IDH mutations with high chemoresistance and relapse rates despite recent therapeutic advances. Besides being associated with global promoter hypermethylation, IDH1 mutation facilitated changes in 3D DNA-conformation by CTCF-anchor methylation and upregulated oncogene expression in glioma, correlating with poor prognosis. Here, we investigated the role of IDH1 p.R132H mutation in altering 3D DNA-architecture and subsequent oncogene activation in AML. Using public RNA-Seq data, we identified upregulation of tyrosine kinase PDGFRA in IDH1-mutant patients, correlating with poor prognosis. DNA methylation analysis identified CpG hypermethylation within a CTCF-anchor upstream of PDGFRA in IDH1-mutant patients. Increased PDGFRA expression, PDGFRA-CTCF methylation and decreased CTCF binding were confirmed in AML CRISPR cells with heterozygous IDH1 p.R132H mutation and upon exogenous 2-HG treatment. IDH1-mutant cells showed higher sensitivity to tyrosine kinase inhibitor dasatinib, which was supported by reduced blast count in a patient with refractory IDH1-mutant AML after dasatinib treatment. Our data illustrate that IDH1 p.R132H mutation leads to CTCF hypermethylation, disrupting DNA-looping and insulation of PDGFRA, resulting in PDGFRA upregulation in IDH1-mutant AML. Treatment with dasatinib may offer a novel treatment strategy for IDH1-mutant AML.Acute myeloid leukemia (AML) is characterized by complex molecular alterations and driver mutations. Elderly patients show increased frequencies of  FLT3, KRAS, NRAS), nucleophosmin (NPM1), myeloid transcription factors , chromatin regulators (ASXL1) or DNA methylation-associated genes and epigenetic regulators  [Acute myeloid leukemia (AML) describes a myeloid disorder that is characterized by oncogenic transformation and uncontrolled clonal expansion of poorly differentiated cells of myeloid origin . Among tIDH1 mutations occur in 6-10% of AML cases, whereas elderly AML patients show a significantly higher IDH1 mutation frequency of ~17% accompanied by global DNA hypermethylation [IDH1 and IDH2 catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) in the cytoplasm (IDH1) or the mitochondria (IDH2) as part of the citrate cycle. α-KG further activates enzymes of the ten-eleven translocation (TET) family in the nucleus that are essential mediators of DNA demethylation and subsequent transcriptional activation of target genes [IDH1 is p.R132H, resulting from a G to A base shift that leads to an amino acid shift from arginine at position 132 to histidine and a subsequent conformation change in the IDH1 protein [IDH mutations to the inhibition of myeloid differentiation [IDH1) and enasidenib (IDH2) by the Food and Drug Administration (FDA) has been an important advance in the treatment of patients with AML patients containing IDH mutations [In younger adults, hylation . IDH1 anet genes . The mos protein , 9. Studutations .IDH1 mutations may contribute to tumorigenesis. In glioma, the presence of IDH1 mutation was associated with CpG hypermethylation of binding sites for the insulator protein CTCF (CCCTC-binding factor), disabling CTCF binding and correct establishment of DNA loops. As a result, 3D conformational changes of the DNA-architecture affect long-range interactions and subsequently deregulate gene expression [IDH1-mutated AML has not been explored. A better understanding of these mechanisms will potentially unravel novel therapeutic targets in the treatment of AML with IDH1 mutation, contributing to overcoming resistance and improving patient outcome.In addition to a differentiation block mediated by hypermethylated gene promotors, recent research suggests an alternative mechanism of action through which pression . Dysregupression , 14. HowIDH1 p.R132H mutation by CTCF site hypermethylation, disruption of 3D DNA conformation and subsequent gene expression changes in AML. Differential gene expression was analyzed in public AML RNA-Seq [IDH1 mutation. Differential expression results were then integrated with previously generated DNA methylation data [IDH1-mut AML.The aim of this study was to investigate the impact of the  RNA-Seq , 16 and  RNA-Seq  in orderion data  and publKG1a myeloid leukemia cells contain a FGFR1OP2-FGFR1 gene fusion but lack other known driver mutations or genetic alterations and were therefore selected as cellular model. Cells were obtained from the German Collection of Microorganisms and Cell Cultures , maintained at 0.2–2.0 million cells/ml in RPMI-1640 culture medium  with 20% fetal bovine serum and checked regularly for mycoplasma.IDH1 point mutation in KG1a cells, we applied CRISPR base editing [PDGFRA in IDH1-mut cell clones and knockout of the PDGFRA-CTCF-anchor site in IDH1-wt clones, synthetic sgRNAs were purchased from Synthego  and electroporated together with Cas9 RNP. A detailed description can be found in the To generate an  editing . For CRIA detailed description can be found in the Differential gene expression was analyzed in R v4.0.3 with VST values generated from RNA-Seq raw counts using DESeq2 . A detaiIDH1-mut and IDH1-wt AML from the Study Alliance Leukemia (SAL) elderly AML cohort (n = 79) was assessed using the Infinium® HumanMethylation450 BeadChip platform and combined with the TCGA AML cohort (n = 194) for comprehensive methylation analysis as described previously [DNA methylation of patients with A detailed description can be found in the A detailed description can be found in the A detailed description can be found in the In vivo experiments were performed using busulfan-pretreated NSG mice –26 basedIDH1 mutation after failure of 4 lines of therapy, including allogeneic hematopoietic stem cell transplantation. IDH1-positive AML was treated with venetoclax/azacytidine, DLI and cytarabine (araC)/mitoxantrone but resulted in refractory disease. Therefore, therapy with dasatinib was initiated as compassionate use while approval of ivosidenib treatment as compassionate use was awaited. An executive summary of the patient history is provided in Supplementary Table Peripheral blood from a 54-years old patient with AML enrolled in the Study Alliance Leukemia (SAL) registry was collected for analysis of neutrophil and leukemic blast counts after informed written consent in accordance with the declaration of Helsinki approved by the local institutional review boards. The patient acquired an All experiments were performed at minimum in triplicate. Graphs were generated in Microsoft Excel 2016. Error bars represent the standard deviation of the mean. Statistical analysis was performed in R v4.0.3. . Data were checked for normal distribution and two-sided Students t-test or ANOVA was performed when normally distributed whereas Wilcoxon or Mann–Whitney U test was used when not normally distributed. The significance level was 0.05.IDH1-mut AML that contribute to IDH1-driven leukemogenesis, we analyzed differential gene expression between patients with IDH1-wt (n = 153) and IDH1-mut (n = 28) AML with hotspot R132 mutation within the BeatAML RNA-Seq dataset [p-adjusted < 0.05) in patients with IDH1-mut compared to IDH1-wt AML  as the most upregulated oncogene in IDH1-mut AML  activity as well as growth factor binding  or low (below median) PDGFRA expression (n = 8 versus n = 8) as well as IDH1-wt patient with high or low PDGFRA (n = 68 versus n = 68) using published clinical data by Bamopoulos et al. [PDGFRA expression among IDH1-mut but not IDH1-wt groups . In contrast, no survival difference with respect to PDGFRA expression was found in IDH2-mut versus IDH2-wt groups  and TCGA cohorts (n = 194), analyzed as described [IDH1/2-mut AML as distinct subgroup characterized by a global hypermethylation phenotype. Here, we further identified CpG hypermethylation within a CTCF-anchor region upstream of the PDGFRA gene locus in patients with IDH1-mut AML particularly  and the demethylating agent 5-azacytidine. Cell viability was assessed using WST assay. Mono-treatment with venetoclax and araC treatment reduced cell viability of both IDH1-mut and IDH1-wt cell clones  dasatinib may present an additional treatment option for IDH1-mut AML. IDH1-mut cell clones displayed increased sensitivity towards dasatinib compared to IDH1-wt clones, which was comparable to sensitivity towards IDH1-mut specific inhibitor ivosidenib  [HOXA7 and HOXA9 genes was reported to prevent DNA looping and alter expression of posterior HOXA genes [IDH1-mutant AML, Wilson et al. recently investigated differentially methylated regions (DMR) with regard to enhancer association and identified high enrichment for enhancers in hypermethylated IDH1-mut specific DMRs [IDH1-mut AML to upregulated oncogene expression, providing novel insights into 3D DNA alterations as additional leukemogenesis-driving mechanism in AML.Aberrant 3D DNA architecture has generally been reported for different malignancies when compared to healthy controls , 28–33. BCP-ALL) , 34, 35.XA genes . In IDH1fic DMRs . HoweverPDGFRA is associated with altered 3D DNA conformation in IDH1-mut AML and therefore may represent a novel therapeutic target in IDH1-mut AML. PDGFRA is involved in cell proliferation, migration and invasion, cell survival and chemotaxis [PDGFRA expression in connection with altered DNA conformation as potential leukemogenic mechanism in IDH1-mut AML has not been suggested. We provide evidence for decreased CTCF binding at the CTCF-anchor region upstream of PDGFRA due to increased DNA methylation, suggesting disrupted DNA loop formation and insulation and subsequent upregulation of PDGFRA expression. Upregulated PDGFRA-CTCF methylation and PDGFRA expression was also confirmed upon exogenous treatment with 2-HG, confirming 2-HG mediated CTCF hypermethylation as mechanism of action upregulating PDGFRA. This finding is supported by the work from the B. Bernstein research group showing disrupted DNA looping and subsequent activation of PDGFRA expression in IDH1-mut glioma [PDGFRA expression in connection with altered DNA looping in IDH1-mut AML now extends this mechanism of action beyond the context of glioma. As observed in clinical data of IDH1-mut AML patients, IDH1-mut glioma patients with high PDGFRA expression also showed significantly worse survival rates, supporting a strong clinical relevance of this mechanism. These data strengthen the hypothesis that the correlation between IDH1 mutation-dependent DNA conformational changes and upregulated PDGFRA expression may be a more global mechanism across different cancer types.Here we show that upregulation of tyrosine kinase emotaxis . In the emotaxis –40. Yet,t glioma . Our finIDH1-mut AML, the small-molecule inhibitor ivosidenib presents an effective therapeutic agent, leading to remissions in IDH1-mut AML patients [PDGFRA as novel target may not only be of biological or prognostic [Concerning treatment options for patients . Howeverpatients . For thiognostic  but alsoIDH1 p.R132H mutation. While PDGFRA knockout reduced dasatinib sensitivity in IDH1-mut clones, knockout of the PDGFRA-CTCF site and exogenous 2-HG treatment increased dasatinib sensitivity in IDH1-wt clones and naïve KG1a cells, confirming the specific correlation of dasatinib response to PDGFRA expression. The antileukemic effect of dasatinib was confirmed in an in vivo mouse model of IDH1-wt/-mut KG1a and also observed in a single patient with IDH1-mut AML, supporting dasatinib as potential treatment option for IDH1-mut AML. Additionally, our in vitro data also provide a base for the combination of dasatinib and ivosidenib as potential treatment approach.Here, we provide evidence that treatment with tyrosine kinase inhibitor dasatinib may present a novel treatment option for AML patients with IDH1-mut cells while venetoclax monotherapy resulted in a similar response of IDH1-mut and IDH1-wt KG1a cells. This suggests that inhibition of the ABCB1/MDR1/P-GP efflux pump may be specifically relevant in this IDH1-mut KG1a model for the observation of mutation-specific sensitivity differences as reported previously [With regard to combinational therapies, clinical trials have shown promising effects of combination of the demethylating agent 5-azacytidine or BCL2-inhibitor venetoclax with ivosidenib , 44. In IDH1-mut versus IDH1-wt cells to 5-azacytidine could be confirmed, which supports data from recent clinical trials suggesting 5-azacytidine as combinational partner for ivosidenib treatment of patients with IDH1-mut AML [PDGFRA-CTCF site [IDH1-mut AML specifically.In addition, increased sensitivity of -mut AML . HoweverTCF site , TreatmeIDH1 mutant-specific treatment is supported by data from Tavor et al. [IDH1 mutations (33.3%) versus IDH1-wt AML samples (14.3%) among those responding to dasatinib ex vivo . Tavor and colleagues also reported an enrichment of FLT3-ITD and IDH2-mut samples among dasatinib responders in this study. However, our analysis of the underlying RNA-Seq data (BeatAML) [PDGFRA in IDH2-mut versus IDH1-wt AML samples   revealed-mut AML . SpecifiIDH1-mut specificity of dasatinib is further supported by the reduced leukemic blast count that was observed in a patient with refractory IDH1-mut AML after dasatinib treatment. As ivosidenib or 5-azacytidine pre-treatment did not abrogate the beneficial effect of dasatinib in IDH1-mut AML cells and as the combination of dasatinib and ivosidenib appeared to be beneficial, our data suggest dasatinib as novel treatment option, also in combinations, for patients with IDH1 p.R132H mutant AML.IDH1 p.R132H mutation induces changes in the 3D DNA architecture leading to alteration of long-range gene interactions in AML. We show that methylation-dependent disruption of CTCF binding at the CTCF-anchor upstream of PDGFRA leads to upregulated PDGFRA expression. As PDGFRA overexpression correlated with decreased survival of patients with IDH1-mutant AML, we reason that treatment with TKI dasatinib may present a novel therapeutic option for improved treatment for patients with IDH1 p.R132H mutant AML. This may be of particular interest for elderly patients with mutant IDH1, which present a large proportion of patients and show a particularly high IDH mutation frequency and, so far, poor outcome with high resistance and relapse rates.In conclusion, our data suggest that Supplementary MaterialSupplementary Table 4


Fms-like tyrosine kinase 3 (FLT3) gene mutations occur in approximately 30% of all patients with acute myeloid leukemia (AML). Internal tandem duplication (ITD) in the juxtamembrane domain and point mutations within the tyrosine kinase domain (TKD) are two distinct types of FLT3 mutations. FLT3-ITD has been determined as an independent poor prognostic factor, but the prognostic impact of potentially metabolically related FLT3-TKD remains controversial. Hence, we performed a meta-analysis to investigate the prognostic significance of FLT3-TKD in patients with AML.A systematic retrieval of studies on FLT3-TKD in patients with AML was performed in PubMed, Embase, and Chinese National Knowledge Infrastructure databases on 30 September 2020. Hazard ratio (HR) and its 95% confidence intervals (95% CIs) were used to determine the effect size. Meta-regression model and subgroup analysis were used for heterogeneity analysis. Begg’s and Egger’s tests were performed to detect potential publication bias. The sensitivity analysis was performed to evaluate the stability of findings in meta-analysis.Twenty prospective cohort studies  on the prognostic effect of FLT3-TKD in AML were included: 9,744 subjects with FLT3-WT and 1,226 subjects with FLT3-TKD. We found that FLT3-TKD revealed no significant effect on disease-free survival (DFS)  and overall survival (OS)  in general. However, meta-regressions demonstrated that patient source contributed to the high heterogeneity observed in the prognosis of FLT3-TKD in AML. To be specific, FLT3-TKD represented a beneficial prognosis of DFS  and OS  for Asians, whereas it represented an adverse prognosis of DFS for Caucasians with AML .FLT3-TKD revealed no significant effects on DFS and OS of patients with AML, which is consistent with the controversial status nowadays. Patient source (Asians or Caucasians) can be partially explained the different effects of FLT3-TKD in the prognosis of patients with AML. Acute myeloid leukemia (AML) is the most common acute leukemia in adults with the features that poorly differentiated cells from hematopoietic system infiltrate in bone marrow, blood, and other tissues . NowadayFms-like tyrosine kinase 3 (FLT3) is a potential prognostic genetic marker, which encodes a class 3 receptor tyrosine kinase to plays a crucial role in hematopoiesis. FLT3 gene mutations occur in approximately 30% of all patients with AML . There aTwo independent investigators implemented a systematic search in PubMed, Embase, and Chinese National Knowledge Infrastructure (CNKI) databases systematically, with the last search updated on 30 September 2020. The following terms “(acute myeloid leukemia) or (acute myeloblastic leukemia) or (acute myelocytic leukemia) or (acute myelogenous leukemia) or (acute nonlymphoblastic leukemia) or AML”, “FLT3 or CD135 or (fms-like tyrosine kinase 3) or  or  or (human stem cell tyrosine kinase-1)”, “(tyrosine kinase domain mutation) or (TKD mutation) or D835 or I836”, and “prognosis or prognoses or (prognostic factors) or (prognostic implication) or (prognostic element)” were retrieved in PubMed entries in the National Institutes of Health and European Embase databases without any limitation applied. The key words “急性髓系白血病or急性髓性白血病or急性非淋巴细胞性白血病”, “FLT3”, and “TKD突变” were retrieved in CNKI. The reference lists in retrieved studies and relevant reviews were also manually searched for more eligible studies.All literature studies involved in AML and FLT3-TKD were electronically retrieved for the next filter. Afterward, prospective cohort studies were identified according to their titles and abstracts. The full texts of the literature studies that fulfilled the inclusion criteria were perused to validate their eligibility. Inclusion criteria were as follows: (a) the evaluation of association between prognosis of AML and FLT3-TKD; (b) untreated patients with AML were included in study; (c) complete original materials with specific explanation of sample size; (d) they provided data of all enrolled subjects on either or both of overall survival (OS) and disease-free survival (DFS) after a period of follow-up in the study; (e) with survival information based on the FLT3 status: FLT3-TKD and wild type; and (f) prospective cohort study focusing on human being. Exclusion criteria were as follows: (a) not conforming to inclusion criteria; (b) abstract, review article, letter, comment, and editorial; (c) duplicate publication of previous publications; (d) family-based studies of pedigrees; (e) without detailed FLT3 status data (FLT3-TKD and wild type); (f) with incomplete specific explanation or without specific explanation of sample size; and (g) studies were excluded if they focused exclusively on acute promyelocytic leukemia (APL) (M3). For multiple publications from a same population, the largest study was included only to exclude duplicate studies or overlapping data. According to the inclusion and exclusion criteria, the two independent investigators accomplished study selection independently by screening title, abstract, and full text. Any dissent was solved by discussion. If agreement could not be reached, then a third researcher was consulted. Four studies were discussed and excluded after discussion –14.The data of the eligible studies were extracted in duplicate by two independent researchers. The data extracted comprised first author’s name, publication year, diagnostic criteria for AML, the resource of the subjects, genotyping methods, the number of subjects, the FLT3 status (FLT3-TKD or wild type) of subjects, the number of OS and/or DFS from all subjects (if any), hazard ratio (HR) with 95% confidence interval (CI) of OS and/or DFS, and the baseline data of all subjects in all included prospective cohort studies  Table S1The Newcastle-Ottawa (NOS) scale was used to score the strength of all included studies by the three independent investigators . The scaPotential publication bias was checked by Begg’s funnel plots  and Egge2 statistic  (2 > 50%), the random-effects model was used for assessing information; otherwise, the fixed-effects was conducted  . When thonducted . HeterogThe study selection process for the meta-analysis about the prognosis of FLT3-TKD in AML is shown in 2 = 70.7%, p = 0.000)   and 0.008 (p = 0.299) in patients of AML for DFS and OS, respectively, and the coefficient of patient source is −1.004 (p = 0.015) and −0.358 (p = 0.324) in patients of AML for DFS and OS, respectively Figure 32 = = 72.9%, p = 0.000) in the Caucasian subgroup, and the pooled HR of DFS is 0.56  in the Asian subgroup  in the Caucasian subgroup, and the pooled HR of OS is 0.63  in the Asian subgroup  of all participants included Table S1Begg’s and Egger’s tests have been used to detect any publication bias that indicated that there was no significant bias between studies of FLT3-TKD in prediction of DFS (p = 0.499 of Begg’s and p = 0.233 of Egger’s) and also of OS (p = 0.488 of Begg’s and p = 0.061 of Egger’s). Symmetrical Begg’s funnel plot was obtained Figure 52 = 70.7%, p = 0.000) and OS was 0.98 , which revealed no significant effect of FLT3-TKD on DFS and OS of patients with AML by random effect models. However, meta-regressions demonstrated that patient source associated with the prognosis effect of FLT3-TKD in patients with AML. To be specific, FLT3-TKD represented a beneficial prognosis of DFS  and OS  for Asians with AML, whereas FLT3-TKD represented an adverse prognosis of DFS for Caucasians with AML . However, the results of DFS from Caucasians ought to be interpreted with caution due to the heterogeneity .In this meta-analysis, 20 prospective cohort studies  on FLT3-TKD in AML were included: 9,744 subjects with FLT3-WT and 1,226 subjects with FLT3-TKD . The incIn general, FLT3-TKD reveals no significant effects on DFS and OS of patients with AML, which is consistent with the controversial statue nowadays. The controversies of prognostic impacts on FLT3-TKD in patients with AML were supported by two-sided laboratory evidence of FLT3-TKD. On one hand, many studies indicated that FLT3-TKD associated a beneficial prognosis of AML. In some cases, patients with FLT3-TKD companying with other mutations, such as NPM1 mutation, showed a favorable prognosis  with the2 = 72.9%, p = 0.000), and the pooled HR of OS was 1.11  . In this situation, if related confounding factors were well controlled, then the conclusion might be more convinced. However, it was indicated that Caucasians reveals a distinct genetic alteration profiles of AML than Eastern Asian population  Figure 4pulation , but whapulation , 49. Cytountries , 49. Henountries , 50. Howountries , 50. Conountries –53. Whatountries . Hence, ountries . Currentvia the activation of STAT3 , which may attribute to the fact that Caucasians outnumbered Asians: 635 Asians from four different countries and 10,335 Caucasians from eight different countries in this meta-analysis. In our opinion, the heterogeneity from Caucasian group was from some unknown confounding factors in the united prospective cohorts of Caucasians. In this situation, if related confounding factors were well controlled, then the conclusion might be more convinced. In addition, we failed to get the specific information about chemotherapy from 10,970 participants including chemotherapy regimens, chemotherapy dose, and chemotherapy time, because different types of treatment may exert distinct impacts on the prognosis of the patients with AML. However, FLT3-TKD does not result from general chemotherapies, so the FLT3-TKD in AML in this study is regarded as being from individuals’ genetic backgrounds. Furthermore, because of the limitation from the extracted data, we were unable to perform more stratification analysis according to other confounding factors. Although confounding factors work on the ending events, it is considered as a common problem in clinical research studies because we cannot predict everything before the start.There are several limitations of this study that should be acknowledged. First, there was a heterogeneity in Caucasians (I2 + daunorubicin of 50 mg/m2) in conventional “3 + 7” induction, so that they could receive a better prognosis of AML for survivals. An adequately designed prospective study including a large population with AML with clear FLT3 gene statue is needed to confirm our results.In conclusion, our results showed that FLT3-TKD revealed no significant effect on DFS and OS of patients with AML. However, meta-regressions demonstrated that patient source associated with the prognosis effect of FLT3-TKD in patients with AML. To be specific, FLT3-TKD represented a beneficial prognosis of DFS and OS for Asians with AML, whereas FLT3-TKD represented an adverse prognosis of DFS for Caucasians with AML. However, the results of DFS from Caucasians ought to be interpreted with caution due to the heterogeneity. This meta-analysis provided new information about the distinct prognosis of patients with AML between Asians and Caucasians. From our perspectives, the Caucasians with FLT3-TKD at the initial diagnostic stage of AML could be recommended the Asians dose of cytarabine and daunorubicin (cytarabine of 100 mg/mThe funder of the study supported the study design, data analysis, and publication of the manuscript.The original contributions presented in the study are included in the article/SL was involved in project conceiving, data collection, data curation, literature assessment, investigation, formal analysis, software performance, visualization, and original manuscript writing. NL was involved in data collection, data curation, and literature assessment. YC was involved in project conceiving, study supervision, and manuscript editing. ZZ was involved in study supervision and funding acquisition. YG was involved in project conceiving, literature assessment, study supervision, investigation, funding acquisition, and manuscript editing. All of authors read and approved the final manuscript.


Recent publications suggest that Ca2+-permeable nonselective classical transient receptor potential (TRPC) proteins—especially TRPC1 and 6—are crucially involved in acute and sustained hypoxic responses and the pathogenesis of CHPH. The aim of our study was to investigate whether the simultaneous deletion of TRPC proteins 1, 3 and 6 protects against CHPH-development and affects HPV in mice. We used a mouse model of chronic hypoxia as well as isolated, ventilated and perfused mouse lungs and PASMC cell cultures. Although right ventricular systolic pressure as well as echocardiographically assessed PVR and right ventricular wall thickness (RVWT) were lower in TRPC1, 3, 6-deficient mice, these changes were not related to a decreased degree of pulmonary vascular muscularization and a reduced proliferation of PASMCs. However, both acute and sustained HPV were almost absent in the TRPC1, 3, 6-deficient mice and their vasoconstrictor response upon KCl application was reduced. This was further validated by myographical experiments. Our data revealed that 1) TRPC1, 3, 6-deficient mice are partially protected against development of CHPH, 2) these changes may be caused by diminished HPV and not an altered pulmonary vascular remodeling.Chronic hypoxia-induced pulmonary hypertension (CHPH) is a severe disease that is characterized by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) leading to pulmonary vascular remodeling. The resulting increase in pulmonary vascular resistance (PVR) causes right ventricular hypertrophy and ultimately right heart failure. In addition, increased PVR can also be a consequence of hypoxic pulmonary vasoconstriction (HPV) under generalized hypoxia. Increased proliferation and migration of PASMCs are often associated with high intracellular Ca Chronic hypoxia induced pulmonary hypertension (CHPH) is a serious disease that affects a wide range of people worldwide—with increasing yearly prevalence . HoweverVascular remodeling in CHPH largely affects the tunica media, which is primarily composed of pulmonary arterial smooth muscle cells (PASMCs) . Elevati2+concentration ([Ca2+]i) is hypothesized to be one of the reasons for the altered PASMC phenotype as increased [Ca2+]i is crucial for proliferation, migration and apoptosis of PASMCs and therefore, highly involved in vascular remodeling  proteins, which are non-specific cation channels permeable for both Ca2+ and Na+. As an increase in Na+ activates voltage-gated Ca2+ channels, which also increases cytosolic Ca2+  channels. We hypothesize that TRPC1/3/6−/− mice would be protected against the development of CHPH. To answer this question, we performed investigations in chronically hypoxic mice, isolated, ventilated and perfused lungs and on the cellular level in PASMCs.Amongst others i due to the simultaneous deletion of TRPC1, 3 and 6. However, since TRPC and TRPC1 channels in particular are permeable for both Ca2+ and Na+ we cannot exclude a Na+-mediated impact on vasoreactivity. However, it was previously demonstrated that TRPC1 is crucial for Ca2+ signaling and proper channel function, thus emphasizing the importance of TRPC1-dependent Ca2+−permeability  pulmonary vasoconstriction, which is associated with a partial protection against CHPH. The present study thus offers further evidence for TRPC1, 3 and 6 being crucially involved in the pathomechanisms of CHPH. Further studies are needed to confirm whether TRPC1, 3 and 6 might be possible treatment targets against CHPH.


Since ATP-powered Na+ and Ca2+ pumping activities are increased following stimulation to restore ion gradients and are estimated to consume most neuronal ATP, we aimed to determine if they are coupled to neuronal glycolysis activation. By using two-photon imaging of fluorescent biosensors and dyes in dentate granule cell somas of acute mouse hippocampal slices, we observed that production of cytoplasmic NADH, a byproduct of glycolysis, is strongly coupled to changes in intracellular Na+, while intracellular Ca2+ could only increase NADH production if both forward Na+/Ca2+ exchange and Na+/K+ pump activity were intact. Additionally, antidromic stimulation-induced intracellular [Na+] increases were reduced >50% by blocking Ca2+ entry. These results indicate that neuronal glycolysis activation is predominantly a response to an increase in activity of the Na+/K+ pump, which is strongly potentiated by Na+ influx through the Na+/Ca2+ exchanger during extrusion of Ca2+ following stimulation.Cellular ATP that is consumed to perform energetically expensive tasks must be replenished by new ATP through the activation of metabolism. Neuronal stimulation, an energetically demanding process, transiently activates aerobic glycolysis, but the precise mechanism underlying this glycolysis activation has not been determined. We previously showed that neuronal glycolysis is correlated with Ca Ca2+ is handled by a diverse set of active transporters, including the plasma membrane Ca2+-ATPase (PMCA), which exports 1 Ca2+ across the plasma membrane per ATP (2+-ATPase (SERCA), which pumps 2 Ca2+ into the endoplasmic reticulum lumen per ATP  (+ gradient (built by the Na+/K+ pump) to actively export 1 Ca2+ by importing 3 Na+. Any increase to neuronal Na+ or Ca2+ will increase the activity of their respective ion transporters.There are several ion-ATPases  that regulate neuronal Na changes . Na+ is  per ATP , and the per ATP . Aside fer (NCX) , a secon2+ (Ca2+CYT) induced by excitation are positively correlated with transient increases in subsequent NADHCYT production interpreted as glycolysis being driven primarily by an increase in the activities of Ca2+ pumps (PMCA and SERCA) as Ca2+ is pumped from the cytoplasm. However, this readout of neuronal NADHCYT and Ca2+CYT lacks information about stimulation-induced increases to intracellular Na+, which also likely covary with stimulation strength, so a contribution of increased Na+/K+ pump activity to glycolysis activation cannot be excluded.Transient increases to neuronal cytoplasmic Caoduction ; in othe+/K+ pump is primed to be the predominant driving force underlying neuronal glycolysis activation. Many reports estimate that the Na+/K+ pump consumes ~50% of total brain energy  within acute brain slices by using two-photon fluorescence imaging of genetically encoded fluorescent biosensors and fluorescent dyes. The data provide compelling evidence indicating that increased activity of the Na+/K+ pump is the predominant driver of neuronal glycolysis activation, whereas increased activities of Ca2+ pumps appear to be negligibly coupled to glycolysis.To shed light on the mechanism of neuronal glycolysis activation, we investigated the coupling of Na+ (NADHCYT) and free [Ca2+], respectively (+) or RCaMP (Ca2+) alters each sensor’s fluorescence lifetime (LT), the average time that the fluorophore spends in the excited state prior to its return to the ground state  but not by AOA (an inhibitor of MAS) and were not affected by impaired NADH production in mitochondria . Simultaneous removal of both Na+ and Ca2+ from the bath solution  decreased the Peredox LT from its baseline in ACSF . At this point, with Ca2+CYT elevated, we removed 0.5 mM external Ca2+ and re-applied external Na+ to facilitate strong Na+ influx via forward NCX transport (in exchange for Ca2+CYT), which promptly increased NADHCYT. Following the external Na+ application, Ca2+CYT returned to baseline levels .The decrease to Peredox LT by removing external Naany DGCs , show thlication , which s2+CYT regulation in DGCs are dependent on forward NCX transport. A Ca2+CYT increase (from reverse NCX transport) returned faster to baseline levels if external Na+ was present  increased both the NADHCYT and Ca2+CYT transients evoked by antidromic electrical stimulations in ACSF  still failed to increase NADHCYT  from Ca2+ entry through the mitochondrial Ca2+ uniporter (MCU)  . Could Ced RCaMP , indicatCYT production is closely coupled to the flux of Na+, and that a Ca2+CYT elevation is not sufficient to elevate NADHCYT regardless of whether it is pumped by SERCA, pumped by PMCA, or taken up into mitochondria via MCU. They also show that NADHCYT production is activated more strongly by conditions that favor forward NCX transport that exchanges Ca2+CYT for Na+, and that regulation of both DGC Ca2+CYT and Ca2+MITO depends on Na+. These observations suggest that neuronal glycolysis is both strongly coupled to the activity of the Na+/K+ pump and sensitive to Na+ import via forward NCX transport, which would become activated following an excitatory event where Ca2+CYT must be removed.In summary, these ion substitution experiments show that neuronal NADHCYT by transferring reducing equivalents from NADHCYT to mitochondria  is a major pathway that recycles NADHchondria . MAS actevations , which l courses . But theCa2+MITO , so it rhibition .CYT recycling and consequent elevation of the cytoplasmic NADH:NAD+. This strong increase to Peredox LT, which approaches the upper end of its dynamic range, precludes the ability to measure further NADHCYT increases, so we applied exogenous pyruvate (1.5 mM) to promote the oxidation of NADH to NAD+ via lactate dehydrogenase and restore the Peredox LT approximately back to its baseline LT in ACSF. We then removed external Na+ and Ca2+, which decreased the Peredox LT from the AOA and pyruvate condition  ouabain, which inhibits only the rodent Na+/K+ pump α2- and α3-isozymes that have higher inhibitor sensitivities . The mean ∆Peredox/∆RCaMP ratio  was reduced to nearly 0 after blocking forward NCX transport by removing external Na+  or after blocking the Na+/K+ pump with ouabain .A scatter plot of the transient Peredox LT and RCaMP LT changes that were evoked by Ca2+ puffs  illustraMP ratio  for Ca2+2+CYT can activate glycolysis in the DGC soma if it is traded for Na+ through forward NCX transport, which increases the activity of the Na+/K+ pump. The lack of Ca2+-puff-induced NADHCYT responses when either forward NCX transport or the Na+/K+ pump was blocked further substantiates that any increase in the activity of Ca2+ pumps has a negligible effect on glycolysis activation.These data demonstrate that CaCYT production by Ca2+CYT through coupling between forward NCX transport and the Na+/K+ pump  and RCaMP LT from a DGC bathed in ACSF show that antidromic stimulation evoked a transient decrease to SBFI ∆F/F (note the inverted y-axis), reflecting an increase to [Na+], and a transient increase to RCaMP LT, reflecting an increase to Ca2+CYT  was reduced to 54.6±19.3% of the original response and the Ca2+ transient (as ∆RCaMP LT) was reduced to 14.5±12.5% (n=13)  .2+  could be reversed by strophanthidin at low micromolar concentrations , which regulates DGC resting membrane potential and excitability  ATP consumption less than the ATP consumed by Na+/K+ pumps.The sensitivity of glycolysis to Caycolysis . This maactivity . Even if2+ pumps have an apparent K0.5 for Ca2+ activation of ~0.1 μM and relatively slow maximal turnover rates of ~100 s–1  but could rapidly respond to stimulation-induced Ca2+CYT increases and exchange it for Na+. Unfortunately, most NCX inhibitors are unspecific or do not block the forward transport mode .The NCX has an apparent Kof ~1 μM  and a trof ~1 μM . This meort mode , so the CYT production evoked by Ca2+ puffs in 40 mM Na+ and 0 Ca2+ was eliminated when external Na+ was completely removed  or when ouabain was added  is more influential on the rate of glycolysis than any putative strong activation of Ca2+ pumps by a Ca2+CYT increase.Strong coupling between the NCX, the Naransport . For revrt 3 Na+ . This me2+ puffs in 40 mM Na+ and 0 Ca2+  compared to the external [Na+] during electrical stimulation in ACSF (147 mM), which would slow down the kinetics of Ca2+CYT-dependent Na+ influx through forward NCX transport, consequently leading to a less potent activation of the Na+/K+ pump; both the quantity and the rate of Na+ influx are likely to be important determinants of the NADHCYT transient amplitude and time course.The ∆Peredox/∆RCaMP ratio evoked by Ca2+ ~0.1,  is ~40–52+ ~0.1, , meaning+] per se, but rather by the downstream effect of [Na+] on Na+/K+ pump activity. If glycolytic enzymes could be directly stimulated by an increase to [Na+], then complete inhibition of the Na+/K+ pump should have facilitated stronger NADHCYT responses. But this was not the case, as NADHCYT responses evoked by Ca2+ puffs were strongly attenuated in the presence of 5 mM ouabain , which means that the plasma membrane Na+ gradient in the presence of 5 μM ouabain remains sufficient to prevent a reversal of NCX transport. Second, the stimulation-induced Ca2+CYT transients were increased, which is, perhaps, reflective of an effect on synaptic release.Glycolysis does not appear to be influenced by intracellular  of 13 mM, which is the reported value for hippocampal slice CA1 neurons  and α3 activity ~9-fold .The mean peak amplitude of antidromic stimulation-induced SBFI ∆F/F transients evoked in ACSF was –25.6±4.6% , reflect/K+ pump . Under prnal Na+ , a 25 mM2+ entry reduced the antidromic stimulation-induced peak SBFI ∆F/F to –11.5±5.5%  would increase α1β1 activity ~1.5-fold  and α3 activity ~3-fold .Blocking Ca1.5±5.5% , a ~55% + entry on glycolysis activation could be dependent on the stimulation paradigm. Antidromic stimulation in the presence of synaptic blockers does not involve entry of Na+ through NMDA or AMPA receptors. This means that most channel-mediated Na+ influx that occurs from this stimulation method is through voltage-gated channels, which, in DGCs, are highly localized to the axon initial segment with weak, if any, staining in the soma , during which an increase to the activity of Na+/K+ pumps would be sustained. Both the sensitivity of NADHCYT production to Na+ and the ablation of Ca2+CYT-evoked NADHCYT transients by inhibition of either forward NCX transport or the Na+/K+ pump demonstrate that pumping Na+ is the predominant activity that drives the transient activation of aerobic glycolysis.Neuronal stimulation immediately increases both intracellular  will not be substantially buffered by the ~0.5–1 mM [SBFI] in our recordings  is nearly diffusion-controlled, with very rapid rates of formation and dissociation  equipped with an Olympus LUMPLFLN 60×/W (NA 1.0) objective lens, hybrid photodetectors R11322U-40 , and a Chameleon Vision-S tunable Ti-Sapphire mode-locked laser . The excitation wavelength was 790 nm. Fluorescence emission light from the Peredox and RCaMP biosensors was split with an FF562-Di03 dichroic mirror and bandpass filtered for green (FF01-525/50) and red (FF01-641/75) light; a red 670/50 bandpass filter was used for experiments where SBFI and RCaMP were multiplexed. The photodetector and laser sync signals were preamplified and then digitized at 1.25 GHz using a field-programmable gate array board . A modified version of the ScanImage software written in Matlab   convolved with a Gaussian for the impulse response function . FluoresFluorescence images were analyzed offline using Matlab R2014b software. Regions of interest (ROIs) were defined around individual DGC somas and photon statistics were calculated for all pixels within the ROI. LT values were calculated by fitting the photon arrival histograms with a biexponential decay function . Individual transient fluorescence intensity traces were baseline subtracted and the peak ∆F/F was determined as the minimum intensity value.RCaMP signals were unmixed from Peredox as previously described . RCaMP sAverage time traces of transient fluorescence LT or intensity changes were created using Python. Data acquisition times for individual fluorescence traces were binned to the nearest second for data acquired before and after the stimulation event (which were acquired every 10–60 s) or the nearest 10 ms for data acquired during the stimulation (which were acquired every ~250 ms). Traces from all stimulation-induced transients in the data set were interpolated and merged onto a single time axis, from which the means, standard deviations, and standard error of the means were determined.α=0.05) using a Shapiro-Wilk test. Data sets that met normality criteria were compared using a paired t-test, or two-sample t-test. Data sets that did not meet normality criteria were compared using a paired sample Wilcoxon test or Mann-Whitney test for unpaired samples. Data are reported as mean ± standard deviation (unless otherwise indicated). For box plots, the means are indicated by the filled square, medians are indicated by the horizontal bar, and 5–95% ranges are indicated by whiskers.Statistical analysis was performed using Origin 8.1 software. Data sets were tested for normality .All chemical reagents used to make the brain slicing and artificial cerebrospinal fluid (ACSF) solutions were obtained from Sigma-Aldrich . +, not Ca2+, and the activity of the Na+/K+ pump drive glycolysis. The study is conceptually significant as it seeks to determine how neuronal glycolysis is coupled to electrical activity. The study is thoughtful, uses sophisticated fluorescence lifetime imaging technology and clever experimental designs, and as such provides new insights into how electrical activity regulates glycolysis.The authors investigate mechanisms that regulate glycolytic ATP production in neurons. They conclude that the cytosolic Na public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:+/K+ pump dominates control of glycolysis in hippocampal dentate granule cells" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: James B Hurley (Reviewer #1).Thank you for submitting your article "The NaThe reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. It would be helpful to be able to show definitively that the increased level of NADH is not caused by a decrease of NADH consumption (as might occur if metabolism switched from inefficient glycolysis to much more efficient oxidative phosphorylation). I think the experiments evaluating the role of the malate aspartate shuttle do address this, but this specific question (could more efficient consumption of NADH lower the demand and increase steady state NADH) should be addressed in the discussion.+ rather than the activity of the Na+/K+ ATPase that is stimulating glycolysis. The data in Figure 4B-D suggest that the changes in intracellular [Na+] are substantial and perhaps they could influence the activity of glycolytic enzymes. The ouabain experiment in Figure 3D does help to show that consumption of ATP by the pump is the driving force. But that set of experiments is complicated by the need for very high concentrations of ouabain that could produce non-specific effects. The authors suggest based on a 1979 paper and a review that oubain cannot fully inhibit the Na+/K+ ATPases in mouse neurons but they have not shown that directly for the cultured neuron preparation they use in this paper. I think that the interpretations of the ouabain experiments would be more convincing if the authors could show for certain that μM ouabain is not inhibiting the NaK ATPase activity at μM concentrations in the neurons used in this paper. Would it be possible to do that by measuring ATPase activity as was done it the Marks and Seeds 1979 paper that was cited?2. It would be helpful to test whether it is just the intracellular concentration of Na3. Please rewrite the last paragraph on p. 19. It seems to be one long sentence and I had to read it several times to decipher the points the authors are making.+ ratio is used throughout as the only measurement reflecting glycolytic flux.4. The NADH/NAD+/K+ pump) is meant to provide localized ATP to power these pumps. How does bulk glycolysis (monitored with NADH/NAD+ ratio) relate to localized/compartmentalized glycolysis?5. It has been hypothesized that the close association of glycolytic enzymes with ion transporters  is a good method to study localized/compartmentalized glycolysis, which is thought to be closely coupled to the pumps.10. It is not clear why monitoring bulk glycolysis , in the absence of electrical activity. It is not immediately obvious why electrical activity, which occurs at much faster time scales, would follow the same rules. Indeed SERCA inhibition during activity elevated NADH levels but had no effect at baseline . How do the authors explain these discrepancies?2+/Na+ alone.12. Pharmacological inhibition of the NCX transporter is needed. Many of the conclusions about its role are based on manipulation of extracellular Ca+ being the only ion that regulates glycolysis. This is an interesting finding and there is no need to explain it away. In fact, it may be that it is local microdomains of Ca2+ that activate glycolysis (raise NADH level) rather than bulk Ca2+.13. The finding that inhibition of SERCA during stimulation actually elevates cytosolic NADH level argues against Na+ elevation lasts longer than Ca2+ in the cell. This could be an artefact of the SBFI on/off rate relative to RCaMP. In fact, prolonged elevation of cytosolic Na+ would make neurons refractive to depolarization in AP trains.14. The finding that "SBFI ΔF/F transients were longer in duration than the RCaMP LT transient" does not necessarily mean that Na+/K+ pump on glycolytic activity.15. Monensin might be useful to show the effect of a fairly direct activation of the Na16. In addition to altering the ionic compositions, chemical blocking og NCX (SEA0400?) might be useful to validate the role of NCX in the proposed pathway.+ + 0.5 Ca2+ to 147 Na+ + 0 Ca2+ led to a sustained NADHcyto increase. Cytoplasmic [Na+] shows only a transient rise when Ca2+ entry is inhibited . Please explain.17. Switching back from 0 Nav blocker.18. Please show NADHcyto during antidromic stimulation in presence of Ca2+ response is lower after Ca2+ puff in 0Na+ and with Ouabain treated condition. What could be the possible explanation?19. The cyto-Ca2+ activates the glycerol-phosphate shuttle, which also plays role in recycling NADHcyto. I failed to find any experimental data or discussion in the manuscript on how this shuttle could influence the finding.20. Ca Essential revisions:It would be helpful to be able to show definitively that the increased level of NADH is not caused by a decrease of NADH consumption (as might occur if metabolism switched from inefficient glycolysis to much more efficient oxidative phosphorylation). I think the experiments evaluating the role of the malate aspartate shuttle do address this, but this specific question (could more efficient consumption of NADH lower the demand and increase steady state NADH) should be addressed in the discussion.+ ratio is used throughout as the only measurement reflecting glycolytic flux.The NADH/NAD2+ activates the glycerol-phosphate shuttle, which also plays role in recycling NADHcyto. I failed to find any experimental data or discussion in the manuscript on how this shuttle could influence the finding.CaCYT occur regardless of NADH dynamics in mitochondria  and that the NADHCYT responses arise from increased glycolysis rather than a change in the malate-aspartate shuttle , so an increase to NADHCYT due to changes in mitochondrial metabolism or decreased NADH consumption seems unlikely. We have now added a paragraph on this to the first section of the Discussion.We have previously shown that transient increases to NADHRegarding the glycerol-phosphate shuttle: a recent paper using cultured neurons indicates that it does not make a significant contribution to NADH recycling when MCU and/or MAS remain active , so the G3P shuttle should not have much influence on our results since we never inhibited MCU and MAS simultaneously.+ rather than the activity of the Na+/K+ ATPase that is stimulating glycolysis. The data in Figure 4B-D suggest that the changes in intracellular [Na+] are substantial and perhaps they could influence the activity of glycolytic enzymes. The ouabain experiment in Figure 3D does help to show that consumption of ATP by the pump is the driving force. But that set of experiments is complicated by the need for very high concentrations of ouabain that could produce non-specific effects. The authors suggest based on a 1979 paper and a review that ouabain cannot fully inhibit the Na+/K+ ATPases in mouse neurons but they have not shown that directly for the cultured neuron preparation they use in this paper. I think that the interpretations of the ouabain experiments would be more convincing if the authors could show for certain that μM ouabain is not inhibiting the NaK ATPase activity at μM concentrations in the neurons used in this paper. Would it be possible to do that by measuring ATPase activity as was done it the Marks and Seeds 1979 paper that was cited?It would be helpful to test whether it is just the intracellular concentration of Na+ alone directly stimulates glycolysis. If this were the case then inhibition of the Na+/K+ pump (which otherwise limits increases to intracellular Na+) during the Ca2+ puff experiments should have increased the metabolic transient, but the results show the opposite. Ouabain is a specific inhibitor of the Na+/K+ pump and applying it at mM concentrations is commonly used to inhibit the more-ouabain-resistant Na+/K+ pump isozymes , though we cannot completely rule out possible non-specific effects.We do not believe that elevated intracellular NaThe Marks and Seeds paper reports multiple ouabain binding affinities in freshly obtained brain tissue from mouse, which is a very close match to the acute brain slice tissue (not cultures) used in our experiments.Please rewrite the last paragraph on p. 19. It seems to be one long sentence and I had to read it several times to decipher the points the authors are making.We have improved the readability of this paragraph.+ pump) is meant to provide localized ATP to power these pumps. How does bulk glycolysis (monitored with NADH/NAD+ ratio) relate to localized/compartmentalized glycolysis?It has been hypothesized that the close association of glycolytic enzymes with ion transporters (such as the Na+/K+ ratio) is a good method to study localized/compartmentalized glycolysis, which is thought to be closely coupled to the pumps.It is not clear why monitoring bulk glycolysis .Even if ATP is produced by glycolysis within a submembrane domain, it is likely that the NADH and NADRelated to point 5, most of the peredox measurements in the paper have been made at baseline, in the absence of electrical activity. Therefore, it is not clear how the findings relate to activity-driven glycolysis.Related to point 2, most of the peredox measurements in the paper have been made at baseline , in the absence of electrical activity. It is not immediately obvious why electrical activity, which occurs at much faster time scales, would follow the same rules. Indeed SERCA inhibition during activity elevated NADH levels but had no effect at baseline . How do the authors explain these discrepancies?2+ and Na+. It is true that rapid changes might obey somewhat different rules than the slower changes we are able to make by ion exchange, but we think that the absence of a response to Ca2+ when Na+ is unavailable for exchange via NCX is quite informative. Combined with the observation in the previous paper showing that Ca2+ influx is normally an important contributor to the increased glycolysis during stimulation, we believe this makes a good case for our conclusions: that Na+ is required for converting the Ca2+ signal into energy demand and a glycolytic response. We also corroborated the NADHCYT responses that we observed in our ion substitution experiments with the Ca2+ puff experiments to test the effect of a faster change in free cytoplasmic Ca2+, more similar to what is achieved through electrical stimulation. The amplitudes of the Ca2+ increases that we achieve with both the ion substitution experiments and Ca2+ puff experiments are also similar to the typical amplitudes of Ca2+ increases when evoked by electrical stimulation. Unfortunately, it is impossible to produce either synaptic or antidromic stimulation of the neurons in the absence of extracellular Na+.We have employed the ion substitution experiments to mimic the primary cellular effects of electrical stimulation, the strong changes in intracellular concentrations of both CaWe have now added a paragraph with this information to the Discussion.2+] transients, and it also produces an increase in the Peredox response, normalized to the RCaMP response. As mentioned above (response #4), this can be explained if a larger than normal fraction of the intracellular Ca2+ is removed by NCX because of the absence of SERCA-mediated uptake into ER.The effect of thapsigargin in the context of electrical stimulation  is to increase the size of the intracellular  = 5 mM, is in the low microsecond range, meaning that it will achieve nearly its entire response in ≤ 50 μsec. We can expect the kinetics of Na+ complexation with SBFI, which is a crown ether with several fluorophores attached, to be similar. We have added these citations to the Methods section on single-cell electroporation and use of SBFI.It is highly unlikely that the prolonged Na+ alone (to the levels seen here) should not cause neurons to be refractory to firing; refractoriness typically occurs in the setting of prolonged depolarization and consequent inactivation of NaV channels.Prolonged elevation of cytosolic Na1/2 decay of 410 ms . We have added this citation to the last section of the Discussion.Also, RCaMP has a tTo ensure that the interpretation of changes in cytosolic NADH are valid with respect to glycolysis, it is important to measure dynamic changes in other metabolites such as glucose.CYT signal originates from glycolysis .Measurement of intracellular [glucose] would not help validate the conclusions or refine the interpretation of the NADH signal, since an increase in glycolytic rate is compatible with any change in the level of glucose: up, down, or no change. Levels of glucose and other metabolites are dependent on their own intrinsic, and variable, rates of import/production and export/consumption, so the interpretations of measuring the levels of additional metabolites on the rate of glycolysis is not straightforward. We have also previously reported two extensive characterizations showing the NADH2+/Na+ alone.Pharmacological inhibition of the NCX transporter is needed. Many of the conclusions about its role are based on manipulation of extracellular CaIn addition to altering the ionic compositions, chemical blocking of NCX (SEA0400?) might be useful to validate the role of NCX in the proposed pathway.Although there are many published inhibitors of NCX transport, we are not aware of any that is really effective at specifically inhibiting the forward transport mode of the transporter, which would be needed for informative results in our experiments. Most reported inhibitors of the NCX are only mildly effective at inhibiting the reverse mode but have other non-specific effects and variable isoform selectivity .2+ channel function, so interpreting the results from stimulation in the presence of SEA0400 would also be complicated. We have now added a brief sentence about NCX pharmacology in the Discussion.SEA0400 does not effectively inhibit the forward transport mode of NCX , and it affects Ca+/K+ pump on glycolytic activity.Monensin might be useful to show the effect of a fairly direct activation of the Na+/H+ exchanger, so monensin-induced changes in Na+ would be accompanied by pH changes. This would complicate the interpretation of any monensin-induced effect on glycolysis, since the activities of several glycolytic enzymes are pH-sensitive.Monensin is a Na+ + 0.5 Ca2+ to 147 Na+ + 0 Ca2+ led to a sustained NADHcyto increase. Cytolasmic [Na+] shows only a transient rise when Ca2+ entry is inhibited . Please explain.Switching back from 0 Na+ + 0.5 Ca2+ to 147 Na+ + 0 Ca2+ is an external ion manipulation that is designed to evoke very long-lasting changes in intracellular ion concentrations, while the cytoplasmic [Na+] transients are recorded under a more physiological condition with briefer stimulation via an electrode.These are different experimental paradigms that cannot be compared directly. The switching from 0 Nav blocker.Please show NADHcyto during antidromic stimulation in presence of CaeLife.64821), which is cited in this report’s Discussion.This is shown and described in detail within our earlier report (Figure 3 of Diaz-Garcia et al. 2021 doi:10.7554/2+ response is lower after Ca2+ puff in 0 Na+ and with Ouabain treated condition. What could be the possible explanation?The cyto-Ca2+ transient amplitude are not due to applying ouabain or to removing Na+, but are simply due to the varied effectiveness of the Ca2+ puff during each recording. The scatter plot of the Ca2+ puff data shows a clear comparison of data across each condition for a wide range of Ca2+ increases.These are not paired responses. The differences in Ca2+ puff recordings in different external conditions are not paired. It is also stated in the Figure 3 legend that the ∆Peredox/∆RCaMP ratio significance test is for unpaired samples using a Mann-Whitney test.We have added more clarity to the Results section to indicate that the Ca


At the same time, for a number of particles, the limitations of their use, associated with their size, are shown. The use of nanoparticles with a diameter of less than 10 nm leads to their short life-time in the bloodstream and rapid removal by the liver. Nanoparticles larger than 200 nm activate the complement system and are also quickly removed from the blood. The effects of different-sized SeNPs on brain cells have hardly been studied. Using the laser ablation method, we obtained SeNPs of various diameters: 50 nm, 100 nm, and 400 nm. Using fluorescence microscopy, vitality tests, PCR analysis, and immunocytochemistry, it was shown that all three types of the different-sized SeNPs have a cytoprotective effect on brain cortex cells under conditions of oxygen-glucose deprivation (OGD) and reoxygenation (R), suppressing the processes of necrotic death and inhibiting different efficiency processes of apoptosis. All of the studied SeNPs activate the Ca2+ signaling system of astrocytes, while simultaneously inducing different types of Ca2+ signals. SeNPs sized at 50 nm- induce Ca2+ responses of astrocytes in the form of a gradual irreversible increase in the concentration of cytosolic Ca2+ ([Ca2+]i), 100 nm-sized SeNPs induce stable Ca2+ oscillations without increasing the base level of [Ca2+]i, and 400 nm-sized SeNPs cause mixed patterns of Ca2+ signals. Such differences in the level of astrocyte Ca2+ signaling can explain the different cytoprotective efficacy of SeNPs, which is expressed in the expression of protective proteins and the activation of reactive astrogliosis. In terms of the cytoprotective efficiency under OGD/R conditions, different-sized SeNPs can be arranged in descending order: 100 nm-sized > 400 nm-sized > 50 nm-sized.It is known that selenium nanoparticles (SeNPs) obtained on their basis have a pleiotropic effect, inducing the process of apoptosis in tumor cells, on the one hand, and protecting healthy tissue cells from death under stress, on the other hand. It has been established that SeNPs protect brain cells from ischemia/reoxygenation through activation of the Ca The microelement selenium has a pleiotropic effect and has cytoprotective effects on healthy tissues under stress, but, on the other hand, it has an anticancer effect, causing apoptosis. In this regard, selenium is of great interest for use in medicine. Selenium has a relatively high photoconductivity; catalytic activity; oxidative reactions; high piezoelectric, thermoelectric, nonlinear optical properties; and a low melting point. In addition, the electrical conductivity of selenium can be increased by several orders of magnitude when exposed to visible light. All the listed properties of selenium are also preserved in nanoparticles synthesized on its basis. A widely used physical method for obtaining SeNPs is the laser ablation method, which was used to obtain nanoparticles in this study. This method allows for quick creation of high-quality nanoparticles, without any chemical contamination, in one stage ,2.2+ homeostasis, as well as promote cell survival in the penumbra zone i in the astrocyte causes a Ca2+-dependent secretion of ATP and a lactate release via Cx43 connexin chemichannels. As a result, paracrine activation of the entire network of astrocytes and suppression of the OGD-induced hyperexcitation of neurons occurs. Prolonged incubation of cerebral cortex cells with SeNPs leads to inhibition of the OGD-induced global increase in [Ca2+]i and necrotic death through activation of reactive astrogliosis and expression of the genes encoding cytoprotective proteins i without returning the concentration of intracellular Ca2+ to the baseline. As can be seen in 2+ signals averaged over several tens of astrocytes, upon the addition of any of the studied concentrations of SeNPs, leads to a slow increase in [Ca2+]i, which may be a sign of impaired Ca2+ homeostasis of cells.We have previously shown that the application of SeNPs induces the generation of Cam the ER ; therefo10 μg/mL A–F cause2+ signals in astrocytes (2+ responses and the percentage (2+ responses of cells to the addition of SeNPs (100 nm) are of a cardinally different nature—they are predominantly signals in the form of Ca2+ oscillations that occur without an increase in the base level of [Ca2+]i, which is clearly seen in The application of SeNPs with a diameter of 100 nm also causes the generation of Catrocytes A–F, and rcentage H of resp2+ responses that also start at a 0.25 μg/mL addition  are somewhere between 50 nm-sized SeNPs in diameter and 100 nm-sized SeNPs. In particular, most cells react with Ca2+ oscillations, similar to the application of 100 nm-sized SeNPs, but these oscillations occur with an increase in the base level of [Ca2+]i.The largest SeNPs, 400 nm in diameter, added to astrocytes at various concentrations elicit Caaddition A, and thaddition A–F, simuaddition H, also i signals G showed 2+ signals exclusively in astrocytes of the cerebral cortex; however, the nature of the Ca2+ response of cells is significantly different. The addition of SeNPs with a diameter of 50 nm causes predominantly transient Ca2+ signals, accompanied by a slow increase in the base level of [Ca2+]i during the entire time of fluorescence registration, whereas SeNPs with a diameter of 100 nm cause Ca2+ oscillations in astrocytes that occur without an increase in the basic [Ca2+]i level. The largest of the investigated nanoparticles (400 nm) lead to the generation of Ca2+ oscillations, similarly to the 100 nm SeNPs, but in this case, a moderate increase in the base level of [Ca2+]i occurs. Such differences in the behavior of the Ca2+ signaling system of astrocytes depending on the size of the SeNPs can lead to different physiological effects, due to the involvement of Ca2+ ions in the regulation of most cellular vital processes.Thus, the application of SeNPs of any of the studied diameters leads to a dose-dependent generation of Ca2+ responses of astrocytes to the application of different-sized SeNPs on their concentration showed that astrocytes of the mouse cerebral cortex are most sensitive to the application of SeNPs with a diameter of 100 nm, since the EC50 value for them is 0.58 µg/mL  at least changed shape, ceasing to be transient or oscillatory, but were preserved  activate cell Ca2+ signals through the only mechanism of endocytosis, clathrin-dependent, while larger SeNPs are simultaneously involved in the generation of Ca2+ signals, such as clathrin-dependent endocytosis and micropinocytosis.Thus, the activation of the CaIschemia-like conditions , followed by 24-h reoxygenation, cause a massive death of cortical cells due to the induction of early stages (18%) and late (16%) stages of apoptosis B,C, but By staining cells with more specific vital probes, it was possible to confirm the greater efficiency of 100 nm-sized SeNPs in suppressing OGD/R-induced cell death in the cerebral cortex. After 24 h of OGD/R, up to 40% of the cells died along the apoptotic pathway (Apopxin Green staining), and more than 50% of the cells died along the necrotic pathway (staining with 7-aminoactinomycin D) B. All ofThus, all of the studied different-sized SeNPs have a pronounced cytoprotective effect under the conditions of ischemia/reoxygenation, but SeNPs with a diameter of 100 nm have the most pronounced effect, suppressing OGD/R-induced late stages of apoptosis and necrosis to the control level. Larger (400 nm) or smaller (50 nm) nanoparticles were characterized by a high percentage of cells in the early stages of apoptosis, which are considered reversible ,18.2+-dependent damage to brain cells, which occurs as a result of a biphasic increase in Ca2+ ions in the cytosol of both neurons  incubation of cerebral cortex cells with 3 μg/mL SeNPs (50 nm) does not lead to inhibition of the first and especially the second phase of the OGD-induced increase in [Ca2+]i in neurons  effectively inhibited the global increase in i concentration regulate almost all intracellular processes and the expression of proteins that will determine a cell’s fate at rest and under stress. Our experiments showed that SeNPs of all studied sizes induce the generation of Ca2+ signals; however, the profile of the intracellular increase in Ca2+ has a number of fundamental differences. Thus, small (50 nm-sized) SeNPs induce transient Ca2+ responses in astrocytes of the cerebral cortex, and the key difference from larger SeNPs is a gradual increase in the base concentration of [Ca2+]i. SeNPs of a 100 nm size lead to the generation of Ca2+ oscillations without a baseline increase in [Ca2+]i, while the largest SeNPs induce transient Ca2+ oscillations with a moderate increase in the baseline [Ca2+]i concentration. It is known that an increase in the basic level of [Ca2+]i can lead to cell death in the case when such an increase is global and irreversible [2+ oscillations without increasing the base level of [Ca2+]i, on the contrary, increased the expression of protective genes while suppressing pro-apoptotic and pro-necrotic ones. Such a correlation of stable Ca2+ oscillations with the activation of protective signaling pathways was shown by us for white adipocytes i increase, and small SeNPs only increased the percentage of cells with a reduced global [Ca2+]i increase. At the same time, PCR analysis showed suppression of the baseline and OGD/R-induced expression of pro-apoptotic genes using 100 nm-sized SeNPs, which is explained by Ca2+-dependent activation of protective genes. In addition, during ischemia/reoxygenation, ER stress occurs and, as a result, improper folding of selenium-containing proteins and increased cell death, while SeNPs are able to significantly suppress ER stress, which may be another mechanism of their neuroprotective action [2+]i. Thus, it was shown that SELENOK can interact with palmitoyltransferase (DHHC6) and regulate Ca2+ ion fluxes in the ER through IP3R palmitoylation i, cell cultures were loaded with Fura-2 . The cells were stained with the probe dissolved in Hank’s balanced salt solution (HBSS) composed of (mM): 156 NaCl, 3 KCl, 2 MgSO4, 1.25 KH2PO4, 2 CaCl2, 10 glucose, and 10 HEPES, pH 7.4. To measure [Ca2+]i, we used the system based on an inverted motorized microscope Leica DMI6000B with a high-speed monochrome CCD-camera HAMAMATSU C9100. For excitation and registration of Fura-2 fluorescence, we used an FU-2 filter set  with excitation filters BP340/30 and BP387/15, beam splitter FT-410, and emission filter BP510/84. Illuminator Leica EL6000 with a high-pressure mercury lamp was used as a source of excitation light. To distinguish between neurons and astrocytes, we used short-term applications of 35 mM KCl and 10 µM ATP before the main experiments. This method was described in detail in our previous work i and activation of reactive astrogliosis. Whereas 50 nm-sized and 400 nm-sized SeNPs, while also exerting a protective effect, regulate the expression of a smaller number of protective proteins and markers of reactive astrogliosis, they do not eliminate the OGD-induced hyperexcitation of neurons, but rather limit the global increase in [Ca2+]i. One of the explanations for this difference in the effectiveness of the cytoprotective action of different-sized SeNPs is the presence of two endocytosis pathways for large SeNPs (100 and 400 nm) and the presence of some toxic effects in 50 nm-sized SeNPs.SeNPs with a diameter of 100 nm have a more pronounced cytoprotective effect on the cells of the cerebral cortex under conditions of ischemia/reoxygenation (OGD/R) through an increased baseline and an OGD/R-induced expression of genes encoding protective proteins, suppression of the OGD-induced global increase in [Ca


ETFA and ETFB led to increased mitochondrial activity, mitochondrial stress, and apoptosis in AML cells, but had little to no effect on normal human CD34+ cells. These studies identify a set of proteins that have not previously been associated with leukemia and may ultimately serve as potential targets for therapeutic manipulation to hinder AML progression and help contribute to our understanding of the disease.Acute myeloid leukemia (AML) is a devastating cancer affecting the hematopoietic system. Previous research has relied on RNA sequencing and microarray techniques to study the downstream effects of genomic alterations. While these studies have proven efficacious, they fail to capture the changes that occur at the proteomic level. To interrogate the effect of protein expression alterations in AML, we performed a quantitative mass spectrometry in parallel with RNAseq analysis using AML mouse models. These combined results identified 34 proteins whose expression was upregulated in AML tumors, but strikingly, were unaltered at the transcriptional level. Here we focus on mitochondrial electron transfer proteins ETFA and ETFB. Silencing of  Acute myeloid leukemia (AML) affects the hematopoietic system through abnormal proliferation of myeloid cells in the bone marrow (BM) and ultimately suppresses the production of healthy blood cells. AML is the leading cause of death among adult leukemia patients, although it represents only 35% of those diagnosed . DiseaseMLL1) gene, contains domains essential for transcriptional activity and chromatin modifications during early hematopoiesis, and fusion proteins generated from each translocation lead to aberrant transcription of MLL target genes . As. AsETFA val Fig. . A cohorvel Fig. . These fMLL::AF9 translocation whereas HL-60 cells do not contain common AML translocations [ETFA and ETFB in MOLM-13 cells did not alter mitochondrial localization or expression of TOM20   Fig. .Fig. 5InETFA and ETFB alters mitochondrial function. To evaluate mitochondrial metabolism, we performed Seahorse to determine mitochondrial respiration. We found that silencing of ETFA and ETFB in AML cell lines led to increased oxygen consumption rate (OCR), respiration, extracellular acidification rate (ECAR), ATP production, and spare respiratory capacity  . Depletion led to increased expression of Mac1 (CD11b), a marker of mature myeloid cells  Fig. . Of notells Fig. . MOLM-13ice Fig. . Mice trger Fig. . These sETFA or ETFB reduced the IC:50 to 4.1 nM and 4.7 nM, respectively, and lead to increased apoptosis  protein is highly expressed and plays a role in AML survival by binding pro-apoptotic proteins and inhibiting mitochondrial-induced apoptosis. Currently, venenoclax, a BCL-2 inhibitor has proven efficacious, however some patients develop resistance . To detesis Fig. . TogetheETFA and ETFB on human HSPC, we performed knockdown experiments in human CD34+ cells isolated from mobilized PB. Similarly to the AML cell lines, we utilized shRNAs targeting ETFA and ETFB, and cultured the cells in media containing cytokines to promote proliferation, but maintained the CD34+ population. We observed similar knockdown efficiency in CD34+ cells (Fig. ETFA and ETFB did not lead to differentiation, loss of colony-forming potential, or impacted proliferation (Fig. AML cells were sensitive to depletion of ETFA and ETFB; however, it is important to understand the impact on normal HSPC. In order to determine the effect of silencing lls Fig. . Howeverion Fig. . In addiion Fig. . TogetheETFA and ETFB leads to mitochondrial stress, differentiation, apoptosis, and increased sensitivity to venetoclax. These cellular processes combined most likely impede disease initiation and engraftment of the AML cells in NSG mice.Combining mass spectrometry and RNA-seq analyses in mouse models of AML, we were able to identify a sub-set of proteins that have limited or no alterations in transcript levels compared to their normal counterparts. These upregulated proteins play a role in RNA splicing and mitochondrial processes – two processes currently under investigation for therapeutic targeting and are relied on for AML survival , 45–47. Some of the additional differentially expressed proteins identified from this study correspond with regulators of splicing and spliceosome components. Recently, mutations in genes coding for components of the spliceosome have been found to correlate with certain hematopoietic malignancies, including AML , 53–57. ETFA and ETFB had minimal impact on survival, maintenance, and differentiation to the HSPC population, suggesting that targeting ETFA and ETFB would not lead to hematopoietic failure. Although further validation and examination is needed, these hits may ultimately serve as potential therapeutic targets in future studies relating to AML.Overall, the hits identified from our RNA-seq and MS analyses allow for a continued effort to investigate the roles of these proteins in AML and other hematopoietic malignancies and to understand how they may contribute to the dysregulation of cancer cells. Importantly, silencing of supplemental material


Numerous skin pathologies including dermatitis, anhidrotic ectodermal dysplasia, hyperhidrosis, hair loss and cancer are now linked to dysfunction in SOCE proteins. Principal amongst these are the stromal interaction molecules (STIMs) that sense Ca2+ depletion and Orai channels that mediate Ca2+ influx. In this review, the roles of STIM, Orai and other store-operated channels are discussed in the context of keratinocyte differentiation, melanogenesis, and eccrine sweat secretion. We explore not only STIM1-Orai1 as drivers of SOCE, but also independent actions of STIM, and emerging signal cascades stemming from their activities. Roles are discussed for the elusive transient receptor potential canonical channel (TRPC) complex in keratinocytes, Orai channels in Ca2+-cyclic AMP signal crosstalk in melanocytes, and Orai isoforms in eccrine sweat gland secretion.The skin is a complex organ that acts as a protective layer against the external environment. It protects the internal tissues from harmful agents, dehydration, ultraviolet radiation and physical injury as well as conferring thermoregulatory control, sensation, immunological surveillance and various biochemical functions. The diverse cell types that make up the skin include 1) keratinocytes, which form the bulk of the protective outer layer; 2) melanocytes, which protect the body from ultraviolet radiation by secreting the pigment melanin; and 3) cells that form the secretory appendages: eccrine and apocrine sweat glands, and the sebaceous gland. Emerging evidence suggests that store-operated Ca Each adopt a unique Ca2+ signalling toolkit to support their specific cellular functions. In recent decades, store-operated Ca2+ entry (SOCE), whereby depletion of intracellular Ca2+ stores triggers Ca2+ influx across the plasma membrane, has emerged as a critical process regulating differentiation, melanogenesis and sweat secretion. SOCE dysfunction in skin is linked to anhidrotic ectodermal dysplasia, hyperhidrosis, dermatitis, hair loss and cancer  channels  by the Ca2+-sensing receptor (CaSR), a promiscuous G-protein coupled receptor that couples to several pathways  and reduce keratin one expression when cells are switched to 1.8 mM Cao2+. Additionally, HaCaT cells lacking STIM1 or Orai1 exhibit diminished proliferative responses to changes in Cao2+ and PLCγ1  on nearby melanocytes to stimulate melanogenesis and proliferation through both Gαs signalling via cyclic AMP (cAMP) and Gβγ signalling via MAPK pathway activation , and to a lesser extent, adrenocorticotropic hormone (ACTH), both of which are produced by neighbouring keratinocytes by cleaving proopiomelanocortin (POMC) . αMSH activation . Point moduction . Thus, αpression . As suchadiation .2+-dependent signalling proteins has only recently been proposed for this signalling network. 2+ mobilisation, and Ca2+ release could be blocked by inhibitors of either AC or PLC, interlinking PLC activity and Ca2+ store depletion with the cAMP production caused by MC1R activation  which then activates MITF  Figure . The roltivation . It is utivation . STIM1 koduction . This reoduction . Intriguoduction . Taken ts signal .BR) to trigger a distinct pathway in response to UV exposure (BR stimulation activates PLC to generate diacylglycerol (DAG) and IP3-mediated Ca2+ store mobilisation, which concurrently stimulate the RAS/MAPK signalling pathway to drive MITF activation and melanogenesis  activates the melanocyte endothelin B receptor , followed by the activation of Cl− transport across the epithelium from the plasma to the glandular lumen  directly onto the skin surface, whilst the latter two secrete more viscous lipid- and protein-containing secretions (apocrine sweat and sebum respectively) into the hair follicular space . The pro− transport process have been characterised at the molecular level, but it is only recently that insights into the protein components involved in generating the Ca2+ signal have been reported. Cl− influx is driven by the basolateral Na+-K+-2Cl- co-transporter NKCC1 (− efflux via the Ca2+-activated Cl− channels (CaCCs) TMEM16A (Anoctamin-1) and bestrophin 2  and hyperthermia at high ambient temperatures due to an intrinsic incapacity for evaporative cooling . Further TMEM16A .2+]i response following stimulation with the muscarinic agonist carbachol was 2-fold that of cells isolated from non-hyperhidrotic individuals , showed that the [Caividuals . Expressxpressed . Thapsig2+ entry channels to control their routine physiological functions. Here, the roles of SOCE channels have been summarised for a selection of key skin functions: keratinocyte differentiation, melanocyte proliferation and melanogenesis, and sweat secretion from ‘clear’ cells in the eccrine sweat gland. This selection is by no means exhaustive, with cells from the immune and nervous systems resident in the integumentary system playing vital roles governed by their own unique Ca2+ entry systems. STIM1 and Orai1 have been revealed as key drivers of all the skin functions discussed here, both through their well-characterised SOCE process, the independent actions of STIM1, and the uncharacterised signal cascades stemming from their activities. Clearly, much is yet to be determined about the role of non-Orai store-operated Ca2+ channels such as the elusive TRPC complex in keratinocytes, the role of Orai channels in Ca2+-cAMP signal crosstalk in melanocytes, and the interaction of different Orai isoforms in driving eccrine sweat gland fluid secretion.Numerous skin cell types rely on Ca


Glycine max is a calcium-loving crop. The external application of calcium fertilizer is beneficial to the increase of soybean yield. Indeed, calcium is a vital nutrient in plant growth and development. As a core metal ion in signaling transduction, calcium content is maintained in dynamic balance under normal circumstances. Now, eight transporters were found to control the uptake and efflux of calcium. Though these calcium transporters have been identified through genome-wide analysis, only a few of them were functionally verified. Therefore, in this study, we summarized the current knowledge of soybean calcium transporters in structural features, expression characteristics, roles in stress response, and prospects. The above results will be helpful in understanding the function of cellular calcium transport and provide a theoretical basis for elevating soybean yield. In. In2+ tr and 7-8  56]. Ac. Ac2+ trCaCA have been reported to be regulated by diverse stimuli  is the first soybean CaCA gene isolated. RT-PCR assay verified that the expression of GmCAX5 is ubiquitous in different tissues and induced by PEG (polyethylene glycol), ABA (abscisic acid), and metal ion  treatments. Overexpression of GmCAX5 in Arabidopsis enhanced high CaCl2, NaCl, and LiCl tolerance at the germination stage, with lower Na+ and Li+ accumulation (2+ transport activity still needs further verification . Acc, and IVb2+ . The seq2+ . The con2+ .GmCNGC2/5/7/8/9/12/13/14/25/26/30/31/37/39 were widely expressed in detected tissues . In contrast, GmCNGC29 showed greater expression during seed development, GmCNGC20 displayed specific expression in flowers, GmCNGC33 was only detected in root nodules, and GmCNGC15/24/34 represented specific expression in roots  channel, and transports Ca2+ from vacuole to cytoplasm. TPC, CNGC, and GLR are the only three ion channels, which function as ligand receptors. Structurally, soybean TPCs contain 12 transmembrane domains, two ion transport domains, and two EF-hand domains (PF13499) (2+ fluctuations. EF-hand 2 (with conserved Asp-X3-Asp-X7-Glu) operates as a Ca2+ sensor and regulates the channel open in a voltage-dependent manner when cytosolic Ca2+ binds to this site [Voltage-gated ion channels contain three related topologies, including single voltage-domain channels, four-domain channels, and TPCs. TPC is a ubiquitously expressed channel protein with very few family members. Plant central vacuole is a huge CaPF13499) . SimilarPF13499) . The secPF13499) . HoweverPF13499) . Two EF-his site   didn’t affect AtTPC1 and MpTPCs. According to RNA-seq data, only GmTPC2 was ubiquitously expressed across soybean growth and development and induced by salt and drought stress  genes exist in all photosynthetic organisms and share highly similar amino acid sequences with mammalian ionotropic glutamate receptors (iGluRs) . Furthera patens , Arabidothaliana , Solanumpersicum , Oryza sa sativa , Glycinecine max , Pyrus bhneideri , Medicaguncatula , respectuncatula . It is we groups . As repoe groups . Accordi2+ in whole Arabidopsis seedlings [2+ concentration in rice roots and regulating stomatal movement [3, GdCl3, CNQX, and DNQX) were also active to plant GLRs. Further expression assay suggested that the active agonists and antagonists only affected Ca2+ flow, but did not alter the transcript of GLRs [SlGLR1.1 and SlGLR3.5 overexpressed Arabidopsis displayed similar morphological phenotype as Ca2+ deficiency  and hypersensitive to additional K+/Na+. However, Ca2+ supplementation rescued this sensitivity. These results may be due to the competitive absorption of K+/Na+ mediated by SlGLR1.1 and SlGLR3.5, resulting in Ca2+ deficiency [GLR3.5 and GLR3.7 were involved in Glu-induced stomatal closure. When plants suffered stresses, the content of plant signaling molecule Glu increased and then bound to GLR protein to promote Ca2+ influx, and subsequently, CPK  was activated and phosphorylated SLAC (Slow Anion Channel-Associated), finally leading to stomatal closure. These studies provide a theoretical basis to reveal the function of soybean GLR.Both direct biochemical and crystal structural analyses have shown a diversity of amino acid agonists  could increase intracellular Caeedlings . Howevermovement ,90. Anta of GLRs . SlGLR1.ficiency . In ArabGmGLR varied a lot under different conditions  ion channel is a way for cells to perceive external physical stimulation. Plant MS ion channels consist of five groups: hyperosmolality-gated calcium-permeable channel (OSCA), mid1-complementing activity (MCA), MscS -like (MSL), two-pore potassium (TPK), and piezo channel . Among tbidopsis ,93. Grad. sativa , Z. mays[Z. mays , and G.  [G. max ,94 prote [G. max . These rSaccharomyces cerevisiae and Homo sapiens OSCAs) from clade IV displayed conserved osmotically gated Ca2+ conductance in Chinese Hamster Ovary (CHO) cells and electrophysiological characteristics in Xenopus oocytes [2+ increase induced by stimulus [AtOSCA3.1 (early known as ERD4 (early response to dehydration) from clade III) knockout mutants displayed similar hyperosmolality-induced free calcium increase as wild type, indicating their different role from AtOSCA1.1 [AtOSCA1.3 is an immune receptor-associated cytosolic kinase BIK1-activated Ca2+-permeable channel, which controls stomatal closure during the immune signalling pathway. This activation relies on the recognition and phosphorylation of the Ser-X2-Leu motif in the N terminus of OSCA1.3 by BIK1 [ZmOSCA2.4 (clade II) overexpressed Arabidopsis exhibited enhanced drought resistance with high chlorophyll and proline content, increased drought tolerance-associated gene expression, and decreased senescence-associated gene expression [Characterization studies proved that AtOSCA1.2 from clade I, ScYLR241W, and HsCSC1  is also a member of plant MS ion channels . Similarproteins . Arabido2+ accumulation in single mutant mca2 and double mutant mca1mca2 than in WT and mca1 indicated the main role of AtMCA2 in plant root Ca2+ uptake. Additionally, the Ca2+ absorption is sensitive to ion channel inhibitors GdCl3 and LaCl3 [AtMCA1 and AtMCA2 are involved in cold-induced cytoplasm Ca2+ increase [mca1, mca2, and mca1mca2 displayed dramatically lower cold-induced cytoplasm Ca2+ increase. Mutants mca1mca2 exhibited chilling and freezing sensitivity. In addition, AtMCA1 and AtMCA2 overexpression led to hypersensitivity to increased gravity, suppressing the elongation growth at lower gravity levels [NtMCA1 and NtMCA2 could also rescue the Ca2+ uptake activity of yeast mutant mid1. Subcellular location and expression data analysis suggested their roles in Ca2+-dependent cell proliferation and mechanical stress-induced gene expression by regulating the Ca2+ influx [MCA gene [MCAs, OsMCA1 and ZmMCA  were located on the plasma membrane and could rescue the mid1 phenotype as well [OsMCA1 overexpression and suppression lines indicated that OsMCA1 was a positive regulator in Ca2+ uptake and NADPH oxidase-mediated ROS generation induced by hypo-osmotic stress in rice [mca mutant exhibited deficiency in cell number, size, and differentiation [The observation of lower Cand LaCl3 . Both Atincrease . Comparey levels . Similar+ influx . InteresMCA gene . The sam as well ,108,109.ntiation .MCAs have been identified in soybean with conserved C-terminal PLAC8 domain and only one transmembrane section, which were the same functional domains as reported in AtMCA1/2, NtMCA1/2, OsMCA1, and ZmMCA -D/E) and five short α-helices  and six calcium influx transporters  have been identified  as well. Therefore, further direct evidence is needed to verify their ion transport features. Technical advances enabled us to monitor intracellular calcium fluctuation in real-time, such as non-invasive micro-test technology (NMT), and patch clamp technique. What’s more, two photon-total internal refraction fluorescence (TIRF) microscopy and stimulated emission depletion (STED) microscopy can visualize Ca2+ changes within a single cell with the help of Ca2+ indicators . Also, we can generate direct mutation of key functional residues of these calcium transporters involved in the binding or transport selectivity by using the CRISPR/Cas system, which offers a quick and better way to investigate the calcium transport in soybean rather than testing their transport function in a heterologous system [Thanks to the release of soybean genome sequence data, two calcium efflux transporters (Caentified . They ars system ,131. StrThough the transcripts of some calcium transporters were found to be regulated by biotic and abiotic stresses, only GmCAX5 and GmACA1 have been functionally analyzed. Future, there is still a lot we need to investigate and verify. For example, whether these calcium transporters function by forming homo- or heterodimers, their relationship with transcription and protein phosphorylation in soybean, their roles in increasing soybean yields, and so on. Also, more calcium transporters are likely to be identified through genome studies. Currently, the research on soybean calcium transporters provides us with valuable genetic resources and new ideas to improve soybean output. Furthermore, we can apply CRISPR-Cas technology to design soybeans with high yield, high calcium content, and strong stress resistance, which will be of great practical significance to promote the increase of soybean yield ,131.