Thus, there is a strong rationale supporting HDACi therapy in myeloid malignancies. However, despite initial promising results in phase I trials, HDACi in monotherapy as well as in combination with other drugs, have failed to improve responses or survival. This review provides an overview of the rationale for HDACi in myeloid malignancies, clinical results and speculations on why clinical trials have thus far not met the expectations, and how this may be improved in the future.Myeloid hematological malignancies are clonal bone marrow neoplasms, comprising of acute myeloid leukemia (AML), the myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia (CMML), the myeloproliferative neoplasms (MPN) and systemic mastocytosis (SM). The field of epigenetic regulation of normal and malignant hematopoiesis is rapidly growing. In recent years, heterozygous somatic mutations in genes encoding epigenetic regulators have been found in all subtypes of myeloid malignancies, supporting the rationale for treatment with epigenetic modifiers. Histone deacetylase inhibitors (HDACi) are epigenetic modifiers that, in vitro, have been shown to induce growth arrest, apoptotic or autophagic cell death, and terminal differentiation of myeloid tumor cells. These effects were observed both at the bulk tumor level and in the most immature CD34 MDS is  unclear ,7.Janus kinase 2 (JAK2) V617F mutation occurs in around 95% of PV, and 50% of ET and MF, causing auto-phosphorylation of cytokine receptors and increased JAK-STAT pathway activation [The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) comprises of polycythemia vera (PV), essential thrombocytemia (ET), and myelofibrosis (MF) . The rartivation . Althougtivation .Chronic myelomonocytic leukemia (CMML) is the largest group of MDS/MPN overlap syndromes , where pKIT gene. There are two clinical phenotypes, indolent SM with a normal life expectancy and aggressive SM with a poor prognosis [Systemic mastocytosis (SM) is a rare myeloid malignancy . Over 90rognosis . In the rognosis ,13, howerognosis .Overall, 30% of MDS and CMML patients progress to acute myeloid leukemia (AML). AML is defined by over 20% myeloblasts in the bone marrow, and may be primary , secondary  or treatment related e.g., after chemotherapy of hematological or other tumors (10% of cases). Subtypes of AML are well defined according to cell of origin, specific cytogenetic or other aberrations outlined in the WHO diagnostic criteria . The 5-yIDH1 and 2 are common in AML, and TET2 mutations are common in all myeloid malignancies , a mutation causes a partial tandem duplication (PTD) that confers excessive tri-methylation of H3K4 . MLL, wh MLL-AF9 , thus thTP53 gene encoding the P53 tumor suppressor occurs in approximately 10% of AML and MDS patients, and is associated with a dismal prognosis. The TP53 gene requires coactivator CBP/p300 acetylation for full transcriptional activation, where the KAT p300 acetylates p53. P53 is normally de-acetylated by HDAC1. In addition, in AML with inv(16) or t, p53 activity is inhibited via interactions between the inv(16) fusion protein CBFβ-SMMHC with HDAC8, where HDAC8 aberrantly deacetylates p53, which promotes leukemia. Inhibition of HDAC8 restores p53 and induces apoptosis selectively in leukemic inv(16)+ CD34+ cells but spares normal CD34+cells [Mutations in the 34+cells .HDAC1/2 knockdown in early leukemogenesis expands the leukemia, whereas in leukemic phase, knockdown of the same HDAC1/2 cause differentiation and apoptosis of APL cells, and increased survival of APL mice [EZH2, SUZ12, JARID2 and also mutations in ASXL1 coding for the PRC2 associated protein ASXL1, have been found to be mutated in myeloid malignancies , where a translocation t15;17) generates the PML-RARα fusion protein. Normally, RAR (retinoic acid receptor) is a transcription factor and when retinoic acid is absent, RAR associates with HDAC1/2 containing complex SMRT/N-CoR and represses transcription. Retinoic acid causes release of the corepressor complex and leads to transcription. In APL cells, retinoic acid does not release the corepressor complex, resulting in a differentiation block. However, pharmacological doses of retinoic acid, as used in the clinic, degrade the fusion protein and the cells differentiate. Thus, HDAC play an important role in APL, however their role varies over time as APL mice . The polgnancies  [17,18,1 generate+ progenitor compartment of patients with MF, there is an increase in HDAC levels [HDAC enzymes (KDACs) are commonly mutated in solid tumors, with 30% of endometrial tumors having HDAC mutations, however only 2% of patients with AML have HDAC mutations . A thoroC levels . InteresC levels , and manC levels , and as In general, HDACi treatment alters the expression of 5–10% of transcribed genes, depending on cell type. The specific antitumor activity of HDACi varies between tumor types, and the antitumor activity on a specific tumor type may vary between different HDACi. The general mechanisms for HDACi induced cell death include apoptosis or autophagy, increasing ROS production and decreasing scavengers, increasing DNA damage and decreasing DNA repair, decreasing oncoprotein expression and stability and stimulating immunogenic cell death ,40,41. ITRAIL by activating the TNFS10 gene that encodes TRAIL, triggering death signal via the extrinsic pathway, and additionally RNA interference against TRAIL blocked downstream caspase activation and inhibited MS275 mediated apoptosis, suggesting that at least in AML cells, MS275 mechanism of action is via TRAIL [c-FLP and Bcl.2/Bcl-xL anti-apoptotic factors, as well as sensitized cells to TRAIL/Apo2L mediated apoptosis, thus acting on both the intrinsic and extrinsic apoptotic pathways [TRAIL, DR5, FasL and Fas in leukemic cells but not normal progenitors, thus for the sensitivity of HDACi to leukemia a transformed phenotype is required [Induction of apoptosis is the major route for HDACi induced cell death, and it may be via either the intrinsic (mitochondria) or extrinsic (death receptor) pathway. The specific changes in gene expression leading to apoptosis in myeloid malignancies varies between the different HDACi, but frequently, extrinsic pathway via TRAIL is used. In AML, HDACi MS275 treatment induces the expression of ia TRAIL . In K562pathways . In APL required .+ progenitor cells from AML and MDS patients, vorinostat induced gene expressions of COX2, p15, cFOS, genes that are downregulated in MDS and AML, and suppressed overexpressed genes cyclin D1 and c-MYC [Nur77 expression in AML cell lines and in AML patient leukemia cells, especially in the leukemic stem/very early CD34+/38− progenitors, and induce apoptosis, presenting a novel mechanism of action of HDACi and suggesting that Nur77 may be a biomarker for HDACi apoptotic effect [In a recent study of several AML cell lines as well as CD33nd c-MYC . This lend c-MYC . Recentlc effect . Thus, Hc effect .JAK2 mutated cells, normalizing splenomegaly and blood counts in JAK2 mutant knock-in mice [+ progenitor cells with panobinostat induces apoptosis and inhibits JAK2 expression and activity, subsequently reducing pSTAT3, pSTAT5, pAKT and pGATA1, and partially inhibiting the binding between HSP90 and JAK2, suggesting that acetylation of HSP90 could mediate JAK2 degradation [In AML/ETO, single agent valproic acid inhibits not only the mature leukemic cells but also immature progenitors by targeting the AML1/ETO-HDAC complex SMRT/N-CoR, inducing differentiation . In MPN,-in mice  and promradation . The samKIT D816V mutated primary patient mast cells whereas normal mast cells are unaffected [HDACi therapy in SM was first assessed in a canine model of SM . Our groaffected . To suppaffected . However+ cells were more sensitive than normal CD34+ cells to the treatment, indicating a specific anti-leukemic effect and that normal progenitors are spared. In a molecular study of clinical samples from the clinical trial of Tan et al., using azacitidine and panobinostat for MDS and AML [Nur77, p15 and p21 in the clinical patient samples, and found that restored levels of Nur77 and p21 correlated with clinical responses to the combination therapy [Nur77 as a biomarker of HDACi mediated apoptosis also in the leukemic stem cell compartment [There is a large body of preclinical evidence showing synergistic effects of various first and second generation HDACi in combination with azacitidine or decitabine in MDS and AML cell lines or ex vivo cultured patient cells. These include enhanced growth arrest, inhibition of DNA synthesis and loss of clonogenic potential, and synergistic effects in re-expressing silenced genes ,59,60. W and AML , Liu et partment . In MPN,partment .clinicaltrials.gov, using a plethora of HDACi agents (Single agent first and second generation HDACi have been tested in several small phase I and II studies for MDS and AML, showing low overall response rates and 0–10% partial or complete remissions, reviewed by Morabito et al. and by Stahl et al. ,64, withi agents . A review of valproic acid effects on AML cells conclude that single agent valproic acid may stabilize disease in the many old and fragile AML patients that are unfit for more intensive therapy, however as of yet, no randomized studies have been conducted . HoweverA number of HDACi have been investigated in clinical trials in MPN, recently reviewed by Bose and Verstovsek . OverallFour recent reviews on the combination trials of azacitidine or decitabine with HDACi conclude that there may be still a future for the drug combination, despite the lack of beneficial results in phase II trials ,83,84,85Despite a theoretical rationale and profound preclinical proof of HDACi efficacy in myeloid malignancies, all phase II randomized clinical trials have failed, except for the combination of HDACi with JAK2 inhibitor ruxolitinib in MF. However, optimizing combination treatment strategy, e.g., sequential treatment and not simultaneous, will be a key issue to avoid pharmacological antagonism, and requires further basic in vitro studies of optimizing drug scheduling and doses, as well as biomarkers to follow in vivo drug effects. In addition, novel, more selective HDACi should be preferred, avoiding off target effects. In conclusion, there may still be a role for HDACi in myeloid malignancies, beyond the promising combination therapy of HDACi and ruxolitinib for MF.I apologize to all authors that have made significant contributions to the field but was not cited in the current review, due to practical space limitations.


In this study, we treated herbaceous peony  with ethyl glycol tetraacetic acid (EGTA), an effective Ca2+ chelator, and used morphology indicators, spectroscopic analysis, histochemical staining, electron microscopy, and proteomic techniques to investigate the role of Ca2+ in inflorescence stem mechanical strength. The EGTA treatment reduced the mechanical strength of inflorescence stems, triggered the loss of Ca2+ from cell walls, and reduced lignin in thickened secondary walls in xylem cells as determined by spectroscopic analysis and histochemical staining. Electron microscopy showed that the EGTA treatment also resulted in significantly fewer xylem cell layers with thickened secondary walls as well as in reducing the thickness of these secondary walls. The proteomic analysis showed 1065 differentially expressed proteins (DEPs) at the full-flowering stage (S4). By overlapping the Kyoto encyclopedia of genes and genomes (KEGG) and gene ontology (GO) analysis results, we identified 43 DEPs involved in signal transduction, transport, energy metabolism, carbohydrate metabolism, and secondary metabolite biosynthesis. Using quantitative real-time polymerase chain reaction (qRT-PCR) analysis, we showed that EGTA treatment inhibited Ca2+ sensors and secondary wall biosynthesis-related genes. Our findings revealed that EGTA treatment reduced the inflorescence stem mechanical strength by reducing lignin deposition in xylem cells through altering the expression of genes involved in Ca2+ binding and secondary wall biosynthesis.The mechanical strength of inflorescence stems is an important trait in cut flowers. Calcium ions , a chemical that binds to and removes calcium ions. This resulted in the reduced deposition of a structural protein called lignin in the walls of xylem cells and more pliable stems. Further analysis revealed altered expression of 43 proteins, including those involved in calcium-ion sensing and construction of ‘secondary’ walls in xylem cells within the stem. In plants, Ca2+ signals transient increases in cytosolic free Ca2+. Stimulus-induced increases in the concentration of free Ca2+ in the cytosol often occur as repetitive oscillations or spiking of cytosolic-free Ca2+. Ca2+ encodes stimulus-specific information within a so-called Ca2+ signature, and thus defines the nature and magnitude of the response7. Recently, Ca2+ has received increasing attention due to its positive correlation with the straightness of herbaceous peony  inflorescence stems, which is a very important quality-assessment parameter for cut flowers8. Inflorescence stem straightness is mainly determined by its mechanical strength9. After analyzing the Ca2+ concentration of inflorescence stems in 76 different P. lactiflora cultivars, Li et al.10 found a significant positive correlation between Ca2+ content and mechanical strength in inflorescence stems. Spraying stems with exogenous 4% calcium chloride (CaCl2) also enhanced inflorescence stem mechanical strength11. However, although the exogenous Ca2+ treatment enhances inflorescence stem mechanical strength, no studies have reported the effects of Ca2+ deprivation on P. lactiflora inflorescence stem mechanical strength.Calcium ions  flowers, inflorescence stem bending was associated with the absence of lignin deposition in sclerenchyma cells. This has also been verified in rice (Oryza sativa L.) brittle culm 3 mutants15. In addition to lignin, cellulose synthesis is also essential for proper secondary wall construction16.The mechanical strength in 2+ plays a positive role in pollen tube cell wall formation in apple 17 and in the fruit cell walls of jujube (Zizyphus jujuba Mill. cv. Dongzao)18. However, studies on the effects of Ca2+ on cell wall formation and mechanical strength have focused on the physiological level, and the underlying molecular mechanisms remain unclear. Here, we investigated the hypothesis that a lack of Ca2+ causes inflorescence stem bending due to reduced mechanical strength. Ethyl glycol tetraacetic acid (EGTA), a Ca2+ chelator that binds Ca2+20, was used for Ca2+ deprivation treatment. The relationship with mechanical strength was explored by studying the morphological indices of inflorescence stems and flowers, the extension of inflorescence stem secondary walls, inflorescence stem cell wall composition, protein changes in the inflorescence stem, and expression changes of secondary wall biosynthesis-related genes in the inflorescence stem.CaP. lactiflora growth and development. Specifically, the upper part of the P. lactiflora inflorescence stems was less straight after EGTA treatment compared to the control at all four flower-development stages (flower-bud stage (S1), pigmented stage (S2), unfold-petal stage (S3), and full-flowering stage (S4); Fig. P. lactiflora morphological changes induced by EGTA treatment, five morphological indices, including the upper inflorescence stem mechanical strength, diameter and weight, and the flower diameter and weight at these four flower developmental stages were measured. Values for all five morphological indices were significantly lower in plants treated with EGTA compared to controls. The upper inflorescence stem mechanical strength, which was an average of 28% lower than the control, was consistent with the results observed by the naked eye  and Fourier-transform infrared spectroscopy (FTIR) were performed. Peaks for carbon1s (C1s), nitrogen1s (N1s), oxygen1s (O1s), and calcium2p (Ca2p) were identified in both control and EGTA-treated cell walls , 1640 (amide I C = O), 1510 , 1465, 1425, 1325 (lignin), 1375 (CH band), 1245 (amide III in protein), and 1160, 1107, 1060 and 899 cm-1 (CHO)22. Although the FTIR spectra of cell walls in control and EGTA-treated plants were essentially similar, the relative absorbances of the peaks were different with EGTA treatment compared to the control . The walls in the xylem cells thickened from S2 to S4 Fig. . With EGTo investigate the underlying molecular mechanisms in response to EGTA treatment, control and EGTA-treated inflorescence stems at S4 were sampled for proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ). As a result, 256,853 spectra with 19,326 matching known peptides were obtained, and 5045 proteins were identified. Under EGTA treatment, 1065 differentially expressed proteins (DEPs) compared to the control were found. Of these, 533 were upregulated and 532 were downregulated. To further explore the functions of these DEPs, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were implemented.2+ sensor involved in Ca2+ signature transduction, and ATP-binding cassette, subfamily G, member 2 (ABCG2) plays an important role in transport.Of the 1065 DEPs, 554 were annotated via GO analysis. A total of 1186 GO terms were searched, of which 746 were involved in biological processes, 143 in cellular components, and 297 in molecular function categories (Supplementary Table P<0.05) in four pathways, including ribosome (63 DEPs), glycosylphosphatidylinositol (GPI)-anchor biosynthesis (3 DEPs), phenylalanine metabolism (12 DEPs), and phenylpropanoid biosynthesis , which inhibits the signal transduction of abscisic acid, was observed. The remainder of the upregulated DEPs, such as NADH dehydrogenase Fe-S protein (Ndufs), V/T-type H+-transporting ATPase, and 6-phosphofructokinase 1 (PFK), were involved in oxidative phosphorylation and sugar metabolism. Of the downregulated proteins, DELLA protein, which acts as a negative regulator of gibberellin biosynthesis, was most significantly suppressed by EGTA treatment. CAD, peroxidase (POD), and ATP-binding cassette (ABC) enzymes were involved in lignin biosynthesis, and cellulose synthase A catalytic subunit 7 (CESA7), which is involved in cellulose biosynthesis, was also inhibited by EGTA treatment. Additionally, the Ca2+ sensor CML and MYB transcription factor were also suppressed by EGTA treatment.In addition, we found 43 DEPs (17 upregulated and 26 downregulated) from the overlapping KEGG and GO analysis results Fig. . These D2+-binding proteins related to secondary wall biosynthesis were identified through proteomic analyses. To characterize the effect of EGTA treatment on Ca2+ binding and secondary wall biosynthesis in inflorescence stems, we examined the expression of genes corresponding to these proteins by quantitative real-time polymerase chain reaction (qRT-PCR). These genes included calmodulin (CaM)/CML and calcium-dependent protein kinase (CDPK), which are involved in Ca2+ binding, and an MYB gene that acts as a transcription factor regulating secondary wall biosynthesis. Additionally, phenylalanine ammonia-lyase gene (PAL), 4-coumarate-CoA ligase (4CL), and CAD, which are involved in the phenylpropanoid pathway and important for monolignol synthesis, were analyzed, as well as POD and ABC genes (such as ABCC2 and ABCG2), which are related to monolignol transport. We also analyzed the expression of calcineurin B-like protein-interacting protein kinase (CIPK), cinnamate 4-hydroxylase (C4H), cinnamoyl-CoA reductase (CCR), caffeoyl shikimate esterase (CSE), caffeic acid 3-O-methyltransferase (COMT), and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), which are related to Ca2+-binding proteins and secondary wall biosynthesis.CaCDPK, CAM, and ABCC2 were expressed at the highest levels, whereas PAL, C4H, 4CL, and POD were expressed at the lowest levels  mutants is notably thinner than those in wild-type nodes, and similar results were found in the flexible culm 1 mutants16. In our study, similar results were found for secondary walls, which were thinner with EGTA treatment compared to the control. The thinner thickened secondary walls distributed around the xylem are important for stem mechanical strength in P. lactiflora, but the thickened secondary walls of cells under the epidermal layer are not. Interestingly, we found a nearly 50% decrease in the number of xylem cells in thickened secondary walls after EGTA treatment compared to controls. The reduction in xylem cells in thickened secondary walls  was more important than the reduction of these thickened secondary walls in causing loss of mechanical strength.In ornamental plants, morphological characteristics, especially inflorescence stem mechanical strength, play crucial roles in the quality of cut flowers. Inflorescence stem mechanical strength is correlated with internode number, stem length, weight, and diameter26. Wang et al.21 reported that among the three genotypes of wheat (Triticum aestivum L.), the genotype with the lowest bending stress contained the least lignin. In O. sativa, decreased accumulation of lignin was also observed in brittle culm 5 nodes24. In ornamental plants, similar results have been reported for G. jamesonii14 and P. lactiflora8. In our study, we also found less lignin accumulation in EGTA-treated plants compared to controls. The weakened inflorescence stems were primarily due to the decreased lignin accumulation induced by EGTA treatment.Secondary walls are composed mainly of the polysaccharides cellulose and hemicellulose, as well as lignin and cell wall proteins. Mechanically, secondary walls can be compared to reinforced concrete, with the cellulose microfibrils that are embedded in an amorphous matrix of lignin 25 being analogous to steel rods . Therefore, stem mechanical strength is determined mainly by the properties of the secondary walls, especially ligninNAC and MYB transcription factors. These regulators together activate downstream transcription factors and secondary wall biosynthetic genes27. In this study, both the NAC and MYB transcription factors had lower expression in response to EGTA treatment compared to the control. NAC transcription factors are the master switches that activate secondary wall biosynthesis in vessels and fibers28. The MYB46 and MYB83 transcription factors are second-level master switches that activate the expression of many lignin biosynthetic genes29. The suppression of NAC and MYB transcription factors by EGTA treatment affected the expression of downstream transcription factors and secondary wall biosynthetic genes and eventually influenced secondary wall deposition. However, the functions of NAC and MYB in this study were not conclusive, and additional studies are needed.The transcriptional regulation of secondary wall deposition is a multi-leveled, hierarchical network, mainly regulated by 31. The lignin monomer-biosynthesis pathway is well established, and several genes for lignin biosynthesis have been identified, including PAL, C4H, 4CL, CCR, CAD, p-hydroxycinnamoyl-CoA:quinate/shikimate p-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), CSE, ferulate 5-hydroxylase (F5H), COMT, and CCoAOMT 34, 4CL in hybrid poplar 35, CCR in birch (Betula platyphylla × Betula pendula)36, CAD in Arabidopsis thaliana37, and a CCoAOMT homolog in tobacco (Nicotiana tabacum)38 leads to varying degrees of reduction in lignin content compared to controls. The suppression of these genes also affects plant development, especially the repression of CCR, which reduces the thickness of secondary walls. The expression level of COMT is lower in Chlorogalum grandiflorum varieties with a poor vase life, which is consistent with reduced lignin content and pedicel rigidity39. The CSE-2 loss-of-function mutant shows the typical phenotype of lignin-deficient mutants40. Low expression of lignin biosynthesis-related genes in EGTA-treated inflorescence stems caused a reduction in lignin accumulation.Lignin is a phenolic component of cell walls that plays a role in maintaining cell wall rigidity and can be categorized as hydroxyphenyl (H), guaiacyl (G), or syringyl (S) ligninOMT Fig. 25,32. In2+, and it does not cross the cell membrane41. In our study, the concentration of Ca in inflorescence stem cell walls was lower in plants treated with EGTA, indicating that EGTA chelated Ca2+ in the cell wall. Ca2+ binds with pectin and plays an important role in cell wall structural rigidity. Chelation of cell wall-associated Ca2+ reduced the Ca2+ concentration in cell walls and caused a low Ca2+ environment in the cytoplasm. We surmised that like stimuli such as mechanical injury, cold or ABA, which induce spontaneous Ca2+ oscillations, EGTA treatment may also induce Ca2+ oscillations and generate a Ca2+ signature. Plants have a unique repertoire of Ca2+-binding proteins to decode Ca2+ signatures in the gene families of CAMs/CMLs, CDPKs, and CIPKs43. These proteins sense the Ca2+ signature and activate downstream transcription factor activity and gene expression to alter biochemical function through reversible phosphorylation7. We found that the expression levels of proteins involved in oxidative phosphorylation changed in response to EGTA treatment. The expression of H+-transporting ATPases and several NADH dehydrogenase enzymes was upregulated, whereas the expression of NADH dehydrogenase Fe-S proteins and V/F-type H+-transporting ATPases was downregulated, which suggested that EGTA application affected the phosphorylation of proteins involved in the TCA cycle and glycolysis in inflorescence stems and was also related to signal transduction. CDPK, a Ca2+ sensor, plays an important role in plant cell wall development and secondary growth. For example, Zhao et al.44 found a major role for CDPK11/24 function in plant pollen-tube growth in double mutant lines, and Matschi et al.45 reported that secondary growth is inhibited by a single CDPK (AtCPK28) loss-of-function mutant in Arabidopsis. CAM and CML, members of the NAC, MYB, and WRKY TF families, regulate secondary wall biosynthesis and interact with Ca2+/CaM46. In our study, the expression levels of these Ca2+ signal sensors were different in treated plants compared to controls, suggesting that EGTA application generated Ca2+ signatures that were sensed by CDPK, CAM/CML, and CIPK. The differences in the expression levels of these proteins influenced downstream secondary wall development and thereby reduced the inflorescence stem mechanical strength in P. lactiflora.EGTA chelates cell wall-associated Ca2+ deprivation inhibits photosynthesis by reducing water absorbance and regulation, resulting in leaf wilting and closure of the stomata on the leaves. This prevents gas exchange and leads to a reduction in the cellular carbon dioxide (CO2) concentration and concomitantly rubisco carboxylase activity19. Pn, Tr, and Gs decreased in Japanese morning glory  after EGTA treatment47, which is consistent with the results of our study. Therefore, Ca2+ plays an important role in photosynthesis, and Ca2+ deprivation would suppress photosynthesis in leaves. The reduction in photosynthesis would cause the yield of photosynthetic products available for secondary wall biosynthesis to also be lower. In this study, the expression levels of proteins functioning in sugar metabolism were upregulated, whereas those of proteins that function in decomposition were downregulated. This result suggested that the reduced dry-matter accumulation was due to the lower rates of photosynthesis and altered sugar metabolism, which affected the synthesis of secondary walls.Ca2+ chelator, caused Ca2+ deprivation in P. lactiflora and inhibited plant development, including the mechanical strength of inflorescence stems. We have put forward a hypothetical mechanism by which EGTA application reduced the mechanical strength of inflorescence stems , was used as the experimental material. Specimens were planted in 60 rows with two plants per row in the field and divided into two groups with 30 rows per group. One group was sprayed with 10 mmolL-1 EGTA, and the other was sprayed with distilled water as a control. Treatments were done once a week from the leaf-expansion stage on 30 March to the bloom stage, with a total of five applications. During the development of P. lactiflora, morphological indices and photosynthetic characteristics were measured every 8 days from 7 April to 1 May, including four developmental stages (S1 to S4), and the upper parts (12 cm) of thirty inflorescence stems were collected as samples. Subsequently, 5- to 8-cm lengths of the upper part of inflorescence stems were cut into 5-mm sections and fixed with 2.5% glutaraldehyde (GA) solution in 0.1 molL-1 potassium phosphate buffer solution . Other samples were fixed with formalin-aceto-alcohol solution for microstructure observation. The remainder of the inflorescence stem samples was immediately frozen in liquid nitrogen and stored at -80 °C until examination.12. Fully expanded leaves at the fourth apical node were selected to measure photosynthetic characteristics, including Pn, Tr, and Gs, using a portable photosynthesis system  between 8:00 and 10:00 am, before each collection.Morphological indices were measured as described previously48. X-ray photoelectron spectroscopy determination of cell wall elements was performed on a spectrometer  using a set monochromatic radiation A1Kα source with a background pressure of 5 × 10-10 mbar. Each analysis consisted of a wide survey scan  FTIR determination of cell wall materials and was analyzed by an infrared spectroscopy (IR) spectrometer  using the KBr pellet technique. Spectra were taken in the wave number range from 4000 to 400 cm-1 with a resolution of 4 cm−1 and 32 scans per sample. All spectra were normalized and baseline-corrected with OPUS management software.Cell wall materials were extracted from inflorescence stems as described previouslyStem sections were fixed with formalin-aceto-alcohol for 2 h at room temperature and at 4 °C for a minimum of 1 day. Afterwards, the samples were dehydrated in a graded ethanol series, dehydrated with a xylene–ethanol mixture solution , and finally dehydrated with a xylene–chloroform mixture  twice. After infiltration and embedding in paraffin, 8 μm sections were cut using a rotary microtome  and mounted on glass slides. After the paraffin was removed with xylene, sections were rehydrated in a graded ethanol series followed by distilled water and finally air dried. To detect lignin, the sections were stained with 2% (w/v) phloroglucinol  for 2 min and then treated with 18% hydrochloric acid. The stained sections were observed immediately under a light microscope . For cellulose, the sections were stained with 0.1% calcofluor white M2R in 0.05% Evans blue  for 2 min and observed under a fluorescence microscope with a UV excitation filter SZX2-FUV, a GFP high-performance filter SZX2-FGFPHQ, and an RFP filter SZX2-RFP1 for calcofluor staining .-1 PBS (pH 7.8) for 2 h at room temperature and at 4 °C for more than 1 day. They were rinsed with the same buffer three times (15 min each). For SEM observation, the samples were dehydrated and critical-point dried, sputter-coated with gold, and observed with an SEM  under 5 kV. For TEM observation, the samples were post-fixed in 1% osmium tetroxide in 0.1 molL-1 PBS (pH 7.8) for 4 h at 4 °C after GA. After dehydration in an ethanol series, the samples were infiltrated and embedded in butyl-methyl methacrylate. Seventy-nanometer sections were cut, stained with uranyl acetate and lead citrate, and viewed with a TEM .Inflorescence stem sections were fixed with 2.5% GA in 0.1 molL49, with a few modifications. Briefly, the extraction and purification of protein followed the manufacturer’s instructions. The control samples were labeled with iTRAQ tags 114, 115N, and 115C, and the EGTA-treated samples were labeled with 117C, 118N, and 118C. The combined peptide mixtures were lyophilized. After trypsin digestion, the peptides were desalted on a C18 column (Phenomenex) and dried in a spin vacuum. The desalted peptides (100 μg) were labeled with iTRAQ 6-plex reagent in 200 mmolL-1 triethylamonium bicarbonat (TEAB) according to the manufacturer’s instructions. The peptides labeled with different reagents were combined, vacuum-dried, and separated on an LC-20AB HPLC Pump system  coupled with a high-pH RP column. Subsequently, the peptides were injected into the tandem mass spectrometer Q EXACTIVE  for data-dependent acquisition detection by nano-electrospray ionization.Samples at S4 were used to perform proteome analyses. Protein extraction, quantification, iTRAQ labeling and strong cation-exchange fractionation, LC-ESI-MS/MS analysis, database search, quantification bioinformatics, and annotations were performed as described previouslyt test was used to analyze the significance of protein geometric ratios, and the Benjamini-Hochberg multiple hypothesis test correction was employed to correct the P values. DEPs were screened out using 1.2 normalized protein geometric ratios at a P < 0.05 level with 5% false discovery rate (FDR) correction.Raw data were processed with OpenMS software and searched against the SwissProt and common MS contaminant database using Mascot software. Trypsin was chosen as the enzyme with one missed cleavage allowed. The fixed modification carbamidomethylation was set for cysteine and the variable modification oxidation for methionine. The peptide tolerance was set as 0.05 Da, and the MS/MS tolerance was set as 0.1 Da. The ratio of each protein is given by the geometric mean of the protein ratios measured from all replicates. Student’s -△△Ct comparative threshold cycle method50.Gene transcript levels were analyzed using qRT-PCR with a BIO-RAD CFX ConnectTM Optics Module . cDNA was synthesized from RNA using the PrimeScript® RT Reagent Kit With gDNA Eraser . All gene-specific primers in this study are shown in Supplementary Table All experiments described here were repeated three times in a completely randomized design. Primers were designed using the Primer 5.0 program. All data were means of three replicates with standard deviations. The results were analyzed for variance using the SAS/STAT statistical analysis package .Supplementary Table S1-S5


Vacuolization is a frequently found morphological feature in acute myeloid leukemia (AML) blasts. Subcellular origin and biological function as well as prognostic impact are currently unknown. The aim of this study was to evaluate whether vacuolization correlates with clinically relevant AML features.Bone marrow smears of patients diagnosed with AML at the University Hospital Frankfurt between January 2011 and August 2013 were analyzed for blast vacuolization and correlated with clinically relevant AML features. Patients undergoing standard induction chemotherapy were further analyzed for molecular and cytogenetic features as well as treatment response and survival.14 of 100 patients diagnosed with AML receiving standard induction chemotherapy had evidence of blast vacuolization. Positivity for vacuolization correlated with a CD15 positive immunophenotype and with a higher incidence of high-risk AML according to the European LeukemiaNet risk stratification. AML patients with blast vacuolization had a poor blast clearance after standard induction chemotherapy and poor survival.In conclusion, our findings demonstrate that vacuolization can easily be determined in myeloid leukemia blasts and may be a useful biomarker to predict AML risk groups as well as early treatment response rates and survival. These m therapy , AML-M4Evacuoles - needs to be confirmed by further immunophenotypic analysis, since the discrimination between mature B-ALL (Burkitt lymphoma/leukemia) and Pro-B-ALL has impact on the choice of therapy and prognosis [We observed blast vacuolization in a subset of AML patients. A literature review revealed only limited information about the frequency and no information about the biological function or the clinical significance of blast vacuolization in AML patients. Gervais et al. analyzed 33 AML patients with MYST histone acetyltransferase 3 (MYST3) rearrangements for its most relevant characteristics. Cytoplasmic vacuoles were noted in 21 of these cases (no pictures) with no further information . Studiesrognosis , 10.Although light microscopy has been an established diagnostic tool for decades in diagnosing AML, to our knowledge there are no reports about the clinical role or biological function of vacuoles in myeloid blasts. In this work we evaluated the association of positivity for vacuolization in AML blasts with molecular and cytogenetic AML features, as well as with early treatment response and survival rates.In this retrospective single center study bone marrow smears from AML patients diagnosed at the University Hospital Frankfurt between January 2011 and August 2013 were analyzed for vacuolization. After bone marrow puncture at time of diagnosis bone marrow smears were prepared using May-Grünwald-Giemsa staining . Blast c7+3-regime; cytarabine 100mg/m2 given intravenous (IV) continuously for 7 days is combined with daunorubicin 60mg/m2 given as a 30minute IV infusion on days 3, 4 and 5 [7+3 if early blast clearance was achieved on day 15 bone marrow evaluation or with high-dose cytarabine using the HAM protocol  if blast clearance was not achieved on day 15 bone marrow evaluation [2), if the first induction chemotherapy cycle was not sufficient to achieve bone marrow blast clearance on day 15 [Standard intensive induction chemotherapy was the so-called  4 and 5 . Patientn day 15 . Poor ren day 15 .t-test or the Wilcoxon-Mann-Whitney test accordingly. Fishers exact test was used for binary variables. Death rates were analyzed by Kaplan-Meier method and curves were compared by log-rank test. For assessment of independent predictors of blast clearance on day 15 bone marrow evaluation, a binary logistic regression was used. For assessment of independent predictors of survival, a multivariate Cox regression hazard model with forward stepwise (likelihood ratio) entry was used, factors with p<0.1 in the univariate analysis were included. Statistical analysis was performed with SPSS .This study was designed as a retrospective cohort study. Patients were followed till death or last contact. Continuous variables are shown as median ± range and categorical variables are reported as frequencies and percentages. All continuous variables were tested for normality and were analyzed by using the Student´s In this retrospective single center study we analyzed bone marrow smears from 134 AML patients diagnosed at the University Hospital Frankfurt between January 2011 and August 2013 for vacuolization. Vacuolization of AML blasts was found in 16 of 134 AML patients (11.9%) while 118 patients had AML without blast vacuolization. 34 patients went on to receive a non-intensive chemotherapy due to advanced age or bad performance status. After exclusion of those patients, 14 patients (14%) with vacuolized AML blasts and 86 patients (86%) with nonvacuolized AML blasts were further investigated.Table 1).Median age was 64.5 years (range 34–74) in patients with vacuolized AML blasts and 58.5 years (range 18–81) in AML patients without evidence of blast vacuolization (p = 0.344). At time of diagnosis there was no significant difference between the two groups with respect to gender, lactate dehydrogenase (LDH), WHO classification, peripheral blood cell counts or blast count in the bone marrow or in the peripheral blood. . The appearance can range from approximately 50 to over 1000 vacuoles per 100 AML blasts. The distribution within AML blasts can differ from few single vacuoles per blast cell to groups of >35 vacuoles vacuoles per blast cell. The median percentage of blasts with at least one vacuole was 0% (range 0–8) in AML patients without blast vacuolization and 29% (range 11–91) in AML patients with blast vacuolization. The size of a vacuole ranges from 0.5–2.0 μm. In AML patients without vacuolization generally no vacuoles can be found in any views of different bone marrow smears .The vacuoles seen in AML blasts localize mostly in the cytoplasma, rarely vacuoles project above the nucleus (Table 2). However all AML patients with blast vacuolization who were tested for CD15 expression were positive .No significant difference was seen in the expression of the myeloid markers , the progenitor cell markers  or the lymphatic markers  between patients with and without vacuolized AML blasts (Table 3). Cytogenetic aberrations were found in 8 patients with vacuolized AML blasts and in 32 AML patients without blast vacuolization . A significantly higher incidence of adverse-risk AML was found in AML patients with blast vacuolization when cytogenetic and molecular genetic data was combined into genetic risk groups according to the 2010 ELN guidelines  [Table 4.Molecular aberrations were equally distributed between patients with and without vacuolized AML blasts (= 0.026) . DetaileTable 5). Consequently, only for two (22.2%) of the patients with vacuolized AML blasts a standard induction chemotherapy with the 7+3 protocol was sufficient to achieve a complete remission (CR), whereas for 40 (64.5%) of the AML patients without blast vacuolization the 7+3 protocol was sufficient for CR achievement (p = 0.0289).Day 15 bone marrow evaluation was performed for 12 of the AML patients being positive for vacuolization and in 81 of the other AML patients. Missing day 15 bone marrow evaluation (7 patients) was due to death (4 patients), due to bone marrow smears with insufficient quality for cytological examination (1 patient), due to refusal of consent for performance of bone marrow aspiration (1 patient) and in one patient day 15 bone marrow evaluation was not performed due to treatment on intensive care unit for myocardial infarction. Patients with vacuolized AML blasts had a significantly worse blast clearance rate on day 15 after induction chemotherapy  (Those patients not achieving a blast clearance on d15 went on for a second more intensive induction chemotherapy (HAM) to salvage for a CR. Cytological CR rates after completion of induction chemotherapy were equal between the two groups .Table 6vacuolization of AML blasts was an independent risk factor for poor blast clearance on day 15 bone marrow evaluation after induction chemotherapy (odds ratio (OR) = 8.042, 95% confidence interval (CI) 1.590–40.692, p = 0.012).To further analyze vacuolization of AML blasts as a predictor for incomplete blast clearance on day 15 bone marrow evaluation a binary logistic regression model with forward stepwise likelihood ratio was performed. The nominal dichotome variables, age above 60, gender, vacuolization of AML blasts, abnormal karyotype and adverse-risk AML were included in this model. As shown in Fig 2. Here, AML patients with blast vacuolization showed a poor survival rate. To further analyze blast vacuolization as a prognostic parameter in AML patients undergoing intensive induction chemotherapy a multivariate Cox regression model with forward stepwise likelihood ratio was performed. The nominal dichotome variables age above 60 years, male sex, adverse-risk AML, allogenic SCT as consolidation therapy, CR after induction chemotherapy and vacuolization of AML blasts were included in this model. As shown in Table 7age above 60 years, allogenic SCT as consolidation therapy, CR after induction chemotherapy and vacuolization of AML blasts were independently associated with OS.We analyzed the survival of both patient cohorts. Kaplan-Meier estimates for OS are displayed in During our routinely cytological diagnostic we repeatedly noticed the appearance of vacuoles in myeloid leukemia blasts. We found description of this morphological feature in some but not all reports written by the examining cytologist when diagnosing AML. Our literature review revealed very limited data about the appearance of blast vacuolization in AML patients and no data about its etiology or clinical role –8. To ouet al. published a large retrospective analysis of 460 AML patients—173 being positive for CD15—and demonstrated CD15 to be of independent prognostic value for CR achievement. Those patients tend to have a superior survival rate, although this analysis missed statistical significance [In our study vacuolization of AML blasts at diagnosis correlates with a CD15-positive immunophenotype. CD15 is a cluster of differentiation antigen mediating chemotaxis and phagocytosis . Its proificance . This racup-like acute myeloid leukemia with mutations of FLT3 and NPM1 [Recent studies identified a new cytomorphological feature in AML blasts and found a correlation of the so-called and NPM1 –24. Thus7+3 protocol. However, blast persistence on day 15 was successfully salvaged with high dose cytarabine combined with mitoxantrone (HAM) also in AML patients with blast vacuolization, suggesting a sensitivity to cytarabine of the vacuolized AML subgroup. As a result both populations had equal CR rates after admission of the second induction chemotherapy cycle. Further prospective analysis will have to investigate whether an initial treatment with HAM should be favored over the standard induction chemotherapy with 7+3 in AML patients with blast vacuolization.We analyzed the impact of AML blast vacuolization on day 15 blast clearance and complete remission rates after induction chemotherapy. AML patients being positive for blast vacuolization had a significantly reduced blast clearance on day 15 bone marrow evaluation and consequently a poor response rate to standard induction chemotherapy with the The Kaplan-Meier estimates for OS showed poor survival for AML patients with blast vacuolization and our multivariate analysis identified blast vacuolization to be independently associated with OS. A limitation of our analysis is the limited number of AML patients and the low percentage of patients being positive for blast vacuolization. Retrospective analyses of a larger cohorts are required to address the impact of blast vacuolization in AML patients on OS in more detail.in vitro when treating AML U937 cell lines with lapatinib and demonstrated that these vacuoles appear with lapatinib-induced autophagic cell death [In conclusion, vacuolization can easily be determined in myeloid leukemia blasts by light microscopy. AML patients with blast vacuolization in bone marrow smears have reduced responsiveness to standard induction chemotherapy and impaired survival. Whether vacuoles are also found in the peripheral blood was not analyzed in our study. We hypothesize that vacuoles in AML blasts may represent dysfunctionally enlarged autophagosomes or other cellular organelles. Indeed, Chen et al. (2016) detected a similar blast vacuolization ll death . NeverthS1 Table(SAV)Click here for additional data file.


Because of its slow forward rate for binding, EGTA is thought to not capture calcium ions in very proximity to a channel, whereas it does capture calcium ions at the remote distance. However, in this study, our reaction diffusion simulations (RDSs) of Ca2+ combined with a release calculation using vesicular sensor models indicate that a high concentration of EGTA decreases Ca2+ and vesicular release in the nanodomain of single channels. We found that a key determinant of the effect of EGTA on neurotransmitter release is the saturation of the vesicular sensor. When the sensor is saturated, the reduction in the Ca2+ concentration by EGTA is masked. By contrast, when the sensor is in a linear range, even a small reduction in Ca2+ by EGTA can decrease vesicular release. In proximity to a channel, the vesicular sensor is often saturated for a long voltage step, but not for a brief Ca2+ influx typically evoked by an action potential. Therefore, when EGTA is used as a diagnostic tool to probe the coupling distance, care must be taken regarding the presynaptic Ca2+ entry duration as well as the property of the vesicular Ca2+ sensor.The exogenous Ca Calcium ions entering through a channel pore diffuse to vesicular Ca2+ sensors. During this diffusion, part of calcium ions binds to Ca2+ buffers present in the cytosol. Thus, the probability of vesicular release, i.e., the output from the presynaptic nerve terminal, is determined by the combination of Ca2+ influx, the properties of Ca2+ buffers and Ca2+ sensors, and the distance between VGCCs and Ca2+ sensors. This coupling distance between VGCCs and Ca2+ sensors is an important determinant of the [Ca2+]i sensed by the Ca2+ sensor, because the spatial concentration gradient of Ca2+ formed around the open channel is steep. This gradient was first postulated using a mathematical model of Ca2+ diffusion, referred to as the “Ca2+ microdomain” ethane-N,N,N′,N′-tetraacetic acid] have similar binding affinities, but the forward binding kinetics of BAPTA is ∼40 times faster than that of EGTA  indicated that EGTA can inhibit vesicular release in the immediate vicinity (∼20 nm) of VGCCs, in the nanodomain coupling distance , 2018. Ael pores , little 2+]i around an open VGCC using linearized buffer approximation  method using the Java-based simulation environment D3D. The total simulation volume was set to 1.0 (x) × 1.0 (y) × 1.0 (z) μm, which was divided into the elemental simulation voxels of 10 × 10 × 10 nm for finite difference calculations. With this voxel size, a concentration of 1 mM is equal to 602 molecules per voxel. The time step of simulation was 0.06 μs, and the resultant Ca2+ wave outputs were at 100 kHz. In this dimension, one simulation trial (10 ms in duration) typically took ∼7 h using a Windows 10 PC equipped with Intel Xeon CPU .To estimate i waves were imported into IgorPro 8 (WaveMetrics) and analyzed using NeuroMatic  = 40 . To focuuroMatic .2+-dependent vesicle fusion derived from experiments in the mature calyx of Held (2+](t) generated from RDS. The probability over time of arriving in the fused state is considered the probability that a single vesicle will be released as a function of time (single vesicular release rate). The cumulative release rate, which is equivalent to the vesicular release probability (Pv), was estimated from the integral of the single vesicular release rate over a window from the onset of VGCC opening to 10 ms after closing. With the assumption that the Ca2+ sensors for vesicular release are juxtaposed to the release face membrane, Pvs were calculated only in the voxels on the synaptic surface (z = 0).A five-site model of Ca of Held  was used2+]i gradient around a single open VGCC, I first used the analytical solution. When a calcium ion enters a channel pore, it is unbound (free ion) and starts diffusing. The increment in [Ca2+]i (Δ[Ca2+]i) by diffusion is inversely proportional to the distance (r) from the channel pore. Assuming that Ca2+ is diffusing into a hemisphere adjacent to the plasma membrane, it is calculated by:To quantify the i, and 10 mM BAPTA  strongly decreased [Ca2+]i to 4.8 μM even at a distance of 20 nm (in the presence of ATP).According to Eqs 2 and 3, τ and λ for 0.1 mM EGTA were 1.63 ms and 599 nm, respectively. Thus, when the basal resting [Ca2+]i at an arbitrary distance from the VGCC, it has several limitations because of its assumptions. First, under low-buffered conditions, LBA does not provide reliable estimates because Ca2+ influx could substantially alter the free buffer concentration. Second, as LBA assumes an instantaneous establishment of steady-state Δ[Ca2+]i gradient after channel opening and calculates the steady-state Δ[Ca2+]i, it does not provide for time-dependent changes in [Ca2+]i. To circumvent these limitations, I next estimated local [Ca2+]i gradients using RDS. To simulate time-dependent [Ca2+]i changes, I used a single channel current of 0.3 pA . This difference became apparent in the plot showing entire spatial [Ca2+]i gradients  with that in the presence of 10 mM chelators. As LBA cannot reliably predict [Ca2+]i under low-buffered control conditions, I used [Ca2+]i calculated from RDS. As predicted, both EGTA and BAPTA nearly completely inhibited Δ[Ca2+]i at more distal locations . By contrast, at a 10 nm distance from the VGCC, EGTA inhibited Δ[Ca2+]i by only 15%, whereas BAPTA inhibited it by 83%. Thus, EGTA was much less potent in inhibiting Δ[Ca2+]i than BAPTA at locations close to the channel . When the EGTA concentration is, for example, 10 mM, τ is 16 μs (this is quite fast and approaches the assumption of LBA). However, because each free calcium ion randomly moves in a Brownian motion, the establishment of steady-state [Ca2+]i might take longer at more distal locations. To examine this possibility, the 50% rise time of [Ca2+]i transients was measured. At EGTA concentrations of <0.1 mM, [Ca2+]i transients never reached steady state during the 5 ms channel opening, both at 20 nm and at 100 nm distances  against λ (Eq. 3) on the same graph , squares2+ sensors on vesicles might also act as EFBs. Calcium ions bound by mobile buffers retain their mobility because they diffuse together with their shuttle, whereas those bound by an EFB cannot diffuse. The EFB retards the distribution of calcium ions and delays the establishment of steady-state [Ca2+]i by competing with mobile buffers. In LBA, the contribution of EFB is omitted because once the [Ca2+]i reaches steady state, the standing [Ca2+]i gradients are identical to those without an EFB  and affinities. Because EFB κ is low in most presynaptic terminals, I used a κ of 15 (KD = 100 μM) increased the time to reach steady state without affecting the steady-state amplitude of [Ca2+]i  to 100 (10 mM EFB) increased the time constant for the [Ca2+]i transient at all distances , the EFB concentration was concomitantly adjusted in this simulation. The lowest KD (100 μM) slowed the rise kinetics the most. At the 20 nm distance, the rise time increased ∼20-fold from 3.4 to 66 μs  reduced the peak [Ca2+]i from 41 μM to 34 μM at 20 nm from VGCC  reached 1 under the control condition. The addition of 10 mM EGTA reduced [Ca2+]i and slightly slowed the rise of Pv, but the Pv similarly reached and remained at 1 , this decrease in [Ca2+]i did not affect the Pv. At distal locations (>50 nm), the decrease in [Ca2+]i by EGTA was seen as a decrease in the Pv, because the vesicular Ca2+ sensor entered a linear range.I next examined the effect of EGTA on the Pv, which was estimated from the waveform of i from 41 μM to 35 μM (by 15%) and the Pv from 0.07 to 0.03 (by 52%) at a 20 nm distance from the VGCC  was already in or near the linear range of the vesicular sensor, even a small decrease in [Ca2+]i directly decreased the Pv. To examine how EGTA differently affects the Ca2+ release relationship for voltage steps and action potentials, the change in Pv (EGTA/control) was plotted against that in [Ca2+]i in each voxel location. For voltage step-evoked release, the decrease in the Pv occurred when 10 mM EGTA reduced the [Ca2+]i to 60% or less (2+]i (to ∼80%) decreased the Pv (circles on solid green line). For lower EGTA concentrations, the decreases in Pv and [Ca2+]i were smaller (triangles and diamonds on dashed lines).Next, Pv with action potentials was examined. For this brief Cathe VGCC . The Pv the VGCC . For actturation . As the 2+]i transients saturate vesicular sensors masking the effect of EGTA on release. By contrast, action potential-induced brief Ca2+ influx drives the Pv within a linear range of the sensor, revealing the effect of EGTA on the inhibition of vesicular release. At the same distance, the magnitude of inhibition of release by EGTA varies depending on the duration of Ca2+ influx  and the low-affinity EFB (4 mM) were included in this simulation. The peak [Ca2+]i produced a spatiotemporal gradient  covered the upper left corners of the graphs, in the proximity of the VGCC, and expanded along the temporal axis. An increase in the EGTA concentration decreased the Pv, especially at distal locations for long channel openings. By contrast, at close locations, EGTA minimally affected Pv.To more systematically examine how the duration of channel opening influences the effect of EGTA, the step duration was varied from 0.1 to 30 ms. In this simulation, i around VGCCs. This simulation has several advantages, including the incorporation of the time course of Ca2+ entry and the property of endogenous buffers, including EFBs. Moreover, by combining release simulations, we can incorporate the property of the vesicular sensor to estimate the effect of EGTA on release. In this study, I focused on the Ca2+ microdomain formed around a single channel  in climbing fiber-Purkinje synapses , granule2+]i at distal locations is not sufficient to trigger vesicular release under high-EGTA conditions  does not affect the fast release components for responses at cerebellar mossy fiber-granule synapses , Purkinjnditions . In resp2+ influx-induced, vesicular release. In mammalian synapses, EGTA is typically tested at concentrations ranging between 1 and 30 mM, and the high concentration (>10 mM) resulted in some inhibition of release. There are several possible reasons for the difference between squid and mammalian synapses. First, the forward binding of EGTA is slower in the squid synapse because of the lower temperature and higher ionic concentration. Thus, it is possible that the [Ca2+]i gradient in the presence of EGTA in squid synapses is less steep than that in mammalian terminals. Second, the time course of Ca2+ entry during a presynaptic action potential (∼1 ms; 2+] causes a larger single channel current, enhancing the saturation of sensors. All these factors might contribute to the reduced effect of EGTA at the squid synapse. Although The most famous example showing a lack of an effect of EGTA was at the squid giant synapse, in which ∼80 mM EGTA had no effect on transmitter release but BAPTA diminished it . Althougl ∼1 ms;  is longel ∼1 ms; , increas2+ influx thorough single VGCC hardly induce detectable Pv at distal locations from the channel (2+]i range for synchronous release. To investigate the coupling distance for asynchronous, delayed, and spontaneous release, other Ca2+ sensor models incorporating low [Ca2+]i (Our simulations indicate that complete inhibition of release by EGTA is robust evidence for “microdomain” coupling. If the sensor is located beyond 100 nm from a channel, EGTA completely inhibits vesicular release regardless of how long the channel is open . However channel . For syn channel , and asy channel  and reti channel . Althoug channel , it is i channel  used in  [Ca2+]i  might be [Ca2+]i .2+ sensors (At most central synapses, high concentrations of EGTA partially inhibit vesicular release. This experimental observation and its underlying mechanism should be interpreted with caution because the coupling distance generating partial inhibition ranges several tens of nanometers , coverin sensors , but cou sensors  because  sensors . Thus, f2+ influx during an action potential is 0.36 ms (2+ influx prevents the sensor from becoming saturated; thus the reduction in presynaptic [Ca2+]i by EGTA immediately affects vesicular release. Indeed, it was shown that Ca2+ sensors are not saturated at these synapses because higher external [Ca2+] increases Pv (2+ uncaging experiments can also provide information on vesicular Ca2+ sensors (Partial inhibition by EGTA has also been reported for action potential-evoked release. The first example reported in a central synapse was for the calyx of Held, in which presynaptic loading with 10 mM EGTA from a patch pipette reduced action potential-evoked release . Althoug 0.36 ms . This breases Pv . As with sensors .2+ channels triggering release (2+ influx during an action potential is short (0.58 ms; A robust inhibitory effect of EGTA was also reported for the hippocampal mossy fiber-CA3 pyramidal cell synapse, in which 10 mM EGTA reduces EPSCs to merely 8% . Combine release . As the 0.58 ms; , the cou2+ chelator EGTA can access Ca2+ ions to decrease [Ca2+]i in the proximity of a channel, and the saturation of the vesicular sensor masks the effect of EGTA on vesicular release. When Ca2+ influx is triggered by an action potential, a small reduction of [Ca2+]i by EGTA causes an immediate reduction of vesicular release because the vesicular Ca2+ sensor is in a linear range. Thus, the experimental observation that EGTA inhibits synaptic transmission alone does not directly indicate that the coupling is within the microdomain. A more accurate interpretation requires the consideration of additional biophysical parameters, of which, the most important is the duration of Ca2+ influx.The present study sheds light on two points that have received little attention: the slow CaAll datasets generated for this study are included in the manuscript/YN conceived the research, performed the simulation, wrote the manuscript, and prepared the figures.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In addition, mitochondrial Ca2+ uptake contributes to cytosolic [Ca2+] shaping thus impinging on specific Ca2+-dependent events. Mitochondrial Ca2+ concentration is controlled by influx and efflux pathways: the former controlled by the activity of the mitochondrial Ca2+ uniporter (MCU), the latter by the Na+/Ca2+ exchanger (NCLX) and the H+/Ca2+ (mHCX) exchanger. The molecular identities of MCU and of NCLX have been recently unraveled, thus allowing genetic studies on their physiopathological relevance. After a general framework on the significance of mitochondrial Ca2+ uptake, this review discusses the structure of the MCU complex and the regulation of its activity, the importance of mitochondrial Ca2+ signaling in different physiological settings, and the consequences of MCU modulation on organ physiology.Mitochondrial Ca By targeting the Ca2+-sensitive photoprotein aequorin to mitochondria, Pozzan and coworkers demonstrated that a rapid mitochondrial Ca2+ peak, reaching values well above those of the bulk cytosol, parallels the [Ca2+]cyt rise evoked by cell stimulation  that greatly exceed the values measured in the cytosol due to the close contacts (< 200 nm) between the mitochondria and the ER -phenyl-hydrazone (FCCP), that collapses the ΔΨm, abolishes mitochondrial Ca2+ uptake. [Ca2+]m is additionally regulated by Ca2+ efflux pathways through the mitochondrial Na+/Ca2+ (NCLX) and H+/Ca2+ (mHCX) exchangers cyt cyt increases, the MCU complex is subjected to a cooperative activation that ensures the prompt response of the mitochondria to cell challenge. Overall, the regulation of MCU complex activity by MICU1-MICU2 heterodimers is possible thanks to the ability of these proteins to sense Ca2+ concentration through their EF-hand domains cyt, MICU2 inhibits MCU activity, thus representing the genuine gatekeeper of the channel. On the other hand, MICU1 senses high [Ca2+] and it allows the cooperative activation of MCU during cytosolic Ca2+ increases. Along these lines, MICU1 silencing causes Ca2+ overload due to the loss of the gatekeeper MICU2 and reduces the maximal activation of MCU due to the loss of cooperativity cyt transients, due to a low gating of the channel accompanied by a low cooperativity. In this way, the beat-to-beat Ca2+ transients that occur in the heart cause an integrative Ca2+ accumulation. On the other hand, the high MICU1:MCU ratio, as observed in the liver, confers high cooperative activation of the channel but strengthens the threshold of activation. Therefore, subthreshold [Ca2+] fluctuations are not sufficient to trigger mitochondrial Ca2+ uptake. Moreover, each single sustained cytosolic Ca2+ increase is effectively transmitted to the mitochondria cyt , 77. In  domains , 77. Nonto 40 μM , 115, wh~ 300 nM . These drativity , 111. Noangement . Furtherangement . Indeed,chondria .2+ uptake in HEK293T cells  generated at the SR/mitochondria contacts that allow the massive entrance of Ca2+ into these organelles. Indeed, a fraction of Ca2+ released during systole enters the mitochondria and is released back into the cytoplasm during diastole, resulting in a significant buffering of Ca2+ peaks. In addition, the modulation of MCU protein levels by silencing or overexpression enhances or decreases the amplitude of cytoplasmic Ca2+ oscillation, respectively, and the opposite effect takes place into the mitochondria. Furthermore, mitochondrial Ca2+ uptake in the heart mitochondria is controlled by a low MICU1:MCU ratio, as discussed above. This property has been hypothesized to ensure beat-to-beat mitochondrial Ca2+ accumulation at low frequency, while allowing an integrative matrix Ca2+ accumulation when frequency increases , consistent with the loss of mitochondrial buffering. This cytosolic Ca2+ increase is partially rescued by the addition of ATP, suggesting that these cardiomyocytes display an extramitochondrial adaptation that depends on the reduced ATP availability. Importantly, similar to the MCU−/− model, DN-MCU hearts are not protected against I-R injury.The first heart-specific model generated was a transgenic mouse expressing a dominant-negative MCU isoform, MCU/− model . When exmulation , althougchondria . Moreove2+ accumulation, although the basal mitochondrial [Ca2+] was not affected. Similar to DN-MCU mice and total MCU−/− mice, cardiac-specific inducible MCU−/− mice are indistinguishable from WT in normal conditions and after cardiac pressure overload. Cardiomyocytes derived from these mice present normal respiration rate in basal conditions, although a decrease in oxygen consumption rate was detected after isoproterenol treatment ) ) .2+ uptake in skeletal muscle physiology is being vigorously investigated, as detailed hereafter. The MCU−/− mice which, as explained above, are characterized by a mild phenotype, the most affected tissue is the skeletal muscle  that might be crucial for insulin exocytosis and to open Ca2+-activated K+ channels . Theref release . Specifiactivity . Respiraactivity , and chevity  levels and even small defects in Ca2+ homeostasis can lead to destructive consequences and alter normal neuronal activity, such as in aging cyt. Overall, a complex and highly compartmentalized Ca2+ signaling system in neuronal cells allows the activation of different spatially separated Ca2+-dependent processes at the same time  near the open pores [2+ include Ca2+ influx through ligand-gated glutamate receptors, such as N-methyl-d-aspartate (NMDA) receptor (NMDAR) or various voltage-dependent Ca2+ channels (VDCCs), as well as the release of Ca2+ from intracellular stores [Neurons require extremely precise spatial-temporal control of Catabolism . Neuronsin aging  and neurin aging . Ca2+ iname time . The widen pores . The majr stores . The relr stores .2+ entry through neuron plasma membrane channels, the activation of presynaptic neurons leads to the release of neurotransmitters into the synaptic cleft via the entry of Ca2+ through the voltage-operated Ca2+ channels (VOCCs). The released neurotransmitters in the synaptic cleft, in turn, activate receptors in the postsynaptic PM, thus initiating signal transmission. In postsynaptic neurons, the activation of neurotransmitter receptors results in the generation of Ca2+ signals that trigger responses that are specific to the type of receptor (reviewed in [As for the Caiewed in ).2+ signaling also plays essential roles in the neurogenesis from neural stem/progenitor cells which proliferate, migrate, and ultimately differentiate into billions of neurons and glia that populate the brain. There is increasing evidence that Ca2+ signaling controls specific genetic programs that establish the structures of the nervous system through Ca2+-dependent signaling pathways such as the calcineurin-NFAT signaling axis that has been shown to be critical for axonal growth as well as presynaptic development, dendritogenesis, and neuronal survival .2+ entry into the mitochondria guarantees activity-dependent regulation of cellular energy metabolism [2+-dependent increase in OXPHOS [Neurons are responsible for a huge oxygen consumption in resting conditions in humans. Indeed, the brain uses about 20% of the total oxygen consumed at rest but represents only 2% of the body mass . Importatabolism . As for tabolism , also nen OXPHOS .2+ in a defined subcellular domain, promoting large local cytoplasmic Ca2+ rises. Importantly, Ca2+ sequestration by the mitochondria profoundly affects neurotransmitter release, being strategically located in the proximity of Ca2+ channels such as NMDAR at the synaptic terminal [The mitochondria exert other neuron-specific functions. Indeed, their cellular distribution contributes to the accumulation of a large amount of Caterminal , 62. In terminal .2+ uptake impinges on the neurotoxic effects of the excitatory neurotransmitter glutamate. In this respect, it was clear that the cause of neuronal dysfunction and death was the excessive Ca2+ influx in neuron through the NMDA subtype of glutamate receptor. Experiments performed by modulating MCU allowed to directly determine the role of mitochondrial Ca2+ uptake in response to excitotoxic stimuli [2+ and CaM kinase-dependent induction of the transcription factor Npas4 [For a long time, one of the outstanding questions among neurologists has been whether modulation of mitochondrial Ca stimuli . In detaor Npas4  Fig. 5)2+ uptakeDrosophila melanogaster established that MCU-mediated mitochondrial Ca2+ uptake during development is of fundamental importance for olfactory memory formation but not for learning. Decreased mitochondrial Ca2+ accumulation, triggered by overexpression of a dominant-negative isoform of MCU in mushroom body neurons, causes axon lengthening accompanied by decreased synaptic vesicle content [2+ uptake in dopaminergic neurons. Silencing of D. melanogaster CG4495 gene, identified as MICU1 homolog, in dopaminergic neurons impaired climbing activity, which was worsened with aging, and shortened life span [Following a report of a siRNA library screening that identified MCU and MICU1 as important factors for proper memory formation , a study content . A recenife span  Fig. 5)Drosophil2+ uptake has been extensively studied in the last few years thanks to the availability of transgenic animal models. Either constitutive or conditional deletion of MCU, as well as overexpression of MCUb, has been achieved in vivo. In addition, tissue-specific transgenic animals have been produced. The effects of MCU activity modulation on organs particularly relying on mitochondrial metabolism for their energy demand, including the heart, skeletal muscle, neurons, and pancreas, have been dissected. These studies have highlighted the importance of MCU in physiologic organ functions and in the protection from damaging insults, but also the existence of compensatory mechanisms. Intriguingly, cardiac-specific deletion or overexpression of the NCLX demonstrates the essential role of proper mitochondrial Ca2+ efflux for heart function and, in general, the requirement of fine-tuned mitochondrial Ca2+ dynamics for proper organ physiology. In the future, comprehensive studies will unravel still obscure aspects of mitochondrial Ca2+ homeostasis on cell and tissue functions.The physiological role of mitochondrial Ca


Leishmania species but it is toxic, a Nano-liposomal form of AmB with a size of about 100nm (Lip-AmB) was developed and showed to be effective against Leishmania major, and Leishmania tropica in vitro and against L. major in vivo in animal model. This study was designed to check the irritancy Draize test in rabbits and was completed in the Center for Research and Training in Skin Diseases and Leprosy, TUMS, in 2012.Development of a topical treatment for cutaneous leishmaniasis (CL) is an important step in the improvement of lesion management. Amphotericin B (AmB) is effective against Twenty rabbits in 3 steps were housed individually with artificial lighting (12/12h light/dark). SinaAmpholeish cream or empty liposomes , was applied on a gauze patch and the patches were placed on the designated sites of the skin in the back of the rabbits. At 48 and 72h later, the erythema and oedema were checked, scored and recorded.The erythema score in rabbits was 0.83+0.41 for the SinaAmpholeish and 0.5+0.55 for empty liposomes (P= 0.16). The average score for oedema was 0.67+0.52 for SinaAmpholeish and 0.33+0.52 for empty liposomes (P= 0.16).Based on skin irritancy reactions the topical formulation of SinaAmpholeish is safe and could be further checked in human trials. Cutaneous leishmaniasis (CL) is endemic in 88 countries (72 of them are developing countries and 16 developed ones) and 90% of CL cases are reported from 6 countries including Brazil, Peru, Afghanistan, Saudi Arabia, Syria and Iran . PentavaStreptomyces nodosus. Several formulations of AmB have been marketed and used to treat fungal infections and visceral leishmaniasis, but the main drawback is toxicity. AmBisome™ is a liposomal form of AmB which is the first line treatment for visceral leishmaniasis in some endemic areas but is not as effective against CL (Leishmania major and Leishmania tropica in vitro and against L. major in vivo in a murine model of leishmaniasis (Amphotericin B (AmB) is a polyene antibiotic produced from ainst CL –11. A namaniasis . Large-sDraize test was initially introduced to test various medical, cosmetic, and chemical substances, but was also later used to check the safety of several other products such as cream, lipstick, mascara, face lotion, soap, shampoo, powder, hairspray, toothpaste, lacquer, detergent and anything, which could possibly induce skin irritation . It has The in vivo rabbit eye irritation/corrosion test developed from Draize skin test since 1981. It is used for two types of substances that either might be exposed to the eye due to regular use in or around the eyes (such as eye creams) or through accidental exposure of the eyes to the products that are not originally designed to be in contact with eyes (such as shampoos or skin creams). The evaluation of eye irritation potential for cosmetic substances is necessary to assure that the products are safe for consumers if eyes are exposed to intentionally or accidentally. Usually, 0.1 ml  of the test substance is placed into one eye and the other eye is not treated and kept as control. The treated eye and the control eye are checked regularly after application of the product, usually 1, 24, 48 and 72h after exposure or more frequently if required. Eye irritation is defined as induction of pathological changes in the eye fully recovered within 21d or tissue damages in the eye which might not fully recover even within 21d , 15.The objective of this study was to evaluate the safety and irritancy potential of SinaAmpholeish in rabbit model (Draize test) as well as an eye irritation test, as a step prior to clinical trials in humans.The study was conducted in 2012 in the Center for Research and Training in Skin Diseases and Leprosy, TUMS, Tehran, Iran. The proposal of this study was approved by the Ethical Committee of Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, IranFive-month-old male and female rabbits were purchased from Razi Vaccine and Serum Research Institute, Hesarak, Iran and kept in the animal house of CRTSDL. Rabbits were housed individually with artificial lighting  and free access to conventional laboratory diets and an unrestricted supply of drinking water with a collar fitted and fastened around the rabbit’s neck.Step 1) Healthy white rabbits with no hair loss were purchased and hairs on the back of the animals were shaved. After 24h, 6 rabbits with no sign of abrasion, erythema or oedema due to shaving were selected, 500mg of either 0.4% SinaAmpholeish gel or empty liposomes , was applied on a gauze patch and the patches were placed on the designated sites of the skin in the back of the rabbits, secured and moistened using 0.5 ml of phosphate buffer saline (PBS). The positive control site was covered with a patch containing 0.5ml of 1% of sodium lauryl sulfate (SLS). The negative control site was covered with a patch containing 0.5ml of PBS. Then the entire back of the rabbits was covered for 24h.After 24h of wrapping, the patches were removed. At one hour after removal of the patches and at 48 and 72h later, the erythema and oedema were checked, scored and recorded .Step 2) Healthy New Zealand Albino rabbits were screened and rabbits with no hair loss were selected. The hairs on the back of the rabbits were clipped.After 24h, 6 rabbits with no signs of abrasion, erythema or oedema due to shaving were selected and the following materials were applied on their back: SinaAmpholeish gel, empty liposome (as placebo), SLS 1% as a positive control and PBS as negative control similar to step 1.Step 3) In a pilot study, first white but not New Zealand Albino rabbits were screened and 2 healthy rabbits with no ocular lesion was selected, after gently pulling the lower lid away from the right eyeball, 100mg of SinaAmpholeish gel was placed in the conjunctival sac, then lids were then gently held together for about one second in order to prevent loss of the material. The left eye was untreated as a control. The eyes of the rabbits were regularly checked for 24h and in 2 of them daily for one week. As there was no sign of pain , no anaesthesia was required for the other rabbits.The irritation test was performed in 3 steps:Than 6 New Zealand Albino rabbits with no sign of ocular lesions were selected and treated similarly to the pilot study in white rabbits.In 2 rabbits the right eyes were covered for one hour. The eyes of the rabbits were regularly checked for the first 24h and photos were taken at 24, 48 and 72h.The Wilcoxon signed-rank test was used for comparing reaction between the two arms of study, P-value of less than 0.05 was considered significant.Step one results of the irritancy potential test of the substances were checked in 6 white rabbits , the results showed no reaction in any of the rabbits, no erythema, and no oedema.In step 2, irritancy potential test was checked in New Zealand Albino rabbits, as it is shown in No sign of eye irritation was seen during 72h observation in 8 rabbits (two from the pilot study and 6 from the main study) and two-week observation in 2 rabbits .Treatment of CL is sometimes a dilemma, the only standard treatment is to use pentavalent antimonite which needs multiple injections, accompanies side effects and moreover is not always effective. Development of new more acceptable treatment for CL is urgently needed to increase compliance and facilitate control of anthroponotic form of CL. Topical formulation is an ideal treatment for CL, several topical ointment have been developed and checked in phase 3 clinical trials , 15–17.L. major and L. tropica in vitro and against L. major in vivo in a murine model of leishmaniasis .The risk of topical formulations such as cosmetic and therapeutic to induce irritation or corrosion is usually checked in rabbit using Draize test –14. An aAccording to our previous unpublished data, usually New Zealand Albino rabbit is more sensitive to irritancy test than white rabbits, so first 6 white rabbits were included and no erythema and no oedema was seen in 25, 48 and 72h exposure to either SinaAmpholeish or empty liposomes. When the same procedure was used in 6 New Zealand Albino rabbits the average of erythema and oedema scores was 0.83+0.41 and 0.67+0.52, respectively for SinaAmpholeish gel and 0.5+0.55 and 0.33+ 0.52 for erythema and oedema for empty liposomes, respectively, although there was no significant difference between irritancy test between SinaAmpholeish gel and empty liposomes and control groups (P=0.16), but New Zealand Albino rabbits were more sensitive than white rabbits but the formulation was completely safe and could be further checked in human trials.Based on skin irritancy reactions the topical formulation of SinaAmpholeish is safe and could be further checked in human trials.


V11.1 on cell migration of pancreatic ductal adenocarcinoma (PDAC) cells, focusing on its effects on f-actin organization and dynamics. Cells were cultured either on fibronectin (FN) or on a desmoplastic matrix (DM) with the addition of a conditioned medium produced by pancreatic stellate cells (PSC) maintained in hypoxia (Hypo-PSC-CM), to better mimic the PDAC microenvironment. KV11.1 was essential to maintain stress fibers in a less organized arrangement in cells cultured on FN. When PDAC cells were cultured on DM plus Hypo-PSC-CM, KV11.1 activity determined the organization of cortical f-actin into sparse and long filopodia, and allowed f-actin polymerization at a high speed. In both conditions, blocking KV11.1 impaired PDAC cell migration, and, on cells cultured onto FN, the effect was accompanied by a decrease of basal intracellular Ca2+ concentration. We conclude that KV11.1 is implicated in sustaining pro-metastatic signals in pancreatic cancer, through a reorganization of f-actin in stress fibers and a modulation of filopodia formation and dynamics.Cell migration exerts a pivotal role in tumor progression, underlying cell invasion and metastatic spread. The cell migratory program requires f-actin re-organization, generally coordinated with the assembly of focal adhesions. Ion channels are emerging actors in regulating cell migration, through different mechanisms. We studied the role of the voltage dependent potassium channel K Cell migration is a central feature of many physiological processes such as embryonic development, organ differentiation after birth, and the function of immune cells ,2. Conve2+ concentration ([Ca2+]i) [2+ concentrations exert their main effects on cytoskeletal dynamics via motor proteins such as myosin or dynein [2+]i is therefore one of the main determinants underlying cell motility programs. It is therefore not surprising that different types of Ca2+ channels, both voltage-dependent and voltage-independent such as TRP channels, as well as those channels, mainly K+ channels, which regulate Ca2+ homeostasis through the resting potential (Vm) and hence the driving force for Ca2+, are functionally linked to the motility machinery i) . For exar dynein . The finachinery ,16. In aachinery . For exaachinery . Notablyachinery ,20.V11.1  i was determined by Fura-2 imaging, as in [2+]i was followed, acquiring pictures every 10s, for 10 min. Data relative to the last 2 min in control conditions (CTR) and after the application of E4031 are shown in 2+ of cells seeded either on FN or in DM plus Hypo-PSC-CM. In the latter condition, cells showed a slightly higher [Ca2+]i.Finally, we analyzed whether the above effects of Kg, as in . After 1Cell migration is a key process in cancer, as it underlies invasion and metastatic spread, which in turn determine tumor malignancy. Hence, defining the cellular and molecular mechanisms that trigger and sustain cell migration is crucial especially in those cancers, such as PDAC, whose malignancy mainly relies to the early occurrence of metastases, and thus to the activation of pro-migratory programs.V11.1 promotes cell motility programs in PDAC cells, through a complex regulation of f-actin assembly and dynamics, both in stress fibers and in filopodia.In the present paper, we provide evidence that the voltage dependent potassium channel K2+ concentrations i, through the regulation of the cell resting potential, which affects the driving force for Ca2+. This would imply the involvement of plasma membrane Ca2+ channels, in addition to those mediating Ca2+ release from intracellular stores. Indeed, E4031 significantly decreased [Ca2+]i in cells cultured onto either FN or DM plus Hypo-PSC-CM, and the effect started very quickly after the drug was added. Furthermore, KV11.1 is known to form a signaling complex with the β1 subunit of integrin receptors. We confirmed that KV11.1 co-localizes and co-immunoprecipitates with β1 integrin in PDAC cells, in both the culture conditions used in the present paper. Interestingly, KV11.1 mainly localizes to focal adhesions in cells cultured on FN and in filopodia when cells are cultured on DM plus Hypo. In both conditions, KV11.1 is adjacent, or even complexed with the β1 integrin. Moreover, we previously demonstrated that KV11.1 co-immunoprecipitates with Rho GTPases, and its activity regulates Rac i was measured for another 10 min. At the end of each experiment, the measurements were calibrated by applying 2 µM ionomycin  containing Ringer’s solution with 5 mM EGTA or 5 mM Ca2+. The [Ca2+]i was calculated as described earlier [The intracellular free Cap-values were determined by a Mann–Whitney test (significant level set to p < 0.05), after proving the deviation from normality of each condition using a Kolmogorov–Smirnov test. A two sample ANOVA test (significant level set to p < 0.05) was used otherwise. All + channel-dependent molecular mechanism of regulation of cell mobility and f-actin dynamics, we provide evidence that KV11.1 is implicated in sustaining pro-metastatic signals in pancreatic cancer, through a reorganization of f-actin in stress fibers and a modulation of filopodia formation and dynamics.The present study was aimed to shed light on the K