Extensive genome analysis of Coxiella burnetii reveals limited evolution within genomic groups

This is the final version. Available on open access from BMC via the DOI in this recordAvailability of data and materials: Whole genome sequences were deposited in NCBI under BioProjects PRJNA430350 and PRJNA506366, as well as in the Sequence Read Archive as studies SRP130048 and SRP170036. Individual GenBank accession numbers for the WGS data are as follows: Q532 = PPFQ00000000.1 ; Q540 = PPFP00000000.1 ; Q545 = PPFO00000000.1 ; Q556 = PPFN00000000.1 ; Q559 = PPFM00000000.1 ; Cb_D1 = RQJU00000000.1; Cb_D2 = RQJT00000000.1 ; Cb_D8 = RQJS00000000.1 ; and Cb_D10 = RQJR00000000.1 .The authors declare that all other data supporting the findings of this study are available within the article and its supplementary information files.Background: Coxiella burnetii is a zoonotic pathogen that resides in wild and domesticated animals across the globe and causes a febrile illness, Q fever, in humans. An improved understanding of the genetic diversity of C. burnetii is essential for the development of diagnostics, vaccines and therapeutics, but genotyping data is lacking from many parts of the world. Sporadic outbreaks of Q fever have occurred in the United Kingdom, but the local genetic make-up of C. burnetii has not been studied in detail. Results: Here, we report whole genome data for nine C. burnetii sequences obtained in the UK. All four genomes of C. burnetii from cattle, as well as one sheep sample, belonged to Multi-spacer sequence type (MST) 20, whereas the goat samples were MST33 (three genomes) and MST32 (one genome), two genotypes that have not been described to be present in the UK to date. We established the phylogenetic relationship between the UK genomes and 67 publically available genomes based on single nucleotide polymorphisms (SNPs) in the core genome, which confirmed tight clustering of strains within genomic groups, but also indicated that sub-groups exist within those groups. Variation is mainly achieved through SNPs, many of which are non-synonymous, thereby confirming that evolution of C. burnetii is based on modification of existing genes. Finally, we discovered genomic-group specific genome content, which supports a model of clonal expansion of previously established genotypes, with large scale dissemination of some of these genotypes across continents being observed. Conclusions: The genetic make-up of C. burnetii in the UK is similar to the one in neighboring European countries. As a species, C. burnetii has been considered a clonal pathogen with low genetic diversity at the nucleotide level. Here, we present evidence for significant variation at the protein level between isolates of different genomic groups, which mainly affects secreted and membrane-associated proteins. Our results thereby increase our understanding of the global genetic diversity of C. burnetii and provide new insights into the evolution of this emerging zoonotic pathogen.Defence Science and Technology Laboratory (DSTL

An integrated computational-experimental approach reveals Yersinia pestis genes essential across a narrow or a broad range of environmental conditions

This is the final version. Available from BMC via the DOI in this recordAvailability of data and materials: The datasets supporting the conclusions of this article are available at the NCBI GEO website https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE100226.BACKGROUND: The World Health Organization has categorized plague as a re-emerging disease and the potential for Yersinia pestis to also be used as a bioweapon makes the identification of new drug targets against this pathogen a priority. Environmental temperature is a key signal which regulates virulence of the bacterium. The bacterium normally grows outside the human host at 28 °C. Therefore, understanding the mechanisms that the bacterium used to adapt to a mammalian host at 37 °C is central to the development of vaccines or drugs for the prevention or treatment of human disease. RESULTS: Using a library of over 1 million Y. pestis CO92 random mutants and transposon-directed insertion site sequencing, we identified 530 essential genes when the bacteria were cultured at 28 °C. When the library of mutants was subsequently cultured at 37 °C we identified 19 genes that were essential at 37 °C but not at 28 °C, including genes which encode proteins that play a role in enabling functioning of the type III secretion and in DNA replication and maintenance. Using genome-scale metabolic network reconstruction we showed that growth conditions profoundly influence the physiology of the bacterium, and by combining computational and experimental approaches we were able to identify 54 genes that are essential under a broad range of conditions. CONCLUSIONS: Using an integrated computational-experimental approach we identify genes which are required for growth at 37 °C and under a broad range of environments may be the best targets for the development of new interventions to prevent or treat plague in humans.This work was funded by the Defence Science and Technology Laboratory, award DSTLX-1000060221 (WP1)

Protection against Experimental Melioidosis with a Synthetic manno-Heptopyranose Hexasaccharide Glycoconjugate

This is the final version of the article. Available from the publisher via the DOI in this record.Melioidosis is an emerging infectious disease caused by Burkholderia pseudomallei and is associated with high morbidity and mortality rates in endemic areas. Antibiotic treatment is protracted and not always successful; even with appropriate therapy, up to 40% of individuals presenting with melioidosis in Thailand succumb to infection. In these circumstances, an effective vaccine has the potential to have a dramatic impact on both the scale and the severity of disease. Currently, no vaccines are licensed for human use. A leading vaccine candidate is the capsular polysaccharide consisting of a homopolymer of unbranched 1→3 linked 2-O-acetyl-6-deoxy-β-d-manno-heptopyranose. Here, we present the chemical synthesis of this challenging antigen using a novel modular disaccharide assembly approach. The resulting hexasaccharide was coupled to the nontoxic Hc domain of tetanus toxin as a carrier protein to promote recruitment of T-cell help and provide a scaffold for antigen display. Mice immunized with the glycoconjugate developed IgM and IgG responses capable of recognizing native capsule, and were protected against infection with over 120 × LD50 of B. pseudomallei strain K96243. This is the first report of the chemical synthesis of an immunologically relevant and protective hexasaccharide fragment of the capsular polysaccharide of B. pseudomallei and serves as the rational starting point for the development of an effective licensed vaccine for this emerging infectious disease.This work was funded by the United Kingdom Ministry of Defence. The mass spectral data described here were acquired on an Orbitrap Fusion mass spectrometer funded by National Institutes of Health grant 1S10OD010645-01A1

A Noise Trimming and Positional Significance of Transposon Insertion System to Identify Essential Genes in Yersinia pestis

This is the final version of the article. Available from Springer Nature via the DOI in this record.Massively parallel sequencing technology coupled with saturation mutagenesis has provided new and global insights into gene functions and roles. At a simplistic level, the frequency of mutations within genes can indicate the degree of essentiality. However, this approach neglects to take account of the positional significance of mutations - the function of a gene is less likely to be disrupted by a mutation close to the distal ends. Therefore, a systematic bioinformatics approach to improve the reliability of essential gene identification is desirable. We report here a parametric model which introduces a novel mutation feature together with a noise trimming approach to predict the biological significance of Tn5 mutations. We show improved performance of essential gene prediction in the bacterium Yersinia pestis, the causative agent of plague. This method would have broad applicability to other organisms and to the identification of genes which are essential for competitiveness or survival under a broad range of stresses.This work was supported by the Defence Science and Technology Laboratory under contract DSTLX-1000060221 (WP1)

The GRADE System for Rating Clinical Guidelines

Brian Kavanagh critiques the GRADE system of grading guidelines, arguing that even though it has evolved through the Evidence-Based Medicine movement, there is no evidence that GRADE itself is reliable

Prevalence of Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma spp. and Leishmania infantum in apparently healthy and CVBD-suspect dogs in Portugal - a national serological study

<p>Abstract</p> <p>Background</p> <p>Canine vector-borne diseases (CVBDs) are caused by a wide range of pathogens transmitted to dogs by arthropods including ticks and insects. Many CVBD-agents are of zoonotic concern, with dogs potentially serving as reservoirs and sentinels for human infections. The present study aimed at assessing the seroprevalence of infection with or exposure to <it>Dirofilaria immitis, Ehrlichia canis, Borrelia burgdorferi </it>sensu lato, <it>Anaplasma </it>spp. and <it>Leishmania infantum </it>in dogs in Portugal.</p> <p>Methods</p> <p>Based on 120 veterinary medical centres from all the regions of mainland and insular Portugal, 557 apparently healthy and 628 CVBD-suspect dogs were sampled. Serum, plasma or whole blood was tested for qualitative detection of <it>D. immitis </it>antigen and antibodies to <it>E. canis, B. burgdorferi </it>s. l., <it>Anaplasma </it>spp. and <it>L. infantum </it>with two commercial in-clinic enzyme-linked immunosorbent assay kits. Odds ratios (OR) were calculated by logistic regression analysis to identify independent risk factors of exposure to the vector-borne agents.</p> <p>Results</p> <p>Total positivity levels to <it>D. immitis, E. canis, B. burgdorferi, Anaplasma </it>spp., <it>L. infantum</it>, one or more agents and mixed agents were 3.6%, 4.1%, 0.2%, 4.5%, 4.3%, 14.0% and 2.0% in the healthy group, and 8.9%, 16.4%, 0.5%, 9.2%, 25.2%, 46.3% and 11.6% in the clinically suspect group, respectively. Non-use of ectoparasiticides was a risk factor for positivity to one or more agents both in the apparently healthy (OR = 2.1) and CVBD-suspect (OR = 1.5) dogs. Seropositivity to <it>L. infantum </it>(OR = 7.6), <it>E. canis </it>(OR = 4.1) and <it>D. immitis </it>(OR = 2.4) were identified as risk factors for the presence of clinical signs compatible with CVBDs. Positivity to mixed agents was not found to be a risk factor for disease.</p> <p>Conclusions</p> <p>Dogs in Portugal are at risk of becoming infected with vector-borne pathogens, some of which are of zoonotic concern. CVBDs should be considered by practitioners and prophylactic measures must be put in place to protect dogs and limit the risk of transmission of vector-borne agents to humans. This study is expected to give veterinary and public health authorities an increased awareness about CVBDs in Portugal and to serve as a reference for future investigations and control actions.</p

Clinical knee findings in floor layers with focus on meniscal status

<p>Abstract</p> <p>Background</p> <p>The aim of this study was to examine the prevalence of self-reported and clinical knee morbidity among floor layers compared to a group of graphic designers, with special attention to meniscal status.</p> <p>Methods</p> <p>We obtained information about knee complaints by questionnaire and conducted a bilateral clinical and radiographic knee examination in 134 male floor layers and 120 male graphic designers. After the exclusion of subjects with reports of earlier knee injuries the odds ratio (OR) with 95% confidence intervals (CI) of knee complaints and clinical findings were computed among floor layers compared to graphic designers, using logistic regression. Estimates were adjusted for effects of body mass index, age and knee straining sports. Using radiographic evaluations, we conducted side-specific sensitivity analyses regarding clinical signs of meniscal lesions after the exclusion of participants with tibiofemoral (TF) osteoarthritis (OA).</p> <p>Results</p> <p>Reports of knee pain (OR = 2.7, 95% CI = 1.5–4.6), pain during stair walking (OR = 2.2, 95% CI = 1.3–3.9) and symptoms of catching of the knee joint (OR = 2.9, 95% CI = 1.4–5.7) were more prevalent among floor layers compared to graphic designers. Additionally, significant more floor layers than graphic designers had clinical signs suggesting possible meniscal lesions: a positive McMurray test (OR = 2.4, 95% CI = 1.1–5.0) and TF joint line tenderness (OR = 5.4, 95% CI = 2.4–12.0). Excluding floor layers (n = 22) and graphic designers (n = 15) with radiographic TF OA did not alter this trend between the two study groups: a positive McMurray test (OR = 2.2, 95% CI = 1.0–4.9), TF joint line tenderness (OR = 5.0, 95% CI = 2.0–12.5).</p> <p>Conclusion</p> <p>Results indicate that floor layers have a high prevalence of both self-reported and clinical knee morbidity. Clinical knee findings suggesting possible meniscal lesions were significant more prevalent among floor layers compared to a group of low-level exposed graphic designers and an association with occupational kneeling could be possible. However, causality cannot be confirmed due to the cross-sectional study design.</p

Evidence and argument in policymaking: development of workplace smoking legislation

<p>Abstract</p> <p>Background</p> <p>We sought to identify factors that affect the passage of public health legislation by examining the use of arguments, particularly arguments presenting research evidence, in legislative debates regarding workplace smoking restrictions.</p> <p>Methods</p> <p>We conducted a case-study based content analysis of legislative materials used in the development of six state workplace smoking laws, including written and spoken testimony and the text of proposed and passed bills and amendments. We coded testimony given before legislators for arguments used, and identified the institutional affiliations of presenters and their position on the legislation. We compared patterns in the arguments made in testimony to the relative strength of each state's final legislation.</p> <p>Results</p> <p>Greater discussion of scientific evidence within testimony given was associated with the passage of workplace smoking legislation that provided greater protection for public health, regardless of whether supporters outnumbered opponents or vice versa.</p> <p>Conclusion</p> <p>Our findings suggest that an emphasis on scientific discourse, relative to other arguments made in legislative testimony, might help produce political outcomes that favor public health.</p

Applying unmixing to gene expression data for tumor phylogeny inference

<p>Abstract</p> <p>Background</p> <p>While in principle a seemingly infinite variety of combinations of mutations could result in tumor development, in practice it appears that most human cancers fall into a relatively small number of "sub-types," each characterized a roughly equivalent sequence of mutations by which it progresses in different patients. There is currently great interest in identifying the common sub-types and applying them to the development of diagnostics or therapeutics. Phylogenetic methods have shown great promise for inferring common patterns of tumor progression, but suffer from limits of the technologies available for assaying differences between and within tumors. One approach to tumor phylogenetics uses differences between single cells within tumors, gaining valuable information about intra-tumor heterogeneity but allowing only a few markers per cell. An alternative approach uses tissue-wide measures of whole tumors to provide a detailed picture of averaged tumor state but at the cost of losing information about intra-tumor heterogeneity.</p> <p>Results</p> <p>The present work applies "unmixing" methods, which separate complex data sets into combinations of simpler components, to attempt to gain advantages of both tissue-wide and single-cell approaches to cancer phylogenetics. We develop an unmixing method to infer recurring cell states from microarray measurements of tumor populations and use the inferred mixtures of states in individual tumors to identify possible evolutionary relationships among tumor cells. Validation on simulated data shows the method can accurately separate small numbers of cell states and infer phylogenetic relationships among them. Application to a lung cancer dataset shows that the method can identify cell states corresponding to common lung tumor types and suggest possible evolutionary relationships among them that show good correspondence with our current understanding of lung tumor development.</p> <p>Conclusions</p> <p>Unmixing methods provide a way to make use of both intra-tumor heterogeneity and large probe sets for tumor phylogeny inference, establishing a new avenue towards the construction of detailed, accurate portraits of common tumor sub-types and the mechanisms by which they develop. These reconstructions are likely to have future value in discovering and diagnosing novel cancer sub-types and in identifying targets for therapeutic development.</p