Non-canonical NF-κB signaling limits the tolerogenic ß-catenin-Raldh2 axis in gut dendritic cells to exacerbate intestinal pathologies.

Dendritic cell (DC) dysfunction is known to exacerbate intestinal pathologies, but the mechanisms compromising DC-mediated immune regulation in this context remain unclear. Here, we show that intestinal dendritic cells from a mouse model of experimental colitis exhibit significant levels of noncanonical NF-κB signaling, which activates the RelB:p52 heterodimer. Genetic inactivation of this pathway in DCs alleviates intestinal pathologies in mice suffering from colitis. Deficiency of RelB:p52 diminishes transcription of Axin1, a critical component of the ß-catenin destruction complex, reinforcing ß-catenin-dependent expression of Raldh2, which imparts tolerogenic DC attributes by promoting retinoic acid synthesis. DC-specific impairment of noncanonical NF-κB signaling leads to increased colonic numbers of Tregs and IgA+ B cells, which promote luminal IgA production and foster eubiosis. Experimentally introduced ß-catenin haploinsufficiency in DCs with deficient noncanonical NF-κB signaling moderates Raldh2 activity, reinstating colitogenic sensitivity in mice. Finally, inflammatory bowel-disease patients also display a deleterious noncanonical NF-κB signaling signature in intestinal DCs. In sum, we establish how noncanonical NF-κB signaling in dendritic cells can subvert retinoic acid synthesis to fuel intestinal inflammation.

Collinsella aerofaciens linked with increased ethanol production and liver inflammation contribute to the pathophysiology of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is an emerging global health problem and a potential risk factor for metabolic diseases. The bidirectional interactions between liver and gut made dysbiotic gut microbiome one of the key risk factors for NAFLD. In this study, we reported an increased abundance of Collinsella aerofaciens in the gut of obese and NASH patients living in India. We isolated C. aerofaciens from the fecal samples of biopsy-proven NASH patients and observed that their genome is enriched with carbohydrate metabolism, fatty acid biosynthesis, and pro-inflammatory functions and have the potency to increase ethanol level in blood. An animal study indicated that mice supplemented with C. aerofaciens had increased levels of circulatory ethanol, high levels of hepatic hydroxyproline, triglyceride, and inflammation in the liver. The present findings indicate that perturbation in the gut microbiome composition is a key risk factor for NAFLD.

Microbiota and epigenetics: Health impact.

Epigenetic changes associated with disease development and progressions are of increasing importance because of their potential diagnostic and therapeutic applications. Several epigenetic changes associated with chronic metabolic disorders have been studied in various diseases. Epigenetic changes are mostly modulated by environmental factors, including the human microbiota living in different parts of our bodies. The microbial structural components and the microbially derived metabolites directly interact with host cells, thereby maintaining homeostasis. Microbiome dysbiosis, on the other hand, is known to produce elevated levels of disease-linked metabolites, which may directly affect a host metabolic pathway or induce epigenetic changes that can lead to disease development. Despite their important role in host physiology and signal transduction, there has been little research into the mechanics and pathways associated with epigenetic modifications. This chapter focuses on the relationship between microbes and their epigenetic effects in diseased pathology, as well as on the regulation and metabolism of the dietary options available to the microbes. Furthermore, this chapter also provides a prospective link between these two important phenomena, termed "Microbiome and Epigenetics."

Gut microbiome dysbiosis in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex multi-factorial chronic relapsing disease of the digestive tract where dysbiosis of autochthonous intestinal microbiota, environmental factors and host genetics are implicated in the disease development, severity, course and treatment outcomes. The two clinically well-defined forms of IBD are Crohn's disease (CD) and ulcerative colitis (UC). The CD affects the local immune response of the entire gastrointestinal tract whereas the inflammation in UC is mainly restricted to the colonic mucosa. Prolong progressive inflammation due to CD and UC often lead to colonic cancer. In healthy individuals, the enormous taxonomic diversity and functional potency of gut microbiota including members from the bacterial and fungal microbiota tune the host immunity and keep the gastric environment beneficial and protective. However, expansion of pathobionts, autochthonous microbes with the potency of pathogenicity in dysbiotic condition, in the gastrointestinal tract and subsequently enriched inflammatory microbial products in the gastrointestinal milieu attract different immune cells and activate aberrant host immune response which leads to excessive production and secretion of different cytokines that damage the colonic epithelial cells and manifest chronic inflammatory digestive disease. In the current chapter, we provided our updated understanding about the different bacterial and fungal pathobionts, their genomic and metabolic signatures, and geo-specific diversity of gut microbes linked with IBD across the globe at the molecular resolution. An improved understanding of IBD and the factors associated with the disease will be a boost for therapeutic development and disease management.

Human microbiome and cardiovascular diseases.

A number of microorganisms are co-evolved with the host, among which bacteria are the predominant organisms in the colonic site. The human microbiota contributes to various physiological functions, including the digestion and degradation of food components, harvesting of inaccessible nutrients, immune system regulation, maintenance of gut barrier function, and regulation of brain function and behavior. Microbes in the gut produce a wealth of metabolites from the exogenous dietary substances or endogenous metabolic compounds produced by the host and the resident microorganisms. These microbial-derived metabolites are the major factors in the host-microbiota cross-talk and influence the host's cardiometabolic health directly or indirectly depending on the structure and function of the microbial community. Evidence suggests that the perturbation in the composition and function of gut microbiota (referred to as gut dysbiosis) is associated with the development of several diseased conditions such as that of the gastrointestinal tract or colorectal cancer, metabolic diseases such as obesity, diabetes, immune disorders e.g. asthma, allergies, depression, anxiety and cardiometabolic disease. Several pathological conditions in the gastrointestinal tract may impair the intestinal barrier that allows translocation of bacteria and their metabolites to a remote organ such as the heart, which may ultimately be associated with systemic inflammation and the development of CVDs. In this chapter, we will discuss various gut microbiota-dependent metabolites, which have a significant role in cardiovascular diseases' pathologic processes and their risk factors. Finally, we will discuss the therapeutic potential of the gut-metabolite-heart axis as a novel target for the treatment of CVD and highlight the current updates and exciting directions for future research.

Region-specific genomic signatures of multidrug-resistant Helicobacter pylori isolated from East and South India.

Helicobacter pylori is a ubiquitous bacterium and contributes significantly to the burden of chronic gastritis, peptic ulcers, and gastric cancer across the world. Adaptive phenotypes and virulence factors in H. pylori are heterogeneous and dynamic. However, limited information is available about the molecular nature of antimicrobial resistance phenotypes and virulence factors of H. pylori strains circulating in India. In the present study, we analyzed the whole genome sequences of 143 H. pylori strains, of which 32 are isolated from two different regions (eastern and southern) of India. Genomic repertoires of individual strains show distinct region-specific signatures. We observed lower resistance phenotypes and genotypes in the East Indian (Kolkata) H. pylori isolates against amoxicillin and furazolidone antibiotics, whereas higher resistance phenotypes to metronidazole and clarithromycin. Also, at molecular level, a greater number of AMR genes were observed in the east Indian H. pylori isolates as compared to the southern Indian isolates. From our findings, we suggest that metronidazole and clarithromycin antibiotics should be used judicially in the eastern India. However, no horizontally acquired antimicrobial resistance gene was observed in the current H. pylori strains. The comparative genome analysis shows that the number of genes involved in virulence, disease and resistance of H. pylori isolated from two different regions of India is significantly different. Single-nucleotide polymorphisms (SNPs) based phylogenetic analysis distinguished H. pylori strains into different clades according to their geographical locations. Conditionally beneficial functions including antibiotic resistance phenotypes that are linked with faster evolution rates in the Indian isolates.

Trifecta and Pentafecta Outcomes in Laparoscopic and Robotic Nephron-Sparing Surgery for Highly Complex Renal Tumors: A Propensity Score-Matched Cohort Analysis.

Purpose: To compare the trifecta and pentafecta outcomes of laparoscopic partial nephrectomy (LPN) and robotic partial nephrectomy (RPN) in highly complex renal tumors (RENAL nephrometry score ≥10) using a matched cohort analysis. Methods: Patients undergoing LPN or RPN for renal tumors with RENAL score ≥10 between January 2014 and December 2019 were matched using 1:2 propensity score match analysis based on age, body mass index, gender, laterality of tumor, RENAL score, and American Society of Anesthesiologists (ASA) score. The two groups were compared for trifecta and pentafecta outcomes. Results: Thirty patients undergoing LPN (Group A) were matched with 60 patients undergoing RPN (Group B). The mean age (standard deviation) was 53.7 (12.9) years. The median (interquartile range) RENAL score was 10 (10-11). In comparison, the mean warm ischemia time in Group A was significantly longer than that in Group B (26.2 vs 23.0 minutes, p = 0.013). The overall complication rate was 36.7% in Group A as compared with 20% in Group B (p = 0.440). The trifecta outcomes could be achieved in 11 patients (36.7%) in Group A compared with 40 patients (66.7%) in Group B (p = 0.012). Moreover, 10 patients (33.3%) in Group A and 28 patients (46.7%) in Group B achieved pentafecta outcomes (p = 0.227). Conclusions: In a matched cohort of patients undergoing nephron-sparing surgery for highly complex renal tumors (RENAL score ≥10), the robotic approach offers a superior advantage in the achievement of trifecta outcomes as compared with the laparoscopic approach. However, both LPN and RPN can achieve similar pentafecta outcomes.

TLR22-mediated activation of TNF-α-caspase-1/IL-1ß inflammatory axis leads to apoptosis of Aeromonas hydrophila-infected macrophages.

Toll-like receptors (TLRs) represent first line of host defence against microbes. Amongst different TLRs, TLR22 is exclusively expressed in non-mammalian vertebrates, including fish. The precise role of TLR22 in fish-immunity remains abstruse. Herein, we used headkidney macrophages (HKM) from Clarias gariepinus and deciphered its role in fish-immunity. Highest tlr22 expression was observed in the immunocompetent organ - headkidney; nonetheless expression in other tissues suggests its possible involvement in non-immune sites also. Aeromonas hydrophila infection up-regulates tlr22 expression in HKM. Our RNAi based study suggested TLR22 restricts intracellular survival of A. hydrophila. Inhibitor and RNAi studies further implicated TLR22 induces pro-inflammatory cytokines TNF-α and IL-1ß. We observed heightened caspase-1 activity and our results suggest the role of TLR22 in activating TNF-α/caspase-1/IL-1ß cascade leading to caspase-3 mediated apoptosis of A. hydrophila-infected HKM. We conclude, TLR22 plays critical role in immune-surveillance and triggers pro-inflammatory cytokines leading to caspase mediated HKM apoptosis and pathogen clearance.

Low Bifidobacterium Abundance in the Lower Gut Microbiota Is Associated With Helicobacter pylori-Related Gastric Ulcer and Gastric Cancer.

Helicobacter pylori infection in stomach leads to gastric cancer, gastric ulcer, and duodenal ulcer. More than 1 million people die each year due to these diseases, but why most H. pylori-infected individuals remain asymptomatic while a certain proportion develops such severe gastric diseases remained an enigma. Several studies indicated that gastric and intestinal microbiota may play a critical role in the development of the H. pylori-associated diseases. However, no specific microbe in the gastric or intestinal microbiota has been clearly linked to H. pylori infection and related gastric diseases. Here, we studied H. pylori infection, its virulence genes, the intestinal microbiota, and the clinical status of Trivandrum residents (N = 375) in southwestern India by standard H. pylori culture, PCR genotype, Sanger sequencing, and microbiome analyses using Illumina Miseq and Nanopore GridION. Our analyses revealed that gastric colonization by virulent H. pylori strains (vacAs1i1m1cagA+) is necessary but not sufficient for developing these diseases. Conversely, distinct microbial pools exist in the lower gut of the H. pylori-infected vs. H. pylori-non-infected individuals. Bifidobacterium (belonging to the phylum Actinobacteria) and Bacteroides (belonging to the phylum Bacteroidetes) were present in lower relative abundance for the H. pylori+ group than the H. pylori- group (p < 0.05). On the contrary, for the H. pylori+ group, genus Dialister (bacteria belonging to the phylum Firmicutes) and genus Prevotella (bacteria belonging to the phylum Bacteroidetes) were present in higher abundance compared to the H. pylori- group (p < 0.05). Notably, those who carried H. pylori in the stomach and had developed aggressive gastric diseases also had extremely low relative abundance (p < 0.05) of several Bifidobacterium species (e.g., B. adolescentis, B. longum) in the lower gut suggesting a protective role of Bifidobacterium. Our results show the link between lower gastrointestinal microbes and upper gastrointestinal diseases. Moreover, the results are important for developing effective probiotic and early prognosis of severe gastric diseases.

Gastric microbiome of Indian patients with Helicobacter pylori infection, and their interaction networks.

The gastric microbiome is suspected to have a role in the causation of diseases by Helicobacter pylori. Reports on their relative abundance vis-à-vis H. pylori are available from various ethnic and geographic groups, but little is known about their interaction patterns. Endoscopic mucosal biopsy samples from the gastric antrum and corpus of 39 patients with suspected H. pylori infection were collected and microbiomes were analyzed by 16S rDNA profiling. Four groups of samples were identified, which harbored Helicobacter as well as a diverse group of bacteria including Lactobacillus, Halomonas and Prevotella. There was a negative association between the microbiome diversity and Helicobacter abundance. Network analyses showed that Helicobacter had negative interactions with members of the gastric microbiome, while other microbes interacted positively with each other, showing a higher tendency towards intra-cluster co-occurrence/co-operation. Cross-geographic comparisons suggested the presence of region-specific microbial abundance profiles. We report the microbial diversity, abundance variation and interaction patterns of the gastric microbiota of Indian patients with H. pylori infection and present a comparison of the same with the gastric microbial ecology in samples from different geographic regions. Such microbial abundance profiles and microbial interactions can help in understanding the pathophysiology of gastric ailments and can thus help in development of new strategies to curb it.

Mutational analysis of the (p)ppGpp synthetase activity of the Rel enzyme of Mycobacterium tuberculosis.

Rel(Mtb), a GTP pyrophosphokinase encoded by the Mycobacterium tuberculosis (Mtb) genome, catalyzes synthesis of (p)ppGpp from ATP and GDP(GTP) and its hydrolysis to GDP(GTP) and pyrophosphate to mediate stringent response, which helps bacteria to survive during nutrient limitation. Like other members of Rel_Spo homologs, Rel(Mtb) has four distinct domains: HD, Rel_Spo (RSD), TGS and ACT. The N-terminal HD and RSD are responsible for (p)ppGpp hydrolysis and synthesis, respectively. In this study, we have dissected the rel(Mtb) gene function and determined the minimal region essential for (p)ppGpp synthetic activity. The Rel(Mtb) and its truncated derivatives were expressed from an arabinose inducible promoter (P(BAD)), and in vivo functional analyses were done in a (p)ppGpp null Escherichia coli strain. Our results indicate that only 243 amino acids (188-430 residues) containing fragment are sufficient for Rel(Mtb) (p)ppGpp synthetic activity. The results were further confirmed by in vitro assays using purified proteins. We further characterized the RSD of Rel(Mtb) by substituting several conserved amino acids with structurally related residues and identified six such residues, which appeared to be critical for maintaining its catalytic activity. Furthermore, we have also extended our analysis to an RSD encoding gene rv1366 of Mtb, and experimental results indicated that the encoded protein Rv1366 is unable to synthesize (p)ppGpp.

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene.

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli, V. choleraeDeltarelADeltaspoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae. By repeated subculturing of a V. choleraeDeltarelADeltaspoT mutant, a suppressor strain with (p)ppGpp(0) phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene (VC1224), which codes for a small protein (approximately 29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV. Using E. coliDeltarelA or DeltarelADeltaspoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (DeltarelADeltaspoT relV676), which seems to be responsible for the (p)ppGpp(0) phenotype. Analysis of a V. choleraeDeltarelADeltaspoTDeltarelV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.

Molecular characterization of vibrio cholerae DeltarelA DeltaspoT double mutants.

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae DeltarelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA (+ )background, but unlike E. coli, several V. cholerae DeltarelA DeltaspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli DeltarelA DeltaspoT mutant (ppGpp(0) strain), the V. cholerae DeltarelA DeltaspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of DeltarelA DeltaspoT mutants could be reverted back to DeltarelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae.