A Perspective Review on Diet Quality, Excess Adiposity, and Chronic Psychosocial Stress and Implications for Early-Onset Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide. Although the overall incidence of CRC has been decreasing over the past 40 y, early-onset colorectal cancer (EOCRC), which is defined as a CRC diagnosis in patients aged >50 y has increased. In this Perspective, we highlight and summarize the association between diet quality and excess adiposity, and EOCRC. We also explore chronic psychosocial stress (CPS), a less investigated modifiable risk factor, and EOCRC. We were able to show that a poor-quality diet, characterized by a high intake of sugary beverages and a Western diet pattern (high intake of red and processed meats, refined grains, and foods with added sugars) can promote risk factors associated with EOCRC development, such as an imbalance in the composition and function of the gut microbiome, presence of chronic inflammation, and insulin resistance. Excess adiposity, particularly obesity onset in early adulthood, is a likely contributor of EOCRC. Although the research is sparse examining CPS and CRC/EOCRC, we describe likely pathways linking CPS to tumorigenesis. Although additional research is needed to understand what factors are driving the uptick in EOCRC, managing body weight, improving diet quality, and mitigating psychosocial stress, may play an important role in reducing an individual's risk of EOCRC.

Effect of Diets Varying in Iron and Saturated Fat on the Gut Microbiota and Intestinal Inflammation: A Crossover Feeding Study among Older Females with Obesity.

Obesity is considered an independent risk factor for colorectal cancer (CRC). Altered nutrient metabolism, particularly changes to digestion and intestinal absorption, may play an important role in the development of CRC. Iron can promote the formation of tissue-damaging and immune-modulating reactive oxygen species. We conducted a crossover, controlled feeding study to examine the effect of three, 3-week diets varying in iron and saturated fat content on the colonic milieu and systemic markers among older females with obesity. Anthropometrics, fasting venous blood and stool were collected before and after each diet. There was a minimum 3-week washout period between diets. Eighteen participants consumed the three diets (72% Black; mean age 60.4 years; mean body mass index 35.7 kg/m2). Results showed no effect of the diets on intestinal inflammation (fecal calprotectin) or circulating iron, inflammation, and metabolic markers. Pairwise comparisons revealed less community diversity between samples (beta diversity, calculated from 16S rRNA amplicon sequences) among participants when consuming a diet low in iron and high in saturated fat vs. when consuming a diet high in iron and saturated fat. More studies are needed to investigate if dietary iron represents a salient target for CRC prevention among individuals with obesity.

Formation of secondary allo-bile acids by novel enzymes from gut Firmicutes.

The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B-cis orientation in which the sterol rings are "kinked", as well as small quantities of A/B-trans oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7α-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5α-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-Δ4-LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals.

Diversity and distribution of sulfur metabolic genes in the human gut microbiome and their association with colorectal cancer.

BACKGROUND: Recent evidence implicates microbial sulfidogenesis as a potential trigger of colorectal cancer (CRC), highlighting the need for comprehensive knowledge of sulfur metabolism within the human gut. Microbial sulfidogenesis produces genotoxic hydrogen sulfide (H2S) in the human colon using inorganic (sulfate) and organic (taurine/cysteine/methionine) substrates; however, the majority of studies have focused on sulfate reduction using dissimilatory sulfite reductases (Dsr). RESULTS: Here, we show that genes for microbial sulfur metabolism are more abundant and diverse than previously observed and are statistically associated with CRC. Using ~ 17,000 bacterial genomes from publicly available stool metagenomes, we studied the diversity of sulfur metabolic genes in 667 participants across different health statuses: healthy, adenoma, and carcinoma. Sulfidogenic genes were harbored by 142 bacterial genera and both organic and inorganic sulfidogenic genes were associated with carcinoma. Significantly, the anaerobic sulfite reductase (asr) genes were twice as abundant as dsr, demonstrating that Asr is likely a more important contributor to sulfate reduction in the human gut than Dsr. We identified twelve potential pathways for reductive taurine metabolism and discovered novel genera harboring these pathways. Finally, the prevalence of metabolic genes for organic sulfur indicates that these understudied substrates may be the most abundant source of microbially derived H2S. CONCLUSIONS: Our findings significantly expand knowledge of microbial sulfur metabolism in the human gut. We show that genes for microbial sulfur metabolism in the human gut are more prevalent than previously known, irrespective of health status (i.e., in both healthy and diseased states). Our results significantly increase the diversity of pathways and bacteria that are associated with microbial sulfur metabolism in the human gut. Overall, our results have implications for understanding the role of the human gut microbiome and its potential contributions to the pathogenesis of CRC. Video abstract.

Bile Acids, Gut Microbes, and the Neighborhood Food Environment-a Potential Driver of Colorectal Cancer Health Disparities.

Bile acids (BAs) facilitate nutrient digestion and absorption and act as signaling molecules in a number of metabolic and inflammatory pathways. Expansion of the BA pool and increased exposure to microbial BA metabolites has been associated with increased colorectal cancer (CRC) risk. It is well established that diet influences systemic BA concentrations and microbial BA metabolism. Therefore, consumption of nutrients that reduce colonic exposure to BAs and microbial BA metabolites may be an effective method for reducing CRC risk, particularly in populations disproportionately burdened by CRC. Individuals who identify as Black/African American (AA/B) have the highest CRC incidence and death in the United States and are more likely to live in a food environment with an inequitable access to BA mitigating nutrients. Thus, this review discusses the current evidence supporting diet as a contributor to CRC disparities through BA-mediated mechanisms and relationships between these mechanisms and barriers to maintaining a low-risk diet.

Support policies that foster a healthy food environment and incentivize healthy food purchases to mitigate cancer inequities.

The COVID-19 pandemic has highlighted the inequitable access to resources, leading to a disproportionate burden of disease in vulnerable communities in the USA. However, these inequities in health outcomes are not limited to COVID-19. Approximately 18% of cancers are related to dietary behaviors and excess body weight. Underserved communities, such as minority racial/ethnic groups living in neighborhoods of low socioeconomic status, experience barriers to healthy eating including lack of access to high-quality healthy foods and higher availability of unhealthy foods and beverages in local retail food outlets. Strikingly, these same populations are more likely to die from cancers related to dietary intake and obesity like colorectal, liver, and pancreatic cancers. To reduce cancer inequities, policy makers can act by supporting programs that incentivize healthy food purchases and improve the local food environment in underserved communities.

Introducing the Microbes and Social Equity Working Group: Considering the Microbial Components of Social, Environmental, and Health Justice.

Humans are inextricably linked to each other and our natural world, and microorganisms lie at the nexus of those interactions. Microorganisms form genetically flexible, taxonomically diverse, and biochemically rich communities, i.e., microbiomes that are integral to the health and development of macroorganisms, societies, and ecosystems. Yet engagement with beneficial microbiomes is dictated by access to public resources, such as nutritious food, clean water and air, safe shelter, social interactions, and effective medicine. In this way, microbiomes have sociopolitical contexts that must be considered. The Microbes and Social Equity (MSE) Working Group connects microbiology with social equity research, education, policy, and practice to understand the interplay of microorganisms, individuals, societies, and ecosystems. Here, we outline opportunities for integrating microbiology and social equity work through broadening education and training; diversifying research topics, methods, and perspectives; and advocating for evidence-based public policy that supports sustainable, equitable, and microbial wealth for all.

Completion of the gut microbial epi-bile acid pathway.

Bile acids are detergent molecules that solubilize dietary lipids and lipid-soluble vitamins. Humans synthesize bile acids with α-orientation hydroxyl groups which can be biotransformed by gut microbiota to toxic, hydrophobic bile acids, such as deoxycholic acid (DCA). Gut microbiota can also convert hydroxyl groups from the α-orientation through an oxo-intermediate to the ß-orientation, resulting in more hydrophilic, less toxic bile acids. This interconversion is catalyzed by regio- (C-3 vs. C-7) and stereospecific (α vs. ß) hydroxysteroid dehydrogenases (HSDHs). So far, genes encoding the urso- (7α-HSDH & 7ß-HSDH) and iso- (3α-HSDH & 3ß-HSDH) bile acid pathways have been described. Recently, multiple human gut clostridia were reported to encode 12α-HSDH, which interconverts DCA and 12-oxolithocholic acid (12-oxoLCA). 12ß-HSDH completes the epi-bile acid pathway by converting 12-oxoLCA to the 12ß-bile acid denoted epiDCA; however, a gene(s) encoding this enzyme has yet to be identified. We confirmed 12ß-HSDH activity in cultures of Clostridium paraputrificum ATCC 25780. From six candidate C. paraputrificum ATCC 25780 oxidoreductase genes, we discovered the first gene (DR024_RS09610) encoding bile acid 12ß-HSDH. Phylogenetic analysis revealed unforeseen diversity for 12ß-HSDH, leading to validation of two additional bile acid 12ß-HSDHs through a synthetic biology approach. By comparison to a previous phylogenetic analysis of 12α-HSDH, we identified the first potential C-12 epimerizing strains: Collinsella tanakaei YIT 12063 and Collinsella stercoris DSM 13279. A Hidden Markov Model search against human gut metagenomes located putative 12ß-HSDH genes in about 30% of subjects within the cohorts analyzed, indicating this gene is relevant in the human gut microbiome.

Berberine alters gut microbial function through modulation of bile acids.

BACKGROUND: Berberine (BBR) is a plant-based nutraceutical that has been used for millennia to treat diarrheal infections and in contemporary medicine to improve patient lipid profiles. Reduction in lipids, particularly cholesterol, is achieved partly through up-regulation of bile acid synthesis and excretion into the gastrointestinal tract (GI). The efficacy of BBR is also thought to be dependent on structural and functional alterations of the gut microbiome. However, knowledge of the effects of BBR on gut microbiome communities is currently lacking. Distinguishing indirect effects of BBR on bacteria through altered bile acid profiles is particularly important in understanding how dietary nutraceuticals alter the microbiome. RESULTS: Germfree mice were colonized with a defined minimal gut bacterial consortium capable of functional bile acid metabolism (Bacteroides vulgatus, Bacteroides uniformis, Parabacteroides distasonis, Bilophila wadsworthia, Clostridium hylemonae, Clostridium hiranonis, Blautia producta; B4PC2). Multi-omics (bile acid metabolomics, 16S rDNA sequencing, cecal metatranscriptomics) were performed in order to provide a simple in vivo model from which to identify network-based correlations between bile acids and bacterial transcripts in the presence and absence of dietary BBR. Significant alterations in network topology and connectivity in function were observed, despite similarity in gut microbial alpha diversity (P = 0.30) and beta-diversity (P = 0.123) between control and BBR treatment. BBR increased cecal bile acid concentrations, (P < 0.05), most notably deoxycholic acid (DCA) (P < 0.001). Overall, analysis of transcriptomes and correlation networks indicates both bacterial species-specific responses to BBR, as well as functional commonalities among species, such as up-regulation of Na+/H+ antiporter, cell wall synthesis/repair, carbohydrate metabolism and amino acid metabolism. Bile acid concentrations in the GI tract increased significantly during BBR treatment and developed extensive correlation networks with expressed genes in the B4PC2 community. CONCLUSIONS: This work has important implications for interpreting the effects of BBR on structure and function of the complex gut microbiome, which may lead to targeted pharmaceutical interventions aimed to achieve the positive physiological effects previously observed with BBR supplementation.

Hydration Biomarkers Are Related to the Differential Abundance of Fecal Microbiota and Plasma Lipopolysaccharide-Binding Protein in Adults.

INTRODUCTION: Prevalence of chronic hypohydration remains elevated among adults in the USA; however, the health effects of hypohydration in regards to human gut health have not been explored. METHODS: This study examined the relationship between total water intake, hydration biomarkers (first-morning urine specific gravity [FMUsg], first-morning urine volume [FMUvol], and plasma copeptin), fecal microbiota, and plasma lipopolysaccharide-binding protein (LBP) in adults (25-45 years, 64% female). Fecal microbiota composition was assessed using 16S rRNA gene sequencing (V4 region). Immunoassays quantified plasma copeptin and LBP in fasted venous blood samples. Dietary variables were measured using 7-day food records. Linear discriminant analysis effect size (LEfSe) analyzed differentially abundant microbiota based on median cutoffs for hydration markers. Multiple linear regressions examined the relationship between LBP and copeptin. RESULTS: LEfSe identified 6 common taxa at the genus or species level that were differentially abundant in FMUsg, total water (g/day), or plasma copeptin (µg/mL) groups when split by their median values. Uncultured species in the Bacteroides, Desulfovibrio, Roseburia, Peptococcus, and Akkermansia genera were more abundant in groups that might indicate poorer hydration status. Multivariate linear analyses revealed a positive relationship between plasma copeptin and LBP when controlling confounding variables (F(6,52) = 4.45, p = 0.002, R2 = 0.34). CONCLUSIONS: Taxa common between markers are associated with the intestinal mucus layer, which suggests a potential link between hydration status and intestinal mucus homeostasis. The relationship between LBP and copeptin indicates that copeptin may be sensitive to metabolic endotoxemia and potentially gut barrier function.

Effects of taurocholic acid metabolism by gut bacteria: A controlled feeding trial in adult African American subjects at elevated risk for colorectal cancer.

Colorectal cancer (CRC) is the third leading cause of cancer and second leading cause of cancer death in the United States. Recent evidence has linked a high fat and animal protein diet and microbial metabolism of host bile acids as environmental risk factors for CRC development. We hypothesize that the primary bile salt taurocholic acid (TCA) is a key, diet-controlled metabolite whose use by bacteria yields a carcinogen and tumor-promoter, respectively. The work is motivated by our published data indicating hydrogen sulfide (H2S) and secondary bile acid production by colonic bacteria, serve as environmental insults contributing to CRC risk. The central aim of this study is to test whether a diet high in animal protein and saturated fat increases abundance of bacteria that generate H2S and pro-inflammatory secondary bile acids in African Americans (AAs) at high risk for CRC. Our prospective, randomized, crossover feeding trial will examine two microbial mechanisms by which an animal-based diet may support the growth of TCA metabolizing bacteria. Each subject will receive two diets in a crossover design- an animal-based diet, rich in taurine and saturated fat, and a plant-based diet, low in taurine and saturated fat. A mediation model will be used to determine the extent to which diet (independent variable) and mucosal markers of CRC risk and DNA damage (dependent variables) are explained by colonic bacteria and their functions (mediator variables). This research will generate novel information targeted to develop effective dietary interventions that may reduce the unequal CRC burden in AAs.

Quercetin Alleviates Intestinal Oxidative Damage Induced by H2O2 via Modulation of GSH: In Vitro Screening and In Vivo Evaluation in a Colitis Model of Mice.

The gastrointestinal tract is exposed to pro-oxidants from food, host immune factors, and microbial pathogens, which may induce oxidative damage. Oxidative stress has been shown to play an important role in the onset of inflammatory bowel disease. This study aimed to use a novel model to evaluate the effects of a screened natural component and explore its possible mechanism. An in vitro oxidative stress Caco2 cell model induced by H2O2 was established using a real-time cellular analysis system and verified by addition of glutathione (GSH). A variety of plant components were chosen for the screening. Quercetin was the most effective phytochemical to alleviate the decreased cell index caused by H2O2 among the tested plant components. Furthermore, quercetin ameliorated dextran sulfate sodium salt (DSS)-induced colitis and further increased the serum GSH. The mechanism of quercetin protection was explored in Caco2. Reversed H2O2-induced cell damage and decreased reactive oxygen species and apoptosis ratio were observed in quercetin-treated cells. Also, quercetin increased expression of the glutamate-cysteine ligase catalytic subunit (GCLC), the first rate-limiting enzyme of glutathione synthesis, and increased intracellular GSH concentration under H2O2 treatment. This effect was abolished by the GCLC inhibitor buthionine sulfoximine. These results indicated that quercetin can improve cell proliferation and increase intracellular GSH concentrations by upregulating transcription of GCLC to eliminate excessive reactive oxygen species (ROS). Increased extracellular H2O2 concentration induced by quercetin under oxidative stress was related to the inhibition of AQP3 and upregulation of NOX1/2, which may contribute to the observed protective effects of quercetin. Moreover, the novel H2O2-induced oxidative stress cell model based on the real-time cellular analysis system was an effective model to screen natural products to deal with intestinal oxidative damage and help accelerate the discovery of new drugs for inflammatory bowel disease (IBD).

The 'in vivo lifestyle' of bile acid 7α-dehydroxylating bacteria: comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice.

The formation of secondary bile acids by gut microbes is a current topic of considerable biomedical interest. However, a detailed understanding of the biology of anaerobic bacteria in the genus Clostridium that are capable of generating secondary bile acids is lacking. We therefore sought to determine the transcriptional responses of two prominent secondary bile acid producing bacteria, Clostridium hylemonae and Clostridium hiranonis to bile salts (in vitro) and the cecal environment of gnotobiotic mice. The genomes of C. hylemonae DSM 15053 and C. hiranonis DSM 13275 were closed, and found to encode 3,647 genes (3,584 protein-coding) and 2,363 predicted genes (of which 2,239 are protein-coding), respectively, and 1,035 orthologs were shared between C. hylemonae and C. hiranonis. RNA-Seq analysis was performed in growth medium alone, and in the presence of cholic acid (CA) and deoxycholic acid (DCA). Growth with CA resulted in differential expression (>0.58 log2FC; FDR < 0.05) of 197 genes in C. hiranonis and 118 genes in C. hylemonae. The bile acid-inducible operons (bai) from each organism were highly upregulated in the presence of CA but not DCA. We then colonized germ-free mice with human gut bacterial isolates capable of metabolizing taurine-conjugated bile acids. This consortium included bile salt hydrolase-expressing Bacteroides uniformis ATCC 8492, Bacteroides vulgatus ATCC 8482, Parabacteroides distasonis DSM 20701, as well as taurine-respiring Bilophila wadsworthia DSM 11045, and deoxycholic/lithocholic acid generating Clostridium hylemonae DSM 15053 and Clostridium hiranonis DSM 13275. Butyrate and iso-bile acid-forming Blautia producta ATCC 27340 was also included. The Bacteroidetes made up 84.71% of 16S rDNA cecal reads, B. wadsworthia, constituted 14.7%, and the clostridia made up <.75% of 16S rDNA cecal reads. Bile acid metabolomics of the cecum, serum, and liver indicate that the synthetic community were capable of functional bile salt deconjugation, oxidation/isomerization, and 7α-dehydroxylation of bile acids. Cecal metatranscriptome analysis revealed expression of genes involved in metabolism of taurine-conjugated bile acids. The in vivo transcriptomes of C. hylemonae and C. hiranonis suggest fermentation of simple sugars and utilization of amino acids glycine and proline as electron acceptors. Genes predicted to be involved in trimethylamine (TMA) formation were also expressed.

Educational video intervention improves knowledge and self-efficacy in identifying malnutrition among healthcare providers in a cancer center: a pilot study.

PURPOSE: Cancer is the second leading cause of death in the USA, and malnutrition secondary to cancer progression and treatment side effects is common. While abundant evidence indicates that nutrition support improves patient outcomes, it is estimated that up to half of malnutrition cases are misclassified or undiagnosed. The use of a multidisciplinary team to assess nutrition status has been observed previously to reduce delays in nutritional support. Hence, educating all members of the oncology healthcare team to assess nutrition status may encourage earlier diagnosis and lead to improved patient outcomes. Thus, the objective was to perform a pilot study to assess change in knowledge and self-efficacy among oncology team members after watching an educational video about malnutrition. METHODS: A pre-test post-test educational video intervention was given to 77 ambulatory oncology providers during weekly staff meetings at a community ambulatory oncology center in central Illinois. Change in knowledge and self-efficacy in malnutrition assessment and diagnosis was measured and acceptability of the brief educational video format was also observed. RESULTS: Mean test scores improved by 1.95 ± 1.48 points (p < 0.001). Individual occupational groups improved scores significantly (p ≤ 0.005) except for specialty clinical staff. Self-efficacy improved from 38 to 70%. 90.8% of participants indicated the educational video improved their confidence in assessing malnutrition. CONCLUSIONS: The educational video was well accepted and improved knowledge and self-efficacy of malnutrition assessment and diagnosis among ambulatory oncology providers. Wider implementation of such an educational intervention and longitudinal testing of knowledge retention and behaviors change is warranted.

Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7α-dehydroxylating anaerobes. While this organism is well-studied relative to bile acid metabolism, little is known about the basic nutrition and physiology of C. scindens ATCC 35704. To determine the amino acid and vitamin requirements of C. scindens, the leave-one-out (one amino acid group or vitamin) technique was used to eliminate the nonessential amino acids and vitamins. With this approach, the amino acid tryptophan and three vitamins (riboflavin, pantothenate, and pyridoxal) were found to be required for the growth of C. scindens In the newly developed defined medium, C. scindens fermented glucose mainly to ethanol, acetate, formate, and H2. The genome of C. scindens ATCC 35704 was completed through PacBio sequencing. Pathway analysis of the genome sequence coupled with transcriptome sequencing (RNA-Seq) under defined culture conditions revealed consistency with the growth requirements and end products of glucose metabolism. Induction with bile acids revealed complex and differential responses to cholic acid and deoxycholic acid, including the expression of potentially novel bile acid-inducible genes involved in cholic acid metabolism. Responses to toxic deoxycholic acid included expression of genes predicted to be involved in DNA repair, oxidative stress, cell wall maintenance/metabolism, chaperone synthesis, and downregulation of one-third of the genome. These analyses provide valuable insight into the overall biology of C. scindens which may be important in treatment of disease associated with increased colonic secondary bile acids.IMPORTANCEC. scindens is one of a few identified gut bacterial species capable of converting host cholic acid into disease-associated secondary bile acids such as deoxycholic acid. The current work represents an important advance in understanding the nutritional requirements and response to bile acids of the medically important human gut bacterium, C. scindens ATCC 35704. A defined medium has been developed which will further the understanding of bile acid metabolism in the context of growth substrates, cofactors, and other metabolites in the vertebrate gut. Analysis of the complete genome supports the nutritional requirements reported here. Genome-wide transcriptomic analysis of gene expression in the presence of cholic acid and deoxycholic acid provides a unique insight into the complex response of C. scindens ATCC 35704 to primary and secondary bile acids. Also revealed are genes with the potential to function in bile acid transport and metabolism.

Zinc Supplementation, via GPR39, Upregulates PKCζ to Protect Intestinal Barrier Integrity in Caco-2 Cells Challenged by Salmonella enterica Serovar Typhimurium.

Background: Zinc has been shown to improve intestinal barrier function against Salmonella enterica serovar Typhimurium (S. typhimurium) infection, but the mechanisms involved in this process remain undefined.Objective: We aimed to explore the roles of G protein-coupled receptor (GPR)39 and protein kinase Cζ (PKCζ) in the regulation by zinc of intestinal barrier function.Methods: A Transwell Caco-2 monolayer was pretreated with 0, 50, or 100 µM Zn and then incubated with S. typhimurium for 0-6 h. Afterward, cells silenced by the small interfering RNA for GPR39 or PKCζ were pretreated with 100 µM Zn and incubated with S. typhimurium for 3 h. Finally, transepithelial electrical resistance (TEER), permeability, tight junction (TJ) proteins, and signaling molecules GPR39 and PKCζ were measured.Results: Compared with controls, S. typhimurium decreased TEER by 62.3-96.2% at 4-6 h (P < 0.001), increased (P < 0.001) permeability at 6 h, and downregulated (P < 0.05) TJ protein zonula occludens (ZO)-1 and occludin by 104-123%, as well as Toll-like receptor 2 and PKCζ by 35.1% and 75.2%, respectively. Compared with S. typhimurium-challenged cells, 50 and 100 µM Zn improved TEER by 26.3-60.9% at 4-6 h (P < 0.001) and decreased (P < 0.001) permeability and bacterial invasion at 6 h. A total of 100 µM Zn increased ZO-1, occludin, GPR39, and PKCζ 0.72- to 1.34-fold (P < 0.05); however, 50 µM Zn did not affect ZO-1 or occludin (P > 0.1). Silencing GPR39 decreased (P < 0.05) zinc-activated PKCζ and blocked (P < 0.05) the promotion of zinc on epithelial integrity. Furthermore, silencing PKCζ counteracted the protective effect of zinc on epithelial integrity but did not inhibit GPR39 (P = 0.138).Conclusion: We demonstrated that zinc upregulates PKCζ by activating GPR39 to enhance the abundance of ZO-1, thereby improving epithelial integrity in S. typhimurium-infected Caco-2 cells.

Race-dependent association of sulfidogenic bacteria with colorectal cancer.

OBJECTIVE: Colorectal cancer (CRC) incidence is higher in African Americans (AAs) compared with non-Hispanic whites (NHWs). A diet high in animal protein and fat is an environmental risk factor for CRC development. The intestinal microbiota is postulated to modulate the effects of diet in promoting or preventing CRC. Hydrogen sulfide, produced by autochthonous sulfidogenic bacteria, triggers proinflammatory pathways and hyperproliferation, and is genotoxic. We hypothesised that sulfidogenic bacterial abundance in colonic mucosa may be an environmental CRC risk factor that distinguishes AA and NHW. DESIGN: Colonic biopsies from uninvolved or healthy mucosa from CRC cases and tumour-free controls were collected prospectively from five medical centres in Chicago for association studies. Sulfidogenic bacterial abundance in uninvolved colonic mucosa of AA and NHW CRC cases was compared with normal mucosa of AA and NHW controls. In addition, 16S rDNA sequencing was performed in AA cases and controls. Correlations were examined among bacterial targets, race, disease status and dietary intake. RESULTS: AAs harboured a greater abundance of sulfidogenic bacteria compared with NHWs regardless of disease status. Bilophila wadsworthia-specific dsrA was more abundant in AA cases than controls. Linear discriminant analysis of 16S rRNA gene sequences revealed five sulfidogenic genera that were more abundant in AA cases. Fat and protein intake and daily servings of meat were significantly higher in AAs compared with NHWs, and multiple dietary components correlated with a higher abundance of sulfidogenic bacteria. CONCLUSIONS: These results implicate sulfidogenic bacteria as a potential environmental risk factor contributing to CRC development in AAs.

Zinc enhances intestinal epithelial barrier function through the PI3K/AKT/mTOR signaling pathway in Caco-2 cells.

Zinc plays an important role in maintaining intestinal barrier function as well as modulating cellular signaling recognition and protein kinase activities. The phosphatidylinositol 3-kinase (PI3K) cascade has been demonstrated to affect intercellular integrity and tight junction (TJ) proteins. The current study investigated the hypothesis that zinc regulates intestinal intercellular junction integrity through the PI3K/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. A transwell model of Caco-2 cell was incubated with 0, 50 and 100 µM of zinc at various time points. Transepithelial electrical resistance (TEER), paracellular permeability, TJ proteins, cell proliferation, differentiation and cell damage were measured. Compared with controls, 50 and 100 µM of zinc increased cell growth at 6, 12 and 24 h and the expression of proliferating cell nuclear antigen at 24 h. Zinc (100 µM) significantly elevated TEER at 6-24 h and reduced TJ permeability at 24 h, accompanied by the up-regulation of alkaline phosphatase (AP) activity and zonula occludens (ZO)-1 expression. In addition, zinc (100 µM) affected the PI3K/AKT/mTOR pathway by stimulating phosphorylation of AKT and the downstream target mTOR. Inhibition of PI3K signaling by LY294002 counteracted zinc promotion, as shown by a decrease in AP activity, TEER, the abundance of ZO-1 and phosphorylation of AKT and mTOR. Additionally, TJ permeability and the expression of caspase-3 and LC3II (markers of cell damage) were increased by addition of PI3K inhibitor. In conclusion, the activation of PI3K/AKT/mTOR signaling by zinc is involved in improving intestinal barrier function by enhancing cell differentiation and expression of TJ protein ZO-1.

H2 metabolism is widespread and diverse among human colonic microbes.

Microbial molecular hydrogen (H2) cycling is central to metabolic homeostasis and microbial composition in the human gastrointestinal tract. Molecular H2 is produced as an endproduct of carbohydrate fermentation and is reoxidised primarily by sulfate-reduction, acetogenesis, and methanogenesis. However, the enzymatic basis for these processes is incompletely understood and the hydrogenases responsible have not been investigated. In this work, we surveyed the genomic and metagenomic distribution of hydrogenase-encoding genes in the human colon to infer dominant mechanisms of H2 cycling. The data demonstrate that 70% of gastrointestinal microbial species listed in the Human Microbiome Project encode the genetic capacity to metabolise H2. A wide variety of anaerobically-adapted hydrogenases were present, with [FeFe]-hydrogenases predominant. We subsequently analyzed the hydrogenase gene content of stools from 20 healthy human subjects. The hydrogenase gene content of all samples was overwhelmingly dominated by fermentative and electron-bifurcating [FeFe]-hydrogenases emerging from the Bacteroidetes and Firmicutes. This study supports that H2 metabolism in the human gut is driven by fermentative H2 production and interspecies H2 transfer. However, it suggests that electron-bifurcation rather than respiration is the dominant mechanism of H2 reoxidation in the human colon, generating reduced ferredoxin to sustain carbon-fixation (e.g. acetogenesis) and respiration (via the Rnf complex). This work provides the first comprehensive bioinformatic insight into the mechanisms of H2 metabolism in the human colon.