Candida albicans and Crohn’s disease (CD) was intended to assess the effect of a 6-month course of the antifungal fluconazole (FCZ) on post-operative recurrence of CD. Methods: Mycological samples (mouth swabs and stools) and serum samples were collected from 28 CD patients randomized to receive either FCZ (n = 14) or placebo (n = 14) before surgical resection. Serological analysis focused on levels of calprotectin, anti-glycan antibodies, and antibody markers of C. albicans pathogenic transition. Levels of galectin-3 and mannose binding lectin (MBL) involved in C. albicans sensing and inflammation were also measured. Results: 1, 2, 3, and 6 months after surgery, endoscopy revealed recurrence in 5/12 (41.7%) patients in the FCZ group and 5/9 (55.6%) in the placebo group, the small cohort preventing any clinical conclusions. In both groups, surgery was followed by a marked decrease in C. albicans colonization and biomarkers of C. albicans pathogenic transition decreased to non-significant levels. Anti-glycan antibodies also decreased but remained significant for CD. Galectin-3 and calprotectin also decreased. Conversely, MBL levels, which inversely correlated with anti-C. albicans antibodies before surgery, remained stable. Building biostatistical multivariate models to analyze he changes in antibody and lectin levels revealed a significant relationship between C. albicans and CD. Conclusion: Several combinations of biomarkers of adaptive and innate immunity targeting C. albicans were predictive of CD recurrence after surgery, with area under the curves (AUCs) as high as 0.86. FCZ had a positive effect on biomarkers evolution. ClinicalTrials.gov ID: NCT02997059, 19 December 2016. University Hospital Lille, Ministry of Health, France. Effect of Fluconazole on the Levels of Anti-Saccharomyces cerevisiae Antibodies (ASCA) After Surgical Resection for Crohn’s Disease. Multicenter, Randomized, and Controlled in Two Parallel Groups Versus Placebo.Background and aims: This study prompted by growing evidence of the relationship between the yeast  Candida albicans is a frequent commensal of humans that can colonize all segments of the digestive tract and vagina. Its role in intestinal homeostasis remains largely unknown in contrast to its deleterious effects, which are well-documented. These range from mucocutaneous candidiasis, with vaginal infections occurring in 75% of women at least once in their lifetime, to systemic infections originating in the gut of hospitalized patients. The main risk factors are immunodeficiency related to the primary pathology and the medico-surgical procedures used to manage it, including broad-spectrum antibiotics [C. albicans undergoes saprophytic-pathogenic transition. Candida species now rank fourth among the causes of nosocomial infections with candidemia associated with mortality rates as high as 40–60% [The yeast ibiotics . In thess 40–60% . ConsideSaccharomyces cerevisiae, ref. [S. cerevisiae antibodies” (ASCA), ref. [C. albicans by showing a correlation between ASCA and C. albicans colonisation in FDRs [C. albicans, when growing in human tissues, ref. [C. albicans mannan but extended to other important immunologically active components of the C. albicans cell wall, such as glucans and chitin. Combined serological and mycological studies in patients with CD or invasive Candida infection (ICI) showed that anti-laminaribioside (ALCA) and anti-chitobioside (ACCA), respectively, described as CD biomarkers, ref. [C. albicans-gut interactions confirmed that Dextran Sodium Sulfate (DS)induced inflammation in mice promoted C. albicans colonization, which in turn increased inflammation, generating ASCA [C. albicans, such as galectin-3  [The interest of the gastroenterology community started with the yeast ae, ref.  a harmleae, ref. . MedicalA), ref.  and theiA), ref. . FurtherA), ref.  vs. 6% oA), ref. . These aA), ref. ,9. From  in FDRs . It was es, ref.  was resprs, ref.  could alrs, ref. . The deving ASCA . Major p   and toll   were alsC. albicans and balance invasion control and inflammatory damage [C. albicans and cross-reactive Th17 cells, highlighting anti-C. albicans immunity as a central mechanism for the induction of the human antifungal Th17 response [C. albicans gut biotope, is coherent with many observations suggesting a link between Th17 exacerbation in inflammatory bowel disease (IBD) and C. albicans [Considerable progress has been made in our understanding of the complex immunological mechanisms that finely tune the response against y damage ,17,18. Iresponse . Furtherresponse ,20. Thisalbicans . Candida species, which might explain why candidiasis [Over the past decade, next generation sequencing (NGS) methods of intestinal microbiota analysis have gradually replaced methods of living yeast isolation and identification. Due to the relatively low abundance of fungi among bacteria, ref.  focus ondidiasis  is the mdidiasis ,27. Thesdidiasis .C. albicans in triggering or exacerbating CD [Thus, a substantial body of clinical, experimental, mycological, and immunological evidence supports the role of ating CD ,29. Howen = 28) and the use of suboptimal doses of FCZ, the study was not conclusive clinically. However, careful patient follow-up over 6 months with regular mycological and serological sampling provided unique samples to follow the evolution of C. albicans colonization together with measurement of the adaptive and innate immunity parameters linked to both C. albicans growth and CD inflammation. These biological parameters were scored, and their relevance analyzed individually before multivariate analysis. Our results show that surgical reduction of inflammation and FCZ have an impact on C. albicans survival and decrease immune stimulation, while multivariate analysis suggested the influence of fluconazole on biomarkers linked to recurrence.We carried out a pilot clinical study to assess the effect of fluconazole (FCZ)  on post-This prospective, randomized, double-blind, placebo-controlled study was registered at ClinicalTrials.gov under the ID: NCT02997059, 19 December 2016  where al®, Marnes la Coquette, France) and incubated at 37 °C under standard conditions. The isolated yeasts were identified by MALDI-TOF mass spectrometry  as described previously [Oral swabs and stools were seeded onto chromogenic agar plates  designed on the basis of previous studies; the limit of detection of this kit is 400 ng/mL with median values of 8892 ng and 1318 ng/mL in CD patients and controls, respectively . The tes® panel , comprising ASCA, AMCA, ALCA, and ACCA kits involving S. cerevisiae mannan, mannobioside, laminaribioside, and chitobioside antigens, respectively [Anti-glycan antibodies were detected with the IBDXectively . All assCandida mannan antibodies were measured with the PlateliaTM Candida Ab Plus ELISA kit  [Anti- France) . The cutC. albicans pathogenic phase IgG antibodies were detected by ELISA using recombinant Hyphal wall protein-1 Hwp1 produced in Escherichia coli, as described previously [Anti-eviously . The cutβ-D-glucan (BDG) through a specific biochemical test derived from the Limulus test for endotoxin detection. It is recommended worldwide as a surrogate marker for the diagnosis of ICI and is used routinely in our laboratory [Circulating fungal glucans were detected with the Fungitell asssay . This test detects the presence of fungal cell wall boratory . Some paboratory ,38.α-1,2 anomery, rare in the living world but largely expressed on the C. albicans surface, was discovered by our group [Gal-3 is a lectin of innate immunity with a variety of important roles. Its ability to sense mannosides with a ur group ,15,39. Gur group .α-mannosides expressed at high levels on the S. cerevisiae and C. albicans yeast surface. Levels of MBL were determined with the MBL oligomer ELISA kit  involving a monoclonal antibody against the MBL carbohydrate-binding domain, according to manufacturer’s instructions. Despite the absence of consensus, it is generally agreed that MBL concentrations ≤500 ng/mL reflect deficiency and that values <100 ng/mL are associated with increased susceptibility to infection [Mannose binding lectin (MBL) is an important lectin of innate immunity triggering many defense mechanisms including the complement system. This lectin senses the ubiquitous nfection ,42.All statistics and figures were computed with R statistical open-source software, SAS 9.4 , and GraphPad Prism v6.0. Software GraphPad Software Inc., San Diego, CA, USA Categorical variables are expressed as number (percentage). Quantitative variables are described as median and interquartile range (IQR). Normality was assessed by the Shapiro-Wilks test combined with graphical evaluation (QQplot).t test in case of normality and the non-parametric rank signed test otherwise. These analyses were adjusted for the level at inclusion. In case of non-normality of biological parameters, these analyses were performed on log-transformed data in case of log-normality and on rank data otherwise. Correlations between biological parameters are given by the Pearson or Spearman coefficient of correlation depending on the distribution of the parameters. Considering the exploratory nature of theses analyses, all statistical tests were performed at the two-tailed α level of 0.05 and the results should be interpreted as hypothesis-generating. First, exploratory univariate analyses were performed to assess the evolution of each biomarker after surgery by comparing the value at inclusion with the value at the end of the study (6 months) using the paired Student’s t-test according to normality and sample variance distribution. Adjusted p-value below 0.05 was considered as statistically significant according to the Benjamini and Hochberg approach [a is the calculated coefficient and j is the biomarker number.as follows:Second, statistics and figures were computed with the “R/Bioconductor” statistical open-source software . Biomarkapproach . Correlaapproach . The equContingency tables have been performed from multivariate mROC biomarkers combination results and chi-squared tests were performed on these tables.C. albicans (n = 16), C. tropicalis (n = 3), and C. lusitaniae (n = 1). Post-operatively, patients were characterized by a gradual reduction in C. albicans isolates and a broader spectrum of species isolated, which comprised C. krusei, C. parapsilosis, C. glabrata, C. lambica, C. norvegiensis, and S. cerevisiae .The main results are shown in revisiae A. Quantirevisiae B showed Whatever the treatment, an analysis of the number of colonized patients showed that the decrease in yeast colonization at 6 months was associated with a transient increase at 1 month in patients who had a recurrence of CD, suggesting that this temporal variation could be critical for the outcome.C. albicans oral and fecal loads is observed during the 6 months post-surgery.Together these results show that, whatever the treatment and endoscopic evolution, an important and gradual decrease in p = 0.76).CRP positive values (>3) did not differ significantly between the initial visit and the 6-month visit (p = 0.002). Due to the limited number of patients in each group, the differences were not significantly associated with treatment (p = 0.09) or recurrence (p = 0.202); however, it is worth noting that the only two obvious increases at the end of the survey corresponded to patients with recurrence of CD.The change in serum calprotectin levels is shown in p < 0.0001). However, all patients except two remained above the positivity threshold at the end of follow-up. For ACCA, the decreasing trend was less systematic, but significant (p = 0.04). No significant trend was observed for ALCA (p = 0.11), or AMCA (p = 0.33). No association was found between the decrease in levels, treatment, or recurrence. According to the clinical interpretation of the ASCA, ALCA, and ACCA tests, based on the recommended cut-off values, the few patients who evolved from a positive to a negative status  had initial values slightly above the cut-off values. The fluctuations in AMCA were of higher magnitude, decreasing or increasing, leading to more changes from a positive to a negative status. However, consideration of the results from the entire IBD panel would not have affected the diagnosis of CD.C. albicans antibodies, produced during tissue invasion by C. albicans, is used to survey patients at risk of nosocomial ICI. Our results are shown in Candida antibody levels decreased during the survey and reached non-significant levels at 6 months. Anti-Candida mannan antibodies (p < 0.0001). As for yeast colonization, no influence other than surgery  was significantly related to this decrease. When considering the relationship between the decrease in anti-C. albicans antibodies and anti-glycan markers of CD, the decrease in anti-C. albicans mannan antibodies tended to be associated with a decrease in ASCA (p = 0.069).The measurement of anti-tibodies E and anttibodies F exhibitOf note, a comparison of BDG levels before surgery and 6 months later gave non-significant results (data not shown).C. albicans surface mannosides. As shown in p < 0.0001). As for anti-C. albicans antibodies and yeast colonization, no influence other than surgery could be established for this decrease. Gal-3 and calprotectin levels correlated before surgery and at the end of the survey . During the survey, the decreases in serum Gal-3 and calprotectin were correlated (p = 0.0001). MBL is another soluble lectin playing an important role in innate immunity with regard to C. albicans. It senses the massive bulk of cell wall mannan surface α-mannosides. Almost half of CD patients  had low or very low MBL levels (<1000) at inclusion. As shown in p = 0.283). When the correlation between MBL levels and other biomarkers was analyzed, a significant relationship was found between low MBL levels and high levels of anti-C. albicans antibodies before surgery , suggesting a compensation by adaptive immunity for the innate MBL defect. After surgery and a decrease in Candida colonization this relation disappeared . The results from the analysis of the ability of combined Candida and CD biomarkers to discriminate between surgery and 6 months later are summarized in Gal-3 is a cell membrane-associated pattern recognition receptor binding Candida were affected by clinical CD remission after surgery, we first analyzed whether one biomarker, or a combination, could discriminate patient status before and 6 months after surgery using multivariate ROC curves. As a reference marker of systemic inflammation, we used calprotectin which provided acceptable results in terms of specificity and sensitivity (AUC = 0.76) . When combined with anti-C. albicans antibodies the AUC was an impressive 0.86 suggesting that, as for colonization, a cause-to-effect relationship exists in relation to the reduction of inflammation by surgery. Of note, although BDG values analyzed individually provided non-significant results, their combination with anti-C. albicans mannan antibodies and Gal-3 gave an impressive AUC = 0.84 supporting the impact of CD surgery on C. albicans biomarkers.Considering that the biomarkers related to  = 0.76) . HoweverC. albicans antibodies, circulating BDG, and calprotectin could predict relapses (the threshold is represented by a dotted line). Interestingly, patients were not “equal” in this analysis since biomarker levels already differed at the beginning of the survey; the most discriminating time in the evolution was 2 months.We then analyze whether some factors could be predictive of relapses over time. In this respect, we analyzed the evolution of different combinations of markers at each visit. As shown in C. albicans, Gal-3 and MBL, appeared more determinant for sensing a potential role of antifungals on disease evolution  at each visit .2 test; p = 0.007). The number of recurrences appeared to be lower in patients treated with FCZ compared to placebo.When patients who were positive for the Gal-3 + MBL + calprotectin combination were considered  and the true negatives (negative test in placebo patients)) there was a significant difference in terms of recurrence between the two populations . Thus, the role of adaptive immunity was less noticeable than that of innate immunity regarding relapses in relation to FCZ.When considering patients who respond positively to an association of adaptive immunity biomarkers with lectins , the comC. albicans could have an evolution that is predictive of CD recurrence, and that antifungals with the same target  also have an influence on recurrence.Altogether it appears that biomarkers of adaptive or innate immunity targeting C. albicans colonization in CD patients, with 18/23 (78%) patients sampled before surgery colonized. This is in accordance with several clinical [C. albicans in the gut is favored by inflammation. C. albicans was the predominant species isolated, sometimes associated with C. tropicalis. C. tropicalis has been described in some NGS mycobiota analyses [C. albicans. Of note, during this survey involving more than 200 samples, S. cerevisiae was only isolated once, in contrast to a study that attributed an important deleterious role for this species on CD evolution [C. albicans colonization. Interestingly, this decrease in colonization was also observed in the oral cavity, suggesting a systemic effect.This study describes the high rate of clinical  and expeclinical ,18 studianalyses ,51 as a analyses  which clvolution ,63. Globvolution . This isCandida interplay to assess whether some biomarkers could reflect disease evolution.The clinical conclusions of this pilot study are restricted by the limited number of CD patients and the absence of endoscopic documentation in some of them. Only a very slight trend towards a higher proportion of recurrences in the placebo group was observed. In contrast to clinical analysis, for which the study was obviously underpowered, patient and medical team investment during the study led to the collection of an impressive number of samples. We therefore focused on a biological analysis of the host-Fecal calprotectin is a recognized biomarker of IBD ,65. SomeC. albicans infection [albicans stimulation [C. albicans infection . Before surgery, these antibodies were present in up to 16% of patients at levels not found in the control population [C. albicans tolerance, or conditions promoting the virulence of this species. It is worth noting that surgery and the associated reduction in Candida colonization led, in all patients, to these antibodies falling to levels not discriminating colonization from infection. It is therefore possible that this decrease in anti-C. albicans antibodies may account, at least in part, for the decrease in anti-glycan antibodies known to be generated by the pathogenic phase of C. albicans [C. albicans antibodies and the IBDX panel seem to support this hypothesis, which could result from reduction in C. albicans burden.ASCA were the first antibodies directed against yeast glycans to be associated with CD . After tnfection . We obsemulation . In the pulation ,35,70. Talbicans ,49. The C. albicans α-1,2 oligomannosides acting as immunomodulatory adhesins [α production after cross-talk with TLR-2 [C. albicans gut burden. In a previous study, Netea et al. showed that higher levels of Gal-3 were associated more with Candida infection than with inflammatory diseases; however, CD was not among the inflammatory diseases studied [C. albicans and gut inflammation/fibrosis although the expression of C. albicans α-mannosides, reduced in vivo, may have a conflicting role [The rationale for exploring Gal-3 levels in this study was twofold. First, Gal-3 has been identified by our group as the receptor for adhesins . This inth TLR-2  and dectth TLR-2 . Second,th TLR-2 ,71 incluth TLR-2 ,73. It i studied . Some obing role .C. albicans-CD interplay [α-mannoside residues at the C. albicans cell wall surface led to early interest in this lectin being able to activate the complement system after binding to the yeast and mediate opsonization for clearance; this mechanism is more effective for Candida than for bacteria [C. albicans infection [C. albicans antibodies observed before surgery disappeared after 6 months, suggesting the absence of a need for compensatory mechanisms when the high gut burden of C. albicans is reduced.Like Gal-3, MBL is a lectin playing a dual role in the nterplay . The probacteria . Severalnfection ,77. Withnfection ,57. It hnfection . Althougnfection . It is nC. albicans could have an evolution that, in parallel with the reduction in colonization, accounts for differences in patient status before and after surgery. Furthermore, we showed that biomarkers of adaptive or innate immunity targeting C. albicans could have an evolution that is predictive of CD recurrence. As far as differences between patients, with recurrence or not, were visible early in follow up  [C. albicans in T-cell activation, was confirmed by another study that showed a role for C. albicans in GvHD through its interaction with dectin-1 [C. albicans has never been investigated in IBDs.Interestingly, this conclusion about an effect of FCZ on immune functions via e (GvHD) . This obdectin-1 . DespiteC. albicans pathogenic traits has reached a very a high standard over the past few decades following the declaration of C. albicans as a major human pathogen due to improvements in medical practices. As this increased incidence in ICI was concomitant with the increased incidence of IBD, it is surprising that few mycologists have investigated the role of C. albicans in IBDs despite information gained from analysis of the mycobiota. This first translational study on the evolution of myco-immunological parameters associated with CD after surgical resection highlights a strong relationship between C. albicans survival, its immune control, and inflammation.Basic research on C. albicans colonization-living load. This provides a logical counterargument to the numerous observations that inflammation increases C. albicans colonization, which in turn increases inflammation. Interestingly, the biomarkers studied, either related to C. albicans pathogenicity, or related to both ICI and CD, also decreased. Finally, building multivariate biostatistical models has established significant links between CD recurrence, immune sensing of C. albicans, and the positive effects of antifungal treatment. This fulfilled the aims of this pilot study, namely to define how an anti-C. albicans treatment could interfere with CD evolution. The results suggest that prospective studies intended to assess the effect of new generations of antifungal drugs on CD evolution would be worthwhile. More generally, cumulative data on the deleterious role of C. albicans in CD also raise the question of the value of an anti-Candida vaccine strategy to limit these effects.From a mycological point of view, this is the first study to report that decreasing inflammation by intestinal resection decreases


Burkholderia gladioli is known to cause respiratory tract infections in cystic fibrosis patients. Here, we describe the annotation of the 38,038-bp genome sequence of Mana, a P2-like phage of B. gladioli. Understanding the genomic characteristics of phages infecting pathogens like B. gladioli can lead to advancements in phage therapy. Burkholderia gladioli is known to cause respiratory tract infections in cystic fibrosis patients. Here, we describe the annotation of the 38,038-bp genome sequence of Mana, a P2-like phage of B. gladioli. Understanding the genomic characteristics of phages infecting pathogens like B. gladioli can lead to advancements in phage therapy. Burkholderia gladioli is a ubiquitous Gram-negative bacterium . The genome sequence was then assembled using SPAdes v3.5.0  (GU911303) and KL3 (GU911304), respectively   and 34 aectively . Most ofodophage . The lysMT701591.1 and BioSample accession no. SAMN14609638. The BioProject accession number is PRJNA222858, and the SRA accession number is SRR11558334.The genome sequence of phage Mana was deposited under GenBank accession no.


Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. However, due to the heterogeneity of CRC, the clinical therapy outcomes differ among patients. There is a need to identify predictive biomarkers to efficiently facilitate CRC treatment and prognosis.Methods: The expression profiles from Gene Expression Omnibus (GEO) database were used to identify cancer hallmarks associated with CRC outcomes. An accurate gene signature based on the prognosis related cancer hallmarks was further constructed.Results: Hypoxia was identified to be the primary factor that could influence CRC outcomes. Sixteen hypoxia-related genes were selected to construct a risk gene signature (HGS) associated with individuals’ prognosis, which was validated in three independent cohorts. Further, stromal and immune cells in tumor microenvironment (TME) were found to be associated with hypoxia. Finally, among the 16 hypoxia-related genes, six genes  were identified as the most attributable ones to drug resistance.Conclusion: HGS can accurately predict CRC prognosis. The expression of the drug resistance-related genes is critical in CRC treatment decision-making. Colorectal cancer (CRC) is the second leading cause of cancer-related death. In 2020, there were approximately 1 million CRC-related deaths worldwide.Metastasis and uncontrolled growth of solid tumors often require new blood vessels. The newly-formed microvasculature is highly abnormal in structure and function, leading to a hypoxia tumor microenvironment (TME) , known tIn the present study, we confirmed that hypoxia was of dominant prognostic significance for CRC. And we established a hypoxia-related gene signature (HGS) and validated the prognosis prediction model in three independent cohorts. Finally, we explored the correlation of hypoxia with stromal and immune cells, and identified its role in drug resistance.Expression profile of CRC tissues in GSE39582, GSE38832, and GSE161158 datasets were downloaded from the GEO database .2 TCGAR package “GSVA” was used to perform single sample gene set enrichment analysis (ssGSEA). The ssGSEA was applied to explore the enrichment of tumor-related pathways and immune cell infiltration in the GSE39582 database. Tumor-related data sets were obtained from hallmark gene sets in the MSigDB database .4 The β = 4). The minimum number of genes was set as 30 for the high reliability of the results. For further quantification of hypoxia related genes and modules, genes with p value of less than .001 were retained for subsequent analysis.The weighted gene co-expression networks analysis (WGCNA) were constructed using the top 25% most differently expressed genes in the GSE39582 dataset. Among all the soft threshold values, we chose the β value with the highest mean connectivity . In the training cohort and all validation cohorts, we separately created a chip expression profile and a sample data file and then imported them into GSEA software. The univariate Cox regression analysis was conducted to identify recurrence free survival (RFS) and overall survival (OS) related cancer hallmarks. To select hypoxia related genes associated with prognosis, Lasso penalized Cox regression analysis was applied. Finally, the LASSO Cox regression model was employed to identify hypoxia highly correlated genes and construct the prognostic HGS. For each patient, the coefficients of Logistic Regression were used to calculate the HGS score. The formula is: HGS score = ∑ (coefficient × mRNA expression).Hypoxia score and relevant clinical parameters were used to construct a nomogram, using the survival and the “rms” package of R. The nomogram was constructed to estimate 1-, 3-, and 5-year survival probabilities. The calibration curve and C index were used to evaluate the performance of the model.We used xCell to estimate the cellular composition of stromal cells and immune cells in the tumor in the GSE39582 dataset. Immune and stromal cell scores for each sample were calculated. The CIBERSORTx online website was used to evaluate the infiltration of 22 immune cells in each sample.The GSCALite database is used for drug sensitivity analysis.3 cells per well) were seeded in 96-well plates overnight. Then, the cells were exposed to different concentrations of oxaliplatin for 48 h. The cell viability was evaluated using CCK-8 reagent (MedChemExpress). For gene expression assay, we extracted the total mRNA of HCT116 and HCT116/L-OHP cells and reversely transcribed them to cDNA. We performed Real-time quantitative PCR (qPCR) using AceQ qPCR SYBR Green Master Mix (Vazyme). ABI Stepone Real-Time PCR System (Applied Biosystems) was used to measure the fluorescence. The primers were listed in Human colorectal cancer cells (HCT116) and their corresponding oxaliplatin-resistant cells (HCT116/L-OHP) were incubated in RPMI1640 containing 10% fetal bovine serum (FBS), supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml) . For celp < .05; **p < .01; ***p < .001; NS, not significant.Data was analyzed using the R software. For forest plots, the hazard ratio (HR) was generated by univariate Cox or multivariate Cox proportional hazard regression. Kaplan-Meier approach was used for the survival analysis. Differences between groups were assessed using the Wilcoxon test. Statistically significant differences were showed as follow: *p = .01) and epithelial-mesenchymal transition (EMT)  were strongly associated with poor RFS (p = .03), hypoxia , angiogenesis , and EMT  were significantly associated with shorter OS  were identified and used to construct the optimal HGS  (p < .0001)  . GSEA re< .0001) . The are< .0001) . Furtherutations . The newp < .0001) (p = .009) . We found that the patients with worse prognosis exerted higher HGS scores . The HGS< .0001) . GSEA re = .009) . Multiva = .009) . Further = .009) . The are = .009) .A nomogram for predicting individual RFS probability of CRC patients was developed by combining HGS and clinicopathological characteristics  of the patients . The areTo clarify the potential role of hypoxia in TME, we carried out correlation analysis between HGS and immune and stromal cells. The results showed that both Immune score and Stromal score were positively correlated with HGS score . A corre+ memory T cells, CD8+ naïve T cells, NKT cells, plasma cells, Th1 cells, Th2 cells, and pDCs were significantly higher in HGS-low group individuals, whereas M1 macrophages, M2 macrophages, DCs and cDCs were significantly higher in HGS-high group individuals , fibroblasts, endothelial cells, and adipocytes among others. Experimental evidence revealed that hypoxia promoted recruitment of MSCs to primary breast tumors as well as tumor metastasis to lymph nodes and lungs . Hypoxia+ T cells. Th1 cells activates CD8+ T cells and NKT cells by expressing IFN-γ. Th2 cells exerts their anti-tumor property by recruiting eosinophils through the expression of IL-4 and IL-13   . NKT celes (ROS) . Neverthes (ROS) . We founDCs are classified into two major lineages, myeloid or conventional DCs (cDCs), which are functioned in antigen processing and presenting, and plasmacytoid DCs (pDCs), which produce massive IFNs . We founTumor-associated macrophages (TAMs) are the primary tumor-infiltrating cell types. M1 and M2 are the major functional subtypes of macrophages . In thisPD1, PDL1, PDL2, CTLA4, TIM3, CD47, B7-H3, VISTA, VTCN1, CD70, and LAG3. In related studies, hypoxia significantly increased the expression of PD-L1 on DCs, macrophages and tumor cells, and induced the apoptosis of T cells (Our analyses further revealed that hypoxia increases the expression of several immune checkpoints including  T cells . Besides T cells . These fSnail and Slug to promote EMT (2 for ATP generation. Under hypoxic conditions, oxidative phosphorylation slows down and glycolysis increases to reduce O2 consumption. It was reported that HIF-1 reprogram glucose metabolism and promote mitochondrial autophagy to switch cancer cells from oxidative to glycolytic metabolism (Other molecular pathways also involve in hypoxia related poor prognosis and drug resistance. We found that hypoxia showed positive correlation with angiogenesis, EMT and p53 pathway, and showed negative correlation with E2F targets, oxidative phosphorylation, DNA repair and G2/M checkpoint. Uncontrolled proliferating tumors need much blood supply and develop hypoxic microenvironment. Hypoxia stimulates angiogenesis, resulting in the formation of immature, abnormal, and leaky neovessels. However, these neovessels are often insufficient to meet the elevated nutrients and oxygen demand of more cancer cells, which in turn aggravate hypoxia. As a result, the highly hypoxic and disordered vascular microenvironment promotes cancer cells’ invasion and distant metastasis . EMT is mote EMT . Interesmote EMT . Tumor-amote EMT . Hypoxiamote EMT . In retumote EMT . E2F fammote EMT . It was mote EMT . Oxidatitabolism . Hypoxiatabolism . It is atabolism . The gentabolism . Cell cytabolism . As a reDCBLD2, a biomarker with largest risk to develop chemoresistance, was reported to promote CRC progression, and mediate drug resistance to 5-FU, a first-line drug for CRC (PLEC, a cytoskeletal gene, contributed to the invasiveness of neuroblastoma and resistance to cisplatin (S100A11, a member of calcium-binding proteins, promoted resistance of multiple cancers to 5-FU and cisplatin (PLAT participated in proliferation, invasion and migration of non-small cell lung cancer as well as well as gefitinib resistance (PPAP2B, a lipid phosphate phosphohydrolase, promoted invasion of breast cancer (LAMC2 is a component of epithelial basement membrane shown to promote chemoresistance in several cancers (Hypoxia has been shown to promote acquired resistance to conventional chemo/radio therapy . In the  for CRC . PLEC, aisplatin . S100A11isplatin . Moreovesistance . PPAP2B,t cancer . LAMC2 i cancers .The decision of postoperative chemotherapy for CRC patients has been based on clinicopathologic characteristics for several decades. Molecular biomarkers have great advantages in predicting disease outcome and thus may guide postoperative chemotherapy. Several commercial platforms, such as CRCassigner and ColoGuidePro, have been utilized in clinic for predicting patients’ prognosis and response to chemotherapy. Based on multiple and large cohorts, HGS shows its superiority in broad application and quantitative evaluation against the commercial platforms . NeverthIn summary, a novel gene signature based on 16 hypoxia-related genes was constructed, which could efficiently predict CRC prognosis. Besides, we found a strong association between hypoxia and stromal cells and immune status in the TME, which might be the underling mechanisms of hypoxia to determine CRC prognosis. Finally, we identified a list of six genes, which were highly correlated to drug resistance. HGS could be a useful tool in predicting CRC prognosis, as well as guiding personalized chemotherapy.


Ahnfeltia  is a globally widespread genus with 11 accepted species names. Two of the most widespread species in this genus, A. plicata and A. fastigiata, may have diverged genetically due to past geographic changes and subsequent geographic isolation. To investigate this genomic and genetic diversity, we generated new plastid (ptDNAs) and mitochondrial genomes (mtDNAs) of these Ahnfeltia species from four different regions (A. plicata - Chile and UK and A. fastigiata - Korea and Oregon). Two architecture variations were found in the Ahnfeltia genomes: in ptDNA of A. fastigiata Oregon, the hypothetical pseudogene region was translocated, likely due to recombination with palindromic repeats or a gene transfer from a red algal plasmid. In mtDNA of A. fastigiata Korea, the composition of the group II intronic ORFs was distinct from others suggesting different scenarios of gain and loss of group II intronic ORFs. These features resulted in genome size differences between the two species. Overall gene contents of organelle genomes of Ahnfeltia were conserved. Phylogenetic analysis using concatenated genes from ptDNAs and mtDNAs supported the monophyly of the Ahnfeltiophycidae. The most probable individual gene trees showed that the Ahnfeltia populations were genetically diversified. These trees, the cox1 haplotype network, and a dN/dS analysis all supported the theory that these Ahnfeltia populations have diversified genetically in accordance with geographic distribution.The agarophyte  Ahnfeltia and the recently added microscopic Pihiella  red algal class that has been divided into five subclasses: the Hidenbrandiophycidae, Nemaliophycidae, Corallinophycidae, Rhodymeniophycidae, and Ahnfeltiophycidae . Among tPihiella . Species pattern .Ahnfeltia, the subject of this study, is commercially important as it is a valuable agarophyte, known for high-quality agar production with a low sulfate ratio  and speciation events took place 2-5 Mya  and Northwest (NW) Pacific  since these methods can examine and analyze extensive arrays of data. This is particularly useful for data obtained from organelle genomes . In prevdatabase . In ordeAhnfeltia thalli were sampled by snorkeling or collecting from tidepools in intertidal zones in the following regions. A. plicata populations were sampled from Seno Skyring, Magallanes, Chile (52°32′45.1″S 71°56′46.3″W), and Freshwater West, Pembrokeshire, Wales, UK (51°39′35.9″N 5°03′52.2″W). A. fastigiata samples were collected from Baeknyeong island in Yellow Sea, Republic of Korea (37°59′01.3″N 124°41′54.0″E), and Seal Rock, Oregon, USA (44°29′48.5″N 124°05′07.5″W) (Four populations of fresh ′07.5″W) . The colrbcL genes were determined before genome sequencing. The rbcL marker, which encodes the ribulose-1,5-bisphosphate carboxylase large subunit, is the most widely used in species identification in the red algae (rbcL primers were F57 and rbcLrevNew described in https://www.geneious.com). For phylogenetic analysis, published rbcL sequences of Rhodymeniophycidae and Ahnfeltia were obtained from NCBI  protocol . To confed algae . The rbchttps://www.geneious.com). Assembled genomes were corrected by Pilon  for whole genome sequencing. PacBio Sequel I  and Illumina Nova Seq 6000  platforms were used to generate long- and short-read raw data, respectively. Organelle genome assembly for each population was conducted with two methods using short and long reads data. Before using Illumina data, low-quality reads were eliminated using Trimmomatic v0.39 . NOVOPlaby Pilon . Primaryby Pilon . Protein plicata . Annotat plicata . rRNAs w plicata .A. plicata (NC_031145). Because the published A. plicata ptDNA was based on a collection from Magallanes, Chile, and generated based on the short-read Ion Torrent PGM platform (A. plicata ptDNA (NC_031145). The direction of the SSC region was confirmed by PCR and Sanger sequencing. Specific primers were manually produced using Geneious prime 2020.0.4 (https://www.geneious.com). Detailed information on PCR locations and primers for IR regions is provided in the supplementary information  region of ptDNAs was opposite to the published ptDNA of platform  by this ormation . PCRs weormation . Howeverhnfeltia .Ahnfeltia genomes as well as all available organelle genomes of Florideophyceae and Bangiophyceae as an outgroup  using MMseqs2 , synonymous substitutions (dS), and dN/dS ratio were calculated to estimate the evolutionary selection pressure on CDSs in organelle genomes of T v7.310 . AlignedT v7.310 . CalculaT v7.310 . Outlierhttps://github.com/mossmatters/KewHybSeqWorkshop). All individual gene trees were first constructed with the outgroup Rhodymeniophycidae using IQ-TREE v1.6.8 with 1,000 fast bootstrap replications (-b 1000) and model test option (-m TEST)  . A coale-m TEST)  and pie -m TEST) . Lastly,cox1 haplotype network to evaluate genetic variation based on geographic distribution. We compared cox1 sequences of the four new Ahnfeltia populations with the COI-5P results of A. plicata and 30 sequences of A. fastigiata. Alignment was conducted with MAFFT v7.310  between 16S rRNA (rrn16) and 23S rRNA (rrn23) in an inverted repeat region.The genome sizes of the four assembled ptDNAs of  plicata . OverallAhnfeltia samples were almost identical, but one regional difference was found in A. fastigiata Oregon. In the Mauve whole genome alignment, the hypothetical protein pseudogene was translocated to the LSC region in A. fastigiata Oregon but was located in the SSC region in other strains. A. fastigiata Oregon. In addition, genome expansion mainly occurred around the hypothetical protein pseudogene regions in A. fastigiata , 32,819 bp (A. plicata UK), 33,492 bp (A. fastigiata Korea), and 33,309 bp (A. fastigiata Oregon). The number of CDSs was 26 in A. plicata Chile, A. plicata UK, and A. fastigiata Oregon, but there were only 25 CDSs in A. fastigiata Korea due to a pseudogenized intronic ORF in cox1 (orf780 in this study). GC contents were very similar, 32.9–33.4%. The numbers of rRNA, tRNA, and introns were the same in all taxa, 2, 24, and 3, respectively (The mtDNAs size of ectively .Ahnfeltia was in the group II intronic ORF composition in 23S rRNA (rrn23) and cox1. In A. plicata Chile, A. plicata UK, and A. fastigiata Oregon, a group II intronic ORF, a maturase, existed in rrn23, and another group II intronic ORF, orf729, was present in cox1. In contrast, in A. fastigiata Korea, the maturase was absent in rrn23, but two group II intronic ORFs, orf729 and orf780, existed in cox1. Additionally, there was genome expansion in cox1 group II intronic ORF (orf729) in both A. fastigiata Oregon and Korea. The nonsense mutation was found in orf780 in cox1 of A. fastigiata Korea  were supported in the rbcL gene phylogeny with the currently available six sequences in the NCBI database. Within the A. fastigiata clade, there was distinct rbcL divergence between sequences from Korea and Oregon . However, in most cases, the outliers likely resulted from biases caused by their short sequence length  and the highly conserved genes within species . In contrast, psaE showed a strong positive selection (dN/dS > 1)  . Synonym strains . Overall strains . dN and  plicata .Ahnfeltia: the translocation of the pseudogenized hypothetical protein region in ptDNA of A. fastigiata Oregon, and the different composition of group II intronic ORF in mtDNA of A. fastigiata Korea. These genomic variations were distinct features of the organelle genomes of Ahnfeltia, but variations in organelle genome structures did not occur between the two Ahnfeltia species, A. fastigiata and A. plicata. This suggested a relatively recent speciation event perhaps 2-5 Mya  were different, suggesting different intraspecific selection pressures on different organelles. Based on these results, we postulate that geographic distribution in the North Pacific may have strongly influenced the divergence of organelle genomes of A. fastigiata. Several biogeographic studies have revealed high genetic diversity of marine organisms in the North Pacific and have shown a deep genetic separation within amphi-North Pacific taxa  revealed a distinct genetic signature in populations from the Yellow Sea and Japan  with a nonsense mutation. This suggests that a hypothetical protein was horizontally introduced by plasmids into the ancestor of the two species; then, the extension of the intergenic region (including homologous tandem repeat copies) in A. fastigiata led to the translocation of the hypothetical proteins in A. fastigiata Oregon. These sudden plasmid-mediated events are distinguished from conventional cumulative mutations  could have lost maturase from rrn23 group II introns of A. fastigiata Korea, and then also had a minimum of two independent orf780 gene losses in cox1 . Most group II introns in organelle genomes have lost their self-splicing ability because of the accumulation of nonsense transcripts  (The second scenario is one gain and one loss in ta Korea . In thisI intron . Becausephyceae) , gene trAhnfeltia revealed that genome sizes of both ptDNAs and mtDNAs in A. fastigiata are a bit larger than those in A. plicata. One clear point is that the genome size difference of Ahnfeltia is due to lengths of intergenic regions of the ptDNAs and intronic regions of the mtDNAs  of A. fastigiata remained ca. 800 bp longer than that of A. plicata   had occurred in sequences that originated externally in non-coding regions. Then the repair system operated differently in each taxon when they speciated and diversified. The finding of plasmid-derived hypothetical protein pseudogenes in ptDNAs and pseudogenized group II intronic-derived ORF (orf780) in mtDNAs of A. fastigiata also indicates that the exotic genes will be degraded in organelle genomes.Organelle genome expansion in non-coding regions  has been reported from diverse algal lineages . Organele) . Moreovee)  suggestsAhnfeltia mtDNAs and ptDNAs were relatively conserved compared to their genome architecture variation. In dN/dS analysis, most coding genes were under purifying selection as synonymous substitution (dS) rates were higher than those of non-synonymous substitution (dN)  (Table S2). psaE is a short gene coding for one of the subunits of the photosystem I complex (PSI), consisting of 62 amino acids. PsaE subunit joins the docking of ferredoxin and flavodoxin on the stromal side of the membrane  . One casmembrane . It has membrane . Therefod genome , while id genome , suggestd genome .rbcL gene phylogeny and cox1 haplotype network indicated that regional genetic diversification had occurred in the four Ahnfeltia populations , A. plicata Chile and UK belonged to two different haplotypes separated by three haplotype steps, and A. fastigiata Korea and Oregon showed a similar genetic distance between two different haplotypes. Tree topology analysis using individual genes also indicated that Ahnfeltiophycidae is highly conserved and most of the ptDNAs and mtDNAs genes supported this subclass as an independent lineage. Around half of ptDNA genes and mtDNA genes supported the topology of this tree with >80% bootstrap supports, although some genes contradicted the relationship   .Ahnfeltia clearly demonstrates that Ahnfeltia organelle genomes have been dynamically changing, and genetic divergence of Ahnfeltia has enabled it to adapt to factors encountered during its global expansion. Widely separated populations of both A. fastigiata and A. plicata showed numerous genetic adaptations. The amphi-North Pacific species A. fastigiata showed more genomic divergence than the bipolar species, A. plicata. In A. fastigiata, genome structure variations were present. In the Oregon population, tandem repeats and a plasmid-mediated translocation event in ptDNA had occurred; in the Korean population, a different group II intron ORFs composition in mtDNA was present. On the other hand, stable genomic structures were shown in two A. plicata populations: Chile and UK. Overall, our data suggests that geographic distribution has affected the genomic and genetic characteristics of Ahnfeltia. To further confirm our geographic theory, a future whole-genomic study at the population-level is needed that incorporates more extensive worldwide sampling along with comparisons of both historic and present day geological and ecological data in each area.Our primary genomic study of


Helicobacter (H.) pylori targets mitochondria directly. However, mitochondrial genome (mtDNA) polymorphism (haplogroup) is not yet considered an important factor for H. pylori infection. Here, we clarified the association of mitochondrial haplogroups with H. pylori prevalence and the ability to perform damage. Seven mtDNA haplogroups were identified among 28 H. pylori-positive subjects. Haplogroup B was present at a higher frequency and haplotype D at a lower one in the H. pylori population than in that of the H. pylori-negative one. The fibroblasts carrying high-frequency haplogroup displayed a higher apoptotic rate and diminished mitochondrial respiration following H. pylori infection. mtDNA mutations were accumulated more in the H. pylori-positive population than in that of the H. pylori-negative one in old age. Among the mutations, 57% were located in RNA genes or nonsynonymous protein-coding regions in the H. pylori-positive population, while 35% were in the H. pylori-negative one. We concluded that gastric disease caused by Helicobacter virulence could be associated with haplogroups and mtDNA mutations.Mitochondria are essential organelles that are not only responsible for energy production but are also involved in cell metabolism, calcium homeostasis, and apoptosis. Targeting mitochondria is a key strategy for bacteria to subvert host cells’ physiology and promote infection.  Helicobacter pylori is a gram-negative, microaerophilic bacterium that colonizes the gastric mucosa of at least half of all humans worldwide [H. pylori can induce their imbalance, resulting in gastric diseases, such as chronic gastritis, peptic ulceration, gastric carcinoma, and lymphoma [H. pylori in the global population was steadily decreasing, it was recently estimated to be approximately 50% [orldwide . The intlymphoma ,3. Althotely 50% ,5,6.H. pylori induces the instability of mtDNA in stomach tissue [H. pylori. Polymorphisms of the host nuclear genome were demonstrated to be associated with the prevalence of H. pylori and related diseases [H. pylori activity [H. pylori infection, but also the response to treatment [Mitochondria play essential roles in normal cellular function in eukaryotes, such as energy production, calcium homeostasis, apoptotic activation, and cell death ,8. Severh tissue ,10. Indediseases ,12,13. Treatment .H. pylori.Mitochondrial haplogroups are collections of similar haplotypes defined by combinations of single-nucleotide polymorphisms in mtDNA inherited from the mother . SpecifiH. pylori. We also measured mitochondrial respiratory function and apoptosis in the cultured fibroblasts with selected haplogroups.Here, we determined mtDNA haplogroup in each individual and investigated mtDNA mutations in gastric tissue infected with H. pylori test was performed on both the antrum and body because the H. pylori positivity rate in the antrum and body may be different depending on the patient’s gastric acid secretion level. The positive or negative of H. pylori in mucosal biopsy specimens were confirmed by culturing on Brucella broth agar. This study was approved by the Institutional Review Board for Human Research at Asan Medical Center (IRB number: 2020-0108).All subjects were recruited from Asan Medical Center, Seoul, Korea. Written informed consent was obtained from each subject. Two mucosal biopsy specimens from 43 subjects, 45–78-year-old of Korean people, were obtained from the gastric antrum and corpus greater curvature using standard-sized biopsy forceps during endoscopy. The H. pylori strain ATCC 43504  [H. pylori strains were cultured on Brucella broth agar, which was supplemented with 5% sheep blood and vancomycin (10 µg/mL), trimethoprim (5 µg/mL), amphotericin B (5 µg/mL), and polymyxin B (2.5 IU). The H. pylori strains were cultured under microaerophilic conditions . All stock was maintained at −70 °C in Brucella broth supplemented with 15% glycerol.Cultured fibroblasts were infected with VA, USA) . H. pylohttps://www.mitomap.org, accessed on 25 February 2021) and homoplasmic variants were used to establish haplogroup. The locus of variants in mtDNA and GB frequency of each variant (frequency of variants reported in GenBank) are also available in Biopsied gastric tissues were collected during endoscopy in patients with gastric disease and extracted total genomic DNA using Gentra DNA Extraction Kit  in accordance with the manufacturer’s protocol. The whole mtDNA was amplified using TaKaRa LA Taq , and mtDNA sequencing was performed using Miseq  as previously described, with minor modification . Briefly4 cells were plated onto a 6-well cell culture plate and culture in F12/DMEM with 10% FBS , 100 units/mL penicillin and 100 μg/mL streptomycin (Hyclone), 100 μM β-mercaptoethanol , and 100 μM nonessential amino acids  under 5% CO2 at 37 °C in a humidified incubator for 5 days. The media were changed every 2 days. On day 5 of cell culture, bacterial cells were added to fibroblasts at a multiplicity of infection (MOI) of 50–300 bacteria for total H. pylori. For 24 h of H. pylori infection, skin fibroblasts were washed three times with PBS and incubated in an antibiotic-free medium. Cultures were maintained at 37 °C under an atmosphere of 10% CO2.Fibroblasts, which were already analyzed for mtDNA haplogroups for an independent mitochondrial project, were used to study apoptosis and mitochondrial function. Approximately 5 × 10H. pylori infection using the Dead Cell Apoptosis Kit (Invitrogen), in accordance with the manufacturer’s protocol. Briefly, harvested cells (~1 × 106 cells/mL) were incubated in annexin-binding buffer mixed with Alexa Fluor® 488 annexin V and PI solution for 15 min at room temperature. After the incubation period, stained cells were analyzed by flow cytometry, measuring the fluorescence emission at 530 nm and 575 nm using 488 nm excitation.Apoptosis assay was performed 24 h after the completion of H. pylori infection using the XF Cell Mito Stress Test Kit in an XF24 Extracellular Flux Analyzer , as previously described [Mitochondrial respiratory function was measured 24 h after the completion of escribed . The mitp < 0.05 was considered significant. Data were analyzed using regression analysis, independent-group t-test, or Fisher’s exact tests for two-group comparisons and ANOVA with Tukey for multiple group comparisons (GraphPad Prism).Assessment of mitochondrial respiratory functions was triplicated, and assessment of apoptosis was repeated four times. The data are presented as mean ± SEM or SD. https://www.mitomap.org, accessed on 25 February 2021), with homoplasmic variants in Miseq sequencing results. A total of 43 subjects, H. pylori-positive (n = 28), -negative population (n = 15), were investigated their mtDNA haplogroups and mutations. Further, the apoptosis and mitochondrial dysfunction due to H. pylori infection were evaluated in fibroblasts with selected haplogroups , -positive populations (n = 28), and the Korean population  in the H. pylori-positive population than in that of the Korean population and the H. pylori-negative population , whereas haplogroup B showed a significantly higher proportion . The remaining haplogroup displayed no significant difference in the frequency among the groups  and low-frequency haplogroups (haplogroup D) were infected with H. pylori with an MOI of 50, 100, 150, and 300 for 24 h  following H. pylori infection at 300 MOI, which possibly resulted from severe mitochondrial dysfunction under H. pylori. Therefore, we performed H. pylori infection at a reduced MOI to induce less mitochondrial damage in fibroblasts. When the cells were infected with H. pylori at 150 MOIs, most of them still showed abnormal OCR measurements  and H. pylori-negative subjects (n = 15). The number of mtDNA mutations in functional regions (coding regions and RNAs) was significantly higher (p < 0.05) in the H. pylori-positive population than in that of the -negative population at the age of 60 years or older, while similar rates were found in the D-loop as a noncoding region  in the H. pylori-positive population than in that of the negative one, and the mean heteroplasmy of mtDNA mutations above 15% heteroplasmy was significantly higher in the H. pylori-positive population  or sex in the ulations D. Males pulation E.H. pylori-positive population, 43% were nonsynonymous substitutions in the coding regions of mtDNA proteins, resulting in amino acid changes  was detected in the D-loop region as a noncoding region, and only 26% and 9% of mutations were nonsynonymous substitutions and located in RNA genes, respectively.Among 42 mutations in the  changes F. 14% weH. pylori-positive population. We focused on haplogroups D and B because of the significant changes in their frequency after H. pylori infection ((p < 0.05) in haplogroup B than in that of haplogroup D , which leads to acidosis in cells [H. pylori infection at high MOI could induce hyper acidosis in a culture dish, which would disturb the measurement of mitochondrial respiration. Therefore, the MOI of H. pylori was reduced for infecting cells, which enabled mitochondrial respiration to be measured normally, suggesting the need for an optimized MOI to measure mitochondrial function upon H. pylori infection. Under these optimized conditions, we demonstrated that mitochondrial dysfunction associated with H. pylori infection differed depending on the mtDNA haplogroup. The haplogroup that showed a higher frequency in the H. pylori-positive population was revealed to be associated with diminished mitochondrial respiration upon H. pylori infection. Meanwhile, the haplogroup with a lower frequency in the H. pylori-positive population showed less dysfunction upon H. pylori infection, suggesting different severity of mitochondrial dysfunction upon H. pylori infection, depending on the mtDNA haplogroup.Mitochondrial dysfunction was also previously reported to be induced by nfection . H. pyloic cells . Furtherutations . In thised cells . H. pyloin cells ,35. H. pH. pylori was induced epithelial apoptosis and elevated oxidative stress and oxidative damage to mitochondria in vivo in humans [H. pylori infection showed comparable results to that of in vivo infection, such as the induction of apoptosis and mitochondrial dysfunction, suggesting that H. pylori infection in vitro could represent the infection in vivo. Once H. pylori induces mitochondrial dysfunction and acidosis progresses, apoptosis subsequently occurs [H. pylori-positive population displayed severe mitochondrial dysfunction, which could result in a higher rate of apoptosis upon H. pylori infection. Virulence factors of H. pylori, such as urease subunit A (UreA), cytotoxin-associated gene A protein (CagA), and lipopolysaccharide (LPS), can induce cell apoptosis and increase oxidative stress [H. pylori-infected cells could be induced apoptosis and decreased mitochondrial function by those of virulence factors of H. pylori.n humans ,37. In ty occurs ,38. The e stress . H. pyloH. pylori infection [However, fibroblasts used for the functional study were not genetically identical except for the mtDNA haplogroup, which could affect susceptibility to nfection ,14. TherH. pylori could have a system to control the host immune response and protect the host against autoimmune diseases, asthma, and esophageal adenocarcinoma, which could also be considered a potential factor of cell cellular damage in the result of our study.To establish the chronic infection of the host, bacteria should modulate the immune system of the host, which could enhance resistance and virulence in response to each other between hosts and pathogens . H. pyloH. pylori with mtDNA mutations and individual mtDNA haplogroup. This study revealed that the number of mtDNA mutations was higher in H. pylori-positive gastric tissue than in that of the H. pylori-negative one in old age, and that mtDNA mutations were more frequently distributed in functional regions in the H. pylori-positive population. mtDNA mutations were not related to sex, whereas the antrum of the stomach tended to accumulate more mtDNA mutations in the H. pylori-positive population, but this was not the case in the body of the stomach. These results imply that mtDNA mutations are associated with the prevalence of H. pylori infection and that different anatomical positions within the stomach have different levels of susceptibility to H. pylori infection [We investigated the potential associations of nfection ,40.H. pylori inhabits the mucosal epithelium of the stomach, causing unnoticed chronic gastritis in all carriers, and severe gastric diseases, such as peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma, in 10–15% of infected individuals [H. pylori infection causes DNA damage such as oxidized bases, and single- and double-strand breaks [H. pylori infection and mtDNA mutations accumulated in H. pylori-positive patients [H. pylori-positive and -negative populations with the results showing that the number of mtDNA mutations was higher in H. pylori-positive patients at the age of 60 years or older, with such mutations being particularly located in functional regions (coding regions and RNA genes), which could constitute evidence that H. pylori affects mitochondrial function. The number of mtDNA mutations under 15% heteroplasmy was increased and heteroplasmy was increased in mtDNA mutations above 15% heteroplasmy by H. pylori infection, suggesting that H. pylori is an important factor inducing mtDNA mutations potentially.ividuals . H. pylod breaks . The inspatients ,10. ThisH. pylori-positive population, but the biopsy positions of the stomach appeared to be associated with the accumulation of mtDNA mutations. The antrum harbored more mtDNA mutations after H. pylori infection compared with that of the body of the stomach. This could be caused by the higher distribution of H. pylori or a stronger inflammatory reaction in the antrum upon infection with H. pylori [H. pylori-positive population, more mtDNA mutations in this group were nonsynonymous substitutions in protein-coding regions or located in RNA genes than those in the H. pylori-negative population, suggesting the association of H. pylori with the abnormal function of mitochondria.mtDNA mutations showed no sex bias within the . pylori . RegardiH. pylori infection, but there was no consideration of haplogroup in H. pylori patients [H. pylori-positive population, which could support understanding the association between the prevalence of H. pylori and mtDNA haplogroup.Previous studies reported the accumulation of mtDNA mutation by patients ,9,10. WeH. pylori impair mitochondrial DNA repair mechanisms lead to gastric epithelial cells being susceptible to the accumulation of mitochondrial DNA instability, which contributes to gastric carcinogenesis [H. pylori affects mitochondrial DNA replication [ogenesis ,10. Furtlication . HaplogrH. pylori, however, other factors, such as epigenetic factors, could be complementary with mtDNA mutation for the prevalence or pathogenesis in H. pylori [H. pylori injection.Although this study focused only on mtDNA mutation associated with . pylori . Genetic. pylori , and theH. pylori patients could be related to the modulated immune system by the bacterial pathogen [Further, the accumulation of mtDNA mutations in pathogen , which aH. pylori-positive subjects, each H. pylori subject comes from a different environment and family, and therefore, we speculated on the possibility of different and diverse strains in each dividual [H. pylori heterogeneity would explain the low heritability and difficulty in replicating the results of H. pylori association studies [For 28 dividual . A previ studies ,46, whicH. pylori infection, further studies are needed to confirm the association between H. pylori and mtDNA haplogroups with the large-scale screening of mtDNA haplogroup in H. pylori patients. Further, if the finding of a high frequency of haplogroup in H. pylori-infected patients is replicated in other populations, it would provide powerful evidence for the association between H. pylori infection and mtDNA haplogroup, and the prevalence of H. pylori in genetically sensitive individuals could be reduced by introducing preventive strategies to minimize environmental causes.For a deeper insight into the impact of mtDNA haplogroup upon H. pylori-positive population, resulting from higher susceptibility to H. pylori. This haplogroup was associated with greater damage to mitochondrial function and higher apoptosis upon H. pylori infection. However, there were limitations, as this was a pilot study with small sample size and the use of fibroblasts and a commercial H. pylori strain. Further studies on virulence factors of H. pylori strains isolated from H. pylori patients are required.Our study demonstrated that mtDNA haplogroup B was present at a high frequency in the


Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is a tiny natural egg parasitoid of various agricultural pest insects, including Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. However, how T. pretiosum seek and localise host insect eggs is still not clear. The olfactory system is critical in guiding insect behaviours, including mating, feeding and oviposition, which play pivotal roles in the interactions between parasitoid wasps and their hosts. This project aimed to investigate T. pretiosum major olfactory tissue (antennae) and the olfactory genes, including odorant binding proteins (OBPs) and odorant receptors (ORs). T. pretiosum adult antennae were examined under scanning electron microscopy, and four types of olfactory sensilla were observed. Using T. pretiosum genome, 22 OBPs and 105 ORs were identified, which were further compared with olfactory genes of other Hymenoptera insect species. The expression patterns of OBPs between T. pretiosum male and female adults were examined to identify female- or male-specific OBPs. This study enriches our knowledge of T. pretiosum olfactory system and will help better use it in the integrated pest management (IPM) for many insect pest species.Parasitic wasps are the major natural enemies of many organisms, and therefore they are broadly used in the biological control of numerous agricultural and horticultural pests. For example, Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae) is a tiny natural egg parasitoid of several agricultural pest insects, which has been widely used in the biological control for Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. However, limited studies have been conducted on T. pretiosum olfactory system, which is critical in regulating insect behaviours. In this study, T. pretiosum adult antennae were investigated under ascanning electron microscopy (SEM). Four types of olfactory sensilla were observed, including chaetica sensilla (CS), trichoid sensilla (TS), faleate sensilla (FS) and placoid sensilla (PS). Using T. pretiosum genome, 22 putative odorant binding proteins (OBPs) and 105 odorant receptors (ORs) were identified, which were further compared with olfactory genes of Apis mellifera, Nasonia vitripennis and Diachasma alloeum. The expression patterns of OBPs between T. pretiosum male and female adults were examined by quantitative real time PCR (qRT-PCR) approaches. Three female-specific OBPs  were identified, which may play crucial roles in T. pretiosum host-seeking and oviposition behaviours. This study enriches our knowledge of T. pretiosum olfactory genes and improves our understanding of its olfactory system. Insect behaviours, including mating, foraging, host-finding and oviposition, are guided by their olfactory systems . Hair-liTrichogramma pretiosum is a minute (≤0.5 mm long) wasp and female adults lay eggs into a number of lepidopteran eggs, including Plutella xylostella, Helicoverpa armigera, Spodoptera frugiperda and Ectomyelois ceratoniae. After T. pretiosum eggs hatch, the larvae devour the developing caterpillar, pupate and grow into adult wasps inside the host eggs. Adult wasps emerge by chewing holes in the host eggs and are then ready to parasitise other moth eggs. T. pretiosum have been used to control caterpillar pests in a wide range of horticultural and field crops [Parasitic wasps develop in or on various life stages of other arthropod hosts, and chemical signals are critical in guiding their mating, host-seeking and oviposition behaviours. For example, after mating, female wasps utilise host-associated chemical cues, including host pheromones or herbivore-induced plant volatiles to localise hosts ,16. Tricld crops .T. pretiosum use volatile host pheromones to locate host eggs [Helicoverpa zea and synthetic H. zea female pheromone components increased parasitisation rates by T. pretiosum [T. pretiosum responds to calling H. zea females [T. pretiosum olfactory system, which plays a critical role in semiochemical detection and guiding T. pretiosum host-seeking behaviours. The completion of the whole genome sequences of T. pretiosum provides an invaluable resource for us to annotate and analyse olfactory genes in this minute parasitoid wasp [Generally, egg parasitoid wasps rely on chemical cues originating from the adult host, host products, or the plant that host feeding on to seek host eggs , which aost eggs . Volatilretiosum . Later,  females . Howeveroid wasp .T. pretiosum antennae and sensilla were investigated by using scanning electron microscopy (SEM). Then T. pretiosum genome database was used to annotate the key olfactory genes, including OBPs and ORs, which were further analysed using bioinformatics, phylogenetics and molecular approaches.In this study, firstly, T. pretiosum pupae were sourced from Bugs for Bugs™  and kept in the lab at 25 ± 1 °C, 70–80% relative humidity (R.H.) and 16:8 h (Light:Dark) photoperiod. Emerged adults were collected immediately using vacuum traps and anaesthetised using carbon dioxide  for five minutes, and then sexed under a stereomicroscope  based on antennae structures. Emerged adults were allocated for RNA extraction and scanning electron microscopy (SEM) .T. pretiosum adults were immediately stored in liquid nitrogen and then homogenised using a disposable homogenising pestle . T. pretiosum adult body sizes are tiny (<0.5 mm), so we did not dissect different tissues such as antennae and palps for RNA extraction. Total RNA was extracted using the Qiagen RNeasy mini kit  following the manufacturer’s protocol. The purified total RNA was treated by DNase I  to remove genomic DNA, quantified, quality checked using NanoDrop™ ND-2000  and stored at −80 °C in the Western Australia State Agricultural and Biotechnology Centre .After sexing, collected T. pretiosum adults were preserved in 3% glutaraldehyde in 0.025 M pH 7.0 phosphate buffer for 24 h and then used for fixation with a Pelco Biowave processor . A critical point drying apparatus  was used in the preparation process at the critical point of CO2 = 31.1 °C and 1071 psi. Dried samples were mounted on SEM stubs under a SZH10 microscope  and then sputtered with 10 nm gold using a Polaron Sputter coater SC 7640  with argon gas . Samples were examined and photographed under a Zeiss 1555 VP-FESEM SEM instrument  operated at 10 kV, high current, 10–12 mm working distance, and 30 µm aperture. Sample preparation and examination were conducted at the Centre for Microscopy, Characterization, and Analysis (CMCA) at the University of Western Australia. Five male and five female antennae were observed under SEM (n = 5).T. pretiosum OBPs, and ORs in the genome (NCBI: PRJNA297592) were identified using BLAST (blastn) searches with reported D. melanogaster and Apis mellifera OBP and OR genes as queries, as previously described [T. pretiosum genome according to exon/intron splice site of GT-AG rule. The identified OBP and OR amino acid sequences . Calculated molecular weights (MW) and isoelectric points (pIs) were obtained using the ExPASy proteomics server  [http://gsds.cbi.pku.edu.cn/index.php, accessed on 5 September 2021).Encoded TpreOBPs  were alier 2021) . The amiA. mellifera, Nasonia vitripennis and Diachasma alloeum OBP protein sequences [T. pretiosum, A. mellifera, N. vitripennis and D. alloeum ORs.TpreOBP amino acid sequences  were useequences ,25,26,27T. pretiosum cDNA templates were prepared from total RNA samples using the SuperScript™ VILO™ cDNA Synthesis Kit  following the manufacturer’s protocol. Quantitative Real-Time PCR (qRT-PCR) was performed using gene-specific primers . The reference housekeeping gene, glyceraldehyde-phosphate dehydrogenase (GAPDH), was selected here because it has been shown as one of the best candidate reference genes in qPCR analysis [® Green PCR Master Mix  as follows: 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 15 s, and 65 to 95 °C in increments of 1.0 °C for 5 s [−ΔΔCt method for relative quantification [t-test . primers , which wanalysis . A 2-Ste for 5 s . For eacfication . Three bT. pretiosum adult antennae are sexually dimorphic  in T. pretiosum is higher than D. alloeum (15 OBPs), A. mellifera (21 OBPs), but lower than N. vitripennis (90 OBPs). The number of ORs (105 ORs) in T. pretiosum is significantly lower than in A. mellifera (163 ORs), D. alloeum (187 ORs), and N. vitripennis (301 ORs).A total of 22 OBPs and 105 ORs were identified from the m genome , which wTrichogrammadendrolimi. TpreOBP3 showed 97% identify to OBP3 (Sequence ID ASA40277.1) from Trichogramma japonicum. No signal peptide was predicted from TpreOBP7, while all the other 21 TpreOBPs carry signal peptides. The mature  TpreOBPs range from 109 to 132 amino acids, and their molecular weights range from 12,279 to 15,033 Da  from 5,033 Da . The iso5,033 Da . The ali5,033 Da a. Most oectively b. TpreOBT. pretiosum and three other Hymenopteran species: A. mellifera, N. vitripennis and D. alloeum (N. vitripennis OBPs (NvitOBPs)  , suggestretiosum . There aretiosum . One “doretiosum , as descT. pretiosum-specific OBP was observed, which did not show similarity to any other ORs from A. mellifera, N. vitripennis and D. alloeum. As one of the most conserved genes in various insect species, TpreORCO shared 72.8% identity with N. vitripennis ORCO (NvitOR1), 60.8% identity with A. mellifera ORCO (AmelOR2) and 51.3% identity with D. alloeum ORCO  at the amino acid level. Various TpreORs were clustered closely with N. vitripennis ORs (NvitORs)  , just asOBPs are involved in the first step of odorant detection, so they are our major targets in the expression profile study. Expression profiles between male and female insects can help identify the female- or male-specific OBPs and build the links between OBPs and their potential functions. Here, TpreOBPs were examined between male and female adults by qRT-PCR. To test the designed primers, reverse transcriptase (RT)-PCR was conducted first, and the products were analysed using electrophoresis. All 22 TpreOBPs were successfully amplified, and their band sizes were the same as expected, suggesting the primers were designed appropriately.To study the expression levels, the qRT-PCR approach was utilised to compare TpreOBP expression levels between males and females with the TpreGADPH gene as a reference gene . The resType 2 are male-specific OBPs, including TpreOBP22, TpreOBP5, TpreOBP10 and TpreOBP17, which demonstrated significantly higher expression levels (>5) in male adults than in female adults b. For exT. pretiosum, is frequently used as biological control agent against major lepidopteran pests, including P. xylostella, H. armigera and E. ceratoniae. However, the mechanism of how T. pretiosum localise host eggs is not fully understood yet. Without this knowledge, it is difficult for us to better understand T. pretiosum host-seeking and parasitising behaviours and refine our current application by using it as an efficient and effective biological control agent. This study on T. pretiosum olfactory system provided us with insights into this field.Parasitoid wasps constitute a large group of hymenopteran superfamilies, which lay eggs on or in the bodies of other arthropods, resulting in the death of their hosts. Trichogrammatids are some of the smallest parasitoid wasps that grow up within-host insect eggs. One of them, T. pretiosum adult antennae were examined using SEM because they are the major insect olfactory organs. Male and female T. pretiosum showed very different antennae structures. Furthermore, the faleate sensilla (FS) was only observed in female but not male antennae, suggesting significant differences between their olfactory systems. Interestingly, this was not observed in another P. xylostella parasitoid wasp, Diadegma semiclausum, which lays eggs into P. xylostella larvae specifically (Unpublished data).Firstly, T. pretiosum genome project is a significant step towards further understanding its olfactory system and potential applications in pest control. T. pretiosum genome shows rapid genome evolution compared to other hymenopterans , reflecting adaptations to miniaturisation and to life as a specialised egg parasitoid. In the T. pretiosum genome, 22 OBPs and 105 ORs were identified, and compared with olfactory proteins of A. mellifera, N. vitripennis and D. alloeum. A. mellifera is one of the model species for Hymenoptera, whose olfaction and social behaviours have been extensively studied. N. vitripennis is the most widely studied of the parasitoid wasps, which is a generalist and parasitises a wide range of dipteran hosts, including blowflies, fleshflies and houseflies. D. alloeum is a specialist parasitoid of the fruit fly, Rhagoletis pomonella. The number of OBPs and ORs exhibited significant differences in these four Hymenopteran species, supporting the differences among their chemosensory systems and biology. For example, A. mellifera has 163 ORs but only ten gustatory receptors (GRs). Presumably a large number of ORs can enhance A. mellifera olfactory ability and facilitate the typical foraging and social behaviours while honeybees have limited need for GRs for plant secondary metabolite discrimination since flowering plants have evolved visual and olfactory cues to attract bees [T. pretiosum has the lowest numbers of ORs in these four species. It may be due to that most of its life stages are inside the host eggs and its tiny body size. A number of OBPs and ORs exhibited high similarities at amino acid level between T. pretiosum and N. vitripennis, suggesting these proteins may play similar roles in both two generalist parasitoid wasps. A recent study compared the OBPs and ORs between T. pretiosum and D. semiclausum because both are important parasitoid wasps and widely used in the biological control for P. xylostella, one of the most destructive insect pests of cruciferous plants [T. pretiosum, a natural egg parasitoid that lay eggs to various host insect eggs, D. semiclausum lay eggs into P. xylostella larvae specifically and selectively. A number of OBPs and ORs were identified showing high similarities between these two wasps , which may function similarly in these two species. More conserved OBPs or ORs will be identified from D. semiclausum after its genome was available. Further functional characterisation of these OBPs or ORs will provide insights to their roles in T. pretiosum olfactory system and behaviours.Completing the act bees . T. prets plants . Unlike T. pretiosum adults were used for RNA extraction rather than antennae because T. pretiosum is too tiny (<0.5 mm), and the antennae collection is extremely time-consuming, which may cause RNA degradation. Therefore, the qRT-PCR results only exhibited the expression profile of TpreOBPs between male and female adult bodies, but not antennae. OBPs have been reported to be expressed in non-olfactory organs rather than antennae, and they may have other functions [Aedes aegypti OBP22 is produced in the sperm and transferred to females during mating [OBPs are involved in the first step of odorant detection, so the characterisation of their sex-specific expression will help build the links between OBPs and their potential functions. For example, female-specific OBPs are more likely to play a significant role in the parasitism behaviours. Male-specific OBPs may be involved in the sex pheromone detection and mating behaviours. Here the whole bodies of male and female unctions . For exag mating . TherefoT. pretiosum, indicating they may play similar roles in both. For example, they are detecting flower odors for nectar-sucking, which are a major sugar resource for insect species. These results help us link the functions of T. pretiosum OBPs for future functional characterisation studies. Further, in vitro  and in vivo  functional characterisation will help demonstrate their roles in T. pretiosum olfactory system. For example, after the knock-down or knock-out of the target OBPs or ORs, the treated insect responses and behaviours will be observed to various chemical compounds [TpreOBP19, 15 and 3, are female-specific OBPs, which are the candidate OBPs to assist in parasite behaviours. TpreOBP22, TpreOBP5, TpreOBP10 and TpreOBP17 showed male-specific expression, suggesting they may contribute to female seeking and mating behaviours. Other TrepOBPs showed similar expression levels between male and female ompounds ,35. On tompounds .T. pretiosum, which were further studied by phylogenetic and bioinformatics methods. The sex-specific expression patterns of T. pretiosum OBPs were analysed using qRT-PCR between male and female adults. This study advances our understanding of the chemosensory system of T. pretiosum at the molecular level and provides a foundation for further research on the olfactory system in T. pretiosum.In summary, 22 OBPs and 105 ORs were identified from the genome sequence of


Helicobacter pylori (H. pylori) infection are reportedly related. However, the mechanism is not fully understood. This study aimed to elucidate the relationship between H. pylori infection and pyloric incompetence in a non-operated stomach. A total of 502 non-operated participants who underwent an upper intestinal endoscopy were prospectively enrolled. Endoscopic findings , sex, age, gastrin, pepsinogen (PG) I and PG II levels were evaluated. PG I/PG II ratio, anti-H. pylori-Ab positivity, and atrophic gastritis status were significantly different between the normal and incompetent pylori . Open-type atrophic gastritis was significantly higher in the incompetent pylori. Incompetence of the pylorus and EAC were moderately correlated (Cramer’s V = 0.25). Multivariate analysis revealed that the presence of anti-H. pylori-Ab was the only independent factor associated with the incompetence of the pylorus, with an adjusted odds ratio of 2.70 . In conclusion, pyloric incompetence was associated with H. pylori infection and moderately correlated to the severity of atrophic gastritis in non-operated stomachs.Duodenogastric reflux (DGR) causes bile reflux gastritis (BRG) and may develop into gastric cancer. DGR is classified as primary in non-operated stomachs or secondary to surgical intervention. Primary DGR and  Among reflux diseases in the gastrointestinal (GI) tract, the competence of the junctions is critical. Incompetence of the esophagogastric sphincter is well known to cause gastroesophageal reflux disease (GERD) . The nexHelicobacter pylori (H. pylori) and chronic gastritis in 1983 [H. pylori in the 1990s [H. pylori eradication [H. pylori infection. However, the relationship has still been controversial [H.pylori infection for DGR remains unclear. In this study, we aimed to elucidate the relationship between H. pylori infection and pyloric incompetence in a non-operated stomach.After Warren and Marshall’s first report of  in 1983 , some inoversial ,28. The H. pylori eradication therapy. They did not have any particular symptoms.This study was a single-center, prospective, observational study conducted between August and December 2013 in Yutaka Fukuda Clinic . The inclusion criteria were as follows: (i) participants who underwent upper gastrointestinal endoscopy for healthy check-up without any antispastics, including scopolamine butylbromide and glucagon, and underwent blood tests; (ii) those who were under 80 years of age; and (iii) those without a history of surgery for the stomach and Finally, 502 participants  were continuously enrolled. One expert endoscopist, who did not receive the blood test results, assessed the following items: endoscopic atrophic degree, nodular gastritis, xanthoma, fundic gland polyp, and pyloric incompetence. The endoscopic atrophy classification (EAC) was assessed using the Kimura and Takemoto classification . We defiH. pylori immunoglobulin G antibody (anti-H. pylori-Ab) was measured using an enzyme immunoassay kit using antigens derived from Japanese individuals ; the serum levels of pepsinogen I and II  and gastrin were measured using chemiluminescent enzyme radioimmunoassay  and radioimmunoassay kit , respectively. An H. pylori antibody titer of ≥10 U/mL was classified as H. pylori-positive [The blood test was conducted on the same day as upper gastroenteroscopy following overnight fasting. The serum anti-positive . The valpositive ,32. We pt-test and chi-squared test were applied to compare the two groups. The logistic regression model was applied for multivariate analyses. The adjusted odds ratio (OR) with a 95% confidence interval (CI) was also calculated. The Cochran–Armitage test and Cramer’s V were applied to examine the correlation between two categorical variables. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were also investigated. All statistical analyses were performed using R (Ver. 4.0.3) or StatFlex . Values were expressed as mean with standard deviation (SD). Statistical significance was set at p < 0.05.Welch’s H. pylori-positive, and 55 (11.0%) participants’ pylori were deemed as endoscopically incompetent.We first divided the participants into incompetent pylorus (n = 55) and normal pylorus (n = 447) groups. The age, serum gastrin level, PG I and PG II levels, PG I/PG II, and endoscopic atrophic classification were evaluated. Endoscopic atrophic gastritis was divided into closed type or open type by EAC. Open-type atrophic gastritis was observed in 87 (19.5%) and 23 (41.8%) participants in the normal and incompetent pylorus groups, respectively.p = 0.043), participants whose anti-H. pylori-Ab was positive , and participants who had atrophic gastritis  were significantly different between the incompetent pylorus and the normal pylorus group   and strongly-related (Cramer’s V = 0.73) to the incompetent pylorus group.Furthermore, we analyzed EAC between the two groups. The presence of incompetent pylori was higher with open-type atrophic gastritis   . There wH. pylori-Ab positivity ; therefore, we excluded these factors. The presence of anti-H. pylori antibodies was the only independent factor associated with pyloric incompetence, with an adjusted odds ratio of 2.70 .Thereafter, we conducted a multivariate analysis to distinguish independent factors related to pyloric incompetence from other confounding factors and calculated the adjusted odds ratio (OR) with 95% CI . PG I/PGH. pylori infection. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for predicting H. pylori infection in known predictive endoscopic findings  and pyloric incompetence . Indeed, endoscopic pyloric incompetence is not diagnostic of DGR; however they are considered to be closely associated [H. pylori-Ab positive group and had a strong correlation to the incompetent pylorus group. Atrophic gastritis is the first step in Correa’s cascade [H. pylori infection and a high bile acid concentration in the gastric juice. These data suggest that both H. pylori infection and the resulting atrophic gastritis may affect the competence of the pylorus.Matsuhisa et al.  reportedsociated . Moreove cascade . Patholo cascade . Li et a cascade  demonstrH. pylori infection induces open-type atrophic gastritis, wherein severe atrophy impairs the function of the pylorus; the incompetent pylorus consequently causes DGR. We hypothesized that H. pylori infection in patients with DGR [H. pylori infection [H. pylori infection. Our study findings may have partially fulfilled the goals of the 50-year investigations on the mechanism of primary DGR/BRG. Interestingly, Szőke et al. demonstrated that smoking is associated with with DGR . Smokingwith DGR ,22. Smoknfection . We did H. pylori infection is a leading cause of gastric cancer [H. pylori infection may cause progression to gastric cancer that may not be through the traditional process: inflammation–atrophy–metaplasia [It is estimated that 0.1% to 0.3% of atrophic gastritis cases develop into gastric adenocarcinomas annually . Patientc cancer ,43. The taplasia H. pylori is decreasing, especially in developed countries with the expanded indication of H. Pylori eradication therapy, it is estimated that approximately 4.4 billion people are still infected with H. pylori worldwide [H. pylori infection and diagnose the severity of atrophic gastritis.Although the prevalence of orldwide . TherefoThe Kyoto classification of gastritis was advocated at the 85th Congress of the Japan Gastroenterological Endoscopy Society to standardize endoscopic findings of gastritis. The Kyoto classification included categories of xanthoma, nodularity, and fundic gland polyp ,46. HoweH. pylori infection. The sensitivity of pyloric incompetence was higher than those of nodular gastritis, xanthoma, and fundic gland polyp in predicting H. pylori infection. On the other hand, its specificity was lower. Incompetence of the pylorus may be a good predictor of H. pylori infections.Our data demonstrated that the severity of atrophic gastritis was moderately related to pyloric incompetence. Therefore, we also investigated the sensitivity and specificity of pyloric incompetence for H. pylori eradication therapy at another hospital or may have experience natural resolution of the infection. Third, there might be false-negative cases in diagnosing H. pylori infection with serum anti-H. pylori-Ab. It was reported that in a population with no history of eradication therapy, 17% of participants whose anti-H. pylori-Ab titer was 3–9.9 U/mL were false negative [H. pylori infection and incompetency of the pylorus.This study has several limitations. First, this is a single-center study, which enrolled only a small number of participants. Second, the study might have included participants who had unintentionally undergone negative . Fourth,In this study, we focused on the competence of the pylorus. In future studies, we may also focus on the last junction in the GI tract, which is the ileocecal valve; its incompetency causes “backwash ileitis” in ulcerative colitis . It can H. pylori infection and moderately correlates to the severity of atrophic gastritis in non-operated stomachs. Pyloric incompetence can also be a predictive endoscopic finding of H. pylori infection.In this study, we elucidated that the incompetence of the pylorus is associated with