Moving from information and collaboration to action: report from the 3rd International Dog Health Workshop, Paris in April 2017

Abstract Background Breed-related health problems in dogs have received increased focus over the last decade. Responsibility for causing and/or solving these problems has been variously directed towards dog breeders and kennel clubs, the veterinary profession, welfare scientists, owners, regulators, insurance companies and the media. In reality, all these stakeholders are likely to share some responsibility and optimal progress on resolving these challenges requires all key stakeholders to work together. The International Partnership for Dogs (IPFD), together with an alternating host organization, holds biennial meetings called the International Dog Health Workshops (IDHW). The Société Centrale Canine (French Kennel Club) hosted the 3rd IDHW, in Paris, in April, 2017. These meetings bring together a wide range of stakeholders in dog health, science and welfare to improve international sharing of information and resources, to provide a forum for ongoing collaboration, and to identify specific needs and actions to improve health, well-being and welfare in dogs. Results The workshop included 140 participants from 23 countries and was structured around six important issues facing those who work to improve dog health. These included individualized breed-specific strategies for health and breeding, extreme conformations, education and communication in relation to antimicrobial resistance, behavior and welfare, genetic testing and population-based evidence. A number of exciting actions were agreed during the meeting. These included setting up working groups to create tools to help breed clubs accelerate the implementation of breed-health strategies, review aspects of extreme conformation and share useful information on behavior. The meeting also heralded the development of an online resource of relevant information describing quality measures for DNA testing. A demand for more and better data and evidence was a recurring message stressed across all themes. Conclusions The meeting confirmed the benefits from inclusion of a diverse range of stakeholders who all play relevant and collaborative parts to improve future canine health. Firm actions were set for progress towards improving breed-related welfare. The next international workshop will be in the UK in 2019 and will be organized by the UK Kennel Club

Genome landscapes and bacteriophage codon usage

Across all kingdoms of biological life, protein-coding genes exhibit unequal usage of synonmous codons. Although alternative theories abound, translational selection has been accepted as an important mechanism that shapes the patterns of codon usage in prokaryotes and simple eukaryotes. Here we analyze patterns of codon usage across 74 diverse bacteriophages that infect E. coli, P. aeruginosa and L. lactis as their primary host. We introduce the concept of a `genome landscape,' which helps reveal non-trivial, long-range patterns in codon usage across a genome. We develop a series of randomization tests that allow us to interrogate the significance of one aspect of codon usage, such a GC content, while controlling for another aspect, such as adaptation to host-preferred codons. We find that 33 phage genomes exhibit highly non-random patterns in their GC3-content, use of host-preferred codons, or both. We show that the head and tail proteins of these phages exhibit significant bias towards host-preferred codons, relative to the non-structural phage proteins. Our results support the hypothesis of translational selection on viral genes for host-preferred codons, over a broad range of bacteriophages.Comment: 9 Color Figures, 5 Tables, 53 Reference

Characterisation of Muta™Mouse λgt10-lacZ transgene: evidence for in vivo rearrangements

The multicopy λgt10-lacZ transgene shuttle vector of Muta™Mouse serves as an important tool for genotoxicity studies. Here, we describe a model for λgt10-lacZ transgene molecular structure, based on characterisation of transgenes recovered from animals of our intramural breeding colony. Unique nucleotide sequences of the 47 513 bp monomer are reported with GenBank® assigned accession numbers. Besides defining ancestral mutations of the λgt10 used to construct the transgene and the Muta™Mouse precursor (strain 40.6), we validated the sequence integrity of key λ genes needed for the Escherichia coli host-based mutation reporting assay. Using three polymerase chain reaction (PCR)-based chromosome scanning and cloning strategies, we found five distinct in vivo transgene rearrangements, which were common to both sexes, and involved copy fusions generating ∼10 defective copies per haplotype. The transgene haplotype was estimated by Southern hybridisation and real-time–polymerase chain reaction, which yielded 29.0 ± 4.0 copies based on spleen DNA of Muta™Mouse, and a reconstructed CD2F1 genome with variable λgt10-lacZ copies. Similar analysis of commercially prepared spleen DNA from Big Blue® mouse yielded a haplotype of 23.5 ± 3.1 copies. The latter DNA is used in calibrating a commercial in vitro packaging kit for E.coli host-based mutation assays of both transgenic systems. The model for λgt10-lacZ transgene organisation, and the PCR-based methods for assessing copy number, integrity and rearrangements, potentially extends the use of Muta™Mouse construct for direct, genomic-type assays that detect the effects of clastogens and aneugens, without depending on an E.coli host, for reporting effects

The Generation of Promoter-Mediated Transcriptional Noise in Bacteria

Noise in the expression of a gene produces fluctuations in the concentration of the gene product. These fluctuations can interfere with optimal function or can be exploited to generate beneficial diversity between cells; gene expression noise is therefore expected to be subject to evolutionary pressure. Shifts between modes of high and low rates of transcription initiation at a promoter appear to contribute to this noise both in eukaryotes and prokaryotes. However, models invoked for eukaryotic promoter noise such as stable activation scaffolds or persistent nucleosome alterations seem unlikely to apply to prokaryotic promoters. We consider the relative importance of the steps required for transcription initiation. The 3-step transcription initiation model of McClure is extended into a mathematical model that can be used to predict consequences of additional promoter properties. We show in principle that the transcriptional bursting observed at an E. coli promoter by Golding et al. (2005) can be explained by stimulation of initiation by the negative supercoiling behind a transcribing RNA polymerase (RNAP) or by the formation of moribund or dead-end RNAP-promoter complexes. Both mechanisms are tunable by the alteration of promoter kinetics and therefore allow the optimization of promoter mediated noise.Comment: 4 figures, 1 table. Supplemental materials are also include

Diminished mental- and physical function and lack of social support are associated with shorter survival in community dwelling older persons of Botswana

<p>Abstract</p> <p>Background</p> <p>Mortality rates for older persons in Botswana have been unavailable and little is known of predictors of mortality in old age. This study may serve as a precursor for more detailed assessments.</p> <p>The objective was to assess diminished function and lack of social support as indicators of short term risk of death.</p> <p>Methods</p> <p>A national population based prospective survey was undertaken in Botswana; twelve rural areas and three urban centers were included.</p> <p>372 community-dwelling persons aged sixty years and over, were included; 265 were followed-up. Sixteen subjects were deceased at follow-up.</p> <p>Subjects were interviewed and clinically assessed at home. Measures of cognitive function, depression and physical function and sociodemographic information were collected. Subjects were followed-up at average 6.8 months after baseline.</p> <p>Results</p> <p>Overall mortality rate was 10.9 per 100 person years. Age-adjusted odds ratios (OR) for death during follow-up were; 4.2 (CI 1.4–12.5) and 3.6 (CI 1.0–12.7) for those with diminished physical- and cognitive function, respectively.</p> <p>Indicators of limited social support; household with only 1 or 2 persons and eating alone, yielded age adjusted ORs of 4.3 (CI 1.5–12.5) and 6.7 (CI 2.2–20), respectively, for death during follow-up.</p> <p>Conclusion</p> <p>Older community dwelling persons with diminished cognitive- or physical function, solitary daily meals and living in a small household have a significantly increased risk of rapid deterioration and death.</p> <p>Health policy should include measures to strengthen informal support and expand formal service provisions to older persons with poor function and limited social networks in order to prevent premature deaths.</p

Morphogenesis of the T4 tail and tail fibers

Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study

Comparative (Meta)genomic Analysis and Ecological Profiling of Human Gut-Specific Bacteriophage φB124-14

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome

DNA Damage and Reactive Nitrogen Species are Barriers to Vibrio cholerae Colonization of the Infant Mouse Intestine

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants

DNA repair, genome stability and cancer: a historical perspective

The multistep process of cancer progresses over many years. The prevention of mutations by DNA repair pathways led to an early appreciation of a role for repair in cancer avoidance. However, the broader role of the DNA damage response (DDR) emerged more slowly. In this Timeline article, we reflect on how our understanding of the steps leading to cancer developed, focusing on the role of the DDR. We also consider how our current knowledge can be exploited for cancer therapy

Collagen-Like Proteins in Pathogenic E. coli Strains

The genome sequences of enterohaemorrhagic E. coli O157:H7 strains show multiple open-reading frames with collagen-like sequences that are absent from the common laboratory strain K-12. These putative collagens are included in prophages embedded in O157:H7 genomes. These prophages carry numerous genes related to strain virulence and have been shown to be inducible and capable of disseminating virulence factors by horizontal gene transfer. We have cloned two collagen-like proteins from E. coli O157:H7 into a laboratory strain and analysed the structure and conformation of the recombinant proteins and several of their constituting domains by a variety of spectroscopic, biophysical, and electron microscopy techniques. We show that these molecules exhibit many of the characteristics of vertebrate collagens, including trimer formation and the presence of a collagen triple helical domain. They also contain a C-terminal trimerization domain, and a trimeric α-helical coiled-coil domain with an unusual amino acid sequence almost completely lacking leucine, valine or isoleucine residues. Intriguingly, these molecules show high thermal stability, with the collagen domain being more stable than those of vertebrate fibrillar collagens, which are much longer and post-translationally modified. Under the electron microscope, collagen-like proteins from E. coli O157:H7 show a dumbbell shape, with two globular domains joined by a hinged stalk. This morphology is consistent with their likely role as trimeric phage side-tail proteins that participate in the attachment of phage particles to E. coli target cells, either directly or through assembly with other phage tail proteins. Thus, collagen-like proteins in enterohaemorrhagic E. coli genomes may have a direct role in the dissemination of virulence-related genes through infection of harmless strains by induced bacteriophages