Cell calcium最新文献

{"title":"Regulation of K+-dependent Na+/Ca2+-exchanger subtype 4, NCKX4, by palmitoylation","authors":"By Maryam Al-Khannaq ,&nbsp;Jonathan Lytton","doi":"10.1016/j.ceca.2025.103069","DOIUrl":"10.1016/j.ceca.2025.103069","url":null,"abstract":"<div><div>Mammalian K⁺-dependent Na⁺/Ca²⁺ exchangers (NCKX), encoded by the SLC24 gene family, are crucial for maintaining Ca²⁺ homeostasis. NCKX4, widely expressed in the brain and sensory neurons, plays a key role in neuronal satiety and enamel formation. Despite its importance, the regulatory mechanisms of NCKX4 remain largely unexplored. This study investigates how palmitoylation, a post-translational modification affecting membrane proteins, regulates NCKX4 and influences its cellular localization and function.</div><div>Using Acyl-RAC and palmitate-based click-chemistry, we found that approximately 14% of NCKX4 is palmitoylated at steady-state in both endogenous and transfected systems. The level of this modification is highly dynamic, being regulated by inhibitors of palmitoylation (2-bromopalmitate) and depalmitoylation (palmostatin B), resulting in greater than a two-fold decrease or increase, respectively. Site-directed mutagenesis of six cysteine residues revealed two key sites (Cys118 and Cys425) critical for NCKX4 palmitoylation.</div><div>The subcellular distribution of palmitoylated NCKX4 was examined via proximity ligation and click-chemistry. NCKX4 was found across multiple membrane compartments, with a higher fraction localizing to the plasma membrane when palmitoylation was inhibited by 2-bromopalmitate. However, a Ca²⁺ imaging assay in HEK293T cells showed no significant change in aggregate cellular NCKX4-mediated Ca²⁺ transport upon modulation of palmitoylation status. These data suggest palmitoylation promotes internalization of the NCKX4 protein while also activating it, counter-acting effects that result in unchanged NCKX4-mediated cellular Ca²⁺ transport activity.</div><div>In summary, NCKX4 is subject to dynamic palmitoylation, which influences both distribution across cellular compartments and intrinsic Ca²⁺ transport activity. These findings contribute to our understanding of the regulation and functional roles of NCKX4 in cellular physiology.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"131 ","pages":"Article 103069"},"PeriodicalIF":4.0,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144889882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"NAADP-mediated calcium release promotes angiopoietin 2 secretion by regulating Rab46-dependent Weibel-Palade body trafficking","authors":"Ryan D. Murray ,&nbsp;Melissa Rose ,&nbsp;Katarina T. Miteva ,&nbsp;David J. Beech ,&nbsp;Lynn McKeown","doi":"10.1016/j.ceca.2025.103068","DOIUrl":"10.1016/j.ceca.2025.103068","url":null,"abstract":"<div><div>Angiopoietin2 (Ang2), a regulator of angiogenesis, is stored with other pro-inflammatory and pro-thrombotic mediators, in endothelial-specific vesicles called Weibel-Palade bodies (WPBs). Acute stimulation of endothelial cells with histamine, delays Ang2 secretion by activating Rab46-specific trafficking of Ang2-containing WPBs to the microtubule organising centre (MTOC), where they persist until Ca²⁺ binds to the EF-hand of Rab46, enabling detachment. Here, using Ca²⁺ imaging and high-resolution light microscopy, we pharmacologically investigated the contribution of endolysosomal two-pore channel proteins (TPC) to the Ca²⁺ signal necessary for WPB detachment and Ang2 secretion. We show an increase in the histamine-evoked clustering of Rab46 (and thus WPBs) at the MTOC in the presence of TPC inhibitors Ned-19 and tetrandrine, and a decrease in the presence of a TPC2 agonist, TPC2-A1-N. Histamine-evoked secretion of Ang2 was decreased by pharmacological inhibition of TPC channels but potentiated in the presence of TPC2-A1-N. These data suggest that histamine-mediated Ca²⁺ release via TPC2 channels is necessary for the Rab46-dependent detachment of Ang2-positive WPBs from the MTOC and thus Ang2 secretion.</div></div><div><h3>Summary</h3><div>Ca²⁺ binding to the EF-hand of Rab46 in endothelial cells has previously been reported but the molecular mechanisms and functional relevance are unclear. Here, the authors show that Ca²⁺ released from TPC channels regulates the detachment of Rab46-positive WPBs from the MTOC, which thereby promotes secretion of Ang2.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"131 ","pages":"Article 103068"},"PeriodicalIF":4.0,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144892187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Histamine 1 receptors and reverse-mode Na+/Ca2+ exchanger drive extracellular Na+-dependent intracellular Ca2+ oscillations in human cerebrovascular endothelial cells","authors":"Valentina Brunetti ,&nbsp;Roberto Berra-Romani ,&nbsp;Nayeli Coyotl-Santiago ,&nbsp;Yair Esquitin-Gonzalez ,&nbsp;Giorgia Chinigò ,&nbsp;Gerardo Rosario Biella ,&nbsp;Francesco Moccia ,&nbsp;Giorgia Scarpellino","doi":"10.1016/j.ceca.2025.103067","DOIUrl":"10.1016/j.ceca.2025.103067","url":null,"abstract":"<div><div>Cerebrovascular endothelial cells represent the core component of the blood-brain barrier, (BBB) which plays a critical role in regulating the local ionic microenvironment around the synapses. Therefore, cerebrovascular endothelial cells experience dramatic changes in the extracellular concentrations of potassium and sodium ions during intense neuronal firing or pathological conditions, such as spreading depression.</div><div>Herein, we assessed the mechanisms by which a reduction in extracellular sodium concentration ([Na⁺]_o) triggers complex Ca²⁺ signals in the hCMEC/D3 cell line, which is the most widespread model of human BBB.</div><div>We demonstrate that lowering the [Na⁺]_o elicits a variety of Ca²⁺ signals, including monotonic increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and repetitive oscillations in [Ca²⁺]_i, which are triggered by the reverse-mode Na⁺/Ca²⁺ exchanger and histamine 1 receptor (H1R). Furthermore, we provide the first evidence that H1R may play a critical role in translating a reduction in [Na⁺]_o into the activation of phospholipase C and following production of inositol triphosphate (InsP₃), thereby inducing the rhythmic activation of InsP₃ receptors on the endoplasmic reticulum (ER) and progressive depletion of the ER Ca²⁺ pool. The fall in the ER Ca²⁺ concentration leads to quick Store-Operated Ca²⁺ Entry activation, which maintains the intracellular Ca²⁺ oscillations by rapidly refilling the ER Ca²⁺ store. The endothelial Ca²⁺ oscillations induced by the reduction in [Na⁺]_o may then lead to nitric oxide release.</div><div>These findings, therefore, shed novel light on the mechanisms whereby G_q protein coupled receptors (G_qPCRs) can shape endothelial Ca²⁺ signaling and Ca²⁺-dependent events at the human neurovascular unit.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"131 ","pages":"Article 103067"},"PeriodicalIF":4.0,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144886537","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Chemical but not mechanical stimulation reduce TRPA1 channel lateral mobility","authors":"Alicia Sampieri ,&nbsp;Alexander Asanov ,&nbsp;Aaron Pavel Rodríguez-Hernández ,&nbsp;Ileana Tobías-Juárez ,&nbsp;Daniel Martínez-Flores ,&nbsp;Luis Vaca","doi":"10.1016/j.ceca.2025.103059","DOIUrl":"10.1016/j.ceca.2025.103059","url":null,"abstract":"<div><div>The transient Receptor Potential Ankyrin 1 (TRPA1) is a member from the TRP superfamily of ion channels. TRPA1 channels are calcium-permeable nonselective cation channels, which are highly conserved throughout the animal kingdom. Mammals have only one member (TRPA1), while zebrafish has two (TRPA1a and TRPA1b). TRPA1 channels are activated by a plethora of stimuli, including noxious cold, mechanical stimulation, calcium, pH, reactive oxygen, and carbonyl species. In the present study we characterize the modulation of TRPA1b channel lateral mobility by Allyl isothiocyanate (AITC) and mechanical stimulation. We show that only AITC stimulation alters channel diffusion at the plasma membrane.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"131 ","pages":"Article 103059"},"PeriodicalIF":4.0,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144772087","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calmodulin enhancement of mitochondrial calcium uniporter function in isolated mitochondria","authors":"Sara A. Garcia ,&nbsp;Anne M. Neumaier ,&nbsp;Michael Kohlhaas ,&nbsp;Anton Xu ,&nbsp;Alexander Nickel ,&nbsp;Katharina J. Ermer ,&nbsp;Luzia Enzner ,&nbsp;Christoph Maack ,&nbsp;Vasco Sequeira ,&nbsp;Christopher N. Johnson","doi":"10.1016/j.ceca.2025.103056","DOIUrl":"10.1016/j.ceca.2025.103056","url":null,"abstract":"<div><div>Mitochondrial calcium (Ca²⁺) uptake and factors that regulate this process have been an area of immense interest given the roles in cellular energetics. Here, we have investigated the ability of the Ca²⁺ sensing protein Calmodulin (CaM) to modify the function of the Mitochondrial Ca²⁺ Uniporter (MCU). Our data leveraged recombinantly produced CaM and mitochondria isolated from healthy and MCU impaired/diseased mice (Barth syndrome model). We found CaM enhanced Ca²⁺ uptake in both the absence and presence of CaMKII inhibition (KN93 as well as AIP). Mitochondria lacking function MCU (Barth syndrome model) validated that MCU was responsible for Ca²⁺ uptake in our experiments. Control experiments demonstrate that the observed CaM enhancement does not arise from CaM Ca²⁺ buffering. Fitting the Ca²⁺fluorescence data supported a monophasic decay process where the presence of CaM yielded enhanced kinetic rates of Ca²⁺ uptake. This CaM enhancement effect persisted in the presence of PTP impairment (cyclosporin), and subtle modification to the CaM protein sequence (D131E) revealed that an intact CaM-C domain Ca²⁺ binding was required for enhancement of MCU function.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"131 ","pages":"Article 103056"},"PeriodicalIF":4.0,"publicationDate":"2025-07-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144772086","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Copine A is essential for calcium homeostasis in Dictyostelium","authors":"Amber D. Ide ,&nbsp;Cody T. Morrison ,&nbsp;Christer A. Carne ,&nbsp;Cynthia K. Damer","doi":"10.1016/j.ceca.2025.103055","DOIUrl":"10.1016/j.ceca.2025.103055","url":null,"abstract":"<div><div>Copines are a family of calcium-dependent phospholipid-binding proteins found in most eukaryotic organisms. The expression of multiple copine genes is dysregulated in various types of human cancers. Yet, a common mechanistic function for copines remains enigmatic. We are studying copines in <em>Dictyostelium</em>, which has six copine genes (<em>cpnA-cpnF</em>). Cells lacking <em>cpnA</em> (<em>cpnA-</em>) exhibit many phenotypes including defects in development, chemotaxis, adhesion, and contractile vacuole (CV) function. In this study, we identified a novel link between CpnA and calcium homeostasis. We found that <em>cpnA-</em> cells have more phosphatidylserine (PS) exposed in the outer leaflet of the plasma membrane due to having an increased intracellular calcium concentration. The PS exposure defect and the enlarged CV defect in <em>cpnA-</em> cells were rescued by chelating calcium. We further investigated the role of PatA, a CV-localized Ca²⁺-ATPase responsible for pumping calcium into the CV. Although <em>cpnA-</em> cells expressed normal levels of <em>patA</em>, immunofluorescence revealed reduced PatA localization to the CV membrane. Notably, <em>patA</em> knockdown (<em>patA^KD</em>) cells phenocopied <em>cpnA-</em> cells, displaying enlarged CVs, elevated intracellular calcium, and increased PS exposure. Taken together, our findings suggest that CpnA promotes calcium sequestration into the CV, likely by regulating PatA localization or activity. This role in calcium homeostasis provides a mechanistic framework for understanding copine function and offers insight into how calcium dysregulation associated with copines may contribute to cancer progression.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"130 ","pages":"Article 103055"},"PeriodicalIF":4.3,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144687286","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Calcium imaging: Unraveling the neurobiological mechanisms of depression across cellular and circuit dimensions","authors":"Xu Han ,&nbsp;Jinfang Song ,&nbsp;Zihui Geng ,&nbsp;Runxin Li ,&nbsp;Bingjin Li","doi":"10.1016/j.ceca.2025.103054","DOIUrl":"10.1016/j.ceca.2025.103054","url":null,"abstract":"<div><div>Calcium imaging has emerged as a pivotal technique for monitoring neuronal and glial activity, gaining widespread recognition in neuroscience research. This method primarily utilizes genetically encoded calcium indicators (GECIs) or synthetic fluorescent dyes to detect physiologically relevant calcium dynamics. Despite being one of the most prevalent mental disorders, depression's pathogenesis remains poorly understood. Calcium imaging serves as a powerful tool to identify depression-related cell types and neural circuits. This review systematically summarizes the evolution of calcium indicators and their integration with behavioral paradigms, electrophysiology, optogenetics, and chemogenetics to elucidate cellular and circuit mechanisms underlying depression. In addition, calcium imaging in depression and other disease comorbidities is also discussed. These synthesized findings establish a framework for developing precision-targeted antidepressant interventions.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"130 ","pages":"Article 103054"},"PeriodicalIF":4.3,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144596591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dietary calcium intake controls epithelial expression of TRPV6 independent of 1,25(OH)2D3 endocrine signaling","authors":"Hinata Tanishige ,&nbsp;Atsushi Uekawa ,&nbsp;Hitoki Yamanaka ,&nbsp;Shigeaki Kato ,&nbsp;Ritsuko Masuyama","doi":"10.1016/j.ceca.2025.103053","DOIUrl":"10.1016/j.ceca.2025.103053","url":null,"abstract":"<div><div>Dietary calcium intake modifies the action of active vitamin D [1,25(OH)₂D₃], which promotes the expression of transient receptor potential vanilloid (TRPV) 6, an epithelial calcium channel, to initiate intestinal calcium absorption in response to biological requirements. However, it is unclear whether the change caused by dietary intake results from endocrine regulation or the direct responses to luminal contents. In this study, to reveal the underlying mechanisms of intestinal calcium transport in response to dietary intake, we assessed the early postprandial responses in mice.</div><div>Although mice lacking intestinal vitamin D receptor function (<em>Int Vdr-</em>) exhibited severe calcium deficiency, a high-calcium diet (1 % calcium) containing 2-fold calcium compared to a control diet reversed impaired calcium absorption and compensated for the mechanisms of 1,25(OH)₂D₃-dependent transcellular calcium transport. Additionally, the calcium-sensing receptor (CaSR) was abundantly present at the basolateral site in the intestine and the signals were emphasized by a high-calcium diet.</div><div>To examine the direct response of intestinal epithelium to dietary intake, wild-type (<em>Int Vdr+</em>) and <em>Int Vdr-</em> mice were fed a control or high-calcium diet for 30- or 60-min after 23 h fasting. Serum glucose levels increased 30 min post-feeding in either genotype. TRPV6 expression increased 30 min post-feeding, whereas serum calcium levels were unaltered, suggesting that dietary intake stimulates TRPV6 expression.</div><div>These data suggest that the regulation of calcium absorption activated immediately after feeding differs from the mechanism involving endocrine responses. Factors altered in the early phase of feeding, such as glucose, may contribute to the regulation of calcium absorption.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"130 ","pages":"Article 103053"},"PeriodicalIF":4.3,"publicationDate":"2025-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144665828","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Macrophage migration inhibitory factor induces phospholamban phosphorylation in cardiac muscle","authors":"Zihan Tang ,&nbsp;Feng Liu ,&nbsp;Miyuki Nishi ,&nbsp;Fabienne Mackay ,&nbsp;Mutsuo Harada ,&nbsp;Hiroshi Takeshima","doi":"10.1016/j.ceca.2025.103051","DOIUrl":"10.1016/j.ceca.2025.103051","url":null,"abstract":"<div><div>The pleiotropic cytokine macrophage migration inhibitory factor (MIF) elevates sarcoplasmic reticulum (SR) Ca²⁺ content and enhances Ca²⁺ transient in cardiac muscle. Our imaging and immunoblot data indicated that the MIF-evoked effect is caused mainly by the phosphorylation of the SR Ca²⁺-pump regulator phospholamban (PLN). Gene expression data suggested that the cluster of differentiation 74 (CD74) and the C-X-C motif chemokine receptor 7 (CXCR7) form a major MIF receptor complex in cardiomyocytes, but CXCR7 activation alone seemed sufficient to exert the MIF-evoked effect. Our pharmacological assessments suggested that phosphoinositide 3-kinase (PI3K), AKT kinase and endothelial nitric oxide synthase (eNOS) were continuously stimulated in the downstream of CXCR7 activation. Furthermore, NO thus generated likely reacted to activate Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), leading to PLN phosphorylation and subsequent SR Ca²⁺-pump activation. Therefore, the CXCR7-PI3K-AKT-eNOS-CaMKII-PLN axis is proposed as a central pathway for MIF-evoked potentiation of cardiac Ca²⁺ signaling.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"130 ","pages":"Article 103051"},"PeriodicalIF":4.3,"publicationDate":"2025-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144654931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"VDAC1 as Janus in cell death and survival: Annexin A5 to the rescue","authors":"Ophélie Champion ,&nbsp;Jacek J. Litewka ,&nbsp;Pawel E. Ferdek ,&nbsp;Geert Bultynck","doi":"10.1016/j.ceca.2025.103052","DOIUrl":"10.1016/j.ceca.2025.103052","url":null,"abstract":"<div><div>VDAC1, a large conductance channel in the outer mitochondrial membrane, plays a crucial role in mitochondrial physiology. VDAC1 supports cellular metabolism and survival by serving as a mitochondrial Ca²⁺-uptake and ATP-exit system. Conversely, VDAC1 also contributes to apoptosis by forming oligomeric pores mediating cytochrome c release. Recently, Oflaz et al., EMBO J, 2025, identified the Ca²⁺-binding protein Annexin A5 as a dynamic, Ca²⁺-dependent switch that enhances VDAC1’s Ca²⁺-transport function while at the same time preventing pro-apoptotic VDAC1 oligomer formation.</div></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":"130 ","pages":"Article 103052"},"PeriodicalIF":4.3,"publicationDate":"2025-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144572386","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}