ChemFOnt: the chemical functional ontology resource.

The Chemical Functional Ontology (ChemFOnt), located at https://www.chemfont.ca, is a hierarchical, OWL-compatible ontology describing the functions and actions of >341 000 biologically important chemicals. These include primary metabolites, secondary metabolites, natural products, food chemicals, synthetic food additives, drugs, herbicides, pesticides and environmental chemicals. ChemFOnt is a FAIR-compliant resource intended to bring the same rigor, standardization and formal structure to the terms and terminology used in biochemistry, food chemistry and environmental chemistry as the gene ontology (GO) has brought to molecular biology. ChemFOnt is available as both a freely accessible, web-enabled database and a downloadable Web Ontology Language (OWL) file. Users may download and deploy ChemFOnt within their own chemical databases or integrate ChemFOnt into their own analytical software to generate machine readable relationships that can be used to make new inferences, enrich their omics data sets or make new, non-obvious connections between chemicals and their direct or indirect effects. The web version of the ChemFOnt database has been designed to be easy to search, browse and navigate. Currently ChemFOnt contains data on 341 627 chemicals, including 515 332 terms or definitions. The functional hierarchy for ChemFOnt consists of four functional 'aspects', 12 functional super-categories and a total of 173 705 functional terms. In addition, each of the chemicals are classified into 4825 structure-based chemical classes. ChemFOnt currently contains 3.9 million protein-chemical relationships and ∼10.3 million chemical-functional relationships. The long-term goal for ChemFOnt is for it to be adopted by databases and software tools used by the general chemistry community as well as the metabolomics, exposomics, metagenomics, genomics and proteomics communities.

Unfermented ß-fructan Fibers Fuel Inflammation in Select Inflammatory Bowel Disease Patients.

BACKGROUND & AIMS: Inflammatory bowel diseases (IBD) are affected by dietary factors, including nondigestible carbohydrates (fibers), which are fermented by colonic microbes. Fibers are overall beneficial, but not all fibers are alike, and some patients with IBD report intolerance to fiber consumption. Given reproducible evidence of reduced fiber-fermenting microbes in patients with IBD, we hypothesized that fibers remain intact in select patients with reduced fiber-fermenting microbes and can then bind host cell receptors, subsequently promoting gut inflammation. METHODS: Colonic biopsies cultured ex vivo and cell lines in vitro were incubated with oligofructose (5 g/L), or fermentation supernatants (24-hour anaerobic fermentation) and immune responses (cytokine secretion [enzyme-linked immunosorbent assay/meso scale discovery] and expression [quantitative polymerase chain reaction]) were assessed. Influence of microbiota in mediating host response was examined and taxonomic classification of microbiota was conducted with Kraken2 and metabolic profiling by HUMAnN2, using R software. RESULTS: Unfermented dietary ß-fructan fibers induced proinflammatory cytokines in a subset of IBD intestinal biopsies cultured ex vivo, and immune cells (including peripheral blood mononuclear cells). Results were validated in an adult IBD randomized controlled trial examining ß-fructan supplementation. The proinflammatory response to intact ß-fructan required activation of the NLRP3 and TLR2 pathways. Fermentation of ß-fructans by human gut whole microbiota cultures reduced the proinflammatory response, but only when microbes were collected from patients without IBD or patients with inactive IBD. Fiber-induced immune responses correlated with microbe functions, luminal metabolites, and dietary fiber avoidance. CONCLUSION: Although fibers are typically beneficial in individuals with normal microbial fermentative potential, some dietary fibers have detrimental effects in select patients with active IBD who lack fermentative microbe activities. The study is publicly accessible at the U.S. National Institutes of Health database (clinicaltrials.gov identification number NCT02865707).

Neutrophils promote T-cell activation through the regulated release of CD44-bound Galectin-9 from the cell surface during HIV infection.

The interaction of neutrophils with T cells has been the subject of debate and controversies. Previous studies have suggested that neutrophils may suppress or activate T cells. Despite these studies, the interaction between neutrophils and T cells has remained a largely unexplored field. Here, based on our RNA sequencing (RNA-seq) analysis, we found that neutrophils have differential transcriptional and functional profiling depending on the CD4 T-cell count of the HIV-infected individual. In particular, we identified that neutrophils in healthy individuals express surface Galectin-9 (Gal-9), which is down-regulated upon activation, and is consistently down-regulated in HIV-infected individuals. However, down-regulation of Gal-9 was associated with CD4 T-cell count of patients. Unstimulated neutrophils express high levels of surface Gal-9 that is bound to CD44, and, upon stimulation, neutrophils depalmitoylate CD44 and induce its movement out of the lipid raft. This process causes the release of Gal-9 from the surface of neutrophils. In addition, we found that neutrophil-derived exogenous Gal-9 binds to cell surface CD44 on T cells, which promotes LCK activation and subsequently enhances T-cell activation. Furthermore, this process was regulated by glycolysis and can be inhibited by interleukin (IL)-10. Together, our data reveal a novel mechanism of Gal-9 shedding from the surface of neutrophils. This could explain elevated plasma Gal-9 levels in HIV-infected individuals as an underlying mechanism of the well-characterized chronic immune activation in HIV infection. This study provides a novel role for the Gal-9 shedding from neutrophils. We anticipate that our results will spark renewed investigation into the role of neutrophils in T-cell activation in other acute and chronic conditions, as well as improved strategies for modulating Gal-9 shedding.

HMDB 5.0: the Human Metabolome Database for 2022.

The Human Metabolome Database or HMDB (https://hmdb.ca) has been providing comprehensive reference information about human metabolites and their associated biological, physiological and chemical properties since 2007. Over the past 15 years, the HMDB has grown and evolved significantly to meet the needs of the metabolomics community and respond to continuing changes in internet and computing technology. This year's update, HMDB 5.0, brings a number of important improvements and upgrades to the database. These should make the HMDB more useful and more appealing to a larger cross-section of users. In particular, these improvements include: (i) a significant increase in the number of metabolite entries (from 114 100 to 217 920 compounds); (ii) enhancements to the quality and depth of metabolite descriptions; (iii) the addition of new structure, spectral and pathway visualization tools; (iv) the inclusion of many new and much more accurately predicted spectral data sets, including predicted NMR spectra, more accurately predicted MS spectra, predicted retention indices and predicted collision cross section data and (v) enhancements to the HMDB's search functions to facilitate better compound identification. Many other minor improvements and updates to the content, the interface, and general performance of the HMDB website have also been made. Overall, we believe these upgrades and updates should greatly enhance the HMDB's ease of use and its potential applications not only in human metabolomics but also in exposomics, lipidomics, nutritional science, biochemistry and clinical chemistry.

Chemotherapy-induced weight gain in early-stage breast cancer: a prospective matched cohort study reveals associations with inflammation and gut dysbiosis.

BACKGROUND: Early-stage breast cancer patients treated with chemotherapy risk the development of metabolic disease and weight gain, which can result in increased morbidity and reduced quality of life in survivorship. We aimed to analyze changes within the gastrointestinal microbiome of early-stage breast cancer patients treated with and without chemotherapy to investigate a potential relationship between dysbiosis, a systemic inflammatory response, and resultant anthropomorphic changes. METHODS: We undertook an a priori analysis of serially collected stool and plasma samples from 40 patients with early-stage breast cancer who underwent adjuvant endocrine therapy only, adjuvant chemotherapy only, or both. Gut microbiota were assessed by metagenomic comparison of stool samples following deep sequencing. Inflammatory biomarkers were evaluated by proteomic analysis of plasma and measurement of fecal calprotectin. Body composition was investigated by dual-energy X-ray absorptiometry to determine biomass indices. RESULTS: As opposed to treatment with endocrine therapy only, chemotherapy resulted in statistically and clinically significant weight gain and an increase in the android to gynoid ratio of fat distribution. Patients treated with chemotherapy gained an average of 0.15% total mass per month, as opposed to a significantly different loss of 0.19% in those patients who received endocrine-only therapy. Concurrently, a twofold increase in fecal calprotectin occurred after chemotherapy that is indicative of interferon-dependent inflammation and evidence of colonic inflammation. These anthropomorphic and inflammatory changes occurred in concert with a chemotherapy-dependent effect on the gut microbiome as evidenced by a reduction in both the abundance and variety of microbial species. CONCLUSIONS: We confirm the association of chemotherapy treatment with weight gain and potential deleterious anthropometric changes and suggest that alterations of bacterial flora may contribute to these phenomena through the induction of systemic inflammation. Consequently, the gut microbiome may be a future target for intervention in preventing chemotherapy-dependent anthropometric changes.

Transcriptional responses to Fusarium oxysporum f. sp. lycopersici (Sacc.) Snyder & Hansen infection in three Colombian tomato cultivars.

BACKGROUND: Fusarium oxysporum f. sp. lycopersici (Fol) is a compendium of pathogenic and non-pathogenic fungal strains. Pathogenic strains may cause vascular wilt disease and produce considerable losses in commercial tomato plots. To gain insight into the molecular mechanisms mediating resistance to Fol in tomato, the aim of our study was to characterize the transcriptional response of three cultivars (CT1, CT2 and IAC391) to a pathogenic (Fol-pt) and a non-pathogenic (Fo-npt) strain of Fo. RESULTS: All cultivars exhibited differentially expressed genes in response to each strain of the fungus at 36 h post-inoculation. For the pathogenic strain, CT1 deployed an apparent active defense response that included upregulation of WRKY transcription factors, an extracellular chitinase, and terpenoid-related genes, among others. In IAC391, differentially expressed genes included upregulated but mostly downregulated genes. Upregulated genes mapped to ethylene regulation, pathogenesis regulation and transcription regulation, while downregulated genes potentially impacted defense responses, lipid transport and metal ion binding. Finally, CT2 exhibited mostly downregulated genes upon Fol-pt infection. This included genes involved in transcription regulation, defense responses, and metal ion binding. CONCLUSIONS: Results suggest that CT1 mounts a defense response against Fol-pt. IAC391 exhibits an intermediate phenotype whereby some defense response genes are activated, and others are suppressed. Finally, the transcriptional profile in the CT2 hints towards lower levels of resistance. Fo-npt also induced transcriptional changes in all cultivars, but to a lesser extent. Results of this study will support genetic breeding programs currently underway in the zone.

CD71+VISTA+ erythroid cells promote the development and function of regulatory T cells through TGF-ß.

Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- ß, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-ß compared to CD71+VISTA- erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells-compared to CD71+VISTA- and CD71+ erythroid cells from the VISTA KO mice-significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-ß in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-ß1, TGF-ß2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond.

Lower methane emissions were associated with higher abundance of ruminal Prevotella in a cohort of Colombian buffalos.

BACKGROUND: Ruminants burp massive amounts of methane into the atmosphere and significantly contribute to the deposition of greenhouse gases and the consequent global warming. It is therefore urgent to devise strategies to mitigate ruminant's methane emissions to alleviate climate change. Ruminal methanogenesis is accomplished by a series of methanogen archaea in the phylum Euryarchaeota, which piggyback into carbohydrate fermentation by utilizing residual hydrogen to produce methane. Abundance of methanogens, therefore, is expected to affect methane production. Furthermore, availability of hydrogen produced by cellulolytic bacteria acting upstream of methanogens is a rate-limiting factor for methane production. The aim of our study was to identify microbes associated with the production of methane which would constitute the basis for the design of mitigation strategies. RESULTS: Moderate differences in the abundance of methanogens were observed between groups. In addition, we present three lines of evidence suggesting an apparent higher abundance of a consortium of Prevotella species in animals with lower methane emissions. First, taxonomic classification revealed increased abundance of at least 29 species of Prevotella. Second, metagenome assembly identified increased abundance of Prevotella ruminicola and another species of Prevotella. Third, metabolic profiling of predicted proteins uncovered 25 enzymes with homology to Prevotella proteins more abundant in the low methane emissions group. CONCLUSIONS: We propose that higher abundance of ruminal Prevotella increases the production of propionic acid and, in doing so, reduces the amount of hydrogen available for methanogenesis. However, further experimentation is required to ascertain the role of Prevotella on methane production and its potential to act as a methane production mitigator.

Fibroblast Growth Factor 2 Enhances Zika Virus Infection in Human Fetal Brain.

Zika virus (ZIKV) is an emerging pathogen that can cause microcephaly and other neurological defects in developing fetuses. The cellular response to ZIKV in the fetal brain is not well understood. Here, we show that ZIKV infection of human fetal astrocytes (HFAs), the most abundant cell type in the brain, results in elevated expression and secretion of fibroblast growth factor 2 (FGF2). This cytokine was shown to enhance replication and spread of ZIKV in HFAs and human fetal brain explants. The proviral effect of FGF2 is likely mediated in part by suppression of the interferon response, which would represent a novel mechanism by which viruses antagonize host antiviral defenses. We posit that FGF2-enhanced virus replication in the fetal brain contributes to the neurodevelopmental disorders associated with in utero ZIKV infection. As such, targeting FGF2-dependent signaling should be explored further as a strategy to limit replication of ZIKV.

Human pegivirus-1 associated leukoencephalitis: Clinical and molecular features.

Etiologic diagnosis is uncertain in 35% to 50% of patients with encephalitis, despite its substantial global prevalence and disease burden. We report on 2 adult female patients with fatal leukoencephalitis associated with human pegivirus-1 (HPgV-1) brain infection. Neuroimaging showed inflammatory changes in cerebral white matter. Brain-derived HPgV-1 RNA sequences clustered phylogenetically with other pegiviruses despite an 87-nucleotide deletion in the viral nonstructural (NS)2 gene. Neuropathology disclosed lymphocyte infiltration and gliosis predominantly in brain white matter. HPgV-1 NS5A antigen was detected in lymphocytes as well as in astrocytes and oligodendrocytes. HPgV-1 neuroadaptation should be considered in the differential diagnosis of progressive leukoencephalitis in humans. Ann Neurol 2018;84:789-795.

Complete nucleotide sequence of jasmine virus H, a new member of the family Tombusviridae.

Jasmine virus H (JaVH) is a novel virus associated with symptoms of yellow mosaic on jasmine. The JaVH genome is 3,867 nt in length with five open reading frames (ORFs) encoding a 27-kDa protein (ORF 1), an 87-kDa replicase protein (ORF 2), two centrally located movement proteins (ORF 3 and 4), and a 37-kDa capsid protein (ORF 5). Based on genomic and phylogenetic analysis, JaVH is predicted to be a member of the genus Pelarspovirus in the family Tombusviridae.

Effect of Oral Capsule- vs Colonoscopy-Delivered Fecal Microbiota Transplantation on Recurrent Clostridium difficile Infection: A Randomized Clinical Trial.

Importance: Fecal microbiota transplantation (FMT) is effective in preventing recurrent Clostridium difficile infection (RCDI). However, it is not known whether clinical efficacy differs by route of delivery. Objective: To determine whether FMT by oral capsule is noninferior to colonoscopy delivery in efficacy. Design, Setting, and Participants: Noninferiority, unblinded, randomized trial conducted in 3 academic centers in Alberta, Canada. A total of 116 adult patients with RCDI were enrolled between October 2014 and September 2016, with follow-up to December 2016. The noninferiority margin was 15%. Interventions: Participants were randomly assigned to FMT by capsule or by colonoscopy at a 1:1 ratio. Main Outcomes and Measures: The primary outcome was the proportion of patients without RCDI 12 weeks after FMT. Secondary outcomes included (1) serious and minor adverse events, (2) changes in quality of life by the 36-Item Short Form Survey on a scale of 0 (worst possible quality of life) to 100 (best quality of life), and (3) patient perception on a scale of 1 (not at all unpleasant) to 10 (extremely unpleasant) and satisfaction on a scale of 1 (best) to 10 (worst). Results: Among 116 patients randomized (mean [SD] age, 58 [19] years; 79 women [68%]), 105 (91%) completed the trial, with 57 patients randomized to the capsule group and 59 to the colonoscopy group. In per-protocol analysis, prevention of RCDI after a single treatment was achieved in 96.2% in both the capsule group (51/53) and the colonoscopy group (50/52) (difference, 0%; 1-sided 95% CI, -6.1% to infinity; P < .001), meeting the criterion for noninferiority. One patient in each group died of underlying cardiopulmonary illness unrelated to FMT. Rates of minor adverse events were 5.4% for the capsule group vs 12.5% for the colonoscopy group. There was no significant between-group difference in improvement in quality of life. A significantly greater proportion of participants receiving capsules rated their experience as "not at all unpleasant" (66% vs 44%; difference, 22% [95% CI, 3%-40%]; P = .01). Conclusions and Relevance: Among adults with RCDI, FMT via oral capsules was not inferior to delivery by colonoscopy for preventing recurrent infection over 12 weeks. Treatment with oral capsules may be an effective approach to treating RCDI. Trial Registration: clinicaltrials.gov Identifier: NCT02254811.

The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana.

Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.

PmrB Y358N, E123D amino acid substitutions are not associated with colistin resistance but with phylogeny in Escherichia coli.

Colistin resistance in Escherichia coli is of public health significance for its use to treat multidrug-resistant Gram-negative infections. Amino acid variations in PmrB have been implicated in colistin resistance in E. coli. In this cross-sectional study, 288 generic E. coli isolates from surveillance of broiler chicken and feedlot cattle feces, retail meat, wastewater, and well water were whole-genome sequenced. Phylogroup designation and screening for two amino acid substitutions in PmrB putatively linked to colistin resistance (Y358N, E123D) were performed in silico. Three additional data sets of publicly available E. coli assemblies were similarly scrutinized: (i) E. coli isolates from studies identifying the Y358N or E123D substitutions, (ii) colistin-susceptible E. coli isolates reported in the literature, and (iii) a random sampling of 14,700 E. coli assemblies available in the National Center for Biotechnology Information public database. Within all data sets, ≥95% of phylogroup B1 and C isolates have the PmrB Y358N variation. The PmrB E123D amino acid substitution was only identified in phylogroup B2 isolates, of which 94%-100% demonstrate the substitution. Both PmrB amino acid variations were infrequent in other phylogroups. Among published colistin susceptible isolates, colistin minimum inhibitory concentrations (MICs) were not higher in isolates bearing the E123D and Y358N amino acid variations than in isolates without these PmrB substitutions. The E123D and Y358N PmrB amino acid substitutions in E. coli appear strongly associated with phylogroup. The previously observed associations between Y358N and E123D amino acid substitutions in PmrB and colistin resistance in E. coli may be spurious. IMPORTANCE: Colistin is a critical last-resort treatment for extensively drug-resistant Gram-negative infections in humans. Therefore, accurate identification of the genetic mechanisms of resistance to this antimicrobial is crucial to effectively monitor and mitigate the spread of resistance. Examining over 16,000 whole-genome sequenced Escherichia coli isolates, this study identifies that PmrB E123D and Y358N amino acid substitutions previously associated with colistin resistance in E. coli are strongly associated with phylogroup and are alone not sufficient to confer a colistin-resistant phenotype. This is a critical clarification, as both substitutions are identified as putative mechanisms of colistin resistance in many publications and a common bioinformatic tool. Given the potential spurious nature of initial associations of these substitutions with colistin resistance, this study's findings emphasize the importance of appropriate experimental design and consideration of relevant biological factors such as phylogroup when ascribing causal mechanisms of resistance to chromosomal variations.

In Silico Analysis of Changes in Predicted Metabolic Capabilities of Intestinal Microbiota after Fecal Microbial Transplantation for Treatment of Recurrent Clostridioides difficile Infection.

IMPORTANCE: Although highly effective in treating recurrent Clostridioides difficile infection (RCDI), the mechanisms of action of fecal microbial transplantation (FMT) are not fully understood. AIM: The aim of this study was to explore microbially derived products or pathways that could contribute to the therapeutic efficacy of FMT. METHODS: Stool shotgun metagenomic sequencing data from 18 FMT-treated RCDI patients at 4 points in time were used for the taxonomic and functional profiling of their gut microbiome. The abundance of the KEGG orthology (KO) groups was subjected to univariate linear mixed models to assess the significance of the observed differences between 0 (pre-FMT), 1, 4, and 12 weeks after FMT. RESULTS: Of the 59,987 KO groups identified by shotgun metagenomic sequencing, 27 demonstrated a statistically significant change after FMT. These KO groups are involved in many cellular processes, including iron homeostasis, glycerol metabolism, and arginine regulation, all of which have been implicated to play important roles in bacterial growth and virulence in addition to modulating the intestinal microbial composition. CONCLUSION: Our findings suggest potential changes in key KO groups post-FMT, which may contribute to FMT efficacy beyond the restored microbial composition/diversity and metabolism of bile acids and short-chain fatty acids. Future larger studies that include a fecal metabolomics analysis combined with animal model validation work are required to further elucidate the molecular mechanisms.

Agarose/crystalline nanocellulose (CNC) composites promote bone marrow-derived mast cell integrity, degranulation and receptor expression but inhibit production of de novo synthesized mediators.

Introduction: Mast cells are highly granulated tissue-resident leukocytes that require a three-dimensional matrix to differentiate and mediate immune responses. However, almost all cultured mast cells rely on two-dimensional suspension or adherent cell culture systems, which do not adequately reflect the complex structure that these cells require for optimal function. Methods: Crystalline nanocellulose (CNC), consisting of rod-like crystals 4-15 nm in diameter and 0.2-1 µm in length, were dispersed in an agarose matrix (12.5% w/v), and bone marrow derived mouse mast cells (BMMC) were cultured on the agarose/CNC composite. BMMC were activated with the calcium ionophore A23187 or immunoglobulin E (IgE) and antigen (Ag) to crosslink high affinity IgE receptors (FcεRI). Results: BMMC cultured on a CNC/agarose matrix remained viable and metabolically active as measured by reduction of sodium 3'-[1-[(phenylamino)-carbony]-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT), and the cells maintained their membrane integrity as analyzed by measuring the release of lactate dehydrogenase (LDH) and propidium iodide exclusion by flow cytometry. Culture on CNC/agarose matrix had no effect on BMMC degranulation in response to IgE/Ag or A23187. However, culture of BMMC on a CNC/agarose matrix inhibited A23187-and IgE/Ag-activated production of tumor necrosis factor (TNF) and other mediators such as IL-1ß, IL-4, IL-6, IL-13, MCP-1/CCL2, MMP-9 and RANTES by as much as 95%. RNAseq analysis indicated that BMMC expressed a unique and balanced transcriptome when cultured on CNC/agarose. Discussion: These data demonstrate that culture of BMMCs on a CNC/agarose matrix promotes cell integrity, maintains expression of surface biomarkers such as FcεRI and KIT and preserves the ability of BMMC to release pre-stored mediators in response to IgE/Ag and A23187. However, culture of BMMC on CNC/agarose matrix inhibits BMMC production of de novo synthesized mediators, suggesting that CNC may be altering specific phenotypic characteristics of these cells that are associated with late phase inflammatory responses.

RNA-based immunity terminates viral infection in adult Drosophila in the absence of viral suppression of RNA interference: characterization of viral small interfering RNA populations in wild-type and mutant flies.

Replication of viral RNA genomes in fruit flies and mosquitoes induces the production of virus-derived small interfering RNAs (siRNAs) to specifically reduce virus accumulation by RNA interference (RNAi). However, it is unknown whether the RNA-based antiviral immunity (RVI) is sufficiently potent to terminate infection in adult insects as occurs in cell culture. We show here that, in contrast to robust infection by Flock house virus (FHV), infection with an FHV mutant (FHVΔB2) unable to express its RNAi suppressor protein B2 was rapidly terminated in adult flies. FHVΔB2 replicated to high levels and induced high mortality rates in dicer-2 and argonaute-2 mutant flies that are RNAi defective, demonstrating that successful infection of adult Drosophila requires a virus-encoded activity to suppress RVI. Drosophila RVI may depend on the RNAi activity of viral siRNAs since efficient FHVΔB2 infection occurred in argonaute-2 and r2d2 mutant flies despite massive production of viral siRNAs. However, RVI appears to be insensitive to the relative abundance of viral siRNAs since FHVΔB2 infection was terminated in flies carrying a partial loss-of-function mutation in loquacious required for viral siRNA biogenesis. Deep sequencing revealed a low-abundance population of Dicer-2-dependent viral siRNAs accompanying FHVΔB2 infection arrest in RVI-competent flies that included an approximately equal ratio of positive and negative strands. Surprisingly, viral small RNAs became strongly biased for positive strands at later stages of infection in RVI-compromised flies due to genetic or viral suppression of RNAi. We propose that degradation of the asymmetrically produced viral positive-strand RNAs associated with abundant virus accumulation contributes to the positive-strand bias of viral small RNAs.

Prevotella: A Key Player in Ruminal Metabolism.

Ruminants are foregut fermenters that have the remarkable ability of converting plant polymers that are indigestible to humans into assimilable comestibles like meat and milk, which are cornerstones of human nutrition. Ruminants establish a symbiotic relationship with their microbiome, and the latter is the workhorse of carbohydrate fermentation. On the other hand, during carbohydrate fermentation, synthesis of propionate sequesters H, thus reducing its availability for the ultimate production of methane (CH4) by methanogenic archaea. Biochemically, methane is the simplest alkane and represents a downturn in energetic efficiency in ruminants; environmentally, it constitutes a potent greenhouse gas that negatively affects climate change. Prevotella is a very versatile microbe capable of processing a wide range of proteins and polysaccharides, and one of its fermentation products is propionate, a trait that appears conspicuous in P. ruminicola strain 23. Since propionate, but not acetate or butyrate, constitutes an H sink, propionate-producing microbes have the potential to reduce methane production. Accordingly, numerous studies suggest that members of the genus Prevotella have the ability to divert the hydrogen flow in glycolysis away from methanogenesis and in favor of propionic acid production. Intended for a broad audience in microbiology, our review summarizes the biochemistry of carbohydrate fermentation and subsequently discusses the evidence supporting the essential role of Prevotella in lignocellulose processing and its association with reduced methane emissions. We hope this article will serve as an introduction to novice Prevotella researchers and as an update to others more conversant with the topic.

Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences.

CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.

The effect of fecal microbial transplant on intestinal microbial composition in short-bowel neonatal piglets.

BACKGROUND: Short-bowel syndrome (SBS) in neonates is associated with microbial dysbiosis due to intestinal surgery, prolonged hospitalization, enteral nutrition, and repeated antibiotic exposure. Sepsis and liver disease, leading causes of morbidity and mortality in SBS, may relate to such intestinal dysbiosis. We investigated the safety and feasibility of fecal microbial transplant (FMT) to alter intestinal microbial composition in SBS piglets. METHODS: Following a 75% distal small-intestinal resection, piglets were fed parenteral nutrition with an elemental diet and randomized to saline (SAL; n = 12) or FMT (n = 12) treatments delivered by gastric tube on day 2 (d2). The FMT donor was a healthy adult pig. Comparisons were also made to healthy sow-fed littermate controls (SOW; n = 6). Stool samples were collected daily, and tissue samples were collected at baseline and termination. Microbial DNA was extracted from stool and analyzed using 16S ribosomal RNA sequencing. RESULTS: All piglets survived to the end point. On d2-d4, FMT piglets had some differences in microbiota composition compared with SAL, SOW, and donor counterparts. Between base and term, there were transitory changes to alpha and beta diversity in FMT and SAL. CONCLUSION: FMT treatment in postsurgical neonatal piglets with SBS appears safe, with no increase in sepsis and no mortality. In SBS piglets, FMT induced transient changes to the intestinal microbiota. However, these changes did not persist long-term.