Linked mutations within the pathogenicity locus of Clostridioides difficile increase virulence.

BACKGROUND: The clinical manifestations of Clostridioides difficile infections range from diarrhoea to pseudomembranous colitis (PMC) and death. We evaluated the association between gene content in C. difficile clinical isolates and disease severity. METHODS: Fifty-three C. difficile isolates were subjected to Sanger sequencing, clinical data were used to analyse the association of gene content with disease severity, and 83 non-duplicate isolates were collected to confirm the results. Virulence was further examined by functional in vitro and in vivo experiments. RESULTS: Among the 53 C. difficile isolates, ribotypes 017 (n = 9, 17.0%) and 012 (n = 8, 15.1%) were predominant. Fifteen strains exhibited a correlation between mutations of pathogenicity locus genes (tcdB, tcdC, tcdR, and tcdE) and were named linked-mutation strains. Ribotypes are not associated with clinical PMC and Linked-mutation strains. The proportion of patients with PMC was higher in the group infected with linked-mutation strains than in the non-linked-mutation group (57.14% vs. 0%, p < 0.001). The linked-mutation rate of C. difficile was higher in patients with PMC than in patients without PMC (89.47% vs. 7.8%, p < 0.0001). Linked-mutation strains showed greater cytotoxicity in vitro and caused more severe tissue damage in a mouse model. CONCLUSIONS: Linked-mutation strains are associated with high virulence and PMC development. This result will help monitor the clinical prognosis of C. difficile infection and provide key insights for developing therapeutic targets and monoclonal antibodies.

PARP2 downregulation in T cells ameliorates lipopolysaccharide-induced inflammation of the large intestine.

Introduction: T cell-dependent inflammatory response with the upregulation of helper 17 T cells (Th17) and the downregulation of regulatory T cells (Treg) accompanied by the increased production of tumor necrosis alpha (TNFa) is characteristic of inflammatory bowel diseases (IBD). Modulation of T cell response may alleviate the inflammation thus reduce intestinal damage. Poly(ADP-ribose) polymerase-2 (PARP2) plays role in the development, differentiation and reactivity of T cell subpopulations. Our aim was to investigate the potential beneficial effect of T cell-specific PARP2 downregulation in the lipopolysaccharide (LPS) induced inflammatory response of the cecum and the colon. Methods: Low-dose LPS was injected intraperitoneally to induce local inflammatory response, characterized by increased TNFa production, in control (CD4Cre; PARP2+/+) and T cell-specific conditional PARP2 knockout (CD4Cre; PARP2f/f) mice. TNFa, IL-1b, IL-17 levels were measured by ELISA, oxidative-nitrative stress was estimated by immunohistochemistry, while PARP1 activity, p38 MAPK and ERK phosphorylation, and NF-kB expression in large intestine tissue samples were examined by Western-blot. Systemic & local T cell subpopulation; Th17 and Treg alterations were also investigated using flowcytometry and immunohistochemistry. Results: In control animals, LPS induced intestinal inflammation with increased TNFa production, while no significant elevation of TNFa production was observed in T cell-specific PARP2 knockout animals. The absence of LPS-induced elevation in TNFa levels was accompanied by the absence of IL-1b elevation and the suppression of IL-17 production, showing markedly reduced inflammatory response. The increase in oxidative-nitrative stress and PARP1-activation was also absent in these tissues together with altered ERK and NF-kB activation. An increase in the number of the anti-inflammatory Treg cells in the intestinal mucosa was observed in these animals, together with the reduction of Treg count in the peripheral circulation. Discussion: Our results confirmed that T cell-specific PARP2 downregulation ameliorated LPS-induced colitis. The dampened TNFa production, decreased IL-17 production and the increased intestinal regulatory T cell number after LPS treatment may be also beneficial during inflammatory processes seen in IBD. By reducing oxidative-nitrative stress and PARP1 activation, T cell-specific PARP2 downregulation may also alleviate intestinal tissue damage.

Inflammatory bowel disease is causally related to irritable bowel syndrome: a bidirectional two-sample Mendelian randomization study.

Introduction: Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are lifelong digestive diseases that severely impact patients' quality of life. The existence of a causal association between IBS and IBD remains unclear. This study aimed to determine the direction of causality between IBD and IBS by quantifying their genome-wide genetic associations and performing bidirectional two-sample Mendelian randomization (MR) analyses. Methods: Genome-wide association studies (GWAS) among a predominantly European patient cohort identified independent genetic variants associated with IBS and IBD. Two separate databases (a large GWAS meta-analysis and the FinnGen cohort) for both IBS and IBD were consulted to retrieve statistics on instrument-outcome associations. MR analyses included inverse-variance-weighted, weighted-median, MR-Egger regression, MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) methods, and sensitivity analyses were performed. The MR analyses were carried out for each outcome data, followed by a fixed-effect meta-analysis. Results: Genetically predicted IBD was associated with an increased risk of IBS. Odds ratios (95% confidence intervals) for samples of 211,551 (17,302 individuals with IBD), 192,789 (7,476 Crohn's disease cases), and 201,143 (10,293 ulcerative colitis cases) individuals were 1.20 (1.00, 1.04), 1.02 (1.01, 1.03), and 1.01 (0.99, 1.03), respectively. After outlier correction using MR-PRESSO, the odds ratio for ulcerative colitis was 1.03 (1.02, 1.05) (p = 0.001). However, an association between genetically influenced IBS and IBD was not identified. Discussion: This study confirms that IBD is causally related to IBS, which may interfere with the diagnosis and treatment of both diseases.

Leaky Gut Plays a Critical Role in the Pathophysiology of Autism in Mice by Activating the Lipopolysaccharide-Mediated Toll-Like Receptor 4-Myeloid Differentiation Factor 88-Nuclear Factor Kappa B Signaling Pathway.

Increased intestinal barrier permeability, leaky gut, has been reported in patients with autism. However, its contribution to the development of autism has not been determined. We selected dextran sulfate sodium (DSS) to disrupt and metformin to repair the intestinal barrier in BTBR T+tf/J autistic mice to test this hypothesis. DSS treatment resulted in a decreased affinity for social proximity; however, autistic behaviors in mice were improved after the administration of metformin. We found an increased affinity for social proximity/social memory and decreased repetitive and anxiety-related behaviors. The concentration of lipopolysaccharides in blood decreased after the administration of metformin. The expression levels of the key molecules in the toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-nuclear factor kappa B (NF-κB) pathway and their downstream inflammatory cytokines in the cerebral cortex were both repressed. Thus, "leaky gut" could be a trigger for the development of autism via activation of the lipopolysaccharide-mediated TLR4-MyD88-NF-κB pathway.

Global trends in Akkermansia muciniphila research: A bibliometric visualization.

Background: Akkermansia muciniphila is a member of the gut microbiome, using mucin as sources of carbon, nitrogen, and energy. Since the first discovery of this unique bacterium in 2004, A. muciniphila has been extensively studied. It is considered a promising "next-generation beneficial microbe." The purpose of this paper is to sort out the research status and summarize the hotspots through bibliometric analysis of the publications of A. muciniphila. Methods: The publications about A. muciniphila from January 2004 to February 2022 were obtained from the Web of Science Core Collection. Visualization analyses were performed using three bibliometric tools and GraphPad Prism. Results: A total of 1,478 published documents were analyzed. Annual publication number grew from 1 in 2004 to 336 in 2021, with China being the leading producer (33.36%). De Vos, Willem M was the most productive author with the highest H-index (documents = 56, H-index = 37), followed by Cani, Patrice D (documents = 35, H-index = 25). And Scientific Reports published the most papers. PNAS was the keystone taxa in this field, with high betweenness centrality (0.11) and high frequency. The keywords with high frequency in recent years include: oxidative stress, diet, metformin, fecal microbiota transplantation, short-chain fatty acids, polyphenols, microbiota metabolites and so on. The keyword "oxidative stress" was observed to be increasing in frequency recently. Conclusion: Over time, the scope of the research on the clinical uses of A. muciniphila has gradually increased, and was gradually deepened and developed toward a more precise level. A. muciniphila is likely to remain a research hotspot in the foreseeable future and may contribute to human health.

Gastrointestinal symptoms have a minor impact on autism spectrum disorder and associations with gut microbiota and short-chain fatty acids.

Children with autism spectrum disorder (ASD) experience gastrointestinal (GI) issues more frequently and severely than children who are typically developing (TD). The connections between gastrointestinal problems, microbiota, and short-chain fatty acids (SCFAs) in ASD are still being debated. We enrolled 90 children, 45 of whom were diagnosed with ASD, and examined the impact of GI disorders on ASD. The six-item GI Severity Index questionnaire was used to evaluate gastrointestinal symptoms, while the Social Responsiveness Scale was used to evaluate autism symptoms. Further, the Children's Sleep Habits Questionnaire and the Children's Eating Behavior Questionnaire are used to assess sleep and eating disorders in children. We assessed fecal microbiota by 16S rRNA gene sequencing, and SCFA concentrations by gas chromatography/mass spectrometry. The results revealed that children with ASD exhibited a high rate of gastrointestinal issues (78%), as well as higher rates of social impairment and poor sleeping habits, compared to TD children. However, GI disturbances have a minor impact on autism. In addition, the levels of propionic acid, butyric acid, and valeric acid were significantly higher in the ASD group. Besides, the ASD, TD, and GI subgroups possessed distinct microbiome profiles. These findings suggest that gastrointestinal disturbances have no discernible effect on the core symptoms of autism. Although autism may not cause an increase in GI symptoms directly, alterations in metabolites, such as SCFAs, may cause GI symptoms.

Global trends in gut microbiota and clostridioides difficile infection research: A visualized study.

BACKGROUND: Clostridioides (clostridium) difficile infection (CDI) is the most common cause of nosocomial diarrheal disease, which has become a public health problem worldwide; gut dysbiosis plays a central role in its pathophysiology. This study conducted a bibliometric analysis of publications on gut microbiota and CDI to summarize the current status of research including research hotspots. METHODS: Relevant publications from January 2004 to February 2022 were identified from the Web of Science Core Collection. Three bibliometric tools were used to perform visualization analyses. RESULTS: A total of 1983 publications were analyzed. Annual publications increased from 11 in 2004-237 in 2021, with the US being the leading producer (47.55 % of all papers). EG Pamer had the highest average citations per article (average citations per item = 153.03, H-index = 29). Frontiers in Microbiology published the most papers. The main research foci were "fecal microbiota transplantation," "colonization resistance," and "multidrug-resistant bacteria." The keywords with the highest frequency in recent years include: gut dysbiosis, antibiotic resistance, bile-acids, 16 s sequencing, multidrug-resistant bacteria, and short chain fatty acids. CONCLUSIONS: Gut microbiota and CDI is likely to remain a prominent area of research in the foreseeable future. Current research hotspots ("fecal microbiota transplantation," "colonization resistance," and "multidrug-resistant bacteria") should receive even more attention in future studies.

Effect of metformin in autistic BTBR T + Itpr3tf/J mice administered a high-fat diet.

The biological mechanisms linking diet-related obesity and autism-related behaviors remain unclear. We aimed to characterize these interactions, focusing on gut microbiota, 5-hydroxytryptamine (5-HT) levels, and autistic behaviors in an animal model for autism; a high-fat diet (HFD) BTBR T + Itpr3tf/J (BTBR) mouse. In this model, we also examined the medication effects of metformin (Met) which is known to ameliorate several symptoms of autism spectrum disorder (ASD). Therefore, we hypothesized that HFD exacerbates BTBR autistic symptoms, which can be alleviated by Met, and the effects are associated with serotonin and the microbiota. As expected, compared with mice fed a normal diet, ten-week HFD-fed mice showed increased body weight, adiposity, and glucose levels. HFD consumption markedly aggravated repetitive behaviors in the self-grooming test. Met reduced HFD-induced hyperactivity. Notably, HFD intervention rescued sociability in the three-chamber sociability test. Furthermore, HFD stimulated tryptophan production, which was inhibited by Met. In contrast, 5-HT levels were lower in the gut and higher in the cortex in the HFD group. Moreover, Met suppressed inflammation in the hippocampus of HFD-fed mice by significantly downregulating the expression of pro-inflammatory cytokines (NF-κB, IL-17A, and IL-6). HFD increased the Firmicutes/Bacteroidetes ratio, and Met supplementation decreased richness while increasing bacterial diversity. We found that the abundance of gut microbiota (Lachnoclostridium, Anaerotruncus, Mucispirillum, and Lactococcus) was correlated with behavior scores and 5-HT levels. Overall, HFD consumption improved sociality in BTBR mice, which was related to the modulation of 5-HT levels and the composition of the microbiota. Met did not show any significant positive effects on the autism phenotype associated with HFD.

Promotion of Interface Fusion of Solid Polymer Electrolyte and Cathode by Ultrasonic Vibration.

All-solid-state polymer lithium batteries have good safety, stability, and high energy densities and are employed in wireless sensors. However, the solid contact between the polymer electrolyte and the cathode leads to high interface resistance, limiting the broad application of solid-state lithium batteries. This paper proposes an ultrasonic fusion method to reduce the interface resistance between the polymer electrolyte and the cathode. The method applied a high-frequency ultrasonic vibration technique to impact the polymer electrolyte/cathode structure, melting the electrolyte at the interface and thus generating good contact at the interface. The experimental results showed that the ultrasonic fusion method decreased the interface resistance between the polymer electrolyte and the cathode by 96.2%. During the ultrasonic fusion process, high-frequency ultrasonic vibrations generated high temperatures at the interface, and the polymer electrolyte became molten, improving the contact between the electrolyte and the cathode. The ultrasonic fusion method eliminated the gaps at the interface, and the interface became more compact. Furthermore, ultrasonic vibrations made the molten electrolyte fill the holes in the cathode, and the contact area was enhanced, providing more Li+ ions transmission paths.

Accurate Quantitative Histomorphometric-Mathematical Image Analysis Methodology of Rodent Testicular Tissue and Its Possible Future Research Perspectives in Andrology and Reproductive Medicine.

Infertility is increasing worldwide; male factors can be identified in nearly half of all infertile couples. Histopathologic evaluation of testicular tissue can provide valuable information about infertility; however, several different evaluation methods and semi-quantitative score systems exist. Our goal was to describe a new, accurate and easy-to-use quantitative computer-based histomorphometric-mathematical image analysis methodology for the analysis of testicular tissue. On digitized, original hematoxylin-eosin (HE)-stained slides (scanned by slide-scanner), quantitatively describable characteristics such as area, perimeter and diameter of testis cross-sections and of individual tubules were measured with the help of continuous magnification. Immunohistochemically (IHC)-stained slides were digitized with a microscope-coupled camera, and IHC-staining intensity measurements on digitized images were also taken. Suggested methods are presented with mathematical equations, step-by-step detailed characterization and representative images are given. Our novel quantitative histomorphometric-mathematical image analysis method can improve the reproducibility, objectivity, quality and comparability of andrological-reproductive medicine research by recognizing even the mild impairments of the testicular structure expressed numerically, which might not be detected with the present semi-quantitative score systems. The technique is apt to be subjected to further automation with machine learning and artificial intelligence and can be named 'Computer-Assisted or -Aided Testis Histology' (CATHI).

Metformin Alleviates Autistic-Like Behaviors Elicited by High-Fat Diet Consumption and Modulates the Crosstalk Between Serotonin and Gut Microbiota in Mice.

The biological mechanisms linking diet-related obesity and autistic behaviors remain unclear. Metformin has proven to be beneficial in the treatment of many syndromes, including autism spectrum disorder. Therefore, the aim of this study was to assess whether metformin treatment could ameliorate metabolic and behavioral alterations in C57BL/6 mice kept on a high-fat diet (HFD), and whether these changes were related to modifications in the gut microbiota and 5-HT levels. As expected, ten weeks of HFD ingestion increased body weight, adiposity, and glucose levels. HFD-fed mice showed a marked aggravation of repetitive behaviors (marble burying and self-grooming), and this was prevented by metformin administration. In addition, HFD-fed mice increased the total distance travelled in the open field test. This hyperactivity was counteracted by metformin cotreatment. In the elevated plus maze test, HFD-fed mice showed a reduced number of entries into the open arms. Interestingly, both HFD and metformin cotreatment increased social interactions in the three-chamber test. HFD increased the levels of intestinal tryptophan and 5-hydroxyindoleacetic acid. Metformin stimulated gut tryptophan and promoted the synthesis of 5-HT in the HFD group. Lactococcus, Trichococcus, Romboutsia, and Faecalibaculum were enriched in HFD-fed mice, whereas the HFD group cotreated with metformin was enriched in Intestinimonas and L. reuteri. Faecalibacterium was positively correlated with sociability and 5-HT pathway components in mice that received metformin. In summary, HFD consumption elicited a complex phenotype comprising higher levels of anxiety-like and repetitive behaviors but also increased sociability. Metformin could potentially improve HFD-induced disorders in the autistic spectrum through a mechanism involving positive modulation of 5-HT levels in the gut and its microbiota composition.

Metformin Exerts Anti-inflammatory and Mucus Barrier Protective Effects by Enriching Akkermansia muciniphila in Mice With Ulcerative Colitis.

The present study aimed to determine if metformin exerts anti-inflammatory and mucus-protective effects via the gut microbiota. Metformin has extensive benefits including anti-inflammatory effects. Previous studies showed that metformin changed the gut microbiota composition and increases the number of goblet cells. Intestinal dysbiosis and goblet cell depletion are important features of ulcerative colitis (UC). The underlying mechanism and whether metformin can improve the mucus barrier in UC remain unclear. Metformin (400 mg/kg/day) was administered to mice with dextran sulfate sodium (DSS)-induced UC for 2 wk to investigate the effects of metformin on the intestinal mucus barrier. The gut microbiota was depleted, using antibiotics, to explore its role in the mucus-protecting effects of metformin. Akkermansia muciniphila (A. muciniphila), which was enriched in metformin-treated mice, was administered to mice to investigate the effects of the bacteria on UC and the mucus barrier. Metformin attenuated DSS-induced UC in mice, as evidenced by the alleviation of diarrhea, hematochezia, and the decrease in body weight. The expression of mucin2, a prominent mucus barrier protein, was increased in the metformin-treated group compared to the DSS-treated group. Furthermore, fecal 16S rRNA analysis showed that metformin treatment changed the gut microbiota composition by increasing the relative abundance of Lactobacillus and Akkermansia species while decreasing Erysipelatoclostridium at the genus level. Antibiotic treatment partly abolished the anti-inflammatory and mucus-protecting effects of metformin. Administration of A. muciniphila alleviated the colonic inflammation and mucus barrier disruption. Metformin alleviated DSS-induced UC in mice and protected against cell damage via affecting the gut microbiota, thereby providing a new mechanism for the therapeutic effect of metformin in patients with UC. This study also provides evidence that A. muciniphila as a probiotic has potential benefits for UC.

Left Ventricular SGLT1 Protein Expression Correlates with the Extent of Myocardial Nitro-Oxidative Stress in Rats with Pressure and Volume Overload-Induced Heart Failure.

Myocardial sodium-glucose cotransporter 1 (SGLT1) has been shown to be upregulated in humans with heart failure (HF) with or without diabetes. In vitro studies have linked SGLT1 to increased nitro-oxidative stress in cardiomyocytes. We aimed to assess the relation between left ventricular (LV) SGLT1 expression and the extent of nitro-oxidative stress in two non-diabetic rat models of chronic heart failure (HF) evoked by either pressure (TAC, n = 12) or volume overload (ACF, n = 12). Sham-operated animals (Sham-T and Sham-A, both n = 12) served as controls. Both TAC and ACF induced characteristic LV structural and functional remodeling. Western blotting revealed that LV SGLT1 protein expression was significantly upregulated in both HF models (both p < 0.01), whereas the phosphorylation of ERK1/2 was decreased only in ACF; AMPKα activity was significantly reduced in both models. The protein expression of the Nox4 NADPH oxidase isoform was increased in both TAC and ACF compared with respective controls (both p < 0.01), showing a strong positive correlation with SGLT1 expression (r = 0.855, p < 0.001; and r = 0.798, p = 0.001, respectively). Furthermore, SGLT1 protein expression positively correlated with the extent of myocardial nitro-oxidative stress in failing hearts assessed by 3-nitrotyrosin (r = 0.818, p = 0.006) and 4-hydroxy-2-nonenal (r = 0.733, p = 0.020) immunostaining. Therefore, LV SGLT1 protein expression was upregulated irrespective of the nature of chronic hemodynamic overload, and correlated significantly with the expression of Nox4 and with the level of myocardial nitro-oxidative stress, suggesting a pathophysiological role of SGLT1 in HF.

The appropriate cutoff gastric pH value for Helicobacter pylori eradication with bismuth-based quadruple therapy.

BACKGROUND: This study aimed to investigate whether an increased proton pump inhibitor (PPI) dose enhanced the efficacy of Helicobacter pylori (H. pylori) eradication and determine the appropriate cutoff intragastric pH value that could predict H. pylori eradication with bismuth-based quadruple therapy. MATERIALS AND METHODS: A total of 207 H. pylori infected, treatment naive patients were enrolled in this prospective, open-label, randomized controlled trial. Patients were randomly allocated into Eso40-group (esomeprazole 40 mg bid) and Eso20-group (esomeprazole 20 mg bid), and their CYP2C19 genotyping status was assessed. The 24-h intragastric pH monitoring on day 7 was performed, and percentage of time gastric pH ≥ 3, ≥4, ≥5, and ≥6 (pH holding time ratios; HTRs) were measured. H. pylori eradication was evaluated using 13 C-urea breath test. RESULTS: No significant difference in the eradication rates was observed between two groups. The median 24-h intragastric pH value was not significant different between two groups but the Eso40 Group had a significant higher pH4 HTRs (91.11% [95%CI: 87.50%-95.83%] vs. 95.83% [95.83%-100%]; p = .002). Additionally, the median 24-h intragastric pH value showed significantly difference between two groups in EM genotype (Eso20 Group 6.00 [95%CI; 5.75-6.15] vs. Eso40 Group 6.30 [6.05-6.30]; p = .019). Similar results were observed in pH4 HTRs. There were significant differences in intragastric pH value (6.10 [95%CI: 4.40-7.00] vs. 5.65 [4.85-5.95], p = .038) and in pH4 HTRs (96% [95%CI: 92.00%-96.00%] vs. 87.5% [67.00%-100.0%], p = .019) between eradication-successful and eradication-failed patients. Statistical analysis suggested that the median intragastric pH = 5.7 could identify the success of H. pylori eradication. CONCLUSIONS: Bismuth-based quadruple therapy resulted in high H. pylori eradication rates either in PPI standard or double doses. Double dose of esomeprazole is associated with better intragastric acid suppression. A median 24-h intragastric pH of 5.7 could be appropriate cutoff value for predicting the successful H. pylori eradication.