Role of Candida albicans Tem1 in mitotic exit and cytokinesis

Journal ArticleResearch Support, Non-U.S. Gov'tCopyright © 2014 Elsevier Inc. All rights reserved.Candida albicans demonstrates three main growth morphologies: yeast, pseudohyphal and true hyphal forms. Cell separation is distinct in these morphological forms and the process of separation is closely linked to the completion of mitosis and cytokinesis. In Saccharomyces cerevisiae the small GTPase Tem1 is known to initiate the mitotic exit network, a signalling pathway involved in signalling the end of mitosis and initiating cytokinesis and cell separation. Here we have characterised the role of Tem1 in C. albicans, and demonstrate that it is essential for mitotic exit and cytokinesis, and that this essential function is signalled through the kinase Cdc15. Cells depleted of Tem1 displayed highly polarised growth but ultimately failed to both complete cytokinesis and re-enter the cell cycle following nuclear division. Consistent with its role in activating the mitotic exit network Tem1 localises to spindle pole bodies in a cell cycle-dependent manner. Ultimately, the mitotic exit network in C. albicans appears to co-ordinate the sequential processes of mitotic exit, cytokinesis and cell separation.BBSR

Understanding the life experiences of people with multiple complex needs: peer research in a health needs assessment

Background Multiple complex needs (MCN) describe a population experiencing a combination of homelessness, substance use, offending and/or mental ill-health. Using peer researchers, this study aimed to explore the perspectives of individuals with lived experience of MCN with regards to (i) issues leading to MCN and (ii) key intervention opportunities. Methods As part of a health needs assessment in Gateshead (North East England), trained peer researchers interviewed 27 adults (aged ≥18 years) with experience of MCN, identified using purposive sampling methods. Peer researchers designed a topic guide for interviews which were audio recorded and thematically analyzed. Results Interviewees reported adverse childhood experiences leading to MCN including abuse, bereavement, parental imprisonment, family break-up and inadequate support. Mental ill-health, substance use, poverty, early experiences of unstable housing and acute homelessness were identified as major precedents for adulthood experiences of MCN. Between 16 and 20 years, access to housing, social and mental health support was perceived as having the potential to prevent circumstances worsening. Individuals perceived removing barriers to mental health, housing and welfare and financial supports could help. Conclusions This study highlights the perceived role austerity, adverse childhood events and current service provision have in current and future experiences of MCN. Individuals expressed a need for future interventions and support to be judgement free and provided by workers who are educated about MCN and related adversity. Involving peer researchers and individuals with experience of MCN in future research and service provision could ensure appropriate measures and supports are put in place

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general

Leber Congenital Amaurosis Associated with AIPL1: Challenges in Ascribing Disease Causation, Clinical Findings, and Implications for Gene Therapy

Leber Congenital Amaurosis (LCA) and Early Childhood Onset Severe Retinal Dystrophy are clinically and genetically heterogeneous retinal disorders characterised by visual impairment and nystagmus from birth or early infancy. We investigated the prevalence of sequence variants in AIPL1 in a large cohort of such patients (n = 392) and probed the likelihood of disease-causation of the identified variants, subsequently undertaking a detailed assessment of the phenotype of patients with disease-causing mutations. Genomic DNA samples were screened for known variants in the AIPL1 gene using a microarray LCA chip, with 153 of these cases then being directly sequenced. The assessment of disease-causation of identified AIPL1 variants included segregation testing, assessing evolutionary conservation and in silico predictions of pathogenicity. The chip identified AIPL1 variants in 12 patients. Sequencing of AIPL1 in 153 patients and 96 controls found a total of 46 variants, with 29 being novel. In silico analysis suggested that only 6 of these variants are likely to be disease-causing, indicating a previously unrecognized high degree of polymorphism. Seven patients were identified with biallelic changes in AIPL1 likely to be disease-causing. In the youngest subject, electroretinography revealed reduced cone photoreceptor function, but rod responses were within normal limits, with no measurable ERG in other patients. An increasing degree and extent of peripheral retinal pigmentation and degree of maculopathy was noted with increasing age in our series. AIPL1 is significantly polymorphic in both controls and patients, thereby complicating the establishment of disease-causation of identified variants. Despite the associated phenotype being characterised by early-onset severe visual loss in our patient series, there was some evidence of a degree of retinal structural and functional preservation, which was most marked in the youngest patient in our cohort. This data suggests that there are patients who have a reasonable window of opportunity for gene therapy in childhood

Role of the Candida albicans MNN1 gene family in cell wall structure and virulence

Background The Candida albicans cell wall is the first point of contact with the host, and its outer surface is heavily enriched in mannoproteins modified through the addition of N- and O-mannan. Previous work, using mutants with gross defects in glycosylation, has clearly identified the importance of mannan in the host-pathogen interaction, immune recognition and virulence. Here we report the first analysis of the MNN1 gene family, which contains six members predicted to act as α-1,3 mannosyltransferases in the terminal stages of glycosylation. Findings We generated single null mutants in all members of the C. albicans MNN1 gene family, and disruption of MNN14 led to both in vitro and in vivo defects. Null mutants in other members of the family demonstrated no phenotypic defects, suggesting that these members may display functional redundancy. The mnn14Δ null mutant displayed hypersensitivity to agents associated with cell wall and glycosylation defects, suggesting an altered cell wall structure. However, no gross changes in cell wall composition or N-glycosylation were identified in this mutant, although an extension of phosphomannan chain length was apparent. Although the cell wall defects associated with the mnn14Δ mutant were subtle, this mutant displayed a severe attenuation of virulence in a murine infection model. Conclusion Mnn14 plays a distinct role from other members of the MNN1 family, demonstrating that specific N-glycan outer chain epitopes are required in the host-pathogen interaction and virulence

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

The Regulation of the Hog1 Stress Activated Protein Kinase in Candida albicans

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Imaging the Selective Binding of Synapsin to Anionic Membrane Domains

Synapsins are membrane-associated proteins that cover the surface of synaptic vesicles and are responsible for maintaining a pool of neurotransmitter-loaded vesicles for use during neuronal activity. We have used atomic force microscopy (AFM) to study the interaction of synapsin I with negatively charged lipid domains in phase-separated supported lipid bilayers prepared from mixtures of phosphatidylcholines (PCs) and phosphatidylserines (PSs). The results indicate a mixture of electrostatic binding to anionic PS-rich domains as well as some nonspecific binding to the PC phase. Interestingly, both protein binding and scanning with synapsin-coated AFM tips can be used to visualize charged lipid domains that cannot be detected by topography alone.NRC publication: Ye

Interaction of synapsin I with membranes

The synapsins (I, II, and III) comprise a family of peripheral membrane proteins that are involved in both regulation of neurotransmitter release and synaptogenesis. Synapsins are concentrated at presynaptic nerve terminals and are associated with the cytoplasmic surface of synaptic vesicles. Membrane-binding of synapsins involves interaction with both protein and lipid components of synaptic vesicles. Synapsin I binds rapidly and with high affinity to liposomes containing anionic lipids. The binding of bovine synapsin I to liposomes was studied using fluoresceinphosphatidyl-ethanolamine (FPE) to measure membrane electrostatic potential. Synapsin binding to liposomes caused a rapid increase in FPE fluorescence, indicating an increase in positive charge at the membrane surface. Synapsin I binding to monolayers resulted in a substantial increase in monolayer surface pressure. At higher initial surface pressures, the synapsin-induced increase in monolayer surface pressure is dependent on the presence of anionic lipids in the monolayer. Synapsin I also induced rapid aggregation of liposomes, but did not induce leakage of entrapped carboxyfluorescein, while other aggregation-inducing agents promoted extensive leakage. These results are in agreement with the presence of amphipathic stretches of amino acids in synapsin I that exhibit both electrostatic and hydrophobic interactions with membranes, and offer a molecular explanation for the high affinity binding of synapsin I to liposomes and for stabilization of membranes by synapsin I.NRC publication: Ye

Two-component mediated peroxide sensing and signal transduction in fission yeast

Two-component related proteins play a major role in regulating the oxidative stress response in the fission yeast, Schizosaccharomyces pombe. For example, the peroxide-sensing Mak2 and Mak3 histidine kinases regulate H(2)O(2)-induced activation of the Sty1 stress-activated protein kinase pathway, and the Skn7-related response regulator transcription factor, Prr1, is essential for activation of the core oxidative stress response genes. Here, we investigate the mechanism by which the S. pombe two-component system senses H(2)O(2), and the potential role of two-component signaling in the regulation of Prr1. Significantly, we demonstrate that PAS and GAF domains present in the Mak2 histidine kinase are essential for redox-sensing and activation of Sty1. In addition, we find that Prr1 is required for the transcriptional response to a wide range of H(2)O(2) concentrations and, furthermore, that two-component regulation of Prr1 is specifically required for the response of cells to high levels of H(2)O(2). Significantly, this provides the first demonstration that the conserved two-component phosphorylation site on Skn7-related proteins influences resistance to oxidative stress and oxidative stress-induced gene expression. Collectively, these data provide new insights into the two-component mediated sensing and signaling mechanisms underlying the response of S. pombe to oxidative stress. Antioxid. Redox Signal. 15, 153-165