Establishing how bacterial cells position the division site

University of Technology, Sydney. Faculty of Science.In virtually all bacteria cell division is essential and tightly regulated both temporally and spatially to ensure that cells divide precisely at the centre between segregated chromosomes. Failure to do so can lead to cell death. The earliest event in bacterial cell division is the polymerization of the highly conserved tubulin-like protein, FtsZ, to form a contractile structure called the Z ring, on the inner side of the cytoplasmic membrane at midcell and between chromosomes. The Z ring subsequently contracts causing the cell envelope to invaginate, generating two newborn cells. Thus the Z ring defines the position of the division site in bacterial cells. How the Z ring is positioned precisely at midcell is a controversial topic that remains unresolved. Division site positioning has long been believed to occur via the combined action of two factors: the Min system and nucleoid occlusion. Both factors have been proposed to prevent Z ring assembly along the length of the cell, allowing it to assemble only once chromosomes segregate and nucleoid occlusion is relieved specifically at midcell. The research described in this thesis challenges this paradigm, providing compelling evidence that other mechanisms in addition to nucleoid occlusion and the Min system act to position the Z ring at midcell in B. subtilis. Moreover, this work also shows that nucleoid occlusion and the Min system do not define the Z ring position at midcell but rather ensure that the midcell division site is utilized efficiently. A clue to an additional mechanism for positioning the Z ring has emerged from studies investigating the relationship between chromosome replication and Z ring position. The nature of this relationship has remained obscure for years. Part of this thesis involves a closer examination of this relationship. It was found that the ability to position the Z ring at midcell is linked specifically to the progress of the initiation stage of DNA replication, such that the frequency of Z rings at midcell increases as this stage of DNA replication is progressively completed. Moreover, this link was found to be nucleoid occlusion independent. Spatial and temporal control of Z ring assembly has been widely attributed to the Min system and nucleoid occlusion. While inactivating both systems substantially affects cell division, it is currently unknown whether their absence affects precise midcell Z ring positioning. This thesis deals with this question, and it was found that the combined effect of MinCD and Noc proteins actually affects the timing and efficiency of Z ring assembly, but not its spatial precision between nucleoids at midcell. If Noc and MinCD proteins do not position the Z ring at midcell, what other factores may play this role? Two hypotheses were proposed to help explain the precise Z ring positioning observed in absence of nae and minCD: 1) Noc-independent nucleoid occlusion or 2) factors completely independent of nucleoid occlusion position the Z ring at midcell. Experiments designed to discriminate between these hypotheses showed that they are actually both valid: while the data obtained suggests that factors completely independent of nucleoid occlusion (Noc inclusive) and the Min system position the Z ring at midcell, it also suggested that other Noc-independent nucleoid occlusion factors prevent the Z ring from assembling at midcell over unreplicated DNA

The ParB homologs, Spo0J and Noc, together prevent premature midcell Z ring assembly when the early stages of replication are blocked in Bacillus subtilis

© 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd Precise cell division in coordination with DNA replication and segregation is of utmost importance for all organisms. The earliest stage of cell division is the assembly of a division protein FtsZ into a ring, known as the Z ring, at midcell. What still eludes us, however, is how bacteria precisely position the Z ring at midcell. Work in B. subtilis over the last two decades has identified a link between the early stages of DNA replication and cell division. A recent model proposed that the progression of the early stages of DNA replication leads to an increased ability for the Z ring to form at midcell. This model arose through studies examining Z ring position in mutants blocked at different steps of the early stages of DNA replication. Here, we show that this model is unlikely to be correct and the mutants previously studied generate nucleoids with different capacity for blocking midcell Z ring assembly. Importantly, our data suggest that two proteins of the widespread ParB family, Noc and Spo0J are required to prevent Z ring assembly over the bacterial nucleoid and help fine tune the assembly of the Z ring at midcell during the cell cycle

Patient outcomes up to 15 years after stroke: survival, disability, quality of life, cognition and mental health

BACKGROUND: The global epidemiological shift of disease burden towards long-term conditions means understanding long-term outcomes of cardiovascular disease is increasingly important. More people are surviving stroke to experience its long-term consequences, but outcomes in people living more >10 years after stroke have not been described in detail. METHODS: Data were collected for the population-based South London Stroke Register, with participants followed up annually until death. Outcomes were survival, disability, activity, cognitive impairment, quality of life, depression and anxiety. FINDINGS: Of 2625 people having first-ever stroke, 262 (21%) survived to 15 years. By 15 years, 61% (95% CI 55% to 67%) of the survivors were male, with a median age of stroke onset of 58 years (IQR 48-66). 87% of the 15-year survivors were living at home and 33.8% (26.2% to 42.4%) had mild disability, 14.3% (9.2% to 21.4%) moderate disability and 15.0% (9.9% to 22.3%) severe disability. The prevalence of disability increased with time but 1 in 10 of the 15-year survivors had lived with moderate-severe disability since their stroke. At 15 years, the prevalence of cognitive impairment was 30.0% (19.5% to 43.1%), depression 39.1% (30.9% to 47.9%) and anxiety 34.9% (27.0% to 43.8%), and survivors reported greater loss of physical than mental quality of life. CONCLUSIONS: One in five people live at least 15 years after a stroke and poor functional, cognitive and psychological outcomes affect a substantial proportion of these long-term survivors. As the global population of individuals with cardiovascular long-term conditions grows, research and health services will need to increasingly focus on preventing and managing the long-term consequences of stroke

Development of a Highly Differentiated Human Primary Proximal Tubule MPS Model (aProximate MPS Flow)

\ua9 2023 by the authors.The kidney proximal tubule (PT) mediates renal drug elimination in vivo and is a major site of drug-induced toxicity. To reliably assess drug efficacy, it is crucial to construct a model in which PT functions are replicated. Current animal studies have proven poorly predictive of human outcome. To address this, we developed a physiologically relevant micro-physiological system (MPS) model of the human PT, the aProximate MPS Flow platform (Patent No: G001336.GB). In this model, primary human PT cells (hPTCs) are subjected to fluidic media flow and a shear stress of 0.01–0.2 Pa. We observe that these cells replicate the polarity of hPTCs and exhibit a higher expression of all the key transporters of SLC22A6 (OAT1), SLC22A8 (OAT3), SLC22A2 (OCT2), SLC47A1 (MATE1), SLC22A12 (URAT1), SLC2A9 (GLUT9), ABCB1 (MDR1), ABCC2 (MRP2), LRP2 (megalin), CUBN (cubilin), compared with cells grown under static conditions. Immunofluorescence microscopy confirmed an increase in OAT1, OAT3, and cilia protein expression. Increased sensitivity to nephrotoxic protein cisplatin was observed; creatinine and FITC-albumin uptake was significantly increased under fluidic shear stress conditions. Taken together, these data suggest that growing human PT cells under media flow significantly improves the phenotype and function of hPTC monolayers and has benefits to the utility and near-physiology of the model

Patient, carer and public involvement in major system change in acute stroke services: The construction of value

BACKGROUND: Patient and public involvement is required where changes to care provided by the UK National Health Service are proposed. Yet involvement is characterized by ambiguity about its rationales, methods and impact. AIMS: To understand how patients and carers were involved in major system changes (MSCs) to the delivery of acute stroke care in 2 English cities, and what kinds of effects involvement was thought to produce. METHODS: Analysis of documents from both MSC projects, and retrospective in-depth interviews with 45 purposively selected individuals (providers, commissioners, third-sector employees) involved in the MSC. RESULTS: Involvement was enacted through consultation exercises; lay membership of governance structures; and elicitation of patient perspectives. Interviewees' views of involvement in these MSCs varied, reflecting different views of involvement per se, and of implicit quality criteria. The value of involvement lay not in its contribution to acute service redesign but in its facilitation of the changes developed by professionals. We propose 3 conceptual categories-agitation management, verification and substantiation-to identify types of process through which involvement was seen to facilitate system change. DISCUSSION: Involvement was seen to have strategic and intrinsic value. Its strategic value lay in facilitating the implementation of a model of care that aimed to deliver evidence-based care to all; its intrinsic value was in the idea of citizen participation in change processes as an end in its own right. The concept of value, rather than impact, may provide greater traction in analyses of contemporary involvement practices

A two-step transport pathway allows the mother cell to nurture the developing spore in Bacillus subtilis

© 2017 Ramírez-Guadiana et al. One of the hallmarks of bacterial endospore formation is the accumulation of high concentrations of pyridine-2,6-dicarboxylic acid (dipicolinic acid or DPA) in the developing spore. This small molecule comprises 5–15% of the dry weight of dormant spores and plays a central role in resistance to both wet heat and desiccation. DPA is synthesized in the mother cell at a late stage in sporulation and must be translocated across two membranes (the inner and outer forespore membranes) that separate the mother cell and forespore. The enzymes that synthesize DPA and the proteins required to translocate it across the inner forespore membrane were identified over two decades ago but the factors that transport DPA across the outer forespore membrane have remained mysterious. Here, we report that SpoVV (formerly YlbJ) is the missing DPA transporter. SpoVV is produced in the mother cell during the morphological process of engulfment and specifically localizes in the outer forespore membrane. Sporulating cells lacking SpoVV produce spores with low levels of DPA and cells engineered to express SpoVV and the DPA synthase during vegetative growth accumulate high levels of DPA in the culture medium. SpoVV resembles concentrative nucleoside transporters and mutagenesis of residues predicted to form the substrate-binding pocket supports the idea that SpoVV has a similar structure and could therefore function similarly. These findings provide a simple two-step transport mechanism by which the mother cell nurtures the developing spore. DPA produced in the mother cell is first translocated into the intermembrane space by SpoVV and is then imported into the forespore by the SpoVA complex. This pathway is likely to be broadly conserved as DPA synthase, SpoVV, and SpoVA proteins can be found in virtually all endospore forming bacteria

Patient experience of centralised acute stroke care pathways

Background: In 2010, Greater Manchester (GM) and London centralized acute stroke care services into a reduced number of hyperacute stroke units, with local stroke units providing on‐going care nearer patients’ homes. / Objective: To explore the impact of centralized acute stroke care pathways on the experiences of patients. / Design: Qualitative interview study. Thematic analysis was undertaken, using deductive and inductive approaches. Final data analysis explored themes related to five chronological phases of the centralized stroke care pathway. / Setting and participants: Recruitment from 3 hospitals in GM (15 stroke patients/8 family members) and 4 in London (21 stroke patients/9 family members). / Results: Participants were impressed with emergency services and initial reception at hospital: disquiet about travelling further than a local hospital was allayed by clear explanations. Participants knew who was treating them and were involved in decisions. Difficulties for families visiting hospitals a distance from home were raised. Repatriation to local hospitals was not always timely, but no detrimental effects were reported. Discharge to the community was viewed less positively. / Discussion and conclusions: Patients on the centralized acute stroke care pathways reported many positive aspects of care: the centralization of care pathways can offer patients a good experience. Disadvantages of travelling further were perceived to be outweighed by the opportunity to receive the best quality care. This study highlights the necessity for all staff on a centralized care pathway to provide clear and accessible information to patients, in order to maximize their experience of care

Field evaluation of the intermittent preventive treatment of malaria during pregnancy (IPTp) in Benin: evolution of the coverage rate since its implementation

Background: Malaria is an important public health problem in Africa. Pregnant women are a vulnerable population and this disease can underlie an increased risk of low-birth weight newborns (< 2500 g); these women therefore need management during pregnancy. This was previously provided by chloroquine treatment, which, because of compliance problems and drug resistance, was replaced by intermittent preventive treatment with sulfadoxine-pyrimethamine (ITPp-SP) with two single doses taken after 16 weeks of amenorrhea, at least 4 weeks apart. This protocol was recommended by the World Health Organization (WHO) in 1998 and was initiated in Benin in 2006 after its political adoption in 2004. A retrospective longitudinal study was conducted in eight maternity hospitals in two geographical areas in Benin (in the south and north). The study investigated 2420 women who gave birth from 2005 to 2009. The antenatal cards of those women were randomly selected over 5 years with the aim of analyzing the IPT coverage in the study's maternity hospitals. Results: The rate of IPT-SP coverage evolved from 3.7% in 2005 to 87.8% in 2009 for women who had received at least one dose and from 2.7% to 68.4% from 2005 to 2009 for those who had received complete ITP (two doses). Variability in the results was observed depending on the geographical area (north/south) and the type of area (rural/urban). Conclusions: In total, application of IPT-SP 2-doses has rapidly evolved since 2005, but the objective of 80% IPT coverage has not yet been achieved throughout the country. Moreover, problems of drug shortage recurring in the field (reported by health staff) remain to be resolved