Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen.

Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens.

Then there were seven: a commentary on creating a public involvement strategy group for a policy research unit in behavioural science.

The National Institute for Health and Care Research (NIHR) Policy Research Unit in Behavioural Science (PRU-BS) was funded to inform government on the application of behavioural science in health and social care policy. What makes this unit different to other topic specific ones, was the wide range of its brief. Because of this, the PPI group would need to include a wide range of experience and expertise and be prepared to learn. We were a different type of public group for a different type of task. This paper deals with how we approached this. In this paper we outline how the PPI plan in the funding proposal for the PRU-BS was adapted to real world challenges. We describe the stages in the formation of the PPI Strategy Group and how a virtual platform was created to ensure good communication. We discuss our pragmatic approach of developing Terms of Reference and a PPI strategy document. Given the restrictions imposed by the Covid-19 pandemic we explain how we tackled PPI SG member induction sessions, meetings and training sessions. To illustrate how the group operates we provide an example of our involvement in a PRU-BS project. Central to our paper is the lessons we learned. We hope the challenges we met in forming the unique PPI SG, how these were overcome, and our recommendations will help others faced with a similar task.

The Policy Research Unit in Behavioural Science (PRU-BS) was formed in early 2019, funded by the National Institute for Health and Care Research (NIHR). The aim of the unit is to advise the government on the use of behavioural science (the study of human behaviour) to inform health and social care policy. From the outset the aim was to embed PPI in all aspects of the unit's work from the governance and direction of the unit to the individual research projects it conducts. As behavioural science cuts across all aspects of health, recruiting members of the public to work within the PRU-BS required careful thought. In this paper we describe the processes of recruiting to our PPI Strategy Group, the induction and training, and the ways in which we worked to develop the group and become embedded within the unit. Lastly, we present several recommendations based on our experiences of forming the PPI Strategy Group. Although this is aimed primarily at those contemplating setting up a group whose remit extends beyond a single research project, we hope this will be a useful resource for the public, researchers and others working in PPI.

Mental health nurses' attitudes towards risk assessment: An integrative systematic review.

WHAT IS KNOWN ON THE SUBJECT?: Risk assessment and risk management are considered to be important practices carried out by mental health nurses. Risk assessment can help keep mental health service users' safe, but some nurses see it as a 'tick the box' exercise. Some studies have looked at nurses' attitudes to risk assessment but no one has systematically described all the studies. WHAT THE ARTICLE ADDS TO EXISTING KNOWLEDGE?: Mental health nurses' attitudes towards risk assessment are diverse with regard to its legitimacy, conduct and value. This study provides an organised framework to help understand the areas in which these different attitudes occur. WHAT ARE THE IMPLICATIONS FOR PRACTICE?: Since attitudes can influence clinical practice, nurses need to reflect on how they view risk assessment. Further research is required to investigate whether particular attitudes are positive or negative and whether attitudes can be changed. ABSTRACT: INTRODUCTION: Understanding nurses' attitudes towards risk assessment could inform education and practice improvements. AIM/QUESTION: To explore mental health nurses' attitudes towards risk assessment. METHOD: An integrative systematic review (PROSPERO: CRD42023398287). Multiple databases (PubMed, CINAHL, MEDLINE, EMBASE and PsycINFO) were searched for primary studies of mental health nurses' attitudes towards risk assessment. Qualitative studies were subject to inductive coding and thematic analysis; quantitative data were integrated with emerging themes. RESULTS: Eighteen articles were included. Qualitative studies commonly lacked rigorous analyses. Four themes emerged: underlying purpose and legitimacy of risk assessment (philosophical orientation); use of structured approaches (technical orientation); value of intuition (intuitive orientation); and service user involvement (relationships orientation). There were contradictory study findings in each thematic category indicating different attitudes among mental health nurses. DISCUSSION: Mental health nurses' attitudes towards risk assessment vary in four key domains. Survey studies suggest they are more approving of structured approaches to risk assessment than many qualitative studies suggest. There is a need to develop a valid measure of attitudes to risk assessment. IMPLICATIONS FOR PRACTICE: This review could help health organisations to develop strategies to improve their risk assessment policies and practice. There is a need to develop structured training and education programmes.

Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells.

Embryonic Stem (ES) cells have the potential to form every cell of the body and thus are of great promise for tissue transplantation. One of the rising techniques that allows studying the differentiation state of ES cells is quantitative RT-PCR (qRT-PCR). When relative quantification by qRT-PCR is applied, accurate normalization is necessary, since differentiated embryonic stem cells and developing embryos contain heterogeneous cell populations. Corrections for variations in the qRT-PCR reaction are needed to allow comparisons between different samples. We applied the normalization tools geNorm and Normfinder to ten reference genes identifying the most stable ones for relative quantification of gene expression during differentiation of human ES cells, as well as in differentiated mouse ES cells and in the developing mouse embryo. For relative quantification by qRT-PCR in these systems, we advise to use normalization factors based on multiple stable reference genes. However, when the use of several reference genes would be unpractical, a single reference gene in each experimental setup could be sufficient. When looking for single stable reference genes, beta-actin works best in both mouse embryo and ES cell experiments and glyceraldehyde-3-phosphate-dehydrogenase can be applied in both mouse and human ES cell experiments.

Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies.

Whole-mount in situ hybridization (WISH) is a technique widely used in developmental biology to study the localization of RNA sequences in intact tissues or whole organisms. In this chapter we present a detailed protocol that was optimized for gene expression analysis in early stage mouse embryos (5.5-10.5 days post-coitum) and embryoid bodies formed by differentiating embryonic stem cells and can be used for the detection of up to two distinct RNA sequences simultaneously. The initial steps of the procedure are the generation of the labeled riboprobe(s) and the embryo or embryoid body preparation, which can be completed in less than 2 days. The actual WISH procedure, comprised of the hybridization, the post-hybridization washes, and the immunological staining, can be completed in 3 days.

Systems biology: a new tool for farm animal science.

It is rapidly emerging that the tender meat phenotype is affected by an enormous amount of variables, not only tied to genetics (livestock breeding selection), but also to extrinsic factors, such as feeding conditions, physical activity, rearing environment, administration of hormonal growth promotants, pre-slaughter handling and stress. Proteomics has been widely accepted by meat scientists over the last years and is now commonly used to shed light on the postmortem processes involved in meat tenderization. This review discusses the latest findings with the use of proteomics and systems biology to study the different biochemical pathways postmortem aiming at understanding the concerted action of different molecular mechanisms responsible for meat quality. The conversion of muscle to meat postmortem can be described as a sequence of events involving molecular pathways controlled by a complex interplay of many factors. Among the different pathways emerging are the influence of apoptosis and lately also the role of autophagy in muscle postmortem development. This review thus, focus on how systems-wide integrated investigations (metabolomics, transcriptomics, interactomics, phosphoproteomics, mathematical modeling), which have emerged as complementary tools to proteomics, have helped establishing a few milestones in our understanding of the events leading from muscle to meat conversion.

Advances in apoptotic mediated proteolysis in meat tenderisation.

Meat tenderness is considered to be one of the most important attributes of meat quality; however it is also one of the most variable. Ultimate meat tenderness is influenced by the amount of intramuscular connective tissue, the length of the sarcomere and also the proteolytic potential of the muscle. Post-mortem proteolysis by endogenous proteases causes the weakening of myofibril structures and associated proteins, which results in tenderisation. The caspase proteolytic system was first identified to be a potential contributor to post-mortem proteolysis and tenderisation in 2002. Since then research has both supported and challenged this hypothesis. The purpose of this review is to examine the experimental evidence available for caspases' involvement in post-mortem proteolysis, and to highlight cross-talk between this proteolytic system and the calpain system, a known contributor to meat tenderisation.

Exposure of Salmonella enterica serovar Typhimurium to high level biocide challenge can select multidrug resistant mutants in a single step.

BACKGROUND: Biocides are crucial to the prevention of infection by bacteria, particularly with the global emergence of multiply antibiotic resistant strains of many species. Concern has been raised regarding the potential for biocide exposure to select for antibiotic resistance due to common mechanisms of resistance, notably efflux. METHODOLOGY/PRINCIPAL FINDINGS: Salmonella enterica serovar Typhimurium was challenged with 4 biocides of differing modes of action at both low and recommended-use concentration. Flow cytometry was used to investigate the physiological state of the cells after biocide challenge. After 5 hours exposure to biocide, live cells were sorted by FACS and recovered. Cells recovered after an exposure to low concentrations of biocide had antibiotic resistance profiles similar to wild-type cells. Live cells were recovered after exposure to two of the biocides at in-use concentration for 5 hours. These cells were multi-drug resistant and accumulation assays demonstrated an efflux phenotype of these mutants. Gene expression analysis showed that the AcrEF multidrug efflux pump was de-repressed in mutants isolated from high-levels of biocide. CONCLUSIONS/SIGNIFICANCE: These data show that a single exposure to the working concentration of certain biocides can select for mutant Salmonella with efflux mediated multidrug resistance and that flow cytometry is a sensitive tool for identifying biocide tolerant mutants. The propensity for biocides to select for MDR mutants varies and this should be a consideration when designing new biocidal formulations.

Tenderness--an enzymatic view.

One of the most common causes of unacceptability in meat quality is toughness. Toughness is attributed to a range of factors including the amount of intramuscular connective tissue, intramuscular fat, and the length of the sarcomere. However, it is apparent that the extent of proteolysis of key proteins within muscle fibres is significant determinant of ultimate tenderness. The objective of this manuscript is to describe the main endogenous proteolytic enzyme systems that have the potential to be involved in muscle post-mortem proteolysis and whether the experimental evidence available supports this involvement.

Wnt-induced proteolytic targeting.

Misregulation of the Wnt pathway is a common route to cancer, including primary breast cancers. In this issue of Genes & Development, Miranda-Carboni and colleagues (3121-3134) demonstrate that the cyclin-dependent kinase inhibitor p27(Kip1) is ubiquitylated for proteasomal degradation in Wnt10b-induced mammary tumors exclusively by the Cul4A E3 ligase, which is strongly induced by Wnt signaling. The discovery of a new Wnt-induced proteolytic targeting system has important implications for the mechanism of Wnt-initiated tumorigenesis.

An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies.

Determining the precise expression pattern of a gene of interest at various stages of development is essential to understanding its biological function in embryology. This protocol describes a sensitive method for whole-mount in situ hybridization (WISH) to mouse embryos, using cRNA probes. Adaptations are provided that allow the protocol to be applied to embryonic stages ranging from blastocysts to postimplantation stage embryos, and to embryoid bodies. We also describe an in situ method for differential detection of two probes. Probe labeling and dissection and preparation of the embryos can be performed in 2 d. The actual WISH procedure can be completed in another 3 d.

Expression of Frizzled5, Frizzled7, and Frizzled10 during early mouse development and interactions with canonical Wnt signaling.

Wnt signaling has been shown to be important in the patterning of the gastrulating mouse embryo, especially in axis formation. To this date, there is no clear indication that the Wnt receptors, Frizzleds (Fzds), are involved in such early specification. Moreover, at the gastrulation stage, the only Fzd with a known characterized expression pattern is Fzd8, which is expressed in the anterior visceral endoderm (aVE) (Lu et al. [2004] Gene Expr Patterns 4:569-572). Following a real time RT-PCR study to evaluate Fzd expression in the gastrulating embryo, we used whole-mount in situ hybridization to reveal new expression domains for Fzd5, Fzd7, and Fzd10. Fzd5 is expressed in the aVE and Fzd7 expression is restricted to the epiblast of the gastrulating embryo. The expression pattern of Fzd10 in the primitive streak of the gastrula suggests it has a role in mesoderm induction. We also show that the purified, secreted forms of the extracellular cysteine-rich domains (CRDs) of FZD5, Fzd7, and Fzd8 can antagonize Wnt3a-induced beta-Catenin accumulation in L-cells, whereas in mouse embryonic stem cells, these CRDs can inhibit spontaneous mesoderm formation and promote neural differentiation. Our data demonstrate that Fzd5, Fzd7, and Fzd10 are expressed in distinct domains of the gastrulating embryo, and that the CRDs of FZD5, Fzd7, and Fzd8 can regulate Wnts, indicating that Fzds interpret Wnt signals during embryonic mesoderm and neural induction.

The transcription factor hepatocyte nuclear factor-6 controls the development of pancreatic ducts in the mouse.

BACKGROUND & AIMS: A number of hereditary polycystic diseases are associated with formation of cysts within the pancreatic ducts. The cysts result from abnormal tubulogenesis, but how normal pancreatic duct development is controlled remains poorly understood. Here, we investigate the transcriptional mechanisms that control pancreatic duct development by addressing the role of the transcription factor hepatocyte nuclear factor (HNF)-6. METHODS: Using immunostaining, we have determined the expression pattern of HNF-6 in pancreatic ducts during mouse development. Hnf6 null mice at various stages of development were studied by immunolocalization methods to assess the morphology, differentiation, and proliferation status of ductal cells. The expression of genes involved in hereditary polycystic diseases was determined by real-time, reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: We show that HNF-6 is expressed in the pancreatic duct epithelium throughout development and that, in the absence of HNF-6, duct morphogenesis is perturbed. Although development of the intercalated ducts is normal, cysts appear within the interlobular and intralobular ducts. This is associated with abnormal development of primary cilia at the apical pole of the duct cells and with reduced expression of a set of genes involved in polycystic diseases, namely those coding for HNF-1beta and for the cilium-associated proteins polyductin/fibrocystin and cystin. CONCLUSIONS: We identify HNF-6 as the first transcriptional regulator of pancreatic duct development and reveal the existence of different regulatory mechanisms in distinct duct compartments. HNF-6 controls a network of genes involved in cilium formation and in hereditary polycystic diseases. Finally, HNF-6 deficiency represents a genetically defined model of pancreatic cystic disease.

Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development.

In this extensive study, real-time reverse transcriptase-polymerase chain reaction was used to analyze the expression levels of all 19 Wnt genes and their 11 potential antagonists in mouse blastocysts, pregastrula, gastrula, and neurula stages. By complementing these results with in situ hybridization, we revealed new expression domains for Wnt2b and Sfrp1, respectively, in the future primitive streak at the posterior side and in the anterior visceral endoderm before the initiation of gastrulation. Moreover, the anterior visceral endoderm expresses three secreted Wnt antagonists (Sfrp1, Sfrp5, and Dkk1) in partially overlapping domains. We also identified expression patterns for the Wnt1, Wnt3a, Wnt6, Wnt7b, Wnt9a, Wnt10b, and Sfrp1 genes at the blastocyst stage. In particular, the expression of Wnt1 and Sfrp1 predominantly in the inner cell mass and of Wnt9a in the mural trophoblast and inner cell mass cells surrounding the blastocoele suggests new roles for the Wnt pathway in preimplantation development. This article is the first report on the regional expression of Wnt genes in the mouse blastocyst.