Two active states of the Ras-related Bud1/Rsr1 protein bind to different effectors to determine yeast cell polarity

Cells of budding yeast organize their cytoskeleton in a highly polarized manner during vegetative growth. Selection of a site for polarization requires a group of proteins including a Ras-like GTPase, Bud1, and its regulators. Another group of proteins, which includes a Rho-like GTPase (Cdc42), its guanine nucleotide exchange factor (Cdc24), and Bem1, is necessary for organization of the actin cytoskeleton and for cell polarization. We have proposed previously that the Bud1 protein, through its GTPase cycle, determines the localization of one or more of the cell polarity proteins to the bud site. Herein we demonstrate that Bud1 directly interacts with Cdc24 and Bem1: Bud1 in its GTP-bound form associates preferentially with Cdc24, whereas the GDP-bound form of Bud1 associates with Bem1. We also present subcellular fractionation data for Bud1 that is consistent with the idea that Bud1 can travel between the site for budding on the plasma membrane and the cytosol. We propose that Bud1 can exist in two active states for association with different partners and that the switch from Bud1–GTP to Bud1–GDP provides a regulatory device for ordered assembly of a macromolecular complex at the bud site.     

An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

We have developed a limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse using a miniturized stroma- dependent bone marrow culture assay in vitro. The cells were overlaid on irradiated stromal layers in microtiter wells in a range of concentrations, and frequencies of cobblestone area-forming cells (CAFC) were calculated by employing Poisson statistics. The production of secondary granulocyte/macrophage colony-forming units (CFU-G/M) in the adherent layer of individual wells was correlated with the presence of such cobblestone areas. CAFC frequencies were determined in bone marrow cell suspensions that were either enriched for marrow repopulating ability (MRA) in vivo, while depleted for spleen colony- forming units (CFU-S), or vice versa. The separation of bone marrow cells (BMC) was either based on centrifugal elutriation, or monoclonal antibody-mediated magnetic depletion of cells carrying cell surface differentiation antigens, and subsequent sorting on the basis of light scatter and rhodamine-123 retention as a measure of mitochondrial activity. In addition, 5-fluorouracil-resistant BMC were studied. Our investigations show that a time-dependent cobblestone area formation exists that reflects the turnover time and primitiveness of CAFC. The frequency of precursors forming cobblestone areas on day 28 after overlay is proposed to be a measure for MRA, whereas the day-7 CAFC frequency closely corresponds with day-12 CFU-S numbers in the suspensions tested.     

Role of the sialophorin (CD43) receptor in mediating influenza A virus- induced polymorphonuclear leukocyte dysfunction

Polymorphonuclear leukocytes (PMNLs) exposed to influenza A virus (IAV) undergo activation of the respiratory burst followed by depression of cell function when subsequently exposed to particulate or soluble stimuli. The effect of IAV on PMNLs is likely to be mediated through the attachment of IAV to one or more specific receptors. Recently, IAV has been shown to bind to the sialophorin protein (CD43) receptor on PMNL plasma membranes. The present study was performed to determine if the sialophorin receptor was responsible for IAV-induced PMNL dysfunction. When PMNLs were incubated with IAV or CD43 monoclonal antibody (MoAb) for 30 minutes and then exposed to a secondary particulate (opsonized zymosan) or soluble (FMLP or phorbol 12- myristate 13-acetate) stimulus, there was significant depression of the PMNL chemiluminescence response compared with the equivalent control (P < .05). When PMNL were incubated with the CD43 MoAb and then cross- linked with a goat antimouse IgG antibody, no depression of PMNL function occurred upon secondary stimulation. Exposure of cells to IAV aggregates also eliminated the PMNL dysfunction that normally occurs due to the virus. Similar to IAV, PMNL dysfunction due to the CD43 MoAb could be overcome by priming the cells with granulocyte-macrophage colony-stimulating factor. These findings indicate that IAV-induced PMNL dysfunction is mediated, at least in part, through the sialophorin receptor.     

Estimating the prevalence of any impairing childhood mental disorder in the national health interview survey

This study investigates whether the six‐item Strengths and Difficulties Questionnaire SDQ (five symptoms and one impact item) included in the National Health Interview Survey (NHIS) can be used to construct models that accurately estimate the prevalence of any impairing mental disorder among children 4–17 years old as measured by a shortened Child/Adolescent or Preschool Age Psychiatric Assessment (CAPA or PAPA). A subsample of 217 NHIS respondents completed a follow‐up CAPA or PAPA interview. Logistic regression models were developed to model presence of any child mental disorder with impairment (MDI) or with severe impairment (MDSI). Models containing only the SDQ impact item exhibited highly biased prevalence estimates. The best‐performing model included information from both the five symptom SDQ items and the impact item, where absolute bias was reduced and sensitivity and concordance were increased. This study illustrates the importance of using all available information from the six‐item SDQ to accurately estimate the prevalence of any impairing childhood mental disorder from the NHIS. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a patient-derived explant model for prediction of drug responses in endometrial cancer

ObjectiveTo undertake a pilot study to develop a novel Patient-Derived-Explant (PDE) model system for use in endometrial cancer (EC) that is capable of monitoring differential drug responses in a pre-clinical setting.MethodsFresh tumour was obtained post-hysterectomy from 27 patients with EC. Tumours were cut into 1–3 mm³ explants that were cultured at the air-liquid interface for 16–24 h in culture media. Explants were cultured in different media conditions to optimise viability. Explants were also treated with carboplatin/paclitaxel or pembrolizumab for 24 h and processed into histology slides. Multiplexed immunofluorescence for Ki67 (proliferation marker), cPARP (apoptosis marker) and CAM 5.2 (tumour mask) was performed followed by image analysis and quantitation of biomarker expression.ResultsEC samples are amenable to PDE culture with preserved histological architecture and PDE viability for up to 48 h, with the addition of autologous serum in culture media facilitating EC-PDE viability. Our PDE platform provides evidence of differential drug-response to conventional chemotherapeutics and immune checkpoint inhibition, and these responses can be assessed in the context of a preserved tumour microenvironment.ConclusionsOur PDE platform represents a rapid, low-cost pre-clinical model which can be easily integrated into drug development pipelines. PDE culture preserves original tumour architecture and enables evaluation of spatial relationships in the tumour microenvironment. PDE culture has the potential for personalised drug-testing in a pre-clinical setting which is increasingly important in an era of personalised medicine in the treatment of EC.     

Laparoscopic Excision of a Renal Subcapsular Abscess Presenting as a Subcapsular Haematoma

Renal subcapsular abscess is a very rare entity that is defined by a suppurative process localized to a space between the renal capsule and the renal parenchyma. The pathogenesis and aetiology of this entity remain speculative. To our knowledge, only five cases have been reported in the English literature. We describe a 74-year old woman with renal subcapsular abscess treated with laparoscopic removal and do a review of the literature.