Long-term stability of microbiome diversity and composition in fecal samples stored in eNAT medium.

Fecal samples collected for microbiome analyses are typically frozen to avoid postcollection changes in microbial composition. eNAT is a guanidine thiocyanate-based medium that stabilizes microbial DNA and allows safe specimen handling and shipping by inactivating microorganisms. We collected fecal samples (n = 50) from children undergoing hematopoietic stem cell transplantation. We divided samples into three aliquots: (a) stored in RNAlater and immediately transferred to -80°C; (b) stored in eNAT medium and immediately transferred to -80°C; and (c) stored in eNAT medium at ambient temperature (~20°C) for 30 days prior to transfer to -80°C. Mean (standard deviation) Shannon diversity and Chao1 indices in sample aliquots were 2.05 (0.62) and 23.8 (16.6), respectively. Comparing samples frozen immediately in RNAlater to samples frozen immediately in eNAT, there were no differences in Shannon diversity (p = .51), Chao1 richness (p = .66), and overall microbiome composition (p = .99). Comparing eNAT samples frozen immediately to samples stored at ambient temperature, we identified no differences in Shannon diversity (p = .65), Chao1 richness (p = .87), and overall microbiome composition (p = .99). Storage of fecal samples in eNAT at ambient temperature for 30 days did not alter microbiome richness, diversity, or composition. eNAT may be a useful medium for fecal microbiome studies, particularly when cold chain storage is unavailable.

Fetal exposure to the maternal microbiota in humans and mice.

Previous studies have demonstrated the presence of microbial DNA in the fetal environment. However, it remains unclear whether this DNA represents viable bacteria and how it relates to the maternal microbiota across body sites. We studied the microbiota of human and mouse dyads to understand these relationships, localize bacteria in the fetus, and demonstrate bacterial viability. In human preterm and full-term mother-infant dyads at the time of cesarean delivery, the oral cavity and meconium of newborn infants born as early as 24 weeks of gestation contained a microbiota that was predicted to originate from in utero sources, including the placenta. Using operative deliveries of pregnant mice under highly controlled, sterile conditions in the laboratory, composition, visualization, and viability of bacteria in the in utero compartment and fetal intestine were demonstrated by 16S rRNA gene sequencing, fluorescence in situ hybridization, and bacterial culture. The composition and predicted source of the fetal gut microbiota shifted between mid- and late gestation. Cultivatable bacteria in the fetal intestine were found during mid-gestation but not late gestation. Our results demonstrate a dynamic, viable mammalian fetal microbiota during in utero development.

Early-life skin microbiota in hospitalized preterm and full-term infants.

BACKGROUND: The infant skin microbiota may serve as a reservoir of bacteria that contribute to neonatal infections and stimulate local and systemic immune development. The objectives of our study were to characterize the skin microbiota of preterm and full-term infants during their birth hospitalization and describe its relationship to the microbiota of other body sites and the hospital environment. RESULTS: We conducted a cross-sectional study of 129 infants, including 40 preterm and 89 full-term infants. Samples were collected from five sites: the forehead and posterior auricular scalp (skin upper body); the periumbilical region, inguinal folds, and upper thighs (skin lower body); the oral cavity; the infant's immediate environment; and stool. Staphylococcus, Streptococcus, Enterococcus, and enteric Gram-negative bacteria including Escherichia and Enterobacter dominated the skin microbiota. The preterm infant microbiota at multiple sites had lower alpha diversity and greater enrichment with Staphylococcus and Escherichia than the microbiota of comparable sites in full-term infants. The community structure was highly variable among individuals but differed significantly by body site, postnatal age, and gestational age. Source tracking indicated that each body site both contributed to and received microbiota from other body sites and the hospital environment. CONCLUSION: The skin microbiota of preterm and full-term infants varied across individuals, by body site, and by the infant's developmental stage. The skin harbored many organisms that are common pathogens in hospitalized infants. Bacterial source tracking suggests that microbiota are commonly exchanged across body sites and the hospital environment as microbial communities mature in infancy.

The severity of nonalcoholic fatty liver disease is associated with gut dysbiosis and shift in the metabolic function of the gut microbiota.

UNLABELLED: Several animal studies have emphasized the role of gut microbiota in nonalcoholic fatty liver disease (NAFLD). However, data about gut dysbiosis in human NAFLD remain scarce in the literature, especially studies including the whole spectrum of NAFLD lesions. We aimed to evaluate the association between gut dysbiosis and severe NAFLD lesions, that is, nonalcoholic steatohepatitis (NASH) and fibrosis, in a well-characterized population of adult NAFLD. Fifty-seven patients with biopsy-proven NAFLD were enrolled. Taxonomic composition of gut microbiota was determined using 16S ribosomal RNA gene sequencing of stool samples. Thirty patients had F0/F1 fibrosis stage at liver biopsy (10 with NASH), and 27 patients had significant F≥2 fibrosis (25 with NASH). Bacteroides abundance was significantly increased in NASH and F≥2 patients, whereas Prevotella abundance was decreased. Ruminococcus abundance was significantly higher in F≥2 patients. By multivariate analysis, Bacteroides abundance was independently associated with NASH and Ruminococcus with F≥2 fibrosis. Stratification according to the abundance of these two bacteria generated three patient subgroups with increasing severity of NAFLD lesions. Based on imputed metagenomic profiles, Kyoto Encyclopedia of Genes and Genomes pathways significantly related to NASH and fibrosis F≥2 were mostly related to carbohydrate, lipid, and amino acid metabolism. CONCLUSION: NAFLD severity associates with gut dysbiosis and a shift in metabolic function of the gut microbiota. We identified Bacteroides as independently associated with NASH and Ruminococcus with significant fibrosis. Thus, gut microbiota analysis adds information to classical predictors of NAFLD severity and suggests novel metabolic targets for pre-/probiotics therapies.

The role of HuR in the post-transcriptional regulation of interleukin-3 in T cells.

Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells.

Association of plasma endotoxin, inflammatory cytokines and risk of colorectal adenomas.

BACKGROUND: Recent studies suggest that bacterial endotoxins may be associated with various chronic diseases, including colorectal adenomas and cancer. Given the evidence linking inflammation and colorectal cancer, we sought to determine if plasma endotoxin concentrations are associated with indicators of systemic or local inflammation and colorectal adenomas. METHODS: This cross-sectional study consisted of participants who underwent screening colonoscopies and included adenoma cases (n=138) and non-adenoma controls (n=324). Plasma concentrations of endotoxin were measured with Limulus Amebocyte Lysate (LAL) assay. We quantified concentrations of inflammatory cytokines, interleukin-4 (IL-4), IL-6, IL-8, IL-10, IL-12, tumor necrosis factor-alpha (TNF-α), and interferon-γ (IFN-γ) in plasma by ELISA and mRNA expression levels in rectal mucosal biopsies by quantitative RT-PCR. Interleukin-17 was evaluated only in the rectal mucosa. RESULTS: Compared to subjects with low plasma endotoxin concentrations, those with higher concentrations were more likely to have adenomas (OR 1.4, 95% CI 1.0-2.1). Among subjects with adenomas, those with villous histology were more likely to have higher endotoxin concentrations (5.4 vs. 4.1EU/mL, p=0.05) and lower plasma IFN-γ (0 vs. 1.64 pg/mL, p=0.02) compared to those with only tubular adenomas. Cases showed a trend of having higher plasma TNF-α levels than controls (p=0.06), but none of the other plasma or rectal mucosal cytokine levels differed between cases and controls. Elevated mucosal IL-12 levels were associated with having multiple adenomas (p=0.04). Higher concentrations of plasma endotoxin predicted increased plasma IL-12 levels (OR 1.5, 95% CI 1.0-2.2) and rectal mucosal IL-12 (OR 1.9, 95% CI 1.0-3.7) and IL-17 gene expression (OR 2.2, 95% CI 1.0-4.6). CONCLUSIONS: These findings suggest that interactions between elevated plasma endotoxin concentrations and inflammatory cytokines may be relevant to the development of colorectal adenomas.

Fusobacterium is associated with colorectal adenomas.

The human gut microbiota is increasingly recognized as a player in colorectal cancer (CRC). While particular imbalances in the gut microbiota have been linked to colorectal adenomas and cancer, no specific bacterium has been identified as a risk factor. Recent studies have reported a high abundance of Fusobacterium in CRC subjects compared to normal subjects, but this observation has not been reported for adenomas, CRC precursors. We assessed the abundance of Fusobacterium species in the normal rectal mucosa of subjects with (n = 48) and without adenomas (n = 67). We also confirmed previous reports on Fusobacterium and CRC in 10 CRC tumor tissues and 9 matching normal tissues by pyrosequencing. We extracted DNA from rectal mucosal biopsies and measured bacterial levels by quantitative PCR of the 16S ribosomal RNA gene. Local cytokine gene expression was also determined in mucosal biopsies from adenoma cases and controls by quantitative PCR. The mean log abundance of Fusobacterium or cytokine gene expression between cases and controls was compared by t-test. Logistic regression was used to compare tertiles of Fusobacterium abundance. Adenoma subjects had a significantly higher abundance of Fusobacterium species compared to controls (p = 0.01). Compared to the lowest tertile, subjects with high abundance of Fusobacterium were significantly more likely to have adenomas (OR 3.66, 95% CI 1.37-9.74, p-trend 0.005). Cases but not controls had a significant positive correlation between local cytokine gene expression and Fusobacterium abundance. Among cases, the correlation for local TNF-α and Fusobacterium was r = 0.33, p = 0.06 while it was 0.44, p = 0.01 for Fusobacterium and IL-10. These results support a link between the abundance of Fusobacterium in colonic mucosa and adenomas and suggest a possible role for mucosal inflammation in this process.

Somatic gene mutations in African Americans may predict worse outcomes in colorectal cancer.

BACKGROUND AND OBJECTIVE: African Americans have worse outcomes in colorectal cancer (CRC) than Caucasians. We sought to determine if KRAS, BRAF and PIK3CA mutations might contribute to the racial differences in CRC outcome. METHODS: DNA was extracted from tissue microarrays made from CRC samples from 67 African Americans and 237 Caucasians. Mutations in KRAS, BRAF, and PIK3CA were evaluated by PCR sequencing. We also examined microsatellite instability (MSI) status. Associations of mutation status with tumor stage and grade were examined using a logistic regression model. Cox proportional hazards models were used to estimate the all-cause mortality associated with mutational status, race and other clinicopathologic features. RESULTS: KRAS mutations were more common in African Americans than among Caucasians (37% vs 21%, p=0.01) and were associated with advanced stage (unadjusted odds ratio (OR)=3.31, 95% confidence interval (CI) 1.03-10.61) and grade (unadjusted OR=5.60, 95% CI 1.01-31.95) among African Americans. Presence of BRAF mutations was also positively associated with advanced tumor stage (adjusted OR=3.99, 95%CI 1.43-11.12) and grade (adjusted OR=3.93, 95%CI 1.05-14.69). PIK3CA mutations showed a trend toward an association with an increased risk of death compared to absence of those mutations (adjusted for age, sex and CRC site HR=1.89, 95% CI 0.98-3.65). Among African Americans, the association was more evident (adjusted for age, sex and CRC site HR=3.92, 95% CI 1.03-14.93) and remained significant after adjustment for MSI-H status and combined education-income level, with HR of 12.22 (95%CI 1.32-121.38). CONCLUSIONS: Our results suggest that African Americans may have different frequencies of somatic genetic alterations that may partially explain the worse prognosis among African Americans with CRC compared to whites.

Differences in microbial signatures between rectal mucosal biopsies and rectal swabs.

There is growing evidence the microbiota of the large bowel may influence the risk of developing colorectal cancer as well as other diseases including type-1 diabetes, inflammatory bowel diseases and irritable bowel syndrome. Current sampling methods to obtain microbial specimens, such as feces and mucosal biopsies, are inconvenient and unappealing to patients. Obtaining samples through rectal swabs could prove to be a quicker and relatively easier method, but it is unclear if swabs are an adequate substitute. We compared bacterial diversity and composition from rectal swabs and rectal mucosal biopsies in order to examine the viability of rectal swabs as an alternative to biopsies. Paired rectal swabs and mucosal biopsy samples were collected in un-prepped participants (n = 11) and microbial diversity was characterized by Terminal Restriction Fragment Length polymorphism (T-RFLP) analysis and quantitative polymerase chain reaction (qPCR) of the 16S rRNA gene. Microbial community composition from swab samples was different from rectal mucosal biopsies (p = 0.001). Overall the bacterial diversity was higher in swab samples than in biopsies as assessed by diversity indexes such as: richness (p = 0.01), evenness (p = 0.06) and Shannon's diversity (p = 0.04). Analysis of specific bacterial groups by qPCR showed higher copy number of Lactobacillus (p < 0.0001) and Eubacteria (p = 0.0003) in swab samples compared with biopsies. Our findings suggest that rectal swabs and rectal mucosal samples provide different views of the microbiota in the large intestine.

Increased rectal microbial richness is associated with the presence of colorectal adenomas in humans.

Differences in the composition of the gut microbial community have been associated with diseases such as obesity, Crohn's disease, ulcerative colitis and colorectal cancer (CRC). We used 454 titanium pyrosequencing of the V1-V2 region of the 16S rRNA gene to characterize adherent bacterial communities in mucosal biopsy samples from 33 subjects with adenomas and 38 subjects without adenomas (controls). Biopsy samples from subjects with adenomas had greater numbers of bacteria from 87 taxa than controls; only 5 taxa were more abundant in control samples. The magnitude of the differences in the distal gut microbiota between patients with adenomas and controls was more pronounced than that of any other clinical parameters including obesity, diet or family history of CRC. This suggests that sequence analysis of the microbiota could be used to identify patients at risk for developing adenomas.

Differential gene expression between African American and European American colorectal cancer patients.

The incidence and mortality of colorectal cancer (CRC) is higher in African Americans (AAs) than other ethnic groups in the U. S., but reasons for the disparities are unknown. We performed gene expression profiling of sporadic CRCs from AAs vs. European Americans (EAs) to assess the contribution to CRC disparities. We evaluated the gene expression of 43 AA and 43 EA CRC tumors matched by stage and 40 matching normal colorectal tissues using the Agilent human whole genome 4x44K cDNA arrays. Gene and pathway analyses were performed using Significance Analysis of Microarrays (SAM), Ten-fold cross validation, and Ingenuity Pathway Analysis (IPA). SAM revealed that 95 genes were differentially expressed between AA and EA patients at a false discovery rate of ≤5%. Using IPA we determined that most prominent disease and pathway associations of differentially expressed genes were related to inflammation and immune response. Ten-fold cross validation demonstrated that following 10 genes can predict ethnicity with an accuracy of 94%: CRYBB2, PSPH, ADAL, VSIG10L, C17orf81, ANKRD36B, ZNF835, ARHGAP6, TRNT1 and WDR8. Expression of these 10 genes was validated by qRT-PCR in an independent test set of 28 patients (10 AA, 18 EA). Our results are the first to implicate differential gene expression in CRC racial disparities and indicate prominent difference in CRC inflammation between AA and EA patients. Differences in susceptibility to inflammation support the existence of distinct tumor microenvironments in these two patient populations.

Genomic hotspots for adaptation: the population genetics of Müllerian mimicry in Heliconius erato.

Wing pattern evolution in Heliconius butterflies provides some of the most striking examples of adaptation by natural selection. The genes controlling pattern variation are classic examples of Mendelian loci of large effect, where allelic variation causes large and discrete phenotypic changes and is responsible for both convergent and highly divergent wing pattern evolution across the genus. We characterize nucleotide variation, genotype-by-phenotype associations, linkage disequilibrium (LD), and candidate gene expression patterns across two unlinked genomic intervals that control yellow and red wing pattern variation among mimetic forms of Heliconius erato. Despite very strong natural selection on color pattern, we see neither a strong reduction in genetic diversity nor evidence for extended LD across either patterning interval. This observation highlights the extent that recombination can erase the signature of selection in natural populations and is consistent with the hypothesis that either the adaptive radiation or the alleles controlling it are quite old. However, across both patterning intervals we identified SNPs clustered in several coding regions that were strongly associated with color pattern phenotype. Interestingly, coding regions with associated SNPs were widely separated, suggesting that color pattern alleles may be composed of multiple functional sites, conforming to previous descriptions of these loci as "supergenes." Examination of gene expression levels of genes flanking these regions in both H. erato and its co-mimic, H. melpomene, implicate a gene with high sequence similarity to a kinesin as playing a key role in modulating pattern and provides convincing evidence for parallel changes in gene regulation across co-mimetic lineages. The complex genetic architecture at these color pattern loci stands in marked contrast to the single casual mutations often identified in genetic studies of adaptation, but may be more indicative of the type of genetic changes responsible for much of the adaptive variation found in natural populations.