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The role of store-operated Ca

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1f/f) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1f/f-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1f/f-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1f/f-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1f/f-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.

AIM To assess the effectiveness and safety of levetiracetam when used as first-line treatment of neonatal seizures. METHOD Four electronic databases, Medline, Embase, Web of Science, and ClinicalTrials.gov were systematically searched from inception until 20th November 2020. Randomized controlled trials (RCTs) and observational studies that included neonates born preterm and term were eligible for inclusion. The primary outcome measure was levetiracetam effectiveness, defined as seizure cessation within 24 hours of starting treatment. Secondary outcomes included short-term adverse events, mortality before discharge, and long-term neurodevelopmental outcomes. RESULTS Fourteen studies assessing 1188 neonates were included: four RCTs, three observational trials with phenobarbital as the control arm, and seven observational studies of levetiracetam with no control arm. Pooled efficacy of levetiracetam from observational studies was 45% (95% confidence interval [CI] 34-57%) (GRADE - very low). Meta-analysis of RCTs evaluating levetiracetam versus phenobarbital showed that both were equally effective (risk ratio [95% CI] 0.6 [0.30-1.20]) (GRADE - very low). Levetiracetam resulted in a lower risk of short-term adverse events compared to phenobarbital (risk ratio [95% CI] 0.24 [0.06-0.92]) (GRADE - moderate). INTERPRETATION Very low certainty of evidence suggests levetiracetam might not be more effective than phenobarbital. Moderate certainty of evidence indicates levetiracetam is associated with a lower risk of adverse events. Future trials on neonatal antiseizure medication therapy should include continuous electroencephalogram (EEG) monitoring as standard of care and enrol a homogenous population with similar seizure aetiology. What this paper adds Levetiracetam is effective in 45% of neonatal seizures. Levetiracetam might not be more effective than phenobarbital. Levetiracetam is likely to be safer than phenobarbital. Evidence available is limited and of very low certainty.

To determine the intrinsic role of Orai1 in osteoclast development, Orai1-floxed mice were bred with LysMcre mice to delete Orai1 from the myeloid lineage. PCR, in situ labelling and Western analysis showed Orai1 deletion in myeloid-lineage cells, including osteoclasts, as expected. Surprisingly, bone resorption was maintained in vivo, despite loss of multinucleated osteoclasts; instead, a large number of mononuclear cells bearing tartrate resistant acid phosphatase were observed on cell surfaces. An in vitro resorption assay confirmed that RANKL-treated Orai1 null cells, also TRAP-positive but mononuclear, degraded matrix, albeit at a reduced rate compared to wild type osteoclasts. This shows that mononuclear osteoclasts can degrade bone, albeit less efficiently. Further unexpected findings included that Orai1(fl/fl)-LysMcre vertebrae showed slightly reduced bone density in 16-week-old mice, despite Orai1 deletion only in myeloid cells; however, this mild difference resolved with age. In summary, in vitro analysis showed a severe defect in osteoclast multinucleation in Orai1 negative mononuclear cells, consistent with prior studies using less targeted strategies, but with evidence of resorption in vivo and unexpected secondary effects on bone formation leaving bone mass largely unaffected.

Background Seizures are the most significant neonatal emergency, with implications on neurodevelopment and mortality. Evidence for the best management of them still remains limited. Phenobarbital is currently the most used drug for neonatal seizure management. The use of Levetiracetam as an alternative is increasing. It is hypothesised to have a better safety profile. Objectives Assess the effectiveness and safety of levetiracetam when used as the first line treatment of neonatal seizures. Methods Three electronic databases; MEDLINE, EMBASE, and Web of Science were systematically searched from inception until 20th November 2020. Randomized controlled trials (RCTs) and observational studies that included term and preterm neonates were eligible for inclusion. The primary outcome measure was effectiveness of levetiracetam, defined as seizure cessation within 24 hours of starting treatment. Secondary outcomes included short-term adverse events, mortality before discharge and long-term neurodevelopmental outcomes. Results 14 studies assessing 1,188 neonates were included. Four were RCTs, three observational trials with phenobarbital as the control arm and seven observational studies of levetiracetam with no control arm. Pooled efficacy of levetiracetam from observational studies was 45% (95% CI- 34%-57%). Meta-analysis of RCTs evaluating levetiracetam versus phenobarbital showed that both were equally effective [RR (95% CI) - 0.6 (0.3–1.20)] (GRADE – Very low). Levetiracetam resulted in a lower risk of short-term adverse events compared to phenobarbital [RR (95% CI) - 0.24 (0.06–0.92)] (GRADE – Moderate). Conclusions Very low-quality evidence suggests that levetiracetam might not be more effective than phenobarbital. Moderate quality evidence indicates levetiracetam is associated with a lower risk of adverse events.

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid β-oxidation into the reducing equivalents NADH and FADH(2). Although mitochondrial matrix uptake of Ca(2+) enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca(2+) flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca(2+), mitochondrial bioenergetics and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1(R97W) resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca(2+). We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca(2+), likely because of increased MICU1 expression but resulted in changes in mitochondrial morphology. TCA cycle substrate–dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca(2+) flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca(2+) overload during periods of nutrient stress.

While the zinc finger transcription factors EGR1, EGR2, and EGR3 are recognized as critical for T‐cell function, the role of EGR4 remains unstudied. Here, we show that EGR4 is rapidly upregulated upon TCR engagement, serving as a critical “brake” on T‐cell activation. Hence, TCR engagement of EGR4(−/−) T cells leads to enhanced Ca(2+) responses, driving sustained NFAT activation and hyperproliferation. This causes profound increases in IFNγ production under resting and diverse polarizing conditions that could be reversed by pharmacological attenuation of Ca(2+) entry. Finally, an in vivo melanoma lung colonization assay reveals enhanced anti‐tumor immunity in EGR4(−/−) mice, attributable to Th1 bias, Treg loss, and increased CTL generation in the tumor microenvironment. Overall, these observations reveal for the first time that EGR4 is a key regulator of T‐cell differentiation and function.

Ca2+ signals, which facilitate pluripotent changes in cell fate, reflect the balance between cation entry and export. We found that overexpression of either isoform of the Ca2+-extruding plasma membrane calcium ATPase 4 (PMCA4) pump in Jurkat T cells unexpectedly increased activation of the Ca2+-dependent transcription factor nuclear factor of activated T cells (NFAT). Coexpression of the endoplasmic reticulum-resident Ca2+ sensor stromal interaction molecule 1 (STIM1) with the PMCA4b splice variant further enhanced NFAT activity; however, coexpression with PMCA4a depressed NFAT. No PMCA4 splice variant dependence in STIM1 association was observed, whereas partner of STIM1 (POST) preferentially associated with PMCA4b over PMCA4a, which enhanced, rather than inhibited, PMCA4 function. A comparison of global and near-membrane cytosolic Ca2+ abundances during store-operated Ca2+ entry revealed that PMCA4 markedly depressed near-membrane Ca2+ concentrations, particularly when PMCA4b was coexpressed with STIM1. PMCA4b closely associated with both POST and the store-operated Ca2+ channel Orai1. Furthermore, POST knockdown increased the near-membrane Ca2+ concentration, inhibiting the global cytosolic Ca2+ increase. These observations reveal an unexpected role for POST in coupling PMCA4 to Orai1 to promote Ca2+ entry during T cell activation through Ca2+ disinhibition.

Continuous positive airway pressure (CPAP) has consistently improved depressive symptoms of Obstructive Sleep Apnoea (OSA) patients. Yet, only one study included OSA patients using antidepressants. We assessed the effects of CPAP on depressive symptoms in OSA patients, including those using antidepressants. Also, evaluating if antidepressant users differ in presentation to sleep clinics or response to CPAP. At initial sleep consultations, 101 participants completed the Public Health Questionnaire-9 (PHQ 9) to assess depressive symptoms. Also, the Epworth Sleepiness Scale, Fatigue Severity Scale, and Massachusetts General Hospital Antidepressant Treatment Resistance Questionnaire to define antidepressant treatment. 96 participants completed a diagnostic sleep study, with severity assessed by Apnoea-Hypopnoea Index (AHI). 43 were diagnosed with OSA and began CPAP treatment. After 6-8 weeks of CPAP, 30 repeated the questionnaires. 58.4% of all participants and 60.5% of those with OSA had significant depressive symptoms. The 32.7% using antidepressants were significantly more depressed (p The highly prevalent depressive symptoms in OSA patients improve significantly with CPAP therapy, even after resistance to antidepressant medication. The reduction in OSA severity from antidepressants may prevent these patients being treated with CPAP and achieving an improvement in depressive symptoms.

Uncertainty still clouds the future of the University of Papua New Guinea's award-winning journalism training newspaper Uni Tavur, suspended in the second semester 1997 due to lack of staff and funding. - Letters by Jean Nuia (PNG); Robert Foster (USA); John Henningham (Australia); Laurie Breen (UK); Rogert Patching (Australia); Wendy, Bacon, Chris Nash, David McKnight, Penny O'Donnell and Brenda Mattick (Australia); Murray Burt (UK); Robert Hooper (USA); and Joe Chika Anyanwu (Australia).

Antigen presentation to the T-cell receptor leads to sustained cytosolic Ca2+ elevation, which is critical for T-cell activation. We previously showed that in activated T cells, Ca2+ clearance is inhibited by the endoplasmic reticulum Ca2+ sensor stromal interacting molecule 1 (STIM1) via association with the plasma membrane Ca2+/ATPase 4 (PMCA4) Ca2+ pump. Having further observed that expression of both proteins is increased in activated T cells, the current study focused on mechanisms regulating both up-regulation of STIM1 and PMCA4 and assessing how this up-regulation contributes to control of Ca2+ clearance. Using a STIM1 promoter luciferase vector, we found that the zinc finger transcription factors early growth response (EGR) 1 and EGR4, but not EGR2 or EGR3, drive luciferase activity. We further found that neither STIM1 nor PMCA4 is up-regulated when both EGR1 and EGR4 are knocked down using RNA interference. Further, under these conditions, activation-induced Ca2+ clearance inhibition was eliminated with little effect on Ca2+ entry. Finally, we found that nuclear factor of activated T-cell (NFAT) activity is profoundly attenuated if Ca2+ clearance is not inhibited by STIM1. These findings reveal a critical role for STIM1-mediated control of Ca2+ clearance in NFAT induction during T-cell activation.—Samakai, E., Hooper, R., Martin, K. A., Shmurak, M., Zhang, Y., Kappes, D. J., Tempera, I., Soboloff, J. Novel STIM1-dependent control of Ca2+ clearance regulates NFAT activity during T-cell activation.

Ca(2+) is a ubiquitous, dynamic and pluripotent second messenger with highly context-dependent roles in complex cellular processes such as differentiation, proliferation, and cell death [1]. These Ca(2+) signals are generated by Ca(2+)-permeable channels located on the plasma membrane (PM) and endoplasmic reticulum (ER) and shaped by PM- and ER-localized pumps and transporters. Differences in the expression of these Ca(2+) homeostasis proteins contribute to cell and context-dependent differences in the spatiotemporal organization of Ca(2+) signals and, ultimately, cell fate. This review focuses on the Early Growth Response (EGR) family of zinc finger transcription factors and their role in the transcriptional regulation of Stromal Interaction Molecule (STIM1), a critical regulator of Ca(2+) entry in both excitable and non-excitable cells.

Background: The management of IPF has changed since the introduction of anti-fibrotic agents Pirfenidone (Pirf) and Nintedanib (Nin) however, there are no studies on head to head comparison of these agents. Aims: To examine the real-world tolerability, efficacy and safety of Pirf and Nin in patients with a diagnosis of IPF Methods: We conducted a retrospective single centre records review on 71 patients who received Pirf or Nin for IPF between Sep 2013 and Aug 2016. Data was collected on diagnosis, demographics, pulmonary function testing, adverse events and reasons for withdrawal or switching from the medication. We also compared the tolerability and efficacy of these two agents. Results: 36 patients were started on Pirf and 35 on Nin. The mean age for the Pirf group was 73.6 ± 7.1 (86.1% males) while for Nin this was 73.9 ± 5.8 (85.7% males). In the Pirf group 15 (41.7) were withdrawn from treatment, 9 (25%) switched to Nin and 10 (27.8) died while in the Nin group the figures were 14 (40 %); 2 (5.7) and 7 (20%) respectively. There was no difference in the decline in FVC or TLCO over time between the two medications. Conclusions: There was no difference in the rate of decline in lung functions (FVC and TLCO) or withdrawal rates between the two drugs, the number of patients who switched from Pirfenidone to Nintedanib was higher compared contrariwise suggesting better tolerability to Nintedanib..

Despite pharmacological prophylaxis using calcineurin (CN) inhibitors (i.e., cyclosporin A and Tacrolimus), graft-versus-host disease (GVHD) remains a major barrier to the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Upon antigen stimulation, activated T cell receptor (TCR) signaling triggers rapid Ca2+ influx. This induces both CN-mediated NFAT activation, and increase of mitochondrial Ca2+ content, a major driver of metabolic activity. However, mitochondrial Ca2+ overload triggers opening of the mitochondrial permeability transition pore and cell death. We hypothesize that pharmacologically increasing mitochondrial Ca2+ load may decrease T cell survival capability, thereby reducing the GVH reaction. If this can be accomplished, it may lead to new strategies for inhibition of GVHD, which is conceptually different from the use of CN inhibitors. To test this hypothesis, we employed a high throughput drug screening system with the proliferation and cytotoxicity of TCR-activated human T cells as the readout, and screened the NPL-800 library (https://www.timtec.net) composed of 800 pure natural compounds. With a stringent criterion in consideration of dose-dependent effect, 26 compounds stood out for reducing the count of activated human T cells with a reduction rate of at least 30% at both 1.0uM and 10.0uM. Positive hits included inhibitors of DNA synthesis, the Na-K-ATPase and mitochondrial metabolism. We were particularly interested in artesunate (ART), which is a derivative of artemisinin that has been used for treating malaria in patients. While artemisinin acts by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in P. falciparum malaria, which causes passive endoplasmic reticulum (ER) Ca2+ depletion and the subsequent cytosolic Ca2+ influx, ART did not inhibit SERCA in T cells. Ex vivo culture assays showed that ART dose-dependently reduced the survival of TCR-activated murine T cells. This inhibitory effect of ART was abrogated by inhibiting Ca2+ influx using BTP2, a potent inhibitor of store-operated Ca2+ channels. Furthermore, treatment of murine CD8 T cells with ART induced significant increases in mitochondrial Ca2+ loading upon TCR activation. These data suggest that inhibition of T cell survival by ART was dependent on TCR activation-induced Ca2+ influx and associated with enhanced mitochondrial Ca2+ uptake. We examined the impact of ART on GVHD in Balb/c mice receiving C57BL/6 (B6) mouse T cell-depleted bone marrow (TCD-BM) and CD4+ T cells. Intraperitoneal injection of ART (10 mg/kg, every other day) from day 1 to day 28 after transplantation reduced clinical signs of GVHD in these recipients and significantly improved their overall survival. Similar inhibition effects of ART on GVHD were observed in miHA-mismatched B6 anti-Balb/b and haplo-identical B6 anti-BDF1 mouse models of GVHD. Further investigations showed that in vivo administration of ART caused significant decreases in the number of host-reactive donor T cells in the spleen and liver of Balb/c mice 7 days after transfer of B6 TCD-BM plus CD4+ T cells. ART treatment did not affect the capacity of donor T cells to produce effector cytokines (e.g., IFN-g and TNF-α) in individual cells. Importantly, in vivo administration of ART preserved anti-leukemia activity of donor T cells and did not impair the reconstitution of hematopoiesis and lymphocytes. Collectively, our findings indicate that pharmacologically increasing mitochondrial Ca2+ loading may have significant implications in the development of novel strategies to prevent GVHD and other T cell-mediated inflammatory disorders in a broad context. Since ART therapy has been clinically approved, this work could be immediately translated into patients. Disclosures No relevant conflicts of interest to declare.

Orai channels at the plasma membrane mediate store-operated Ca(2+) entry in response to Ca(2+) depletion of the endoplasmic reticulum. Orai channels are gated by stromal-interacting molecule proteins, which act as Ca(2+) sensors, and the association of these proteins is enhanced in cholesterol-rich lipid rafts. In research published in Science Signaling, Derler et al. report that cholesterol inhibits Orai function through direct association with the channel amino terminus.

TPCs (two-pore channels) have recently been identified as targets for the Ca2+-mobilizing messenger NAADP (nicotinic acid–adenine dinucleotide phosphate). TPCs have a unique structure consisting of cytosolic termini, two hydrophobic domains (I and II) each comprising six transmembrane regions and a pore, and a connecting cytosolic loop; however, little is known concerning how these channels are assembled. In the present paper, we report that both domain I and II of human TPCs are capable of independent insertion into membranes, whereas the loop linking the domains fails to insert. Pairs of transmembrane regions within domain I of TPC1 are also capable of insertion, consistent with sequential translational integration of hydrophobic regions. Insertion of the first two transmembrane regions, however, was inefficient, indicating possible interaction between transmembrane regions during translation. Both domains, and each pair of transmembrane regions within domain I, were capable of forming oligomers, highlighting marked redundancy in the molecular determinants driving oligomer formation. Each hydrophobic domain formed dimers upon cross-linking. The first four transmembrane regions of TPC1 also formed dimers, whereas transmembrane regions 5 and 6, encompassing the pore loop, formed both dimers and tetramers. TPCs thus probably assemble as dimers through differential interactions between transmembrane regions. The present study provides new molecular insight into the membrane insertion and oligomerization of TPCs.

Graft-versus-host disease (GVHD) remains a major barrier for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have identified the central role of the histone methyltransferase Ezh2 in regulating allogeneic T-cell expansion, differentiation and function. Conditional loss of Ezh2 in donor T cells inhibits GVHD in mice due to the inability of alloreactive T cells to persist. However, the molecular mechanism by which Ezh2 deficiency causes alloreactive T cell death remains unknown. Here we demonstrate that genetic deletion of Stromal Interaction Molecule (Stim) 1, a dynamic endoplasmic reticulum Ca2+ sensor and regulator of Ca2+ signaling, rescues antigen-activated Ezh2-null (Ezh2-/-) T cells, leading to restored persistence of alloreactive effector T cells in mice and severe GVHD. Using RNA-sequencing analysis, we found Ezh2-deficiency led to the upregulation of multiple genes (e.g., Ifng, Prf1, Ccl5, Ccl4, Upp1 and Spp1) known to be regulated by Ca2+ signals through calcineurin (CN), the primary target of the immunosuppressant cyclosporine A (CsA). This reverse correlation between Ezh2 inhibition and CsA-treatment for gene expression suggests that Ezh2 may antagonize Ca2+ signaling in activated T cells. Calcium signaling assays revealed higher cytosolic Ca2+ uptake and more frequent Ca2+ oscillations in Ezh2-/- T cells. Moreover, Ezh2-/- T cells exhibited significantly increased polarization of Stim1 and Orai1 in the cellular membrane. These data reveal an unexpected role of Ezh2 as a negative regulator of Ca2+ entry, thereby serving as a 'brake' for Ca2+ signaling. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we found significantly fewer Ezh2-/- or Stim1-/- IFN-g-secreting effector T cells compared to the WT counterparts on day 8 or 14 post-transplantation. In contrast, deleting Stim1 from Ezh2-/- donor T cells rescued the cells in the spleen and liver, producing even more donor T cells and IFN-g-secreting effector T cells compared to WT T cells and inducing severe GVHD. We further examined the cell autonomous effect of Stim1 deletion on the rescue of Ezh2-/- T cells by mixing WT T cells (B6/SJL, CD45.1) with Ezh2- and/or Stim1- conditional knockout T cells (i.e., Ezh2-/-, Stim1-/- or Ezh2-/- x Stim1-/- B6 T cells (CD45.2)) at a ratio of 1:1 before transferring into the Balb/c mice. While loss of either Ezh2 or Stim1 led to lower frequency of IFN-g+IL-2+ effector T cells, combined deletion of both genes restored the frequency and number of IFN-g+IL-2+ effector T cells to that of WT T cells. Thus, Stim1-mediated Ca2+ signals are crucial for mediating cell death in alloantigen-driven Ezh2-/- effector T cells. To further determine whether the inhibition of CN-NFAT contributes to the rescue, we treated T cell receptor (TCR)-activated Ezh2-/- T cells with CsA or the calcium release-activated channel specific inhibitor BTP2, respectively, in vitro. While BTP2 dramatically improved the survival of IFN-g-producing effector T cells, CsA did not, suggesting the involvement of CN-NFAT-independent pathways. Ca2+ overload is known to impair mitochondrial function and cause massive cell death. As compared to TCR-activated WT T cells, activated Ezh2-/- T cells displayed significantly less ATP, lower mitochondrial membrane potential, enlarged mitochondrial mass, and decreased capacity to upregulate oxidative phosphorylation. Stim1 deletion largely reversed the metabolic defect in Ezh2-/- T cells, indicating the critical role of mitochondrial metabolism in rescuing these T cells. Considered together, our findings identify the remarkable coordination between Ezh2- and Stim1-regulated effector T cell persistence. As such, these investigations may lead to new approaches to inhibit GVHD, with broad implications to defining fundamental mechanisms of T cell differentiation for control of adaptive immunity, such as tumor immunity and autoimmunity. Disclosures Reshef: Incyte: Consultancy; Takeda Pharmaceuticals: Consultancy; Pfizer: Consultancy; Kite Pharma: Consultancy; Atara Biotherapeutics: Consultancy; Bristol-Myers Squibb: Consultancy.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca(2+) release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca(2+) stores, fuelling speculation that archetypal intracellular Ca(2+) channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca(2+) influx independent of ryanodine receptors and that it activates a Ca(2+)-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca(2+) channel.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the two-pore channel (TPC) family in mammals are the long sought after target channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, a key driver of melanoma invasiveness, induces Ca2+ signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive cell lines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca(2+) homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca(2+) release was also impaired using either a Rab binding-defective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease.

Almost a decade has passed since first STIM, and later Orai, proteins were identified as the molecular constituents of store-operated calcium entry (SOCE). Their roles in immune function have been intensely investigated, but the roles of STIM and Orai in neuronal cells have been much less clear. Lalonde et al. show that when neurons are hyperpolarized or "at rest," constitutive endoplasmic reticulum (ER) Ca(2+) release leads to SOCE-mediated activation of neuronal transcription factors. Precisely why ER Ca(2+) release is constitutive in neurons remains an important question. Irrespective of the answer, this observation provides an intriguing new perspective on why a relatively low-abundance, small-conductance channel such as Orai1 would be important in neurons, which contain a relative abundance of voltage-operated Ca(2+) channels.

T lymphocytes are key cellular effectors of adaptive immunity able to recognize a virtually limitless number of antigenic peptides and mount an immune response. Ca(2+) signals are crucial to the development and activation of T cells and Stromal Interaction Molecule 1 (STIM1) has been identified as a critical modulator of intracellular Ca(2+) levels in T cells. Although the role of STIM1 in T cell activation has been extensively investigated, the role of STIM1 in T cell development has been somewhat controversial. Indeed, deficiencies in STIM1 expression and function lead to both developmental defects associated with the development of autoimmunity yet also interfere with T cell activation leading to severe combined immunodeficiency signifying a multifaceted role of STIM1 in T cell physiology and pathophysiology.

TPCs (two-pore channels) are NAADP (nicotinic acid–adenine dinucleotide phosphate)-sensitive Ca2+-permeable ion channels expressed on acidic organelles. In the present study we show that deletion of the N-terminal region redirects TPC1 to the ER (endoplasmic reticulum). The introduction of fluorophores at the N-terminus of TPC1 does not affect its subcellular location, but does reversibly abolish NAADP sensitivity. Our results reveal a dual role for the N-terminus in localization and function of TPC1.

Stromal interaction molecules (STIM1 and STIM2) are critical components of store-operated calcium entry. Sensing depletion of endoplasmic reticulum (ER) Ca(2+) stores, STIM couples with plasma membrane Orai channels, resulting in the influx of Ca(2+) across the PM into the cytosol. Although best recognized for their primary role as ER Ca(2+) sensors, increasing evidence suggests that STIM proteins have a broader variety of sensory capabilities than first envisaged, reacting to cell stressors such as oxidative stress, temperature, and hypoxia. Further, the array of partners for STIM proteins is now understood to range far beyond the Orai channel family. Here we discuss the implications of STIM's expanding role, both as a stress sensor and a general modulator of multiple physiological processes in the cell.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent intracellular Ca(2+)-mobilising messenger. Much evidence indicates that NAADP targets novel Ca(2+) channels located on acidic organelles but the identity of these channels has remained obscure. Recent studies have converged on a novel class of ion channels, the two-pore channels (TPCs) as likely molecular targets. The location of these channels to the endo-lysosomal system and their sensitivity to NAADP match closely those of endogenous NAADP-sensitive channels in both mammalian cells and sea urchin eggs, where the effects of NAADP were discovered. Moreover, the functional coupling of TPCs to archetypal endoplasmic reticulum (ER) Ca(2+) channels is also matched. Biophysical analysis in conjunction with site-directed mutagenesis demonstrates that TPCs are pore-forming subunits of NAADP-gated ion channels. TPCs have a unique two-repeat structure, are regulated by N-linked glycosylation and harbor an endo-lysosomal targeting motif in their N-terminus. Knockdown studies have shown TPCs to regulate smooth muscle contraction, differentiation and endothelial cell activation consistent with previous studies implicating NAADP in these processes. Thus multiple lines of evidence indicate that TPCs are the likely long sought targets for NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an agonist-generated second messenger that releases Ca(2+) from intracellular acidic Ca(2+) stores. Recent evidence has identified the two-pore channels (TPCs) within the endolysosomal system as NAADP-regulated Ca(2+) channels that release organellar Ca(2+) in response to NAADP. However, little is known about the mechanism coupling NAADP binding to calcium release. To identify the NAADP binding site, we employed a photoaffinity labeling method using a radioactive photoprobe based on 5-azido-NAADP ([(32)P-5N(3)]NAADP) that exhibits high affinity binding to NAADP receptors. In several systems that are widely used for studying NAADP-evoked Ca(2+) signaling, including sea urchin eggs, human cell lines (HEK293, SKBR3), and mouse pancreas, 5N(3)-NAADP selectively labeled low molecular weight sites that exhibited the diagnostic pharmacology of NAADP-sensitive Ca(2+) release. Surprisingly, we were unable to demonstrate labeling of endogenous, or overexpressed, TPCs. Furthermore, labeling of high affinity NAADP binding sites was preserved in pancreatic samples from TPC1 and TPC2 knock-out mice. These photolabeling data suggest that an accessory component within a larger TPC complex is responsible for binding NAADP that is unique from the core channel itself. This observation necessitates critical evaluation of current models of NAADP-triggered activation of the TPC family.

Buprenorphine is an opioid receptor ligand whose mechanism of action is incompletely understood.Using Ca(2+) imaging, we assessed the effects of buprenorphine, β-endorphin, and morphine on cytosolic Ca(2+) concentration [Ca(2+)](i), in rat striatal neurons.Buprenorphine (0.01-1 μM) increased [Ca(2+)](i) in a dose-dependent manner in a subpopulation of rat striatal neurons. The effect of buprenorphine was largely reduced by naloxone, a non-selective opioid receptor antagonist, but not by μ, κ, δ or NOP-selective antagonists. β-Endorphin (0.1 μM) increased [Ca(2+)](i) with a lower amplitude and slower time course than buprenorphine. Similar to buprenorphine, the effect of β-endorphin was markedly decreased by naloxone, but not by opioid-selective antagonists. Morphine (0.1-10 μM), did not affect [Ca(2+)](i) in striatal neurons.Our results suggest that buprenorphine and β-endorphin act on a distinct type/subtype of plasmalemmal opioid receptors or activate intracellular opioid-like receptor(s) in rat striatal neurons.

Rapid improvements in communications infrastructure and sophistication of commercial hand-held devices provide a major new source of information for assessing extreme situations such as environmental crises. In particular, ad hoc collections of humans can act as "soft sensors" to augment data collected by traditional sensors in a net-centric environment (in effect, "crowd-sourcing" observational data). A need exists to understand how to task such soft sensors, characterize their performance and fuse the data with traditional data sources. In order to quantitatively study such situations, as well as study distributed decision-making, we have developed an Extreme Events Laboratory (EEL) at The Pennsylvania State University. This facility provides a network-centric, collaborative situation assessment and decision-making capability by supporting experiments involving human observers, distributed decision making and cognition, and crisis management. The EEL spans the information chain from energy detection via sensors, human observations, signal and image processing, pattern recognition, statistical estimation, multi-sensor data fusion, visualization and analytics, and modeling and simulation. The EEL command center combines COTS and custom collaboration tools in innovative ways, providing capabilities such as geo-spatial visualization and dynamic mash-ups of multiple data sources. This paper describes the EEL and several on-going human-in-the-loop experiments aimed at understanding the new collective observation and analysis landscape.

Summary Two levels of audience, commitment, choice, and time were varied in a 2 X 2 X 2 X 2 repeated measures factorial design in an attempt to examine the effects of these variables on attitude change both inside and outside the laboratory. Commitment and choice had no effect on attitude change. Time had a significant effect (p < .001), with a large amount of change occurring immediately after the experimental manipulation and very little change occurring three weeks later. Audience and time also produced a significant interaction (p < .01), with a large amount of attitude change occurring in the audience condition immediately after the manipulation and very little change three weeks later. In the no audience condition some attitude change occurred immediately after the manipulation, but little change remained three weeks later. These findings were interpreted within the framework of Katz's classic conception of an attitude, and it was argued that pressure on the conative dimension of an attitude was not...

The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. While easy to treat if diagnosed early, once the cancer has metastasized and spread, it is extremely resistant to current therapies and patient prognosis is poor. Wnt5A has been shown to be a driver of melanoma cell invasion and metastasis. Changes in cytosolic Ca2+ concentration are a key component of Wnt5A signaling and can contribute to both cell proliferation and migration. The main Ca2+ store in the cell is the endoplasmic reticulum (ER). Depletion of ER Ca2+ during cell signaling is sensed by the ER membrane protein STIM1, which interacts with the plasma membrane channel Orai1 to initiate store-operated calcium entry (SOCE) and replenish ER Ca2+. Therefore, we investigated differences in the expression and function of STIM1 and Orai1 in a range of patient-derived malignant melanoma cell lines, previously characterized as either highly invasive (metastatic) or non-invasive. We find that SOCE is diminished in invasive melanoma cells compared to non-invasive cells, although expression levels and localization of STIM and Orai are not perturbed. This represents a novel finding and suggests that aberrant SOCE is a key contributor to melanoma cell invasion and metastasis. Current investigations are focused on defining the mechanism(s) responsible for SOCE suppression in metastatic melanoma, focusing on both how STIM/Orai function is modulated and assessing the potential role of Wnt5A signaling in this process. Completion of these studies will provide critical new insight into cellular mechanisms controlling STIM/Orai function as well as redefining the role of Ca2+ signaling in tumor progression.