Vascular smooth muscle cells (VSMC) switch to macrophage‐like cells after cholesterol loading, and this change may play an important role in atherogenesis. Muscleblind‐like splicing regulator 1 (MBNL1) is a well‐known splicing factor that has been implicated in many cellular processes. However, the role of MBNL1 in VSMC macrophage‐like transdifferentiation is largely unknown. In this study, we aim to characterize the role of MBNL1‐induced gene splicing during atherogenesis.The expression of MBNL1 and Abelson interactor 1 (Abi1) splice variants (Abi1‐e10 and Abi1‐Δe10) was compared between artery tissues from healthy donors and atherosclerosis patients. Regulatory mechanisms of MBNL1‐induced Abi1 gene splicing were studied, and the signal pathways mediated by Abi1 splice variants were investigated in VSMC.Loss of MBNL1 was found in the macrophage‐like VSMC (VSMC‐M) in artery wall from atherosclerosis patients. In vitro and in vivo evidence confirmed that Abi1 is one of the MBNL1 target genes. Loss of MBNL1 significantly induces the Abi1‐Δe10 isoform expression. Compared to the known actin organization activities of the Abi1 gene, we discovered a novel action of Abi1‐Δe10, whereby Abi1‐Δe10 activates Rac1 independent of upstream stimulation and triggers the Rac1‐NOX1‐ROS pathway, which results in increased expression of transcription factor Kruppel‐like factor 4 (KLF4). While Abi1‐Δe10 inhibits contractile VSMC biomarkers expression and cell contraction, it stimulates VSMC proliferation, migration and macrophage‐like transdifferentiation.Loss‐of‐function of MBNL1 activates VSMC‐M transdifferentiation to promote atherogenesis through regulating Abi1 RNA splicing. Loss of MBNL1 significantly induces the Abi1‐Δe10 isoform expression by alternative splicing in the macrophage‐like VSMC (VSMC‐M) in artery wall from atherosclerosis patients. A novel action of Abi1‐Δe10, whereby Abi1‐Δe10 actives Rac1 independent of upstream stimulation, was discovered. Abi1‐Δe10 triggers the Rac1‐NOX1‐ROS pathway to upregulate KLF4, which results in inhibition of contractile VSMC biomarkers expression and cell contraction and stimulation VSMC proliferation, migration and macrophage‐like transdifferentiation. Through analysing RNA‐seq data, we found that an Abi1 splicing variant that lacking exon10 (Abi1‐Δe10) is upregulated in VSMC‐M. Abi1‐Δe10 splicing is downregulated by splicing factor MBNL1 which expression was decreased in VSMC‐M. This splicing event activates Rac1 in absence of upstream stimulating and promotes KLF4 expression through a NADPH oxidase 1 (NOX1)‐dependent pathway. Our research provides a novel insight into the splicing regulatory mechanism in VSMC phenotype modulation during atherogenesis.22.1Artery samples were collected from peripheral artery atherosclerosis patients who received thigh amputation, and control arteries were obtained from healthy people who underwent amputation by accidents.2.2Tissues were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X‐100 and blocked with 1% BSA for 1 hour at room temperature. Cells were then incubated with the primary antibody, washed with PBS with 0.1% Triton X‐100 and incubated with FITC‐conjugated secondary antibody (1:1000 in PBST containing 1% BSA). Tissue imaging was captured by Zeiss fluorescent microscope (Carl Zeiss).2.3An Abi1‐e10‐specific RISH probe targeting the bp of Abi1‐e10 mRNA, an Abi1‐Δe10‐specific RISH probe targeting the bp of Abi1‐Δe10 mRNA and a negative control probe (targeting the dapB gene from bacteria) were designed. RISH assays were performed by using the QuantiGene ViewRNA ISH Assay Kit (Panomics) following the manufacture's protocol.2.4Tissues were digested by collagenase and cells were suspended with 1X TrypLE Express (Gibco) individually, blocked in PBS (Sigma) containing 0.1% FBS (Gibco) and stained with fluorescent antibodies against CD68 or CD45. PBS‐treated cells served as a control.2.5Human primary aortic smooth muscle cells were purchased from American Type Medium (ATCC PCS‐100‐030) with Vascular Smooth Muscle Cell Growth Kit (ATCC PCS‐100‐04). Lipofectamine 3000 (Invitrogen) and SuperFect Transfection Reagents (Qiagen) were used for plasmid and siRNA transfection.2.6Real‐time qPCR assays were performed as described.2.7Cell proliferation and migration assays were described previously.2.8Whole‐cell lysates were extracted by the buffer containing 50 mM of Tris pH8.0, 150 mM of NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, and proteinase and phosphatase inhibitors (Roche). Immunoblotting assays follow the standard protocol as reported.2.9The human genomic BAC clone (RP11‐75E16) was used as the template for PCR to amplify exon9, exon10, exon11, and their flanking intron regions (~300‐400 base pairs) of Abi1 gene (NM_001012751) by Platinum Taq DNA Polymerase High Fidelity (Invitrogen). The Abi1‐minigene with mutant was generated using the original Abi1‐minigene as the template. The integrity of the final construct was confirmed by DNA sequence.2.10RNA pulldown assay was performed as previously described.2.11RNA immunoprecipitation assay was performed as previous described.2.12To generate VSMC lines stably expressing Abi1‐e10 and1Abi1‐Δe10, the Abi1‐e10 and Abi1‐Δe10 cDNA were cloned into the FU(GW)BW‐blasticidin‐resistant lentivirus vector (purchased from Addgene) using Gateway Technology (Invitrogen) according to the manufacture's instruction. 293T packaging cells were co‐transfected with viral vectors, VSV‐G‐encoding plasmid and pCMV R8.9. The supernatants of transfected 293T cells containing viruses were used to infect VSMC cells in the presence of 10%FBS. The infected LNCaP cells were selected in the presence of 5μg/ml blasticidin. Expression of Abi1‐e10 and Abi1‐Δe10 was confirmed by both Western blot and real‐time qPCR.2.13Rac1 activity was measured by GTP pull‐down assay. Briefly, VSMC were lysed with buffer containing 50 mmol/L of Tris‐HCl . Lysates were incubated with glutathione S‐transferase/p21‐activated protein kinase/Rac interactive binding domain (GST‐PAK‐CRIB) and glutathione Sepharose‐4B beads (Thermo Fisher Scientific). Beads were then briefly washed. Bound proteins were eluted by boiling in 2× SDS sample buffer and then subjected to 12% SDS‐PAGE followed by immunoblotting with the Rac1 antibody. Blots were analysed with densitometry.2.14In cultured VSMC, the ROS levels were determined immediately after sample collection. Cellular ROS levels were assessed by measuring CM‐H2DCFDA (Thermo Fisher) fluorescence. All values were normalized to VSMC protein concentration.2.15t test. The level of significance was set at P <.05 as *, P <.01 as **.Statistical analysis was carried out by the GraphPad Prism 8.0 software. Differences among groups were compared by Student's 33.1It has been reported that vascular smooth muscle cells (VSMC) will transdifferentiate into a macrophage‐like phenotype (VSMC‐M) during atherogenesis. To identify the phenotype of VSMC with the use of cell‐specific markers, we first isolated the intima of control and atherosclerotic artery as described in the Material and Methods section. By double immunofluorescence staining for macrophage biomarker CD68 and smooth muscle biomarker αSMA, we found that the amount of CD68+ VSMC was significantly increased in the thickened intima of ASO arteries Figure . To eval3.2Splicing of Abi1 has been reported to be associated with several splicing factors. We profiled the mRNA level of these factors and found that the change of MBNL1 mRNA level was the most significant between control and atherosclerosis arteries Figure . This reTo confirm that MBNL1 regulates Abi1 splicing, we constructed an Abi1 minigene reporter in which exon10 and is flanking ~ 300bp nucleotides were inserted between exons 9 and 11 and transfected into VSMC. MBNL1 depletion upregulated Abi1‐Δe10 but downregulated Abi1‐e10 mRNA derived from the minigene reporter Figure . In vivo3.3Because MBNL1 is strongly expressed in VSMC but barely expressed in VSMC‐M, we introduced shRNA targeting MBNL1 into VSMC by lentivirus to study functions of MBNL1 Figure A‐B. MBNL3.4Rac1 and Abi1 are components of the WAVE2 complex. To understand further the role of Rac1 and Abi1, we examined levels of activated Rac1 in VSMC expression different Abi1 isoforms. Both G‐LISA Rac1 activation assay and PAK‐CRIB pulldown assay showed that the VSMC(Abi1‐e10) had lower levels of activated Rac1 than did the VSMC(Abi1‐Δe10) Figure . Co‐IP a3.5Recent evidence extends VSMC plasticity to transdifferentiation into macrophage‐like cells during atherogenesis that is dependent on KLF4, a transcription factor that induces cell dedifferentiation. Rac1 may promote VSMC transdifferentiation through modulating the expression and function of KLF4. Specifically, Rac1 activates the NADPH oxidase catalytic subunits 1 (NOX1) which in turn increases reactive oxygen species (ROS) production to generate oxidized phospholipids and induced KLF4 expression Figure . We show4In this manuscript, we report a novel action of Abi1 that contributes to VSMC progression to VSMC‐M. Functionally reprogrammed by the decrease of splicing factor MBNL1, the splice variant of Abi1‐Δe10 is highly expressed in VSMC‐M. Abi1‐Δe10 can interact with Rac1 to trigger the Rac1‐NOX1‐ROS signalling, resulting in the upregulation of KLF4 expression and function, acceleration of VSMC‐M transdifferentiation. These findings highlight the key roles of RNA splicing mechanisms in regulating VSMC phenotype modulation to facilitate atherosclerosis development.Consensus on the epidemiology of the macrophage‐like cell within the atherosclerotic lesions has not been reached. Multiple hypotheses have been proposed including that this cell originates from blood monocyte‐derived macrophages or VSMC through phenotype modulation.Abi1 is a known adapter protein that has been implicated in actin cytoskeletal remodelling, intercellular adhesion, and smooth muscle migration and contraction upon upstream stimulation.KLF4 is a well‐defined key regulator of cell dedifferentiation. Expression of KLF4 can be induced by oxidized phospholipids.Our study established that loss of MBNL1‐induced Abi1 splicing contributes to VSMC‐M progression. The adapter protein Abi1 is known to mediate various cellular functions triggered by RTKs such as EGFR and PDGFR through downstream effectors such as Rac1.5Loss‐of‐function of MBNL1 induced Abi1 gene splicing and generation of the Abi1‐Δe10 isoform, which activates VSMC‐M transdifferentiation to promote atherogenesis through Rac1‐NOX1‐ROS‐KLF4 pathway.The authors declared they do not have anything to disclose regarding conflict of interest with respect to this manuscript.Zhang L involved in concept and design. Li Y, Guo X and Xue G involved in acquisition of data. Li Y and Guo X involved in analysis and interpretation of data. Li Y, Ye M, Guo X and Xue G drafted the manuscript. Ye M, Zhao Y and Xue G critically revised the manuscript. Li Y, Ye M, Guo X and Xue G involved in statistical analysis. Li Y, Zhang L and Ni Q obtained funding. Wang H, Wang Y, Wang W, Yang S, Ni Q, Chen J, Lv L, Zhao Y and Xue G involved in administrative, technical or material support.All procedures were approved by the research ethics committee of Renji Hospital (RA‐2020‐071). All patients included in this research have signed the consents.All authors of the manuscript have read and agreed to its content and are accountable for all aspects of the accuracy and integrity of the manuscript in accordance with ICMJE criteria. The manuscript or portions thereof are not under consideration by any other journal that these findings have not been previously published.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


However, disruption of Ca2+ homeostasis will cause a series of kidney diseases, such as acute kidney injury (AKI), chronic kidney disease (CKD), renal ischemia/reperfusion (I/R) injury, autosomal dominant polycystic kidney disease (ADPKD), podocytopathy, and diabetic nephropathy. During the progression of kidney disease, Ca2+ signaling plays key roles in various cell activities such as necrosis, apoptosis, eryptosis and autophagy. Importantly, there are complex Ca2+ flux networks between the endoplasmic reticulum (ER), mitochondria and lysosomes which regulate intracellular Ca2+ signaling in renal cells and contribute to kidney disease. In addition, Ca2+ signaling also links the crosstalk between various cell deaths and autophagy under the stress of heavy metals or high glucose. In this regard, we present a review of Ca2+ signaling in cell death and crosstalk with autophagy and its potential as a therapeutic target for the development of new and efficient drugs against kidney diseases.The kidney is an important organ for the maintenance of Ca With a wide variety of biological functions in living creatures, calcium ions (Cato death .2+ is primarily stored in bones in the form of CaPO3 (hydroxyapatite), where it plays a structural role and also can be dissolved to serve as a source of Ca2+ in the blood [2+ is a ubiquitous, multifunctional signaling molecule that controls a wide variety of life processes including muscle contraction, neuronal delivery, hormone secretion, organelle communication, cell movement, fertilization, and cell growth. Because of the critical role of Ca2+ in these vital activities, cellular Ca2+ concentration is strictly regulated, and dysfunction of cellular Ca2+ homeostasis is closely associated with a great number of diseases such as heart and hepatic diseases, aging, and type 2 diabetes, as well as certain cancers [Ca cancers ,5.2+ homeostasis; the Ca2+ signaling system in kidney cells regulates cellular processes and decides cell fate, including cell proliferation, apoptosis, necrosis and autophagy, all of which are associated with kidney disease [2+ homeostasis has a certain influence on renal function and the occurrence and development of a series of nephropathies including acute kidney injury (AKI), chronic kidney disease (CKD), renal ischemia/reperfusion (I/R) injury, autosomal dominant polycystic kidney disease (ADPKD), podocytopathy, and diabetic nephropathy, which will be discussed in the following sections.The kidney is an indispensable organ for maintaining body Ca disease ,7. Ca2+ 2+ signaling in the regulation of cell fate in the context of kidney disease, this review focuses on cell death determined by Ca2+ signaling in kidney cells and erythrocytes. We present a general overview of Ca2+ signaling in kidney disease, as well as some types of cell death that are regulated by Ca2+ signaling. In addition, targets of Ca2+ signaling to prevent kidney disease are discussed based on the available data.Based on the significance of Ca2+ not only regulates the normal life activities of cells and organisms, but has also been found to play an important role in the regulation of various cell deaths. Early death of necrotic cells is associated with intracellular Ca2+ overload. It has also been found that apoptosis is controlled by Ca2+ signaling, and other cell activity, including cell death of erythrocytes (eryptosis), has also been shown to be regulated by Ca2+. Moreover, Ca2+ signaling is an important mediator of autophagy, which plays dual roles in cell survival and death. The numerous Ca2+ channels on cell membranes, endoplasmic reticulum (ER), and mitochondria, as well as alterations in lysosomes, modulate cytosolic Ca2+ levels associated with cell necrosis, apoptosis, eryptosis and autophagy. We searched for original research articles from the past two decades using the keywords “Ca2+” and “kidney disease” or “kidney injury”, and “necrosis”, or “apoptosis”, or “eryptosis”, or “autophagy”, and/or “endoplasmic reticulum (ER)”, and/or “mitochondrial”, and/or “lysosome” in the PubMed database, then sorted by “best match” with the “summary” format. For review articles, we referenced the information as mentioned in the study by Aromataris et al., such as by number of databases sourced and searched, number of studies, type and country of origin of the studies included in each review, and method of synthesis/analysis employed to synthesize the evidence [Caevidence .2+ overload, and has been found to be regulated by intracellular Ca2+ release and/or extracellular Ca2+ influx. Methylglyoxal, a physiological glucose metabolite, induces necrotic cell death in Madin-Darby canine kidney (MDCK) renal tubular cells by stimulating both extracellular Ca2+ influx and ER Ca2+ release [2+ release in isolated rabbit kidney cortex microsomes, and TBHP-induced necrotic cell death is reduced by an ER Ca2+ reuptake pump inhibitor, thapsigargin [Bothrops leucurus venom increases cytosolic Ca2+ in a concentration-dependent manner, participating predominantly in necrotic cell death, which contributes to the acute renal failure and nephrotoxicity in the isolated perfused kidney and cultured renal tubular epithelia [2+ release mediates necrotic cell death induced by stress in kidney cells.Necrosis is early cell death associated with intracellular Ca release . Oxidantsigargin . Bothroppithelia . This su2+-influx channel, is upregulated in the renal tubules of patients with acute tubular necrosis, which is significantly associated with high incidence of renal function recovery [2+ entry (SOCE), which causes prolonged Ca2+ entry and leads to a continuous rise in intracellular Ca2+ levels [2+ levels, leading to enhanced ROS production and a dose-dependent increase in apoptotic and necrotic cell death in the porcine renal proximal tubule cell line, LLC-PK1 [2O2-induced necrosis in proximal tubular cells and reduce subsequent acute kidney injury (AKI), which is mediated by transient receptor potential canonical (TRPC)-dependent receptor-operated Ca2+ entry [2+-influx channels which mediate Ca2+ entry have dual roles in renal epithelial cells. Sustained extracellular Ca2+ entry can contribute to renal cell necrosis, while modulation of appropriate extracellular Ca2+ entry may protect against kidney injury.Transient receptor potential ankyrin 1 (TRPA1), a redox-sensing Carecovery . Interna+ levels . This sh LLC-PK1 . However2+ entry . Due to +/Ca2+ exchange is one of the main factors in reversal of intracellular Ca2+ overload in animals with ischemia/reperfusion injury and contrast-induced acute kidney injury [+/Ca2+ exchange, significantly attenuates renal damage and Ca2+ deposition in necrotic tubular epithelium, suggesting that KB-R7943 may be an effective therapeutic agent for acute kidney injury. The use of chemotherapy with ifosfamide is limited by the nephrotoxicity caused by its metabolite chloroacetaldehyde. Chloroacetaldehyde induces sustained elevation of intracellular free Ca2+ by inhibiting the Na+/Ca2+ exchanger, which contributes to necrotic rather than apoptotic cell death and finally leads to nephrotoxicity [+-dependent extrusion of intracellular Ca2+; however, the former did not induce nephrotoxicity, which might due to different Ca2+ load levels in the kidney cells.In addition, Nay injury ,17. KB-Rtoxicity . Both KB2+ signaling acts as an important regulator of apoptosis. Ca2+ overload leads to necrotic or apoptotic death in renal ischemia–reperfusion (I/R) injury [2+ release induces ER stress and oxidative stress and leads to apoptosis in glomerular mesangial cells, which contributes to the progression of CKD [2+ homeostasis, and plays important roles in kidney cells and cancers [2+ levels, which inhibits apoptosis in the kidneys induced by high glucose and ameliorates glomerular endothelial cell and podocyte injury in type 2 diabetes-associated diabetic nephropathy [2+ can control the apoptosis of kidney cells.Apoptosis, or autonomous programmed cell death, is an important process for the maintenance of the stability of the internal environment, and helps an organism better adapt to its living environment. In kidney injury, apoptosis usually occurs in association with necrosis, and Ca) injury . Sustainn of CKD . CaSR is cancers . Activathropathy ,23. This2+ in cells, and it plays an important role in regulating intracellular Ca2+ balance; however, its dysregulation causes cell apoptosis. The ER Ca2+ homeostasis is mainly controlled by two Ca2+ releasing channels, inositol 1,4,5-trisphosphate receptor (IP3R) and ryanodine receptor (RyR), and one Ca2+ reuptake channel, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). Dysfunctions of these ER Ca2+ channels cause a variety of kidney diseases, including ischemia/reperfusion (I/R)-induced tubular injury, autosomal dominant polycystic kidney disease (ADPKD), podocytopathy, and diabetic nephropathy [2+ release from the ER store [2+ release in cystic cells, which contributes to increased apoptosis in ADPKD [2+ efflux through leaky RyR2 which in turn activates cytosolic protease calpain 2, leading to podocyte injury and podocyte apoptosis [2+ channels in the ER play crucial roles in maintaining intracellular Ca2+ homeostasis in kidney cells, and that it is for this reason that renal cell apoptosis and kidney dysfunction contribute to the development of kidney disease.It is well known that the ER is the largest pool of Cahropathy . It is rER store . In addiin ADPKD . Stressepoptosis . The evipoptosis . These r2+ store, and Ca2+ signaling mediates the crosstalk between the ER and the mitochondria. Ca2+ released by IP3R under ER stress is sequestered by mitochondria for mitochondrial respiration and ATP production, and for regulating cell survival. Mitochondrial release of calpain or caspase-3 promotes the change of ER Ca2+ channels, including cleavage IP3R or oxidizing RyR2. These changes may dramatically increase mitochondrial and cytosolic Ca2+ levels and augment apoptotic cell death [2+ concentration. The sustained increase of Ca2+ level feeds back to enhance cytochrome c release and amplification of apoptotic signals [2+ signaling plays a key role in the increased apoptosis found in kidney disease. In diabetic nephropathic mice the impaired activity and expression of SERCA2 causes ER Ca2+ depletion, which triggers mitochondria-mediated apoptotic pathways [2+ channel polycystin 2 enhances the expression of ER-mitochondrial tethering protein mitofusin 2, which increases ER-mediated mitochondrial Ca2+ transfer and apoptosis and thereby contributes to ADPKD [2+ release channel IP3R3 at ER–mitochondrial contact sites, while stabilized IP3R3 prolongs Ca2+ signaling to the mitochondria and enhances apoptosis [2+ signaling between the ER and mitochondria which is associated with apoptosis and plays an important role in kidney disease.Mitochondria represent another intracellular Call death . In addi signals . ER-mitopathways . Downregto ADPKD . In bilapoptosis . These r2+-entry and increased cytosolic Ca2+ levels [2+ entry, which may contribute to anemia in end stage renal disease [43− intoxication in the kidney also induces eryptosis through increased cytosolic Ca2+ levels, which could contribute to the development of anemia in chronic renal failure [2+ concentration in freshly drawn erythrocytes, suggesting that the effect of vitamin D treatment is presumably not effective in stimulating Ca2+-entry [2+ entry or increasing cytosolic Ca2+ levels could contribute to eryptosis, which consequently causes kidney disease.Eryptosis, also known as suicidal erythrocyte death, is characterized by cell shrinkage and phosphatidylserine (PS) exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Eryptosis is the key event in eliciting renal anemia associated with CKD and anemia in end stage renal disease . The myc+ levels . In addi disease ,35. Vana failure . A vitam2+-entry . These s2+, as an important messenger molecule that regulates cell death, is also involved in the regulation of autophagy [Macroautophagy/autophagy, an evolutionarily conserved process in eukaryotes, plays an important role in intracellular material recycling. In the process of autophagy, some damaged organelles and harmful proteins are wrapped by the autophagosomes with a double membrane structure, then sent into lysosomes or vacuoles for degradation and reuse . Ca2+, autophagy .2+ signaling mediates autophagy in renal tubular cells. An in vivo study has shown that the key regulator of the autophagy pathway, mTOR, is involved in tubular repair after AKI [2+ release and decreased mTOR activity [2+ influx in renal proximal tubular cells inhibits mTOR-dependent autophagy, thereby rendering cells more susceptible to death [2+ release or Ca2+ influx controls the development of kidney disease.It has been suggested that intracellular Cafter AKI . In condactivity . Increasto death . This in2+ influx channel in renal cells, plays an important role in such renal diseases as diabetic nephropathy, immune-mediated kidney disease, renal fibrosis, glomerular disease and CKD [2+ influx [2+ channel that conducts Ca2+ flux to regulating autophagy. Increased expression of TRPM3 leads to Ca2+ influx and stimulates autophagy through the CAMKK2/AMPK/ULK1 pathway, which promotes the growth of clear cell renal cell carcinoma (ccRCC) [2+ channels on the cell membrane mediate Ca2+ influx and suppress autophagy, which may contribute to kidney injury or diseases.The canonical transient receptor potential channel 6 (TRPC6), a major Ca and CKD . An in v+ influx . The sam+ influx . The tra (ccRCC) . These r2+ could mediate autophagy, and play important roles in kidney disease. Stromal interaction molecule 1 (STIM1) is a key regulator of ER Ca2+ which can activate store-operated Ca2+ entry (SOCE) upon sensing ER Ca2+ depletion. In podocytes cultured in the serum of diabetic nephrotic rats, silencing STIM1 can reverse decreased autophagy and increased epithelial–mesenchymal transition (EMT) by restoring Ca2+ homeostasis [2+ release to silence high levels of STIM1 [2+ levels [2+-dependent binding of APOL3 to neuronal calcium sensor 1 (NCS-1) and interacting with PI4-kinase IIIB in podocytes, which aggravates kidney inflammation [2+ suggests that there are also complex networks of intracellular Ca2+-regulated autophagy in kidney disease.In addition, intracellular modulators of Caeostasis . In podoof STIM1 ,48,49. I+ levels . The apoammation . The rol2+ contributes to autophagy and is important for lysosomal degradation. Lysosomal degradation is mediated by a class of Ca2+-activated proteases, Calpains (CAPNs), on lysosomes [2+ stores are unaffected, while some extracellular agonist-induced Ca2+ responses may contribute to disease pathogenesis [2+ signaling contributes to the development of kidney diseases also depends on renal cell type.Lysosomal Caysosomes . Lack ofysosomes . Howeverogenesis . Therefo2+ distribution between the ER, mitochondria and lysosomes may affect the function of lysosomal degradation. The mitochondria contact the ER membrane through the mitochondria-associated membrane (MAM), which provides communication between these two organelles and mediates Ca2+ transfer from the ER to the mitochondria. Moreover, MAM-mediated ER–mitochondria Ca2+ crosstalk regulates apoptosis and autophagy, which contributes to the pathogenesis of kidney disease [2+ redistribution from the ER to the mitochondria regulates apoptosis and autophagy and contributes to lead-induced nephrotoxicity in primary rat proximal tubular cells [2+ release channel IP3R and the Ca2+ reuptake pump SERCA, the ER delivers cytosolic Ca2+ to the lysosomes in “piston-like” fashion [2+ uptake by lysosomes is speculated to regulate lysosomal pH [2+/H+ exchange, although this has not been identified in mammalian cells [2+ release from the ER store to the cytosol, which caused disruption of the lysosomal acidic environment, resulting in degradation. However, the Cd-disrupted lysosomal pH was restored by pretreatment with the IP3R inhibitor 2-APB and the SERCA activator CDN1163 [2+ mediates interaction between the ER and mitochondria and lysosomes, and modulates lysosomal function.In addition, intracellular Ca disease . It has ar cells ,57. Alon fashion . Ca2+ upsomal pH . The lysan cells ,61. In h CDN1163 ,63. This2+ efflux from the ER regulates lysosomal Ca2+ levels. The ER Ca2+ channel-like protein transmembrane BAX inhibitor motif containing 6 (TMBIM6) not only mediates ER stress response and apoptosis, but also regulates the local release of Ca2+ through the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel, triggering autophagy induction in the kidney in starved mice [2+ transfer from lysosome to mitochondria to modulate mitochondrial homeostasis [2+ release regulates transcription factor EB (TFEB), which transcriptionally regulates the expression of TRPML1 as well as expression of other autophagic and lysosomal genes [2+ release through TRPML1 channel was inhibited in the podocytes, which may show a contribution to the development of podocytopathy and associated nephrotic syndrome [2+ is regulated in the intracellular Ca2+ store system, which may play important roles in the progression of kidney disease.It has also been suggested that Caved mice . In addieostasis . Moreoveal genes ,67. In pal genes ,69,70. Isyndrome . These r2+ signaling acts as a bridge linking these two types of cellular activities [2+ promote cell proliferation and survival through release of IP3R by the ER; Ca2+ is subsequently transferred to mitochondria to activate mitochondrial metabolism. Mitochondrial Ca2+ homeostasis dysfunction results in mitochondrial degradation by autophagy via activation of AMPK. Mitochondrial Ca2+ overload causes production of reactive oxygen species (ROS) and release of cytochrome c, which eventually leads to cell apoptosis [2+ signaling and Ca2+ subcellular homeostasis may determine the balance between cell survival, apoptosis and autophagy.The relation of cell death and autophagy is complex and occasionally contradictory, but it is critical to cell fate. Intriguingly, Cativities ,74,75,76poptosis ,77,78. T2+ triggered by Trichokonin VI, an antimicrobial peptide, induces autophagy and apoptosis in hepatocellular carcinoma cells. Moreover, siRNA knockdown of autophagy related gene (ATG5) reduces cell apoptosis [2+, which contributes to apoptosis [2+ chelators completely restored cell viability and inhibited Cd-induced apoptosis and autophagy [2+ levels in high-glucose conditions [2+ also plays important roles in ferroptosis [2+ signaling-mediated autophagy may promote cell death and contribute to kidney disease.In some cases, induced autophagy promotes cell death. In the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice, persistent autophagy in kidney proximal tubules was observed. Pharmacological inhibition of autophagy and kidney proximal tubule-specific knockout of autophagy-related 7 (PT-ATG7 KO) suppressed tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration in these mice . This supoptosis . This shpoptosis . In mouspoptosis . In RAW2utophagy . In porcnditions . In addiroptosis , a type roptosis  which haroptosis . These s2+ release through IP3R [In some cases, induced autophagy has protective roles against cell death. It has been shown that high glucose promotes autophagy flux in podocyte cultures and induces LC3B-II expression in podocytes in diabetic mice. Specifically, deletion of ATG5 in podocytes resulted in accelerated diabetes-induced podocytopathy with a leaky glomerular filtration barrier and glomerulosclerosis. Furthermore, the endothelial-specific deletion of ATG5 also resulted in capillary rarefaction and accelerated diabetic nephropathy. Thus, endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis . In mousugh IP3R . In addiugh IP3R . These r2+ signaling may link these two cell activities. In mouse renal tubular cells, Cd-inhibited autophagy flux aggravated apoptosis by inducing elevation of Ca2+ level [2+ between the ER, cytosol and mitochondria [2+ signaling [2+-mediated autophagy process can protect against heavy metal-induced renal cell cytotoxicity and kidney injury [2+ level-mediated inhibition of autophagy may aggravate apoptosis and contribute to kidney injury.However, initial autophagic protection will switch to disruption of autophagic flux, resulting in cell death in renal cells . In othe2+ level . In prim2+ level , which cchondria ,98. Actiignaling . Restoriy injury ,99,100. 2+ microdomains regulating apoptosis, necrosis and autophagy contribute to the development of kidney disease. Under these conditions, Ca2+ signaling determines the fate of kidney cells and the progression of disease. In view of this, therapeutic strategies have to consider whether to target a specific microdomain of Ca2+ signaling in kidney disease.As described above, the Ca2+ signaling-mediated apoptotic pathways in kidney disease could be considered as potential therapeutic targets -induced nephrotoxicity in a rat model [2+ overload. However, MCU inhibitors can protect podocytes from apoptosis and proteinuria induced by Adriamycin or angiotensin II [2+ may also be a potential target for some stress-induced nephropathies. Secondly, modulators targeting mitochondrial Ca therapy . Inhibity injury ,106. In 15 cells . Mitochoat model . Palmitiat model ,110. In at model . In Adriensin II . This su2+ signaling-mediated autophagy provides new therapeutic ways to treat kidney disease  reduced intracellular Ca2+ by activation of SERCA in vascular smooth muscle, which produces vasodilation in human intrarenal arteries [2+ reuptake and cell proliferation in ADPKD cells by inducing autophagy through the CaMKKβ-AMPK-mTOR signaling pathway, which indicates that SSd might be a potential treatment for ADPKD and that SERCA might be a novel target for ADPKD therapy [2+ level through activation of the CaMKKβ-LKB1-AMPK pathway in glomerular endothelial cells and podocytes in the kidney [2+ signaling-mediated autophagy might be a potential target in therapy for metabolic disease-associated kidney diseases.Thirdly, Ca disease . PCV2 inease (2+ . Activattic mice . Ca2+ siarteries . This suarteries . Saikosa therapy . It has  therapy . Activate kidney . Taken t2+ homeostasis in the body; thus, disruption of Ca2+ homeostasis will cause a series of kidney diseases. Regulation of Ca2+ signaling in kidney cells decides cell fate in a way that is closely associated with kidney disease. Ca2+ microdomains including the ER, mitochondria and lysosomes regulate the various modes of cell death, such as necrosis, apoptosis and eryptosis, which when disrupted contributes to the overall development of kidney disease. Moreover, interplay between the Ca2+ microdomains also mediates the crosstalk between these forms of cell death and autophagy. Based on the roles of the Ca2+ microdomains in regulating cell fate in kidney cells, targeting these Ca2+ signaling may lead to novel therapeutic strategies against kidney diseases.Taken together, the kidney plays a key role in regulating Ca


Esophageal squamous cell carcinoma (ESCC) is one of the most lethal gastrointestinal malignancies with high mortality. Recurrence develops within only a few years after curative resection and perioperative adjuvant therapy in 30–50% of these patients. Therefore, it is essential to identify postoperative recurrence biomarkers to facilitate selecting the following surveillance and therapeutic strategies. The general transcription factor IIE subunit beta (GTF2E2) is crucial for physiological and pathological functions, but its roles in the aggression and recurrence of ESCC remain ambiguous. In this study, we found that GTF2E2 was highly expressed in ESCC samples, and elevated GTF2E2 expression predicted early recurrence after surgery for ESCC patients. High expression of GTF2E2 associated with more aggressive clinic features and poor prognosis. GTF2E2 promoted the proliferation and mobility of ESCC cells in vitro and in vivo. We further revealed that miR-139-5p repressed GTF2E2 expression by downregulating its mRNA through binding with Argonaute 2 (Ago2). Rescue assays suggested that miR-139-5p affected GTF2E2-mediated ESCC progression. Moreover, GTF2E2 positively interacted with FUS promoter and regulated FUS expression, and the phenotype changes caused by GTF2E2 manipulation were recovered by rescuing FUS expression in ESCC cells. Additionally, we demonstrated that GTF2E2 promotes ESCC cells progression via activation of the AKT/ERK/mTOR pathway. In conclusion, GTF2E2 may serve as a novel biomarker for recurrence after surgery and a potential therapeutic target for ESCC patients, and it promotes ESCC progression via miR-139-5p/GTF2E2/FUS axis. Esophageal squamous cell carcinoma (ESCC) is one of the most lethal gastrointestinal malignancies and is the primary cause of cancer-related death in China –3. RecenSeveral general transcription factors assemble into a preinitiation complex (PIC) to ensure accurate RNA pol II loading at the transcription start site. Among them, the general transcription factor IIE subunit beta, also known as GTF2E2, is a crucial component for PIC assembly and stabilization required for transcription initiation and promoter opening by facilitating loading and stable binding of TFIIH , 14. PreMicroRNAs (miRNAs) are a class of non-coding RNAs of 19–25 nucleotides in length that regulate post-transcriptional gene expression , 19. InsFused in sarcoma/translocated in liposarcoma (FUS), a nuclear RNA-binding protein, plays an essential role in gene expression, including transcription, splicing, and even mRNA transport . FUS hasIn the present study, we demonstrate that GTF2E2 is significantly upregulated in ESCC and markedly correlated with tumor recurrence after surgery of ESCC. Furthermore, we systematically examined GTF2E2’s potential functions in tumorigenesis and metastasis of ESCC. In addition, our study found that miR-139-5p is an upstream regulator of GTF2E2 by decaying GTF2E2 mRNA in an Ago2-dependent manner. miR-139-5p inhibits the proliferation and mobility of ESCC cells by directly regulating GTF2E2. Interestingly, according to the RNA-seq and ChIP-seq results, GTF2E2 was explored as the direct regulator for the transcription of FUS, which was confirmed by ChIP-PCR and dual-luciferase reporter assays. Rescue assays were conducted to illustrate the role of FUS in GTF2E2-mediated ESCC progression. Collectively, our results indicate that GTF2E2 may serve as a novel recurrence biomarker and potential therapeutic target for ESCC.n = 39) and low expression (n = 40) in ESCC patients from TCGA cohort , we found that GTF2E2 knockdown resulted in upregulation of 2261 genes and downregulation of 2263 genes  analysis in both control and GTF2E2-downregulated cells and identified the differentially expressed genes. Combined with the nes Fig. . A visuanes Fig. . Gene Onnes Fig. .Fig. 7IdWe then studied the genome-wide target sites of GTF2E2 in ESCC cells using the ChIP-seq approach and identified 27,149 peaks corresponding to 7503 RefSeq genes Fig. . The peaNext, we investigated the overlapping gene sets between the differentially expressed genes after GTF2E2 knockdown and ChIP-seq. It was found that 45 upregulated genes were included in the set of GTF2E2 target genes, while 41 downregulated genes were included in the set of GTF2E2 target genes Fig. . InteresFurthermore, we evaluated whether GTF2E2 promotes ESCC progression through FUS. RT-PCR and western blot results showed that GTF2E2 downregulation inhibited and GTF2E2 upregulation enhanced FUS expression level Fig. . Then, wThe dysregulated expression of oncogenes or tumor suppressor genes plays a crucial role in ESCC tumorigenesis and progression. Currently, although surgical resection is still the mainstay treatment for potentially curable esophageal cancer, most tumors recurred within 1 year after surgery, which highlights the significance of exploring promising biomarkers for early detection of recurrence –33. Our As a significant component for RNA transcription initiation, GTF2E2 is involved in various biological processes, and its alterations are related to diverse pathological conditions, such as trichothiodystrophy , 35 and In our research, bioinformatics analysis predicted that the mRNA of GTF2E2 could be targeted by serials of miRNAs. Among these miRNAs, we identified miR-139-5p as an upstream regulator of GTF2E2 by analyzing the expression relevance between miRNAs and GTF2E2 in ESCC cells. GTF2E2 was downregulated, whereas miR-139-5p was upregulated, which were negatively correlated with each other. We further found that miR-139-5p downregulated GTF2E2 expression through binding with Ago2, the core protein of RISC complexes. Our results suggested that upregulated GTF2E2 markedly restored the proliferation, migration, and invasion of ESCC cells inhibited by miR-139-5p overexpression in the in vitro experiment, indicating that the effects of miR-139-5p in ESCC cells were mediated by GTF2E2. miR-139-5p is a well-known tumor suppressor in several types of cancer cells –40. The RNA-seq and ChIP-seq data predicted that FUS is the direct target of GTF2E2, which is a well-known oncogene –30. In tPrevious studies have fully confirmed the signaling pathways involved in tumor cell progression, including mTOR, PI3K-AKT, and MAPK/ERK1/2 –43. To dIn this study, we illustrate for the first time that GTF2E2 plays an oncogenic role in ESCC progression with clinical analysis and both in vivo and in vitro experiments.Data gathered in our study suggested GTF2E2 promotes ESCC progression via the miR-139-5p/GTF2E2/FUS axis. Furthermore, the activation of EMT, MET, and the AKT/ERK/mTOR signaling pathway caused by GTF2E2 manipulation were recovered by rescuing FUS expression in ESCC cells. The miR-139-5p/GTF2E2/FUS axis may affect ESCC cell proliferation and invasion via the acceleration of EMT, MET, and the phosphorylation of AKT/ERK/mTOR signaling pathway components. Altogether, our study provides the evidence that GTF2E2 is a potential biomarker for postoperative recurrence and a therapeutic target in patients with ESCC.In this study, tissue microarray of ESCC samples and adjacent non-tumor tissues were obtained from Shanghai Outdo Biotech Co., Ltd. Patients who experienced recurrence within 1 year after surgery were defined as the early recurrence group and the remaining patients with recurrence were defined as the late recurrence group –33. ThirThe human ESCC cell lines KYSE-150 and TE-1 were purchased from the cell bank of the Chinese Academy of Sciences . Eca-109 was purchased from the National Infrastructure of Cell Line Resource . KYSE-410, KYSE-30, KYSE-450, and normal human esophageal epithelial cell line HEEC were maintained at the Laboratory of Oncology in Tongji Hospital. All cell lines were authenticated recently by short tandem repeats (STRs) profiling and confirmed to be mycoplasma negative. They were maintained in RPMI 1640 , supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin .The miRNA mimics and inhibitors, and normal (scramble) control (NC) oligonucleotides were obtained from Ribobio . A FUS expression construct was created by subcloning the PCR-amplified full-length human FUS cDNA into the pcDNA3.1 vector . miRNA or plasmids transfection was performed using Lipofectamine™ 3000 transfection reagent  according to the manufacturer’s instructions. The short hairpin RNAs (shRNAs) specific to GTF2E2 and FUS (sh-GTF2E2#1/2/3 and sh-FUS#1/2/3) were synthesized and cloned into lentiviral vectors by Genechem Co., Ltd. . Lentivirus containing full-length GTF2E2 cDNA was also synthesized and transfected into ESCC cells to construct GTF2E2-overexpressing cell lines. Cell transfection was performed according to the manufacturer’s protocol. Target sequences are provided in Table 5 cells/well were seeded into 24-well plates. After 24 h, the recombinant plasmid 3′UTR-GTF2E2-WT or 3′UTR-GTF2E2-mutant was co-transfected into cells with miR-139-5p mimic, miR-139-5p inhibitor, or their respective negative control (NC) using Lipofectamine 3000 (Invitrogen). The transfected cells were incubated for 48 h and total protein from cells was extracted by Passive Lysis Buffer (Promega), and the luciferase activity was examined using Dual-Luciferase® Reporter Assay System (Promega) on GloMax 20/20. Firefly luciferase activity was normalized against Renilla luciferase values, and the ratio of firefly/Renilla luciferase activity was presented.To confirm the direct regulating relationship between miR-139-5p and GTF2E2, the 3′-untranslated region (3′UTR) of GTF2E2 or 3′UTR-GTF2E2-mutant was cloned into the pmiR-RB-Report™ vector . About 1 × 10To perform FUS promoter analysis, cells were seeded into 24-well plates. FUS promoter-binding sequence (WT or MUT) was cloned into pPRO-RB-Report vector . Cells were co-transfected with the plasmids containing FUS promoter-binding sequence (WT or MUT) and GTF2E2 upregulation, downregulation, or negative control using Lipofectamine 3000 (Invitrogen). After 48 h, the relative luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega).−ΔCT was calculated and normalized to the 2−ΔCT of 10% input. The primer sequence of GTF2E2-3′ UTR is as follows: forward: TGCATTTACGGGAAAGGGCT; reverse: AGACCCTTCCTTGTCCCACA.The antibody against Ago2 was purchased from Abcam (ab186733). Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit  was applied to enrich Ago2-binding RNA as demonstrated in the manufacturer’s instructions. The enriched RNA was analyzed by RT-PCR. 2ChIP: chromatin immunoprecipitation (ChIP) assay was conducted using the SimpleChIP Enzymatic Chromatin IP Kit  according to the manufacturer’s instructions. In short, cells were crosslinked with 37% formaldehyde, and fragmented chromatin was treated with nuclease and subjected to sonication. Consequently, the supernatants were collected and incubated overnight with anti-GTF2E2 antibody , anti-histone H3 antibody , and normal rabbit IgG antibody  combined with ChIP-Grade Protein G magnetic beads. The beads were washed, and the chromatin complexes were harvested, purified, and reverse-crosslinked, followed by DNA purification, and ChIP-seq and ChIP-PCR detection.ChIP-seq: The immunoprecipitated DNA was analyzed through deep sequencing (ChIP-seq) by SEQHEALTH Biotech Company . ChIP libraries were prepared using ChIP DNA according to the BGISEQ-500ChIP Seq library preparation protocol. After filtering the raw sequencing data (raw data), high-quality sequencing data (clean data) were compared to the human genome (hg19) to obtain the ChIP-seq results. Next, the results were compared to the whole-genome de novo peak calling to explore the protein’s interacting preference in the genome and to perform motif analysis of the binding site.ChIP-PCR: The immunoprecipitated DNA was quantified by real-time PCR with primers for GTF2E2-binding sites in the FUS promoter . Fold enrichment was analyzed based on the threshold cycle (CT) value of the IgG control using the comparative CT method.Experiments have been conducted according to the ethical standards, the Declaration of Helsinki, and national and international guidelines. All animal research was conducted according to the ARRIVE  guidelines and the AVMA  guidelines on euthanasia and was approved by the Animal Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.Tumor formation assay: female BALB/c nude mice of 4–5 weeks old  were maintained at SPF conditions, and given a subcutaneous injection of 5 × 106 cells  into the right flank, respectively (10 mice/group). The mice were randomly divided into groups before injection. Then the subcutaneous xenografts were measured every 2 days. Tumor volume was measured using calipers and calculated according to the formula: V = length × width2/2. After 21 days, the mice were euthanized and the formed tumors were isolated and weighed for the following analyses.Metastasis assay: female BALB/c nude mice of 4–5 weeks old were injected with 100 μL of cell suspension containing 1 × 107 cells via the tail vein (10 mice/group). After 8 weeks of cells injection, the mice were sacrificed, and the lungs and livers were removed to calculate metastatic nodules.The paraffin-embedded tissue samples were sliced into 5-μm‐thick sections, and all slides were stained with GTF2E2 , Ki67 , or FUS  according to the standard procedures. Then GTF2E2 expression was scored according to the staining scope and intensity. Specifically, the staining scope was: 1 (0–25%); 2 (25–50%); 3 (50–75%); and 4 (75–100%), and the staining intensity was 0 (negative); 1 (weakly positive); 2 (moderately positive); and 3 (strongly positive). The overall score was defined by multiplying the staining scope by the staining intensity score . IHC staThe online bioinformatics websites, TargetScan , miRWALKt-test was applied to detect the differential expression of GTF2E2 in cancer tissues compared with adjacent non-malignant tissues, and in early postoperative recurrence samples compared with late postoperative recurrence samples. The relationship between GTF2E2 and clinicopathological characteristics was analyzed using the chi-square test. Survival curves were evaluated using Kaplan–Meier and log-rank tests. The effects of variables on survival were analyzed by the univariate and multivariate Cox proportional hazards models. Two-group or multiple-group comparison was calculated with Student’s t-test or one-way ANOVA, followed by the Dunnett’s post hoc test using GraphPad Prism 5 software . All in vitro experiments were conducted three times. All values were shown as mean ± SD. Differences were defined as statistically significant at p < 0.05 .Data analysis was performed using SPSS Statistics 25.0. The paired supplementaryTable S2


Numerous Ca2+ chelating, storage, and transport mechanisms are required to maintain normal cellular physiology. Ca2+-binding proteins, mainly calmodulin and calbindins, sequester free intracellular Ca2+ ions and apportion or transport them to signaling hubs needing the cations. Ca2+ channels, ATP-driven pumps, and exchangers assist the binding proteins in transferring the ions to and from appropriate cellular compartments. Some, such as the endoplasmic reticulum, mitochondria, and lysosomes, act as Ca2+ repositories. Cellular Ca2+ homeostasis is inefficient without the active contribution of these organelles. Moreover, certain key cellular processes also rely on inter-organellar Ca2+ signaling. This review attempts to encapsulate the structure, function, and regulation of major intracellular Ca2+ buffers, sensors, channels, and signaling molecules before highlighting how cancer cells manipulate them to survive and thrive. The spotlight is then shifted to the slow pace of translating such research findings into anticancer therapeutics. We use the PubMed database to highlight current clinical studies that target intracellular Ca2+ signaling. Drug repurposing and improving the delivery of small molecule therapeutics are further discussed as promising strategies for speeding therapeutic development in this area.Intracellular Ca Low hydration energy, high polarizability, relative flexibility of coordination sites and bond length, and large concentration gradient across cellular membranes (100 nM intracellular to 2 mM extracellular) due to low intracellular levels make it the ion of choice at the core of cellular signaling in prokaryotes and eukaryotes alike i as evident by its role in modulation (activation or inactivation) of Ca2+ pumps (such as PMCA and SERCA) and Ca2+ channels  i gradient required for cellular motility. Migrating endothelial cells have higher expression of PMCA at their leading edges to maintain low basal [Ca2+]i levels thereby, preventing continued activation of Myosin Light Chain Kinase (MLCK) and extended contraction of the cell membrane at the migration frontier [2+]i extrusion allows the proliferation of pancreatic beta cells i signaling was later confirmed by independent work of two groups using high-throughput RNAi screens to identify inhibitors of thapsigargin evoked CRAC current (ICRAC) i can exert anti-migratory and antiangiogenic effects on tumor-derived endothelial cells. Two separate studies have shown that treatment of Breast Tumor-derived Endothelial cells (BTEC) and Renal Tumor-derived Endothelial Cells (RTEC) with 100 mM ATP leads to a biphasic increase in [Ca2+]i—an initial transient release from ER followed by SOCE and then a prolonged release rise due to Ca2+ influx via P2X7R and P2Y11R [2+]i then activates adenylate cyclase10/cAMP/EPAC-1 pathway to disrupt actin cytoskeleton and thus, inhibit migration of tumor-derived endothelial cells.The somewhat controvertible, but the generally accepted, role of ligand-gated Cac for P2 ,229,230.n tumors . ATP binoduction ,232. Theon (EMT) . For insd P2Y11R ,235. Thi2+ ion efflux machinery at the ER-PM junction during tumor formation or progression also requires attention. PMCA and Na+/Ca2+ Exchanger (NCX) lead a concerted effort on a normal cell surface to expel excessive free cytosolic Ca2+ ions and thereby prevent cytotoxicity i transporters too can be leveraged for killing cancer cells. It is well known that calmodulin regulates the slow inactivation of CRAC currents by direct interaction with the STIM1 SOAR domain, thereby disrupting the STIM1-ORAI1 complex [2+ elevation) for tumor survival, growth, migration, and invasion [The anomalous signaling, localization, and mobilization of Caproteins . It is a tissues ,359,360. tissues . It was  tissues . Some of kinases . For ins kinases ,365,366. kinases . Direct  kinases ,369,370. complex . It has  complex . On the  complex . CaM andinvasion ,377,378.2+-binding proteins that are altered by cancer cells [2+ ion-dependent processes including proliferation, differentiation, cell adhesion, migration, intercellular interactions, immune response, and apoptosis [2+ signaling with the Wnt pathway as the potential downstream effector [2+-binding protein is shown to attract dendritic cells and macrophages for phagocytosis [2+ ion buffer which has dominant expression in neurons has so far only been found to protect osteosarcoma cells from apoptotic stimuli [Calreticulin (CRT) and calbindin are other major EF-hand containing Caer cells ,381,382.poptosis ,389,390.poptosis ,394,395.poptosis ,397. Evepoptosis ,399. Thepoptosis ,401. Foreffector ,403. In effector . Studieseffector ,407,408.ocytosis . In glioocytosis . In pancocytosis . Very li stimuli .2+ signaling [2+ ion uptake by mitochondria. Thus, plummeting Ca2+ ion transport from ER to mitochondria in transformed cells portends tumor progression.Aberrant cellular metabolism, a hallmark of a wide variety of tumors, stems from the dysfunction of mitochondrial pathways for ATP production . The shiignaling ,415,416.2+ ion transport, ER stress, ER Ca2+ release, mitochondrial morphology regulation, cellular bioenergetics, apoptosis and survival, autophagy, and ROS generation [2+ ion release, and thus crucial for autophagy and apoptosis induction [2+ ionrelease into mitochondria creates an imbalance in ATP/ADP ratio that derepresses AMPK/mTOR/ULK-1 axis and initiates autophagy flux (based on nutrient starvation) [2+ ion leak channels as less Ca2+ storage in the ER manifests into poor ion supply for ATP production [2+ ion release into the mitochondria, ergo inducing autophagy [2+ ion release into the mitochondria partly by interacting with Bcl2 which then dissociates from IP3R, thereby restoring the ER-mitochondrial Ca2+ ion transfer [2+ ion transport by upregulating the expression of tumor suppressor PML (promyelocytic leukemia) that blocks phosphorylation and inactivation of IP3R by p-Akt [ER-mitochondria junctions or Mitochondrial-Associated Membranes (MAMs) are hubs for proteins that are integral to vital processes such as phospholipid synthesis and translocation, mitochondrial Caneration . IP3R, anduction ,418,419.rvation) . Conversoduction ,422. By oduction ,424,425.oduction ,427. Remoduction . In highoduction ,430,431.utophagy ,433,434.transfer . Chemothby p-Akt ,436.2+ ion transport has emerged as an integral component of the crosstalk between autophagy and apoptosis [2+ ion uptake via MAMs triggers intrinsic apoptosis pathways. Thus, tumor suppressors and proapoptotic molecules like BRCA1 or PTEN bind to IP3R to boost Ca2+ ion uptake at MAMs while tumor promoters and anti-apoptotic molecules such as Bcl2 or Bcl-xL repress mitochondrial Ca2+ ion overload by binding to and inhibiting the channel activity [2+ ion transporting complex at MAMs are also exploited by cancer cells [2+ uptake channel, MCU with its regulators MICU1/2 act as the key mediators of Ca2+-based mitochondrial functions [2+ ion uptake via MCU and its absence causes cell death via mitochondrial overload, tumor cells are widely observed to have elevated levels of these proteins, although expression of MCU is subtype-dependent [The ER-mitochondria Capoptosis . Inhibitactivity ,439. VDAer cells ,441. It er cells ,443. Proer cells ,444. Asier cells ,446,447.unctions ,449. Becependent ,450. Forependent ,452. On 2+ signaling at lysosomal membranes and surfaces of acidic vesicles provides a functional scaffolding for endocytic traffic and autophagy, thereby directly influencing cellular health [Car health ,454.2+ signaling [2+ ion release channels in tumor invasiveness [2+ ion signals via TPCs on lysosomal membranes, and this promoted G0/G1 cell cycle transition, tumor vascularization, focal adhesion kinase (FAK) formation, and migration. Both in vitro and in vivo silencing or pharmacological inhibition of TPC1/2 have also been shown to block invasion and migration of urinary bladder carcinoma (T24), hepatoma (HUH7), and mammary carcinoma [Alteration of such signaling in cancer contributes to cancer hallmarks such as uninhibited growth, angiogenesis, and metastasis ,456,457.ignaling . Initialsiveness . The stuarcinoma ,460. Nguarcinoma ,461.2+ ion release channel expressed on lysosomal membranes and is known to finely control autophagy [2+ activated transcription factor, TFEB is noticeably correlated with cellular malignancy [2+ ion release followed by calcineurin activation that subsequently dephosphorylates TFEB and releases it from 14-3-B-B; free TFEB is then translocated to the nucleus to evoke transcription of autophagy-related genes [TRPML1 is another Cautophagy ,462. Prelignancy ,465,466.ed genes . In shored genes . TFEB uped genes . Althouged genes .2+ signaling involves a smorgasbord of proteins that chelate or transport Ca2+ ions across various cellular compartments and thereby assist in signal induction, relay, or integration. With Ca2+ signaling being pivotal for vital cellular processes, distortion of this machinery can be highly advantageous to cancer cells. Although there has been a steady upward trend in the number of published studies that delineate the functional exploitation of various intracellular Ca2+ signaling components in tumors, limited headway has occurred in translating those research outcomes into clinical applications. To demonstrate this, we first utilized the PubMed database to get a list of all the publications within the realm of intracellular Ca2+ in cancer. Using the advanced search feature, we selected “intracellular Ca2+” and “cancer” as the keywords to look for in all fields  of PubMed articles. We got more than 11,000 studies (not shown). As the published articles between 1960 and 1990 only accounted for less than 0.1 percent of the total, we set our year range for subsequent searches as 1990–2020. To minimize the occurrence of non-relevant articles in our dataset , we used stricter filtration criteria [2+ molecules, Revised Filter 2 was determined to be a sub-set of Revised Filter 1 [2+ signaling molecules in focus was that Revised Filter 2 had a lower percentage of articles on “CaSR or Vitamin D” amongst all the ones with main Ca2+ signaling molecules from the endoplasmic reticulum or its interorganellar junctions [Intracellular Cacriteria A. Based Filter 1 B–D. A no2+ and Vitamin D on cancer (31%) [2+ signaling player in the endoplasmic reticulum or its interorganellar junctions accounted for less than 10 percent of all the published clinical articles. We further evaluated from the main set of published articles the number of studies that used antineoplastic agents (mainly chemotherapeutics) by searching for appropriate keywords. There are 819 articles in the main set that had utilized antineoplastic agent(s) to address a hypothesis related to the role of a major Ca2+ signaling molecule in cancer [2+ signaling molecules as major components of research. Studies with bisphosphonates and not chemotherapeutics were predominant in the antineoplastic group of articles followed by the ones that utilized dietary Ca2+ and/or Vitamin D [2+ signaling.Clinical studies that were extracted from the list of articles in Revised Filter 1 were labeled in PubMed with one or more of these terms— Clinical Study, Clinical Trial, Clinical Trial Phase I, Clinical Trial Phase II, Clinical Trial Phase III, Clinical Trial Phase IV, Comparative Study, Controlled Clinical Trial, Meta-Analysis, Multi-Center Study, Randomized Controlled Trial, Systematic Review. Two-hundred-and-seventy-seven clinical studies were identified with most of them concentrated between year ranges 1991 to 1996 and 2010 to 2017 (with approximately 10 or more studies published each year) A. With oer (31%) B. Transln cancer C. Out ofitamin D D. Among 2+ signaling proteins is currently in the early stages. Some of the examples include RP4010 , Synta66 , CAD204520 , and SKF96365  [2+ signaling could be increased by leveraging the advancements made in the nanocarrier-based targeted drug delivery systems. For example, taking from the design of Antp-LP4 related peptides (VDAC1 inhibitors), Venetoclax (a Bcl2 inhibitor) can be encapsulated in inorganic (such as gold nanoparticle or quantum dots) or organic nanocarriers (such as liposomes) coated with transferrin to specifically target cancer cells that demonstrate significant surface expression of transferrin receptors [2+ signaling [2+ signaling in cancer. Several FDA-approved anticancer agents  and drugs against other disease conditions  can impact Ca2+ signaling machinery [2+ signaling See Research using small molecule drugs targeting numerous Calinical) ,475,476.linical) . Toxicitlinical) ,480,481.eceptors ,482,483.ignaling . Drug reachinery ,485. Som2+ in tumor cells, the vast clinical potential of targeting key players in this area remains underappreciated. Whether it is through redesigning existing therapeutics or developing novel treatments for directly modulating the intracellular Ca2+ levels, greater efforts are needed to find ways to reverse the hijack of intracellular Ca2+ signaling in cancer.Even after decades of research on intracellular Ca


In this paper, a systematic study of the relationship between nonlinear crystal length and intracavity second-harmonic generation (SHG) using MgO-doped periodically-poled lithium niobate (MgO:PPLN) is presented. The experimental results demonstrate a relationship between the maximum SHG power generated and the full-width at half maximum (FWHM) of the crystal’s temperature tuning curve to the length of the nonlinear optical crystal. It was shown that maximum SHG power increases rapidly with the increase of MgO:PPLN length, reaching a saturation length (~ 2 mm), which is much shorter than that predicted by the single-pass SHG theory. This saturation length of the MgO:PPLN crystal is almost independent on 808 nm pump power for typical powers used in continuous wave intracavity SHG lasers. In addition to this saturation effect, a broadening effect was also observed, the FWHM of the temperature tuning curve was shown to have a larger FWHM than that predicted by the single-pass SHG theory for MgO:PPLN shorter than the saturation length. This work has the benefit of allowing engineers to optimize nonlinear crystal length when developing intracavity SHG based diode-pumped solid state (DPSS) lasers. In the past, such DPSS lasers were used for single-pass second harmonic generation configurations to produce continuous-wave (CW) visible lasers17. An intracavity structure is commonly used to increase the effective interaction length in the nonlinear crystals and the fundamental wave intensity. This approach has a number of benefits over the single-pass approach, such as compact form factor, low-cost, and a high conversion efficiency and high output power29.Diode-Pumped Solid-State (DPSS) lasers based on intracavity second harmonic generation (SHG) are highly desirable for a wide range of applications including laser displays29, potassium titanyl phosphate (KTP)21, periodically-poled potassium titanyl phosphate (PPKTP)23, and magnesium-oxide doped periodically-poled lithium niobate (MgO:PPLN)26 have been used in both the single-pass and intracavity SHG. Among these nonlinear crystals, MgO:PPLN has attracted a lot of attention due to its high nonlinear coefficient, large available crystal size and simple wafer-based fabrication process. It is estimated that the raw material cost is only ~ $2/mm for MgO:PPLN. A variety of crystal lengths have been reported for MgO:PPLN crystals in both the single-pass and intracavity SHG configurations. The intracavity structures included nonlinear crystal lengths between 1 and 10 mm, while the single-pass setups employed longer crystals from 10 to 50 mm. For example, MgO:PPLN crystals with lengths of 1, 2, 3, and 10 mm were used in an intracavity setup to achieve maximum SHG powers of 1.28, 2.8, 6.6, and 1.3 W respectively26. However, there has been limited discussion in terms of developing a clear relationship between maximum SHG power and MgO:PPLN crystal length.Nonlinear crystals such as lithium triborate (LBO)31, and the simple cavity structure should serve as a good baseline example of how MgO:PPLN length and fundamental pump power modify the expected maximum SHG output power.It is clear from the reported results that crystal length is an important parameter in determining the output power of SHG lasers. There are also diminishing returns on increasing the crystal length, as the temperature tolerance of the SHG lasers decreases with the increase of crystal length and a longer crystal does not guarantee significantly higher SHG output power. As a result, a given laser cavity will have an optimum crystal length where the output SHG power of the laser effectively saturates. If the crystal length is increased past this “saturation length”, there will be no meaningful increase in power versus the increased cost of fabrication and size of the laser. The argument could be made that this is considered “common knowledge” by those who work with SHG lasers regularly. However, across all the reviewed literature, there was no evidence of an exhaustive study to determine the optimal crystal length for a given laser cavity. We expect that this saturation length will be a function of not only the type of nonlinear crystal used, but also the cavity configuration. There is also incentive to use shorter crystals, if possible, as the temperature tolerance for phase matching becomes stricter as the nonlinear crystal gets longer. This means that lasers with shorter crystals can be more robust and resistant to changes in environmental conditions over lasers with longer crystals. As a result, the main novelty of this paper is to present a systematic study of PPLN length versus maximum SHG output power for compact, watt-level green SHG lasers. Such lasers have strong demand in applications such as laser display and biomedical applications. This is an area our research group has worked on extensively3. This is a result of MgO:PPLN fabrication to produce long crystals with sufficiently large 0.5–1 mm aperture sizes relative to the beam diameters inside the laser cavity, which are typically < 100 microns. The wide body of research available as seen from the previously cited works for MgO:PPLN reinforces the importance of developing empirical relations that can guide the design and optimization of MgO:PPLN-based lasers. Additionally, such relations may be able to generalize to multiple nonlinear crystals, increasing their overall utility and simplifying the design process for SHG lasers.MgO:PPLN is an attractive option for the low-cost, compact watt-level lasers that our research group has worked on for laser displays and biomedical applicationsIn this paper, a compact, continuous wave (CW), intracavity SHG laser based on MgO:PPLN is systematically studied. Several bulk MgO:PPLN crystals of varying lengths were used to examine the impact on intracavity SHG laser performance. Parameters such as the maximum SHG power  temperature) and full width at half maximum (FWHM) of the temperature tuning curve were examined. In addition to varying the nonlinear crystal length, the 808 nm pump power was also varied to illustrate the dependence on pump power. It was shown that the length of the MgO:PPLN crystal where maximum power saturation occurs is consistent across pump powers, and that crystal lengths greater than 2 mm offer little additional SHG output power. In this case, we would state that the saturation length of MgO:PPLN crystal for this cavity configuration is 2 mm, and crystal lengths greater than the saturation length offer minimal benefits. The maximum SHG power versus PPLN crystal length is found to follow a logarithmic function, and an empirical relation for the coefficients and intercept of the logarithmic function are derived. This relation can potentially be used with similar cavity structures to derive an optimal MgO:PPLN length for a given laser cavity, cutting down on fabrication costs by allowing smaller MgO:PPLN crystals to be used for equivalent SHG output powers. The derived empirical relation and experimental results provide an optimized guideline for the use of MgO:PPLN in DPSS SHG lasers such that large temperature tolerance and high maximum SHG powered can be coupled with the reduction of cost and overall laser cavity complexity.4/MgO:PPLN green laser at 532 nm is shown in Fig. 4 crystal as one of the mirrors. The pump source was a 10 W F-mount 808 nm laser diode (LD). The fundamental laser crystal was a 3 × 3 × 5 mm3 a-cut Nd:YVO4 crystal with an Nd doping concentration of 1.1 atm%. The input facet of the laser crystal was anti-reflection (AR) coated at 808 nm and high reflection (HR) coated at 1064 nm to act as one of the end mirrors forming the laser cavity. The output facet of the Nd:YVO4 was AR coated at 1064 nm to minimize loss at the fundamental wavelength. The 5 mol% MgO:PPLN had an aperture of 0.5 × 2 mm2 with a uniform domain inversion period of 6.96 µm. Both facets of the MgO:PPLN were AR coated at 1064 nm and 532 nm. To optimize and further examine performance, the LD, Nd:YVO4, and MgO:PPLN were attached to the cool side of thermo-electric cooling (TEC) devices. A plano-concave output coupling (OC) mirror with a radius of curvature of 100 mm and diameter of 6 mm was used to complete the intracavity structure. The OC mirror was HR coated at 1064 nm and high transmittance (HT) coated at 532 nm. The overall length of this straight cavity remained approximately 40 mm for all experiments.The experimental setup of the end pumped intracavity frequency doubled Nd:YVOThe experimental results were obtained by selecting several MgO:PPLN samples with lengths ranging from 1 to 4 mm. The temperature of the MgO:PPLN was varied in Figure 32. While the model is described in full detail in the cited publication, we will briefly summarize the model here. The model uses the coupled rate equations for a four-level gain medium in a laser cavity to determine the circulating fundamental 1064 nm power in the cavity. This is affected by both the linear loss due to imperfect optical coatings, scattering, absorption, and similar phenomena, as well as an additional nonlinear loss induced by the nonlinear crystal when the fundamental 1064 nm light is converted into 532 nm light. The steady state fundamental power from the cavity equation was used to estimate the generated forward and backward SHG power using the coupled nonlinear wave equations as derived by Boyd33. This generated SHG power was used to update the nonlinear loss term, and the rate equations are used to generate a new steady state power. This process was repeated until the steady state fundamental power, output SHG power, and nonlinear loss are no longer changing between each iteration. The temperature dependent Sellmeier equations were used to generate a temperature tuning curve using the described model. For each theoretical curve in Fig. 32. The circulating fundamental power inside the cavity was measured experimentally by performing the reverse operating, where the measured leaking power was used alongside the transmission of the OC to estimate the intracavity power. The theoretical results were found to be in good agreement with the experimental results. For a more in-depth discussion on the theoretical model, refer to Ref.32.To verify that the saturation length and logarithmic trend extended to other intracavity SHG lasers, we simulated a 561 nm laser based off the 1122 nm laser line in Nd:YAG. It was found that this laser had significantly lower output power compared to the 532 nm laser thanks to the lower gain of this laser line, which is discussed in more detail later in this section. However, the saturation length remained the same at 2 mm, showing that the gain of the laser medium is not a contributing factor to the saturation length. A future investigation will look at various parameters of the laser cavity to determine the underlying physics that govern the saturation length of a nonlinear crystal. To investigate the underlying physics behind the observed features, the SHG power dependence on MgO:PPLN length was simulated using an intracavity SHG theoretical model, with the results plotted in Fig. 32 show that reasonably close results can be obtained for the maximum SHG power using a simplified single mode model that requires significantly less computational power. Additionally, the lack of wavelength selection device in our laser can be attributed to our target application, which is low-cost, compact, watt-level lasers that can be manufactured easily. Etalons are difficult to align and introduce additional linear losses to the laser that decrease the potential output power, and our previous work has shown that the stability without an etalon can be kept below 5%, which is acceptable for laser displays and other applications. A discussion on the stability of the laser as well as the effects of introducing a wavelength selection component are beyond the scope of this paper.It can be observed that our theoretical model in Fig. 34. This laser was selected due to the significantly lower gain of the 1123 nm line, as the stimulated emission cross section of the 1123 nm line is only ~ 1/16th that of the 1064 nm line at 35. We wished to see if similar logarithmic behavior and saturation lengths could be predicted. There is no experimental data to accompany this simulation however, due to a lack of PPLN samples with varying lengths and the appropriate poling period for 561 nm generation. Instead of fitting a logarithm to the experimental data for 561 nm SHG, instead the simulation data was used to fit a logarithm and calculate the derivative of the fit for the saturation length. The maximum SHG power vs. PPLN crystal length is presented in Fig. The simulation was also extended to a 561 nm SHG laser utilizing the 1123 nm transition found in Nd:YAG. This wavelength has several uses in the biomedical sciences, having favorable absorption properties in materials such as hemoglobin and less heat dispersion in the retina over comparable lasersWe can clearly see the same logarithmic trend in the data as observed in Fig. 30. As the PPLN length is increased, the efficiency of the SHG process increases, and so we see an increase in maximum SHG power. However, these gains saturate quickly once the saturation length is reached. This occurs as the increased SHG conversion efficiency from longer crystals is balanced by an equivalent drop in circulating fundamental 1064 nm power due to the increased nonlinear loss in the cavity. Basically, past a certain point, any gain in SHG efficiency is counterbalance by a drop in circulating 1064 nm power that negates most of the benefit of the longer crystal. Therefore, there exists a self-stabilization effect where any increase in the conversion efficiency of the nonlinear crystal has no effect on SHG maximum power as there is always an equal and opposite drop in the fundamental wavelength power. This general trend holds at higher pump powers, showing that while the overall SHG output power increases with 808 nm pump power due to the increased circulating 1064 nm power, the self-stabilization occurs around the same length of PPLN as it does at lower crystal lengths. As a result, for a given range of 808 nm pump powers, there exists an optimal saturation length where there are negligible increases in maximum SHG power when using crystals longer than the saturation length. This matches the general theory developed by Smith which shows that for a given cavity and PPLN length, which draws similar conclusions about how fundamental wave depletion in intracavity structures limits the generation of maximum SHG power33. This allows the optical engineer to design a laser cavity with no “wasted” space, and in “The logarithmic trend can be explained by the physics demonstrated in the model from Ref.Figure As expected, the maximum SHG power is achieved at the optimal QPM temperature. Also, from Fig. It is worth noting that the FWHM from the intracavity SHG shown in Fig. Figure The experimental results of Fig. When developing a laser which will be subject to temperature fluctuations, the power stability is dependent upon the length of the nonlinear crystal governing the SHG process. This is vitally important when selecting an optimized device length as there is a clear trade-off between maximum SHG power (longer device) and a larger FWHM for maximum temperature stability (shorter device).The experimental data for the maximum SHG power as a function of PPLN length in Fig. With the results of the logarithmic relation for each value of 808 nm pump power listed in Table We wish to determine if there is a relation that can be used to predict the coefficient and intercept of the logarithmic equation as a function of 808 nm pump power. The coefficient and intercept were plotted and fit with a second order polynomial, which resulted in an We can see that while there are some minor differences between the two relations, they both show a similar quadratic trend with the values of the coefficient and intercept. Ideally, we would be able to fit more data at higher pump powers to see if the overall trend holds, but we were limited to pump powers up to 5 W due to our limited selection of 808 nm diodes. This empirical relation potentially allows an optical engineer to select the operating pump power and derive a logarithmic relation that describes the SHG output power as a function of PPLN length. This could then be used to determine the optimal PPLN length for a given pump power, or the required pump power for a given desired SHG output power. Future work could expand on this and see if a similar relation can be found for other laser cavity configurations and higher pump powers.A systematic study on the relationships between maximum SHG power and the FWHM of the temperature bandwidth vs. nonlinear crystal length for MgO:PPLN has been presented. There are three main conclusions presented in this paper. First, it has been found that a saturation of the maximum SHG power occurs for an MgO:PPLN length of ~ 2 mm for 808 nm pump powers between 2 and 5 W. This serves as the optimum MgO:PPLN length for compact, watt-level intracavity green SHG lasers. Second, the operating temperature tolerance at the saturation length is ~ 10


How can such an interaction be regulated then? The answer: By facilitating a switch through an allosteric network. Although a number of unbinding mechanisms are being characterized, an extensive computational study by Joswig  Therefore, it is a good strategy to focus on interfaces which have features in common, such as divalent cations , which are cofactors for many proteins. Both of these ions play critical roles in a vast arena of protein functions which are increasingly well characterized at the molecular level. For example, serum calcium can be sensed to exquisite accuracy by the extracellular Ca2+-sensing receptor, which presents a connection between Ca2+ binding and allosteric modulators, agonists, and antagonists for this seven transmembrane G-protein coupled receptor protein. The list of receptors of which Ca2+ and Mg2+ play a role in regulation is growing, including ryanodine, adenosine, and opiod receptors representing other recently studied cases. However, mechanisms can become even more intriguing when another common cellular component is involved. Such is the case with a transmembrane potential in mammalian Ca2+ and voltage-activated K+ channels, or for the protein at the center of this JBC Editor’s pick, the C-type lectin receptor langerin, whose carbohydrate binding is sensitive to both Ca2+ concentration and pH.“Sometimes it’s hard to let go”, is a life truth many people experience. It is also true at the molecular level when complexes between a protein and its ligand or between a protein and another protein or macromolecule need to dissociate as part of a functional cycle. The mechanisms which comprise unbinding processes have only just recently been studied in detail, in part due to the advent of advanced, if not extensive, molecular dynamics (MD) simulations , 2 and e2+ affinity of langerin and other C-type lectins was discovered, but how this pH dependence occurs at the molecular level was a mystery because the Ca2+-binding site is covered up by the carbohydrate ligand. Furthermore, the Asp308, Glu293, and Glu285 residues directly responsible for Ca2+ binding have expected pKa values much lower than the pH associated with the fusion with acidic endosomes (pH of 5.5–6) . When His294 is protonated, it moves out of the way, interacting with neighboring Glu261 or Asn291. The disruption of the contact to His294 forces Lys257 to engage in new interactions. In one state of the lower pH ensemble, Lys257 interacts with Asp308, disrupting interaction with Ca2+ (B). In another state, Lys257 likely no longer supports an optimal positioning of Glu293, another direct Ca2+-binding side chain. An analysis of the trajectories in the form of a Markov model estimates that the two states described above last for 200 to 1300 ns, sufficient time for the Ca2+ to escape the binding pocket. Experimental measurements of Ca2+’s binding affinity in the His294Ala mutant suggest a residual pH sensitivity, indicating the presence of a second pH sensor. Extensive pKa calculations using 10,000 to 30,000 structures extracted from these simulations suggest that in absence of Ca2+, Asp308 and Glu285 can form a protonated dyad with an effective pKa, likely high enough to sense a pH change from 6 to 7. This would amplify the protonation effect of His294. However, constant-pH simulations or quantum mechanics/molecular mechanics simulations should be run to verify this model, as the authors state  in the same low-dimensional space. This space was identified by a principal component analysis on the combined MD trajectories. Careful analysis of distances and populations revealed a molecular switch involving the interaction of uncharged His294 and Lys257 at pH 7 A. When Hith Ca2+ B. In ano2+. A follow-up study will probably be necessary to model the actual dissociation of carbohydrates and Ca2+ from the langerin CRD, either experimentally or using simulations.The model put forward is novel in that it derives from a subset of conformational states discovered during clustering of the MD trajectories. One concern is the lack of analysis of the role of water or solvent ions that may come in to bridge these interactions . Neverth2+ were bound; the langerin crystal structure shows a single Ca2+, but other C-type lectins have up to four. Could additional Ca2+ play a role, perhaps as a Ca2+ rather than a pH sensor? Furthermore, full-length C-type lectin trimers are stabilized by a neck region that brings all three CRDs into close proximity (et al. (There remain additional questions for future investigations. Early on, it was unclear how many Caroximity , 7. Are roximity  in multiroximity . Whether (et al.  will be The author declares that he has no conflict of interest with the contents of this article.