Ruminococcus torques is a keystone degrader of intestinal mucin glycoprotein, releasing oligosaccharides used by Bacteroides thetaiotaomicron.

Symbiotic interactions between humans and our communities of resident gut microbes (microbiota) play many roles in health and disease. Some gut bacteria utilize mucus as a nutrient source and can under certain conditions damage the protective barrier it forms, increasing disease susceptibility. We investigated how Ruminococcus torques- a known mucin-degrader that remains poorly studied despite its implication in inflammatory bowel diseases (IBDs)- degrades mucin glycoproteins or their component O -linked glycans to understand its effects on the availability of mucin-derived nutrients for other bacteria. We found that R. torques utilizes both mucin glycoproteins and released oligosaccharides from gastric and colonic mucins, degrading these substrates with a panoply of mostly constitutively expressed, secreted enzymes. Investigation of mucin oligosaccharide degradation by R. torques revealed strong fucosidase, sialidase and ß1,4-galactosidase activities. There was a lack of detectable sulfatase and weak ß1,3-galactosidase degradation, resulting in accumulation of glycans containing these structures on mucin polypeptides. While the Gram-negative symbiont, Bacteroides thetaiotaomicron grows poorly on mucin glycoproteins, we demonstrate a clear ability of R. torques to liberate products from mucins, making them accessible to B. thetaiotaomicron . This work underscores the diversity of mucin-degrading mechanisms in different bacterial species and the probability that some species are contingent on others for the ability to more fully access mucin-derived nutrients. The ability of R. torques to directly degrade a variety of mucin and mucin glycan structures and unlock released glycans for other species suggests that it is a keystone mucin degrader, which may contribute to its association with IBD. Importance: An important facet of maintaining healthy symbiosis between host and intestinal microbes is the mucus layer, the first defense protecting the epithelium from lumenal bacteria. Some gut bacteria degrade different components of intestinal mucins, but detailed mechanisms used by different species are still emerging. It is imperative to understand these mechanisms as they likely dictate interspecies interactions and may illuminate particular species associated with bacterial mucus destruction and subsequent disease susceptibility. Ruminococcus torques is positively associated with IBD in multiple studies. We identified mucin glycan-degrading enzymes in R. torques and found that it shares mucin degradation products with another gut bacterium implicated in IBD, Bacteroides thetaiotaomicron . Our findings underscore the importance of understanding the mucin degradation mechanisms of different gut bacteria and their consequences on interspecies interactions, which may identify keystone bacteria that disproportionately contribute to defects in mucus protection and could therefore be targets to prevent or treat IBD.

Breast Milk Oligosaccharides Contain Immunomodulatory Glucuronic Acid and LacdiNAc.

Breast milk is abundant with functionalized milk oligosaccharides (MOs) to nourish and protect the neonate. Yet we lack a comprehensive understanding of the repertoire and evolution of MOs across Mammalia. We report ∼400 MO-species associations (>100 novel structures) from milk glycomics of nine mostly understudied species: alpaca, beluga whale, black rhinoceros, bottlenose dolphin, impala, L'Hoest's monkey, pygmy hippopotamus, domestic sheep, and striped dolphin. This revealed the hitherto unknown existence of the LacdiNAc motif (GalNAcß1-4GlcNAc) in MOs of all species except alpaca, sheep, and striped dolphin, indicating the widespread occurrence of this potentially antimicrobial motif in MOs. We also characterize glucuronic acid-containing MOs in the milk of impala, dolphins, sheep, and rhinoceros, previously only reported in cows. We demonstrate that these GlcA-MOs exhibit potent immunomodulatory effects. Our study extends the number of known MOs by >15%. Combined with >1900 curated MO-species associations, we characterize MO motif distributions, presenting an exhaustive overview of MO biodiversity.

Intestinal mucus and their glycans: A habitat for thriving microbiota.

The colon mucus layer is organized with an inner colon mucus layer that is impenetrable to bacteria and an outer mucus layer that is expanded to allow microbiota colonization. A major component of mucus is MUC2, a glycoprotein that is extensively decorated, especially with O-glycans. In the intestine, goblet cells are specialized in controlling glycosylation and making mucus. Some microbiota members are known to encode multiple proteins that are predicted to bind and/or cleave mucin glycans. The interactions between commensal microbiota and host mucins drive intestinal colonization, while at the same time, the microbiota can utilize the glycans on mucins and affect the colonic mucus properties. This review will examine this interaction between commensal microbes and intestinal mucins and discuss how this interplay affects health and disease.

All that glitters is not gold: a stereological study of human donor oocytes.

Here we report a quantitative analysis of human metaphase II (MII) oocytes from a 22-year-old oocyte donor, retrieved after ovarian-controlled hyperstimulation. Five surplus donor oocytes were processed for transmission electron microscopy (TEM), and a stereological analysis was used to quantify the distribution of organelles, using the point-counting technique with an adequate stereological grid. Comparisons between means of the relative volumes (Vv) occupied by organelles in the three oocyte regions, cortex (C), subcortex (SC) and inner cytoplasm (IC), followed the Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction. Life cell imaging and TEM analysis confirmed donor oocyte nuclear maturity. Results showed that the most abundant organelles were smooth endoplasmic reticulum (SER) elements (26.8%) and mitochondria (5.49%). Significant differences between oocyte regions were found for lysosomes (P = 0.003), cortical vesicles (P = 0.002) and large SER vesicles (P = 0.009). These results were quantitatively compared with previous results using prophase I (GV) and metaphase I (MI) immature oocytes. In donor MII oocytes there was a normal presence of cortical vesicles, SER tubules, SER small, medium and large vesicles, lysosomes and mitochondria. However, donor MII oocytes displayed signs of cytoplasmic immaturity, namely the presence of dictyosomes, present in GV oocytes and rare in MI oocytes, of SER very large vesicles, characteristic of GV oocytes, and the rarity of SER tubular aggregates. Results therefore indicate that the criterion of nuclear maturity used for donor oocyte selection does not always correspond to cytoplasmic maturity, which can partially explain implantation failures with the use of donor oocytes.

Mucin utilization by gut microbiota: recent advances on characterization of key enzymes.

The gut microbiota interacts with the host through the mucus that covers and protects the gastrointestinal epithelium. The main component of the mucus are mucins, glycoproteins decorated with hundreds of different O-glycans. Some microbiota members can utilize mucin O-glycans as carbons source. To degrade these host glycans the bacteria express multiple carbohydrate-active enzymes (CAZymes) such as glycoside hydrolases, sulfatases and esterases which are active on specific linkages. The studies of these enzymes in an in vivo context have started to reveal their importance in mucin utilization and gut colonization. It is now clear that bacteria evolved multiple specific CAZymes to overcome the diversity of linkages found in O-glycans. Additionally, changes in mucin degradation by gut microbiota have been associated with diseases like obesity, diabetes, irritable bowel disease and colorectal cancer. Thereby understanding how CAZymes from different bacteria work to degrade mucins is of critical importance to develop new treatments and diagnostics for these increasingly prevalent health problems. This mini-review covers the recent advances in biochemical characterization of mucin O-glycan-degrading CAZymes and how they are connected to human health.

Functions and specificity of bacterial carbohydrate sulfatases targeting host glycans.

Sulfated host glycans (mucin O-glycans and glycosaminoglycans [GAGs]) are critical nutrient sources and colonisation factors for Bacteroidetes of the human gut microbiota (HGM); a complex ecosystem comprising essential microorganisms that coevolved with humans to serve important roles in pathogen protection, immune signalling, and host nutrition. Carbohydrate sulfatases are essential enzymes to access sulfated host glycans and are capable of exquisite regio- and stereo-selective substrate recognition. In these enzymes, the common recognition features of each subfamily are correlated with their genomic and environmental context. The exo-acting carbohydrate sulfatases are attractive drug targets amenable to small-molecule screening and subsequent engineering, and their high specificity will help elucidate the role of glycan sulfation in health and disease. Inhibition of carbohydrate sulfatases provides potential routes to control Bacteroidetes growth and to explore the influence of host glycan metabolism by Bacteroidetes on the HGM ecosystem. The roles of carbohydrate sulfatases from the HGM organism Bacteroides thetaiotaomicron and the soil isolated Pedobacter heparinus (P. heparinus) in sulfated host glycan metabolism are examined and contrasted, and the structural features underpinning glycan recognition and specificity explored.

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota.

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

A single sulfatase is required to access colonic mucin by a gut bacterium.

Humans have co-evolved with a dense community of microbial symbionts that inhabit the lower intestine. In the colon, secreted mucus creates a barrier that separates these microorganisms from the intestinal epithelium1. Some gut bacteria are able to utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, it remains unclear which bacterial enzymes initiate degradation of the complex O-glycans found in mucins. In the distal colon, these glycans are heavily sulfated, but specific sulfatases that are active on colonic mucins have not been identified. Here we show that sulfatases are essential to the utilization of distal colonic mucin O-glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases produced by this species, showing that they are collectively active on all known sulfate linkages in O-glycans. Crystal structures of three enzymes provide mechanistic insight into the molecular basis of substrate specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated O-glycans in vitro and also has a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by a prominent group of gut bacteria, an important process for both normal microbial gut colonization2 and diseases such as inflammatory bowel disease3.

Comprehensive Review of Numerical Chromosomal Aberrations in Chromophobe Renal Cell Carcinoma Including Its Variant Morphologies.

Chromophobe renal cell carcinoma (ChRCC) accounts for 5% to 7% of all renal cell carcinomas. It was thought for many years that ChRCC exhibits a hypodiploid genome. Recent studies using advanced molecular genetics techniques have shown more complex and heterogenous pattern with frequent chromosomal gains. Historically, multiple losses of chromosomes 1, 2, 6, 10, 13, 17, and 21 have been considered a genetic hallmark of ChRCC, both for classic and eosinophilic ChRCC variants. In the last 2 decades, multiple chromosomal gains in ChRCCs have also been documented, depicting a considerably broader genetic spectrum than previously thought. Studies of rare morphologic variants including ChRCC with pigmented microcystic adenomatoid/multicystic growth, ChRCC with neuroendocrine differentiation, ChRCC with papillary architecture, and renal oncocytoma-like variants also showed variable chromosomal numerical aberrations, including multiple losses (common), gains (less common), or chromosomal changes overlapping with renal oncocytoma. Although not the focus of the review, The Cancer Genome Atlas (TCGA) data in ChRCC show TP53, PTEN, and CDKN2A to be the most mutated genes. Given the complexity of molecular genetic alterations in ChRCC, this review analyzed the existing published data, aiming to present a comprehensive up-to-date survey of the chromosomal abnormalities in classic ChRCC and its variants. The potential role of chromosomal numerical aberrations in the differential diagnostic evaluation may be limited, potentially owing to its high variability.

Molecular Genetic Features of Primary Nonurachal Enteric-type Adenocarcinoma, Urachal Adenocarcinoma, Mucinous Adenocarcinoma, and Intestinal Metaplasia/Adenoma: Review of the Literature and Next-generation Sequencing Study.

The diagnosis of primary adenocarcinoma of the urinary bladder may be challenging in routine practice. These tumors may morphologically and immunohistochemically overlap with urachal adenocarcinoma and colorectal adenocarcinoma. Further, their genetic background is poorly understood. We systematically searched the PubMed database for results of complex genetic evaluation of primary bladder adenocarcinoma subtypes. Subsequently, we designed our own series of bladder lesions. We evaluated 36 cases: 16 primary enteric-type adenocarcinomas, 7 urachal enteric adenocarcinomas, 3 primary mucinous/colloid adenocarcinomas, and 10 intestinal-type metaplasia/villous adenoma. Detailed clinical data were collected, and all cases were examined using targeted next-generation sequencing. On the basis of the literature, the first mutated gene in these tumors was reported to be KRAS in 11.3% of cases, followed by TERT promoter mutations in 28.5%. In addition to KRAS and TERT, other genes were also found to be frequently mutated in primary bladder adenocarcinoma, including TP53, PIK3CA, CTNNB1, APC, FBXW7, IDH2, and RB1. In our series, the most frequent gene mutations in primary enteric-type adenocarcinomas were as follows: TP53 (56%); BRCA2, KMT2B (both 33%); NOTCH2, KDR, ARID1B, POLE, PTEN, KRAS (all 28%); in urachal enteric adenocarcinoma they were as follows: TP53 (86%); PTEN, NOTCH (both 43%); in primary mucinous/colloid adenocarcinomas they were as follows: KRAS, GRIN2A, AURKB (all 67%); and, in intestinal-type metaplasia/villous adenoma, they were as follows: APC, PRKDC (both 60%); ROS1, ATM, KMT2D (all 50%). No specific mutational pattern was identified using cluster analysis for any of the groups. Herein, we describe the pathologic features and immunohistochemical staining patterns traditionally used in the differential diagnoses of glandular lesions of the bladder in routine surgical pathology. We outline the mutational landscape of these lesions as an aggregate of published data with additional data from our cohort. Although diagnostically not discriminatory, we document that the most common genetic alterations shared between these glandular neoplasms include TP53, APC (in the Wnt pathway), and KRAS (in the MAPK pathway) mutations.

A Ribose-Scavenging System Confers Colonization Fitness on the Human Gut Symbiont Bacteroides thetaiotaomicron in a Diet-Specific Manner.

Efficient nutrient acquisition in the human gut is essential for microbial persistence. Although polysaccharides have been well-studied nutrients for the gut microbiome, other resources such as nucleic acids and nucleosides are less studied. We describe several ribose-utilization systems (RUSs) that are broadly represented in Bacteroidetes and appear to have diversified to access ribose from a variety of substrates. One Bacteroides thetaiotaomicron RUS variant is critical for competitive gut colonization in a diet-specific fashion. We used molecular genetics to probe the required functions of the system and the nature of the nutrient source(s) underlying this phenotype. Two RUS-encoded ribokinases were the only components required for this effect, presumably because they generate ribose-phosphate derivatives from products of an unlinked but essential nucleoside phosphorylase. Our results underscore the extensive mechanisms that gut symbionts have evolved to access nutrients and the potential for unexpected dependencies among systems that mediate colonization and persistence.

Complex N-glycan breakdown by gut Bacteroides involves an extensive enzymatic apparatus encoded by multiple co-regulated genetic loci.

Glycans are the major carbon sources available to the human colonic microbiota. Numerous N-glycosylated proteins are found in the human gut, from both dietary and host sources, including immunoglobulins such as IgA that are secreted into the intestine at high levels. Here, we show that many mutualistic gut Bacteroides spp. have the capacity to utilize complex N-glycans (CNGs) as nutrients, including those from immunoglobulins. Detailed mechanistic studies using transcriptomic, biochemical, structural and genetic techniques reveal the pathway employed by Bacteroides thetaiotaomicron (Bt) for CNG degradation. The breakdown process involves an extensive enzymatic apparatus encoded by multiple non-adjacent loci and comprises 19 different carbohydrate-active enzymes from different families, including a CNG-specific endo-glycosidase activity. Furthermore, CNG degradation involves the activity of carbohydrate-active enzymes that have previously been implicated in the degradation of other classes of glycan. This complex and diverse apparatus provides Bt with the capacity to access the myriad different structural variants of CNGs likely to be found in the intestinal niche.

Interrogating gut bacterial genomes for discovery of novel carbohydrate degrading enzymes.

Individual human gut bacteria often encode hundreds of enzymes for degrading different polysaccharides. Identification of co-localized and co-regulated genes in these bacteria has been a successful approach to identify enzymes that participate in full or partial saccharification of complex carbohydrates, often unmasking novel catalytic activities. Here, we review recent studies that have led to the discovery of new activities from gut bacteria and summarize a general scheme for identifying gut bacteria with novel catalytic abilities, locating the enzymes involved and investigating their activities in detail. The strength of this approach is amplified by the availability of abundant genomic and metagenomic data for the human gut microbiome, which facilitates comparative approaches to mine existing data for new or orthologous enzymes.

Bacteroides thetaiotaomicron.

This infographic on Bacteroides thetaiotaomicron (Bt) explores the ability of this microbe to digest a broad array of complex carbohydrates, alter its surface features, and its emerging role in gastrointestinal diseases. The infographic of Bacteroides thetaiotaomicron (Bt) illustrates two key facets of its symbiotic lifestyle in the human gut: a broad ability to digest dietary fiber polysaccharides and host glycans, and a dynamic cell-surface architecture that promotes both interactions with and evasion of the host immune system. The starch-utilization system (Sus) is a cell-surface and periplasmic system involved in starch cleavage and transport. Over 80 additional Sus-like systems utilize substrates ranging from host glycans to plant cell wall pectins. Bt has evolved intricate strategies to interact with other microbes or its host, including modification of its surface. Some nutrient utilization pathways select for or directly trigger changes in capsular polysaccharide (CPS) expression. Like other fermentative members of the gut microbiome, Bt produces host absorbable short-chain and organic acids, which can all be absorbed by the host as a source of energy.

Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides.

The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharide utilization loci (PULs). In Bacteroides thetaiotaomicron, a human colonic bacterium, the PULs activated by different pectin domains have been identified; however, the mechanism by which these loci contribute to the degradation of these GalA-containing polysaccharides is poorly understood. Here we show that each PUL orchestrates the metabolism of specific pectin molecules, recruiting enzymes from two previously unknown glycoside hydrolase families. The apparatus that depolymerizes the backbone of rhamnogalacturonan-I is particularly complex. This system contains several glycoside hydrolases that trim the remnants of other pectin domains attached to rhamnogalacturonan-I, and nine enzymes that contribute to the degradation of the backbone that makes up a rhamnose-GalA repeating unit. The catalytic properties of the pectin-degrading enzymes are optimized to protect the glycan cues that activate the specific PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms.

Phenotypic Intratumoral Heterogeneity of Endometrial Carcinomas.

Intratumoral heterogeneity has been shown to play an important role in diagnostic accuracy, development of treatment resistance, and prognosis of cancer patients. Recent studies have proposed quantitative measurement of phenotypic intratumoral heterogeneity, but no study is yet available in endometrial carcinomas. In our study we evaluated the phenotypic intratumoral heterogeneity of a consecutive series of 10 endometrial carcinomas using measures of dispersion and diversity. Morphometric architectural (%tumor cells, %solid tumor, %differentiated tumor, and %lumens) and nuclear [volume-weighted mean nuclear volume ((Equation is included in full-text article.))] parameters, as well as estrogen receptor, progesterone receptor, p53, vimentin, and beta-catenin immunoexpression (H-score) were digitally analyzed in 20 microscopic fields per carcinoma. Quantitative measures of intratumoral heterogeneity included coefficient of variation (CV) and relative quadratic entropy (rQE). In each endometrial carcinoma there was slight variation of architecture from field to field, resulting in globally low levels of heterogeneity measures (mean CV %tumor cells: 0.10, %solid tumor: 0.73, %differentiated tumor: 0.19, %lumens: 0.61 and mean rQE %tumor cells: 18.5, %solid tumor: 20.3, %differentiated tumor: 25.6, %lumens: 21.8). Nuclear intratumoral heterogeneity was also globally low (mean (Equation is included in full-text article.)CV: 0.23 and rQE: 27.3), but significantly higher than the heterogeneity of architectural parameters within most carcinomas. In general, there was low to moderate variability of immunoexpression markers within each carcinoma, but estrogen receptor (mean CV: 0.56 and rQE: 46.2) and progesterone receptor (mean CV: 0.60 and rQE: 39.3) displayed the highest values of heterogeneity measures. Intratumoral heterogeneity of immunoexpression was significantly higher than that observed for morphometric parameters. In conclusion, our study indicates that endometrial carcinomas present a variable but predominantly low degree of phenotypic intratumoral heterogeneity.

GC-MS metabolomics-based approach for the identification of a potential VOC-biomarker panel in the urine of renal cell carcinoma patients.

The analysis of volatile organic compounds (VOCs) emanating from biological samples appears as one of the most promising approaches in metabolomics for the study of diseases, namely cancer. In fact, it offers advantages, such as non-invasiveness and robustness for high-throughput applications. The purpose of this work was to study the urinary volatile metabolic profile of patients with renal cell carcinoma (RCC) (n = 30) and controls (n = 37) with the aim of identifying a potential specific urinary volatile pattern as a non-invasive strategy to detect RCC. Moreover, the effect of some confounding factors such as age, gender, smoking habits and body mass index was evaluated as well as the ability of urinary VOCs to discriminate RCC subtypes and stages. A headspace solid-phase microextraction/gas chromatography-mass spectrometry-based method was performed, followed by multivariate data analysis. A variable selection method was applied to reduce the impact of potential redundant and noisy chromatographic variables, and all models were validated by Monte Carlo cross-validation and permutation tests. Regarding the effect of RCC on the urine VOCs composition, a panel of 21 VOCs descriptive of RCC was defined, capable of discriminating RCC patients from controls in principal component analysis. Discriminant VOCs were further individually validated in two independent samples sets (nine RCC patients and 12 controls, seven RCC patients with diabetes mellitus type 2) by univariate statistical analysis. Two VOCs were found consistently and significantly altered between RCC and controls (2-oxopropanal and, according to identification using NIST14, 2,5,8-trimethyl-1,2,3,4-tetrahydronaphthalene-1-ol), strongly suggesting enhanced potential as RCC biomarkers. Gender, smoking habits and body mass index showed negligible and age-only minimal effects on the urinary VOCs, compared to the deviations resultant from the disease. Moreover, in this cohort, the urinary volatilome did not show ability to discriminate RCC stages and histological subtypes. The results validated the value of urinary volatilome for the detection of RCC and advanced with the identification of potential RCC urinary biomarkers.

Complex pectin metabolism by gut bacteria reveals novel catalytic functions.

The metabolism of carbohydrate polymers drives microbial diversity in the human gut microbiota. It is unclear, however, whether bacterial consortia or single organisms are required to depolymerize highly complex glycans. Here we show that the gut bacterium Bacteroides thetaiotaomicron uses the most structurally complex glycan known: the plant pectic polysaccharide rhamnogalacturonan-II, cleaving all but 1 of its 21 distinct glycosidic linkages. The deconstruction of rhamnogalacturonan-II side chains and backbone are coordinated to overcome steric constraints, and the degradation involves previously undiscovered enzyme families and catalytic activities. The degradation system informs revision of the current structural model of rhamnogalacturonan-II and highlights how individual gut bacteria orchestrate manifold enzymes to metabolize the most challenging glycan in the human diet.