Weisslauridsen0235

The GHB receptor agonist 3-chloropropanoic acid and the antagonist NCS-382 had no effects on glucagon secretion and did not affect stimulation of secretion induced by a drop in glucose from 7 to 1 mM. Inhibition of endogenous GHB formation with the GABA transaminase inhibitor vigabatrin also failed to influence glucagon secretion at 1 mM glucose and did not prevent the suppressive effect of 7 mM glucose. In human islets, GHB tended to stimulate glucagon secretion at 1 mM glucose, an effect mimicked by 3-chloropropanoic acid. We conclude that GHB does not mediate the inhibitory effect of glucose on glucagon secretion. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Mammalian cytochrome P450 enzymes often metabolize many pharmaceuticals and other xenobiotics, a feature that is valuable in a biotechnology setting. However, extant P450 enzymes are typically relatively unstable with T50 values ~30-40 °C. Reconstructed ancestral cytochrome P450 enzymes tend to have variable substrate selectivity compared to related extant forms, but also have higher thermostability and therefore may be excellent tools for commercial biosynthesis of important intermediates, final drug molecules, or drug metabolites. read more The mammalian ancestor of the cytochrome P450 1B subfamily was herein characterized structurally and functionally, revealing differences from the extant human CYP1B1 in ligand binding, metabolism, and potential molecular contributors to its thermostability. While extant human CYP1B1 has one molecule of α-naphthoflavone in a closed active site, we observed that subtle amino acid substitutions outside the active site in the ancestor CYP1B enzyme yielded an open active site with four ligand copies. A structure of the ancestor with 17β-estradiol revealed only one molecule in the active site, which still had the same open conformation. Detailed comparisons between the extant and ancestor forms revealed increases in electrostatic and aromatic interactions between distinct secondary structure elements in the ancestral forms that may contribute to their thermostability.  To the best of our knowledge, this represents the first structural evaluation of a reconstructed ancestral cytochrome P450, revealing key features that appear to contribute to their thermostability. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.In Staphylococcus aureus-caused endocarditis, the pathogen secretes staphylocoagulase (SC), thereby activating human prothrombin (ProT) and evading immune clearance. A previous structural comparison of the SC(1-325) fragment bound to thrombin and its inactive precursor prethrombin 2 has indicated that SC activates ProT by inserting its N-terminal dipeptide Ile1-Val2 into the ProT Ile16 pocket, forming a salt bridge with ProT's Asp194, thereby stabilizing the active conformation. We hypothesized that these N-terminal SC residues modulate ProT binding and activation. Here, we generated labeled SC(1-246) as a probe for competitively defining the affinities of N-terminal SC(1-246) variants preselected by modeling. Using ProT(R155,271,284Q) (ProTQQQ), a variant refractory to prothrombinase- or thrombin-mediated cleavage, we observed variant affinities between ~1 and 650 nM and activation potencies ranging from 1.8-fold that of wild-type SC(1-246) to complete loss of function. Substrate binding to ProTQQQ caused allosteric tightening of the affinity of most SC(1-246) variants, consistent with zymogen activation through occupation of the specificity pocket. Conservative changes at positions 1 and 2 were well tolerated, with Val1-Val2, Ile1-Ala2, and Leu1-Val2 variants exhibiting ProTQQQ affinity and activation potency comparable to wild-type SC(1-246). Weaker binding variants typically had reduced activation rates, although at near-saturating ProTQQQ levels, several variants exhibited limiting rates similar to or higher than that of wild-type SC(1-246). The Ile16 pocket in ProTQQQ appears to favor non-polar, non-aromatic residues at SC positions 1 and 2. Our results suggest that SC variants other than wild-type Ile1-Val2-Thr3 might emerge with similar ProT-activating efficiency. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.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. 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.OBJECTIVE In England, the National Health Service commissioned a National Management Service for children with primary ciliary dyskinesia (PCD). The aims of this study were to describe the health of children seen in this Service and compare lung function to children with cystic fibrosis (CF). DESIGN Multi-centre service evaluation of the English National Management PCD Service. SETTING Four nationally commissioned PCD centres in England. PATIENTS 333 children with PCD reviewed in the Service in 2015; lung function data were also compared with 2970 children with CF. RESULTS Median age at diagnosis for PCD was 2.6 years, significantly lower in children with situs inversus (1.0 vs 6.0 years, p less then 0.001). Compared with national data from the CF Registry, mean (SD) %predicted forced expiratory volume in one second (FEV1) was 76.8% in PCD (n=240) and 85.0% in CF, and FEV1 was lower in children with PCD up to the age of 15 years. Approximately half of children had some hearing impairment, with 26% requiring hearing aids.