One size does not fit all - Trehalose metabolism by Clostridioides difficile is variable across the five phylogenetic lineages.

Clostridioides difficile, the leading cause of antibiotic-associated diarrhoea worldwide, is a genetically diverse species which can metabolise a number of nutrient sources upon colonising a dysbiotic gut environment. Trehalose, a disaccharide sugar consisting of two glucose molecules bonded by an α 1,1-glycosidic bond, has been hypothesised to be involved in the emergence of C. difficile hypervirulence due to its increased utilisation by the RT027 and RT078 strains. Here, growth in trehalose as the sole carbon source was shown to be non-uniform across representative C. difficile strains, even though the genes for its metabolism were induced. Growth in trehalose reduced the expression of genes associated with toxin production and sporulation in the C. difficile R20291 (RT027) and M120 (RT078) strains in vitro, suggesting an inhibitory effect on virulence factors. Interestingly, the R20291 TreR transcriptional regulatory protein appeared to possess an activator function as its DNA-binding ability was increased in the presence of its effector, trehalose-6-phosphate. Using RNA-sequencing analysis, we report the identification of a putative trehalose metabolism pathway which is induced during growth in trehalose: this has not been previously described within the C. difficile species. These data demonstrate the metabolic diversity exhibited by C. difficile which warrants further investigation to elucidate the molecular basis of trehalose metabolism within this important gut pathogen.

Terrisporobacter hibernicus sp. nov., isolated from bovine faeces in Northern Ireland.

A new species of Terrisporobacter, a Gram-positive, spore-forming anaerobic group, proposed name Terrisporobacter hibernicus sp. nov., was isolated in Northern Ireland from bovine faeces collected in 2016. Designated as MCA3T, cells of T. hibernicus sp. nov. are rod shaped and motile. Cells tolerate NaCl from 0.5 to 5.5 % (w/v), with a pH tolerance between pH 6 and 9. The optimal temperature for growth is 35-40 °C, and temperatures from 20 to 30 °C are tolerated. The polar lipid profile displays diphosphatidylglycerol, phosphatidylglycerol, two aminoglycolipids, one glycophospholipid, one aminolipid, three glycolipids, five phospholipids and one lipid. No respiratory quinones are detected. The predominant fatty acid profile includes C16 : 0 at 22.8 %. Strain MCA3T is positive for glucose and maltose acidification, as well as glycerol and sorbitol. The biochemical results from a VITEK2 assay of strain MCA3T, Terrisporobacter petrolearius LAM0A37T and Terrisporobacter mayombei DSM 6539T are also included for the first time. The closed and complete genome of strain MCA3T from a hybrid Oxford Nanopore Technology MinION/Illumina assembly reveals no evidence for known virulence genes. Draft genome sequencing of T. mayombei DSM 6539T and T. petrolearius LAM0A37T, as performed by Illumina MiSeq, provides reference genomes for these respective species of Terrisporobacter for the first time. DNA-DNA hybridization values (d4) of MCA3T to Terrisporobacter glycolicus ATCC 14880T, T. petrolearius LAM0A37T and T. mayombei DSM 6539T are 48.8, 67.4 and 46.3 %, with cutoff value at 70 %. The type strain for T. hibernicus sp. nov. is MCA3T (=NCTC 14625T=LMG 32430T).

Characterization of Fragile X Mental Retardation Protein expression in human nociceptors and their axonal projections to the spinal dorsal horn.

Fragile X Mental Retardation Protein (FMRP) regulates activity-dependent RNA localization and local translation to modulate synaptic plasticity throughout the central nervous system. Mutations in the FMR1 gene that hinder or ablate FMRP function cause Fragile X Syndrome (FXS), a disorder associated with sensory processing dysfunction. FXS premutations are associated with increased FMRP expression and neurological impairments including sex dimorphic presentations of chronic pain. In mice, FMRP ablation causes dysregulated dorsal root ganglion (DRG) neuron excitability and synaptic vesicle exocytosis, spinal circuit activity, and decreased translation-dependent nociceptive sensitization. Activity-dependent, local translation is a key mechanism for enhancing primary nociceptor excitability that promotes pain in animals and humans. These works indicate that FMRP likely regulates nociception and pain at the level of the primary nociceptor or spinal cord. Therefore, we sought to better understand FMRP expression in the human DRG and spinal cord using immunostaining in organ donor tissues. We find that FMRP is highly expressed in DRG and spinal neuron subsets with substantia gelatinosa exhibiting the most abundant immunoreactivity in spinal synaptic fields. Here, it is expressed in nociceptor axons. FMRP puncta colocalized with Nav1.7 and TRPV1 receptor signals suggesting a pool of axoplasmic FMRP localizes to plasma membrane-associated loci in these branches. Interestingly, FMRP puncta exhibited notable colocalization with calcitonin gene-related peptide (CGRP) immunoreactivity selectively in female spinal cord. Our results support a regulatory role for FMRP in human nociceptor axons of the dorsal horn and implicate it in the sex dimorphic actions of CGRP signaling in nociceptive sensitization and chronic pain.

Improving Campus Alcohol Policies Through Assessment and Scoring.

Excessive alcohol consumption is responsible for more than 1,500 deaths annually among college students, of whom more than one in three report having been drunk during the past 30 days. Campus alcohol policies offer a first line of defense against excessive alcohol use but have received little systematic attention in the research literature. The research team previously developed a taxonomy of campus alcohol policies and sanctions, ranked in order of effectiveness, and assessed the accessibility, clarity, and effectiveness of policies at 15 post-secondary educational institutions. Herein we describe the process of reporting those assessments back to the 15 institutions, providing them with recommendations and technical assistance on how to improve their policies, and then re-assessing school alcohol policies for effectiveness and clarity. Conversations with primary points of contact at each school provided further insight into the process of assessing and improving campus alcohol policies. Of the 15 schools assessed, 11 added more effective policies, and four added more effective consequences during the 2 years following receipt of reports on the assessment. Campuses have control over their own policies, and greater attention to them from researchers and practitioners could better maximize their potential for enhancing student health and safety and supporting student success.

Clostridioides difficile as a Potential Pathogen of Importance to One Health: A Review.

Clostridioides difficile (basonym Clostridium) is a bacterial enteropathogen associated with cases of C. difficile infection that can result in pseudomembranous colitis, rapid fluid loss, and death. For decades following its isolation, C. difficile was thought to be a solely nosocomial pathogen, being isolated from individuals undergoing antimicrobial therapy and largely affecting elderly populations. More recently, C. difficile spores have been identified in the broader environment, including in food-producing animals, soil, and food matrices, in both ready-to-eat foods and meat products. Furthermore, evidence has emerged of hypervirulent ribotypes (RTs), such as RT078, similar to those cultured in asymptomatic carriers, also being identified in these environments. This finding may reflect on adaptations arising in these bacteria following selection pressures encountered in these niches, and which occurs due to an increase in antimicrobial usage in both clinical and veterinary settings. As C. difficile continues to adapt to new ecological niches, the taxonomy of this genus has also been evolving. To help understand the transmission and virulence potential of these bacteria of importance to veterinary public health, strategies applying multi-omics-based technologies may prove useful. These approaches may extend our current understanding of this recognized nosocomial pathogen, perhaps redefining it as a zoonotic bacterium. In this review, a brief background on the epidemiological presentation of C. difficile will be highlighted, followed by a review of C. difficile in food-producing animals and food products. The current state of C. difficile taxonomy will provide evidence of Clade 5 (ST11/RT078) delineation, as well as background on the genomic elements linked to C. difficile virulence and ongoing speciation. Recent studies applying second- and third-generation sequencing technologies will be highlighted, and which will further strengthen the argument made by many throughout the world regarding this pathogen and its consideration within a One Health dimension.

Comparison Between Full-Length 16S rRNA Metabarcoding and Whole Metagenome Sequencing Suggests the Use of Either Is Suitable for Large-Scale Microbiome Studies.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter, Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.

Living shorelines achieve functional equivalence to natural fringe marshes across multiple ecological metrics.

Nature-based shoreline protection provides a welcome class of adaptations to promote ecological resilience in the face of climate change. Along coastlines, living shorelines are among the preferred adaptation strategies to both reduce erosion and provide ecological functions. As an alternative to shoreline armoring, living shorelines are viewed favorably among coastal managers and some private property owners, but they have yet to undergo a thorough examination of how their levels of ecosystem functions compare to their closest natural counterpart: fringing marshes. Here, we provide a synthesis of results from a multi-year, large-spatial-scale study in which we compared numerous ecological metrics (including habitat provision for fish, invertebrates, diamondback terrapin, and birds, nutrient and carbon storage, and plant productivity) measured in thirteen pairs of living shorelines and natural fringing marshes throughout coastal Virginia, USA. Living shorelines were composed of marshes created by bank grading, placement of sand fill for proper elevations, and planting of S. alterniflora and S. patens, as well as placement of a stone sill seaward and parallel to the marsh to serve as a wave break. Overall, we found that living shorelines were functionally equivalent to natural marshes in nearly all measured aspects, except for a lag in soil composition due to construction of living shoreline marshes with clean, low-organic sands. These data support the prioritization of living shorelines as a coastal adaptation strategy.

An Economical and Versatile High-Throughput Protein Purification System Using a Multi-Column Plate Adapter.

Protein purification is imperative to the study of protein structure and function and is usually used in combination with biophysical techniques. It is also a key component in the development of new therapeutics. The evolving era of functional proteomics is fueling the demand for high-throughput protein purification and improved techniques to facilitate this. It was hypothesized that a multi column plate adaptor (MCPA) can interface multiple chromatography columns of different resins with multi-well plates for parallel purification. This method offers an economical and versatile method of protein purification that can be used under gravity or vacuum, rivaling the speed of an automated system. The MCPA can be used to recover milligram yields of protein by an affordable and time efficient method for subsequent characterization and analysis. The MCPA has been used for high-throughput affinity purification of SH3 domains. Ion exchange has also been demonstrated via the MCPA to purify protein post Ni-NTA affinity chromatography, indicating how this system can be adapted to other purification types. Due to its setup with multiple columns, individual customization of parameters can be made in the same purification, unachievable by the current plate-based methods.

Yersinia canariae sp. nov., isolated from a human yersiniosis case.

A Gram-negative rod from the Yersinia genus was isolated from a clinical case of yersiniosis in the United Kingdom. Long read sequencing data from an Oxford Nanopore Technologies (ONT) MinION in conjunction with Illumina HiSeq reads were used to generate a finished quality genome of this strain. Overall Genome Related Index (OGRI) of the strain was used to determine that it was a novel species within Yersinia, despite biochemical similarities to Yersinia enterocolitica. The 16S ribosomal RNA gene accessions are MN434982-MN434987 and the accession number for the complete and closed chromosome is CP043727. The type strain is SRR7544370T (=NCTC 14382T/=LMG 31573T).

Assessing Campus Alcohol Policies: Measuring Accessibility, Clarity, and Effectiveness.

BACKGROUND: Excessive alcohol consumption poses significant hazards to health and safety on college campuses. While substantial research exists regarding effective policies for preventing alcohol-related problems in the communities surrounding campuses, on-campus alcohol policies have received far less attention. METHODS: Official campus alcohol policies (CAPs) were retrieved from the websites of the 15 member schools of the Maryland Collaborative to Reduce College Drinking and Related Problems, a voluntary statewide collaborative. CAPs were assessed for accessibility, clarity, and effectiveness. In addition to assessing whether campuses were in compliance with federal regulations for comprehensiveness of policies, a measure of likely policy effectiveness was developed through the use of 2 Delphi panels drawing on alcohol policy researchers and on-campus and community practitioners, respectively. The panels rated 35 potential policies and 13 possible sanctions; lists of policies and sanctions were compiled primarily from what was already in existence at 1 or more member schools. RESULTS: For most campuses, the CAPs could be located within 30 seconds, but tended to be spread across multiple web pages. Language used to communicate the policies tended to be complex and above the reading level of someone with a high school education. At least half of the schools had less than half of the possible policies rated most or somewhat effective by the Delphi panels. Schools were more likely to employ the most effective sanctions, but somewhat and ineffective sanctions were also not uncommon. CONCLUSIONS: CAPs are an important element in reducing negative consequences of alcohol consumption on college campuses. A higher level of research scrutiny is warranted to understand the extent to which CAPs are associated with excessive drinking, but this research describes an evidence- and expert-informed assessment approach that colleges can use to regularly analyze and update their CAPS.

Axonal ribosomes and mRNAs associate with fragile X granules in adult rodent and human brains.

Local mRNA translation in growing axons allows for rapid and precise regulation of protein expression in response to extrinsic stimuli. However, the role of local translation in mature CNS axons is unknown. Such a mechanism requires the presence of translational machinery and associated mRNAs in circuit-integrated brain axons. Here we use a combination of genetic, quantitative imaging and super-resolution microscopy approaches to show that mature axons in the mammalian brain contain ribosomes, the translational regulator FMRP and a subset of FMRP mRNA targets. This axonal translational machinery is associated with Fragile X granules (FXGs), which are restricted to axons in a stereotyped subset of brain circuits. FXGs and associated axonal translational machinery are present in hippocampus in humans as old as 57 years. This FXG-associated axonal translational machinery is present in adult rats, even when adult neurogenesis is blocked. In contrast, in mouse this machinery is only observed in juvenile hippocampal axons. This differential developmental expression was specific to the hippocampus, as both mice and rats exhibit FXGs in mature axons in the adult olfactory system. Experiments in Fmr1 null mice show that FMRP regulates axonal protein expression but is not required for axonal transport of ribosomes or its target mRNAs. Axonal translational machinery is thus a feature of adult CNS neurons. Regulation of this machinery by FMRP could support complex behaviours in humans throughout life.

Regulation of neuronal gene expression by local axonal translation.

RNA localization is a key mechanism in the regulation of protein expression. In neurons, this includes the axonal transport of select mRNAs based on the recognition of axonal localization motifs in these RNAs by RNA binding proteins. Bioinformatic analyses of axonal RNAs suggest that selective inclusion of such localization motifs in mature mRNAs is one mechanism controlling the composition of the axonal transcriptome. The subsequent translation of axonal transcripts in response to specific stimuli provides precise spatiotemporal control of the axonal proteome. This axonal translation supports local phenomena including axon pathfinding, mitochondrial function, and synapse-specific plasticity. Axonal protein synthesis also provides transport machinery and signals for retrograde trafficking to the cell body to effect somatic changes including altering the transcriptional program. Here we review the remarkable progress made in recent years to identify and characterize these phenomena.