Hegelundkoenig5133

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers relies on more than a dozen regulatory subunits (including B/R2, B'/R5, and B/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of the PPA holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase (ERK) signaling and protein kinase A (PKA) substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome-manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain-containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from manchette to sculpt the sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here, we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3-knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examinations revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicated that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues to the underlying pathology of the globozoospermia-like phenotype. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.OBJECTIVE To determine the annual rate of tuberculosis development after a positive tuberculin skin test (TST) or interferon-gamma release assay result (IGRA), or both, among untreated populations with characteristics believed to increase the risk of tuberculosis (at risk populations). DESIGN Systematic review and meta-analysis. DATA SOURCES Embase, Medline, and Cochrane Controlled Register of Trials from 1 January 1990 to 17 May 2019, for studies in humans published in English or French. Reference lists were reviewed. ELIGIBILITY CRITERIA AND DATA ANALYSIS Retrospective or prospective cohorts and randomised trials that included at least 10 untreated participants who tested positive to tuberculosis antigens (contained in TST or IGRA, or both) followed for at least 12 months. Following the preferred reporting items for systematic reviews and meta-analyses (PRISMA) and meta-analyses of observational studies in epidemiology (MOOSE) guidelines, two reviewers independently extracted study data and assessed qualityREGISTRATION PROSPERO CRD42019136608. © Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.INTRODUCTION Antimicrobial stewardship programmes (ASPs) are recommended to improve antibiotic use in healthcare and reduce antimicrobial resistance (AMR). Our aim was to investigate the effectiveness of ASPs in reducing antibiotic consumption, use of broad-spectrum/restricted antibiotics, antibiotic resistance and healthcare-associated infections (HAIs) in neonates. METHODS We searched PUBMED, SCIELO, EMBASE and the Cochrane Database (January 2000-April 2019) to identify studies on the effectiveness of ASPs in neonatal wards and/or neonatal intensive care units (NICUs). Outcomes were as follows reduction of antibiotic consumption overall and of broad-spectrum/target antibiotics, inappropriate antibiotic use, antibiotic resistance and HAIs. ASPs conducted in settings other than acute care hospitals, for children older than 1 month, and ASPs addressing antifungal and antiviral agents, were excluded. RESULTS The initial search identified 53 173 titles and abstracts; following the application of filters and inclusion criteria, a total of six publications were included in the final analysis. selleckchem All studies, of which one was multi-centre study, were published after 2010. Five studies were conducted exclusively in NICUs. Four articles applied multimodal interventions. Reduction of antibiotic consumption overall and/or inappropriate antibiotic use were reported by four articles; reduction of broad-spectrum/targeted antibiotics were reported by four studies; No article evaluated the impact of ASPs on AMR or the incidence of HAI in neonates. CONCLUSION ASPs can be effectively applied in neonatal settings. Limiting the use of broad-spectrum antibiotics and shorting the duration of antibiotic treatment are the most promising approaches. The impact of ASPs on AMR and HAI needs to be evaluated in long-term studies. © Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.