IntS6 and the Integrator phosphatase module tune the efficiency of select premature transcription termination events.

The metazoan-specific Integrator complex catalyzes 3' end processing of small nuclear RNAs (snRNAs) and premature termination that attenuates the transcription of many protein-coding genes. Integrator has RNA endonuclease and protein phosphatase activities, but it remains unclear if both are required for complex function. Here, we show IntS6 (Integrator subunit 6) over-expression blocks Integrator function at a subset of Drosophila protein-coding genes, although having no effect on snRNAs or attenuation of other loci. Over-expressed IntS6 titrates protein phosphatase 2A (PP2A) subunits, thereby only affecting gene loci where phosphatase activity is necessary for Integrator function. IntS6 functions analogous to a PP2A regulatory B subunit as over-expression of canonical B subunits, which do not bind Integrator, is also sufficient to inhibit Integrator activity. These results show that the phosphatase module is critical at only a subset of Integrator-regulated genes and point to PP2A recruitment as a tunable step that modulates transcription termination efficiency.

A Gram-negative-selective antibiotic that spares the gut microbiome.

Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.

Widespread microRNA degradation elements in target mRNAs can assist the encoded proteins.

Binding of microRNAs (miRNAs) to mRNAs normally results in post-transcriptional repression of gene expression. However, extensive base-pairing between miRNAs and target RNAs can trigger miRNA degradation, a phenomenon called target RNA-directed miRNA degradation (TDMD). Here, we systematically analyzed Argonaute-CLASH (cross-linking, ligation, and sequencing of miRNA-target RNA hybrids) data and identified numerous candidate TDMD triggers, focusing on their ability to induce nontemplated nucleotide addition at the miRNA 3' end. When exogenously expressed in various cell lines, eight triggers induce degradation of corresponding miRNAs. Both the TDMD base-pairing and surrounding sequences are essential for TDMD. CRISPR knockout of endogenous trigger or ZSWIM8, a ubiquitin ligase essential for TDMD, reduced miRNA degradation. Furthermore, degradation of miR-221 and miR-222 by a trigger in BCL2L11, which encodes a proapoptotic protein, enhances apoptosis. Therefore, we uncovered widespread TDMD triggers in target RNAs and demonstrated an example that could functionally cooperate with the encoded protein.

Within-host evolutionary dynamics and tissue compartmentalization during acute SARS-CoV-2 infection.

The global evolution of SARS-CoV-2 depends in part upon the evolutionary dynamics within individual hosts with varying immune histories. To characterize the within-host evolution of acute SARS-CoV-2 infection, we sequenced saliva and nasal samples collected daily from vaccinated and unvaccinated individuals early during infection. We show that longitudinal sampling facilitates high-confidence genetic variant detection and reveals evolutionary dynamics missed by less-frequent sampling strategies. Within-host dynamics in both unvaccinated and vaccinated individuals appeared largely stochastic; however, in rare cases, minor genetic variants emerged to frequencies sufficient for forward transmission. Finally, we detected significant genetic compartmentalization of viral variants between saliva and nasal swab sample sites in many individuals. Altogether, these data provide a high-resolution profile of within-host SARS-CoV-2 evolutionary dynamics.IMPORTANCEWe detail the within-host evolutionary dynamics of SARS-CoV-2 during acute infection in 31 individuals using daily longitudinal sampling. We characterized patterns of mutational accumulation for unvaccinated and vaccinated individuals, and observed that temporal variant dynamics in both groups were largely stochastic. Comparison of paired nasal and saliva samples also revealed significant genetic compartmentalization between tissue environments in multiple individuals. Our results demonstrate how selection, genetic drift, and spatial compartmentalization all play important roles in shaping the within-host evolution of SARS-CoV-2 populations during acute infection.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

A noncanonical microRNA derived from the snaR-A noncoding RNA targets a metastasis inhibitor.

MicroRNAs (miRNAs) are small noncoding RNAs that function as critical posttranscriptional regulators in various biological processes. While most miRNAs are generated from processing of long primary transcripts via sequential Drosha and Dicer cleavage, some miRNAs that bypass Drosha cleavage can be transcribed as part of another small noncoding RNA. Here, we develop the target-oriented miRNA discovery (TOMiD) bioinformatic analysis method to identify Drosha-independent miRNAs from Argonaute crosslinking and sequencing of hybrids (Ago-CLASH) data sets. Using this technique, we discovered a novel miRNA derived from a primate specific noncoding RNA, the small NF90 associated RNA A (snaR-A). The miRNA derived from snaR-A (miR-snaR) arises independently of Drosha processing but requires Exportin-5 and Dicer for biogenesis. We identify that miR-snaR is concurrently up-regulated with the full snaR-A transcript in cancer cells. Functionally, miR-snaR associates with Ago proteins and targets NME1, a key metastasis inhibitor, contributing to snaR-A's role in promoting cancer cell migration. Our findings suggest a functional link between a novel miRNA and its precursor noncoding RNA.

Effect of prolonged sedation with dexmedetomidine, midazolam, propofol, and sevoflurane on sleep homeostasis in rats.

BACKGROUND: Sleep disruption is a common occurrence during medical care and is detrimental to patient recovery. Long-term sedation in the critical care setting is a modifiable factor that affects sleep, but the impact of different sedative-hypnotics on sleep homeostasis is not clear. METHODS: We conducted a systematic comparison of the effects of prolonged sedation (8 h) with i.v. and inhalational agents on sleep homeostasis. Adult Sprague-Dawley rats (n=10) received dexmedetomidine or midazolam on separate days. Another group (n=9) received propofol or sevoflurane on separate days. A third group (n=12) received coadministration of dexmedetomidine and sevoflurane. Wakefulness (wake), slow-wave sleep (SWS), and rapid eye movement (REM) sleep were quantified during the 48-h post-sedation period, during which we also assessed wake-associated neural dynamics using two electroencephalographic measures: theta-high gamma phase-amplitude coupling and high gamma weighted phase-lag index. RESULTS: Dexmedetomidine-, midazolam-, or propofol-induced sedation increased wake and decreased SWS and REM sleep (P<0.0001) during the 48-h post-sedation period. Sevoflurane produced no change in SWS, decreased wake for 3 h, and increased REM sleep for 6 h (P<0.02) post-sedation. Coadministration of dexmedetomidine and sevoflurane induced no change in wake (P>0.05), increased SWS for 3 h, and decreased REM sleep for 9 h (P<0.02) post-sedation. Dexmedetomidine, midazolam, and coadministration of dexmedetomidine with sevoflurane reduced wake-associated phase-amplitude coupling (P≤0.01). All sedatives except sevoflurane decreased wake-associated high gamma weighted phase-lag index (P<0.01). CONCLUSIONS: In contrast to i.v. drugs, prolonged sevoflurane sedation produced minimal changes in sleep homeostasis and neural dynamics. Further studies are warranted to assess inhalational agents for long-term sedation and sleep homeostasis.

First Draft Genome Assembly of Root-Lesion Nematode Pratylenchus scribneri Generated Using Long-Read Sequencing.

Root-lesion nematodes (genus Pratylenchus) belong to a diverse group of plant-parasitic nematodes (PPN) with a worldwide distribution. Despite being an economically important PPN group of more than 100 species, genome information related to Pratylenchus genus is scarcely available. Here, we report the draft genome assembly of Pratylenchus scribneri generated on the PacBio Sequel IIe System using the ultra-low DNA input HiFi sequencing workflow. The final assembly created using 500 nematodes consisted of 276 decontaminated contigs, with an average contig N50 of 1.72 Mb and an assembled draft genome size of 227.24 Mb consisting of 51,146 predicted protein sequences. The benchmarking universal single-copy ortholog (BUSCO) analysis with 3131 nematode BUSCO groups indicated that 65.4% of the BUSCOs were complete, whereas 24.0%, 41.4%, and 1.8% were single-copy, duplicated, and fragmented, respectively, and 32.8% were missing. The outputs from GenomeScope2 and Smudgeplots converged towards a diploid genome for P. scribneri. The data provided here will facilitate future studies on host plant-nematode interactions and crop protection at the molecular level.

H3AGWAS: a portable workflow for genome wide association studies.

BACKGROUND: Genome-wide association studies (GWAS) are a powerful method to detect associations between variants and phenotypes. A GWAS requires several complex computations with large data sets, and many steps may need to be repeated with varying parameters. Manual running of these analyses can be tedious, error-prone and hard to reproduce. RESULTS: The H3AGWAS workflow from the Pan-African Bioinformatics Network for H3Africa is a powerful, scalable and portable workflow implementing pre-association analysis, implementation of various association testing methods and post-association analysis of results. CONCLUSIONS: The workflow is scalable-laptop to cluster to cloud (e.g., SLURM, AWS Batch, Azure). All required software is containerised and can run under Docker or Singularity.

Using a multiple-delivery-mode training approach to develop local capacity and infrastructure for advanced bioinformatics in Africa.

With more microbiome studies being conducted by African-based research groups, there is an increasing demand for knowledge and skills in the design and analysis of microbiome studies and data. However, high-quality bioinformatics courses are often impeded by differences in computational environments, complicated software stacks, numerous dependencies, and versions of bioinformatics tools along with a lack of local computational infrastructure and expertise. To address this, H3ABioNet developed a 16S rRNA Microbiome Intermediate Bioinformatics Training course, extending its remote classroom model. The course was developed alongside experienced microbiome researchers, bioinformaticians, and systems administrators, who identified key topics to address. Development of containerised workflows has previously been undertaken by H3ABioNet, and Singularity containers were used here to enable the deployment of a standard replicable software stack across different hosting sites. The pilot ran successfully in 2019 across 23 sites registered in 11 African countries, with more than 200 participants formally enrolled and 106 volunteer staff for onsite support. The pulling, running, and testing of the containers, software, and analyses on various clusters were performed prior to the start of the course by hosting classrooms. The containers allowed the replication of analyses and results across all participating classrooms running a cluster and remained available posttraining ensuring analyses could be repeated on real data. Participants thus received the opportunity to analyse their own data, while local staff were trained and supported by experienced experts, increasing local capacity for ongoing research support. This provides a model for delivering topic-specific bioinformatics courses across Africa and other remote/low-resourced regions which overcomes barriers such as inadequate infrastructures, geographical distance, and access to expertise and educational materials.

Cortical Acetylcholine Levels Correlate With Neurophysiologic Complexity During Subanesthetic Ketamine and Nitrous Oxide Exposure in Rats.

BACKGROUND: Neurophysiologic complexity has been shown to decrease during states characterized by a depressed level of consciousness, such as sleep or anesthesia. Conversely, neurophysiologic complexity is increased during exposure to serotonergic psychedelics or subanesthetic doses of dissociative anesthetics. However, the neurochemical substrates underlying changes in neurophysiologic complexity are poorly characterized. Cortical acetylcholine appears to relate to cortical activation and changes in states of consciousness, but the relationship between cortical acetylcholine and complexity has not been formally studied. We addressed this gap by analyzing simultaneous changes in cortical acetylcholine (prefrontal and parietal) and neurophysiologic complexity before, during, and after subanesthetic ketamine (10 mg/kg/h) or 50% nitrous oxide. METHODS: Under isoflurane anesthesia, adult Sprague Dawley rats (n = 24, 12 male and 12 female) were implanted with stainless-steel electrodes across the cortex to record monopolar electroencephalogram (0.5-175 Hz; 30 channels) and guide canulae in prefrontal and parietal cortices for local microdialysis quantification of acetylcholine levels. One subgroup of these rats was instrumented with a chronic catheter in jugular vein for ketamine infusion (n = 12, 6 male and 6 female). The electroencephalographic data were analyzed to determine subanesthetic ketamine or nitrous oxide-induced changes in Lempel-Ziv complexity and directed frontoparietal connectivity. Changes in complexity and connectivity were analyzed for correlation with concurrent changes in prefrontal and parietal acetylcholine. RESULTS: Subanesthetic ketamine produced sustained increases in normalized Lempel-Ziv complexity (0.5-175 Hz; P < .001) and high gamma frontoparietal connectivity (125-175 Hz; P < .001). This was accompanied by progressive increases in prefrontal (104%; P < .001) and parietal (159%; P < .001) acetylcholine levels that peaked after 50 minutes of infusion. Nitrous oxide induction produced a transient increase in complexity (P < .05) and high gamma connectivity (P < .001), which was accompanied by increases (P < .001) in prefrontal (56%) and parietal (43%) acetylcholine levels. In contrast, the final 50 minutes of nitrous oxide administration were characterized by a decrease in prefrontal (38%; P < .001) and parietal (45%; P < .001) acetylcholine levels, reduced complexity (P < .001), and comparatively weaker frontoparietal high gamma connectivity (P < .001). Cortical acetylcholine and complexity were correlated with both subanesthetic ketamine (prefrontal: cluster-weighted marginal correlation [CW r] [144] = 0.42, P < .001; parietal: CW r[144] = 0.42, P < .001) and nitrous oxide (prefrontal: CW r[156] = 0.46, P < .001; parietal: CW r[156] = 0.56, P < .001) cohorts. CONCLUSIONS: These data bridge changes in cortical acetylcholine with concurrent changes in neurophysiologic complexity, frontoparietal connectivity, and the level of consciousness.

Inactivation of Prefrontal Cortex Attenuates Behavioral Arousal Induced by Stimulation of Basal Forebrain During Sevoflurane Anesthesia.

BACKGROUND: Cholinergic stimulation of prefrontal cortex (PFC) can reverse anesthesia. Conversely, inactivation of PFC can delay emergence from anesthesia. PFC receives cholinergic projections from basal forebrain, which contains wake-promoting neurons. However, the role of basal forebrain cholinergic neurons in arousal from the anesthetized state requires refinement, and it is currently unknown whether the arousal-promoting effect of basal forebrain is mediated through PFC. To address these gaps in knowledge, we implemented a novel approach to the use of chemogenetic stimulation and tested the role of basal forebrain cholinergic neurons in behavioral arousal during sevoflurane anesthesia. Next, we investigated the effect of tetrodotoxin-mediated inactivation of PFC on behavioral arousal produced by electrical stimulation of basal forebrain during sevoflurane anesthesia. METHODS: Adult male and female transgenic rats (Long-Evans-Tg [ChAT-Cre]5.1 Deis; n = 22) were surgically prepared for expression of excitatory hM3D(Gq) receptors or mCherry in basal forebrain cholinergic neurons, and activation of these neurons by local delivery of compound 21, an agonist for hM3D(Gq) receptors. The transgenic rats were fitted with microdialysis probes for agonist delivery into basal forebrain and simultaneous prefrontal acetylcholine measurement. Adult male and female Sprague Dawley rats were surgically prepared for bilateral electrical stimulation of basal forebrain and tetrodotoxin infusion (156 µM and 500 nL) into PFC (n = 9) or bilateral electrical stimulation of piriform cortex (n = 9) as an anatomical control. All rats were implanted with electrodes to monitor the electroencephalogram. Heart and respiration rates were monitored using noninvasive sensors. A 6-point scale was used to score behavioral arousal (0 = no arousal and 5 = return of righting reflex). RESULTS: Compound 21 delivery into basal forebrain of rats with hM3D(Gq) receptors during sevoflurane anesthesia produced increases in arousal score (P < .001; confidence interval [CI], 1.80-4.35), heart rate (P < .001; CI, 36.19-85.32), respiration rate (P < .001; CI, 22.81-58.78), theta/delta ratio (P = .008; CI, 0.028-0.16), and prefrontal acetylcholine (P < .001; CI, 1.73-7.46). Electrical stimulation of basal forebrain also produced increases in arousal score (P < .001; CI, 1.85-4.08), heart rate (P = .018; CI, 9.38-98.04), respiration rate (P < .001; CI, 24.15-53.82), and theta/delta ratio (P = .020; CI, 0.019-0.22), which were attenuated by tetrodotoxin-mediated inactivation of PFC. CONCLUSIONS: This study validates the role of basal forebrain cholinergic neurons in behavioral arousal and demonstrates that the arousal-promoting effects of basal forebrain are mediated in part through PFC.

Near-infrared fluorescent northern blot.

Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive 32P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than 32P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.

Sequencing Framework for the Sensitive Detection and Precise Mapping of Defective Interfering Particle-Associated Deletions across Influenza A and B Viruses.

The mechanisms and consequences of defective interfering particle (DIP) formation during influenza virus infection remain poorly understood. The development of next-generation sequencing (NGS) technologies has made it possible to identify large numbers of DIP-associated sequences, providing a powerful tool to better understand their biological relevance. However, NGS approaches pose numerous technical challenges, including the precise identification and mapping of deletion junctions in the presence of frequent mutation and base-calling errors, and the potential for numerous experimental and computational artifacts. Here, we detail an Illumina-based sequencing framework and bioinformatics pipeline capable of generating highly accurate and reproducible profiles of DIP-associated junction sequences. We use a combination of simulated and experimental control data sets to optimize pipeline performance and demonstrate the absence of significant artifacts. Finally, we use this optimized pipeline to reveal how the patterns of DIP-associated junction formation differ between different strains and subtypes of influenza A and B viruses and to demonstrate how these data can provide insight into mechanisms of DIP formation. Overall, this work provides a detailed roadmap for high-resolution profiling and analysis of DIP-associated sequences within influenza virus populations.IMPORTANCE Influenza virus defective interfering particles (DIPs) that harbor internal deletions within their genomes occur naturally during infection in humans and during cell culture. They have been hypothesized to influence the pathogenicity of the virus; however, their specific function remains elusive. The accurate detection of DIP-associated deletion junctions is crucial for understanding DIP biology but is complicated by an array of technical issues that can bias or confound results. Here, we demonstrate a combined experimental and computational framework for detecting DIP-associated deletion junctions using next-generation sequencing (NGS). We detail how to validate pipeline performance and provide the bioinformatics pipeline for groups interested in using it. Using this optimized pipeline, we detect hundreds of distinct deletion junctions generated during infection with a diverse panel of influenza viruses and use these data to test a long-standing hypothesis concerning the molecular details of DIP formation.

Dicer cleaves 5'-extended microRNA precursors originating from RNA polymerase II transcription start sites.

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

Defining Dysbiosis in Disorders of Movement and Motivation.

The gut microbiota has emerged as a critical player in shaping and modulating brain function and has been shown to influence numerous behaviors, including anxiety and depression-like behaviors, sociability, and cognition. However, the effects of the gut microbiota on specific disorders associated with thalamo-cortico-basal ganglia circuits, ranging from compulsive behavior and addiction to altered sensation and motor output, are only recently being explored. Wholesale depletion and alteration of gut microbial communities in rodent models of disorders, such as Parkinson's disease, autism, and addiction, robustly affect movement and motivated behavior. A new frontier therefore lies in identifying specific microbial alterations that affect these behaviors and understanding the underlying mechanisms of action. Comparing alterations in gut microbiota across multiple basal-ganglia associated disease states allows for identification of common mechanistic pathways that may interact with distinct environmental and genetic risk factors to produce disease-specific outcomes.

Downregulation of the human peripheral myelin protein 22 gene by miR-29a in cellular models of Charcot-Marie-Tooth disease.

The majority of hereditary neuropathies are caused by duplication of the peripheral myelin protein 22 (PMP22) gene. Therefore, mechanisms to suppress the expression of the PMP22 gene have high therapeutic significance. Here we asked whether the human PMP22 gene is a target for regulation by microRNA 29a (miR-29a). Using bioinformatics, we determined that the human PMP22 gene contains the conserved seed sequence of the miR-29a binding site and this regulatory motif is included in the duplicated region in neuropathic patients. Using luciferase reporter assays in HEK293 cells, we demonstrated that transient transfection of a miR-29a mimic is associated with reduction in PMP22 3'UTR reporter activity. Transfecting normal and humanized transgenic neuropathic mouse Schwann cells with a miR-29a expression plasmid effectively lowered both the endogenous mouse and the transgenic human PMP22 transcripts compared with control vector. In dermal fibroblasts derived from neuropathic patients, ectopic expression of miR-29a led to ~50% reduction in PMP22 mRNA, which corresponded to ~20% decrease in PMP22 protein levels. Significantly, miR-29a-mediated reduction in PMP22 mitigated the reduced mitotic capacity of the neuropathic cells. Together, these results support further testing of miR-29a and/or PMP22-targeting siRNAs as therapeutic agents for correcting the aberrant expression of PMP22 in neuropathic patients.

Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7α-dehydroxylating anaerobes. While this organism is well-studied relative to bile acid metabolism, little is known about the basic nutrition and physiology of C. scindens ATCC 35704. To determine the amino acid and vitamin requirements of C. scindens, the leave-one-out (one amino acid group or vitamin) technique was used to eliminate the nonessential amino acids and vitamins. With this approach, the amino acid tryptophan and three vitamins (riboflavin, pantothenate, and pyridoxal) were found to be required for the growth of C. scindens In the newly developed defined medium, C. scindens fermented glucose mainly to ethanol, acetate, formate, and H2. The genome of C. scindens ATCC 35704 was completed through PacBio sequencing. Pathway analysis of the genome sequence coupled with transcriptome sequencing (RNA-Seq) under defined culture conditions revealed consistency with the growth requirements and end products of glucose metabolism. Induction with bile acids revealed complex and differential responses to cholic acid and deoxycholic acid, including the expression of potentially novel bile acid-inducible genes involved in cholic acid metabolism. Responses to toxic deoxycholic acid included expression of genes predicted to be involved in DNA repair, oxidative stress, cell wall maintenance/metabolism, chaperone synthesis, and downregulation of one-third of the genome. These analyses provide valuable insight into the overall biology of C. scindens which may be important in treatment of disease associated with increased colonic secondary bile acids.IMPORTANCEC. scindens is one of a few identified gut bacterial species capable of converting host cholic acid into disease-associated secondary bile acids such as deoxycholic acid. The current work represents an important advance in understanding the nutritional requirements and response to bile acids of the medically important human gut bacterium, C. scindens ATCC 35704. A defined medium has been developed which will further the understanding of bile acid metabolism in the context of growth substrates, cofactors, and other metabolites in the vertebrate gut. Analysis of the complete genome supports the nutritional requirements reported here. Genome-wide transcriptomic analysis of gene expression in the presence of cholic acid and deoxycholic acid provides a unique insight into the complex response of C. scindens ATCC 35704 to primary and secondary bile acids. Also revealed are genes with the potential to function in bile acid transport and metabolism.

Draft Assembly of Elite Inbred Line PH207 Provides Insights into Genomic and Transcriptome Diversity in Maize.

Intense artificial selection over the last 100 years has produced elite maize (Zea mays) inbred lines that combine to produce high-yielding hybrids. To further our understanding of how genome and transcriptome variation contribute to the production of high-yielding hybrids, we generated a draft genome assembly of the inbred line PH207 to complement and compare with the existing B73 reference sequence. B73 is a founder of the Stiff Stalk germplasm pool, while PH207 is a founder of Iodent germplasm, both of which have contributed substantially to the production of temperate commercial maize and are combined to make heterotic hybrids. Comparison of these two assemblies revealed over 2500 genes present in only one of the two genotypes and 136 gene families that have undergone extensive expansion or contraction. Transcriptome profiling revealed extensive expression variation, with as many as 10,564 differentially expressed transcripts and 7128 transcripts expressed in only one of the two genotypes in a single tissue. Genotype-specific genes were more likely to have tissue/condition-specific expression and lower transcript abundance. The availability of a high-quality genome assembly for the elite maize inbred PH207 expands our knowledge of the breadth of natural genome and transcriptome variation in elite maize inbred lines across heterotic pools.

Exercise and the Gut Microbiome: A Review of the Evidence, Potential Mechanisms, and Implications for Human Health.

The gastrointestinal tract contains trillions of microbes (collectively known as the gut microbiota) that play essential roles in host physiology and health. Studies from our group and others have demonstrated that exercise independently alters the composition and functional capacity of the gut microbiota. Here, we review what is known about the gut microbiota, how it is studied, and how it is influenced by exercise training and discuss the potential mechanisms and implications for human health and disease.