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N -acyl lipids are important mediators of several biological processes including immune function and stress response. To enhance the detection of N -acyl lipids with untargeted mass spectrometry-based metabolomics, we created a reference spectral library retrieving N -acyl lipid patterns from 2,700 public datasets, identifying 851 N -acyl lipids that were detected 356,542 times. 777 are not documented in lipid structural databases, with 18% of these derived from short-chain fatty acids and found in the digestive tract and other organs. Their levels varied with diet, microbial colonization, and in people living with diabetes. We used the library to link microbial N -acyl lipids, including histamine and polyamine conjugates, to HIV status and cognitive impairment. This resource will enhance the annotation of these compounds in future studies to further the understanding of their roles in health and disease and highlight the value of large-scale untargeted metabolomics data for metabolite discovery.

The pharmaceutical industry is increasingly embracing laboratory automation to enhance experimental efficiency and operational resilience, particularly through the integration of automated liquid handlers (ALHs). This paper explores the integration of the low-cost Opentrons OT-2 liquid handling robot with F. Hoffmann-La Roche AG's in-house workflow orchestration software, AutoLab, to overcome barriers to lab automation. By leveraging the OT-2's development-oriented interfaces and AutoLab's modular architecture, we achieved a user-friendly, cost-efficient, and flexible automation solution that aligns with FAIR (findable, accessible, interoperable, reusable) data principles. We demonstrate an advanced workflow development methodology, utilizing the software architecture, that facilitates the creation of two flexible pipetting protocols and medium complexity assays. This deep integration approach diminishes the learning curve for novice users while simultaneously enhancing the overall efficiency and reliability of the experimental workflow. Our findings suggest that such integrations can significantly mitigate the challenges associated with lab automation, including cost, complexity, and adaptability, paving the way for more accessible and robust automated systems in pharmaceutical research., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: All authors are employees or contractors at F. Hoffmann-La Roche AG If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Motivation: Sanger sequencing of taxonomic marker genes (e.g. 16S/18S/ITS/rpoB/cpn60) represents the leading method for identifying a wide range of microorganisms including bacteria, archaea, and fungi. However, the manual processing of sequence data and limitations associated with conventional BLAST searches impede the efficient generation of strain libraries essential for cataloging microbial diversity and discovering novel species., Results: isolateR addresses these challenges by implementing a standardized and scalable three-step pipeline that includes: (1) automated batch processing of Sanger sequence files, (2) taxonomic classification via global alignment to type strain databases in accordance with the latest international nomenclature standards, and (3) straightforward creation of strain libraries and handling of clonal isolates, with the ability to set customizable sequence dereplication thresholds and combine data from multiple sequencing runs into a single library. The tool's user-friendly design also features interactive HTML outputs that simplify data exploration and analysis. Additionally, in silico benchmarking done on two comprehensive human gut genome catalogues (IMGG and Hadza hunter-gather populations) showcase the proficiency of isolateR in uncovering and cataloging the nuanced spectrum of microbial diversity, advocating for a more targeted and granular exploration within individual hosts to achieve the highest strain-level resolution possible when generating culture collections., Availability and Implementation: isolateR is available at: https://github.com/bdaisley/isolateR., (© The Author(s) 2024. Published by Oxford University Press.)

Introduction: Cyanobacterial blooms are increasingly common in freshwater sources used for swimming and other recreational water contact activities in Canada. Many species of cyanobacteria can produce toxins that affect human and animal health, but there are limited data on the risk of illness associated with water contact at impacted beaches., Methods and Analysis: This study will investigate the incidence of recreational water illness due to exposure to cyanobacterial blooms and their toxins in four targeted and popular freshwater beaches in Ontario, Manitoba and Nova Scotia, Canada. A prospective cohort design and One Health approach will be used. On-site recruitment of recreational water users will be conducted at two beaches per year during the summers of 2024 and 2025. The population of interest includes recreational water users of any age and their pet dogs. After enrolment, an in-person survey will determine beach exposures and confounding factors, and a 3-day follow-up survey will ascertain any acute illness outcomes experienced by participants or their dogs. The target sample size is 2500 recreational water users. Water samples will be taken each recruitment day and analysed for cyanobacterial indicators (pigments), cell counts and toxin levels. Bayesian regression analysis will be conducted to estimate the association with water contact, cyanobacterial levels and risks of different acute illness outcomes., Ethics and Dissemination: This study has been approved by the Toronto Metropolitan University Research Ethics Board (REB 2023-461). Study results will be published in a peer-reviewed journal and as infographics on a project website., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Human milk oligosaccharides (HMOs) are a set of complex carbohydrates and the third largest solid component of human milk, after lactose and lipids. To date, over 150 HMOs have been identified and the diversity of structures produced by lactating women is influenced by maternal genetics as well as other maternal, infant, and environmental factors. While the concentrations of individual HMOs have been shown to vary between individuals and throughout the course of lactation, the variability of HMO concentration profiles following different pregnancies occurring in the same woman is presently unknown. As such, the objective of this study was to compare HMO concentrations in human milk samples provided by the same women ( n = 34) following repeat pregnancies. We leveraged existing human milk samples and metadata from the UC San Diego Human Milk Research Biorepository (HMB) and measured the concentrations of the 19 most abundant HMOs using high-performance liquid chromatography with fluorescence detection (HPLC-FL). By assessing dissimilarities in HMO concentration profiles, as well as concentration trends in individual structures between pregnancies of each participant, we discovered that HMO profiles largely follow a highly personalized and predictable trajectory following different pregnancies irrespective of non-genetic influences. In conclusion, this is the first study to assess the interactions between parity and time following delivery on variations in HMO compositions.

Treatment advances have greatly improved survival, but myeloma is among the worst of all cancers for delayed diagnosis, causing serious morbidities and early deaths. This delay is largely because the symptom profile of myeloma has very low specificity, and in primary care, myeloma is rare. However, initiating the journey to diagnosis simply requires considering myeloma and sending blood to test for monoclonal immunoglobulin. Laboratory tests reliably detect monoclonal immunoglobulin, which is present in 99% of myeloma cases, so why do health care systems have such a problem with delayed diagnosis? The Myeloma UK early diagnosis programme has brought together diverse expertise to investigate this problem, and this article was prepared by the programme's working group for laboratory best practice. It reviews evidence for test requesting, analysis and reporting, for which there is large variation in practice across the United Kingdom. It presents a 'GP Myeloma diagnostic tool' and how it can be integrated into laboratory practice alongside a laboratory best practice tool. It proposes improved requesting and integration with haematology services for reporting and interpretation. Here the laboratory has a central role in creating efficient and cost-effective pathways for appropriate and timely bone marrow examination for myeloma diagnosis., (© 2024 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Canada experienced a wave of HPAI H5N1 outbreaks in the spring of 2022 with millions of wild and farmed birds being infected. Seabird mortalities in Canada have been particularly severe on the Atlantic Coast over the summer of 2022. Over 7 million birds have been culled in Canada, and outbreaks continue to profoundly affect commercial bird farms across the world. This new H5N1 virus can and has infected multiple mammalian species, including skunks, foxes, bears, mink, seals, porpoises, sea lions, and dolphins. Viruses with mammalian adaptations such as the mutations PB2-E627K, E627V, and D701N were found in the brain of various carnivores in Europe and Canada. To date this specific clade of H5N1 virus has been identified in less than 10 humans. At the ground level, awareness should be raised among frontline practitioners most likely to encounter patients with HPAI., Competing Interests: S Forgie reports support from AMMI Canada to attend their board meeting in Toronto, Canada, April 2023, and a previous leadership role at AMMI Canada. TF Hatchette reports grants from GSK/Pfizer, Dalhousie Pharmacy Fund, Drug Evaluation Alliance of NS, CIHR/CIRN, NSERC, COVID-19 Immunity Task Force, NIH, Genome Atlantic, Nova Scotia COVID-19 Health Research Coalition Funding Competition, NSHA Research Fund, and an IWK Project Grant; consulting fees from RockDoc; speaker fees from Sanofi; and past leadership roles at AMMI Canada and Canadian Public Health Laboratory Network. D Yamamura reports a previous leadership role at AMMI Canada., (© Association of Medical Microbiology and Infectious Disease Canada (AMMI Canada), 2023.)

Beans elicit lower glycemic responses (GRs) than other starchy foods, but the minimum effective dose (MED) to reduce GR is unknown. We sought to determine the MED of beans compared to common starchy foods. Overnight-fasted healthy volunteers consumed ¼c (phase 1, n = 24) or ½c (phase 2, n = 18) of black, cranberry, great northern, kidney, navy and pinto beans and corn, rice, pasta and potato (controls), with blood glucose measured before and for 2 h after eating. GRs (incremental areas under the curves, iAUCs) after beans were consumed were compared to those of controls by ANOVA followed by Dunnett's test. To qualify for MED, beans had to elicit an effective reduction in GR, defined as a statistically significant reduction in iAUC of ≥20% (i.e., a relative glycemic response, RGR, ≤80). Outcomes from in vitro digestion were compared with in vivo RGR. Both doses of all six beans effectively reduced GR versus all four starchy controls, except for ¼c and ½c cranberry and pinto vs. corn, ¼c great northern and navy vs. corn and ¼c navy and pinto vs. potato. MED criteria were met for 18 comparisons of the ¼c servings, with four of the remaining six met by the ½c servings. The overall mean ± SEM RGR vs. controls was similar for the ¼c and ½c servings: 53 ± 4% and 56 ± 3%, respectively. By multiple regression analysis, RGR = 23.3 × RDS + 8.3 × SDS - 20.1 × RS + 39.5 × AS - 108.2 (rapidly digested starch, p < 0.001; slowly digested starch, p = 0.054; resistant starch, p = 0.18; available sugars, p = 0.005; model r = 0.98, p = 0.001). RGR correlated with in vitro glucose release (r = 0.92, p < 0.001). The MED of beans is ¼ cup. For n = 30 comparisons ( n = 24 beans vs. controls, n = 6 controls vs. each other), an effective reduction in GR was predicted from in vitro carbohydrate analysis with 86% sensitivity and 100% specificity.

Objectives: Patients with myeloma often face significant diagnostic delay, with up to one-third of UK patients diagnosed after an emergency presentation (EP). Compared with other routes, patients presenting as an emergency have more advanced disease, increased complications, and poorer prognosis., Methods: An economic model was developed using a decision-tree framework and lifetime time horizon to estimate costs related to different presentation routes (EP, general practitioner [GP] 2-week wait, GP urgent, GP routine, and consultant to consultant) for UK patients diagnosed as having myeloma. After diagnosis, patients received one of 3 first-line management options (observation, active treatment, or end-of-life care). Inputs were derived from UK health technology assessments and targeted literature reviews, or based on authors' clinical experience where data were unavailable. Active treatment, complication, and end-of-life care costs were included., Results: The average per-patient cost of treating myeloma (across all routes) was estimated at £146 261. The average per-patient cost associated with EP (£152 677) was the highest; differences were minimal compared with GP 2-week wait (£149 631) and consultant to consultant (£147 237). GP urgent (£140 025) and GP routine (£130 212) were associated with marginally lower costs. Complication (£42 252) and end-of-life care (£11 273) costs were numerically higher for EP than other routes (£25 021-£38 170 and £9772-£10 458, respectively)., Conclusions: An economic benefit may be associated with earlier diagnosis, gained via reduced complication and end-of-life care costs. Strategies to expedite myeloma diagnosis and minimize EPs have the potential to improve patient outcomes and may result in long-term savings that could offset any upfront costs associated with their implementation., (Copyright © 2023 International Society for Health Economics and Outcomes Research. Published by Elsevier Inc. All rights reserved.)

The human colon is inhabited by a complex community of microbes. These microbes are integral to host health and physiology. Understanding how and when the microbiome causally influences host health will require microbiome models that can be tightly controlled and manipulated. While in vivo models are unrivalled in their ability to study host-microbial interplay, in vitro models are gaining in popularity as methods to study the ecology and function of the gut microbiota, and benefit from tight controllability and reproducibility, as well as reduced ethical constraints. In this set of protocols, we describe the Robogut, a single-stage bioreactor system designed to replicate the conditions of the distal human colon, to culture whole microbial communities derived from stool and/or colonic biopsy samples, with consideration of methods to create culture medium formulations and to build, run, and sample the bioreactor apparatus. Cleaning and maintenance of the bioreactor system are also described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Growth medium preparation Support Protocol 1: Preparing medium supplements Basic Protocol 2: Preparing the bioreactor vessels Support Protocol 2: Making acid and base bottles Support Protocol 3: Preparing the effluent bottles Support Protocol 4: Making acid solution Support Protocol 5: Making base solution Basic Protocol 3: Preparing inoculum and inoculating bioreactors Alternate Protocol 1: Preparing inoculum less than 0.5% (w/v) of vessel volume Alternate Protocol 2: Preparing synthetic community aliquots and inoculation via the septum Alternate Protocol 3: Preparing inoculum from a tissue sample Basic Protocol 4: Sampling the bioreactor vessel Basic Protocol 5: Harvesting bioreactor vessel contents at end of experiment Support Protocol 6: Cleaning and sterilizing sampling needles Basic Protocol 6: Cleaning the bioreactor vessel Support Protocol 7: Cleaning bioreactor support bottles., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

To address concerns about the biocompatibility of novel phenolic immobilization-based food preservatives, their impact on the composition and metabonomic profile of a defined community of human gut microbiota was evaluated. Three phenolics (eugenol, vanillin and ferulic acid) presented in two forms (free or immobilized on different supports) were tested at two concentration levels (0.5 and 2 mg/mL). Free eugenol was the phenolic with the greatest impact on gut microbiota, with a remarkable increase in the abundance of Lachnospiraceae and Akkermansiaceae families. In contrast, immobilized phenolics produced an increase in the abundance of Bacteroides with a reduction in the ratio of Firmicutes to Bacteroidetes. The metabonomic profile was also affected by free and immobilized phenolics differently in terms of fermentation by-products and phenolic biotransformation metabolites. Thus the results suggest the importance of evaluating the impact of new compounds or materials added to food on human gut microbiota and their potential use to modulate microbiota composition., Competing Interests: Declaration of Competing Interest The author declare the following financial interests/personal relationships which may be considered as potential competing interests: Emma Allen-Vercoe is the CSO and co-founder of NuBiyota LLC, a company that is developing human gut microbiota-based live microbial products to treat a range of indications. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The microbes that reside within the equine hindgut create a complex and dynamic ecosystem. The equine hindgut microbiota is intimately associated with health and, as such, represents an area which can be beneficially modified. Synbiotics, supplements that combine probiotic micro-organisms with prebiotic ingredients, are a potential means of influencing the hindgut microbiota to promote health and prevent disease. The objective of the current study was to evaluate the influence of an equine probiotic/prebiotic supplement on characteristics of the microbiota and metabolite production in vitro. Equine cecal fluid and fecal material were collected from an abattoir in QC, CAN. Five hundred milliliters of cecal fluid was used to inoculate chemostat vessels maintained as batch fermenters (chemostat cecal, N = 11) with either 0 g (control) or 0.44 g of supplement added at 12 h intervals. One hundred milliliters of cecal fluid (anaerobic cecal, N = 15) or 5% fecal slurry (anaerobic fecal, N = 6) were maintained in an anaerobic chamber with either 0 g (control) or 0.356 g of supplement added at the time of vessel establishment. Samples were taken from vessels at vessel establishment (0), 24, or 48 h of incubation. Illumina sequencing of the V4 region of the 16S rRNA gene and bioinformatics were performed for microbiome analysis. Metabolite data was obtained via NMR spectroscopy. All statistical analyses were run in SAS 9.4. There was no effect of treatment at 24 or 48h on alpha or beta diversity indices and limited taxonomic differences were noted. Acetate, propionate, and butyrate were higher in treated compared to untreated vessels in all methods. A consistent effect of supplementation on the metabolic profile with no discernable impact on the microbiota of these in vitro systems indicates inoculum microbe viability and a utilization of the provided fermentable substrate within the systems. Although no changes within the microbiome were apparent, the consistent changes in metabolites indicates a potential prebiotic effect of the added supplement and merits further exploration., (© The Author(s) 2023. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In this work, the influence of different forms of presentation of chitosan in the human gut microbiota with a defined bacterial community was evaluated. First, the susceptibility of individual gut bacterial isolates against chitosan was studied within a concentration range between 0.125 and 1 mg/mL. Then, the impact of chitosan (0.25 and 1 mg/mL) on a defined human gut microbial ecosystem was studied by metagenomic and metabonomic analyses. The results showed that chitosan in its free form had a high impact on individual isolates with a minimum inhibitory concentration below 1 mg/mL for most of the strains studied. In comparison, chitosan immobilized in the different carriers displayed a diverse effect on gut microbiota. The most susceptible strains were Agathobacter rectalis strain 16-6-I 1 FAA, Clostridium spiroforme strain 16-6-I 21 FAA and Mediterraneibacter faecis strain 16-6-I 30 FAA. The impact of the different modes of presentation of chitosan was strain-specific and species-specific when compared to results obtained from analysis of other strains within the genera Agathobacter, Clostridium and Mediterraneibacter, and therefore a study using a defined ecosystem was needed to extrapolate the results. Significant decreases in defined community richness and diversity and changes in metabolic profile were observed after exposure to free chitosan. Free chitosan produced significant reductions in the abundance of the genera Lachnoclostridium, Anaerotignum, Blautia, Enterococcus, Eubacterium and Ruthenibacterium together with a slight decrease of the production of SCFAs, among other fermentation by-products. The immobilized chitosan significantly alleviated the impact caused by the antimicrobial polymer and significantly increased the relative abundance of the Bacteroidetes phylum compared to free chitosan. These results suggest the significance of assessing the impact of new ingredients and materials included in food on the human gut microbiota with models that simulate the gastrointestinal environment, such as in vitro bioreactor systems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The equine gastrointestinal (GI) microbiota is intimately related to the horse. The objective of the current study was to evaluate the microbiome and metabolome of cecal inoculum maintained in an anaerobic chamber or chemostat batch fermenter, as well as the fecal slurry maintained in an anaerobic chamber over 48 h. Cecal and fecal content were collected from healthy adult horses immediately upon death. Cecal fluid was used to inoculate chemostat vessels (chemostat cecal, n = 11) and vessels containing cecal fluid (anaerobic cecal, n = 15) or 5% fecal slurry (anaerobic fecal, n = 6) were maintained in an anaerobic chamber. Sampling for microbiome and metabolome analysis was performed at vessel establishment (0 h), and after 24 h and 48 h of fermentation. Illumina sequencing was performed, and metabolites were identified via nuclear magnetic resonance (NMR). Alpha and beta diversity indices, as well as individual metabolite concentrations and metabolite regression equations, were analyzed and compared between groups and over time. No differences were evident between alpha or beta diversity in cecal fluid maintained in either an anaerobic chamber or chemostat. The microbiome of the fecal inoculum maintained anaerobically shifted over 48 h and was not comparable to that of the cecal inoculum. Metabolite concentrations were consistently highest in chemostat vessels and lowest in anaerobic fecal vessels. Interestingly, the rate of metabolite change in anaerobic cecal and chemostat cecal vessels was comparable. In conclusion, maintaining an equine cecal inoculum in either an anaerobic chamber or chemostat vessel for 48 h is comparable in terms of the microbiome. However, the microbiome and metabolome of fecal material is not comparable with a cecal inoculum. Future research is required to better understand the factors that influence the level of microbial activity in vitro, particularly when microbiome data identify analogous communities.

The development of novel food preservatives based on natural antimicrobials such as phenolic compounds is increasing, but their safety should be established before use, including evaluating their impact on the gut microbiota. This work explored the influence of antimicrobial phenolics presented in different forms on selected human gut microbiota members through in vitro susceptibility tests. The bacteria tested exhibited a wide range of susceptibilities to phenolics depending on the molecule structure and mode of administration. Agathobacter rectalis and Clostridium spiroforme, members of the phylum Firmicutes, were the most sensitive strains. Susceptibility was strain- and species-specific, suggesting that it may not be possible to easily extrapolate results across the human microbiome in general. Species of other phyla including Bacteroidetes, Actinobacteria, Proteobacteria and Verrucomicrobia were more resistant than Firmicutes, with growth of some strains even enhanced. Our results provide insights into the biocompatibility of free and immobilized phenolics as potential food additives., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The human gut microbiota is a complex community of prokaryotic and eukaryotic microbes and viral particles that is increasingly associated with many aspects of host physiology and health. However, the classical microbiology approach of axenic culture cannot provide a complete picture of the complex interactions between microbes and their hosts in vivo. As such, recently there has been much interest in the culture of gut microbial ecosystems in the laboratory as a strategy to better understand their compositions and functions. In this review, we discuss the model platforms and methods available in the contemporary microbiology laboratory to study human gut microbiomes, as well as current knowledge surrounding the isolation of human gut microbes for the potential construction of defined communities for use in model systems.

Background: In healthcare, there is increasing recognition of the importance of developing and testing strategies to scale effective interventions. The NHS long-term plan (2019) acknowledges that often a gold standard approach to a problem already exists somewhere within the NHS, however, it has not been replicated widely across the system., Methods: We describe the approach and process measures for national scaling of PROMPT (Practical Obstetric Multi-Professional Training) across 12 obstetric-led maternity units in Wales. PROMPT is an evidence-based training package for local maternity staff, previously associated with improvements in maternal and neonatal outcomes, reduction in litigation related to preventable harm and improved safety culture. PROMPT has previously been disseminated internationally using a train-the-trainer model. However, this has been associated with variations in uptake, fidelity and impact. In Wales, the project was supported by Welsh Government, and a structured scaling plan was developed, encompassing ongoing implementation support from a multi-professional team., Results: PROMPT was successfully implemented in all obstetric led units in Wales, with 326 local PROMPT facilitators trained, and 82.5%-100% of maternity staff attended a local PROMPT course in the first 15 months of the project (January 2019-March 2020). All training courses included evidence-based authentic elements, and 93% of courses in the first year (100/107) were supported by a national implementation team, providing coaching, implementation support and quality assurance., Conclusions: Authentically scaling up complex interventions is a significant challenge. To replicate the improved outcomes demonstrated by PROMPT, intervention reach and fidelity must first be demonstrated.In this national scaling project, our scaling methodology led to the successful implementation of PROMPT across all health boards in Wales. Additionally, we demonstrated reduced variation in adoption, reach, timescale and intervention fidelity between maternity units with varying readiness for change, which had been difficult in two previous large-scale PROMPT implementation projects., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: Assisted vaginal birth (AVB) is a complex intervention involving medical devices, comprising multiple components. This complexity creates difficulties when designing and conducting randomised controlled trials (RCTs), in terms of describing, standardising and monitoring the intervention, and accounting for differing clinician expertise. This review examines the reporting standards of complex interventions involving a medical device, in the context of AVB RCTs., Study Design: Searches were undertaken from the start of indexing to March 2021, and limited to RCTs, feasibility and pilot studies including at least one device for AVB. RCTs were selected if they included participants having an AVB with any device, with or without a comparator group. Reporting details were assessed according to the Consolidating Standards of Reporting Trials extension for non-pharmacological treatments (CONSORT-NPT), focusing on intervention descriptions, standardization, adherence and clinician expertise. Screening of abstracts, full-text articles and data extraction was performed by two independent reviewers., Results: Of 4098 abstracts and 83 full-text articles, 39 papers were included, investigating 80 interventions. Twenty-seven different named devices were identified. Intervention descriptions were provided in 20 (55%) papers with varying levels of detail and with only one covering the entire procedure. Standardization of interventions was mentioned in 25 papers (64%). Only eight (21%) papers reported any form of adherence to the intended procedure. Some data regarding expertise were reported in 25 (64%) papers., Conclusions: Despite some compliance with reporting standards, there is a lack of detail regarding intervention description, standardization, adherence and expertise in RCTs of AVB. This creates difficulties in understanding how intervention delivery was intended and what actually occurred. Clearer guidelines for the reporting of invasive procedures and devices are required., Competing Interests: Declaration of Competing Interest EJH, SR, JFC, TJD are employees of North Bristol NHS Trust, which receives funding from PROMPT Maternity Foundation (PMF) to pay part of their salaries. PMF has received funds from Becton Dickinson, manufacturer of the Odon Device to run pre-clinical simulation studies on the device. EL is an employee of the University of Bristol, which receives funding from PMF to pay part of EL’s salary. NSB is an MRC Clinician Scientist., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Competing Interests: Competing interests The BMJ has judged that there are no disqualifying financial ties to commercial companies. The authors declare the following other interests: none. Further details of The BMJ policy on financial interests are here: https://www.bmj.com/about-bmj/resources-authors/forms-policies-and-checklists/declaration-competing-interests

Metabolomic studies allow a deeper understanding of the processes of a given ecological community than nucleic acid-based surveys alone. In the case of the gut microbiota, a metabolic profile of, for example, a fecal sample provides details about the function and interactions within the distal region of the gastrointestinal tract, and such a profile can be generated in a number of different ways. This unit elaborates on the use of 1D 1 H NMR spectroscopy as a commonly used method to characterize small-molecule metabolites of the fecal metabonome (meta-metabolome). We describe a set of protocols for the preparation of fecal water extraction, storage, scanning, measurement of pH, and spectral processing and analysis. We also compare the effects of various sample storage conditions for processed and unprocessed samples to provide a framework for comprehensive analysis of small molecules from stool-derived samples. © 2020 Wiley Periodicals LLC Basic Protocol 1: Extracting fecal water from crude fecal samples Alternate Protocol 1: Extracting fecal water from small crude fecal samples Basic Protocol 2: Acquiring NMR spectra of metabolite samples Alternate Protocol 2: Acquiring NMR spectra of metabolite samples using Bruker spectrometer running TopSpin 3.x Alternate Protocol 3: Acquiring NMR spectra of metabolite samples by semiautomated process Basic Protocol 3: Measuring sample pH Support Protocol 1: Cleaning NMR tubes Basic Protocol 4: Processing raw spectra data Basic Protocol 5: Profiling spectra Support Protocol 2: Spectral profiling of sugars and other complex metabolites., (© 2020 Wiley Periodicals LLC.)

Background: Smoking rather than injecting heroin has become more common over the last 20 years. Although there is an increasing body of evidence describing high levels of chronic obstructive pulmonary disease (COPD) in people who smoke heroin, there is limited evidence documenting the impact of the long-term condition on this population group., Aim: This study aimed to describe the experiences of people who smoke heroin with COPD in Liverpool, UK., Design & Setting: Participants were purposefully sampled for this qualitative study. They included adults enrolled in an opioid replacement clinic run by Addaction in Liverpool, who had already engaged with spirometry testing for COPD as part of a previous study., Method: Semi-structured interviews were performed with participants with spirometrically confirmed COPD in opioid replacement clinics. Data were analysed using a framework analysis approach., Results: Sixteen potential participants were invited to take part in the study, of which 10 agreed and were interviewed. Three themes common to all interviews were identified: functional measures of lung health that impacted on their activities of daily living; inhaler and medication perceptions with erratic use that was not concordant with their prescription; and the impact of difficulties accessing care., Conclusion: These findings, along with previous studies highlighting the prevalence of COPD in this population, warrant efforts to integrate community COPD and opioid replacement services to improve outcomes for this vulnerable population., (Copyright © 2020, The Authors.)

Background: Heroin smokers have high rates of COPD, respiratory morbidity, hospital admission, and mortality. We assessed the natural history of symptoms and lung function in this population over time., Methods: A cohort of heroin smokers with COPD was followed for 18 to 24 months. At baseline and follow-up, respiratory symptoms were measured by the Medical Research Council Dyspnea Scale (MRC) and the COPD Assessment Tool (CAT), and postbronchodilator spirometry was performed. Frequency of health-care-seeking episodes was extracted from routine health records. Parametric, nonparametric, and linear regression models were used to analyze the change in symptoms and lung function over time., Results: Of 372 participants originally recruited, 161 were assessed at follow-up (mean age, 51.0 ± 5.3 years; 74 women [46%]) and 106 participants completed postbronchodilator spirometry. All participants were current or previous heroin smokers, and 122 (75.8%) had smoked crack. Symptoms increased over time (MRC score increased by 0.48 points per year, P < .001; CAT score increased by 1.60 points per year, P < .001). FEV 1 declined annually by 90 ± 190 mL (P < .001). This deterioration was not associated with change in tobacco or heroin smoking status or use of inhaled medications., Conclusions: Heroin smokers experience a high and increasing burden of chronic respiratory symptoms and a decline in FEV 1 that exceeds the normal age-related decline observed among tobacco smokers with COPD and healthy nonsmokers. Targeted COPD diagnostic and treatment services hosted within opiate substitution services could benefit this vulnerable, relatively inaccessible, and underserved group of people., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Assisted vaginal birth (AVB) can markedly improve maternal and neonatal outcomes arising from complications in the second stage of labour. Historically, both forceps and ventouse devices have been used to assist birth; however, they are not without risk and are associated with complications, such as cephalohaematoma, retinal haemorrhage and perineal trauma. As new devices are developed to overcome the limitations of existing techniques, it is necessary to establish their efficacy and effectiveness within randomised controlled trials (RCTs). A major challenge of evaluating complex interventions (ie, invasive procedures/devices used to assist vaginal birth) is ensuring they are delivered as intended. It can be difficult to standardise intervention delivery and monitor fidelity, and account for the varying expertise of clinicians (accoucher expertise). This paper describes the protocol for a systematic review aiming to investigate the reporting of device standardisation, monitoring and training in trials evaluating complex interventions, using AVB as a case study., Methods and Analysis: Relevant keywords and subject headings will be used to conduct a comprehensive search of MEDLINE, Embase, Cochrane Central Register of Controlled Trials, Cumulative Index of Nursing and Allied Health Literature and ClinicalTrials.gov, for RCTs and pilot/feasibility studies evaluating AVB. Abstracts will be screened and full-text articles of eligible studies reviewed for inclusion. Information relating to the following categories will be extracted: standardisation of device use (ie, descriptions of operative steps, including mandatory/flexible parameters), monitoring of intervention delivery (ie, intervention fidelity, confirming that an intervention is delivered as intended) and accoucher expertise (ie, entry criteria for participation, training programmes and previous experience with the device). Risk of bias of included studies will be assessed., Ethics and Dissemination: Ethical approval is not required because primary data will not be collected. Findings will be disseminated by publishing in a peer-reviewed journal and presentations at relevant conferences., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. Published by BMJ.)

Psychology is integral to the concepts and practice of leadership. Leadership models often have their roots in psychological paradigms, making it intrinsically easy for psychologists to grasp and apply them alongside clinical models. Psychologists' 'second-order' skills have been used to good effect in the changing landscape of military mental healthcare: modelling adaptive responses to change, understanding the non-conscious aspects of relationships in hierarchical organisations, working with systems, the ability to diagnose situational requirements and act accordingly, drawing on a range of psychological theories and leadership styles. The clinical psychologist as a professional who can help others 'reframe' a situation or experience is key to their leadership role within healthcare settings, 'enabling others to achieve shared purpose in the face of uncertainty'. The contribution that clinical psychology has made to leadership in Defence over the past 20 years will be illustrated by personal accounts from 'experts by experience'., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Heroin smoking is associated with deprivation, early onset severe emphysema, premature morbidity and mortality, and high use of health care, but individuals engage poorly with traditional health services., Methods: In this cross-sectional study, we screened a population of heroin smokers, prescribed opiate substitution therapy by community drug services, for airway disease. We assessed drug exposure, respiratory symptoms, health status, and COPD prevalence. Subjects completed spirometry, completed Medical Research Council (MRC) Dyspnea Scale, COPD Assessment Tool (CAT) questionnaire, recorded drug exposure, and provided feedback., Results: A total of 753 people (73% of those approached) completed screening, with 260 participants (35%) having COPD using FEV 1 /FVC < 0.7 and 293 (39%) participants having COPD using the lower limit of normal. A further 112 participants (15%) had asthma-COPD overlap (ACO) with features of COPD and asthma. Compared with those with normal spirometry, participants with COPD were more breathless (MRC score 3.1 vs 1.9; P < .001) and had worse health status (CAT score 22.9 vs 13.4; P < .001), respectively. Individuals with COPD had smoked cigarettes (P < .001), heroin (P < .001), and crack (P = .03) for longer and were more likely to still be smoking heroin (P < .01). Feedback was strongly positive, with 92% of respondents happy for other health-care appointments to be colocated with drug key worker appointments., Conclusions: Most heroin smokers had COPD or ACO, most commonly mild to moderate disease. In high-risk areas, screening this population provides an opportunity to reduce symptoms and risk. Anchoring respiratory health screening to drug center appointments delivers high completion and satisfaction and is an appropriate model for screening other hard-to-reach populations., (Copyright © 2018 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.)

Objective: To determine the effects of low-level laser therapy (LLLT) on clinical outcomes in dogs with cranial cruciate ligament disease treated with tibial plateau leveling osteotomy (TPLO)., Study Design: Randomized, placebo-controlled clinical trial. Owners and surgeons (care providers and assessors) were masked., Animals: Ninety-five dogs were admitted for TPLO and assigned to 1 of 2 groups, laser group (LG; n = 51) or placebo group (PG; n = 44)., Methods: Three treatments (laser or placebo) were administered within a 4-day perioperative period. A fourth treatment was recommended and was accepted in 28.4% of cases (LG, n = 14; PG, n = 13). Dogs in the LG group received laser at wavelengths 660 nm red (100 mW) and at 800, 905 and 970 nm infrared (maximum 15 W continuous wave, 20 W peak pulsed wave), administered simultaneously. Dogs in the PG group received placebo laser (660 nm, 4 mW). Other treatments were identical. Outcomes were measured by difference in clinical metrology instruments (Liverpool Osteoarthritis in Dogs and adjusted Canine Orthopedic Index [COI]), osteotomy healing on a radiographic scale, time to cessation of nonsteroidal anti-inflammatory drug administration, and wound healing by owner questionnaire., Results: The only difference detected between groups consisted of a greater improvement in the gait section of the adjusted COI (ACOI) in the LG group (median [interquartile range; IQR] = 6 [4-7.5]) compared with the PG group (median [IQR] = 4 [2-6]; P < .05)., Conclusion: The laser protocol used in this study was associated with a greater improvement in ACOI gait in dogs treated with TPLO but did not improve any other clinical metrology instrument scores or bone healing., Clinical Significance: This study provides some evidence that LLLT may improve the gait of dogs recovering from a TPLO, as assessed by owners., (© 2018 The American College of Veterinary Surgeons.)

The hypocholesterolemic effect of soy is well-documented and this has led to the regulatory approval of a health claim relating soy protein to a reduced risk of cardiovascular disease (CVD). However, soybeans contain additional components, such as isoflavones, lecithins, saponins and fiber that may improve cardiovascular health through independent mechanisms. This review summarizes the evidence on the cardiovascular benefits of non-protein soy components in relation to known CVD risk factors such as hypertension, hyperglycemia, inflammation, and obesity beyond cholesterol lowering. Overall, the available evidence suggests non-protein soy constituents improve markers of cardiovascular health; however, additional carefully designed studies are required to independently elucidate these effects. Further, work is also needed to clarify the role of isoflavone-metabolizing phenotype and gut microbiota composition on biological effect.

Pulses are highly nutritious foods that are included as part of Canada's Food Guide to promote healthful eating, and they have established health benefits that can contribute to the dietary management of diabetes. A review of studies that have examined the effects of pulse consumption on health outcomes, integral to the management of diabetes, provides credible evidence for improvements in glycemic control, reduction of blood lipids and regulation of body weight. Results from acute feeding trials suggest that postprandial blood glucose response is significantly attenuated by a single pulse serving of between three-quarters and 1 cup. At lower doses, pulses attenuate postprandial blood glucose response more than similar amounts of starchy foods. Long-term pulse consumption of 5 cups per week appears to result consistently in improvements in glycemic control. There is high-quality evidence that supports a role for pulse consumption in the reduction of risk for cardiovascular disease; this provides a sound rationale for the regular incorporation of pulses at about two-thirds of a cup daily in the management of hyperlipidemia in persons with type 2 diabetes. Pulse consumption can contribute to improving satiety, reducing food intake and regulating body weight, which can reduce obesity risk and, in turn, improve diabetes management. Collectively, available evidence provides very good support for a role of regular pulse consumption in the prevention and management of diabetes., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

Heavy training is associated with increased respiratory infection risk and antimicrobial proteins are important in defence against oral and respiratory tract infections. We examined the effect of 14 weeks of vitamin D3 supplementation (5000 IU/day) on the resting plasma cathelicidin concentration and the salivary secretion rates of secretory immunoglobulin A (SIgA), cathelicidin, lactoferrin and lysozyme in athletes during a winter training period. Blood and saliva were obtained at the start of the study from 39 healthy men who were randomly allocated to vitamin D3 supplement or placebo. Blood samples were also collected at the end of the study; saliva samples were collected after 7 and 14 weeks. Plasma total 25(OH)D concentration increased by 130% in the vitamin D3 group and decreased by 43% in the placebo group (both P = 0.001). The percentage change of plasma cathelicidin concentration in the vitamin D3 group was higher than in the placebo group (P = 0.025). Only in the vitamin D3 group, the saliva SIgA and cathelicidin secretion rates increased over time (both P = 0.03). A daily 5000 IU vitamin D3 supplement has a beneficial effect in up-regulating the expression of SIgA and cathelicidin in athletes during a winter training period, which could improve resistance to respiratory infections.

Background: Many medical schools teach the principles of evidence-based medicine (EBM) as part of their undergraduate curriculum. Medical students perceive that EBM is valuable to their undergraduate and postgraduate career. Students may experience barriers to applying EBM principles, especially when searching for evidence or identifying high-quality resources., Context: The UK National Institute for Health and Care Excellence (NICE) Evidence Search is a service that enables access to authoritative clinical and non-clinical evidence and best practice through a web-based portal., Innovation: Evidence-based medicine workshops were organised and delivered by fourth-year medical students, having first received training from NICE to become NICE student champions. The workshops covered the basic principles of EBM and focused on retrieving EBM resources for study through the NICE Evidence Search portal. The scheme was evaluated using a pre-workshop survey and an 8-12 week post-workshop survey. Self-reported confidence in searching for evidence-based resources increased from 29 per cent before the workshop to 87 per cent after the workshop. Only 1 per cent of students rated evidence-based resources as their first preference pre-workshop, compared with 31 per cent post-workshop., Implications: The results show that although many students were aware of evidence-based resources, they tended not to use them as their preferred resource. Despite appreciating the value of evidence-based resources, few students were confident in accessing and using such resources for pre-clinical study. A peer-taught workshop in EBM improved students' confidence with, and use of, evidence-based resources., (© 2014 John Wiley & Sons Ltd.)

Background: Good communication skills are integral to successful doctor-patient relationships. Communication may be verbal or non-verbal, and touch is a significant component, which has received little attention in the primary care literature. Touch may be procedural (part of a clinical task) or expressive (contact unrelated to a procedure/examination)., Aim: To explore GPs' and patients' experiences of using touch in consultations., Design and Setting: Qualitative study in urban and semi-rural areas of north-west England., Method: Participating GPs recruited registered patients with whom they felt they had an ongoing relationship. Data were collected by semi-structured interviews and subjected to constant comparative qualitative analysis., Results: All participants described the importance of verbal and non-verbal communication in developing relationships. Expressive touch was suggested to improve communication quality by most GPs and all patients. GPs reported a lower threshold for using touch with older patients or those who were bereaved, and with patients of the same sex as themselves. All patient responders felt touch on the hand or forearm was appropriate. GPs described limits to using touch, with some responders rarely using anything other than procedural touch. In contrast, most patient responders believed expressive touch was acceptable, especially in situations of distress. All GP responders feared misinterpretation in their use of touch, but patients were keen that these concerns should not prevent doctors using expressive touch in consultations., Conclusion: Expressive touch improves interactions between GPs and patients. Increased educational emphasis on the conscious use of expressive touch would enhance clinical communication and, hence, perhaps patient wellbeing and care.

Background: The aim of this paper was to review and summarize research on prevalence of health risk behaviours, their outcomes as well as risk and protective factors among adolescents in the English-speaking Caribbean., Methods: Searching of online databases and the World Wide Web as well as hand searching of the West Indian Medical Journal were conducted. Papers on research done on adolescents aged 10 - 19 years old and published during the period 1980 - 2005 were included., Results: Ninety-five relevant papers were located. Five papers were published in the 1980s, 47 in the 1990s, and from 2000-2005, 43 papers. Health risk behaviours and outcomes were divided into seven themes. Prevalence data obtained for these, included lifetime prevalence of substance use: cigarettes-24% and marijuana-17%; high risk sexual behaviour: initiation of sexual activity

Objective: The objective was to determine faculty's knowledge of electronic resources, access to a computer, use of electronic resources (both number and frequency) available at the Medical Sciences Library (MSL), and the areas of training needed and to identify areas for further research., Methods: A survey was administered to faculty in medicine, pharmacy, dentistry, and veterinary sciences at The University of the West Indies. The questions covered computer literacy, computer access and location, knowledge and use of electronic resources, and training needs., Results: The response rate was 70%, of whom 97% were computer users. Seventy-three percent used computers daily, and 82% felt that their computer literacy level was average or beyond. Overall, it was found that faculty had high awareness of the electronic resources made available by the MSL but low use of MSL-specific resources supporting the suggested problem of underutilization. Many respondents felt that e-resources were important, and, though many felt that they were competent users, 83% were self-taught and many still expressed a need for training. Over 60% felt that a workshop with a hands-on component was the preferred format for training. It was recommended that there be greater promotion of the library's e-resources.

Background: Multiple randomized trials of breast conservation compared with mastectomy in early breast cancer have validated equivalence of survival. Overwhelmingly the standard management of breast conservation includes surgical removal of the tumour, axillary dissection, postoperative breast irradiation, and adjuvant systemic therapy as appropriate. The outcomes are reviewed of 832 women with early breast cancer treated by local resection and irradiation at Royal Prince Alfred Hospital over an 18 year period, with particular emphasis on the changing patterns of practice., Methods: Between September 1978 and May 1996, 832 women with early stage breast cancer were treated with conservative surgery and radiation therapy. The changes in patient, tumour and treatment factors were analysed over this time period. The outcomes of local recurrence and survival were recorded. Trends in patterns of these variables were evaluated by dividing the 18 years accrual period into three consecutive periods (1978-1983, n = 28; 1984-1990, n = 392; 1991-1996, n = 412)., Results: At a median follow up of 76 months, the 5 and 10 year actuarial local recurrence rates were 4% and 6%, respectively. Half of the local recurrences were at the primary site. Young age, extensive intraduct carcinoma, oestrogen receptor (ER) status and extranodal spread were predictive of local recurrence on multivariate analysis. The 5 and 10 year overall survival rates were 88% and 73%, respectively. Actuarial 5 year local recurrence (4%, 6%, 2%) and survival (96%, 88%, 92%) rates varied little across the three time intervals. There was an increase in median age from 46 to 56 years over the accrual period, with no change in median primary tumour size (1.5 cm). There were significant histopathological improvements in reporting margin status and ER status. Surgically, the median number of axillary lymph nodes retrieved (14) did not alter significantly. With respect to adjuvant therapies, irradiation of lymph nodes regions decreased over time. The proportion of patients receiving adjuvant hormones or chemotherapy increased significantly (18%, 35%, 54%)., Conclusions: The low local recurrence rate and high survival are consistent with published literature for early breast cancer. Changes in practice during the accrual period included improvements in histopathological reporting, a reduction in irradiation of lymph node regions, and an increase in the use of systemic therapy. These changes parallel international recommendations regarding the optimal management of early breast cancer.

When the concept of sentinel lymph node biopsy was described in patients with melanoma, researchers quickly started to use lymphatic mapping techniques in breast cancer patients in an attempt to locate the sentinel node in the axilla. We have been performing mammary lymphoscintigraphy in this role for 6 years and have now studied 159 patients. Like others, we have found that most breast cancers (93%) have lymphatic drainage that includes the axilla, and we have found an average of 1.4 axillary sentinel nodes in these patients. Surgical biopsy of the axillary sentinel nodes accurately staged the node field in 96% of patients. We have also found, however, that the pattern of lymphatic drainage from the cancer site is unpredictable; and in 49% of patients lymphatic drainage occurred across the center line of the breast to axillary or internal mammary sentinel nodes. In more than half of our patients (56%) lymphatic drainage occurred to lymph nodes outside the axilla including the internal mammary (45%), supraclavicular (13%), and interpectoral and intramammary interval nodes (12%). These nodes are also sentinel nodes, and their presence indicates that a sentinel node biopsy procedure that stages only the status of the axillary lymph nodes has the potential to understage about half the patients with breast cancer. High quality lymphoscintigraphy allows accurate mapping of peritumoral lymphatic drainage in most patients with breast cancer. It is possible that in the future accurate nodal staging in each individual will involve biopsy of all sentinel lymph nodes, regardless of their location.

Background: Mammographic screening for breast cancer not only reduces the overall mortality from breast cancer but allows greater opportunities for breast-conserving operations. The predicted degree of breast conservation is not being realized, but is increasing in centres that have published their results., Methods: The operative management of breast cancers diagnosed by BreastScreen Central and Eastern Sydney Screening and Assessment Service were compared between two time periods: January 1988-December 1992 (group 1) and January 1993-December 1995 (group 2). The rate of breast conservation, and other data were compared between the two periods. An attempt was made with multivariate analysis to identify some of the factors that made mastectomy rather than conservation more likely., Results: There were 723 cancers detected that were suitable for analysis (group 1, n = 273; group 2, n = 450). In group 1 the breast conservation rate was 42.9%; this increased significantly to 60.4% in group 2 (P < 0.001). The data were examined to determine if there was any other factor that had changed over the time periods which might account for the increased rate of breast conservation. The use of pre-operative diagnostic techniques such as fine needle aspirate cytology and core biopsy increased significantly. Multivariate analysis comparing the differences in patient age, diagnostic technique, tumour type, grade, size, location and lymph node status, both independently and compositely did not account for the increase in breast conservation in group 2., Conclusion: The increase in breast conservation is due to other factors such as the surgeons' approach and patient attitude. The use of pre-operative, minimally invasive tissue sampling techniques is increasing.

Traction followed by spica casting, the one method used to treat femoral shaft fractures in children that was used in the past, has given way to a multiplicity of methods today. To evaluate the morbidity and costs of these various methods, 85 fractures in 81 patients age six to 16 years were evaluated. Early spica casting gave excellent results with low complications and low costs. All surgical treatments cost approximately the same: 3 times the cost of early spica casting and equivalent to traction followed by spica casting. Intramedullary flexible rods resulted in quicker healing and return to full weight bearing than did external fixation, which had the highest complication rate. One case of avascular necrosis in an 11-year-old girl treated with a reamed intramedullary rod suggests that this method is best reserved for children at or near skeletal maturity.

Background: Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme found in all prokaryotes and eukaryotes. As an enzyme of the thymidylate synthase metabolic cycle, SHMT catalyses the retro-aldol cleavage of serine to glycine, with the resulting hydroxymethyl group being transferred to tetrahydrofolate to form 5, 10-methylene-tetrahydrofolate. The latter is the major source of one-carbon units in metabolism. Elevated SHMT activity has been shown to be coupled to the increased demand for DNA synthesis in rapidly proliferating cells, particularly tumour cells. Consequently, the central role of SHMT in nucleotide biosynthesis makes it an attractive target for cancer chemotherapy., Results: We have solved the crystal structure of human cytosolic SHMT by multiple isomorphous replacement to 2.65 A resolution. The monomer has a fold typical for alpha class pyridoxal 5'-phosphate (PLP) dependent enzymes. The tetramer association is best described as a 'dimer of dimers' where residues from both subunits of one 'tight' dimer contribute to the active site., Conclusions: The crystal structure shows the evolutionary relationship between SHMT and other alpha class PLP-dependent enzymes, as the fold is highly conserved. Many of the results of site-directed mutagenesis studies can easily be rationalised or re-interpreted in light of the structure presented here. For example, His 151 is not the catalytic base, contrary to the findings of others. A mechanism for the cleavage of serine to glycine and formaldehyde is proposed.

As an enzyme of the thymidylate synthase cycle, serine hydroxymethyltransferase (SHMT) has a key role in nucleotide biosynthesis. Elevated activities of SHMT have been correlated with the increased demand for nucleotide biosynthesis in tumors of human and rodent origin, making this enzyme a novel target for cancer chemotherapy. Here the purification and crystallization of recombinant human cytosolic SHMT are reported. Crystals belong to space group P6222 or P6422 with cell parameters a = b = 155.0, c = 235.5 A and diffract to at least 3.0 A resolution.

Risk factors for prevalent infection with verocytotoxigenic Escherichia coli (VTEC) were studied in a random sample of 886 cows and 592 calves under 3 months of age on 80 randomly selected dairy farms in southern Ontario. Fecal-culture supernatants from each animal were screened for verocytotoxicity using a Vero cell assay (VCA) and for verocytotoxin (VT) genes by a polymerase chain reaction (PCR) procedure. Up to 20 F. colt isolates from positive samples were tested for VT production using VCA and PCR. VTEC isolates were serotyped. Farm managers were interviewed using a standardized questionnaire to obtain information on farm- and individual animal-level management practices and characteristics. There was a significant (P < 0.001) positive association between age of calves and their VTEC infection status, and calves were significantly more likely to be infected than cows. The proportion of calves infected on the farm was positively associated with both the use of regular pails for feeding calves (as opposed to nipple bottles or nipple pails) and bringing new animals into the herd in the previous year.

The persistence of Escherichia coli O157:H7 in cattle and the farm environment was investigated on eight Ontario dairy farms positive for E. coli O157:H7 in a longitudinal study commenced one year previously. Faecal samples from cows, calves, humans, cats, rodents, wild birds, a composite fly sample and numerous composite and individual environmental samples were cultured and tested for verotoxin-producing E. coli (VTEC). VTEC isolates were serotyped and E. coli O157:H7 isolates were phage typed. E. coli O157:H7 phage type 34 was isolated from one calf on each of two farms. The same phage type had been isolated on one of these farms 12 months earlier. Most E. coli O157:H7-positive animals and farms became culture-negative within 2 and 3 months, respectively. E. coli O157:H7 was not isolated from any environmental samples, although evidence of VTEC was found in composite samples from calf feeders (19.1%), calf barn surfaces (18%), cow feeders (14.9%), flies (12.5%), cow barn surfaces (11.3%), and individual milk filters (12.5%). VTEC belonging to 21 non-O157 serotypes were isolated from 24 cows (8.2%), 21 calves (18.3%), 2 cow feeder samples (3.0%), and 1 calf feeder sample (4.8%). Shedding of E. coli O157:H7 by infected dairy cattle appears to be transient and persistence of E. coli O157:H7 was not demonstrated from the farm environment sites tested.

Between November 1994 and February 1995 over nine thousand Boer goat embryos were imported into Canada from the Republic of South Africa. This substantial international movement of animal genetics via embryos was achieved through the application of the risk analysis principles prescribed in Section 1.4. of the International Animal Health Code of the Office International des Epizooties (OIE). Integral to the development of the health certification procedures was the application of the fundamental principles of non-discrimination, harmonisation, equivalence and transparency defined in the World Trade Organisation Agreement on Sanitary and Phytosanitary measures. Risk mitigation interventions were founded upon full consideration of the potential for disease transmission by animal embryos as espoused by the International Embryo Transfer Society and the relevant standards contained in Appendix 4.2.3.3. of the OIE International Animal Health Code. All the embryos imported into Canada were implanted into synchronised recipients on arrival. Twenty months later there has been no evidence of disease in either the recipient animals or the resulting animals born in Canada.