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A mysterious feature of Crohn's disease (CD) is the extra-intestinal manifestation of "creeping fat" (CrF), defined as expansion of mesenteric adipose tissue around the inflamed and fibrotic intestine. In the current study, we explore whether microbial translocation in CD serves as a central cue for CrF development. We discovered a subset of mucosal-associated gut bacteria that consistently translocated and remained viable in CrF in CD ileal surgical resections, and identified Clostridium innocuum as a signature of this consortium with strain variation between mucosal and adipose isolates, suggesting preference for lipid-rich environments. Single-cell RNA sequencing characterized CrF as both pro-fibrotic and pro-adipogenic with a rich milieu of activated immune cells responding to microbial stimuli, which we confirm in gnotobiotic mice colonized with C. innocuum. Ex vivo validation of expression patterns suggests C. innocuum stimulates tissue remodeling via M2 macrophages, leading to an adipose tissue barrier that serves to prevent systemic dissemination of bacteria.

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).

Microbes and their metabolic products influence early-life immune and microbiome development, yet remain understudied during pregnancy. Vaginal microbial communities are typically dominated by one or a few well-adapted microbes which are able to survive in a narrow pH range and are adapted to live on host-derived carbon sources, likely sourced from glycogen and mucin present in the vaginal environment. We characterized the cervicovaginal microbiomes of 16 healthy women throughout the three trimesters of pregnancy. Additionally, we analyzed saliva and urine metabolomes using gas chromatography-time of flight mass spectrometry (GC-TOF MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) lipidomics approaches for samples from mothers and their infants through the first year of life. Amplicon sequencing revealed most women had either a simple community with one highly abundant species of Lactobacillus or a more diverse community characterized by a high abundance of Gardnerella, as has also been previously described in several independent cohorts. Integrating GC-TOF MS and lipidomics data with amplicon sequencing, we found metabolites that distinctly associate with particular communities. For example, cervicovaginal microbial communities dominated by Lactobacillus crispatus have high mannitol levels, which is unexpected given the characterization of L. crispatus as a homofermentative Lactobacillus species. It may be that fluctuations in which Lactobacillus dominate a particular vaginal microbiome are dictated by the availability of host sugars, such as fructose, which is the most likely substrate being converted to mannitol. Overall, using a multi-"omic" approach, we begin to address the genetic and molecular means by which a particular vaginal microbiome becomes vulnerable to large changes in composition.IMPORTANCE Humans have a unique vaginal microbiome compared to other mammals, characterized by low diversity and often dominated by Lactobacillus spp. Dramatic shifts in vaginal microbial communities sometimes contribute to the presence of a polymicrobial overgrowth condition called bacterial vaginosis (BV). However, many healthy women lacking BV symptoms have vaginal microbiomes dominated by microbes associated with BV, resulting in debate about the definition of a healthy vaginal microbiome. Despite substantial evidence that the reproductive health of a woman depends on the vaginal microbiota, future therapies that may improve reproductive health outcomes are stalled due to limited understanding surrounding the ecology of the vaginal microbiome. Here, we use sequencing and metabolomic techniques to show novel associations between vaginal microbes and metabolites during healthy pregnancy. We speculate these associations underlie microbiome dynamics and may contribute to a better understanding of transitions between alternative vaginal microbiome compositions.

Inhibition of neuronal nitric oxide synthase (nNOS), an enzyme implicated in neurodegenerative disorders, is an attractive strategy for treating or preventing these diseases. We previously developed several classes of 2-aminoquinoline-based nNOS inhibitors, but these compounds had drawbacks including off-target promiscuity, low activity against human nNOS, and only modest selectivity for nNOS over related enzymes. In this study, we synthesized new nNOS inhibitors based on 7-phenyl-2-aminoquinoline and assayed them against rat and human nNOS, human eNOS, and murine and (in some cases) human iNOS. Compounds with a meta-relationship between the aminoquinoline and a positively charged tail moiety were potent and had up to nearly 900-fold selectivity for human nNOS over human eNOS. X-ray crystallography indicates that the amino groups of some compounds occupy a water-filled pocket surrounding an nNOS-specific aspartate residue (absent in eNOS). This interaction was confirmed by mutagenesis studies, making 7-phenyl-2-aminoquinolines the first aminoquinolines to interact with this residue.

Type 2 diabetes mellitus (T2DM) is a systemic disease, predisposing patients to other inflammatory conditions including periodontitis. The subgingival microbiome, a key player in periodontitis pathogenesis, is not well characterized in T2DM population. To better understand whether the subgingival microbiome is different between T2DM and systemically healthy, nondiabetic (ND) subjects, we performed a longitudinal analysis of the subgingival microbiome in T2DM patients (n = 15) compared with ND subjects (n = 16). Using metagenomic shotgun sequencing, we investigated the microbiome in the healthy periodontal state, periodontitis state, and resolved state after treatment. We found that in the periodontitis state, the shift in the subgingival microbiome from the healthy state was less prominent in T2DM compared with ND subjects, yet the clinical signs of disease were similar for both. Furthermore, we revealed highly correlated presence of pathogenic species in relative abundance not only in the periodontitis state, but also in the healthy state in T2DM, suggesting an elevated risk of progression to periodontitis in this cohort. We further investigated the functional potentials of the subgingival microbiome and identified a set of microbial marker genes associated with the clinical states. These genes were significantly enriched in 21 pathways, some of which are associated with periodontitis and some potentially link T2DM and periodontitis. This study identified the longitudinal changes of the subgingival microbiome associated with periodontitis in T2DM and suggests that T2DM patients are more susceptible to shifts in the subgingival microbiome toward dysbiosis, potentially due to impaired host metabolic and immune regulation.

Abstract Background Microbes and their metabolic products influence early-life immune and microbiome development, yet remain understudied during pregnancy. Vaginal microbial communities are typically dominated by one or a few well adapted microbes, which are able to survive in a narrow pH range. In comparison to other human-associated microbes, vaginal microbes are adapted to live on host-derived carbon sources, likely sourced from glycogen and mucin present in the vaginal environment. Methods Using 16S rRNA and ITS amplicon sequencing, we characterized the cervicovaginal microbiomes of 18 healthy women throughout the three trimesters of pregnancy. Shotgun metagenomic sequencing permitted refinement of the taxonomy established by amplicon sequencing, and identification of functional genes. Additionally, we analyzed saliva and urine metabolomes using GC-TOF and LC-MS/MS lipidomics approaches for samples from mothers and their infants through the first year of life. Results Amplicon sequencing revealed most women had either a simple community with one highly abundant species of Lactobacillus or a more diverse community characterized by a high abundance of Gardnerella , as has also been previously described in several independent cohorts. Integrating GC-TOF and lipidomics data with amplicon sequencing, we found metabolites that distinctly associate with particular communities. For example, cervicovaginal microbial communities dominated by Lactobacillus crispatus also have high mannitol levels, which contradicts the basic characterization of L. crispatus as a homofermentative Lactobacillus species. It may be that fluctuations in which Lactobacillus dominate a particular vaginal microbiome are dictated by the availability of host sugars, such as fructose, which is the most likely substrate being converted to mannitol. Furthermore, indole-3-lactate (ILA) was also indicative of L. crispatus specifically. ILA has immunomodulatory properties through binding the human aryl hydrocarbon receptor (AhR), which may maintain the especially low diversity of L. crispatus dominated communities. Conclusions Overall, using a multi-‘omic approach, we begin to address the genetic and molecular means by which a particular vaginal microbiome becomes vulnerable to large changes in composition.