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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.)

The neurodegenerative movement disorder Parkinson disease (PD) is prevalent in the aged population. However, the underlying mechanisms that trigger disease are unclear. Increasing work implicates both impaired Ca 2+ signalling and lysosomal dysfunction in neuronal demise. Here I aim to connect these distinct processes by exploring the evidence that lysosomal Ca 2+ signalling is disrupted in PD. In particular, I highlight defects in lysosomal Ca 2+ content and signalling through NAADP-regulated two-pore channels in patient fibroblasts harbouring mutations in the PD-linked genes, GBA1 and LRRK2 . As an emerging contributor to PD pathogenesis, the lysosomal Ca 2+ signalling apparatus could represent a novel therapeutic target., Competing Interests: Conflict of interest The author declares no conflict of interest.

Mutations in β-glucocerebrosidase (encoded by GBA1) cause Gaucher disease (GD), a lysosomal storage disorder, and increase the risk of developing Parkinson disease (PD). The pathogenetic relationship between the two disorders is unclear. Here, we characterised Ca(2+) release in fibroblasts from type I GD and PD patients together with age-matched, asymptomatic carriers, all with the common N370S mutation in β-glucocerebrosidase. We show that endoplasmic reticulum (ER) Ca(2+) release was potentiated in GD and PD patient fibroblasts but not in cells from asymptomatic carriers. ER Ca(2+) signalling was also potentiated in fibroblasts from aged healthy subjects relative to younger individuals but not further increased in aged PD patient cells. Chemical or molecular inhibition of β-glucocerebrosidase in fibroblasts and a neuronal cell line did not affect ER Ca(2+) signalling suggesting defects are independent of enzymatic activity loss. Conversely, lysosomal Ca(2+) store content was reduced in PD fibroblasts and associated with age-dependent alterations in lysosomal morphology. Accelerated remodelling of Ca(2+) stores by pathogenic GBA1 mutations may therefore feature in PD., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acidic organelles such as lysosomes serve as non-canonical Ca(2+) stores. The Ca(2+) mobilising messenger NAADP is thought to trigger local Ca(2+) release from such stores. These events are then amplified by Ca(2+) channels on canonical ER Ca(2+) stores to generate physiologically relevant global Ca(2+) signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca(2+) microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca(2+) signals. Ca(2+) microdomains between distinct Ca(2+) stores are thus likely to be integral to the genesis of complex Ca(2+) signals., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.)

Lysosomes are abundant organelles best known for their crucial role in macromolecule turnover. Lysosome dysfunction features in several diseases exemplified by the lysosomal storage disorders and is often associated with marked changes in lysosome structure. Lysosomal morphology may therefore serve as a sensitive readout of endocytic well-being. Here we describe methods for monitoring lysosome morphology in fixed and live cells using fluorescent probes and electron microscopy., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: New guidelines recommend a target glycated hemoglobin (HbA1c) of 7.5% to 8.0% in elderly persons with type 2 diabetes mellitus (T2DM), but real-world data regarding outcomes associated with different HbA1c levels in the elderly are limited. This study assessed outcomes and their association with defined HbA1c thresholds and age ranges in insulin-treated, elderly, patients with T2DM in long-term care (LTC)., Design: Retrospective analysis of medical charts and the Minimum Data Set (MDS) for the period September 2010 through September 2011., Setting: A total of 117 nursing homes in the United States., Participants: Eligible patients had resided in LTC for 3 months or more, had at least 1 full MDS assessment, 2 or more records of insulin dispensing with no pump use, and 1 or more HbA1c measurements., Measurements: Outcomes that were measured included hypoglycemia, ketoacidosis, infections, falls, hospitalization, and emergency room (ER) visits., Results: A total of 583 patients were included (mean age 78.9 years, mean chart observation length 55 days). In all groups, hypoglycemia was lowest in patients with an HbA1c level higher than 9.0%. In patients 75 years or older, infection rates were highest when HbA1c levels were higher than 9.0%. Falls increased by HbA1c level in patients aged 65 to 74 years, but decreased by HbA1c levels in patients 85 years or older. Ketoacidosis, hospitalization, and ER visits were low in all groups., Conclusion: These data suggest that better glycemic levels may not necessarily be associated with better clinical outcomes, and different age groups may exhibit different patterns, thereby supporting the call for individualized glycemic control among elderly patients., (Copyright © 2014. Published by Elsevier Inc.)

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.

Objectives: To examine prevalence, practice patterns, and associated burden of sliding scale insulin (SSI) therapy in elderly adults with type 2 diabetes (T2DM)., Design: A retrospective cross-sectional study assessing merged medical chart data and the Minimum Data Set., Setting: One hundred seventeen long-term care (LTC) facilities in seven U.S. states., Participants: Elderly adults with insulin-treated T2DM (N=2,096) admitted to a LTC facility after January 1, 2009, who had been in the facility for at least 3 months before chart abstraction and had received treatment with non-SSI or SSI regimens. The latter were categorized into SSI-only, basal–SSI, prandial–SSI, basal–prandial–SSI, and multiple regimens with SSI. Data were collected from September 2010 through September 2011., Measurements: Demographic and clinical characteristics were recorded. Fingerstick burden was assessed as the average number of fingersticks per week and average number without subsequent insulin administration. Other measures included glycosylated hemoglobin (HbA1c) and hypoglycemia events., Results: Overall, 73.8% of participants received SSI therapy. SSI-treated participants were more likely to be younger (P=.01), non-white (P=.002), and receiving sulfonylurea (P=.004) than non-SSI treated participants. SSI therapy was associated with a mean±standard deviation of 19.9±7.9 fingersticks per week, of which 12.5±7.6 were not followed by insulin administration. Fewer SSI-treated participants than non-SSI treated participants had one or more HbA1c measurements of 7.0% or less (48.8% vs 57.2%) or 8.5% or less (85.2% vs 87.6%, respectively). Rates of hypoglycemia were similar in both groups (15.0% vs 14.9%)., Conclusion: SSI therapy is widely used in LTC facilities and is associated with a high fingerstick burden. SSI regimens are associated with poorer glycemic control but a rate of hypoglycemia similar to that of non-SSI regimens., (© 2013, Copyright the Authors Journal compilation © 2013, The American Geriatrics Society.)

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.

Objective: To compare drug-utilization patterns and costs in patients with chronic kidney disease (CKD), not on dialysis, yet receiving epoetin alfa (EPO) or darbepoetin alfa (DARB) in a long-term care setting., Design: A retrospective analysis of pharmacy dispensing from January 2007 through March 2009, was conducted using the AnalytiCareSM LTC database., Setting: Long-term care., Patients, Participants: Patients>or=18 years of age, with >or=1 EPO or DARB dose dispensed, were included. Patients dispensed both agents, diagnosed with cancer, receiving chemotherapy, radiation therapy, or renal dialysis, were excluded., Main Outcome Measures: Mean cumulative erythropoiesis-stimulating agent (ESA) dose was used to calculate drug cost (using April 2009 wholesale acquisition cost) and dose ratio (Units EPO:mcg DARB). Results were also stratified by payer types., Results: A total of 2,259 patients were identified (EPO 1,640; DARB 619). EPO patients were slightly older (76.1 vs. 74.8 years of age, P=0.021), with similar proportion of women, compared with DARB patients. Mean (SD) cumulative dose was 98,420 (122,381) Units for EPO and 360 (428) mcg for DARB, resulting in a dose ratio of 273:1 (Units EPO:mcg DARB). The corresponding drug cost was 42% higher with DARB than with EPO ($1,734 vs. $1,217, P<0.001). Stratified analysis by payer types yielded similar results (dose ratios: 299:1 and 270:1 [Units EPO:mcg DARB]); cost premiums: 30% and 44% for Medicare Part A/Facility and Medicare Part D/Medicaid groups, respectively., Conclusions: This study of long-term care CKD patients receiving ESAs reported 42% higher drug cost with DARB compared with EPO and a dose ratio of 273:1.