (1:5120); cANCA (+); anti-MPO 103.9 IU/ml; negative Clinical improvement was observed after two months. Painful indurations regressed and the patient reported no pain in her wrists. There was 1–2 stools/day with sporadic traces of blood and mild diarrhea. The disease was now partially controlled with a combination of corticosteroid, mesalazine, and azathioprine, however, initiation of golimumab was recommended to avoid complications of steroid-dependent UC.Golimumab therapy was initiated with a starting dose of 200 mg (sc.) followed by additional 100 mg sc. 2 weeks later, and 100 mg sc. maintenance dose planned every month. So far the patient reported symptom improvement. Corticosteroid and azathioprine were successfully discontinued without a rebound. The disease is currently controlled with a combination of golimumab and mesalazine.H. pylori eradication with a 3-year follow-up of the progression of the disease that eventually evolved into steroid-dependent UC complicated with enteropathic arthritis that was finally contained with golimumab. Initially, the patient's main concern was mild dyspepsia accompanied with EGD and PHD suggestive of chronic active gastritis with H. pylori invasion. Eradication was an obvious next step based on current guidelines [H. pylori antibiotic sensitivity testing to plan the optimal treatment strategy [C.difficile fecal toxin A and B, O&P)—the patient was diagnosed with ulcerative proctosigmoiditis. Interestingly, a similar pattern of events was observed in a 63-year-old who developed bloody diarrhea upon H. pylori eradication therapy that was initially understood as antibiotic-associated colitis, but the condition didn't resolve on its own following treatment discontinuation [H. pylori eradication therapy, several differences should be emphasized. In [H. pylori eradication regimen, a subsequent urea breath test indicated the infection was still present. As further information regarding H. pylori presence was not reported, the patient might have been H. pylori positive throughout transient UC. In contrast, H. pylori's absence was confirmed in our patient. Furthermore, UC severity and clinical course were less favorable for our patient. Fortunately, the disease is currently under control with golimumab, a reassuring finding considering the benefit of golimumab maintenance therapy was previously observed in patients who responded to golimumab induction [We present a case of an elderly woman developing UC upon idelines . Unfortuidelines , so a sestrategy . Unfortuinuation . Althougized. In , eradicaized. In . Furthernduction , 26.H. pylori infection/exposure and prevalence/incidence of IBD proposed by numerous studies [H. pylori and IBD are ill-equipped to provide a mechanistic explanation for the observed effects. Nevertheless, recent evidence from preclinical experiments suggest chronic H. pylori infection may protect against IBD by inducing systemic immune tolerance and suppressing inflammatory responses [H. pylori infection reduces systemic inflammation and splenic CD4+ T cells and ameliorates colitis-associated histopathological changes in the dextran sulfate sodium-induced mouse model of colitis. Furthermore, in mice infected with Salmonella typhimurium, a co-infection with H. pylori increased mesenteric lymph node interleukin-10 and prevented the TH17 response [H. pylori-induced immunomodulation activation of dendritic cell tolerogenic phenotype and immunosuppressive regulatory T cells were proposed as the most important mechanisms mediating potentially beneficial effects in IBD [The presented case speaks in favor of the inverse association between  studies , 27–33. H. pylori and UC [H. pylori in 56.6% of cases and 20% of controls using polyclonal anti-H. pylori antibodies [Interestingly, some studies reported no association between i and UC , and somtibodies .H. pylori in IBD is that the differences in seroprevalence between IBD and the controls are a consequence of pharmacotherapy. For example, the protective role of 5-aminosalicylic acid on H. pylori infection has been proposed [H. pylori seroprevalence [One possible explanation of the apparently paradoxical findings of the protective/harmful effects of proposed . Similarproposed . Neverthevalence .H. pylori eradication was confirmed; (5) biochemical indicators of disease severity were monitored and reported.Strengths: (1) long-term follow-up; (2) defined early course of the disease (pancolitis evolving to proctosigmoiditis) as colonoscopy of the only other patient  reportedH. pylori eradication (~ 20% of patients do after the age of 60 [H. pylori related. Although the risk of autoimmune diseases (including IBD) is increased in patients with peptic ulcer disease (PUD) who receive anti-H. pylori therapy, the same is true for PUD patients who didn't receive therapy and for patients who received antibiotics for urinary infection [Limitations: We cannot rule-out that (1) our patient would develop UC regardless of the ge of 60 ); (2) UCnfection . Antibionfection , and a lnfection . On contnfection  and sevenfection , 43. Thenfection . Furthernfection . We cannH. pylori eradication therapy, it is to be expected that rare cases of eradication-induced UC might become more prevalent. In our patient, an unfortunate combination of environmental and genetic risk factors , complicated with H. pylori eradication-induced dysbiosis triggered pathophysiological events leading to the development of a serious case of UC. In the future, a better understanding of the molecular mechanisms responsible for the disease development, factors influencing the protective and/or harmful effects of H. pylori , and pe


Background and Objectives: Helicobacter pylori (H. pylori) infection impairs quality of life. However, whether eradication therapy ameliorates gastrointestinal symptoms remains questionable. The main objective of this study was to evaluate the influence of H. pylori eradication therapy on gastrointestinal symptoms. Materials and Methods: A total of 140 patients, 59 women and 81 men, with a mean age of 61 and suffering from H. pylori infection in the University Hospital of Split, Croatia, were enrolled in the study. Patients were randomly assigned to either concomitant or hybrid therapies. The Gastrointestinal Symptom Rating Scale (GSRS) questionnaire was completed by patients prior to and after the eradication therapy. Results: In both groups, the total GSRS score improved significantly after therapy. In the concomitant group, the abdominal pain score, reflux symptoms score and indigestion score decreased significantly after therapy. In the group with hybrid therapy, all five groups of symptoms  significantly decreased after therapy. Patients with adverse events had significantly higher total GSRS scores after eradication therapy. Conclusions: H. pylori eradication therapy could alleviate gastrointestinal symptoms regardless of the treatment used, but the favorable effect seemed to be more pronounced after hybrid therapy. Helicobacter pylori (H. pylori) infection still represents a public health burden. H. pylori remains a crucial element in the development of conditions such as gastritis, gastric and duodenal ulcers, gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (MALT) [H. pylori is a well-known factor in the carcinogenesis of stomach cancer [H. pylori virtually always leads to chronic gastritis, it is asymptomatic in over 80% of cases [H. pylori infection, and it might impair various aspects of a patient’s quality of life (QOL). Current guidelines require the treatment of Helicobacter pylori infection regardless of symptomatology [H. pylori infection has been a subject of numerous studies for decades, the number of studies involving the impact on the quality of life in patients with H. pylori is limited. A causal link between dyspeptic symptoms and H. pylori infection has been suggested mainly through studies of infected patients with uninvestigated or functional dyspepsia. A systematic review of 21 randomized clinical trials concluded that eradication therapy had a small but statistically significant effect in H. pylori-positive non-ulcer dyspepsia (estimated number needed to treat = 14) [H. pylori eradication for functional dyspepsia concluded that efficacy of eradication had symptom-based tendencies in patients with functional dyspepsia (FD) and that it might be effective in the subgroup of FD patients with epigastric pain syndrome [H. pylori [a (MALT) ,2. Furthh cancer . Althougof cases . Howevermatology . Althougat = 14) . A randosyndrome . On the . pylori .H. pylori infection remains scarce [One of the recognized tools for the assessment of gastrointestinal symptoms is the Gastrointestinal Symptom Rating Scale (GSRS) questionnaire. Although it has been frequently used for the assessment of symptomatology in peptic ulcer disease, reflux disease and functional dyspepsia, its use for s scarce ,10,11,12H. pylori infection.As data regarding the comparison of adverse effects between concomitant and hybrid therapies are insufficient, the aim of this study was to determine the influence of different eradication protocols on gastrointestinal symptoms in patients with H. pylori positive were included in the study. In this previous study, a total of 140 patients  with proven H. pylori infection were enrolled [H. pylori eradication therapy; malignant disease of the stomach or any other site; taking proton pump inhibitors (PPI), H2 antagonists, bismuths or antibiotics  during the last month; associated comorbidity ; history of allergies to proton pump inhibitors or antibiotics ; pregnancy and lactation; or refusal to participate in the survey [H. pylori eradication therapy, they were again given the GSRS questionnaire.We conducted a prospective study based on the previous randomized controlled trial comparing concomitant and hybrid therapy . From Juenrolled . Exclusie survey . Patiente survey . Upon a The Gastrointestinal Symptom Rating Scale (GSRS) is a specific, 15-item questionnaire for patients with gastrointestinal symptoms. The questionnaire has a seven-point graded Likert-type scale, where 1 represents absence of symptoms and 7 represents very troublesome symptoms. The reliability and validity of the GSRS are well-documented, and norm values for a general population are available ,14,15,16All participants provided written informed consent. The study was performed in accordance with the principles of good clinical practice from the Declaration of Helsinki, approved by the Ethics Committees of the University Hospital of Split  and the University of Split School of Medicine  and registered as a clinical trial .p < 0.05.The statistical software SPSS version 25 for Windows  was used for statistical data analysis. Data were expressed as medians and interquartile ranges. The Mann-Whitney U test was used for independent samples, and the Wilcoxon test was used for paired samples. The Kolmogorov-Smirnov test was used to test the normality of the data. The statistical significance was defined as n = 69) and hybrid (n = 71) groups. In the previous study, there was no significant difference between groups regarding eradication rates (84.1% (58/69) and 83.1% (59/71), respectively) in the intention-to-treat analysis (p = 0.878) and 96.7% (58/60) and 95.2% (59/62) in the per-protocol analysis (p = 0.675) [p = 0.043) [A total of 140 patients were randomly assigned to concomitant (= 0.675) . However= 0.043) .p < 0.001), as shown in In the concomitant group, the total GSRS result was significantly lower after than prior to eradication therapy (median 23 (IQR 18-28) vs. median 19 (15.75–24); p < 0.001), as shown in In the hybrid group, the total GSRS result was significantly lower after in comparison to before eradication therapy (median 28 (IQR 20–39.5) vs. 22 (IQR 18–31); p = 0.530).When we compared changes in GSRS scores in the concomitant and hybrid groups after therapy in relation to the basal data, there was no significant difference among groups (median 2 (IQR 0–5) vs. median 2 (IQR 0–6.75); p = 0.041). However, comparing changes in GSRS scores prior to and after therapy in relation to the basal data, there was no significant difference between patients who had adverse events and those who didn’t have adverse events (median 2.5 (IQR 0–8) vs. median 2 (IQR 0–5); p = 0.870).Patients with adverse events during eradication therapy had significantly higher total GSRS scores after than prior to eradication therapy: median 22 (IQR 18–31) vs. median 19.5 (IQR 16–25); H. pylori infection, commonly associated with dyspepsia, impairs quality of life [H. pylori infection on gastrointestinal symptoms has been rarely investigated thus far. To date, even less interest has been shown in terms of the possible impact of eradication therapy itself on quality of life and various gastrointestinal symptoms. The intention of this study was to highlight the influence of two different eradication regimes on quality of life in the aspect of gastrointestinal symptoms through a validated questionnaire. Our results indicated the beneficial effect of eradication therapy on quality of life in patients with gastrointestinal symptoms due to H. pylori infection. Both concomitant and hybrid eradication therapies had beneficial impacts on gastrointestinal symptoms in their total GSRS scores. A similar positive effect of eradication therapy has also been observed in earlier studies [H. pylori infected patients demonstrated that quality of life could be improved regardless of eradication outcome [H. pylori infection in the previous study, with a greater than 95% rate of eradication in per-protocol analysis [ of life ,18. Howe studies . Additioanalysis .H. pylori infection. Yet, the only beneficial role of successful eradication therapy, in the case of dyspeptic symptoms, is well-known, as previous studies have shown [H. pylori gastritis can be the cause of dyspepsia in a subset of patients [H. pylori eradication therapy was beneficial for the improvement of upper gastrointestinal symptoms in functional dyspepsia [H. pylori infection and history of eradication did not affect acid-related dyspepsia symptoms [Focusing on individual symptoms, in the concomitant group, a positive effect was demonstrated in the cases of reflux symptoms, abdominal pain and maldigestion. However, in the group with hybrid therapy, a beneficial effect was additionally shown in the case of diarrhea and constipation symptoms. The main difference between concomitant and hybrid therapy is the 7-day shorter administration of clarithromycin and metronidazole, which can result in less adverse events . As expeve shown ,19. H. ppatients ,21. Systyspepsia ,22. On tsymptoms .The impact of eradication therapy on reflux symptoms still remains controversial . On the H. pylori infection and diarrheal syndrome is controversial. An assumption is that hypochlorhydria as a consequence of chronic gastritis increases the risk of diarrhea [The hybrid therapy group showed better outcomes in GSRS scores after eradication in terms of constipation and diarrhea. The link between diarrhea . Diarrhediarrhea ,27. Addidiarrhea . In the H. pylori has been considered a possible factor influencing the gut microbiota [H. pylori infection can contribute to gastric microbial dysbiosis, which may be involved in carcinogenesis [H. pylori eradication potentially restored gastric microbiota to a similar status as found in uninfected individuals and showed beneficial effects on gut microbiota [crobiota ,29,30,31crobiota .H. pylori-associated dyspepsia, by definition, should obtain sustained symptomatic relief for six months after therapy according to the Kyoto definition [H. pylori involved [Our study has several notable limitations. In this study, we evaluated GSRS scores one month after eradication therapy. For some symptoms such as reflux syndrome, we can expect improvement even one year after eradication therapy, as Hirata et al. demonstrated . Meanwhifinition . Howeverinvolved .H. pylori therapy improved quality of life, even one month after therapy. Both concomitant and hybrid therapy had a beneficial role in reflux symptoms, maldigestion and abdominal pain. However, hybrid therapy improved constipation and diarrhea as well, possibly because of less antibiotic usage. Nonetheless, further studies are warranted in order to confirm these inferences.Both concomitant and hybrid eradication of


Fomitiporia mediterranea (63–94%), a fungal pathogen associated with esca. The relative abundance of this fungal species significantly decreased after sodium arsenite treatment in the three vineyards, in particular in white-rot necrotic tissues and their borders (−90%). F. mediterranea was the most sensitive to sodium arsenite among fungi from grapevine woody tissues. These results strongly suggest that the effect of sodium arsenite on GTDs is due to its ability to efficiently and almost specifically eliminate F. mediterranea from white-rot necrotic tissues, allowing saprobic fungi to colonize the tissues previously occupied by this pathogenic fungus.Although sodium arsenite was widely used in Europe until its ban in 2003, its effects on microorganisms is not clearly understood. To improve our understanding of sodium arsenite curative effect on GTDs, grapevines displaying esca-foliar symptoms from different French regions  were treated or not with sodium arsenite, and analyzed for their wood microbiota. Using metabarcoding, we identified the fungal and bacterial taxa composition of microbiota colonizing woody trunk tissues. Large differences in fungal microbiota composition between treated and untreated grapevines were observed while no major impacts were observed on bacteria microbiota. The main fungal species detected in untreated necrotic woody tissues was  Plasmopara parasitica; powdery mildew, Erysiphe necator; gray mold, Botrytis cinerea), there is currently no efficient method against Grapevine Trunk Diseases (GTDs), a threat for viticulture worldwide ever since the ban of sodium arsenite in the 2000s [Phaeomoniella chlamydospora, Phaeoacremonium minimum, Eutypa lata, Fomitiporia mediterranea, and Botryosphaeriaceae species  [P. chlamydospora, for instance, is frequently isolated from black punctuations and sectorial necrotic tissues, whereas F. mediterranea is mainly isolated from sectorial and central white-rot necrotic tissues [Although various fungicides or biocontrol agents control the grapevine foliar diseases  ,7,8. The parvum) . P. chla tissues ,11.Phomopsis spp., responsible for cane and leaf spot diseases [P. chlamydospora and F. mediterranea in trunks from esca-diseased plants [F. mediterranea and Botryosphaeria spp., isolated from esca-diseased grapevine trunks [Arsenic compounds were used in the 18th century as insecticides to protect plants against pests ,13. In vdiseases ,16,17,18diseases ,22,23,24d plants . In addie trunks . These oe trunks  and subsTo improve our understanding of sodium arsenite curative effect on grapevine trunk diseases, we analyzed its impact on fungal and bacterial wood microbiota of grapevines displaying esca-foliar symptoms from three French vineyards . We also assayed the sensitivity of different fungal species isolates from grapevine wood tissues to sodium arsenite. Overall, this study highlights the strong impact of sodium arsenite on the fungal community colonizing woody tissues of grapevine trunks with esca-disease symptomsThe sampling sites were located in three French vineyards, each cultivated with a specific cultivar ‘Gewürztraminer’ with rootstock 33EM, in Alsace ; ‘Chardonnay’ with rootstock SO4 or Fercal, in Champagne , and ‘Merlot’ with rootstock SO4, in Languedoc . These established vineyards, aged 25 for Alsace, 27 for Champagne, and 40 for Languedoc, were examined for esca-foliar symptoms in both 2013 and 2014. In 2013 and 2014, before sodium arsenite treatment in 2014 and 2015, 10 plants expressing esca-foliar symptoms were selected at random in each vineyard. Five of the 10 selected plants were treated with sodium arsenite  at 1250 g/hL until streaming, at the end of winter after pruning, and before bud bursting, BBCH 00 [The DNA extraction protocol used here was similar to that of Bruez et al. ,28. SSCPhttps://submit.ncbi.nlm.nih.gov/subs/sra/SUB8307285/metadata) (accessed on 1 April 2021). In brief, sequences were selected in accordance with the following initial criteria: (i) ≥450 nucleotides in length; (ii) a perfect match to the primers and barcodes; with (iii) no ambiguous base allowed. Although the bacterial primers were designed to amplify bacterial DNA, we verified, using Metaxa software, that no sequences corresponding to chloroplast or mitochondria DNAs were present in the bacterial dataset [https://www.mycobank.org) (accessed on 1 April 2021) and UNITE database [https://www.ncbi.nlm.nih.gov/sra/PRJNA732838) (accessed on 1 June 2021).Fungal and bacterial data were processed using the Quantitative Insights Into Microbial Ecology (QIIME) pipeline (version 1.7.0)   indexes with the EstimateS software package . To compPhaeomoniella chlamydospora, Fomitiporia mediterranea, and Eutypa lata or (ii) saprobes such as Trichoderma spp. and Penicillium spp. [Sodium arsenite  was suspended in sterile water and added to molten malt-agar medium poured into Petri dishes  at a concentration ranging from 1 to 500 mg/L, with MA as control. Each plate was inoculated with a 6 mm diameter mycelial plug from the actively growing margins of each tested fungal strain grown on MA. The culture was left at room temperature in the dark for 7–14 days, depending on the growth rate of the fungus tested. When the margin of the colony growing on MA without sodium arsenite (control) approximately was at 1 or 2 cm from the edge of the Petri dish, mycelial growth was recorded by measuring colony diameter. The growth tests in this study were performed in 2014 on fungi isolated from the trunks of treated and untreated grapevines . The funium spp. ,39. All The vineyards studied were located in Alsace, Champagne, and Languedoc. The grapevine cultivars sampled in each region were ’Gewürztraminer’, ’Chardonnay’, and ’Merlot’, respectively. In 2014 and 2015, 10 plants expressing esca-foliar symptoms were selected at random in each vineyard. Half of the plants were treated with sodium arsenite at the end of winter. All 10 plants, treated or not, were uprooted in September 2014 and 2015, respectively. All untreated plants displayed both esca-foliar symptoms, and central white-rot in woody tissues WR, . WR tissp = 0.0492; p = 0.007; fungal p = 0.049, p = 0.049; fungal index p = 0.049, p = 0.502; bacterial communities p = 0.328; p = 0.062). A comparison of the fungal microbiota between treated and untreated grapevines indicated major differences (p = 0.001). When focus was placed on the different types of tissue , further differences became apparent (p = 0.001). Bacterial communities showed differences for all three regions (p = 0.029). These differences did not depend on the simple distinction between treated and untreated grapevine (p = 0.203), but on the types of tissue involved (p = 0.001).Thirty-six DNA samples  in all wp = 0.492; p = 0.001; p = 0.001) in fungal communities when different types of woody tissue from untreated grapevines were studied. The SSCP fingerprint data of microbiota from white-rot necrosis and sectorial necrosis tissues strongly differed from those of non-necrotic tissues according to the PCA axis 2  and phylogenetic analyses . Most OTUs could be assigned to a single fungal species without ambiguity including all GTD associated fungi detected in these samples . Five OTF. mediterranea was the most abundant fungal species in Alsace and Champagne grapevines , an unknown Agaricales genus (0.3–14%), Cladosporium spp. (0.1–11.4%), and Mucor circinelloides (0–9.8%). White-rot necrotic woody tissues of untreated Alsace and Champagne grapevines were almost exclusively colonized by F. mediterranea . In all these tissues, F. mediterranea was associated with P. chlamydospora as the second most abundant species . These two fungi dominated the necrotic tissues  or tissue (p = 0.218). An additional statistical analysis of each species highlighted significant differences among tissues in the relative abundances of Bradyrhizobium spp., Curtobacterium spp., Erwinia spp., Pantoea spp., Pseudomonas spp., Sphingomonas spp., and Yersina spp. . Differences in relative abundance were plotted for each species/tissue combination by region . The second fungal species displaying a significant reduction in abundance upon sodium arsenite treatment was S. vitis , and by 800-fold the abundance of Aureobasidium pullulans in white-rot necrotic tissues of Champagne grapevines . The taxa compositions of these communities were characterized by using metabarcoding, revealing 718 fungal and 3886 bacterial OTUs. Significant differences in fungal microbiota were observed between treated and untreated grapevines, independently of their region or tissue origin. Sodium arsenite, however, had no significant impact on bacterial microbiota.F. mediterranea, a GTD fungus, was the most abundant species colonizing all trunk woody tissues of diseased grapevines from two of the three vineyards studied  compared to healthy ones, reaching up to 35% of all woody tissues. It should be noted that as this study used drilling for wood sampling, it was not possible to split different types of tissue. Another recent study of young French Cabernet Sauvignon grapevines (Bordeaux vineyard) showed that esca was always associated with the presence of white-rot necrotic tissues in cordons [F. mediterranea (>90%), as observed in the Alsace and Champagne grapevine trunks  and vineyard . Analysis showed that reported ,41. A re cordons . These we trunks . Our res 38-55%, . Still, nes 23%, . This fuapevines ,45,47. Tapevines . HoweverP. chlamydospora was detected in high abundance (>15%), specifically in borders between white-rot necrotic tissues and non-necrotic tissues in all three vineyards. In these borders, P. chlamydospora was the only other fungal species associated with F. mediterranea. No other GTD fungi exceeded 5% abundance in any woody tissues, except for D. seriata. This fungus, D. seriata, was detected in non-necrotic tissues of Alsace and Languedoc grapevines at 5.2% and 7%, respectively, and in sectorial necrosis of Languedoc grapevines up to 18%  than in other tissues  [F. mediterranea was mostly located. We also tested whether in vitro sensitivity to sodium arsenite differed among fungi. F. mediterranea was the most sensitive species among those tested . Sodium arsenite, however, did not affect the bacterial microbiota, either non-pathogenic or the pathogenic including bacteria that could be involved in wood degradation. Based on our results, it would be interesting to develop products as efficient as sodium arsenite on F. mediterranea. One possible way is to design fungicides mimicking sodium arsenite effects on wood microbiota. This goal should include a good delivery of the fungicide to woody tissues including white-rot and a high toxicity toward F. mediterranea.Sodium arsenite was efficiently used to protect and cure grapevines from GTDs before its ban. The present metabarcoding study showed sodium arsenite induced major changes in the fungal microbiota colonizing grapevine woody tissues. Its major effect was to decrease massively the abundance of


Bartonella are transmitted by blood-feeding arthropods and infect a large range of mammals. Although only several species have been identified as causative agents of human disease, it has been proposed that any Bartonella species found in animals may be capable of infecting humans. Within a wide-ranging survey in various geographical regions of the Czech Republic, cadavers of accidentally killed synurbic mammalian species, namely Eurasian red squirrel (Sciurus vulgaris), European hedgehog (Erinaceus europaeus) and Northern white-breasted hedgehog (Erinaceus roumanicus), were sampled and tested for Bartonella presence using multiple PCR reaction approach targeting several DNA loci. We demonstrate that cadavers constitute an available and highly useful source of biological material for pathogen screening. High infection rates of Bartonella spp., ranging from 24% to 76%, were confirmed for all three tested mammalian species, and spleen, ear, lung and liver tissues were demonstrated as the most suitable for Bartonella DNA detection. The wide spectrum of Bartonella spp. that were identified includes three species with previously validated zoonotic potential, B. grahamii, B. melophagi and B. washoensis, accompanied by ‘Candidatus B. rudakovii’ and two putative novel species, Bartonella sp. ERIN and Bartonella sp. SCIER.Free-living animals frequently play a key role in the circulation of various zoonotic vector-borne pathogens. Bacteria of the genus  Bartonella represents an ecologically successful group of bacteria distributed worldwide that has been found in a wide spectrum of blood-feeding arthropods and vertebrates, including humans [Bartonellae are vector-borne, facultative intracellular, fastidious, gram-negative alpha-2-proteobacteria with significant zoonotic potential. The genus g humans ,2,3  have been described, particularly in immunocompromised patients, with mild to serious symptoms  a wide range of mammalian hosts, including humans, carnivores, rodents, bats, ungulates, kangaroos and also marine animals such as sea otters and beluga whales [Bartonella infection has been detected [Bartonella spp. worldwide. The remarkably high infection rate, typically long-lasting bacteremia and vertical transmission observed in some rodents, along with the high level of diversity and frequently mixed infection of different Bartonella species and genotypes, strongly indicate that rodents play a key role as reservoir hosts [a whales ,10,14,15detected . Neverthir hosts ,18,19,20Bartonella DNA. Hedgehogs (Erinaceus europaeus and Erinaceus roumanicus) and squirrels (Sciurus vulgaris) tend towards synurbization, and they are often even more abundant in urban  areas than in natural habitats such as forests  sciurorum fleas [Bartonella infection in hedgehogs. In Algeria, B. elizabethae and B. tribocorum were detected in Atelerix algirus [B. elizabethae and B. clarridgeiae were detected in hedgehog fleas (Archeopsylla erinacei) collected from these hedgehogs [Erinaceus concolor, clustering with B. clarridgeiae and B. rochalimae, but a more precise determination was not possible [B. taylorii were detected in E. roumanicus from Hungary [B. henselae was identified in an A. erinacei flea collected from the same hedgehog species [Synurbic animals, which are frequently infested by ticks and fleas, represent a source of pathogens transmissible to humans. They also constitute a valuable source of biological material for vector-borne pathogen monitoring ,22,23. Is e.g., ,25). The. TheBarts e.g., ,26,27). ,27. Bartd . In the to date ,30,31,32 algirus  and, furedgehogs . In Israpossible . Two seq Hungary , and B.  species .Bartonella detection. Through multiple PCR targeting several DNA loci, the most suitable tissues for Bartonella detection and species identification were determined. The distribution, specificity and zoonotic potential of the detected Bartonella species are discussed considering various hosts worldwide.The main aim of this study was to verify the role of hedgehogs and squirrels as bartonellae hosts and to confirm the suitability of cadavers for Dissection of 55 Eurasian red squirrels, 83 European hedgehogs and 42 Northern white-breasted hedgehogs resulted in a total of 1429 tissue samples .Bartonella DNA using real-time PCR. Ear/skin, muscle, lung, liver, spleen, urinary bladder, kidney and brain tissue and blood were screened from most of the animals , the beta subunit of RNA polymerase (rpoB), and the cell division protein (ftsZ) encoding genes and the 16S–23S rRNA intergenic spacer (ITS). The ITS and gltA-1 were the most sensitive assays  and species identification by gltA-1, gltA-2 and rpoB assays was the most successful  did not allow for accurate species identification , followed by E. roumanicus  and E. europaeus   .Bartonella species were identified by DNA sequencing. Three well-established species, B. washoensis, B. grahamii and B. melophagi, were accompanied by ‘Candidatus B. rudakovii’ and two putative novel species, provisionally designated here as Bartonella sp. ERIN and Bartonella sp. SCIER. No obvious pattern in their geographical distribution was observed in this study , CZ-259 and CZ-262 (rpoB), CZ-23 and CZ-32 (ftsZ) and CZ-175 (ITS); for detailed information about all representative sequences see B. washoensis GenBank sequences. Phylogenetic analysis of the gltA sequences revealed two closely related genotypes represented by CZ-12 and CZ-146 within the B. washoensis clade  and also in two European hedgehog individuals  by amplifying and sequencing the gltA target. The corresponding sequence CZ-87 showed 100% similarity to B. melophagi sequences available in the GenBank (MT635399 and AY724768). ‘Candidatus B. rudakovii’ (EF682090) was the closest match (97.4%–99.1% similarity) for several sequences obtained exclusively from squirrels, represented by CZ-30, CZ-51 and CZ-52, obtained through gltA and rpoB targeted PCR protocols  showed 97.7% similarity to B. grahamii GenBank sequences and clustered to the B. grahamii clade in the phylogenetic analyses  showed 98.5% similarity to the B. grahamii GenBank sequences  and formed a well-supported clade along with B. clarridgeiae (95.6% similarity), B. rochalimae and ‘C. B. rudakovii’  generally aligns with previously published results [Bartonella DNA screening was demonstrated previously by Szekeres et al. [Bartonella spp. diversity) between the two hedgehog species, as none of the analyzed parameters  differed significantly . Our results differed slightly, with the highest detection efficiency for squirrels was found for spleen tissue (85%), followed by liver (71%), lung (70%) and ear (68%) tissues . The skin, muscle, blood, liver and brain tissues from the total of 85 blackbird (Turdus merula) and 11 song thrush (Turdus philomelos) carcasses [Bartonella DNA presence with the same approach used for the mammals in this study. None of these samples were positive, supporting the hypothesis that only mammals (and some reptiles) are hosts for bartonellae, but not birds.The species diversity of  results ,32,34,35s et al. , von Loes et al.  and Lipas . Lipato tissues . Howeverarcasses ,23 were Bartonella spp. identification solely through molecular methods is considered to be a challenging task. It has also been shown that sensitivity varies considerably among different PCR assays, and the primers used may favor detection of specific species in mixed infections [Bartonella species occurring in squirrels . In the comparison of five Bartonella spp. conventional PCR protocols used in this study, the gltA-1 and ITS protocols showed the highest sensitivity (approximately 60%). On the contrary, the ftsZ PCR assay successfully amplified only approximately 6% of the positive samples , and similarly, three species were detected by the rpoB assay. We conclude that the multiple PCR reactions approach revealed higher Bartonella species diversity, which would be missed if only a sole PCR assay were used (fections . Althoug samples . Neverthere used .B. washoensis have been recorded in three cases: two in North America [Otospermophilus beecheyi) seems to be a reservoir [B. washoensis in squirrels in the Netherlands, United Kingdom and Lithuania suggested that S. vulgaris may be a vertebrate reservoir of zoonotic B. washoensis in Europe [B. washoensis bacteria in squirrels strongly support this hypothesis. However, detection of B. washoensis in other mammalian species  indicatB. melophagi has been frequently found in sheep [Melophagus ovinus [Bartonella species is highly host- and vector-specific [B. melophagi infections may cause human disease [Odocoileus virginianus [Hippobosca equina [Haemaphysalis qinghaiensis and Dermacentor everestianus [B. melophagi in a European hedgehog contribute to the discussion about host specificity. in sheep ,44 and ss ovinus ,47,48,49specific ,44,50. Nginianus , forest estianus . Our finBartonella species belong to the B. rochalimae clade  data concerning detection of this candidate species in bank voles (Clethrionomys glareolus) in Western Siberia. According to the sequence similarity, this species was also detected in C. glareolus in Lithuania  detected from Tamiasciurus sp. [Candidatus B. rudakovii’ and likely represents the same species (or its variant). All these sequences were the closest GenBank matches for several sequences obtained from eleven squirrels in this study (represented by CZ-30 and CZ-51 (gltA) and CZ-52 (rpoB)), and thus could be considered as variant(s) of ‘Candidatus B. rudakovii’. Since we were able to detect this Candidatus species by three different PCR protocols  in one individual (squirrel no. 51), we suppose all of the sequences  may constitute a single variant or even genotype. However, mixed infections with several Bartonella species or strains in one animal are common, and there is always the possibility of getting amplicons from the same sample that actually belong to different Bartonella species or genotypes coexisting in the same individual. However, all these sequences also show high similarity with B. rochalimae, a well-recognized zoonotic Bartonella species reported as a causative agent of human disease [B. rochalimae . In oururus sp.  also sho disease ,54. It he  since te  for f humans , cats  worldwide  and several rpoB sequences  obtained from all three targeted mammalian species and designated here as Bartonella sp. SCIER. According to the BLASTn analysis only, these sequences could be considered variants of B. grahamii. On the contrary, phylogenetic analyses indicated Bartonella sp. SCIER as a distinct species , the Northern white-breasted hedgehog (Erinaceus roumanicus) and the Eurasian red squirrel (Sciurus vulgaris) were collected in various geographical regions of the Czech Republic for a project addressing the role of these species in the circulation of vector-borne pathogens and their possible use as sentinel hosts. All of these animals were found dead, mostly as roadkill, killed by other animals  or had died in animal rescue centers. The cadavers were kept at −20 °C until processing. Cadaver collection, dissection, tissue sample preparation and DNA extraction were performed as described previously [A total of 180 cadavers of the European hedgehog  gene [ssrA-F 5′-GCT ATG GTA ATA AAT GGA CAA TGA AAT AA-3′; ssrA-R 5′-GCT TCT GTT GCC AGG TG-3′) and probe (5′-ATTO520-ACC CCG CTT AAA CCT GCG ACG-BHQ1-3′) in 10 μM concentration was used to detect Bartonella spp. in all tissue samples. For real-time PCR, an iQ Multiplex Powermix PCR reagent kit containing iTaq DNA polymerase  was used. Real-time PCR was performed in a LightCycler 480 Real-Time PCR System  under the following conditions: an initial activation of the iTaq DNA polymerase at 95 °C for 5 min, 60 cycles of a 5 s denaturation at 94 °C, followed by a 35 s annealing-extension step at 60 °C (single point measurement at 60 °C), and a cooling cycle of 37 °C for 20 s. The analysis was performed using second derivative calculations for Cp (crossing point) values. Curves were inspected visually in LightCycler 480 software. A negative control (no template) and positive control (B. henselae DNA) were added to each set of samples.All PCR reactions were set up in separate rooms with all required precautions . Real-time PCR targeting a fragment of the NA) gene  with forBartonella-DNA by real-time PCR (at least in one tested tissue sample) were subsequently analyzed by six different conventional PCR assays targeting citrate synthase (gltA), the beta subunit of RNA polymerase (rpoB), and the cell division protein (ftsZ) encoding genes and the 16S–23S rRNA intergenic spacer (ITS)  confirmed the real-time PCR positive results.Hedgehogs and squirrels that tested positive for er (ITS) . For theer (ITS)  per animgltA-1, rpoB, ITS) or second PCR (ftsZ), filled with PCR-grade water. The PCR products were visualized using 1.5% agarose gel electrophoresis under UV light, cleaned with ExoSAP-IT™ PCR Product Cleanup Reagent , and direct Sanger sequencing was provided by SEQme , BaseClear , or Hy Laboratories Ltd. . The sequences were checked manually using Geneious software (version 10.0.6) and compared to sequences from the NCBI GenBank database for Bartonella species identification. All the partial gltA gene sequences of related recognized Bartonella species available in the GenBank database were downloaded and used for phylogenetic analyses . Alignments were generated using MAFFT v.7. The final gltA dataset contained 55 different sequences (representative taxa) and 1199 characters  was used for all conventional PCRs according to the manufacturer’s instructions. A total volume of 20 μL was prepared for each reaction containing 10 μL of the master fco, 10 pmol of each primer, 2 μL of template DNA or 1 μL of PCR product from the first PCR in the case of nested PCR , followed by E. roumanicus (42%) and E. europaeus (24%). The current study extends the known host range of Bartonella species with ascertained zoonotic potential, B. washoensis and B. melophagi. Moreover, two probably new, previously uncharacterized Bartonella species were found in hedgehogs and squirrels. We conclude that hedgehogs and squirrels are suitable hosts for various zoonotic Bartonella spp. and are most likely involved in their maintenance in the environment.In our project, we focused on three vertebrate species: the European hedgehog, the Northern white-breasted hedgehog, and the Eurasian red squirrel and their role as hosts of


Callicarpa integerrima var. chinensis is one of the traditional medical herbs which has the potential for multiple diseases’ treatment. In this study, the complete chloroplast genome sequence of C. integerrima var. chinensis was sequenced and assembled. A typical quadripartite structure was observed in the chloroplast genome of C. integerrima var. chinensis which was 154179 bp in length, including a pair of inverted repeat (IR) regions (each 51396 bp) separated by a large single-copy region (LSC) of 84927 bp and a small single-copy region (SSC) of 17856 bp, and the overall GC contents of the chloroplast genome was 38.08%. Additionally, we annotated 132 genes, including 86 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. Phylogenetic analysis was adopted which confirmed the position of C. integerrima var. chinensis was close to the congeneric C. formosana and C. siongsaiensis. Callicarpa integerrima var. chinensis (C. Pei) S. L. Chen is a herbaceous plant Lamiaceae . Voucher specimen of C. integerrima var. chinensis was deposited at the herbarium of College of Pharmacy, Guilin Medical University  under the voucher number HCP-LJS-202104001). The total genomic DNA was extracted from fresh leaves of the sample using a CTAB method , a small single-copy region  and two inverted repeat regions . The GC content of the chloroplast genome is 38.08%. Annotation results showed the presence of 132 unique genes including 86 protein-coding, 38 tRNA and 8 rRNA genes. Seven protein-coding, seven tRNA and all four rRNA genes were duplicated in the IR regions. The chloroplast genome structure, gene order, GC content and codon usage are similar to typical angiosperm chloroplast genomes (Qian et al. The complete chloroplast genome of C. integerrima var. chinensis, complete chloroplast genome sequences of this taxon and 17 other species selected from the Lamiaceae and one outgroup were used to construct phylogenetic tree. Vitex rotundifolia was selected as the outgroup of the phylogenetic tree. This study also used 58 protein-coding genes to conduct a ML analysis under the GTR + R + I model with 1000 bootstrap replicates. The phylogenetic tree fully resolved C. integerrima var. chinensis in a clade with C. formosana and C. siongsaiensis (C. integerrima var. chinensis reported here contributes to future studies on population genetics, evolutionary, and conservation biology in the Lamiaceae.To confirm the phylogenetic position of saiensis . By comp


Background: Emerging evidence shows that m.5178C>A variant is associated with a lower risk of coronary artery disease (CAD). However, the specific mechanisms remain elusive. Since dyslipidemia is one of the most critical risk factors for CAD and accounts for at least 50% of the population-attributable risk, it is tempting to speculate that the reduced CAD risk caused by the m.5178C>A variant may stem from an improved lipid profile. In order to verify this hypothesis, we conducted the present study to clarify the association of m.5178C>A variant with lipid levels.Methods: By searching ten databases for studies published before 30 June 2021. Thirteen East Asian populations  were included for the analysis.Results: The present study showed that m.5178C>A variant was associated with higher high-density lipoprotein cholesterol (HDL-C)  and total cholesterol (TC)  levels. In subgroup analysis, the association of m.5178C>A variant with higher HDL-C levels were observed in Japanese  and Chinese populations . However, the association of m.5178C>A variant with lower low-density lipoprotein cholesterol (LDL-C) levels were only observed in Japanese populations .Conclusions: The m.5178C>A variant was associated with higher HDL-C and lower LDL-C levels in Japanese populations, which may contribute to decreased CAD risk and longevity of Japanese. The human mitochondrial genome is closely associated with the human lifespan, and each human cell contains hundreds to thousands of copies of the mitochondrial genome, which encode 13 polypeptides .ND2) gene of mitochondrial DNA (mtDNA) and formed by a transversion from cytosine (C) to adenine (A). The mtDNA-ND2 m.5178C>A variant leads to replacing leucine with methionine (p.Leu237Met).The m.5178C>A variant is rare worldwide  and is t−/−) caused severe dyslipidemia [A series of mouse model experiments  showed tipidemia . Togetheipidemia . Moreoveipidemia  reportedipidemia . When coMoreover, it was well documented that ,7,16 varOver the last decade, intensive efforts  have beeThe studies regarding the relationship between m.5178C>A variant and acute myocardial infarction (AMI) were limited but consistent. For instance, Mukae et al.  reportedA comprehensive literature search was performed from 31 March 2021 to 30 June 2021 by using ten databases including PubMed, Medline, Embase, Cochrane Library, Web of Science, Google Scholar, Foreign Medical Journal Service, Excerpta Medica, CNKI, Wanfang. The following keywords were used in the search: ,  and . Additionally, the reference lists of all eligible studies were manually retrieved to obtain more literature.The specific inclusion criteria were listed: (1) The test subjects must be limited to the East Asian population. (2) The studies investigated the association of m.5178C>A variant with serum lipid levels. (3) The studies at least provided one of four parameters in lipid profile (triglyceride (TG), TC, low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C)). (4) The studies provided the frequency of m.5178C>A variant. (5) The studies offered the mean lipid levels with standard deviation (SD) or standard errors (SE) by m.5178C>A variant. (6) The interventional articles provided pre-intervention data. (7) The language of eligible studies was restricted to English or Chinese. The specific exclusion criteria were as follows: (1) The studies did not relate to m.5178C>A variant. (2) The studies did not relate to lipid levels. (3) The studies did not present the frequency of m.5178C>A variant. (4) The studies provided invalid data. (5) The studies provided incomplete data. (6) Pedigree studies. (7) Overlapping studies. (8) Abstract, review, case report, meta-analysis and animal studies.Two authors (Fuqiang Liu and Shengping Wang) extracted the data independently using a standardized data extraction table. The discrepancy in data extracted was resolved by consensus or a discussion with the third author (Zhi Luo). If critical data were absent, e-mail or telephone was used to contact the corresponding author to acquire this information. The following data were extracted from each eligible study: the last name of the first author, year, country , gender , ethnicity , the frequency of m.5178C>A variant, genotyping methods, type of study, type of disease, total sample size, mean lipid levels with SD or SE by m.5178C>A variant.P<0.05 was recognized as statistically significant. The standardized mean difference (SMD) and 95% confidence interval (CI) were used to evaluate the differences in lipid levels.The units of TG, TC, LDL-C and HDL-C were converted into mmol/l. All extracted data were expressed as mean ± SD. All the analyses were performed by STATA software . measurement results among a series of studies were defined as ‘heterogeneity’ [2I statistic and Cochran’s χ2-based Q statistic. Galbraith plots were used to detect the potential sources of heterogeneity. If heterogeneity was significant , the random-effects model (DerSimonian–Laird method) was used to calculate the results [Inevitably, there were differences between the studies included in the meta-analysis. The differences or diversity between participants, interventions, and the geneity’ . Heterog results . OtherwiThe publication bias among the included studies was evaluated by Begg’s funnel plot and Egger’s linear regression test .Subgroup analysis was carried out by ethnicity, gender and health status. The ethnicity was divided into Chinese and Japanese. The health status was divided into type 2 diabetes mellitus (T2DM) patients and healthy subjects. In some studies, the subjects were divided into more than one subpopulation . Each subpopulation was regarded as an independent comparison in the present study.Sensitivity analysis was conducted in the present meta-analysis, in which the comparison was excluded one by one and performed the analysis again after omitting each comparison. If the synthetic results in any study changed substantially to alter the results from significant to non-significant or the other way around. The absence of such a phenomenon usually indicates the robustness and stability of synthetic results.By searching the above ten databases, 70 studies were identified. After the screening, 28 studies were excluded by their title and abstract. Next, 42 studies were estimated by their contents. In which, 23 studies did not provide lipid data, 5 studies  had lipiThe references of the included studies were listed in Supplementary Material. The characteristics of the included studies were presented in Supplementary Table S1. The serum lipid levels by m.5178C>A variant were presented in Supplementary Table S2.P=0.001)  .Then the subgroup analysis was carried out . SubgrouThe analysis that excluded the studies with heterogeneity was also carried out . HoweverP=0.01) changed significantly after excluding these comparisons.In analyzing the association of m.5178C>A variant with lipid levels, significant heterogeneity was detected in TG, TC and HDL-C . Four K. HoweverP=0.66) did not change significantly after excluding this comparison.Sensitivity analysis showed that no comparison might affect the association of m.5178C>A variant with TG A, TC FiB and HDLP=0.78 for TG, P=0.37 for TC, P=0.22 for LDL-C and P= 0.31 for HDL-C).Begg’s funnel plot was used to evaluate the publication bias among the included studies. However, no publication bias was observed in synthetic results A–D, whicThe present study showed that the m.5178C>A variant was significantly associated with higher HDL-C and TC levels. Subgroup analysis showed the significant association of m.5178C>A variant with higher HDL-C levels was primarily in Japanese and Chinese populations. While the significant association of m.5178C>A variant with lower LDL-C levels was only observed in Japanese populations.237Met in mtDNA-ND2 may affect NADH dehydrogenase’s function and thus inhibit ROS release [The mechanisms underlying the association of m.5178C>A variant with lipid levels remain elusive. However, one hypothesis could be proposed to explain our findings, that is, by protecting against oxidative stress. The p.Leu release . Moreove release  showed t release . Therefo release , in whic release ,41–44.In the present study, the significant association of m.5178C>A variant with higher HDL-C and TC levels was observed in Chinese , indicatThe mechanisms underlying the effects of m.5178C>A variant on longevity have not been fully clarified. However, it is now increasingly evidenced that the higher levels of HDL-C ,45–47 anSeveral limitations of the present meta-analysis should be noted. First of all, a large number of genes as well as some environmental factors are involved in dyslipidemia. However, this meta-analysis has not investigated the interactions of m.5178C>A variant with other variant loci or environmental factors on serum lipid levels due to the lack of original data from the included studies. Secondly, a relatively small number of individuals have been included in the lipid association analysis for m.5178C>A variant due to the limited number of studies that met the inclusion criteria, which may reduce the statistic power and even cause type I error . Thirdly, this meta-analysis only included the studies published in English and Chinese as it is very difficult to get the full papers published in various languages.The m.5178C>A variant was associated with higher HDL-C and lower LDL-C levels in Japanese populations, which may contribute to decreased coronary artery disease (CAD) risk and longevity of Japanese.Click here for additional data file.Click here for additional data file.


Helicobacter pylori- (H. pylori-) associated gastric cancer (GC). In this study, an integrated analysis of DNA methylation and gene expression was conducted to identify some potential key epigenetic markers in H. pylori-associated GC. DNA methylation data of 28 H. pylori-positive and 168 H. pylori-negative GC samples were compared and analyzed. We also analyzed the gene expression data of 18 H. pylori-positive and 145 H. pylori-negative GC cases. Finally, the results were verified by in vitro and in vivo experiments. A total of 5609 differentially methylated regions associated with 2454 differentially methylated genes were identified. A total of 228 differentially expressed genes were identified from the gene expression data of H. pylori-positive and H. pylori-negative GC cases. The screened genes were analyzed for functional enrichment. Subsequently, we obtained 28 genes regulated by methylation through a Venn diagram, and we identified five genes  downregulated by hypermethylation. HUS1, GSTO2, and TMEM190 were expressed at lower levels in GC than in adjacent samples (P < 0.05). Moreover, H. pylori infection decreased HUS1, GSTO2, and TMEM190 expression in vitro and in vivo. Our study identified HUS1, GSTO2, and TMEM190 as novel methylation markers for H. pylori-associated GC.Previous studies have shown that abnormal methylation is an early key event in the pathogenesis of most human cancers, contributing to the development of tumors. However, little attention has been given to the potential of DNA methylation patterns as markers for  Helicobacter pylori (H. pylori) is a microaerophilic, spiral-shaped gram-negative bacterium that colonizes the stomach and adheres to the gastric mucosa [H. pylori has been confirmed to have close relationships with gastritis, peptic ulcer, gastric cancer (GC), and mucosa-related lymphoid tissue lymphoma [H. pylori infection is the most definite risk factor for GC [H. pylori infection [H. pylori-induced GC is still unclear. Moreover, specific molecular markers to identify subjects at high risk of H. pylori-associated GC need to be explored.c mucosa . H. pylolymphoma . H. pylor for GC . Out of nfection . CurrentH. pylori infection is related to hypermethylation of gene promoters [H. pylori-associated GC are still scarce. Moreover, the molecular mechanism of the abnormal methylation of gene regulatory regions involved in the generation and development of GC remains elusive.One of the key regulators involved in the environmental response is the methylation of genomic regulatory regions. The methylation of CpG islands in gene promoters is involved in the regulation of gene expression. DNA methylation abnormalities are the main epigenetic mechanism related to gene silencing and play an important role in tumorigenesis , 6. Overromoters . HoweverH. pylori-related GC by analyzing the methylation and gene expression data of The Cancer Genome Atlas (TCGA) database, combined with related experiments.This study is aimed at exploring the potential methylation-related gene markers in https://cancergenome.nih.gov/). The RNA-Seq data and clinical information of 407 samples  were obtained from UCSC Xena (https://xenabrowser.net/datapages/). A flowchart of the process of data acquisition and the subsequent analysis is shown in DNA methylation data from 397 samples  were obtained from TCGA (H. pylori infection information (20 positive and 168 negative) were included and analyzed. The minfi package [P value, and ∣log2FC | >1, P < 0.01 were set as the thresholds to screen for differentially methylated sites.After screening, a total of 188 cases with  package  was usedH. pylori infection were screened from the tumor samples. A total of 163 GC samples (18 with H. pylori and 145 without H. pylori) were included in the subsequent analysis. The analysis used the R language edge package [H. pylori-positive and -negative groups. The threshold of differential gene selection was set as P < 0.01. The results were visualized by volcano map.The samples with clear clinical information of  package  to analyThe genes obtained from differential gene analysis and differential methylation analysis were enriched and analyzed with the R language cluster profiler package , and theH. pylori-associated GC. In this analysis, the methylation sites of each gene in GC patients infected by H. pylori and those not infected by H. pylori, as well as the expression of each gene in the two groups, were visualized by box plots, and the Wilcoxon rank-sum test was performed. Ultimately, 5 genes with significantly upregulated methylation levels and significantly downregulated gene expression levels in the H. pylori-positive group were screened out.The results of the intersection of the differentially expressed genes (DEGs) obtained from the differential gene analysis and the differentially methylated genes (DMGs) obtained from the differential methylation analysis were taken as the genes affected by the methylation of 2. H. pylori 7.13  was cultured on Campylobacter agar plates containing 10% sheep serum under microaerophilic conditions at 37°C. The concentration of the bacteria was determined by spectrophotometry (OD600 nm) after suspension in DMEM/F12. Subsequently, the cell culture medium was replaced with fresh medium without antibiotics, and the cells were cocultured with H. pylori at different times or multiplicities of infection (MOIs).AGS cells (human GC cells) were cultured in DMEM/F12  containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco) at 37°C in an atmosphere of 5% COThe harvested cells were lysed with lysis buffer containing protease inhibitor cocktail . Protein samples were separated by SDS–PAGE and transferred to nitrocellulose membranes. The blocking buffer was prepared with 5% nonfat dry milk in TBST buffer, and the membranes were blocked at room temperature for 1 hour and then incubated with primary antibodies in a shaker at 4°C overnight. Antibodies were anti-GSTO2 , anti-HUS1 , anti-INTS1 , anti-TMEM184A , anti-TMEM190 , and anti-Actin . The membranes were incubated with the corresponding secondary antibodies (1 : 2000) for 1 hour at room temperature the next day, and finally, the proteins were visualized with chemiluminescence solution.H. pylori-positive, and 12 H. pylori-negative GC specimens) were collected from GC patients who underwent gastrectomy at the First Affiliated Hospital of Nanchang University. Gastric mucosal tissues of patients who underwent gastroscopic biopsy at the First Affiliated Hospital of Nanchang University (12 H. pylori-positive and 12 H. pylori-negative patients with gastritis) were collected. The diagnoses of GC and gastritis were confirmed based on histology, and the diagnosis of H. pylori infection was based on immunohistochemistry or culture results. All subjects provided informed consent for obtaining the study specimens. The study protocol was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Nanchang University. The gastric mucosal tissues of the mice were derived from C57BL/6 mice infected with the H. pylori SS1 strain successfully constructed by our research group [H. pylori infection group).Human gastric tissue samples  was then used to extract total tissue RNA, after which SYBR® Premix Ex Taq (TaKaRa) was used for qRT–PCR. The primers used for the detection of human specimens: t-test was used for comparison between two groups. P < 0.05 was considered statistically significant.The Student's M value. As shown in H. pylori-positive and -negative samples were not well distinguished. This might be due to the small number of H. pylori-positive samples.The methylation data were normalized and standardized. As shown in Figure According to the screening threshold, a total of 5609 methylation results with significant differences were obtained. The results without corresponding genes and the results of one methylation corresponding to multiple genes were excluded. Finally, a one-to-one methylation-gene relationship was obtained, and a total of 3298 results were obtained for subsequent analysis. The repeated genes were removed, and 2454 DMGs remained , of whicThe R package Cluster profiler was used for enrichment analysis to investigate the functions of the DMGs. As shown in H. pylori-positive and H. pylori-negative samples were not clearly divided into two clusters  were visualized. Finally, five genes with reduced expression that may be caused by hypermethylation were obtained, namely, GSTO2 , HUS1 (cg10190813), INTS1 (cg07005770), TMEM184A (cg10633906), and TMEM190  . We collected H. pylori-positive and H. pylori-negative chronic nonatrophic gastritis (CNAG) and GC specimens and found that HUS1 and GSTO2 were significantly downregulated (P < 0.05) in the H. pylori-positive group of CNAG samples and GC samples. The mRNA level of TMEM190 was also downregulated in the H. pylori-positive group (P < 0.05), even though the difference was not statistically significant in the GC samples. In addition, we verified these genes at the animal level and found that the mRNA levels of GSTO2 and TMEM190 were significantly downregulated in the gastric mucosa of H. pylori-infected mice (P < 0.05) , HUS1 (cg10190813), INTS1 (cg07005770), TMEM184A (cg10633906), and TMEM190 . Studies have shown that the HUS1 gene is involved in DNA damage repair and apoptosis and has a clear relationship with the occurrence and development of tumors [c mucosa . Similar unclear . This stH. pylori-associated GC will help to improve the accuracy of prognosis prediction. However, this study has some limitations. First, more independent datasets are required to further validate the prognostic value of the genes. Second, additional experimental studies are needed to understand their functional effects.In the process of cancer occurrence and development, DNA methylation changes may be detected earlier than other types of prognostic markers, with higher clinical sensitivity and dynamic range. Therefore, the combination of these candidate genes with other genetic and transcriptional events of H. pylori infection, and that their expression levels were lower in GC. We demonstrated that HUS1, GSTO2, and TMEM190 may play an important role in the pathogenesis and carcinogenesis of H. pylori infection and may be related to the methylation process. This study not only increases the understanding of the potential molecular mechanism of H. pylori-associated GC but also proves the role of abnormal DNA methylation in H. pylori-associated GC.In conclusion, our study reveals five genes affected by methylation using public expression and epigenetic datasets. We further found that HUS1, GSTO2, and TMEM190 can be downregulated by


Scientific Reports 10.1038/s41598-021-82731-1, published online 04 February 2021Correction to: In the original version of this Article, Yasuyuki Watano was omitted as a corresponding author. Correspondence and request for materials should also be addressed to watano@faculty.chiba-u.jp. In addition, the authors Ryosuke Imai and Yoshiaki Tsuda were omitted as equally contributing authors.The original Article and accompanying Supplementary Information file have been corrected.


B. cereus agar was compared to the reference mannitol-yolk polymyxin (MYP) agar (ISO 7932) using inclusion and exclusion test strains (n = 110) and by analyzing naturally contaminated milk and other food samples (n = 64). Subsequently, the panC group affiliation and toxin gene profile of Bacillus cereus senso lato (s.l.) isolates were determined. Our results corroborate that the overall best performing media CHROMagar™ B. cereus  and BACARA®  are more sensitive and specific compared to Brilliance™ B. cereus, MYP and ChromoSelect Bacillus Agar. Both media allow unequivocal detection of B. cereus with low risks of misidentification. Media containing ß-D-glucosidase for the detection of presumptive B. cereus may form atypical colony morphologies resulting in a false negative evaluation of the sample. Naturally contaminated samples presented high numbers of background flora, while numbers of presumptive B. cereus were below the detection limit (<10 CFU g−1 or mL−1). Recovery after freezing resulted in the highest detection of B. cereus s.l. on BACARA® (57.8%), CHROMagar™ B. cereus (56.3%) and MYP agar (54.7%). The panC/toxin profile combination IV/A was the most abundant (33.0%), followed by III/F (21.7%) and VI/C (10.4%). More panC and toxin combinations were present in 15.6% of samples when reanalyzed after freezing. In order to improve detection and confirmation of B. cereus s.l. in food samples, we recommend the parallel use of two complementary selective media followed by molecular characterization . When determining psychrotolerant or thermophilic members of the B. cereus group, the selective agar media should additionally be incubated at appropriate temperatures . If high-risk toxin genes  are detected, the strain-specific ability to produce toxin should be examined to decisively assess risk. In this study, the performance of four alternative selective chromogenic  Bacillus senso latu (s.l.) consists of Gram-positive, rod-shaped, aerobic or facultative anaerobic spore-forming bacteria that are widespread in the environment and commonly found in soil, plant material and in the gut of insects  g−1) was set within the Commission Regulation (EC) No 1441/2007 . In addition, warning values are available, for example, for dried herbs and spices; tofu and bakery products (4 log CFU g−1); or fruits and vegetables, cereals, pasta, mayonnaises, dressings, soups and ready-to-eat instant products (3 log CFU g−1) .In order to protect consumer health, a process criterion for presumptive B. cereus s.l. is detected and enumerated on classical culture media as for example mannitol egg yolk polymyxin (MYP) agar. Chromogenic reactions rely on enzymatic cleavage  of a particular substrate and the release of a chromogen, which is more specific than conventional microbiological growth media. Some chromogenic media additionally detect PLC activity in order to facilitate unambiguous identification. Selectivity is achieved in both media types by the addition of antibiotic substances , which inhibit the growth of undesirable Gram-positive and Gram-negative bacteria [In current practice, presumptive bacteria ,12.B. cereus s.l. counts, strain-specific properties such as toxin gene profiles or other virulence factors need to be investigated for risk characterization efforts. A broad range of phenotypical  and genotypical methods  and panC-typing) are required to accurately identify and group B. cereus s.l. on the species level [Apart from es level ,14,15, rB. cereus s.l. and cereulide. However, database richness is decisive for accurate species identification, and an enhanced cloud-based exchange of spectral data would be necessary for propagation [Fourier transform infrared (FTIR) spectroscopy and Matrix-Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometry have been developed to speed up the identification and characterization of pagation ,18,19,20B. cereus s.l. and accelerate the time to result by easier visual evaluation of morphology and color changes of media.As many innovative methods are established exclusively in expert’s laboratories, there is still the need for rapid and unambiguous isolation and differentiation methods applicable in food and dairy plant laboratories. Contemporary chromogenic media may represent a useful tool to facilitate identification of B. cereus group members by using a bacterial test strain panel and analyzing naturally contaminated samples under everyday conditions. Furthermore, an in-depth molecular-biological characterization of inclusivity test strains and sample isolates was performed to explore strain-specific features.This study was initiated to assess and compare the performance of the ISO standard medium MYP agar with four alternative chromogenic selective plating media for detection and enumeration of food-intoxication and spoilage-associated Bacillus , CHROMagar™ B. cereus , Brilliance™ B. cereus  and BACARA® agar  was evaluated in comparison to the ISO recommended standard medium MYP [Within the scope of this study, the performance of commercially available chromogenic selective media ChromoSelect dium MYP   consisting of B. cereus target organisms  and non-target Bacillus spp.  was compiled. B. cereus s.l. strains originated from emetic and diarrheal outbreaks , environmental samples, fruits and vegetables, cereals, fish, tea, herbs, spices, milk and dairy products  at the specified conditions , selective media were inoculated. In order to obtain a few well-defined bacterial colonies, an isolated single colony from the working culture was transferred onto selective nditions , the pren = 64) from 18 producers were collected from the production chain and retail level. Food samples  belonged to the source categories “fruits and vegetables”, “nuts, nut products and seeds”, “fish and fishery products”, “herbs” and “cocoa and cocoa preparations, coffee, and tea”  were heat treated, except for one raw milk sample (bactofugation) provided by a local dairy  were produced organically. Milk samples were examined after 24 h provocation at 30 °C for enrichment to ensure detection of B. cereus s.l. All food samples were analyzed before and after freezing at −20 °C to trigger outgrowth of spores.Important information regarding processing was gathered, including the type of milk with reference to animal species , agricultural system , processing and predicted shelf-life  . The maj®; Seward Ltd., West Sussex, UK). Ten-fold serial dilutions in sterile Ringer’s solution  were plated in duplicate up to 10−5 on selective agar media by using the spatula method. Following incubation, growth was assessed, and colonies displaying characteristic morphology were enumerated to determine the extent of B. cereus s.l. contamination. Randomly picked colonies with typical and atypical morphology were isolated, subjected to confirmation and characterized with regard to panC group affiliation and toxin gene profile.In order to prepare sample homogenates, 25 mL or 25 g of food product was diluted 1:10 in sterile buffered peptone water ; food samples were additionally mixed for 180 s in a paddle blender (StomacherB. cereus s.l. cultures grown overnight on TSA-Y at 30 °C (Biokar Diagnostics and Merck KGaA) using the Chelex® 100 resin  method as described by Walsh et al. [Bacterial DNA was extracted from h et al. . After eBacillus cereus s.l. strains from culture collections and presumptive isolates from naturally contaminated food products and milk were confirmed as group members by PCR method targeting the gyrase B gene (gyrB) as described by Dzieciol et al. [l et al. .B. cereus s.l. strains were screened for their toxin gene content by conventional PCR assays. Amplification was performed according to Ehling-Schulz et al. [nheA, nheB, hblA and hblD. Furthermore, PCR pre-screening assay were applied for cytK-1/cytK-2. Detection of the emetic toxin cereulide gene ces was performed after Dzieciol et al. [Confirmed z et al.  with minl et al.  with slil et al.   gene were conducted as previously reported [B. cereus s.l. strains to one of the seven major phylogenetic groups  defined by Guinebretière et al. [https://www.tools.symprevius.org/Bcereus/english.php, accessed on 17 December 2021)  of a fragment of the pantothenate synthetase  after overnight incubation at 30 °C  Supplem.In order to differentiate the phenotypic appearance of test strains and evaluate their potential for misidentification, the growth of inclusivity and exclusivity strains was classified in typical and atypical according to their reaction(s) and colony morphology on selective media .B. cereus s.l. in naturally contaminated samples was illustrated in a bar plot . The relative frequency of panC group (II–VI) and toxin gene profile (A–F) combinations among B. cereus s.l. isolates (n = 106) associated with naturally contaminated samples was depicted as pie chart . Graphics were created with open-source statistical computer environment R version 4.1.0 [A mosaic plot was used for visualizing the results of growth and phospholipase C reactions of inclusivity and exclusivity test strains for each of the tested media MYP, HI, BRI, CH and BA . Detectability of on 4.1.0 .n = 110) were assigned to toxin profile C  and A . The ces gene was present in six (5.5%) test strains derived from foodborne outbreaks. Among the target test strains, panC group III , IV  and VI  were the most abundant. The most frequent combination of panC group and toxigenic profile in the entire panel of inclusivity test strains was IV/A , obtained from milk and dried products . Other common combinations were VI/C  isolated from milk, soil and salad, as well as III/D  mainly detectable in strains isolated from protein-rich food .The detailed strain properties of the inclusivity test strains are presented in n = 109–110/110); one B. pseudomycoides strain did not grow on three selective media (n = 14), HI  and CH , resulting in an atypical white phenotype (panC-type/toxin profile VI/C. On chromogenic media (CH and BA), the PLC reaction was more distinct in comparison to MYP agar  and BA . Several non-target organisms were not effectively suppressed by polymyxin B in MYP and  and HI  (n = 110) was also observed on BRI , although we only observed colony morphologies that could not be misidentified as presumptive B. cereus due to their atypical pin-point growth (Best performing media in terms of exclusivity  were CH  97/110) . Comparat growth .Listeria monocytogenes, Paenibacillus polymyxa and Serratia marcescens).PLC reaction typical for the target organisms was observed in three and two exclusivity tests strains on MYP and BA, respectively (n = 51) and food (n = 13) samples analyzed were contaminated with presumptive B. cereus at the limit of detection, resulting in quantitative data below 10 and 100 CFU g−1, respectively. Further details on sample characteristics can be found in B. cereus s.l. colonies grown on selective media test panel are shown in Milk (B. cereus group containing samples with respect to the distribution of the panC group in combination with toxin profiles. The samples were negative in PCR confirmation of the emetic toxin gene (ces); in consequence, the toxin profiles B  and E (nhe and ces gene combination positive) were not detected. The panC/toxin profile combination IV/A was the most abundant in the sample set (33.0%), followed by III/F (21.7%) and VI/C (10.4%). Representatives of panC group IV are described as highly cytotoxic and do generally grow at temperatures ≥10 °C. Toxin profile A represents nhe, hbl and cytK gene (cytK-2) positive isolates. The enterotoxin genes nhe, hbl and cytK-2 are located in the chromosome of different species of the B. cereus group, whereas the cytK-1 gene is harbored exclusively by thermophilic species B. cytotoxicus (panC group VII). Representatives of panC group VII were not detected in any sample. panC group III is considered highly cytotoxic and is representative of B. cereus group grown at temperatures of ≥15 °C. The carriage of nhe gene (non-hemolytic enterotoxin) characterizes toxin profile F. Strains affiliated to panC group VI, which low cytotoxic and grown at ≥5 °C. Toxin profile C is characterized by the presence of nhe and hbl genes [https://www.tools.symprevius.org/bcereus/english.php; accessed on 17 December 2021).bl genes  . Recovery after freezing was tested using the selective media test panel, and it resulted in the highest recovery on BA , CH  and MYP  (n = 6) for presumptive B. cereus s.l. recovery before and after freezing was observed for milk samples of different origin. panC group and toxin gene profile combinations of B. cereus s.l. detected before and after freezing are provided in panC group and toxin profile combinations were identical before and after freezing. In 12 (18.8%) and 16 samples (25.0%), respectively, B. cereus s.l. was detectable either only before or after freezing. In 13 samples (20.3%), different panC and toxin combinations were detectable after freezing in comparison to analysis before freezing.Naturally contaminated samples were initially pre-screened for the presence of presumptive  n = 35) . SupplemB. cereus s.l. is documented among the most prevalent foodborne pathogens, causing one third of food poisoning events in Europe [n Europe .B. cereus s.l. in food depends mainly on the contamination of the raw material, as well as on recontamination during processing and extrinsic and intrinsic growth conditions during storage. This results in an increased likelihood of disease-relevant concentrations in minimally processed foods consumed either raw or unheated or in inadequately stored extended shelf-life (ESL) products  [The presence of e abuse) ,29,30,31e abuse) .B. cereus requires microbiology-trained personnel and is labor-intensive if samples are comprehensively assessed. Most commonly, detection and confirmation are performed using selective culture media such as MYP agar according to ISO 7932 [B. cereus s.l. are missed in the yellow-stained agar, and the sample is often considered false negative by the investigator.The detection of presumptive ISO 7932 . In induB. cereus s.l. detection and enumeration. For this purpose, inclusivity and exclusivity were elicited, and group diversity was determined by using naturally contaminated samples before and after freezing.This study focused on the comparison of alternative chromogenic selective nutrient media to identify the best performer for B. cereus s.l. strains showed an inclusivity of >99% for all media, which is in general very promising. Nevertheless, atypical colony morphologies may occur. The highest rates of atypical β-D-glucosidase negative colonies were observed on BRI , HI  and CH  agar, resulting in a white phenotype. Atypical morphologies were largely related to the milk-derived or soil-derived panC-type/toxin profile combination VI/C. These atypical B. cereus s.l. phenotypes appear to be niche-specific and may possibly be associated with specific panC types with variable exploitability of starch and various carbohydrates in the genetic clade. For instance, panC group IV comprises strains isolated from vegetables indicated limited substrate utilization pathways. Furthermore, a sub-branch within panC group III showed the least carbohydrate fermentation capacity due to a lack of aryl-6-phospho-β-glucosidase-encoding genes in the genome [The study of e genome .B. cereus s.l. colonies on chromogenic B. cereus media manufactured by Oxoid or BMC-Biosynth, which is a concern for a proper evaluation [B. cereus s.l. isolation in this setting [B. cereus colonies by additional lecithinase reaction. All this research shows that B. cereus s.l. analysis is demanding and that current media are not sufficiently selective to analyze the diversity of the group.Previous studies focusing on agar evaluations also reported ß-D-glucosidase-negative aluation ,12,34. Ialuation  showed i setting . In a mo setting . BCCA waB. cereus s.l. Almost all group isolates were PLC-positive in the literature: 96% [Bacillus pseudomycoides (panC group I/toxin profile C) growth was inhibited on MYP, HI and CH  on the tested media  and Gram-negative  non-target strains grew despite the addition of polymyxin B in combination with trimethoprim. Lower growth of cocci was observed on BRI in contrast to MYP and HI. However, the proprietary antibiotic mixtures of CH and BA were even superior in selectivity compared to other media.Cross-reactivity for PI-PLC, PC-PLC and β-D-glucosidase was previously observed for Listeria . In our ed media . On BRI,B. cereus s.l. can be improved by the parallel use of two complementary selective agars, as it is already standard practice in the detection of L. monocytogenes [Salmonella spp. [B. cytotoxicus, as shown in previous reports [The detection and differentiation of presumptive ytogenes  and Salmlla spp. . The comlla spp. . Starch- reports ,46. TherB. cereus s.l. at the limit of detection. In principle, this finding is reassuring, but when assessing the safety of a product throughout the food production chain, including storage to the end of shelf-life, particularly nutrient-rich products contaminated with low levels of B. cereus s.l. lacking competitive flora cannot be considered completely safe. On the one hand, one can assume low level contaminations in the case of fresh produce, which, however, can result in rapid multiplication and accumulation of emetic and enterotoxins when temperature deviations occur. Moreover, low contamination levels of highly processed foods do not preclude the presence of the heat-stable and acid-stable toxin cereulide at the time of consumption posing a health risk to the consumer [B. cereus s.l. populations. In particular, the diversity of milk isolates between manufacturers was distinctive, which could be attributed to processing methods applied (such as microfiltration and high-temperature treatment) and/or the presence of persister cells in the production environment , CH (56.3%) and MYP (54.7%)  and by the influence of matrix components during initial testing [panC and toxin profile combinations.Naturally contaminated samples were pre-screened for the presence of presumptive  (54.7%) . Identic testing . Group s testing ,49. In opanC/toxin profile combination among target strains and naturally contaminated sample isolates was IV/A , followed by VI/C (17.3% and 10.4%), III/F and II/F  and III/D (10.9% target strains) (The predominant strains) .panC group III strains may carry the ces gene encoding for emetic toxin cereulide [B. cereus s.l. strains assigned to panC group VI were often isolated from raw milk (target strain set) and were highly abundant among heat-treated milk samples . Since other studies have found the use of B. thuringiensis biopesticides to be safe or of low risk to public health [Recently, the connection of biopesticidal organism . In our  strains ,52. Thisc health ,54, futuB. cereus s.l. diagnosis, which is especially practiced in routine analysis. We tested a selective media panel using test strains and naturally contaminated samples at the detection limit, which is relevant for practice. The results show that it is necessary to include more than one selective medium in the analysis, comparable to Listeria monocytogens and Salmonella diagnostics in food and animal feed. In order to be able to make a statement about contamination with presumptive B. cereus s.l. at all, it is recommended to perform, e.g., PCR, FTIR or MALDI-based confirmation and subtyping  and to assess growth behavior   .


This study suggests a key mechanism by which RSV infection delays wound healing.The airway epithelium’s ability to repair itself after injury, known as epithelial restitution, is an essential mechanism enabling the respiratory tract’s normal functions. Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections worldwide. We sought to determine whether RSV delays the airway epithelium wound repair process both in vitro and in vivo. We found that RSV infection attenuated epithelial cell migration, a step in wound repair, promoted stress fiber formation, and mediated assembly of large focal adhesions. Inhibition of Rho-associated kinase, a master regulator of actin function, reversed these effects. There was increased RhoA and phospho-myosin light chain 2 following RSV infection. In vivo, mice were intraperitoneally inoculated with naphthalene to induce lung injury, followed by RSV infection. RSV infection delayed reepithelialization. There were increased concentrations of phospho-myosin light chain 2 in  The airway epithelium serves as the first line of defense against inhaled allergens, particles, and viruses and therefore is under constant threat of injury by environmental exposure . Airway Epithelial restitution is a multistep process requiring robust cross talk between major cellular functions. It involves several essential cellular processes including spreading and migration of cells into the wounded area, followed by proliferation and differentiation of epithelial cells . MigratiRespiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in children worldwide, infecting nearly all children by their second birthday . RSV maiOur previous studies revealed that RSV disrupts epithelial cell contacts, known as apical junctional complexes (AJC) –35. RSV The impact of RSV on the restoration processes of an injured airway is unknown. In the current study, we postulated that RSV infection attenuates restoration of the airway epithelia via mechanisms affecting actin cytoskeleton organization and Rho GTPase signaling. We examined the effects of RSV infection on airway epithelial restitution, and in particular its early phases involving cell migration. We analyzed the role of downstream molecular mechanisms by which RhoA signaling induces RSV-mediated hypomotility. In addition, we investigated the effects of RSV infection on airway epithelial restitution in vivo.118 , total focal adhesion kinase , phospho-FAK Tyr397 , phospho-MLC2 , MLC2 , and RhoA . Alexa Fluor 488 (Cat. No. A12379) and 633 (Cat. No. A22284) phalloidin as well as anti-rabbit and anti-mouse secondary antibodies conjugated to Alexa Fluor 488, 568, or 633  dyes were obtained from Thermo Fisher Scientific . The specificity of antibodies against MLC2 and pMLC2 has been reported previously , anti-secretoglobin family 1A member 1 , anti-E-cadherin monoclonal antibody , total paxillin , phospho-PAX Tyreviously –51. ROCKeviously .16HBE14o (henceforth referred to as 16HBE cells) were provided by Dr. Dieter Gruenert . This cell line was isolated from a 1-yr-old male heart-lung patient and immortalized with the origin of replication defective simian virus 40 plasmid. Cells were genotyped by Short Tandem Repeat (STR) analysis to verify cell line authentication and were negative for mycoplasma contamination . Cells wRSV derived from RSV A2, which expresses red fluorescent protein upon replication (rrRSV), was a kind gift from Dr. Mark Peeples  and Dr. Peter Collins  , 56. VirFor immunofluorescent staining, cells were fixed in 4% paraformaldehyde and subjected to immunofluorescence labeling as previously described –35. ImmuAssays were performed as previously described , 57–59. Wound Healing Assays. Cell lysates were collected with RIPA lysis buffer  at 10 min and 2 h postwound along with the control group. Western blot analysis was performed as previously described .An uncropped Western blot figure for each antibody showing the entire lane with molecular weight markers is provided as Supplemental Fig. S1 . For the attachment assay, which uses detection and counting of bound cells , cells wCell proliferation was assessed by two methods: using a commercially available 5-ethynyl-2′-deoxyuridine (EdU) kit (Abcam) and counting. For the EdU assay, cells at 60%−70% confluency were fixed, permeabilized, and incubated with EdU, a thymidine analog that becomes incorporated into newly synthesized DNA according to the manufacturer’s protocol. EdU was covalently cross-linked with fluorescent azide, iFluor 488. DNA was stained with DAPI, and EdU fluorescence was read at an excitation/emission wavelength of 491/520 nm. Photographs were taken under fluorescent light and used to quantify the ratio of EdU-positive cells to total cells. For counting, after 48 h of exposure of epithelial cells to control medium, UV-RSV, or RSV, cells were gently detached with TrypLE Express, stained with trypan blue, and counted by a hemocytometer.day 2 postinjection, received an intranasal inoculation of 9.6 × 106 plaque-forming units of RSV as described in our recent publication  and day 14 . Lung tissue and bronchoalveolar lavage fluid were collected and used to evaluate lung inflammation by quantification of infiltrated leukocytes and protein translocation into the lung. Airway epithelial injury and restitution were evaluated by assessing hematoxylin and eosin-stained paraffin-embedded lung sections and by antigen retrieval and immunolabeling of club cells with a specific marker, CC10/scgb1a1  dissolved in corn oil –67. Studlication . Mice we/scgb1a1 , the panHuman primary epithelial cells were isolated from human tissue from deceased pediatric donors. Tissue was provided by the International Institute for the Advancement of Medicine according to procedures approved by the Cleveland Clinic. As such, the human tissue is exempted from requiring Institutional Review Board approval as the use of this tissue is not considered a human study by the Cleveland Clinic Foundation. All animal procedures used in this study adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Institutional Animal Care and Use Committee (approved protocol 2018-2030) of the Lerner Research Institute at the Cleveland Clinic Foundation. This facility is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (Accreditation No. 000383) and is in compliance with federal law and National Institutes of Health regulations.t test or paired Student’s one-tailed t test were used. For comparison of multiple groups, we performed one-way ANOVA followed by Dunnett’s post hoc test for all groups of the experiment.Data were analyzed using Prism software  and Microsoft Excel. Data are representative of three or more experiments and are presented as means and SD. For comparison of two groups with parametric data, Student’s two-tailed materials and methods. This MOI was chosen based on our previous studies and pilot experiments showing the lowest concentration of RSV infection with a significant impact on wound healing . Animals exposed to both naphthalene and RSV presented with more severe weight loss . Two days later, some of the animals received an intranasal inoculation of 9.6 × 10ght loss A and incght loss B and proght loss C compare , as well as by National Institutes of Health (NIH) Grants K08AI112781 and R01HL148057 (to F. Rezaee) and R01061007 (to G. Piedimonte). This work used the Leica SP8 confocal microscope that was purchased with funding from NIH SIG Grant S10OD019972.The authors have no financial relationship with a biotechnology and/or pharmaceutical manufacturer that has an interest in the subject matter or materials discussed in the submitted manuscript.No conflicts of interest, financial or otherwise, are declared by the authors.F.R. conceived and designed research; D.T.L., N.G., A.R., T.J.H., and F.R. performed experiments; D.T.L., N.G., A.R., T.J.H., G.P., and F.R. analyzed data; D.T.L., N.G., G.P., and F.R. interpreted results of experiments; N.G. and F.R. prepared figures; F.R. drafted manuscript; D.T.L. and F.R. edited and revised manuscript; D.T.L., N.G., A.R., T.J.H., G.P., and F.R. approved final version of manuscript.