IL2RA mRNA expression appeared to be the best candidate as a prognostic biomarker. Therefore, the aim of this study is to characterize the prognostic value of IL2RA mRNA expression and evaluate its potential to refine prognostification in AML.Elevated protein expressions of CD markers such as IL2RA/CD25, CXCR4/CD184, CD34 and CD56 are associated with adverse prognosis in acute myeloid leukemia (AML). However, the prognostic value of mRNA expressions of these CD markers in AML remains unclear. Through our pilot evaluation, IL2RA mRNA expression were measured by TaqMan realtime quantitative PCR. Morphological, cytogenetics and mutational analyses were also performed. In an intermediate-risk AML cohort with 66 patients, the mRNA expression of prognostic biomarkers  were quantified by NanoString technology. A TCGA cohort was analyzed to validate the prognostic value of IL2RA. For statistical analysis, Mann–Whitney U test, Fisher exact test, logistic regression, Kaplan–Meier and Cox regression analyses were used.In a cohort of 239 newly diagnosed AML patients, IL2RA mRNA expression independently predicted shorter relapse free survival  and overall survival  irrespective of age, cytogenetics, FLT3-ITD or c-KIT D816V mutational status. In core binding factor (CBF) AML, high IL2RA mRNA expression correlated with FLT3-ITD status (p = 0.023). Multivariable analyses revealed that high IL2RA expression (p = 0.002), along with c-KIT D816V status (p = 0.013) significantly predicted shorter RFS, whereas only high IL2RA mRNA expression (p = 0.014) significantly predicted shorter OS in CBF AML. In intermediate-risk AML in which multiple gene expression markers were tested by NanoString, IL2RA significantly correlated with ID1 (p = 0.006), FLT3 (p = 0.007), CDKN1B (p = 0.033) and ERG (p = 0.030) expressions. IL2RA (p < 0.001) and FLT3 (p = 0.008) expressions remained significant in predicting shorter RFS, whereas ERG (p = 0.008) and IL2RA (p = 0.044) remained significant in predicting shorter OS. Similar analyses in TCGA intermediate-risk AML showed the independent prognostic role of IL2RA in predicting event free survival (p < 0.001) and OS (p < 0.001).In AML cohort of 239 patients, high IL2RA mRNA expression is an independent and adverse prognostic factor in AML and specifically stratifies patients to worse prognosis in both CBF and intermediate-risk AML.High The online version of this article (10.1186/s12967-019-1926-z) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) is a well-known heterogeneous hematological malignancy with a broad range of prognosis, which is greatly impacted by clinical factors, cytogenetics and molecular characteristics . In the interleukin 2 receptor subunit alpha (IL2RA/CD25) ,,interleu4) [CD34 , 12 and 34 [CD56 , 14 haven system –21. RNA CD34 mRNA and protein expressions were not consistent [By far, the prognostic values of mRNA expression of most CD markers in AML remain elusive. To our knowledge, the only prognostic CD marker, of which the mRNA expression prognostic value has been indicated, is CD34 , 23. Hownsistent –26. In gnsistent , 28 whicnsistent –29. In aIL2RA, CXCR4, CD34 and CD56), analyzed the prognostic values of their mRNA expressions in the TCGA-LAML cohort and determined IL2RA as best candidate gene for further study in larger cohort. Subsequently, in our clinical cohorts, we aimed to systemically evaluate the prognostic value of IL2RA mRNA expression in AML in the context of clinical and laboratory factors with prognostic relevance. We further characterized its prognostic role in core binding factor (CBF) AML in the context of established prognostic factors, as well as in intermediate-risk AML in the context of other mRNA expression prognostic factors . Our study not only consistently reveals independent prognostic value of mRNA expression of IL2RA in AML, particularly in CBF and intermediate-risk AML, but also serves as a proof-of-concept study for future research endeavors to investigate prognostic roles of mRNA expression of other CD biomarkers.In our study, we initially sought to investigate the prognostic value of mRNA expressions of various CD biomarkers in AML and study if they can add independent prognostic value to the established prognostic factors. We conducted a pilot study to evaluate correlations of mRNA/protein expressions of four prognostic CD marker genes (IL2RA using BM samples from 239 adult patients (age range: 15–65) diagnosed with AML between 2012 and 2016 at Institute of Hematology, Wuhan Union Hospital. The patients received intensive induction chemotherapy and consolidation chemotherapy or HCT [IL2RA together with eight other known prognostic genes simultaneously in a multigene panel testing platform by NanoString using BM samples from 66 adult patients diagnosed with intermediate-risk AML. The diagnosis of AML was made according to World Health Organization classification [We analyzed mRNA expression of fication  and FrenConventional cytogenetic analysis was performed on G-banded preparations from 48-h bone marrow cell cultures. The chromosomal aberrations were described according to the International System for Cytogenetic Nomenclature (ISCN) 2009 .Flow cytometry analysis on fresh bone marrow samples were carried out at the time of diagnosis. In addition to monoclonal antibodies (Abs) and isotype control IgGs used for diagnostic immunophenotyping , antibodFMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) [nucleophosmin 1 (NPM1) [CEBPA [c-KIT D816V [Genomic DNA was extracted from diagnostic marrow specimens BM samples using standard method and LT3-ITD) , nucleop1 (NPM1) , CEBPA [) [CEBPA  and c-KIIT D816V  mutationIL2RA gene expression was measured by TaqMan assay Hs00907779_m1 and the CXCR4 gene expression was measured by TaqMan assay as previously described [ABL1 gene was selected as the reference gene to compensate for the variations in mRNA and cDNA, and the ABL1 TaqMan assay was described in previous study [IL2RA, CXCR4 and ABL1 were determined by replicates and the mean CT was used for calculation. The cycle number difference was calculated (ΔCt = Ct target gene − CtABL1) and the relative expression values of target gene were expressed as 2−ΔCT. The PCR efficiencies of IL2RA and ABL1 assays have been evaluated by standard curves. The standard curves for ABL1 assays were generated by tenfold dilution series of four different plasmid concentrations  and the standard curves for IL2RA assays were generated by tenfold dilution series of positive RNA samples . The PCR efficiencies calculated for either ABL1 or IL2RA assays varied between 95.0 and 105.0%. Negative controls and interassay positive controls have been included in the PCR assays. The data were analyzed using the Applied Biosystems 7500 software v2.0.5.BM samples RNA extraction were performed as previously described . Reverseescribed . ABL1 geus study . The comus study . The thrABL1 were designed according to principles described before [ABL1 were used to further normalize the gene counts. For each NanoString assay, about 250 ng sample RNA was used for each reaction. The experimental procedures were carried out on the NanoString Preparation Station and Digital Analyzer according to manufacturer’s instructions [The probes sequences for the prognostic transcripts including the reference gene d before . Six posructions , 41. TheLevel 3 RNA-seq gene expression data  of 151 AML cases were retrieved from the TCGA data portal and clinical data from the cohort were retrieved from Additional file Mann–Whitney U test or Kruskal–Wallis test was used for comparison of continuous variable between different subsets of patients. Fisher exact or Chi-square tests were used for comparison of categorical variables between different subsets of patients. The bivariate Spearman correlation test was used to measure associations among continuous variables. Normalized NanoString counts were log2 transformed before the correlation analysis. Complete remission (CR), RFS and OS were defined as previous described . UnivariIL2RA, CXCR4, CD56 and CD34). The IL2RA mRNA and its protein CD25 levels were assayed in 54 primary AML BM samples by TaqMan RQ-PCR and FCM respectively, in which the mRNA level was shown as 2−∆Ct of IL2RA gene relative to ABL1 gene and the expression level of CD25 protein was illustrated as percentage in leukemia blast cells. A significant association was found between mRNA expression of IL2RA gene and protein expression of CD25 . Among these CD markers, only the mRNA expression of 3. Among IL2RA mRNA expression using BM RNA samples by TaqMan RQ-PCR. The characteristics of patients including clinical, cytogenetics and molecular features in this cohort were described and their correlation with IL2RA mRNA expression level were analyzed and summarized in Table IL2RA mRNA expression were observed in AML with certain molecular abnormalities  mutation  . Based on the results, we set IL2RA mRNA expression cutoff value at 80 percentiles ;FUS-ERG  shorter RFS (p < 0.001) and shorter OS  and cytogenetics risk status  remained significant in predicting CR status, high IL2RA expression , cytogenetics risk status  and c-KIT D816V  remained significant in predicting shorter RFS and high IL2RA expression , cytogenetics risk group  and age  remained significant in predicting shorter OS  cases with high IL2RA expression in intermediate-risk AML and 9 out of 37 (24.3%) cases in poor-risk AML subgroup. Although high IL2RA expression cases appeared to be more frequent in AML of poor risk than in AML of favourable- or intermediate-risk, the difference was not significant ;RUNX1-RUNX1T1 or inv(16);CBFB-MYH11 cytogenetics, since APL were not included. In the AML cohort, there were 9 out of 54 (16.7%) cases with high ;RUNX1-RUIL2RA mRNA expression status was significantly associated with age (p = 0.026) and FLT3-ITD (p = 0.023) status  and shorter OS , along with c-KIT D816V  remained as significant in predicting shorter RFS and high IL2RA mRNA level  alone remained significant in predicting shorter OS in this particular subtype of AML  and shorter OS  as well as IL2RA in an intermediate-risk AML cohort 66 patients. The clinical characteristics of these patients were described in Additional file IL2RA were found to significantly associate with expression of four other prognostic genes including ID1 , ERG  and CDKN1B   and FLT3  mRNA expressions remained significant as predictors for shorter RFS, whereas high ERG  and IL2RA  mRNA expressions remained significant for shorter OS  of TCGA-LAML cohort, we also examined the prognosis value of the nine prognostic genes by uni- and multivariable analyses. In this independent study, high AML1/ETO or with t;PML/RARA [IL2RA gene, among a selected list of CD marker genes, is a significant and independent prognostic biomarker in AML, in particular, in CBF and intermediate-risk AML subtypes.The prognostic value of CD biomarkers has been increasingly recognized in the AML research community over the last decade to help improve the current standard prognostic tools in AML clinical practice , 5, 14. PML/RARA . While tIL2RA mRNA expression in 239 AML excluding APL and showed that IL2RA mRNA is differentially expressed in groups classified by FLT3-ITD, CEBPADM or NPM1+FLT3-ITD− status, the recurrent genetic mutations that are prognostic relevant. Through a minimal p-value approach, we determined the optimal cutoff value for IL2RA mRNA expression that facilitate its use as a prognostic tool. We found that high IL2RA mRNA level is positively or negatively correlated with recurrent mutational and cytogenetic aberrations including FLT3-ITD, NPM1+FLT3-ITD−, and t;FUS/ERG, which was consistent with previous reports on CD25 [IL2RA mRNA expression was correlated with lower CR rate, shorter RFS and OS in AML and the effect is independent on other prognostic factors such as age, cytogenetic, FLT3-ITD and c-KIT D816V status. Therefore, we established the significant and independent role of high IL2RA mRNA expression as an adverse prognostic factor in AML.Our study quantified IL2RA could provided additional prognostic information to cytogenetics in AML, we further studied its prognostic value in AML of distinct cytogenetics risk statuses. CBF AML is a group of AML defined by carrying transcripts t;AML1/ETO or inv(16);CBF/MYH11. Although CBF AML belongs to the favorable-risk group, it still demonstrated considerable clinical, pathophysiological and molecular heterogeneity. In CBF AML, around 85% cases achieve CR after induction therapy, about 40–50% cases relapse after CR and around 50% cases remain alive at 5 years [IL2RA cases was also low in APL  was not significantly different from that in intermediate-risk and poor-risk AML. Further survival analyses within this subgroup of patients demonstrated strong association between high IL2RA expression and shorter RFS and OS, and it is the first time that elevated expression of CD25 biomarker gene are indicated in predicting adverse outcome in CBF AML.While  5 years . In prev 5 years . Althougipts t8;2;AML1/ETOc-KIT, N/KRAS, and FLT3) have been shown as the most frequent additional mutations in CBF AML that confer worse prognosis [c-KIT D816V has been indicated to predict poor prognosis, particularly in AML with t [IL2RA expression and c-KIT D816V status (p = 1.000). Further uni- and multivariable survival analysis indicateded that high IL2RA expression and c-KIT D816V mutation remained as two independent prognostic factors to predict shorter RFS, whereas high IL2RA expression alone remained significant in predicting shorter OS in CBF AML. Moreover, high IL2RA expression significantly correlated with FLT3-ITD mutation in CBF AML (p = 0.023), similar as in intermediate-risk AML. However, it should be noted that since FLT3-ITD mutation was present at a much lower frequency in CBF-AML (4.7% in our study and 7% in literature) than in intermediate-risk AML [IL2RA expression and FLT3-ITD mutation should be rather limited. It remained to be studied if the prognostic significance of high IL2RA mRNA expression in CBF AML is associated with gene mutations involved in activating tyrosine kinase signaling other than FLT3-ITD or c-KIT D816V mutation, or with other mechanisms. Nevertheless, our results suggested the significant potential of high IL2RA mRNA, in coordination with c-KIT D816V mutation, to further refine prognostification in CBF AML and warrant future validation in larger cohort of CBF-AML.Mutations in genes activating tyrosine kinase signaling  , 52. Corrisk AML , the intIL2RA or CD25 in intermediate-AML has been consistently shown in our study and previous studies [IL2RA prognostic value is associated with or dependent on other gene expression prognostic biomarkers which has been well studied in intermediate-risk or normal karyotype AML. In our intermediate-risk AML cohort, a panel of prognostic gene expression markers including BAALC [CDKN1B [ERG [FLT3 [ID1 [IL2RA, MN1 [MECOM/EVI1 [WT1 [IL2RA expression with other mRNA biomarkers were shown, IL2RA and FLT3 mRNA expressions remained significant in predicting shorter RFS, whereas ERG and IL2RA mRNA expressions remained significant in predicting shorter OS by uni- and multivariable analyses. Independent analysis on the same panel of genes in intermediate-risk AML cases in TCGA-LAML cohort consistently demonstrated the significant and independent value of IL2RA mRNA expression in predicting inferior clinic outcome. So far, various mRNA expression gene-panels have been designed for better stratification in AML [IL2RA gene into such multi-gene panel to improve prognostification within intermediate-risk AML.The prognostic value of  studies . Howeverng BAALC , CDKN1B  [CDKN1B , ERG [15N1B [ERG , FLT3 [5RG [FLT3 , ID1 [17LT3 [ID1 , IL2RA, 2RA, MN1 , MECOM/ECOM/EVI1 , and WT1VI1 [WT1 , 55 weren in AML , 47 and IL2RA is prognostic in AML can be implicated by its correlation results with other mRNA expression biomarkers. It is first shown by our study that upregulation of IL2RA was correlated with upregulation of FLT3 tyrosine kinase transcripts [ID1, a key common target of oncogenic tyrosine kinases that contribute to transformation of leukemias [IL2RA expression with tyrosine kinases pathways. Our study also showed significant correlation of IL2RA with ERG and CDKN1B, the expressions of which have been indicated in stem cell-like featured gene signatures [IL2RA/CD25 is involved in the crosstalk of LSC related signalling and support that IL2RA is an indicator of the LSC signature which has been shown as a fundamental adverse prognostic feature in AML [IL2RA mRNA expression such as IL2RA expression regulatory T cells [The mechanisms by which nscripts  and of teukemias , indicatgnatures , 58. Pregnatures , 5, our e in AML . In addi T cells  or IL2/I T cells , which wIL2RA mRNA expression in AML, particularly in CBF and intermediate-risk AML, supporting the application of high IL2RA mRNA expression as a prognostic tool to improve current stratification strategies in AML.In conclusion, our findings established the significant and independent prognostic value of high Additional file 1. Additional tables and figures.


Given the intimate link to both life and death processes, it is imperative that mitochondria tightly regulate intramitochondrial Ca2+ levels with a high degree of precision. Among the Ca2+ handling tools of mitochondria, the leucine zipper EF-hand containing transmembrane protein-1 (LETM1) is a transporter protein localized to the inner mitochondrial membrane shown to constitute a Ca2+/H+ exchanger activity. The significance of LETM1 to mitochondrial Ca2+ regulation is evident from Wolf-Hirschhorn syndrome patients that harbor a haplodeficiency in LETM1 expression, leading to dysfunctional mitochondrial Ca2+ handling and from numerous types of cancer cells that show an upregulation of LETM1 expression. Despite the significance of LETM1 to cell physiology and pathophysiology, the molecular mechanisms of LETM1 function remain poorly defined. In this review, we aim to provide an overview of the current understanding of LETM1 structure and function and pinpoint the knowledge gaps that need to be filled in order to unravel the underlying mechanistic basis for LETM1 function.Mitochondrial calcium (Ca Eukaryotic cells have evolved to use Ca2+ not only as a primary messenger when binding and activating or inactivating enzymes and proteins, but also as a secondary messenger involved in the signal transduction of various pathways. Thus, Ca2+ signaling drives a spectrum of cellular events from cell growth and proliferation to cell cycle arrest and apoptosis . . 102]. TMDM38Δ ,104. AddWHS is a genetic disease most commonly arising from single copy de novo deletion of a portion of the short arm of chromosome 4 prior to birth. WHS is characterized by seizures, impaired cognitive function, congenital heart defects, facial abnormalities including a flat nose and enlarged forehead resulting in the so-called Greek warrior helmet appearance, and delayed growth and development ,66. PatiLETM1, MSX1, a homeobox gene involved in the development of mouth and teeth structures, and WHSC1, a histone H3 lysine methyltransferase that interacts with β-catenin [2+ transport similar to cell cultures with LETM1 knockdown. Collectively, these studies indicate that LETM1 activity may be an important determinant in WHS, where a loss of LETM1-mediated CHE activity in neuronal mitochondria could lead to dysregulated Ca2+ homeostasis and signaling in individuals harboring the deletion in chromosome 4 [A region spanning ~165 kilobases in chromosome 4 represents the region deleted in WHS patients, comprising -catenin ,67,101. -catenin . Additiomosome 4 ,60,66.2+ binding loop of human LETM1. Mechanistically, the substitution of acidic Glu for the basic Lys may affect the electrostatic guidance of Ca2+ into the EF-hand Ca2+ binding loop. Ultimately, a high resolution structure for LETM1 is required to unambiguously reveal the mechanisms whereby these 183 mutations may contribute to LETM1 dysfunction and various cancers.LETM1 has been found to be overexpressed in breast, colon, esophagus, lung, ovarian, rectal, stomach, bladder and uterine cervical cancers . This fi2+ uptake not only influences cytosolic Ca2+ signals involved in myriad cellular processes, but also affects numerous mitochondrial processes integral to both cell survival and cell death. Several studies have shown LETM1 mediates a CHE activity in the mitochondria which is dependent on pH, thus indicating that LETM1 is integral to cytosolic and mitochondrial Ca2+ signaling. However, whether LETM1 directly or indirectly mediates CHE or KHE activity remains an important question in the mitochondrial biology field requiring more research. Associated with this question, LETM1 electrogenicity is incompletely understood. Regardless of the specific type of ion exchanger activity, LETM1 is undoubtedly vital for ion homeostasis within the mitochondrial matrix. Mitochondrial Ca2+ binding EF-hand play in LETM1 function? Does human LETM1 contain a second EF-hand motif, as in hundreds of other EF-hand proteins in nature, and what is the mechanistic role of this second EF-hand? How do post-translational modifications of LETM1 such as acetylation affect the structural mechanisms of exchanger function? How do point mutations in LETM1 associated with cancer affect the structure and function of LETM1? What are the structural mechanisms of LETM1 inhibition by RR, Ru-360 and CPG-37157, if any?High resolution structural information on LETM1 represents a major knowledge gap in the field and may unravel the precise ion exchange activity of LETM1. While a high-resolution structure of the assembled and functional LETM1 oligomer represents the idyllic goal, there are numerous specific structural questions which may be more simply obtainable and represent important structural targets: What mechanistic role does the canonical Ca2+ signaled malignancies. Collectively, the answers to these structural questions may provide insights into precisely how LETM1 functional deficiency contributes or leads to WHS. Importantly, LETM1 structural biology research will undoubtedly guide new therapeutic strategies, diagnostic tests and research tools which will help detect and treat WHS as well as other Ca


FUS‐ERG has a poor prognosis regardless of allo‐hematopoietic stem cell transplantation (HSCT). Maintenance therapy such as azacitidine after allo‐HSCT for AML with FUS‐ERG may be clinically meaningful.Acute myeloid leukemia (AML) with  FUS‐ERG has a poor prognosis regardless of allo‐hematopoietic stem cell transplantation (HSCT). Maintenance therapy such as azacitidine after allo‐HSCT for AML with FUS‐ERG may be clinically meaningful.Acute myeloid leukemia (AML) with  Our patient was able to achieve 2nd CR by AZA for approximately 8 months. We suggested that AZA may be a salvage therapy for AML with FUS‐ERG.We report a case of pediatric relapse AML with FUS‐ERG fusion transcript.FUS‐ERG is estimated to be 1% of all de novo and secondary cases of AML.FUS‐ERG is reportedly associated with a poor prognosis and high rate of relapse.The t16;21) is a nonrandom chromosomal translocation that occurs in acute myeloid leukemia (AML), myelodysplastic syndrome that evolves into AML, blast crisis of chronic myelogenous leukemia, and rare cases of Ewing's tumors.6;21 as treatment for achieving the first complete remission (CR).AML with FUS‐ERG and experienced early relapse after the first CR with allo‐HSCT.Here, we describe the clinical course of a patient who received salvage therapy with azacitidine (AZA) for pediatric AML with t/24/µL. Lactate dehydrogenase and transaminase levels were 2371 IU/L and within normal range, respectively. The bone marrow (BM) examination revealed hypercellular BM. The nucleated cell count was 26.4 × 104/μL with a blast cell count of 71.8%  in BM was 1,608,400 copies/μg RNA. The diagnosis of AML with t/FUS‐ERG and M5a according to the French‐American‐British classification was made based on the BM examination.A previously healthy 10‐year‐old boy was admitted to our hospital because of a fever. The physical examination showed petechial hemorrhage of the precordia and leg, but it did not indicate hepatosplenomegaly or lymphadenopathy. Results of the laboratory examination indicated leucocytosis, anemia, and thrombocytopenia. The following findings were observed: white blood cell count, 127,000/µL; neutrophil count, 2.0%; blast cell count, 94.5%; hemoglobin level, 9.3 g/dL; and platelet count, 1.7 × 102/d on days 2‐4, fludarabine 30 mg/m2/d on days 2‐6, cytarabine 2 g/m2/d on days 2‐6) that included gemtuzumab ozogamicin (3 mg/m2/d on day 7) led to CR, which was followed by peripheral blood stem cell transplantation (PBSCT) from a human leukocyte antigen (HLA)‐matched sibling donor at 11 years of age. Sibling donor was sister. The conditioning regimen consisted of melphalan (L‐PAM) (90 mg/m2/d daily for 2 days) and buslfan (4 mg/kg/d daily for 4 days). Graft‐versus‐host disease (GVHD) prophylaxis was performed with short‐course methotrexate (MTX) and cyclosporine. BM engraftment was achieved 12 days after PBSCT, but veno‐occlusive disease (VOD) and infective endocarditis developed 20 and 68 days after PBSCT, respectively. Although GVHD prophylaxis had been discontinued because of VOD, he did not have acute GVHD.According to the AML‐05 protocol of the JPLSG,WT1‐mRNA . He was diagnosed as having a cytogenetic relapse after PBSCT. At 156 days, AZA was administered .WT1‐mRNA in peripheral blood was significantly reduced to 140 copies/μg RNA after three courses of AZA. For approximately 8 months, eight courses of AZA were administered, but he was diagnosed as having relapse in the BM and central nervous system (CNS) a second time. The hematotoxicity of AZA was neutropenia of Grades III‐IV and febrile neutropenia.At 145 days, the BM examination revealed the presence of blast cells (3%), 46,XY,del(6)(q21),t,der(17)t[1]/46,XX19] in the chromosome banding, and an increased expression of  in the c2/d for 4 days), cytarabine (2 g/m2/d for 4 days), L‐PAM (60 mg/m2/d for 3 days), and low‐dose total body irradiation (4 Gy) with HLA‐matched cord blood transplantation (CBT). GVHD prophylaxis was performed with short‐course methotrexate (MTX) and tacrolimus. Thrombotic microangiopathy developed 9 days after CBT, and primary graft failure (GF) was diagnosed 20 days after CBT. Therefore, he underwent PBSCT with an HLA‐matched sibling donor without conditioning regimen 20 days after CBT because of GF.Although low‐dose Ara‐C and aclarubicin with concomitant use of a granulocyte colony‐stimulating factor regimen and triple intrathecal therapy (TIT) consisted of cytarabine (30 mg), methotrexate (12 mg), and hydrocortisone (25 mg) were administered, a third CR was not achieved. TIT was performed a total of 7×, and cerebrospinal fluid test revealed that the leukemia cells were negative. He underwent cranial irradiation (14 Gy) and subsequently received a reduced‐intensity conditioning regimen consisting of fludarabine  response.Low‐dose AZA maintenance therapy after allo‐HSCT has been reported in patients with AML and myelodysplastic syndrome.ERG is linked with an unfavorable prognosis in a subgroup of leukemic patients with AML and acute T‐lymphoblastic leukemia.DNMT3A, ASXL1, BCOR) by targeted NGS approach in AML with FUS‐ERG cases.ERG‐positive prostate cancer cells have been reported to induce epigenetic activation of Tudor domain‐containing protein 1, and histone deacetylase inhibitors induce apoptosis and affect the acetylation status of p53.It has been reported that a high expression of FUS‐ERG that early relapse occurred after the first CR with allo‐HSCT. The patient was able to maintain CR a second time for approximately 8 months. Although AZA was administered after relapse in our patient, we expected an epigenetic and GVL effect. Our patient may have showed CR because we administered AZA when there were few leukemic cells such as the cytogenetic relapse. Platzbecker et al reported that pre‐emptive therapy with AZA can prevent or substantially delay hematological relapse in measurable residual disease (MRD)‐positive patients with MDS or AML who are at high risk of relapse.FUS‐ERG.We administered AZA for pediatric AML with He developed recurrence in the BM and CNS during AZA therapy. Thus, concurrent treatment for recurrence in the BM and CNS was necessary, and we suggested the use of the TIT during administration of AZA.FUS‐ERG is associated with a poor prognosis, and achieving CR with allo‐HSCT is difficult. A novel therapeutic approach is needed for this clinical condition, and we suggest epigenetic therapy such as AZA as maintenance therapy after allo‐HSCT for AML with FUS‐ERG.In conclusion, expression of the CD56 antigen in AML with All authors declare no conflicts of interest relevant to this article.Dai Keino: conceptualized and designed the study, drafted the initial manuscript, and approved the final manuscript. Takashi Mori, Mizuho Morimoto, Kensuke Kondo, and Tetsuya Mori: performed the initial analyses, and reviewed and revised the manuscript. Akitoshi Kinoshita: designed the data collection instruments, coordinated and supervised data collection, and critically reviewed the manuscript. All authors approved the final manuscript and agreed to be accountable for all aspects of the work.


Women’s self-help groups (SHGs) have increasingly been used as a vehicle for social, political, and economic empowerment as well as a platform for service delivery. Although a growing body of literature shows evidence of positive impacts of SHGs on various measures of empowerment, our understanding of ways in which SHGs improve awareness and use of public services is limited. To fill this knowledge gap, this paper first examines how SHG membership is associated with political participation, awareness, and use of government entitlement schemes. It further examines the effect of SHG membership on various measures of social networks and mobility. Using data collected in 2015 across five Indian states and matching methods to correct for endogeneity of SHG membership, we find that SHG members are more politically engaged. We also find that SHG members are not only more likely to know of certain public entitlements than non-members, they are significantly more likely to avail of a greater number of public entitlement schemes. Additionally, SHG members have wider social networks and greater mobility as compared to non-members. Our results suggest that SHGs have the potential to increase their members’ ability to hold public entities accountable and demand what is rightfully theirs. An important insight, however, is that the SHGs themselves cannot be expected to increase knowledge of public entitlement schemes in absence of a deliberate effort to do so by an external agency. Although they can be found in various forms all over the world , self-heinformation about the schemes among potential beneficiaries to take advantage of the schemes and the ability of potential beneficiaries to hold the public entities accountable. Insights from related work . Women that are part of SHGs, because of regular interactions not only with group members but also with external agents that facilitate these groups, may become more adept at communicating. Often, this experience of being in a group and interacting with other women can in itself boost women’s self-confidence. Family members (mostly husbands) of women in SHGs may also feel more confident about the ability of these women to leave the homestead and engage with the outside world, perhaps even going outside the village (with their group).gram sabha and the mahila gram sabha, described in The ability to hold public entities accountable, the second demand side constraint identified above, is more complex. One can view this as a culmination of factors - improved social networks, greater mobility and greater self-confidence – that may lead to greater political participation, which in turn may lead to greater accountability. Women in SHGs meet regularly for their group meetings, which exposes them to the practice of meeting in groups and may make them more likely to attend village council meetings  more frequently because of the intervention, an SHG-delivered micro-credit program, and that the program contributed to an estimated increase of trust in other villages, elected representatives, or government representatives of between 5% and 15% points.  gram sabha. In addition, we study the awareness and utilization of a range of government entitlement schemes targeted at households, and at women and children. Though the studies discussed above did not look explicitly at awareness and use of government entitlement schemes, it is likely that the same mechanisms that increase women’s political empowerment could also operate to increase their knowledge of their entitlements and their claim on the benefits due to them. For example, by disseminating information about local institutions, governmental programs, policies, and procedures, SHGs may lower the cost of accessing information about community issues  health and nutrition.In this paper, we focus on a subset of these outcomes, notably political participation, improved awareness and utilization of government entitlement schemes, and some mechanisms – mobility, social networks – that could potentially help explain those outcomes. Schemes targeted at households are sometimes restricted to those households with a BPL (Below Poverty Line) card, as with the financial assistance for the construction of houses (the Indira Awas Yojana). Other schemes like the Public Distribution Scheme have different entitlements of foodgrains for households of different degrees of poverty. Finally, workfare schemes like the Mahatma Gandhi National Rural Employment Guarantee Act (MGNREGA) are self-targeting, and are not restricted to any particular income group.Among the schemes aimed at pregnant and lactating mothers and young children, the Integrated Child Development Scheme (ICDS) is the oldest, dating back to 1975. This scheme provides supplementary nutrition to mothers and children through local ICDS centers. In addition, the ICDS performs the role of a crèche, providing pre-school education to children aged 3–6 years. The Janani Suraksha Yojana (JSY), introduced in 2005, is a scheme aimed at improving childbirth in an institutional setting. Mothers and frontline health and ICDS workers are provided financial incentives to deliver the child in a health institution, with the amounts varying both within and across states. Finally, the Janani-Shishu Suraksha Karyakram (JSSK) ensures free-of-cost medical care to pregnant women and newborn children.33.1This study draws on data from a baseline survey conducted from September to December 2015 in eight districts of five states of eastern and central India – Madhya Pradesh, Orissa, Chhattisgarh, Jharkhand and West Bengal. The baseline survey forms the first wave of an impact evaluation of nutrition-intensification efforts being made by an Indian NGO, PRADAN. Three blocks were selected in each district of the study, making a total of 24 blocks. From each of the blocks between five and seven villages were chosen at random from the full list of villages, and from each village 20 women were selected at random from among all ever-married women aged 15–49. The final sample size at baseline was 2744 women. Sample selection was not conditioned on SHG membership, and at baseline approximately 38% of our sample belonged to an SHG. The low level of saturation allows us to compare outcomes across women who belong to an SHG and those who do not.Women’s SHGs in our area of study could be formed by PRADAN, by other NGOs, or by the government under NRLM. Unfortunately, we do not have information in our baseline survey on which organizations, governmental or otherwise, form and support these SHGs; in most cases, our respondents were not able to identify which organization supported the SHG to which they belonged.3.23.2.1SHG members are, on average, about 2.6 years older than non-members and have been married for about 2.9 years longer . The meaThere appear to be some significant differences between SHG members and non-members in terms of wealth and asset ownership. A principal components analysis of wealth that captures home, animal and mobile phone ownership, along with dwelling characteristics, availability of electricity and food security, indicates that SHG members are better-off than non-members, and that a smaller proportion of SHG members fall in the poorest wealth quintile compared to non-SHG members (16.4% versus 22.3%). SHG members are also likely to own a greater mix of assets  compared to non-SHG members, and on average, more likely to report that their household owns more land and livestock than the average in their district. It is hard to establish the causality between SHG membership and wealth. SHG members could be better-off because of their participation in the group, but conversely, better-off women may be more likely to participate in SHGs, because they have the time or adequate resources for regular savings, for example . Since women’s participation (including attendance) was observed to be low in the regular village meetings, which tend to be male-dominated spaces ), these ‘women-only’ gram sabhas were established to provide women with an opportunity to voice their grievances. In the overall sample, 87.4 percent of the respondent women had a voter ID and 86.7 percent of them voted in the last election (mahila gram sabha (adult women’s village meeting) or the gram sabha.9We measure the extent of political participation of the women in our sample by their previous voting behavior and attendance of programs . In addielection . These nmahila gram sabha compared to 4.5 percent of non-members. Similarly, 12.4 percent of SHG members ever participated in the gram sabha, significantly higher than the 3.2 percent of non-members. The political participation score is higher among SHG members by about 10 percentage points and this difference is statistically significant.We find that SHG members are in general more politically active than non-members. Almost 94% of SHG members voted in the last election, as compared to only 82.5% of non-SHG members (unadjusted p < 0.01), and a significantly higher proportion of SHG members, reportedly, made the decision to vote on their own.3.2.3Awareness of entitlement schemes varies in our sample . About 7Although several schemes have been in place for decades, overall utilization of entitlement programs is very low . Only 343.2.4Each respondent was asked several questions about a random sub-sample of 5 women selected from the sample of 20 women in the same village. The respondent was asked whether she knew each of these women, and if yes, whether they were members of the same group, whether she had spoken to or exchanged information with them, and whether she would leave her child with them in case of an emergency.Finally, regarding mobility and confidence, SHG members felt slightly more comfortable speaking in public, on average, compared to non-members, and were also less likely to require permission to go to a village meeting or meeting of an association.4This paper aims to examine the effect of SHG membership on the outcomes of interest. Although one could assess impact by comparing mean outcomes for women who are SHG members to non-members, this approach does not recognize that women who are SHG members are likely to be systematically different from nonmembers. As seen in To eliminate this bias, we must construct a comparison group from among non-members that were similar to SHG members before the SHGs were introduced. The preferred approach to constructing such a comparison group is to randomly provide access to the program among similarly eligible individuals. But because the introduction of such SHGs was not randomly assigned across villages in our sample, this method was not feasible. The absence of “hard” targeting criteria , and for occupation . Characteristics of the marriage include the woman’s marital status, the number of years she has been married (if married), and dummy variables for the presence of the husband at the time of interview. In addition, we control for indicators of financial resources , indicators of work load  indicators of decision-making , and various indicators related to locus of control, self-esteem, and trust.Household-level demographic variables include household size and the number of individuals in various age-sex categories, and dummy variables for the caste of the household head. The probit also includes controls for the household’s relative wealth.i in household h in district d of state s, h, and In addition to presenting the matching estimates, we present the simple ordinary least squares estimates of the relation between SHG membership and the outcomes of interest as follows:5We first examine the association between SHG membership and political participation, awareness, and utilization of government entitlement schemes. We then explore potential mechanisms through which SHG membership could affect these outcomes. The mechanisms explored are social networks and mobility.155.1mahila gram sabha) as well as the gram sabha, which involves both male and female adults, and to believe that the local governing body, the gram panchayat, will take positive action to demands/suggestions made by women and the SHG.Increasing political awareness is one of the key programmatic features of many, although not all, SHGs. Not surprisingly, both OLS and NNM estimates indicate a positive significant association of SHG membership with various indicators of political participation. NNM estimates show that, compared to non-SHG members, SHG members are more likely to have voted in the last election (p < 0.01), to have voted because it was their right (p < 0.01), and to have made the decision of who to vote for on their own (p < 0.01), and, have an overall political participation score that is higher (p < 0.01) . SHG mem5.2Finally, Why might SHG membership not increase awareness of more entitlement schemes? We propose the following explanations. The first possibility is that NGOs working to improve knowledge around entitlement schemes focus their energies on those that are available to the majority of group members, e.g. household-level schemes like the PDS and MGNREGA, rather than those limited to a specific demographic category within those households. Indeed, convergence of the MGNREGA with the NRLM has been pushed by activists and government officials since the inception of the workfare scheme, with SHG women being mobilized to audit the scheme, report irregularities, and in some cases even to maintain the muster rolls. Second, several of the schemes outlined in SHG membership, however, significantly increases utilization of government entitlements . OLS est5.3Connectedness to women within a group does not necessarily mean that the respondent will expand her social circle, however, or the number of people to whom she can turn for financial assistance. OLS estimates show a positive association of SHG membership with whether the respondent woman had a conversation with more than 9 people in her own hamlet in the last 30 days (p < 0.10), and whether she could borrow INR 1000 from at least 10 people from her own hamlet (p < 0.01), however, the latter effect is less significant when matching methods are used  Panel B.Other factors may also affect the woman’s ability to interact socially with other women, such as their husbands’ willingness to allow them to leave the homestead to attend meetings. OLS estimates suggest that SHG membership is positively associated with the probability that the woman does not need permission from her husband and/or other household member to go to a village meeting or meeting of an association they are a member of (including the SHG) (the NNM estimates are not significant) . Both OL6gram sabha and to believe that the gram panchayat would take positive action in response to suggestions made by women and/or the SHG. This last result indicates not only trust but also confidence in women’s collective power.Our findings show a strong positive association of SHG membership with several political participation indicators. Women who are SHG members are more likely to have voted in the last election, and to have decided to do so of their own accord and because they feel it is their right to vote. Being an SHG member also makes these women more likely to attend the In terms of knowledge of different entitlement schemes, we find that SHG members are more likely to have heard about the workfare scheme, MGNREGA, and the housing scheme, IAY but not others. However, despite similar knowledge about entitlements schemes between SHG members and non-members, we find that SHG members are more likely to utilize some of these schemes, for example MGNREGA, AAY and IAY, indicating that SHG members may be more able to translate their information into action, either because of their individual empowerment (e.g. mobility), or because of the strength of the collective .Our findings also show that membership in a SHG has a positive effect on several social network outcomes – with SHG women being more likely to know other women in their village, be part of a social group with them and to talk to them about important matters like health and nutrition. We find that SHG women are slightly more likely to be able to borrow money from someone from a neighboring village, indicating that the social network effect goes beyond the village they live in. Furthermore, SHG members are less likely to need permission from their husbands or other household member to go to a village meeting – indicating improved ability and self-confidence in interacting with those outside their household.Going back to the pathways and the constraints identified above – information and ability to hold public entities accountable – and the channels through which SHG membership may alleviate them, our findings confirm the existence of the cross cutting SHG pathways (identified in gram sabha transcripts, (panchayat and government officials were accountable.Our results are consistent with a growing body of qualitative evidence on how self-help groups contribute to collective social behavior, participatory democracy and governance in India . SHG memscripts,  found thgram sabhas frame their narratives in terms of common issues. Overall, our results indicate the potential for SHGs to empower women both individually and collectively, which may lead to better awareness, accountability, and governance of public entitlement schemes.Greater awareness and utilization of public entitlement schemes among SHG women, as observed in our results, could be a result of the women taking up issues for which they have found common ground. This is consistent with ’s findinThe authors of this article wish to confirm that there are no known conflicts of interest associated with this publication. None of the coauthors has received financial support for this work that could have influenced its outcome.We also confirm that the manuscript has been approved for submission by all seven co-authors, and that all co-authors have approved the order of the listing of the authors. There is no person who contributed significantly to this manuscript but has not been listed as a co-author.


Phenethyl isothiocyanate (PEITC) against Ehrlich ascites carcinoma in-vivo and in-vitro. In-vivo, ascetic fluid volume, body weight, serum malondialdehyde (MDA) level and total antioxidant capacity (TAC) were determined using Ehrlich ascites carcinoma (EAC) bearing mice. In-vitro, MTT assay was used. RT-PCR was used to investigate role of PEITC in apoptosis by analyzing the expression of Bax, caspase-9, and Bcl-2 genes. The effect of PEITC on caspase-9 enzyme activity was also tested. PEITC and/or Doxorubicin (Dox) treatment significantly suppressed EAC growth as compared to EAC/oil control mice. PEITC treatment showed a dose-dependent inhibition of EAC cells as indicated by MTT assay. We found that significant increase in MDA level and decrease in TAC caused by Dox treatment were significantly reduced by combination with PEITC treatment. Bax, caspase-9 genes’ expression and caspase-9 enzymatic activity were significantly increased, while Bcl-2 gene expression was significantly decreased in PEITC treated mice. PEITC may act as a promising anticancer agent either alone or more effectively in combination with Dox through apoptotic cell death induction.The aim of this study is to investigate the antitumor activity and possible molecular mechanism of  Cancer is considered as the second cause of mortality around the world where up to 12.7 million patients have been diagnosed with cancer and this number is expected to reach 21 million within the next 15 years   have been reported to reduce the incidence of different types of cancers , 10. Phe (PEITC)  is an im (PEITC) . PEITC epoptosis .Apoptosis is a self-cellular suicidal mechanism that is controlled by diverse signaling pathways and used to destroy damaged cells . Many gePEITC and/or Dox against Ehrlich ascites carcinoma bearing-mice in-vivo and on Ehrlich ascites carcinoma cells in-vitro. Furthermore, we studied the effect of PEITC and/or Dox on expression of some apoptotic genes in order to elucidate the underlying molecular mechanism of PEITC action.Accordingly, the present work was designed to evaluate the antitumor and cytotoxic effect of Drugs and ChemicalsPEITC, >98% purity was purchased from Abcam . Doxorubicin HCl (Adricin) was purchased from EIMC United Pharmaceuticals . 3- 2,5diphenyl-2H-tetrazolium-bromide (MTT) was purchased from SERVA electrophoresis GmbH . Trypan Blue and Dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich . Roswell Park Memorial Institute (RPMI) - 1640 medium was purchased from Gibco Laboratories . Fetal bovine serum (FBS) and penicillin-streptomycin antibiotic were purchased from Lonza . All other chemicals are of high purity and analytical grade.Animalsad libitum. Ehrlich ascites carcinoma (EAC) induction started 10 days following animals’ accommodation under these standard conditions. All animal experiments carried out in this study were complied with ″Research Ethics Committee″ Faculty of Pharmacy, Mansoura University, Mansoura, Egypt, in accordance with ″Principles of Laboratory Animal Care″ .Adult Swiss female albino mice (25–30 g) were purchased from Theodore Bilharz Institute, Giza, Egypt and kept in animal house of Faculty of Pharmacy, Mansoura University, Egypt. Animals were kept in standard size polycarbonate cages under standard laboratory conditions  and had free access to food and water Ehrlich ascites carcinoma cells6 cells in adult female Swiss albino mice  passage of 1 X 10ino mice . Seven dEhrlich ascites carcinoma model6 EAC cells (0.1 mL/mouse) for tumor induction. On the next day, mice were weighted then randomly divided into 4 groups; each group consists of 10 animals. Treatments were administered for 14 days as follows: On day zero, mice were injected intraperitoneally (IP) with 1 X 10PEITC-treated group that received PEITC at a dose of 60 mg/kg/0.1 mL oil/mouse/day orally  2 h following receiving PEITC . A fifth group of eight mice that received 0.1 mL oil/day/mouse orally was used as normal control group. On day 15, animals were weighted, blood was collected by retro-orbital puncture and then sacrificed. Serum samples were prepared by centrifugation of blood samples that was withdrawn via retro-orbital puncture at 4000 rpm for 10 min at 4 °C. Ascetic fluid was aspirated from all animals for measuring tumor volume as an indication for the antitumor effect. Ascetic fluid samples were centrifuged at 1800 rpm for 5 min at 4 °C. The pellet was flash-frozen in liquid nitrogen then stored in -80 °C till used.EAC/oil control group that received 0.1 mL oil/mouse/day orally, y orally , Dox-trey orally  and PEITMalondialdehyde and total antioxidant capacity assayet al.  and total antioxidant capacity (TAC) colorimetric assay kits from Biodiagnostic Company  were used for spectrophotometric determination of serum MDA levels and serum TAC according to the methods reported by Satoh  and Koraet al. , respectCell culture2 .EAC cells were cultured in RPMI-1640 medium supplied with 10% FBS and 1% (v/v) penicillin-streptomycin antibiotic. They were maintained at 37 °C in a humidified atmosphere with 5% COCell viability assay5 cells/well) were inoculated in 96-well flat bottom tissue culture plate . One day after, cells were treated with three different concentrations of PEITC , Dox (100 µmol/L), a combination of PEITC 20 µmol/L + Dox 100 µmol/L and DMSO with a final concentration of 0.1% that have been used as a control. Cells were incubated for 24 h and incubation period continued for additional 4 h at 37 °C following the addition of 20 μL/well of MTT solution (5 mg/mL in PBS). The formazan crystals formed as a result of MTT reduction were solubilized by treating cells with 100 µL of 0.04 N acidic isopropanol. Absorbance was measured at 540 nm using a microplate reader, . The experiment was repeated three times. Viable cells were calculated as percentage relative to DMSO treated control cells.MTT assay was used for the assessment of cell viability. EAC cells  for Bax, caspase-9 and Bcl-2 gene expression® Reagent  according to the manufacturer’s instructions. The quantity and quality of the isolated RNA was assessed spectrophotometrically at 260 nm and 260/280 nm ratio, respectively, using NanoPhotometer® . One microgram of the total RNA was reverse transcribed into single-stranded complementary DNA using QuantiTect Reverse Transcription Kit  according to the manufacturer’s instructions. The mRNA expression level of apoptosis genes Bax, caspase-9 and Bcl-2 was determined in different EAC cells. For normalization of gene expression, mouse Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was quantified in parallel with target genes. Reactions were performed using HOT FIREPol EvaGreen qPCR Mix  in Rotor-Gene Q . Gene specific primers are summarized in −ΔΔCT method relative to GAPDH. The specificity of the designed primers and the size of amplified PCR products were confirmed by melt curve analysis and 2% agarose gel electrophoresis, respectively.Total RNA was isolated from EAC cells using TRIzolCaspase-9 enzyme activity colorimetric assayEAC cells collected from control and treated groups were lysed using chilled lysis buffer provided by Caspase-9 Colorimetric Assay Kit  and then caspase-9 enzyme activity was determined spectrophotometrically according to the manufacturer’s instructions.Statistical analysisP-value of less than 0.05 was considered statistically significant.Results are presented as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test using GraphPad Prism 6.01  was used to find out statistically significant results. Effect of PEITC on EAC cell growth in-vivoPEITC treatment inhibited EAC cell growth as indicated by the decrease in ascetic fluid volume and the change in body weight. PEITC treatment and Dox treatment showed a significant decrease in ascetic fluid volume by (69.14% and 87.37%), respectively as compared to EAC/oil control group. Furthermore, PEITC/Dox combination therapy significantly decreased ascetic fluid volume by (95.42%) as compared to EAC/oil control group , respectively  for 24 h showed a significant dose-dependent decrease in EAC cell viability by 22.59%, 33.06% and 42.39%, respectively. Dox treatment (100 µmol/L) for 24 h significantly decreased cell viability by 53.41%. Combination of PEITC (20 µmol/L) and Dox (100 µmol/L) treatments for 24 h caused a significant decrease in cell viability by 75.29% MTT assay was used for testing EAC cell viability. As compared to DMSO treated EAC control cells, .Effect of PEITC on malondialdehyde level and total antioxidant capacityP < 0.0001 and P < 0.0001, respectively). On the contrary, serum TAC of EAC/oil control group and mice receiving Dox treatment significantly decreased as compared to normal control group . PEITC treatment significantly decreased serum MDA level and increased serum TAC as compared to EAC/oil control group . PEITC/Dox combination treatment significantly decreased serum MDA level as compared to EAC/oil control group (P < 0.0001) and Dox treated group (P < 0.0001). On the other hand, PEITC/Dox combination treatment significantly increased serum TAC as compared to EAC/oil control group (P < 0.0001) and Dox treated group (P = 0.0006).As shown in Effect of PEITC on caspase-9 enzymatic activityPEITC, Dox and PEITC/Dox treated groups significantly enhanced caspase-9 enzymatic activity as compared to EAC/oil control group by 1.69 (P = 0.02), 1.92 (P = 0.002) and 2.52 (P < 0.0001) fold, respectively. Moreover, PEITC/Dox combination therapy significantly increased caspase-9 enzymatic activity as compared to both PEITC treated group (P = 0.004) and Dox treated group (P = 0.04).As shown in Effect of PEITC on Bax, caspase-9 and Bcl-2 gene expressionPEITC treatment significantly increased expression level of both Bax and caspase-9 genes as compared to their levels in EAC/oil control group by 3.92 (P = 0.006) and 3.2 (P = 0.009) fold, respectively. Similarly, Dox treatment significantly increased expression level of both genes by 4.53 (P = 0.001) and 3.49 (P = 0.002) fold, respectively. PEITC/Dox combination treatment significantly increased expression level of both genes by 12.71 (P < 0.0001) and 14.5 (P < 0.0001) fold, respectively in comparison with their levels in EAC/oil control group. Moreover, combination therapy significantly increased expression level of Bax and caspase-9 genes, as compared to their levels in PEITC treated group (P < 0.0001 and P < 0.0001) and Dox treated group (P < 0.0001 and P < 0.0001), respectively , 0.52 (P = 0.002) and 0.31 (P < 0.0001) fold, respectively. PEITC/Dox combination treatment decreased Bcl-2 expression level as compared to its level in both PEITC treated group and Dox treated group, though that decrease was not statistically significant (On the contrary, nificant .PEITC is a promising anti-cancer agent that showed inhibitory effect against tumor growth in different previous studies (PEITC anti-tumor action, the exact molecular mechanism for apoptosis induction has not been fully investigated (PEITC compared to and in combination with Dox were investigated using Ehrlich ascites carcinoma model in-vivo and in-vitro. studies  and 23. stigated . In thisin-vivo study showed that either PEITC treatment or Dox treatment solely caused a significant inhibition of EAC cell growth as indicated by the significant decrease in ascetic fluid volume and body weight of treated mice as compared to EAC/oil group. Upon combination of both PEITC and Dox treatments, further decrease in ascetic fluid volume was observed as compared to PEITC treatment alone. These results were further supported by in-vitro study, where PEITC treatment showed inhibition of EAC cellular viability in a dose-dependent manner. Interestingly, the cytotoxic effect of highest dose of PEITC treatment when combined with Dox treatment significantly exceeded the cytotoxic effect of each treatment alone. Altogether, our results showed that PEITC and/or Dox treatments effectively inhibit EAC cellular growth in-vivo and in-vitro.Our results from PEITC exerts its antitumor activity, is the induction of apoptosis (PEITC was able to induce apoptosis in different types of cultured human cancer cell lines (One of the possible mechanisms by which poptosis . It has ll lines -28. Apopll lines . It usuall lines . The Bclll lines , 32.PEITC treatment increased the expression level of both Bax and caspase-9 genes and decreased the expression level of Bcl-2 gene as compared to EAC/oil control group. These results were further reinforced by the ability of PEITC treatment to enhance caspase-9 enzymatic activity. Interestingly, PEITC/Dox combination treatment surpassed each treatment alone in increasing the expression level of Bax and caspase-9 genes and in enhancing caspase-9 enzymatic activity but not in decreasing Bcl-2 gene expression level.In agreement with previous studies -36, our PEITC and/or Dox treatment may confirm induction of apoptotic pathway. This could be further explained by the release of cytochrome c from mitochondria as a result of increase in Bax and decrease in Bcl-2 expression. Cytochrome c forms an apoptosome with apoptotic protease activating factor 1 and pro-caspase-9, consequently caspase-9 activation, pro-caspase-3 induction and eventually apoptosis (Alteration of measured pro/anti-apoptotic genes in EAC cells upon poptosis , 38.PEITC antitumor activity, it has been reported that PEITC exerts antioxidant activity (PEITC caused a significant decrease in MDA level and significant increase in TAC as compared to EAC/oil control group. Carcinogenesis is a multistep process that is initiated and promoted by multiple factors; most prominently oxidative stress . Free raactivity . This aget al. (et al. (PEITC treatment was combined with Dox treatment, MDA level was significantly decreased and TAC was significantly increased in comparison with Dox treatment alone. Collectively, PEITC/Dox combination treatment showed a significant anti-tumor effect without causing significant oxidative damage. This could be explained by the ability of PEITC to induce some antioxidant enzymes whose activity was reduced by Dox treatment (On the contrary, MDA level in mice receiving Dox treatment was significantly increased unlike TAC, which was significantly decreased as compared to normal control group. This was in agreement with Lorenzo et al. , who rep (et al.  found threatment , 49, thureatment .PEITC efficaciously suppressed EAC tumor growth in-vivo and in-vitro via alteration of apoptotic genes and its marked antioxidant effect. It can be concluded that PEITC is useful as natural therapy that could be considered for cancer treatment along with Dox. However, further research is required to fully establish the antitumor effect of PEITC/Dox combination before considered for clinical trial.Taken together, our results indicated that


However, the mechanism by which BaCl2 causes muscle injury is unresolved. Because Ba2+ inhibits K+ channels, we hypothesized that BaCl2 induces myofiber depolarization leading to Ca2+ overload, proteolysis, and membrane disruption. While BaCl2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl2 treatment.Local injection of BaClm) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca2+]i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl2 (50–75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant.The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3–4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential  with enhanced cleavage of αII-spectrin. Eliminating Ca2+ from PSS prevented the rise in [Ca2+]i and ameliorated myonuclear staining with PI during BaCl2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl2 and morphological integrity deteriorated through 72 h.Addition of 1.2% BaCl2 injures myofibers through depolarization of the sarcolemma, causing Ca2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.BaCl Whilammation –5, the ate cells , 6–9 , myelinated axons projecting from α motor neurons in the spinal cord terminate and are covered by perisynaptic Schwann cells which overlay postsynaptic clusters of nicotinic acetylcholine receptors . Arterioy supply , it is u2 injures skeletal muscle myofibers and whether motor innervation and microvascular supply are concomitantly disrupted by exposure to BaCl2. We studied the acute effects of BaCl2 exposure on membrane potential (Vm), [Ca2+]i, membrane integrity, αII-spectrin degradation, and force production in extensor digitorum longus (EDL) muscles. Neuromuscular synapses were studied in the tibialis anterior (TA) muscle and the microcirculation was studied in the gluteus maximus (GM) muscle at 0 (control) and 1–3 days post injury (dpi) following local injection of BaCl2.The aim of this study was to determine how BaCln = 31), D2-Tg(S100B-EGFP)1Wjt/J (S100B-GFP) ; and Cdh5-CreERT2:ROSA-mTmG (Cdh5-mTmG) (cross of VE-cadherin-CreERT2 mice  responses were measured as reported i . Isotonic BaCl2 solution resulted in a similar increase in [Ca2+]i (F340/F380 increased from 1.18 ± 0.02 (baseline) to 1.58 ± 0.06 (BaCl2); n = 3). In contrast, adding 1.2% BaCl2 to Ca2+-free PSS had no significant effect on [Ca2+]i . In the absence of BaCl2, Fura 2 fluorescence remained stable at the resting baseline for at least 30 min (n = 3).A primary consequence of myofiber depolarization in healthy muscle is internal release of Carcolemma . The add2 in standard PSS increased resting force from 7.4 ± 0.1 to 11.1 ± 0.4 g over ~ 30 min, which then returned to baseline during the 60 min exposure . Whereas a rise in [Ca2+]i activates the contractile proteins [2+]i stimulates mitochondrial production of reactive oxygen species (ROS), which can impair cross-bridge function i leads to degradation of muscle fibers through proteolysis by Ca2+-activated neutral proteases i leads to proteolysis, membrane disruption, and myofiber death. Moreover, preventing the rise of [Ca2+]i by removing extracellular Ca2+ preserves membrane integrity as demonstrated by the paucity of myonuclei stained with PI under this condition . Infiltons from , 56 throons from .2 is widely used as a method for studying myofiber damage and regeneration i as observed in whole-muscle preparations (Fig. 2-induced myotoxicity for single fibers appear to be less than observed in our experiments using whole muscles. We hypothesize that the absence of fixed attachments at the ends of single myofibers may reduce the damaging membrane stress following Ca2+-induced hypercontraction. Scale bars = 10 μm.


Lung cancer is the leading cause of cancer-related death in the Unites States, and approximately 85% of all lung cancers are classified as non-small cell lung cancer (NSCLC), which is extremely difficult to treat and its survival rate is low. After decades of clinical trials, the most effective treatments are still those that implement the first-generation platinum anticancer agent cisplatin (CDDP) in combination with other drugs. We previously demonstrated that the naturally-occurring compound phenethyl isothiocyanate (PEITC) can be used to sensitize NSCLC cells to CDDP. Furthermore, co-encapsulation of PEITC and CDDP in liposomes enhances their toxicity toward NSCLC cells. We here optimize liposomal-PEITC-CDDP, demonstrate the release of PEITC and CDDP from the nanoparticle, and show that liposomal-PEITC-CDDP is much more toxic toward both A549 and H596 human NSCLC cell lines than toward WI-38 and BEAS-2B human normal lung cell lines. Thus, we have prepared an efficacious therapy that has significantly higher toxicity toward cancer cell lines than normal cell lines. TM is less toxic to patients than free CDDP is, but has a similar efficacy against NSCLC [TM with the βx-tubulin binding agent paclitaxel was similar to that of CDDP with paclitaxel; however, much less toxicity to the patient was observed [Lung cancer is the leading cause of cancer-related death in the United States . Approxist NSCLC . In a raobserved . Not allobserved .In 2010, we showed that naturally occurring isothiocyanates (ITCs) can be used to enhance the efficacy of CDDP ; ITCs arIn a separate report, a synthetic ITC derivative, ethyl 4-isothiocyantobutanoate, was shown to sensitize ovarian cancer cells to CDDP and, moreover, it allowed for CDDP to overcome CDDP-resistance in cell culture . Then, iIn 2014, we reported on the preparation of a liposomal nanoparticle containing both PEITC and CDDP . StudiesWe here expand on our previous work. Again, DSPC was used; however, the optimized liposomal nanoparticles have a 1:3 ratio of CDDP to PEITC, whereas the previously reported nanoparticles had a 1:2 ratio, so less CDDP can be administered. Careful characterization studies were performed and release profiles obtained for liposomal-CDDP, liposomal-PEITC and liposomes containing both CDDP and PEITC: liposomal-PEITC-CDDP. Then, toxicity was tested in two human NSCLC cell lines, A549 and H596, and compared to its toxicity in human normal lung cells, BEAS-2B and WI-38.Blank and drug-encapsulated liposomes were prepared with the combination of DSPC:EPG in the ratio of 4:1. Various quantities of PEITC and/or CDDP were added to the liposomal formulation . PercentParticle size distributions and zeta potentials of blank and PEITC and/or CDDP loaded liposomes were characterized using a Zetasizer. As shown in Drug release of nanoparticles is an important factor in determining the biological efficiency of drug delivery vehicles. Plots of percentage of CDDP and/or PEITC released from liposome formulations versus time are shown in The MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay can be used to measure the change in cell viability caused by a change in metabolic activities. The cytotoxicity of free PEITC and/or CDDP, blank liposomes, drug-loaded liposomes toward human NSCLC cells (H596 and A549) and normal lung cells (WI-38 and BEAS-2B)  were tesp = 1.1 × 10−14) and H596 cell lines (p = 1.55 × 10−7) [In the cytotoxicity study using A549 cells A, the pe × 10−7) . A Dunne × 10−7) .p = 2.09 × 10−5) and H596 (p = 1.75 × 10−6) cell lines (p = 5.86 × 10−6) and H596 (p = 1.15 × 10−3) cell lines. Lipo-PEITC, Lipo-CDDP and Lipo-PEITC-CDDP groups in the two lung cancer lines showed significantly higher cytotoxicity effects than free PEITC, CDDP and CDDP + PEITC groups, respectively, which indicated that liposomes can be used as an effective drug delivery vehicle.The blank liposomes appeared to have no significant effect on cell growth with the percent survival of 92.6 ± 3.7%, 90.7 ± 9.1%, 85.5 ± 19.9%, and 96.0 ± 14.2% for A549, H596, WI-38 and BEAS-2B, respectively. Liposomes containing CDDP (5 µM) or PEITC (15 µM) showed greater toxicities toward A549 than the free drugs. The percent cell survival after treatment with liposomes loaded with CDDP (Lipo-CDDP) was 43.4 ± 4.0%, and for liposomes with PEITC (Lipo-PEITC), the percent cell survival was 64.6 ± 4.2%. Similarly, in H596 cells, the percent survival of the Lipo-CDDP treatment group  was lower than that of the free CDDP group , while cell percent survival of the Lipo-PEITC treatment group  was also lower than free PEITC . A Welch two-sample t-test found that the liposomal formulation of CDDP (Lipo-CDDP) exhibits a significantly higher cytotoxic effect than free CDDP on both A549 (ll lines . Similarp = 1.26 × 10−14) and H596 cell lines (p = 5.58 × 10−10). A Dunnett’s post hoc test accounting for multiple comparisons shows that for both cancer cell lines, the percent survival of the cells treated with Lipo-PEITC-CDDP is significantly lower than the free drug combination (CDDP + PEITC), as well as liposomal formulations of either single drug (Lipo-CDDP and Lipo-PEITC) (When treated with liposomes containing 5 µM of CDDP and 15 µM of PEITC (Lipo-PEITC-CDDP), the A549 cells had a percent survival of 33.3 ± 2.9%, which was greater than that of CDDP + PEITC, Lipo-CDDP and Lipo-PEITC. Like A549 cells, the lowest percent survival of H596 cells (28.6 ± 6.3%) occurred when treated by 5 µM of CDDP and 15 µM of PEITC loaded in liposomes (Lipo-PEITC-CDDP), which was lower than that of CDDP + PEITC, Lipo-CDDP and Lipo-PEITC. There is a significant difference among groups Lipo-PEITC-CDDP, CDDP + PEITC, Lipo-CDDP, and Lipo-PEITC as determined using a one-way ANOVA for both A549 (o-PEITC) . Loaded p = 1.16 × 10−14) between no treatment group and Lipo-PEITC-CDDP group for the A549 cell line. The t-value capturing the effect size of the difference is 68.0. Similarly, for H596, a difference between the two groups was found with a t-value of 23.9 and a p-value of 4.22 × 10−8. On the other hand, the effect sizes of the difference on normal cell lines were much lower  (A Welch two-sample t-test found that there is a significant difference ( × 10−3) . This reBecause of our 3:1 PEITC:CDDP ratio, we were able to use less cisplatin in the Lipo-PEITC-CDDP against NSCLC cells than in our last formulation, which was 2:1 PEITC: CDDP . We werel-α-phosphatidylglycerol  (EPG) were obtained from Avanti Polar Lipids, Inc. . Chloroform, phenethyl isothiocyanate (PEITC), cisplatin (CDDP) and dialysis sacks  were purchased from Sigma-Aldrich . The 1,2-benzenedithiol (BDT) was obtained from Thermo Fisher Scientific . Platinum (1000 ± 3 µg/mL), lutetium (1000 µg/mL) and gold (1000 µg/mL) standards were purchased from High-Purity Standards . The normal lung fibroblast cell line WI-38, normal bronchial epithelial cell line BEAS-2B and NSCLC cell lines H596 and A549 were purchased from the American Type Culture Collection . CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was from Promega .1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and First, 12.6 mg DSPC and 4.1 mg EPG were added to a round-bottom flask with 10, 16 or 20 µL of PEITC and 0.4 mL of chloroform. After 30 min of rotary evaporation, the dry, thin lipid film formed at the bottom of the flask was dissolved with 1 mL of normal saline solution containing 4, 6 or 8.33 µmol of CDDP. Then the mixture was sonicated for 2 min with a water-type sonicator  and vortexed for 30 s at 65 °C to obtain the resultant drug-encapsulated liposomes. Multilamellar liposomes were extruded 10 times through 200 nm and 100 nm polycarbonate membranes at 65 °C using an extruder  to achieve small unilamellar liposomes. The free CDDP and PEITC were removed using 50 kDa ultracentrifugation filter columns  at the speed of 3000 ×g for 20 min. A Zetasizer Nano  was used to investigate the particle size distribution and zeta potential of the liposomes prepared using the dynamic light scattering technique. The structure and morphology of PEITC and CDDP co-encapsulated liposomes were characterized by Field Emission Scanning Electron Microscope (FESEM), Supra 55 VP .2 = 0.98) in the same manner, and concentrations of PEITC in samples were determined based on the standard curve.The amount of PEITC loaded in the liposomes was determined using a 1,2-benzenedithiol (BDT) assay reported by Zhang with minor modifications . To 1 mLThe PEITC drug loading was calculated using the equation:Cisplatin loading was determined using inductively-coupled plasma-mass spectrometry . Liposomal-CDDP and liposomal-PEITC-CDDP were digested with 70% nitric acid and heated at 70 °C overnight to evaporate. Then, the sample was re-dissolved in 70% nitric acid and diluted using deionized water, and the final samples for the ICP-MS test contained 2% nitric acid. Lutetium (20 ppb) was used as an internal standard.The CDDP drug loading was calculated using the equation:v/v methanol. A spin bar was added in the vial to achieve 100 rpm rotation speed. The temperature was set at 37 °C, and aliquots (100 µL) of the release medium were withdrawn for analysis at different time points (up to 24 h) and replaced with fresh medium. The absorbance of PEITC released from Lipo-PEITC and Lipo-PEITC-CDDP was measured using the same BDT assay as described above. The amount of CDDP released from Lipo-CDDP and Lipo-PEITC-CDDP was determined using ICP-MS.Different amounts of PEITC or CDDP were used to prepare liposomal-PEITC or liposomal-CDDP. The formulation that achieved the highest loading (%) of PEITC is referred to as “Lipo-PEITC”, the highest loading (%) of liposomal-CDDP is referred to as “Lipo-CDDP” and the liposomal formulation which combined the highest loading (%) of both PEITC and CDDP is referred to as “Lipo-PEITC-CDDP”. These three liposomal formulations were tested for their in vitro drug release and cytotoxicity. Drug release was studied using a dialysis method. Dialysis sacks were rinsed thoroughly using deionized water for 30 min, then 1 mL of Lipo-PEITC, Lipo-CDDP or Lipo-PEITC-CDDP was placed in the dialysis sack. The sack was fully immersed in a capped glass vial containing 20 mL of 1× PBS with 25% The percentage of release of CDDP or PEITC at different time points was calculated using the equation:2 (standard conditions) atmosphere incubator. For the two normal cell lines, the culture medium used for the WI-38 cells was the minimum essential medium (MEM) containing 100 µg/mL streptomycin, 10% fetal bovine serum (FBS), 2.0 mM l-glutamine and 100 IU/mL penicillin; the culture medium used for the BEAS-2B cells was bronchial epithelial basal medium (BEBM) with 10% FBS and supplements. The culture medium used for the two cancer cell lines, H596 and A549 cells, was Roswell Park Memorial Institute (RPMI) medium containing 100 µg/mL streptomycin, 100 IU/mL penicillin, 10% FBS, and 2.0 mM l-glutamine.All cell studies were carried out in a humidified 37 °C, 5% CO3 cells/mL (100 µL/well) in 96-well plates and allowed to grow for 24 h; after that, the medium was removed and replaced with 100 µL of medium containing the treatment suspensions, which was removed after an exposure time of 24 h and replaced with 100 µL of fresh medium. To each well, 20 µL of MTS solution was added and incubated for 2 h, and the UV-Vis absorbance was read at 490 nm. The percent survival of cells treated was calculated using the following equation:t is the absorbance of cells in treatment groups, Am is the absorbance of medium alone and Ac is the absorbance of cells without treatment. All p values were calculated using Microsoft Excel t-test function .Nine groups (n = 6) were included to test the cytotoxicity toward each cell line. Two control groups were included: group 1, control group with only medium; group 2, control group with non-treated cells. Group 3–9 were cells with treatment: group 3, free CDDP; group 4, free PEITC; group 5, CDDP and PEITC (CDDP + PEITC); group 6, blank liposomes; group 7, Lipo-CDDP; group 8, Lipo-PEITC; group 9, Lipo-PEITC-CDDP. The cells were seeded at 5 × 10R [t-tests were conducted using the t.test function. The Levene test was carried out to test for homogeneity of variances using leveneTest from the car package [multcomp package [All statistical analyses were conducted using R . For the package . For com package .Cisplatin is commonly used against NSCLC in the clinic; however, the negative side-effects associated with this metal-based drug urgently need to be addressed. We encapsulated CDDP and PEITC in liposomes, as this strategy has been shown to increase the circulation time of encapsulated CDDP and reduce its side-effects . Further


In this study, the binding of CaM, its mutants at calcium binding sites , and truncated proteins (N-lobe and C-lobe) to NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM  bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1.Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca V1.1-NaV1.9 and NaX—forming the VGSCs superfamily [V1.1 is widely expressed in cell bodies and axon initial segments of neurons in the brains [V1.1 contains a classical calmodulin (CaM)-binding IQ (isoleucine and glutamine) domain [2+ signal transduction and alters the activity in response to changes in free Ca2+ concentration ([Ca2+]). All ten isoforms of VGSCs contain a unique IQ domain  increases from ~10−7 M during resting state and up to ~10−3 M during active state . The summarized data from the densitometer analyses of replicate gels are shown in Bmax of CaM to IQ was 2.06 (Bmax1 + Bmax2), 0.66 (Bmax1 + Bmax2), 1.08 (Bmax1 + Bmax2), and 1.38 (Bmax1 + Bmax2) mol/mol (CaM/IQ) at ≈free, 100 nM, 500 nM, and 2 mM [Ca2+], respectively (n = 4), indicating that apoCaM had the highest affinity with IQ domain. However, the binding of CaM to IQ domain was in a Ca2+-dependent manner in the presence of Ca2+. The binding affinity estimated as Kd value also showed a Ca2+-dependent increase in the presence of Ca2+ /E (glutamic acid) mutation on the IQ domain of Cawith CaM . In this] into E A. As shoV1.1 channel, we then examined the binding properties of CaM mutants to NaV1.1 IQ domain. This included CaM12 and CaM34   CaM. The maximal binding estimated as Bmax was 1.27, 0.70, 0.82, and 0.91 mol/mol CaM12/IQ (n = 4) at ≈free, 100 nM, 500 nM and 2 mM Ca2+, respectively, showing an obvious Ca2+ dependence in the presence of Ca2+  A 26]. A. AV1.1 c of Ca2+ . It is i34 to IQ domain. As shown in 34 to IQ was also concentration-dependent. The parameters obtained  at ≈free, 100 nM, 500 nM, and 2 mM Ca2+, respectively. It was noted that this profile of [Ca2+] dependence was different from those of wt CaM and CaM12.Next, we examined the binding of CaMobtained  revealed1234 to NaV1.1 IQ domain. As shown in 2+ dependence was totally diminished because of the mutation in four Ca2+ binding sites. As shown in Bmax were 1.06, 1.02, 1.06, and 1.03 mol/mol (CaM1234/IQ) at ≈free, 100 nM, 500 nM, and 2 mM [Ca2+], respectively. The values of Bmax and Kd were not significantly different among different Ca2+ concentrations, confirming the Ca2+-insensitive nature of CaM1234. Our data showed that functional Ca2+ binding sites of either N- or C-lobe were required for Ca2+ dependency in CaM binding to NaV1.1 IQ domain.Furthermore, we examined the binding of CaMV1.1 IQ, we first computationally investigated the interactions between N-lobe or C-lobe of CaM and NaV1.1 IQ domain using Discovery Studio 2017. As shown in V1.1 IQ domain were 11.28 and −23.31 kcal/mol, respectively, whereas these parameters for the best interaction of C-lobe with IQ domain were 10.66 and −26.21 kcal/mol, respectively. In addition, as shown in V1.1 IQ domain, the best ZDock score and lowest E_RDock were 12.78 and −30.72 kcal/mol, respectively.In order to study the effect of specific lobe of CaM on the Na2+ concentrations by pull-down assay. As shown in Bmax for N-lobe were 0.18, 0.16, 0.31, and 0.17 mol/mol (N-lobe/IQ) at ≈free, 100 nM, 500 nM, and 2 mM [Ca2+], respectively (n = 4). Thus the profile of [Ca2+] dependence of N-lobe was similar to that of CaM34.Next, we further examined the bindings of individual (truncated) N- and C-lobe of CaM A to IQ d2+]. The maximal binding presented by Bmax were 0.32, 0.13, 0.13, and 0.15 mol/mol (C-lobe/IQ) at ≈free, 100 nM, 500 nM, and 2 mM [Ca2+], respectively (n = 4) (2+] dependence of C-lobe was similar to those of wt CaM and CaM12.We then examined the binding of C-lobe to IQ domain. As shown in  (n = 4) . Thus, t2+-dependent manner. Kd of N-lobe was lower than that of C-lobe in the presence of Ca2+ ([Ca2+] ≥ 100 nM), whereas Kd of C-lobe was lower than that of N-lobe in the absence of Ca2+. Additionally, Bmax of N-lobe was higher than that of C-lobe in the presence of Ca2+, whereas Bmax of C-lobe was higher than that of N-lobe in the absence of Ca2+, indicating that C-lobe was the predominant domain in apoCaM interacting with NaV1.1 IQ domain, and N-lobe was the predominant domain in Ca2+/CaM interacting with NaV1.1 IQ domain.The parameters  revealedV1.1 have been identified  compared to that in the presence of Ca2+, suggesting that phosphorylation of IQ domain by CaMKII had little effect on its binding with apoCaM. By contrast, the binding of Ca2+/CaM to IQ was increased in the phosphorylated IQ, indicating that the effect of CaMKII on the CaM binding to NaV1.1 IQ domain could exert its regulation only in the presence of Ca2+ . Thus, we mutated three potential CaMKII-mediated phosphorylation sites—T1909, S1918, and T1934—into A  was generated by PCR using the cDNA of human NaV1.1 as the template. The primers were designed using VectorNTI software. The human CaM and its truncated proteins—N-lobe (a.a. 2–80) and C-lobe (a.a. 76–148)—were subcloned from HEK293 cells  was calculated with MaxChelator (http://maxchelator.stanford.edu/index.html). Then, the reaction systems were gently washed twice with the same buffer. Bound CaM (or its mutant) and IQ (or EQ) were resuspended in 5× SDS-PAGE loading buffer and resolved in 15% SDS-PAGE gels. Proteins were stained by Coomassie brilliant blue R (CBB). Protein bands in the SDS-PAGE gels were digitized by the Photoshop software , and the grey level was quantified by Image J software  , whereas at high [Ca2+], N-lobe binds to IQ (lobe switching) and C-lobe binds to III-IV linker, resulting in depolarization of the inactivation curve . Thus, the property difference between the two lobes of CaM might endow the Ca2+-dependent and lobe-specific modulation of NaV1.1 channel. However, we still do not know the reason why CaM binding to the IQ domain is the largest at free Ca2+ and smaller at 100 nM Ca2+. We speculate that C-lobe of CaM might be at least partially occupied with Ca2+ at 100 nM Ca2+. In this scenario, the weaker binding of CaM to the IQ domain would be explained by the fact that the IQ domain has lower affinity for Ca2+/C-lobe than for Ca2+-free C-lobe. This point may be supported by the CaM12 and CaM34 experiments   condition, we found there was no significant difference in the CaM binding to the IQ domain between the phosphorylated and dephosphorylated peptides. One possible reason for this may be that the phosphorylation of NaV1.1 IQ might not affect the conformation of the apoCaM binding region, which interacts mainly with C-lobe of CaM. However, in the presence of Ca2+ CaMKII-mediated phosphorylation of IQ increased the binding of CaM to IQ domain, while CIP-mediated dephosphorylation of IQ decreased the binding of CaM. Thus, it is possible that Ca2+/CaM binding region interacts mainly with N-lobe of CaM, which might be different from the apoCaM binding region. Our data has shown that CaMKII regulates the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain, indicating that CaMKII-mediated phosphorylation of NaV1.1 affects CaM binding to NaV1.1. It is therefore speculated that CaMKII-mediated phosphorylation of NaV1.1 IQ domain might change the conformation of IQ domain, leading to increased binding of Ca2+/CaM to NaV1.1.One of the most intriguing findings of our research is that CaMKII-mediated phosphorylation of Na kinases . CaMKII of VGCCs , CaMKII I sites. ,46. In ant (Q54) ,47. BaseV1.1 in a calcium-dependent manner [2+ condition [2+/CaM/CaMKII on NaV1.1 based on our present study and other studies [2+]—a nonphosphorylated state of Ca2+ concentration—C-lobe is the predominant domain for apoCaM binding to IQ domain, and the binding of apoCaM to NaV1.1 IQ is not affected by CaMKII. At high [Ca2+] but at a nonphosphorylated state of IQ domain, N-lobe becomes the predominant domain since some of the CaM binds with Ca2+. Phosphorylation of IQ by CaMKII modulates the binding of Ca2+/CaM to the channel. Meanwhile, channel activity is maintained to the basal level at 80–100 nM [Ca2+]. At high [Ca2+]—a phosphorylated state of IQ domain—N-lobe is the predominant domain for Ca2+/CaM binding to IQ domain. Meanwhile, the effect of CaMKII-mediated phosphorylation is further promoted, leading to an increased binding of Ca2+/CaM to the channel compared to that at 80–100 nM [Ca2+]. The channel activity will also be increased at high [Ca2+] compared to that at 80–100 nM [Ca2+].A previous study had shown that CaM overexpression in HEK1.1 cells increases the peak current of Nat manner . Our preondition . Thus, w studies ,13: At l2+/CaM to IQ was Ca2+- and concentration-dependent, and apoCaM more preferentially binds to NaV1.1 IQ domain than Ca2+/CaM. In addition, C-lobe of CaM is the predominant domain in apoCaM binding to NaV1.1 IQ domain, whereas N-lobe of CaM is the predominant domain in Ca2+/CaM binding to NaV1.1 IQ domain. In addition, CaMKII-mediated phosphorylation increases the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. Our data provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity, and CaMKII on CaM binding to NaV1.1.In summary, we found that the binding of Ca