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Keith L Davis,1 Wenhui Wei,2 Juliana L Meyers,1 Brett S Kilpatrick,3 Naushira Pandya4 1RTI Health Solutions, Research Triangle Park, NC, USA; 2Sanofi US, Inc, Bridgewater, NJ, USA; 3AnalytiCare, LLC, Glenview, IL, USA; 4Nova Southeastern University College of Osteopathic Medicine, Fort Lauderdale, FL, USA Background: The management of type 2 diabetes mellitus in long-term care (LTC) settings can be complex as a result of age-related complications. Despite guideline recommendations, sliding scale insulin remains commonplace in the LTC setting and data on basal insulin use are lacking.Methods: This retrospective study used medical chart data and the Minimum Data Set from elderly LTC facility patients who received basal insulin (insulin glargine, insulin detemir, or neutral protamine Hagedorn insulin) for the treatment of diabetes, to investigate the practice patterns and associated clinical outcomes.Results: A total of 2,096 elderly, insulin-treated patients in LTC were identified, with 59.5% of them (N=1,247) receiving basal insulin. Of these, more than 50% of patients received sliding scale insulin in co-administration with basal insulin. Despite its ease of use, insulin pen use was very low, at 14.6%. Significant differences were observed between the basal insulin groups for glycated hemoglobin level and dosing frequency. Hypoglycemia was uncommon -17.2% of patients experienced at least one event, and there was no significant difference in the prevalence of hypoglycemia between the groups.Conclusion: These data suggest the underutilization of basal insulin in the LTC setting and worryingly high combinational use with sliding scale insulin. Differences in glycated hemoglobin and dosing frequencies between types of basal insulin warrant further comparative effectiveness studies. Keywords: long-term care, nursing homes, type 2 diabetes mellitus, insulin detemir, insulin glargine, NPH insulin

Lysosomes are acidic Ca 2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca 2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca 2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca 2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca 2+ signals. NH 4 Cl evoked comparable changes in pH but failed to affect Ca 2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca 2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca 2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca 2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca 2+ release. We conclude that Ca 2+ signals evoked by GPN most likely derive from acidic organelles., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Two-pore channels (TPCs) are Ca 2+ -permeable ion channels localised to the endo-lysosomal system where they regulate trafficking of various cargoes including viruses. As a result, TPCs are emerging as important drug targets. However, their pharmacology is ill-defined. There are no approved drugs to target them. And their mechanism of ligand activation is largely unknown. Here, we identify a number of FDA-approved drugs as TPC pore blockers. Using a model of the pore of human TPC2 based on recent structures of mammalian TPCs, we virtually screened a database of ~1500 approved drugs. Because TPCs have recently emerged as novel host factors for Ebola virus entry, we reasoned that Ebola virus entry inhibitors may exert their effects through inhibition of TPCs. Cross-referencing hits from the TPC virtual screen with two recent high throughput anti-Ebola screens yielded approved drugs targeting dopamine and estrogen receptors as common hits. These compounds inhibited endogenous NAADP-evoked Ca 2+ release from sea urchin egg homogenates, NAADP-mediated channel activity of TPC2 re-routed to the plasma membrane, and PI(3,5)P 2 -mediated channel activity of TPC2 expressed in enlarged lysosomes. Mechanistically, single channel analyses showed that the drugs reduced mean open time consistent with a direct action on the pore. Functionally, drug potency in blocking TPC2 activity correlated with inhibition of Ebola virus-like particle entry. Our results expand TPC pharmacology through the identification of approved drugs as novel blockers, support a role for TPCs in Ebola virus entry, and provide insight into the mechanisms underlying channel regulation. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech., (Copyright © 2018. Published by Elsevier B.V.)

Two-pore channels (TPCs) are Ca 2+ -permeable endo-lysosomal ion channels subject to multi-modal regulation. They mediate their physiological effects through releasing Ca 2+ from acidic organelles in response to cues such as the second messenger, NAADP. Here, we review emerging evidence linking TPCs to disease. We discuss how perturbing both local and global Ca 2+ changes mediated by TPCs through chemical and/or molecular manipulations can induce or reverse disease phenotypes. We cover evidence from models of Parkinson's disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes. A need for more drugs targeting TPCs is identified., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca 2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca 2+ -mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca 2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca 2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca 2+ -dependent hubs for signaling., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca 2+ release but their role in Ca 2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca 2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca 2+ release and Ca 2+ entry. Ca 2+ release required lysosomal and ER Ca 2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca 2+ channels, are capable of 'chatter' with ER Ca 2+ channels. Our data identify new modalities for TRPML1 action., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease., (© 2015. Published by The Company of Biologists Ltd.)

Acidic organelles form an important intracellular Ca(2+) pool that can drive global Ca(2+) signals through coupling with endoplasmic reticulum (ER) Ca(2+) stores. Recently identified lysosome-ER membrane contact sites might allow formation of Ca(2+) microdomains, although their size renders observation of Ca(2+) dynamics impractical. Here, we generated a computational model of lysosome-ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca(2+) 'amplifier' and lysosomal leaks as the Ca(2+) 'trigger'. The model qualitatively described global Ca(2+) responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca(2+) mobilising messenger NAADP demonstrated that lysosome-ER microdomains are capable of driving global Ca(2+) oscillations. Interestingly, our simulations suggest that the microdomain [Ca(2+)] need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca(2+). The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome-ER Ca(2+) dynamics., (© 2014. Published by The Company of Biologists Ltd.)

Ca(2+) microdomains are critical for regulating cellular activity and often form at membrane contact sites. Such sites between lysosomes and the ER potentially provide a platform for signaling by the Ca(2+) mobilizing messenger NAADP. However, at present we know little of how Ca(2+) release events are coordinated at these experimentally intractable junctions. We therefore developed a computational model of lysosome-ER microdomains, which suggested that small leaks of Ca(2+) from the lysosome couple to Ca(2+)-sensitive Ins(1,4,5)P 3 receptors on the ER to generate global, microdomain-dependent Ca(2+) signals. Here we discuss how the "mix-and-match" arrangement of different Ca(2+) signaling proteins on the "source" and "target" membranes might generate functionally heterogeneous Ca(2+) microdomains.

Accumulating evidence implicates acidic organelles of the endolysosomal system as mobilisable stores of Ca(2+) but their relationship to the better-characterised endoplasmic reticulum (ER) Ca(2+) store remains unclear. Here we show that rapid osmotic permeabilisation of lysosomes evokes prolonged, spatiotemporally complex Ca(2+) signals in primary cultured human fibroblasts. These Ca(2+) signals comprised an initial response that correlated with lysosomal disruption and secondary long-lasting spatially heterogeneous Ca(2+) oscillations that required ER-localised inositol trisphosphate receptors. Electron microscopy identified extensive membrane contact sites between lysosomes and the ER. Mobilisation of lysosomal Ca(2+) stores is thus sufficient to evoke ER-dependent Ca(2+) release probably through lysosome-ER membrane contact sites, and akin to the proposed mechanism of action of the Ca(2+) mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Our data identify functional and physical association of discrete Ca(2+) stores important for the genesis of Ca(2+) signal complexity.