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[alpha]-heteroaryl acetaldehydes have become important building blocks in the synthesis of synthetic nucleosides. Novel organocatalytic cascades have enabled the rapid generation of nucleosides, which are valuable building blocks in the development of antisense oligonucleotides or as stand-alone antiviral and anticancer therapies, obviating the need for laborious synthetic routes relying on chiral pool starting materials and inefficient synthetic routes. This manuscript describes a robust and scalable protocol to [alpha]-heteroaryl acetaldehydes from readily available building blocks. Key words: nucleoside, heterocycles, Nucleoside analogs are valuable building blocks in the development of antisense oligonucleotides or as stand-alone antiviral and anticancer therapies. However, their syntheses can be laborious owing to a reliance on [...]

Combatting Clostridioides difficile infections, a dominant cause of hospital-associated infections with incidence and resulting deaths increasing worldwide, is complicated by the frequent emergence of new virulent strains. Here, we employ whole-genome sequencing, high-throughput phenotypic screenings, and genome-scale models of metabolism to evaluate the genetic diversity of 451 strains of C. difficile. Constructing the C. difficile pangenome based on this set revealed 9,924 distinct gene clusters, of which 2,899 (29%) are defined as core, 2,968 (30%) are defined as unique, and the remaining 4,057 (41%) are defined as accessory. We develop a strain typing method, sequence typing by accessory genome (STAG), that identifies 176 genetically distinct groups of strains and allows for explicit interrogation of accessory gene content. Thirty-five strains representative of the overall set were experimentally profiled on 95 different nutrient sources, revealing 26 distinct growth profiles and unique nutrient preferences; 451 strain-specific genome scale models of metabolism were constructed, allowing us to computationally probe phenotypic diversity in 28,864 unique conditions. The models create a mechanistic link between the observed phenotypes and strain-specific genetic differences and exhibit an ability to correctly predict growth in 76% of measured cases. The typing and model predictions are used to identify and contextualize discriminating genetic features and phenotypes that may contribute to the emergence of new problematic strains.

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

Background & aimsAlthough Clostridioides difficile infection (CDI) is known to involve the disruption of the gut microbiota, little is understood regarding how mucus-associated microbes interact with C difficile. We hypothesized that select mucus-associated bacteria would promote C difficile colonization and biofilm formation.MethodsTo create a model of the human intestinal mucus layer and gut microbiota, we used bioreactors inoculated with healthy human feces, treated with clindamycin and infected with C difficile with the addition of human MUC2-coated coverslips.ResultsC difficile was found to colonize and form biofilms on MUC2-coated coverslips, and 16S rRNA sequencing showed a unique biofilm profile with substantial cocolonization with Fusobacterium species. Consistent with our bioreactor data, publicly available data sets and patient stool samples showed that a subset of patients with C difficile infection harbored high levels of Fusobacterium species. We observed colocalization of C difficile and F nucleatum in an aggregation assay using adult patients and stool of pediatric patients with inflammatory bowel disease and in tissue sections of patients with CDI. C difficile strains were found to coaggregate with F nucleatum subspecies in vitro; an effect that was inhibited by blocking or mutating the adhesin RadD on Fusobacterium and removal of flagella on C difficile. Aggregation was shown to be unique between F nucleatum and C difficile, because other gut commensals did not aggregate with C difficile. Addition of F nucleatum also enhanced C difficile biofilm formation and extracellular polysaccharide production.ConclusionsCollectively, these data show a unique interaction of between pathogenic C difficile and F nucleatum in the intestinal mucus layer.

Cronkhite-Canada Syndrome (CCS) is a rare, noninherited polyposis syndrome affecting 1 in every million individuals. Despite over 50 years of CCS cases, the etiopathogenesis and optimal treatment for CCS remains unknown due to the rarity of the disease and lack of model systems. To better understand the etiology of CCS, we generated human intestinal organoids (HIOs) from intestinal stem cells isolated from 2 patients. We discovered that CCS HIOs are highly proliferative and have increased numbers of enteroendocrine cells producing serotonin (also known as 5-hydroxytryptamine or 5HT). These features were also confirmed in patient tissue biopsies. Recombinant 5HT increased proliferation of non-CCS donor HIOs and inhibition of 5HT production in the CCS HIOs resulted in decreased proliferation, suggesting a link between local epithelial 5HT production and control of epithelial stem cell proliferation. This link was confirmed in genetically engineered HIOs with an increased number of enteroendocrine cells. This work provides a new mechanism to explain the pathogenesis of CCS and illustrates the important contribution of HIO cultures to understanding disease etiology and in the identification of novel therapies. Our work demonstrates the principle of using organoids for personalized medicine and sheds light on how intestinal hormones can play a role in intestinal epithelial proliferation., Introduction In 1955, Leonard Cronkhite and Wilma Canada documented a rare, nonhereditary disease associated with nonadenomatous cystic polyps in the gastrointestinal (GI) tract, ectodermal abnormalities of alopecia, nail dystrophy, and [...]

Hospital acquired Clostridioides (Clostridium) difficile infection is exacerbated by the continued evolution of C. difficile strains, a phenomenon studied by multiple laboratories using stock cultures specific to each laboratory. Intralaboratory evolution of strains contributes to interlaboratory variation in experimental results adding to the challenges of scientific rigor and reproducibility. To explore how microevolution of C. difficile within laboratories influences the metabolic capacity of an organism, three different laboratory stock isolates of the C. difficile 630 reference strain were whole-genome sequenced and profiled in over 180 nutrient environments using phenotypic microarrays. The results identified differences in growth dynamics for 32 carbon sources including trehalose, fructose, and mannose. An updated genome-scale model for C. difficile 630 was constructed and used to contextualize the 28 unique mutations observed between the stock cultures. The integration of phenotypic screens with model predictions identified pathways enabling catabolism of ethanolamine, salicin, arbutin, and N-acetyl-galactosamine that differentiated individual C. difficile 630 laboratory isolates. The reconstruction was used as a framework to analyze the core-genome of 415 publicly available C. difficile genomes and identify areas of metabolism prone to evolution within the species. Genes encoding enzymes and transporters involved in starch metabolism and iron acquisition were more variable while C. difficile distinct metabolic functions like Stickland fermentation were more consistent. A substitution in the trehalose PTS system was identified with potential implications in strain virulence. Thus, pairing genome-scale models with large-scale physiological and genomic data enables a mechanistic framework for studying the evolution of pathogens within microenvironments and will lead to predictive modeling to combat pathogen emergence.

The National Institutes of Health (NIH) organized a three-day human microbiome research workshop, August 16-18, 2017, to highlight the accomplishments of the 10-year Human Microbiome Project program, the outcomes of the investments made by the 21 NIH Institutes and Centers which now fund this area, and the technical challenges and knowledge gaps which will need to be addressed in order for this field to advance over the next 10 years. This report summarizes the key points in the talks, round table discussions, and Joint Agency Panel from this workshop.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24–48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease. [ABSTRACT FROM AUTHOR]

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe β-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. β-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. β-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that β-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that β-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes. [ABSTRACT FROM AUTHOR]

DNA encoded library (DEL) synthesis represents a convenient means to produce, annotate and store large collections of compounds in a small volume. While DELs are well suited for drug discovery campaigns, the chemistry used in their production must be compatible with the DNA tag, which can limit compound class accessibility. As a result, most DELs are heavily populated with peptidomimetic and sp2‐rich molecules. Herein, we show that sp3‐rich mono‐ and bicyclic heterocycles can be made on DNA from ketochlorohydrin aldol products through a reductive amination and cyclization process. The resulting hydroxypyrrolidines possess structural features that are desirable for DELs and target a distinct region of pharmaceutically relevant chemical space. [ABSTRACT FROM AUTHOR]

Herein we report the automation and scale-up of a photofluorination process key to the production of branched-chain aliphatic radiotracers such as (S)-5-[18F]fluorohomoleucine ((S)-5-[18F]]FHL). (S)-5-[18F]FHL is a leucine analogue that is primarily taken up by the L-type amino acid transporter (LAT or System L). LAT1 expression levels correlate closely with tumor proliferation, angiogenesis, and treatment outcomes, making it an attractive target for molecular imaging of cancer. We have previously synthesized (S)-5-[18F]FHL and tested this tracer in mice bearing PC3 (prostate) or U87 (glioma) xenografts in order to establish its feasibility for detecting and monitoring treatment for a broad range of cancers. In this study, the radiosynthesis of 5-[18F]FHL is demonstrated on an automated DT-PhotoFluor module with a radiochemical yield of 20.1 ± 4.8% (n = 3), radiochemical purity of 94.5 ± 4.9% (n = 3), and a synthesis time of ~ 75 min. The reported DT-PhotoFluor module will allow for higher molar activity, better reproducibility, and reduced radiation exposure for upcoming first-in-human studies. [ABSTRACT FROM AUTHOR]