Doctors' attitudes to patient occupation information in four hospital specialties.

BACKGROUND: Although we do not know how often doctors enquire about their patients' work, evidence suggests that occupation is often not recorded in clinical notes. There is a lack of research into doctors' views on the importance of patient occupation or their educational needs in this area. AIMS: To assess doctors' attitudes to using patient occupation information for care-planning and to determine doctors' need for specific training in occupational health. METHODS: We undertook a cross-sectional survey of doctors in cardiology, obstetrics and gynaecology, oncology and orthopaedics. Our questionnaire explored attitudes of the doctors to asking patients about their occupational status, their training and competency to do so, and their training needs in occupational health. RESULTS: The response rate was 42/46 (91%). Obstetrics and gynaecology 6/9 (67%) and oncology doctors 3/6 (50%) reported enquiring about the nature of patients' occupations' 'most of the time'/'always' and that it rarely influenced clinical decisions. This contrasted with orthopaedic doctors 12/12 (100%) and cardiology doctors 14/15 (93%). Although 19/42 (45%) participants felt it was important to ask patients their occupation, only 10/42 (24%) 'always' asked patients about their work. The majority of participants 29/41 (71%) reported receiving no training in occupational health, but 37/42 (88%) considered that some training would be useful. CONCLUSIONS: Training on the importance of occupation and its' role as a clinical outcome in care-planning, might help doctors feel more competent in discussing the impact of health on work with patients.

The surgical management of sarcomas of the chest wall: A 13-year single institution experience.

INTRODUCTION: Chest wall sarcomas are rare. Resection and reconstruction pose significant anatomical and functional challenges. We present our experience of managing these tumours as plastic surgeons working within a specialist sarcoma MDT. METHODS: All cases of chest wall sarcoma in which a plastic surgeon took part were analysed (2003-2016). Tumours of the breast, abdomen and groin were excluded. Demographics, surgical details and outcomes were analysed. RESULTS: Forty-seven patients were identified. Median age at presentation was 61 years (range 7-91). Thirty-three were male and 14 were female. Chondrosarcoma (n = 16) was the most frequently occurring tumour, followed by myxofibrosarcoma (n = 6), leiomyosarcoma (n = 5) and unclassified sarcomas (n = 5). The majority of tumours were of high (n = 16) or intermediate grade (n = 17) histologically. Wide local excision was carried out in all cases. Twenty-two cases required a mesh and cement reconstruction of the chest wall. Soft tissue reconstruction involved pedicled LD flap +-skin graft (n = 17), direct closure (n = 13), pedicled VRAM (n = 7), free ALT flap (n = 6), and others (n = 4). Clear resection margins were achieved in 32 patients (68%). Fourteen patients underwent adjuvant radiotherapy and four adjuvant chemotherapy. Nine patients (19%) developed a local recurrence, and the median duration from resection to recurrence was 17 months (range 3-72). Nine patients (19%) developed metastasis. Eleven patients died (23.4%), and the median duration of survival 30 months (range 3-92). Thirty-six patients remain well, with a median duration of follow up 57.5 months (range 6-141). Estimated 5 year disease specific survival is 74.2%. CONCLUSION: Plastic surgeons have a vital role in the management of chest wall sarcomas. We present a reconstructive algorithm, which has enabled us to achieve good oncological and functional outcomes and a low complication profile .

Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, causing pneumonia, septicemia, and meningitis. In recent years, it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential for hsdS inversions. In addition, mutants of homologous recombination pathways also undergo hsdS inversions. In this work, we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types, including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by transcriptome sequencing (RNAseq), confirms that the level of creX expression is impacted by these genomic rearrangements. In addition, we have shown that the frequency of hsdS recombination is temperature dependent. Most importantly, we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS, and SPD_0921 are not involved in spnIII recombination, suggesting that a currently unknown mechanism is responsible for the recombination of these phase-variable type I systems.IMPORTANCEStreptococcus pneumoniae is a leading cause of pneumonia, septicemia, and meningitis. The discovery that genetic rearrangements in a type I restriction-modification locus can impact gene regulation and colony morphology led to a new understanding of how this pathogen switches from harmless colonizer to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction-modification enzyme, occur across many different pneumococcal serotypes and sequence types and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal gene regulation and the need for further investigation of mechanisms of site-specific recombination.

Mutation and Selection in Bacteria: Modelling and Calibration.

Temporal evolution of a clonal bacterial population is modelled taking into account reversible mutation and selection mechanisms. For the mutation model, an efficient algorithm is proposed to verify whether experimental data can be explained by this model. The selection-mutation model has unobservable fitness parameters, and, to estimate them, we use an Approximate Bayesian Computation algorithm. The algorithms are illustrated using in vitro data for phase variable genes of Campylobacter jejuni.

Coadministration of the Campylobacter jejuni N-Glycan-Based Vaccine with Probiotics Improves Vaccine Performance in Broiler Chickens.

Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni "supercolonizer mutant" with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.IMPORTANCECampylobacter jejuni is a common cause of human diarrheal disease worldwide and is listed by the World Health Organization as a high-priority pathogen. C. jejuni infection typically occurs through the ingestion of contaminated chicken meat, so many efforts are targeted at reducing C. jejuni levels at the source. We previously developed a vaccine that reduces C. jejuni levels in egg-laying chickens. In this study, we improved vaccine performance in meat birds by supplementing the vaccine with probiotics. In addition, we demonstrated that C. jejuni colonization levels in chickens are negatively correlated with the abundance of clostridia, another group of common gut microbes. We describe new methods for vaccine optimization that will assist in improving the C. jejuni vaccine and other vaccines under development.

Long-term dinoflagellate culture performance in a commercial photobioreactor: Amphidinium carterae case.

The aim of this work was to study the culture performance of a dinoflagellate in a commercial photobioreactor. The results obtained during this long-term experiment allow to confirm that Amphidinium carterae is a promising dinoflagellate that can be exploited successfully in closed systems, in semi-continuous mode in indoor and outdoor environments. The average results in an indoor 5cm light-path 320L photobioreactor were, in terms of specific growth rate (0.29d(-1)), duplication time (3.1d(-1)) and dry biomass productivity (78mgL(-1)d(-1)). Specific compounds production was found including ω3 and ω6 fatty acids and, pigments (Peridinin, ß-carotene). These promising results, besides unique characteristics found during the exploitation period such as resistance to mechanical stress, self-control of contaminant organisms, and quick cells aggregation when the culture is not in turbulence conditions, makes A. carterae one of the new target species suitable for commercially exploitation on an industrial scale.

Evaluation of batch and semi-continuous culture of Porphyridium purpureum in a photobioreactor in high latitudes using Fourier Transform Infrared spectroscopy for monitoring biomass composition and metabolites production.

The culture strategy (batch or semi-continuous) was evaluated for biomass and metabolite formation in Porphyridium purpureum cultures in higher latitudes (>50° N). FTIR was used technology to characterise macromolecule biomass composition and the quality of the metabolites produced. Semi-continuous culture was found to be the most feasible strategy to develop microalgal biomass production facilities in higher latitudes, due to their average results in terms of growth rate (0.27 day(-1)), duplication time (2.5-4 days), maximum cell density achieved (1.43*10(7) cells m L(-1)), biomass productivity of 47.04 mg L(-1) day(-1) and an exopolysaccharides production of 2.1 g L(-1). FTIR technology applied to microalgal production is a valuable and reliable tool to determine on a daily basis not just the evolution of macromolecules composition (lipids, carbohydrates and proteins) but also for the characterisation of the metabolites produced such as phycoerythrin or exopolysaccharides in P. purpureum cultures.

Broad conditions favor the evolution of phase-variable loci.

UNLABELLED: Simple sequence repeat (SSR) tracts produce stochastic on-off switching, or phase variation, in the expression of a panoply of surface molecules in many bacterial commensals and pathogens. A change to the number of repeats in a tract may alter the phase of the translational reading frame, which toggles the on-off state of the switch. Here, we construct an in silico SSR locus with mutational dynamics calibrated to those of the Haemophilus influenzae mod locus. We simulate its evolution in a regimen of two alternating environments, simultaneously varying the selection coefficient, s, and the epoch length, T. Some recent work in a simpler (two-locus) model suggested that stochastic switching in a regimen of two alternating environments may be evolutionarily favored only if the selection coefficients in the two environments are nearly equal ("symmetric") or selection is very strong. This finding was puzzling, as it greatly restricted the conditions under which stochastic switching might evolve. Instead, we find agreement with other recent theoretical work, observing selective utility for stochastic switching if the product sT is large enough for the favored state to nearly fix in both environments. Symmetry is required neither in s nor in sT. Because we simulate finite populations and use a detailed model of the SSR locus, we are also able to examine the impact of population size and of several SSR locus parameters. Our results indicate that conditions favoring evolution and maintenance of SSR loci in bacteria are quite broad. IMPORTANCE: Bacteria experience frequent changes of environment during the infection cycle. One means to rapidly adapt is stochastic switching: a bacterial lineage will stochastically produce a variety of genotypes, so that some descendants will survive if the environment changes. Stochastic switching mediated by simple sequence repeat (SSR) loci is widespread among bacterial commensals and pathogens and influences critical interactions with host surfaces or immune effectors, thereby affecting host persistence, transmission, and virulence. Here, we use the most detailed in silico model of an SSR locus to date, with its phase variation calibrated to match the mod locus of Haemophilus influenzae. The type III restriction-modification system encoded by mod participates in the regulation of multiple other genes; thus, SSR-mediated phase variation of mod has far-reaching cis-regulatory effects. This coupling of phase-variable switching to complex phenotypic effects has been described as the "phasevarion" and is central to understanding the infection cycle of bacterial commensals and pathogens.

Autologous olfactory ensheathing cell transplantation in human paraplegia: a 3-year clinical trial.

Olfactory ensheathing cells show promise in preclinical animal models as a cell transplantation therapy for repair of the injured spinal cord. This is a report of a clinical trial of autologous transplantation of olfactory ensheathing cells into the spinal cord in six patients with complete, thoracic paraplegia. We previously reported on the methods of surgery and transplantation and the safety aspects of the trial 1 year after transplantation. Here we address the overall design of the trial and the safety of the procedure, assessed during a period of 3 years following the transplantation surgery. All patients were assessed at entry into the trial and regularly during the period of the trial. Clinical assessments included medical, psychosocial, radiological and neurological, as well as specialized tests of neurological and functional deficits (standard American Spinal Injury Association and Functional Independence Measure assessments). Quantitative test included neurophysiological tests of sensory and motor function below the level of injury. The trial was a Phase I/IIa design whose main aim was to test the feasibility and safety of transplantation of autologous olfactory ensheathing cells into the injured spinal cord in human paraplegia. The design included a control group who did not receive surgery, otherwise closely matched to the transplant recipient group. This group acted as a control for the assessors, who were blind to the treatment status of the patients. The control group also provided the opportunity for preliminary assessment of the efficacy of the transplantation. There were no adverse findings 3 years after autologous transplantation of olfactory ensheathing cells into spinal cords injured at least 2 years prior to transplantation. The magnetic resonance images (MRIs) at 3 years showed no change from preoperative MRIs or intervening MRIs at 1 and 2 years, with no evidence of any tumour of introduced cells and no development of post-traumatic syringomyelia or other adverse radiological findings. There were no significant functional changes in any patients and no neuropathic pain. In one transplant recipient, there was an improvement over 3 segments in light touch and pin prick sensitivity bilaterally, anteriorly and posteriorly. We conclude that transplantation of autologous olfactory ensheathing cells into the injured spinal cord is feasible and is safe up to 3 years of post-implantation, however, this conclusion should be considered preliminary because of the small number of trial patients.

The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases.

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5'-AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild-type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17-38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.

Bacterial genetic loci implicated in the Pseudomonas putida GR12-2R3--canola mutualism: identification of an exudate-inducible sugar transporter.

Pseudomonas putida GR12-2R3 promotes the emergence and growth of diverse plant species. Analyses of TnphoA insertion mutations are revealing bacterial characteristics pertinent to the plant-microbe interaction. Pseudomonas putida PG269 is a TnphoA insertion derivative of GR12-2R3 that expresses canola seed exudate-inducible alkaline phosphatase (PhoA) activity. It promoted the growth of canola roots, as well as strain GR12-2R3, and outgrew its parent when they were cocultured in the presence of canola roots or in liquid seed exudate medium. (In contrast, mutant PG126 failed to promote canola root growth and was outgrown by its parent strain.) The PhoA activity of strain PG269 was induced by glucosamine and other sugars; glucosamine inhibited the growth of strain GR12-2R3 and stimulated the growth of strain PG269. Strain PG269 contained two TnphoA insertions: seiA1::TnphoA and seiB1::TnphoA. Strain PG312, which contained only insertion seiA1::TnphoA, shared all aspects of the PG269 phenotype, except the ability to outcompete strain GR12-2R3 during coculture. Insertion seiA1::TnphoA interrupted an open reading frame related in sequence to members of the MalF family of sugar transporter subunits. The PhoA-inducing fraction of canola seed exudate was hydrophilic, low in molecular weight, and heat stable. It cochromatographed with basic amino acids and amino sugars, and was inactivated by strains GR12-2R3 and PG269. Gene seiA may encode a subunit of an ABC transporter with broad specificity for glucose and related sugars whose expression can be induced by exudate sugars.

Vaccinia virion protein VP8, the 25 kDa product of the L4R gene, binds single-stranded DNA and RNA with similar affinity.

Vaccinia virus protein VP8 is a 25 kDa product of the L4R gene and is an abundant virion protein that binds single-stranded (ss) and double-stranded (ds) DNA. Binding of ssDNA is preferred at high salt concentrations. Using a recombinant 25 kDa L4R (rL4R) protein and a gel mobility shift assay with radiolabelled oligonucleotides, the Kd for a 45mer oligonucleotide was determined to be 2 nM. The Kd was unaltered by 50 mM KCl but was reduced 35-fold by 100 mM KCl. Multiple rL4R molecules bound to a single 45mer oligonucleotide, and using oligonucleotides of different lengths it was calculated that one rL4R molecule bound every 17 nt. Binding to ssDNA was competed by both deoxyribo- and ribo-polynucleotides. RNA binding was observed for both rL4R and native VP8, purified from virions, using a gel mobility shift with a radiolabelled ssRNA of 130 nt. The Kd of rL4R for this ssRNA substrate was 3 nM in the absence of salt and binding was positively cooperative. The potential roles of L4R protein in vaccinia virus early transcription are discussed.

Retention in situ and spectral analysis of fluorescent vacuole components in sections of plant tissues.

The contents of plant vacuoles vary in different organs and with the health of the plant, but little is known of the cell-to-cell distribution of soluble organic compounds within plant tissues. Soluble fluorescent phenolic compounds can be immobilized in plant tissues using an anhydrous freeze-substitution and resin embedment process. The vacuolar fluorescence can be characterized in fluorescence photomicrographs for variations in color and intensity, or more quantitatively with spectra obtained using a microspectrofluorometer. This is demonstrated here in freeze-substituted roots and leaves of soybean. Excitation and emission spectra of individual vacuoles can be compared with spectra of pure compounds to form profiles of the varied phenolic contents of plant vacuoles. Such analyses will add an important anatomical dimension to the study of plant defense and stress responses.

Stimulation of vaccinia virion DNA helicase I8R, but not A18R, by a vaccinia core protein L4R, an ssDNA binding protein.

Four DNA-dependent ATPases were purified from vaccinia virions and tested for DNA helicase activity on two dsDNA substrates. ATPases D6R and D11L were inactive on both substrates, A18R unwound the substrate with a short 20 bp duplex region and 18R unwound both substrates. In addition, the 18R protein was stimulated to unwind longer DNA duplexes by a 25 kDa protein purified from vaccinia virions, representing the cleaved product of the L4R gene, an ssDNA binding protein. Purified recombinant 25 kDa L4R protein also stimulated 18R DNA helicase activity and maximum activity was observed only when there were < 13 nucleotides of DNA per molecule of L4R protein. The DNA helicase activity of the A18R protein was not stimulated by either recombinant 25 kDa L4R protein or by an E. coli ssDNA binding protein.

Vaccinia virion protein I8R has both DNA and RNA helicase activities: implications for vaccinia virus transcription.

A nucleic acid-dependent ATPase was purified from vaccinia virions and shown to have both DNA:DNA and RNA:RNA helicase activities. This is only the third helicase to be identified that can unwind both DNA and RNA duplexes. The DNA helicase activity copurified with nucleoside triphosphate phosphohydrolase II (NPHII), an RNA helicase encoded by gene I8R (S. Shuman, Proc. Natl. Acad. Sci. USA 89:10935-10939, 1992). Immunodepletion with two antisera to NPHII and analysis of recombinant NPHII protein (C. H. Gross and S. Shuman, J. Virol. 69:4727-4736, 1995) confirmed that the DNA helicase activity was encoded by the I8R gene. The I8R DNA helicase unwound DNA in a 3'-to-5' direction only, unwound duplexes of 35 bp but not 45 bp, and could be stimulated to unwind longer duplexes by the Escherichia coli single-stranded DNA-binding protein. DNA helicase activity was not stimulated by salt and was sensitive to 100 mM NaCl or KCl. The I8R protein has amino acid similarity to human RNA helicase A and to nuclear DNA helicase II, a bovine DNA and RNA helicase. On the basis of the phenotype of I8R temperature-sensitive mutants, it was suggested that the I8R protein is not required for DNA replication but might aid in the extrusion of early mRNA from the virus core. The DNA helicase activity of the I8R protein allows another interpretation of the mutant phenotype, namely, that the I8R DNA helicase activity is required for initiation of early transcription from within vaccinia virions.