PPI-Induced Changes in Plasma Metabolite Levels Influence Total Hip Bone Mineral Density in a UK Cohort.

Proton pump inhibitors (PPIs) are among the most used drugs in the UK. PPI use has been associated with decreased bone mineral density (BMD) and increased fracture risk, although these results have been inconsistent. We hypothesized that PPI could modulate BMD by altering gut and/or host systemic metabolic environments. Using data from more than 5000 British male and female individuals, we confirmed that PPI use is associated with decreased lumbar spine and total hip BMD. This effect was not mediated through the gut microbiome. We suggest here that PPI use may influence total hip BMD, both directly and indirectly, via plasma metabolites involved in the sex hormone pathway. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A Novel approach using O-CVP to treat paraneoplastic NMO spectrum disorder associated with follicular lymphoma.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neuroinflammatory disorder of the central nervous system that very rarely may be a paraneoplastic phenomenon. We describe the case of a woman with a longitudinally extensive transverse myelitis (LETM). We identified a previously undiagnosed, follicular lymphoma and she was treated with the immunochemotherapy regime (obinutuzumab, cyclophosphamide, vincristine and prednisolone; O-CVP) for paraneoplastic NMOSD. Following two cycles, there was almost complete radiological remission of the myelitis and the patient showed some improvement in her neurological function. This case illustrates the heterogeneous aetiology that LETM may have and that O-CVP may be used as therapeutic option in patients with NMOSD driven by follicular lymphoma.

Probable delirium is a presenting symptom of COVID-19 in frail, older adults: a cohort study of 322 hospitalised and 535 community-based older adults.

BACKGROUND: Frailty, increased vulnerability to physiological stressors, is associated with adverse outcomes. COVID-19 exhibits a more severe disease course in older, comorbid adults. Awareness of atypical presentations is critical to facilitate early identification. OBJECTIVE: To assess how frailty affects presenting COVID-19 symptoms in older adults. DESIGN: Observational cohort study of hospitalised older patients and self-report data for community-based older adults. SETTING: Admissions to St Thomas' Hospital, London with laboratory-confirmed COVID-19. Community-based data for older adults using the COVID Symptom Study mobile application. SUBJECTS: Hospital cohort: patients aged 65 and over (n = 322); unscheduled hospital admission between 1 March 2020 and 5 May 2020; COVID-19 confirmed by RT-PCR of nasopharyngeal swab. Community-based cohort: participants aged 65 and over enrolled in the COVID Symptom Study (n = 535); reported test-positive for COVID-19 from 24 March (application launch) to 8 May 2020. METHODS: Multivariable logistic regression analysis performed on age-matched samples from hospital and community-based cohorts to ascertain association of frailty with symptoms of confirmed COVID-19. RESULTS: Hospital cohort: significantly higher prevalence of probable delirium in the frail sample, with no difference in fever or cough. Community-based cohort: significantly higher prevalence of possible delirium in frailer, older adults and fatigue and shortness of breath. CONCLUSIONS: This is the first study demonstrating higher prevalence of probable delirium as a COVID-19 symptom in older adults with frailty compared to other older adults. This emphasises need for systematic frailty assessment and screening for delirium in acutely ill older patients in hospital and community settings. Clinicians should suspect COVID-19 in frail adults with delirium.

A mutation in the major autophagy gene, WIPI2, associated with global developmental abnormalities.

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.

Assessing mammalian autophagy.

Autophagy (self-eating) is a highly conserved, vesicular pathway that cells use to eat pieces of themselves, including damaged organelles, protein aggregates or invading pathogens, for self-preservation and survival (Choi et al., N Engl J Med 368:651-662, 2013; Lamb et al., Nat Rev Mol Cell Biol 14:759-774, 2013). Autophagy can be delineated into three major vesicular compartments (the phagophore, autophagosome, autolysosome, see Fig. 1). The initial stages of the pathway involve the formation of phagophores (also called isolation membranes), which are open, cup-shaped membranes that expand and sequester the cytosolic components, including organelles and aggregated proteins or intracellular pathogens. Closure of the phagophore creates an autophagosome, which is a double-membrane vesicle. Fusion of the autophagosome with the lysosome, to form an autolysosome, delivers the content of the autophagosome into the lysosomal lumen and allows degradation to occur.Autophagy is a dynamic process that is initiated within 15 min of amino acid starvation in cell culture systems (Köchl et al., Traffic 7:129-145, 2006) and is likely to occur as rapidly in vivo (Mizushima et al., J Cell Biol 152:657-668, 2001). To initiate studies on the formation of the autophagosomes, and trafficking to and from the autophagic pathway, an ideal starting approach is to do a morphological analysis in fixed cells. Additional validation of the morphological data can be obtained using simple Western blot analysis. Here we describe the most commonly used morphological technique to study autophagy, in particular, using the most reliable marker, microtubule-associated protein 1A/1B-light chain 3 (LC3). In addition, we describe a second immunofluorescence assay to determine if autophagy is being induced, using an antibody to WD repeat domain, phosphoinositide interacting 2 (WIPI2), an effector of the phosphatidylinositol (3)-phosphate (PI3P) produced during autophagosome formation.

WIPI2B links PtdIns3P to LC3 lipidation through binding ATG16L1.

WIPI proteins, phosphatidylinositol 3-phosphate (PtdIns3P) binding proteins with ß-propeller folds, are recruited to the omegasome following PtdIns3P production. The functions of the WIPI proteins in autophagosome formation are poorly understood. In a recent study, we reported that WIPI2B directly binds ATG16L1 and functions by recruiting the ATG12-ATG5-ATG16L1 complex to forming autophagosomes during starvation- or pathogen-induced autophagy. Our model of WIPI2 function provides an explanation for the PtdIns3P-dependent recruitment of the ATG12-ATG5-ATG16L1 complex during initiation of autophagy.

WIPI2b and Atg16L1: setting the stage for autophagosome formation.

The double-membraned autophagosome organelle is an integral part of autophagy, a process that recycles cellular components by non-selectively engulfing and delivering them to lysosomes where they are digested. Release of metabolites from this process is involved in cellular energy homoeostasis under basal conditions and during nutrient starvation. Selective engulfment of protein aggregates and dysfunctional organelles by autophagosomes also prevents disruption of cellular metabolism. Autophagosome formation in animals is crucially dependent on the unique conjugation of a group of ubiquitin-like proteins in the microtubule-associated proteins 1A/1B light chain 3 (LC3) family to the headgroup of phosphatidylethanolamine (PE) lipids. LC3 lipidation requires a cascade of ubiquitin-like ligase and conjugation enzymes. The present review describes recent progress and discovery of the direct interaction between the PtdIns3P effector WIPI2b and autophagy-related protein 16-like 1 (Atg16L1), a component of the LC3-conjugation complex. This interaction makes the link between endoplasmic reticulum (ER)-localized production of PtdIns3P, triggered by the autophagy regulatory network, and recruitment of the LC3-conjugation complex crucial for autophagosome formation.

WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1.

Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic reticulum-localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of double-membrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12-5-16L1 complex. The molecular mechanisms of Atg12-5-16L1 recruitment and significance of PtdIns(3)P synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs, WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy through recruitment of the Atg12-5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding Salmonella and recruits the Atg12-5-16L1 complex, initiating LC3 conjugation, autophagosomal membrane formation, and engulfment of Salmonella.

Endocytosis and autophagy: Shared machinery for degradation.

Two key questions in the autophagy field are the mechanisms that underlie the signals for autophagy initiation and the source of membrane for expansion of the nascent membrane, the phagophore. In this review, we discuss recent findings highlighting the role of the classical endosomal pathway, from plasma membrane to lysosome, in the formation and expansion of the phagophore and subsequent degradation of the autophagosome contents. We also highlight the striking conservation of regulatory factors between the two pathways, including those regulating membrane budding and fusion, and the role of the lysosome in sensing the nutrient status of the cell, regulating mTORC1 activity, and ultimately the initiation of autophagy. Editor's suggested further reading in BioEssays The evolution of dynamin to regulate clathrin-mediated endocytosis Abstract.

Fission yeast sec3 bridges the exocyst complex to the actin cytoskeleton.

The exocyst complex tethers post-Golgi secretory vesicles to the plasma membrane prior to docking and fusion. In this study, we identify Sec3, the missing component of the Schizosaccharomyces pombe exocyst complex (SpSec3). SpSec3 shares many properties with its orthologs, and its mutants are rescued by human Sec3/EXOC1. Although involved in exocytosis, SpSec3 does not appear to mark the site of exocyst complex assembly at the plasma membrane. It does, however, mark the sites of actin cytoskeleton recruitment and controls the organization of all three yeast actin structures: the actin cables, endocytic actin patches and actomyosin ring. Specifically, SpSec3 physically interacts with For3 and sec3 mutants have no actin cables as a result of a failure to polarize this nucleating formin. SpSec3 also interacts with actin patch components and sec3 mutants have depolarized actin patches of reduced endocytic capacity. Finally, the constriction and disassembly of the cytokinetic actomyosin ring is compromised in these sec3 mutant cells. We propose that a role of SpSec3 is to spatially couple actin machineries and their independently polarized regulators. As a consequence of its dual role in secretion and actin organization, Sec3 appears as a major co-ordinator of cell morphology in fission yeast.