FLT3) gene is mutated in one-third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT kinase, MAP kinases, and STAT5. FLT3 inhibitors have been approved for the treatment of FLT3-mutated AML. However, treatment response to FLT3 inhibitors may be short-lived, and resistance may emerge. Compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with FLT3-mutated AML. Here STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin (PKC412), BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor S63845, and BCL-2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. Synergistic effects on cell viability were detected in both FLT3-mutated and FLT3-wild-type AML cells treated with AC-4-130 in combination with the MCL1 inhibitor S63845. AML patient samples with a strong response to AC-4-130 and S63845 combination treatment were characterized by mutated FLT3 or mutated TET2 genes. Susceptibility of AML cells to AC-4-130, PTC596, trametinib, PKC412, and venetoclax was altered in the presence of HS-5 stroma. Only the MCL1 inhibitor S63845 induced cell death with equal efficacy in the absence or presence of bone marrow stroma. The combination of the STAT5-inhibitor AC-4-130 and the MCL1 inhibitor S63845 may be an effective treatment targeting FLT3-mutated or TET2-mutated AML.The FMS-like tyrosine kinase 3 ( FLT3 gene are detected in around a third of patients with de novo acute myeloid leukemia (AML). Mutated FLT3 variants are constitutively active kinases signaling via AKT and MAP kinases, and as a gain of function via STAT5 [FLT3-mutated AML by the FDA in 2017, and was also authorized for use in the EU. However, treatment response to FLT3 inhibitors may be short-lived, and leukemia relapse is the major cause of treatment failure, as resistance may frequently emerge [FMS-like tyrosine kinase 3 (FLT3) is an inducible growth factor receptor signaling via PI3K-PDK1-AKT and via RAS-RAF-MEK-ERK leading to cell growth and proliferation . Mutatioy emerge . Moreovey emerge .STAT5 refers to two highly related genes, STAT5A and STAT5B, which are both located on human chromosome 17 [FLT3-mutated AML. STAT5 inhibition was reported to be a promising strategy for FLT3-ITD+ AML treatment [osome 17 . The STAreatment . Myeloidreatment . Stromalreatment ,12.FLT3 and TP53 [Compounds directly targeting STAT5 canonical functions may inhibit dimerization, DNA binding, or transcriptional activity. The mechanisms of direct STAT5 inhibition include disruption of tyrosine phosphorylation, dimerization, nuclear translocation, and/or DNA binding. Targeting the SH2 domain was, therefore, the main focus for the design and identification of selective inhibitors . AC-4-13and TP53 .FLT3 mutated or wild-type, NPM1 mutated or wild-type, as well as TP53 mutated or wild-type cells.Here, we assessed the STAT5-inhibitor AC-4-130, FLT3 inhibitor midostaurin, BMI-1 inhibitor PTC596, MEK-inhibitor trametinib, MCL1-inhibitor S63845, and BLC2 inhibitor venetoclax as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells grown in the absence or presence of bone marrow stroma. AML cells represented all major morphologic and molecular subtypes with normal karyotype, including FLT3 mutated or wild-type, NPM1 mutated or wild-type, as well as TP53 wild-type, mutated, hemizygous, and null cells , and other cytokines thereby inducing STAT signaling. The AML cell lines covered the major morphologic and molecular subtypes including, particularly, ll cells .50 of 0.7 µM; intermediate in TP53 wild-type MOLM-13 and ML-2 cell lines, with IC50 values of 3 µM; reduced in the TP53-mutant MOLM-16 and TP53 wild-type cell line OCI-AML3, with IC50 values of 10 µM; and very low in the TP53 null HL-60 cells , and other cytokines thereby inducing STAT signaling. The STAT5 inhibitor AC-4-130 was more effective in SKM-1 , and lesCDKN1A and BCL2 were determined by qRT-PCR , 2 B-ALL, and 1 CML, as well as PBMCs of 4 healthy donors (HD), were subjected to single-compound and combination treatments . TreatmeFLT3 mutated, five TET2 mutated AML, and one AML with IDH2 and DNMT3A mutations. One of the secondary AMLs susceptible to the combination of AC-4-130 and S63845 carried a TP53 mutation with a variant allele frequency of 5%, while two AMLs less susceptible to this combination treatment carried TP53 mutations with a variant allele frequency of 50% and 92%. Of the two B-ALL samples, one was very susceptible to the combination of AC-4-130 and S63845, while the other one had a normal response. However, to determine whether this treatment combination may be effective in B-ALL, a wider array of ALL samples would have to be tested.Compared to the healthy donor cells, the tested patient samples split into two groups with respect to response to combination treatment with AC-4-130 and S63845 . Cell viFLT3-mutated AML. To characterize the susceptibility of various AML cell lines to the STAT5 inhibitor AC-4-130, we performed a dose-escalation screening. Susceptibility to AC-4-130 varied in the tested AML cell lines with elevated susceptibility of the TP53-mutated secondary AML cell line SKM-1, intermediate susceptibility of the TP53 wild-type cell lines MOLM-13 and ML-2, as well as reduced susceptibility of the TP53 wild-type cell line OCI-AML3, the TP53 mutated MOLM-16, and the TP53 null cell line HL-60. The elevated susceptibility of the SKM-1 cell line was unexpected, as there is no mutated FLT3 activating STAT5 signaling in these cells, and the presence of mutated tumor suppressor TP53 is often associated with chemo-resistance. In view of the cell-viability studies in AML patient cells, the susceptibility of SKM-1 cells to AC-4-130 may be due to the presence of a TET2 mutation in this cell line. To investigate the elevated susceptibility of FLT3 wild-type SKM-1 compared to FLT3-mutated MOLM-13 cells to AC-4-130, cell viability was determined in AML cells grown in the presence of bone marrow stroma secreting granulocyte and macrophage colony-stimulating factors , and other cytokines thereby inducing STAT5 signaling. In the presence of bone marrow stroma, AC-4-130 and the BMI1 inhibitor PTC596 were more effective in SKM-1 cells and less effective in MOLM-13 cells, indicating that the elevated susceptibility of SKM-1 cells to AC-4-130 and PTC596 may be due to dominant signaling of cytokine receptors via STAT5 and via PI3K/AKT in this cell line. In addition, co-culture with HS-5 stroma may induce the STAT5 target protein BCL2, as has been suggested for HL-60 cells [As treatment response to FLT3 inhibitors may be short-lived, with leukemia relapse as the major cause of treatment failure, compounds targeting STAT5 may enhance and prolong effects of FLT3 inhibitors in this subset of patients with 60 cells . In cont60 cells . Similar60 cells . The dif60 cells . In our FLT3 wild-type AML cell lines treated with combinations of the STAT5 inhibitor AC-4-130 and the MCL1 inhibitor S63845, and in the FLT3-ITD-positive MOLM-13 cells treated with combinations of the STAT5 inhibitor AC-4-130 and the FLT3 inhibitor midostaurin (PKC412). However, concurrent STAT5 and FLT3 inhibitor treatment was not effective in FLT3-mutated AML patient samples. There were also clearly antagonistic effects observed in MOLM-13 cells treated with a combination of STAT5 inhibitor AC-4-130 and the MEK inhibitor trametinib. Moreover, the effects of concurrent STAT5 and MEK inhibition were rather antagonistic in AML patient samples, independent of FLT3 status. This antagonistic effect in the presence of STAT5 and MEK inhibition may result from induced signaling via PI3K and AKT, thereby preventing induction of apoptosis.To determine which compounds may be effective in AML cells treated in combination with a STAT5 inhibitor, we focused on inhibitors expected to elicit synergistic cytotoxic effects in combination treatments based on previous studies with BMI1, FLT3, MCL1, and MEK inhibitors ,18,19,20FLT3 or mutated TET2, and one case of mutated IDH2 and DNMT3A genes. The susceptibility of FLT3-mutated cells to the STAT5 and MCL1 inhibitor may be based on the gain of function of the mutated FLT3 activating STAT5 [TET2 mutant cells to the STAT5 inhibitor may be related to a cooperativity of TET2 and STAT5 in the activation of downstream targets [TET2 mutated cells to the MCL1 inhibitor may arise due to AKT-induced MCL1 over-activation in these cells, where AKT may no longer be regulated by mutated TET2 protein [IDH mutations, as in TET2 mutated cells. To validate the findings in a translational setting, we applied the STAT5 inhibitor AC-4-130 in combination with BMI1, FLT3, MCL1, MEK, and BCL2 inhibitors to AML patient samples. While treatments with combinations of the STAT5 inhibitor AC-4-130 with the FLT3 inhibitor midostaurin (PKC412), the BMI1 inhibitor PTC596 or the MEK inhibitor trametinib were rather ineffective, the combination of AC-4-130 and the MCL1 inhibitor S63845 led to a significant reduction in cell viability in a larger subset of AML. The susceptible AML patient samples were characterized by the presence of either mutated  targets ,29. The  protein ,23. Leuk protein . As TET2 protein ,32, suscTP53 mutation with a variant allele frequency (VAF) of 5%. Two AML samples less susceptible to the combination of AC-4-130 and S63845 carried TP53 mutations at frequencies of 50% and 92%. TP53 mutation variant allele frequency may be associated with clinical outcome of patients with myelodysplastic syndrome (MDS) and AML [TP53 mutation with VAF > 40% was independently associated with a significantly higher cumulative incidence of relapse and worse relapse-free and overall survival in patients treated with a cytarabine-based regimen.Susceptibility to the combination treatment of AC-4-130 and S63845 was significant in a secondary AML carrying a  and AML ,34. In nIt is important to note that there was a considerable effect of the MCL1 inhibitor S63845 in combination with the STAT5 inhibitor AC-4-130 on cell viability of normal PBMCs of healthy donors. This is in contrast to the marginal effect of the MCL1 inhibitor S63845 in combination with the MEK inhibitor trametinib on normal PBMCs previously described in , indicatCompared to previous studies, we now, for the first time, included the bone marrow stroma in our preclinical testing of AML cells. This is a new approach that introduces a new level of testing of potentially effective combination therapies. New insights may be gained by restating preclinical testing in AML cells grown in the presence of bone marrow stroma.Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland, between 2005 and 2018 were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the local ethics committee of Bern, Switzerland. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treatment. The AML cells were analyzed at the central hematology laboratory of the University Hospital Bern according to state-of-the-art techniques . MutatioFLT3wt, DNMT3A R882C, NPM1mut, TP53wt), MOLM-13 , FLT3-ITD, TP53wt), MOLM-16 , ML-2 , FLT3wt, TP53mut), and HL-60  were supplied by the Leibniz Institute DSMZ, German Collection of Micro-Organisms and Cell Cultures. AML cells were grown in RPMI 1640 media  supplemented with 20% fetal bovine serum  in a standard cell culture incubator at 37 °C with 5% CO2. The human bone marrow stroma cell line HS-5 was obtained from ATCC (ATCC® CRL-11882™). HS-5 cells were grown in DMEM media  supplemented with 10% fetal bovine serum . The HS-5 cells secreted granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), macrophage-CSF (M-CSF), Kit ligand (KL), macrophage-inhibitory protein-1 alpha, interleukin-1 alpha , IL-1beta, IL-1RA, IL-6, IL-8, IL-11, and leukemia inhibitory factor (LIF). For the co-culture assays, HS-5 cells were plated on six-well plates on day 1. On day 2, Nunc 0.4 uM cell culture inserts  were placed over the HS-5 feeder layer and AML cells were filled into the cell culture inserts. On day 3, AML cells were collected from the six-well inserts and replated on 96-well plates, before addition of compounds. Cytotoxicity assays were performed on day 4.Human AML cells lines OCI-AML3 , the MCL1 inhibitor S63845, and the MEK inhibitor trametinib were purchased at MedChemExpress . A stock solution of venetoclax was prepared by dissolving a venclexta tablet  in DMSO. Cell viability was determined after 20 h of treatment using the MTT-based cell-proliferation kit I . This time point was selected because the cellular responses were effectual for the calculation of combination indexes after 20 h of treatment with two compounds in leukemic cells. For AML cell lines, four independent assays  with four measurements  per dosage were performed. For hematological patient samples, two independent assays with three technical replicates per dosage were performed. Statistical analysis was done on GraphPad Prism . Data are depicted as XY graphs, box plots or scatter plots with mean values and SD. For the calculation of combination indexes, three dosages of AC-4-130 and two dosages of the other compounds were applied alone and in combination. Combination indexes were calculated on CompuSyn software .For assays with AML cell lines, cells were plated at a density of 5 × 10RNA was extracted from AML cells and quantified using qPCR. The RNA extraction kit was supplied by Macherey-Nagel, Düren, Germany. Reverse transcription was done with MMLV-RT . Real-time PCR was performed on the QuantStudio 7 Real-Time PCR Instrument using FastStart Universal master mix  and gene-specific probes  Hs00355782_m1 (CDKN1A), Hs04986394_s1 (BCL2), and Hs02758991_g1 (GAPDH). Measurements for CDKN1A and BCL2 were normalized with GAPDH values (ddCt relative quantitation). Assays were performed in three or more independent experiments. Data are depicted in column bar graphs plotting mean with SD values.Staining for apoptosis was done using annexinV-CF488A  in annexinV buffer and Hoechst 33,342 (10 μg/mL) for 15 min at 37 °C, followed by several washes. Propidium iodide was added shortly before imaging on a NC-3000 imager . For cell-cycle analysis, cells were incubated in lysis buffer with DAPI (10 μg/mL) for 5 min at 37 °C and analyzed on NC-3000 imager.


Downregulated METTL3 expression in AML‐MSCs induced an increase in AKT protein, resulting in enhanced MSC adipogenesis, thereby contributing to chemoresistance in AML cells. Therefore, targeting AKT regulation by mRNA modification in MSC adipogenesis might provide a novel therapeutic strategy to overcome AML chemoresistance.Adipogenesis of bone marrow mesenchymal stem cells (MSCs) promotes chemoresistance of acute myeloid leukaemia (AML) cells. MSCs from AML patients (AML‐MSCs) display enhanced adipogenesis compared with bone marrow MSCs from healthy donors. However, the precise molecular mechanism by which adipogenesis of MSCs from AML marrow differs from normal counterparts remains obscure. We found that METTL3 significantly inhibits MSC adipogenesis. Here, we aimed to identify the molecular mechanism linking METTL3 and MSC adipogenesis. Analysis of m 6A modification of AKT‐mRNA transcripts, which prevents reader protein‐mediated AKT‐mRNA decay, thereby increasing mRNA stability and protein expression. Activation of the PI3K/AKT pathway promoted adipogenic differentiation of MSCs, thereby contributing to chemoresistance of AML cells. This study provides new insights into AML chemoresistance and may benefit the treatment of AML.We find that METTL3 reduces the level of m AMLacute myeloid leukaemiaAML‐MSCsMSCs from AML patientsAra‐CcytarabineBMMbone marrow microenvironmentBPbiological processCCK‐8Cell Counting Kit‐8DNRdaunorubicinHD‐MSCsMSCs from healthy donorsKEGGKyoto Encyclopedia of Genes and Genomes6AmN6 methyladenineMNCsmononuclear cellsMSCsmesenchymal stem cellsOROOil Red OqPCRquantitative PCRAcute myeloid leukaemia (AML) cells remain in intimate contact with the stromal microenvironment in the bone marrow (BM) . Remodel6‐methyladenine (m6A) is the most abundant and reversible RNA modification in eukaryotic mRNA ) for 3 days, with adipogenic medium B , for 1 days until adipocytes appeared. The differentiation process continued for 14 days, when lipid droplets became obvious indicating differentiation into adipocytes, which were then stained with Oil Red O  [2O (3 : 2). The cells were rinsed three times with distilled water and examined using a microscope . We quantified triglyceride accumulation by eluting ORO‐stained lipids with 100% isopropanol and then measuring optical density at 450 nm by spectrometry .We cultured MSCs in alternating adipogenic medium A  , 22. Bri5 /well) were cultured with 0.1% gelatine in six‐well plates. When the ratio of fusion reached between 60% and 70%, the medium was discarded and osteogenic differentiation culture medium (2 mL) was added. The cells were fixed in 4% paraformaldehyde 21 days after the induction of differentiation for 15 min [The MSCs  was dissolved in DMSO at a stock concentration of 10 m5/well each; 6‐well plates) were co‐cultured with differentiated adipocytes in RPMI1640 for 24 h, and then, the sensitivity of AML cells to chemotherapy was measured using Cell Counting Kit‐8 (CCK‐8) as described [−1 daunorubicin , 10 μm cytarabine  or PBS (vehicle control). The cell growth inhibition rate (%) was calculated as follows:Differentiated adipocytes were washed twice with normal growth medium and then placed in the same medium. Thereafter, AML cells . The m6A content in total RNA was quantified using EpiQuik m6A RNA Methylation Quantification Kits  [6A levels were quantified by reading the absorbance of each well at 450 nm to create a standard curve [Total RNA was isolated using TRIzol  as described by the manufacturer. The quality of RNA was analysed using a NanoDropNY, USA) . Brieflyrd curve . All samTotal RNA was isolated using TRIzol (Invitrogen) as described by the manufacturer. Total RNA (200 ng) was reverse‐transcribed in 10 μL reaction volumes using cDNA Synthesis Kits , as described by the manufacturer. Quantitative PCR (qPCR) was performed using the cDNA as template, SYBR Green qPCR Master Mix  and a model 7500 Real‐Time PCR System . The endogenous controls were GAPDH or ACTB, and each reaction was run in triplicate. The qPCR comprised 1 μL of cDNA, 5 μL of SYBR Green qPCR Mix, 0.3 μL of PCR primers and 3.4 μL of RNase‐free water. The cycling protocol was 95 °C for 1 min, followed by 35 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The respective forward and reverse primers were as follows (5′→3′):in vitro and in vivo  was analysed using the 2–ΔΔCt method [METTL3: CAGGGCTGGGAGACTAGGAT and CTGGGCTGTCACTACGGAAG; AKT1: TGAGGAGCCCTGGTCTAATGAT and AAGACCCATTCAGAAGAGTTAT; c‐MYC: CCTACCCTCTCAACGACAGC and TTGTTCCTCCTCAGAGTCGC; and GAPDH: GGAGCGAGATCCCTCCAAAAT and GGCTGTTGTCATACTTCTCATGG. Relative expression t method , and thrimagej software .Proteins were extracted using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific Inc.) containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific Inc.), and then, protein concentrations were determined using BCA Protein Assay Kits (Thermo Fisher Scientific Inc.). Protein (30–60 μg/well) was resolved by 10% SDS/PAGE, transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific Inc.) and activated with methanol. The membranes were washed with Tris‐buffered saline plus Tween 20, nonspecific protein binding was blocked with 5% milk, and then the blots were incubated with 1 : 1000‐diluted primary antibodies against METTL3 , GAPDH , AKT , p‐AKT  and PPAR‐γ . Secondary antibodies were 1 : 5000‐diluted HRP‐conjugated goat anti‐mouse or rabbit IgG (H + L) as appropriate . Chemiluminescence signals were visualised using BeyoECL Star Kits (Beyotime Institute of Biotechnology) and detected using the ChemiDoc Touch System . Band intensity was quantified using http://www.addgene.org/19319/), verified by DNA sequencing and transfected into 293T cells. The negative control was a nontargeting empty pLJM1‐EGFP vector. The sequences of short hairpin RNAs (shRNAs) targeting human METTL3 (shRNA #9 and shRNA #12) were used as described [http://www.addgene.org/1864/). Thereafter, MSCs were infected with lentiviral particles. The effects of METTL3 overexpression and knockdown were confirmed by RT‐qPCR and western blotting.The wild‐type METTL3 coding region sequence (METTL3‐CDS) was amplified from HD‐MSC cDNA using PCR and the respective forward and reverse primers (5′→3′): AAATCTAGAATGTCGGACACGTGGAGCTC and TTTGCGATCGCCTATAAATTCTTAGGTTTAGAG. The PCR‐amplified product was subsequently cloned into pLJM1‐EGFP  containing 44 122 mRNA and 12 496 lncRNA degenerate probes. The hybridised arrays were scanned using a G2505C Scanner  [2‐scaled spike‐in RNA intensities. The fold changes between the HD‐MSCs/AML‐MSCs were determined for each transcript, and P‐values were calculated. Differentially m6A‐methylated RNAs were identified using a cut‐off of fivefold (P < 0.05). Differentially m6A‐methylated mRNA transcripts were identified using Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses and Gene Set Enrichment Analysis (GSEA) [Immunoprecipitated RNA samples of the HD‐MSCs and AML‐MSCs were labelled with Cy5 fluorescent dye using Super RNA Labelling Kits  and then purified using RNeasy Mini Kits. The Cy5‐labelled cRNAs were fragmented and hybridised to a human mRNA and lncRNA mCA, USA) . All spos (GSEA) .6A modification site characteristics [6A modifications on AKT1.The SRAMP can predict meristics . The ful6A  and anti‐IgG at 4 °C overnight. The RNA complexes were extracted using proteinase K and phenol/chloroform/isoamyl alcohol, amplified by qRT‐PCR.Immunoprecipitated RNA‐binding protein was assayed using Magna RIP Kits as described by the manufacturer . Harvested MSCs were lysed with RIP lysis buffer on ice and then incubated with the input anti‐METTL3 (Abcam), anti‐mgraphpad prism version 8.0 . The significance of differences between groups was determined using Student t‐tests, and values with P < 0.05 were considered statistically significant.Data are expressed as means ± SD of three independent experiments. All data were analysed using in vitro. The isolated MSCs were verified by flow cytometry as being positive for CD44, CD73, CD90 and CD105, but negative for the haematopoietic markers CD34 and CD45  between HD‐MSCs and AML‐MSCs using principal component analysis. The results revealed distinct clustering of individual HD‐MSCs and AML‐MSCs Fig. , indicat6A methylase METTL3 is important in the adipogenesis of BMSCs in pigs [6A modifications play important roles in the differentiation of human BM MSCs. Global m6A levels were decreased in total RNA isolated from AML‐MSCs compared with HD‐MSCs (Fig. 6A‐related enzymes, METTL3, METTL14, WTAP, FTO and ALKBH5 in MSCs, and found significant differences in the expression of METTL3 (Fig. 6A‐related enzymes was significantly lower in AML‐MSCs than HD‐MSCs (Fig. The epigenetic modification RNA plays key roles in the stem cell differentiation. The m in pigs  and mice in pigs . TherefoSCs Fig. . We evalTL3 Fig. . The fouTL3 Fig. . The expSCs Fig. .We evaluated the adipogenesis and chemosensitivity of co‐cultured AML cells using gene editing to modulate METTL3 expression in AML‐MSCs to determine the effects of METTL3 on MSC adipogenesis. The overexpression of METTL3 significantly inhibited AML‐MSC adipogenesis Fig.  and incrWe further investigated the molecular mechanism through which METTL3 is linked with AML‐MSC adipogenesis. The overexpression and knockdown of METTL3, respectively, decreased and increased AKT1 expression at the mRNA level. The overexpression of METTL3 upregulated the protein expression of p‐AKT and AKT and downregulated that of PPAR‐γ Fig. , whereasOverall, these results showed that METTL3 expression negatively regulates MSC adipogenesis and AML chemoresistance.6A‐methylated RNAs in HD‐MSCs and AML‐MSCs using a microarray of probes for 44,122 mRNAs and 12,496 lncRNAs. We found that 127 mRNAs were differentially weakly methylated between the HD‐MSCs and AML‐MSCs (Fig. 6A levels in mRNAs of genes related to the PI3K/AKT signalling pathways were significantly reduced in the AML‐MSCs compared with HD‐MSCs.We investigated possible targets of METTL3 during MSC adipogenesis by profiling mSCs Fig. . In addiSCs Fig.  and GSEASCs Fig.  showed t6A to inhibit MSC adipogenesis, we overexpressed METTL3 in MSCs. Subsequent western blot findings showed that the protein expression of AKT was remarkably reduced (Fig. AKT1‐mRNA in MSCs (Fig. AKT1‐mRNA in MSCs was modified with m6A (Fig. c‐MYC nor was c‐MYC present in m6A IPs (Fig. c‐MYC as a negative control mRNA, which could rule out false positives of METTL3/m6A IPs. The above results indicated that METTL3 could affect the protein expression of AKT in MSCs by mediating the m6A modification of AKT1‐mRNA.To further explore the mechanism of METTL3 mediating the regulation of AKT expression by METTL3 mced Fig. . FurtherSCs Fig. . Meanwhim6A Fig. . In the IPs Fig. . We sele6A sites in AKT1‐mRNA using SRAMP. The results revealed 27 m6A sites (Fig. 6A sites were concentrated in the CDS of AKT1‐mRNA. Further investigation is needed to determine which of the predicted sites are functional.We also predicted mtes Fig.  of which6A is the most abundant RNA modification in eukaryotic mRNAs, but the role of m6A‐mRNA in tumorigenesis and tumour development has not been investigated from the perspective of the tumour microenvironment of MSCs. To utilise the restructured BMM that controls the differentiation of MSCs into specific lineages for clinical AML treatment, deeper understanding of the molecular mechanism involved in specific lineage differentiation is essential. The present study identified an important mechanism that promotes the differentiation of pluripotent MSCs into adipocytes. We found that METTL3 mediates the m6A modification of AKT1‐mRNA, leading to increased AKT1‐mRNA and protein expression, which renders MSCs more likely to differentiate into adipocytes, thus changing the BMM and causing changes in AML chemoresistance (Fig. The BMMs of AML are remodelled to ensure that AML cells survive and resist the effects of chemotherapy. Adipocytes in BM are mainly differentiated from MSCs. The enhanced adipogenesis of AML‐MSCs can prevent chemotherapy from killing AML cells. Modification of RNA by m6A modification of RNA is the most abundant internal RNA modification in eukaryotes [6A methylase, METTL3, has two‐way effects in different cancers. For example, abundant METTL3 expression in AML [6A‐dependently regulates the differentiation of MSCs.The dynamic and reversible mkaryotes , and it n in AML , 33, 34,n in AML  and glion in AML  promotesn in AML  and prosn in AML  cancer. 6A‐dependently interacts with METTL3. The preliminary results of RNA‐seq and the m6A microarray showed reduced m6A activity in the PI3K/AKT signalling pathway. The m6A modification of mRNAs associated with PI3K/AKT signalling pathways was significantly upregulated, which might have promoted the adipogenic differentiation of MSCs. However, the precise molecular mechanisms require further exploration. Decreases in METTL3 regulate AKT activities and promote the proliferation and tumorigenicity of endometrial cancer, and m6A methylation regulates AKT pathways [The PI3K/AKT pathway plays important roles in mediating the proliferation, apoptosis and differentiation of cells . Overactpathways . However6A modification on mRNA transcription are mediated by specific m6A‐binding proteins called m6A readers [6A in mammals [6A [6A modifications and the YTH domain family are widespread in eukaryotes and play regulatory roles in various biological processes, we propose that m6A‐binding proteins play specific roles in m6A‐mediated adipogenesis. Amount of AKT protein expression negatively correlated with levels of m6A‐modified mRNA. We plan to further investigate whether YTHDF2 promotes AKT degradation and inhibits MSC differentiation. Due to a limited sample size, further study of a larger sample is required to verify the present finding.The effects of m readers . The YTH mammals . For insmals [6A . Because6A modification of mRNA associated with PI3K/AKT signalling pathways. Activation of the PI3K/AKT signalling pathways might promote MSC adipogenesis, which could potentially mediate AML chemoresistance. The present findings provide a theoretical foundation that should help to determine new targets of AML treatment from the perspective of BMMs and provide important insights that will lead to novel clinical strategies for treating AML.We found that METTL3 is associated with the adipogenesis of human MSCs. Decreased METTL3 expression in AML‐MSCs significantly reduced the amount of mThe authors declare no conflicts of interest.H‐FH, Z‐PP and BW conceived and designed the project; ZP, DH, RY and X‐TW acquired the data; ZP and W‐HX analysed and interpreted the data; and Z‐PP and R‐LY wrote the paper.


Previous evidence illustrates that contraction of cerebral arteries from sheep and underlying Ca2+ signaling pathways are modified by age and that long-term hypoxia (LTH) causes aberrations in Ca2+ signaling pathways and downstream effectors impacting vasoregulation. We hypothesize that age and LTH affect the influence of membrane depolarization on whole-cell intracellular Ca2+ oscillations and sub-cellular Ca2+ spark activity in cerebral arteries. To test this hypothesis, we examined Ca2+ oscillatory and spark activities using confocal fluorescence imaging techniques of Fluo-4 loaded basilar arterial myocytes of low- and high-altitude term fetal (∼145 days of gestation) and adult sheep, where high-altitude pregnant and non-pregnant sheep were placed at 3,801 m for >100 days. Ca2+ oscillations and sparks were recorded using an in situ preparation evaluated in the absence or presence of 30 mM K+ (30K) to depolarize myocytes. Myocytes from adult animals tended to have a lower basal rate of whole-cell Ca2+ oscillatory activity and 30K increased the activity within cells. LTH decreased the ability of myocytes to respond to depolarization independent of age. These observations illustrate that both altitude and age play a role in affecting whole-cell and localized Ca2+ signaling, which are important to arterial vasoreactivity and cerebral blood flow.Cerebral arterial vasoreactivity is vital to the regulation of cerebral blood flow. Depolarization of arterial myocytes elicits whole-cell Ca In an age where cardiovascular health is so heavily scrutinized, we still struggle to reduce deaths from cardiovascular diseases. With the identification of modifiable risk factors such as smoking, diet, exercise, and obesity, we are now able to help prevent a significant portion of the morbidity and mortality associated with cardiovascular disease; however, environmental exposures that we have little or no control over are important to cardiovascular development and health . What isGestational long-term hypoxia (LTH) from living at high altitude or placental insufficiency of various etiologies has a marked effect on fetal cerebral vascular development that can have repercussions throughout an individual’s lifetime . DecreasCa) channel activation, which is responsible for membrane hyperpolarization and consequent vasorelaxation . Cerebral arteries dilate in response to acute hypoxia, allowing for distribution of a greater portion of cardiac output to the brain in an attempt to maintain oxygen delivery despite reduced oxygen tensions . Howeverlaxation . Long-tedelivery . Prior tdelivery . Studiesdelivery . Howeverdelivery , suggestdelivery .Ca channel activation. We theorized that in arteries from low altitude fetuses, myocyte depolarization would increase whole-cell Ca2+ oscillations and localized rapid calcium signals (Ca2+ sparks) but that LTH would blunt these responses. We further hypothesized that post-natal development would magnify the increases in whole-cell and localized Ca2+ responses to membrane depolarization. This was examined by interrogating Ca2+ signals in basilar cerebral arterial myocytes of fetal and adult sheep housed at low- or high-altitude.The current series of studies were designed to interrogate the effects of animal age and long-term hypoxia on calcium signals in basilar arterial myocytes under resting conditions and in response to membrane depolarization. We hypothesized that long-term hypoxia due to high altitude exposure would alter calcium signaling in ways that may restrict BKn = 7) and non-pregnant ewes (n = 7) of a mixed Western breed were divided into low altitude (normoxic) and high-altitude long-term hypoxic (LTH) groups. All ewes were obtained from Nebeker Ranch in Lancaster, CA, United States at an elevation near sea level, 720 m. Normoxic control ewes were maintained near sea level at 720 m for the duration of their gestation. Animals for the LTH groups were held at Nebeker Ranch under normoxic conditions until 30 days gestation at which time the pregnant and non-pregnant ewes were transported to the Barcroft Laboratory, White Mountain Research Station in Bishop, CA, United States at an elevation of 3,801 m. Previous work shows that residing at the Barcroft laboratory results in a maternal arterial PO2 of 60 ± 3 Torr and a fetal arterial PO2 of 19 ± 2 Torr  and phenytoin sodium (10 mg/kg).Surgical and experimental procedures were performed in accordance with the regulations of the Animal Welfare Act, the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals, and “The Guiding Principles in the Care and Use of Animals” approved by the Council of the American Physiological Society and by the Institutional Animal Care and Use Committee of Loma Linda University. Pregnant . Basilar arteries then were quickly dissected under normoxic conditions in BSS. Basilar arteries were selected from the same anatomical locations in both fetal and adult sheep to maintain segments of similar function and embryological origin. Because of this, there was a significant difference in diameter between fetal and adult arteries  as described previously  of the following composition (mM): 126 NaCl; 5 KCl; 10 HEPES; 1 MgCleviously . All exp2+ of basilar arterial myocytes was measured in situ with a Ca2+ sensitive fluorescent dye  using a Zeiss 710 NLO laser scanning confocal imaging work station  with an inverted microscope (ZEISS Axio Observer), using procedures based on previous studies  with fine insect dissecting pins and placed into an open bath imaging chamber . Myocytes were illuminated at 488 nm via a Krypton-argon laser. Emitted light was captured with a photomultiplier tube with a band limited spectral grating of range 493–622 nm in both full frame and line scan imaging studies.Intracellular Ca studies . Fluo-4 2+ was recorded, the pinhole was set at an imaging depth of 5.4 μm, which is roughly the depth of an individual smooth muscle cell based on examination of both fixed and live preparations  through equimolar replacement of potassium for sodium was added to the tissues to depolarize the plasma membrane of the myocytes (A time series of 300 full frame Fluo-4 fluorescence images of 512 × 512 pixels were made over 234 s (roughly 780 ms/frame). In order to make sure that the smooth muscle intracellular Caarations . This thmyocytes . 5 min lRegions of interest for full frame fluorescent imaging were automatically detected using the LCPro plug in for Fiji (ImageJ) , followiP < 0.05. The program then analyzes the signal in the time and space domains for several parameters. In the current study, examinations were made of the following parameters: Amplitude (F/F0), which is the maximum amplitude of the signal transient as defined by the global maximum of the F/F0 curve within the time-period of the signal. Duration (s), which is the time interval defined by the period from one-half of the maximal amplitude values before and after the signal peak. Rise Time (s), which is written as “attack” in the program output and is the time interval defined by the period from one-half of the maximal signal amplitude value preceding the signal peak to the signal peak. Decay (s), which is the time interval defined by the time at the maximum amplitude to the time at one-half of the maximal amplitude following the peak. Area under curve (AUC) (F*s/F0), which is the discrete right-sided Riemann sum of the signal area during the period that is above one-half of the maximum signal amplitude  was chosen as the critical spatial cutoff because this distance allowed for the inclusion of most all neighboring cells.Spatiotemporal characteristics of the whole-cell Caby LCPro . The pro 0.1–1.0 . “FriendFluo-4 AM loaded basilar arterial myocytes were recorded with a Zeiss LSM 710 NLO laser scanning confocal imaging workstation on an inverted platform (Zeiss Axio Observer Z1). After being loaded with Fluo-4, the arteries were prepared and imaged as detailed above. Line scan images were captured at 529 lines per second and recordings were made for a duration of 18.9 s. The lateral pixel size ranged from 0.0148 to 0.0911 μm per pixel and the pinhole was adjusted so that the cells were imaged to a depth of 2.5 μm, which is about 50% of the width of a myocyte based on prior morphological studies in live cells .2+ sparks, their frequency, amplitude, and other spatiotemporal characteristics via SparkLab 4.3.1 .2+ oscillatory and spark events in whole-cell and line-scan recordings. A P-value of P < 0.05 was considered significant and were further broken down to P < 0.01 and P < 0.001 where appropriate.Data analysis and the production of graphs was performed using GraphPad Prism 9.1.2 . The graphs present the data as means ± SD. Continuous variable data was tested for normality prior to analysis. These datasets were not normally distributed and as such they were evaluated using non-parametric statistical tests. A Kruskal–Wallis ANOVA with a Dunn’s multiple comparisons test was performed on non-parametric datasets when making examinations between the experimental groups based on animal age, altitude, and treatment condition. The density of cell firing in video recordings was evaluated by drawing 3 boxes of a known size in each recording and counting the number of cells with events inside the box, with each cell only being counted once. For calcium spark analysis the numbers of cells were evaluated for those cells with or without events. The number of cells in videos or lines-scan recordings that had events was summarized. Statistical comparisons of this discrete data was made by performing contingency analysis between groups using Chi-Square analysis to evaluate potential changes in the incidence of Ca2+ oscillations, the number of line scans examined for Ca2+ sparks, and number of Ca2+ events. The percentage of cells firing with Ca2+ sparks was determined by the number of line scans containing Ca2+ events through visual observation relative to the total number of line scans observed.The specific test performed for each data set are depicted in the figure legends. Sample sizes were determined by several different measurements including the number of animals studied, the number of regions of interest showing Ca2+ oscillatory activity and that long-term hypoxia (LTH) would impair oscillatory events in basilar arterial myocytes. LCPro  that release Ca2+ into the cytosol , data that is summarized in 2+ sparks, see The percentage of cells with medium-duration CaThe percentage of cells with calcium events was similar in the fetal and adult groups and unaffected by LTH in the control bathing solution with low (5 mM) extracellular potassium. Membrane depolarization with 30K increased calcium oscillations in normoxic fetal and adult basilar arterial myocytes, an effect that was more pronounced in adult as compared to fetal sheep. Following long term hypoxia, however, membrane depolarization failed to increase cellular calcium oscillations in both fetal and adult basilar arterial preparations.2+ oscillations were then quantified, with summarized data shown in 2+ oscillations. 2+ oscillations were unaffected by maturation. However, in adult normoxic myocytes the AUC was slightly increased relative to that in the adult hypoxic period. Membrane depolarization caused a modest increase in the AUC in adult hypoxic myocytes. The AUC was unaffected by membrane depolarization in fetal normoxic and hypoxic groups; however, the AUC was lower in myocytes from fetal hypoxic and adult normoxic relative to fetal normoxic animals following membrane depolarization. The amplitudes of the Ca2+ responses are shown in 2+ event amplitude in the fetal normoxic and adult hypoxic groups. Event amplitude was also elevated in adult hypoxic relative to adult normoxic myocytes following membrane depolarization. The duration of the Ca2+ events is shown in 2+ rise time during the oscillations was unaffected by LTH or animal age under basal conditions. Membrane depolarization shortened the rise time modestly in only the fetal LTH group. Following membrane depolarization, the rise time was longer in fetal normoxic myocytes as compared to fetal hypoxic and adult normoxic myocytes. 2+ signal. Under basal conditions, LTH modestly shortened the time for the decay of the oscillatory signal in both fetal and adult myocytes. Membrane depolarization shortened the decay in myocytes from adult normoxic animals, which caused the decay duration to become equivalent with that from LTH adult animals. Following membrane depolarization, the time for Ca2+ decay was also shorter in LTH fetal myocytes relative to the normoxic counterparts.The kinetics and magnitude of the medium-duration Ca2+ oscillations were significantly less common than medium duration oscillations or Ca2+ sparks as illustrated by the reduced number of events. The effects of animal age, LTH, and membrane depolarization on these events are presented in Long-duration Ca2+ oscillations among smooth muscle cells were then analyzed for temporal correlations between the Ca2+ transients, or “friendship,” between oscillations at the various ROIs. The larger the correlation coefficient, the greater the level of event “friendship.” Temporally related correlations between the Ca2+ events were then identified by those events that had a correlation coefficient of r ≥ 0.8  are closely associated (neighbors), however, there were some subtle effects of animal age, LTH, and membrane depolarization. There was a slight increase in the percentage of adult normoxic myocyte ROIs who were neighbors relative to those in fetal normoxic myocytes. Although there were fewer Ca2+ oscillatory events in fetal hypoxic myocytes than their normoxic counterparts, a greater proportion were neighbors under control conditions. Membrane depolarization preferentially increased the percentage of friends who were neighbors in normoxic myocytes independent of animal age. 2+ oscillatory events that are spatially related. Unlike the tight spatial relationship between temporally related events, events that occur in nearby regions are not necessarily temporally correlated. Even still, there were influences of LTH and membrane depolarization. Long-term hypoxia preferentially reduced the percentage of neighbors who were friends in fetal myocytes under control conditions. Membrane depolarization preferentially reduced the percentage of neighbors who were friends in myocytes of fetal and adult normoxic animals.The interactions of the Ca r ≥ 0.8 . Results r ≥ 0.8 . Animal 2+ sparks in myocytes from fetuses and adults. 2+ spark tracings from line scan recordings for control groups analyzed with SparkLab  of the Ca2+ spark events.The magnitude and kinetics of Ca2+ oscillations and sparks in basilar arterial myocytes. These studies were performed because of the integral role basilar arteries have in regulating vascular reactivity and providing blood flow to the brain stem, which is critical to autonomic cardiorespiratory functions that are impacted by LTH. Basilar arterial myocytes had robust Ca2+ oscillations and the number of event sites were increased by membrane depolarization. The data also illustrate that animal age and LTH modify the coupling of membrane depolarization to the activation of whole-cell Ca2+ oscillations as well as the generation of localized Ca2+ sparks.The current study was designed to examine the impact of animal age and high altitude induced LTH on cytosolic Ca2+ oscillations by LTH is not unique to the cerebral vasculature. Previously, we reported that Ca2+ oscillatory activity was reduced by LTH in pulmonary arterial myocytes , however, there were fewer temporally related events (friends). On closer examination the neighboring events mainly occurred within individual cells as opposed to adjacent cells. While we have not explored this deeply in the current study, the findings suggest that when myocytes are depolarized the Ca2+ oscillations do not propagate from cell to cell, but rather, the number of events increase through recruitment of unrelated cells across the arterial wall. One explanation for this apparent lack of cell-to-cell communication is that membrane depolarization recruits additional L-Type Ca2+ channels on individual cells in a stochastic manner, which then increases Ca2+ responses in separate cells. Further, the data suggest gap junction connections between myocytes are not being activated, which limits cell-to-cell propagation of Ca2+ signals . Depolarization of the plasma membrane is well regarded to enhance Ca2+ spark activity and the ensuing repolarization of the membrane and vasodilation acts as a negative feedback regulator of vasoreactivity. With regards to the effects of age and LTH, basilar arterial myocytes from adults had far greater Ca2+ spark activity relative to those from fetuses. Furthermore, LTH reduced spark activity in adults to fetal levels and suppressed the depolarization mediated increase in Ca2+ spark activity in adult myocytes. The inability of depolarization to increase Ca2+ spark activity in adults following LTH is interesting and reminiscent of the impact of LTH on fetal pulmonary arterial myocytes , “The Guiding Principles in the Care and Use of Animals” approved by the Council of the American Physiological Society.The animal study was reviewed and approved by the Loma Linda University Health Institutional Animal Care and Use Committee. All study procedures adhered to the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals (SW, CW, AB, and LZ contributed to conception and design of the study. MR and NO performed laboratory experimentation. CW and JLP designed customized software for data analysis. SBC, MR, NO, and CR performed data and statistical analysis. CR wrote the first draft of the manuscript. SC wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to investigate the biological impacts and possible mechanisms of a novel lncRNA, LncSIK1, in AML progression and retinoic acid‐regulated AML cell development.in vivo. RNA or chromatin immunoprecipitation assays were conducted to measure the interaction of E2F1 and LncSIK1 or the LC3 and DRAM promoters. Autophagy was measured by transmission electron microscopy and Western blotting.The expression pattern of LncSIK1 was evaluated by qPCR and fluorescence in situ hybridization. CCK‐8 assay, immunofluorescence, Wright‐Giemsa staining, flow cytometry and Western blotting were performed to assess cell proliferation and differentiation. Bioluminescence imaging and H&E staining were used to detect AML progression in vivo. Mechanistically, in NB4 cells, LncSIK1 recruited the E2F1 protein to the promoters of LC3 and DRAM and induced autophagy‐dependent degradation of the oncoprotein PML‐RARa. However, LncSIK1 blocked E2F1 expression and the E2F1‐mediated transcription of LC3 and DRAM, thereby relieving aggressive autophagy in Molm13 cells.LncSIK1 was silenced in bone marrow mononuclear cells from AML patients compared with those from healthy donors. LncSIK1 strengthened the effect of retinoic acid in inducing cell differentiation and inhibiting cell proliferation in AML cells. Moreover, the silencing of LncSIK1 was critical to maintaining AML leukaemogenesis, as LncSIK1 enhancement retarded AML progression Taken together, these data indicated that LncSIK1 was an important regulator of AML development through regulating the E2F1/autophagy signalling pathway. The hypothetical regulatory network of LncSIK1 in NB4 and Molm13 cells.LncSIK1 exerted its significant positive effect on the sensitivity of AML cells to retinoic acid.in vitro and in vivo.LncSIK1 enhancement retarded AML progression The E2F1/autophagy axis played an important role in LncSIK1‐regulated AML development. Two AML cell lines  were used for this study.22.1g for 25 mins. The mononuclear cells in the interphase layer were carefully transferred into another fresh tube, washed with Hank's balanced salt solution (Beyotime Biotechnology) twice and then applied to subsequent experiments. CD34+ haematopoietic stem/progenitor cells (HSPCs) and CD34− cells were purified from the cord blood of healthy donors from the First Affiliated Hospital of Anhui Medical University using Ficoll‐Paque PLUS and anti‐CD34‐coated magnetic beads (Miltenyi Biotec). Briefly, we isolated mononuclear cells from fresh cord blood as described above. Next, a single cell suspension obtained was incubated with anti‐CD34‐coated beads and selected on autoMACS Pro (Miltenyi Biotec). CD34+ cells were identified by incubation with CD34 antibody  and analysis with flow cytometry .Primary AML leukaemia cells were isolated from the bone marrow of AML patients at diagnosis from the Second Hospital of Anhui Medical University. Briefly, bone marrow blood was slowly layered over Ficoll‐Paque PLUS solution  and centrifuged at 550×2.2em26Cd52Il2rgem26Cd22/Gpt (NCG) immunodeficient mice  were obtained from Gempharmatech , and NOD‐PrkdcscidIl2rgem1/Smoc (M‐NSG) immunodeficient mice  were obtained from the Shanghai Model Organisms Center . Male mice aged of 6–9 weeks were used for the experiments. All animals were fed under a specific pathogen‐free facility  in the Laboratory Animal Center of Anhui Medical University. All experiments on animals were performed in accordance with the institutional ethical guidelines for animal experiments (Ethics Number: LISC20190751).NOD/ShiLtJGpt‐Prkdc7 cells/ml in phosphate buffer saline (PBS) (HyClone). NCG mice were subcutaneously injected with 0.1 ml of the suspension (5 × 106 cells) into either side of the flank area. Mice were sacrificed 2 weeks later, and tumours were weighed, fixed and subjected to haematoxylin and eosin (H&E) staining.For the subcutaneous tumour xenograft study procedure Figure A, select7 cells/mL in PBS and M‐NSG mice were injected with 0.1 ml of the suspension (5 × 106 cells) into the tail vein. Three weeks after inoculation, the mice were sacrificed for analysis. Bioluminescence imaging (BLI) was applied to evaluate human cell infiltration  in mice. H&E staining and flow cytometry were employed to detect cell engraftment in bone marrow, peripheral blood, liver, kidney and spleen as described.For the intravenous bone marrow engraftment study procedure Figure B, M‐NSG 2.3t test, while data with a non‐normal distribution were analysed by the Mann‐Whitney nonparametric test. Comparisons between groups were performed using one‐way ANOVA followed by Tukey's multiple comparison. Correlations between the samples were evaluated statistically through Pearson's correlation coefficient. Error bars depict the mean ± standard error of the mean (SEM). P values of less than.05 were considered as statistically significant.Data were assessed using the software SPSS 19.0 (SPSS). Data with a normal distribution were analysed by Student's Further details of the materials and methods are included in Additional file 33.1n = 6), AML‐M2 (n = 42), AML‐M3 (n = 9), AML‐M4 (n = 15) and AML‐M5 (n = 7) according to the French‐American‐British classification.n = 10), suggesting that LncSIK1 may be implicated in the pathogenesis and progression of AML  or empty vector (EV) followed by ATRA or ATPR incubation. Following infection, LncSIK1 levels were significantly enhanced . The procedure is shown in Figure in vivo.To explore the effect of LncSIK1 on tumour growth, we established a subcutaneous tumour model using NB4 cells. NCG mice received subcutaneous injections of selected NB4 cells , Beclin1 Figure , and theIn conclusion, LncSIK1 induced autophagy in NB4 cells to clear the PML‐RARa oncoprotein, in contrast, the high basal autophagy level in Molm13 cells was reversed by LncSIK1.4In the current study, we found that the silencing of LncSIK1 was critical for maintaining AML, moreover, LncSIK1 enhanced the effect of retinoic acid on AML cells. In terms of mechanism, LncSIK1 rescued autophagy by interacting with E2F1 and recruiting E2F1 to bind with the promoters of LC3 and DRAM in NB4 cells. However, the interaction between LncSIK1 and E2F1 inhibited E2F1 levels and thus suppressed autophagy in Molm13 cells. This different role of autophagy is consistent with published studies, indicating that the role of the autophagy pathway in AML remains a topic of debate and controversy. Whether the lncRNA/E2F1 axis forms a distinctive autophagic regulatory network that plays multiple roles in AML progression is a question we need to consider.In general, the heterogeneity caused by different genetic mutations leads to diverse roles of autophagy in AML. For example, researchers pointed out that AML1‐ETO‐positive patients may benefit from autophagy inhibitors.In addition to the discovery of lncRNAs, microRNAs (miRNAs) are also established regulators of lncRNA‐mediated transcription.In summary, our study concluded that LncSIK1 is silenced during AML leukaemogenesis and that rescue of LncSIK1 retards AML progression. Schematic and hypothetical regulatory networks are represented in the graphical abstract image and provide a potential regulatory mechanism for LncSIK1 in AML.The authors declare no conflict of interest.K.W. and F.C. performed study concept and design; K.W., J.L. and G.D. performed the development of methodology and writing, review and revision of the paper; Z.O., S.L., and X.X. provided acquisition, analysis and interpretation of data, and statistical analysis; M.Z., and X.P. provided technical and material support. All authors read and approved the final paper.Fig S1Click here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.Supplementary MaterialClick here for additional data file.


International Working Group (IWG) and European LeukemiaNet (ELN) adult response definitions are currently used to evaluate the efficacy of new agents for childhood acute myeloid leukemia (AML); however, the criteria are not consistent with consensus definitions used in pediatric trials or the common practice of intensifying treatment prior to full hematopoietic recovery of ANC ≥ 1000 cells/μL and platelets ≥ 100 cells/μL. This retrospective analysis of the two most recent Phase 3 AML trials in the Children’s Oncology Group assesses the incidence, timing, and prognostic significance of count recovery following induction chemotherapy in children with AML. These data confirm that awaiting count recovery to meet adult criteria does not reflect standard practice in pediatric AML and IWG/ELN-defined CR does not have a significant impact on survival in children. Continuing to use adult IWG/ELN count recovery definitions limits childhood AML drug development by underestimating response, and therefore, updated response criteria are needed for pediatric AML patients.International Working Group (IWG) and European LeukemiaNet (ELN) response definitions are utilized to evaluate the efficacy of new agents for childhood acute myeloid leukemia (AML) for regulatory purposes. However, these criteria are not consistent with definitions used in pediatric AML trials or with standard pediatric practice to proceed with subsequent therapy cycles prior to IWG/ELN-defined count recovery. We retrospectively analyzed data from the two most recent Phase 3 pediatric AML clinical trials conducted by the Children’s Oncology Group (COG) to assess the incidence, timing, and prognostic significance of count recovery following induction chemotherapy. Of the patients with fewer than 5% bone marrow blasts at the end of first induction, 21.5% of patients proceeded to a second induction cycle prior to achieving ANC ≥ 500 cells/μL and platelets ≥ 50,000 cells/μL, both well below the IWG/ELN thresholds of ANC > 1000 cells/μL and platelets > 100,000 cells/μL. In these two sequential childhood AML Phase 3 trials, neither ANC nor platelet recovery predicted survival. Intensification of treatment through the initiation of subsequent therapy cycles prior to attainment of IWG/ELN-defined CR is common practice in clinical trials for children with AML, suggesting that updated response definitions are needed for pediatric AML. Acute myeloid leukemia (AML) in children and adults represent a phenotypically heterogeneous and genetically complex subtype of hematopoietic malignancies. There are approximately 20,000 newly diagnosed cases of AML in the United States each year with an average age at diagnosis of 68 years; however, fewer than 500 of these cases occur in children under the age of 15. Given this differential age distribution ,4. AssesIn 2010, Walter et al. demonstrated clearly that in adult AML patients treated 15 to 35 years ago, it was routine to await count recovery at the end of induction to evaluate for residual dysplasia . During n = 6) were excluded from this analysis, as were all patients enrolled in AAML1031’s Arm D expansion cohort (n = 378) because response data were not collected for these patients. Patients who withdrew consent or went off-study for other reasons were censored at that date. We retrospectively analyzed data from the two most recent Phase 3 pediatric clinical trials for de novo AML conducted by the COG in the United States. The eligibility, therapy, and results for these trials have been previously reported ,10. The n = 1645) achieved a complete response and were evaluated for time to and incidence of count recovery. Comprehensive CBC data were not collected for each patient. Instead, investigators were required to report whether patients achieved peripheral ANC of 500 cells/μL and non-transfused platelet count of 50,000 cells/μL prior to Induction II, and the date each parameter was achieved. Both COG trials recommended but did not require that Induction II begin when ANC > 1000 cells/μL and platelets > 75,000 cells/μL  had RD assessed by centrally performed difference-from-normal (ΔN) flow cytometry at the end of induction I, which was previously shown to be superior to morphology in assessing response in children . Those wcells/μL . Survivap values <0.05 were considered significant. Living patients were censored at date of last follow-up. Data were frozen at 31 December 2019.Statistical analyses were performed with SAS version 9.4 . The Kaplan–Meier method was applied to estimate probabilities of survival with standard errors according to Greenwood and compared with the log-rank test . Survivan = 1645), the proportion of patients who proceeded to Induction II prior to recovery of ANC ≥ 500 cells/μL and platelet count ≥ 50,000 cells/μL was 21.5%  or OS (p = 0.896) (p = 0.453) or OS . There was a trend toward decreased outcomes in the multivariable Cox analysis, as shown in Survival analysis based on count recovery group was performed among patients with fewer than 5% bone marrow AML blasts by centralized ΔN flow cytometry, and is reported in = 0.896)  or RR (S= 0.896) . To addrThe standard AML treatment approach over the past 40 years for both children and adults has been anthracyclines combined with cytarabine, though the therapeutic landscape is now evolving to include more targeted therapies. We now have a better understanding of AML biology, response, risk classification, and the impact of each on survival. Accordingly, as we evaluate therapies for children, we must define relevant response criteria in childhood AML to support the development of promising new therapies to advance cures. Delays in count recovery in adults with AML are thought to reflect the persistence of leukemia, often with a myelodysplasia (MDS) phenotype. Childhood AML often has more aggressive biology compared to that in adults and the MDS phenotype is quite rare ,4. Awaitp = 0.063) (The current IWG/ELN criteria for response imply that the absence of full hematopoietic recovery (ANC > 1000 cells/uL and plt > 100 cells/uL) is prognostic in adults with AML. Although retrospective studies have suggested inferior outcomes for adult patients with CRi/CRp compared to those with CR ,18, this= 0.063) . SurvivaOne of the limitations of this study is the lack of detailed end-of-induction CBC data for each patient enrolled in these clinical trials. Therefore, we are unable to directly refute the IWG/ELN CR criteria . As described above, it is not common practice for physicians treating childhood AML to wait for count recovery to these thresholds, and therefore, cut-offs of ANC ≥ 500 of cells/µL and platelets ≥ 50,000 cells/µL were used for data analysis. Data from our colleagues in the International Berlin-Frankfurt-Munster (i-BFM) group confirm this practice, demonstrating that only one-third of patients achieve recovery to IWG/ELN criteria after AML induction . It is possible that strict adherence to waiting for count recovery to the IWG/ELN criteria might show more significant survival differences among these groups. The current IWG and ELN criteria were based upon retrospective data in adults supporting prolonged survival for patients who achieve CR . SpecifiAlthough CR per IWG and ELN criteria has historically been used as a surrogate for survival in adults, recent studies question the reliable association between CR and survival, suggesting it may be time to reconsider these definitions for response evaluation in both adult and pediatric AML . MultiplIn the largest available US pediatric cohort, we are unable to confirm an association between peripheral blood count recovery and survival, despite a comparable sample size to the Walter et al. cohort. Continuing to use adult IWG/ELN response assessment definitions places severe limitations on childhood AML drug development by classifying lack of CR using these guidelines as treatment failure. Additionally, these definitions are not aligned with the standard of care for intensification of pediatric AML therapy and are not followed by pediatric oncologists.Given the data presented here, response criteria must be reconsidered for pediatric patients with AML. International cooperative groups are currently working to compile new, standard definitions for both count recovery thresholds and remission status in order to more accurately define treatment responses in future pediatric AML trials. In the meantime, we propose that CR, CRp and CRi are all valid primary study endpoints.


Indeed, when Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) activity was blocked by cyclopiazonic acid (CPA), the extracellular application of ATP failed to trigger any intracellular Ca2+ waves, indicating that metabotropic purinergic receptors (P2Y) are mainly involved in the mApoE-PA-LIP-induced increase of the Ca2+ wave triggered by ATP. In conclusion, mApoE-PA-LIP modulate intracellular Ca2+ dynamics evoked by ATP when SERCA is active through inositol-1,4,5-trisphosphate-dependent (InsP3) endoplasmic reticulum Ca2+ release. Considering that P2Y receptors represent important pharmacological targets to treat cognitive dysfunctions, and that P2Y receptors have neuroprotective effects in neuroinflammatory processes, the enhancement of purinergic signaling provided by mApoE-PA-LIP could counteract Aβ-induced vasoconstriction and reduction in cerebral blood flow (CBF). Our obtained results could give an additional support to promote mApoE-PA-LIP as effective therapeutic tool for Alzheimer’s disease (AD).In light of previous results, we assessed whether liposomes functionalized with ApoE-derived peptide (mApoE) and phosphatidic acid (PA) (mApoE-PA-LIP) impacted on intracellular calcium (Ca Alzheimer’s disease (AD) is a neurodegenerative disorder, characterized by alterations in memory formation and storage . It is a2+ signaling toolkit, including metabotropic purinergic signaling, is thought to play a role in these changes i) [PA is a potent activator of inositol phosphate production and an important role of PA in cell signaling is the increase of intracellular Ca[Ca2+]i) . PA coul[Ca2+]i) . PA coul[Ca2+]i) , 22. The2+]i plays a crucial role within the NVU  (DSPE-PEG-MAL) and with 5 mol % of phosphatidic acid (PA)  . BrieflymApoE peptide  was covalently attached on mApoE-PA-LIP surface by thiol–maleimide coupling, to give a final peptide: mal-PEG-PE molar ratio of 1.2:1, as previously described .mApoE-PA-LIP size and polydispersity index (PDI) were obtained using a ZetaPlus particle sizer  at 25 °C in H2O by dynamic light scattering (DLS) technique with a 652-nm laser beam. The cell viability of hCMEC/D3 is not affected by mApoE-PA-LIP administration up to 200 μM of total lipids (assessed by MTT assay) . We conf2 1.5 mM; HEPES 10 mM; Glucose 10 mM). Ca2+-free solution (0 [Ca2+]e) was obtained by substituting Ca2+ with 2 mM NaCl and by adding 0.5 mM EGTA.hCMEC/D3 were cultured on coverslips, maintained in a low-profile chamber with physiological salt solution (PSS)  was added to the PSS and 0 [Ca2+]e solutions. Then, mApoE-PA-LIP were dissolved at a final concentration of 0.01mg/ml  in PSS and 0 [Ca2+]e solutions.ATP (50μM) was added to the PSS and 0 e KRB). Cyclopiazonic acid (10μM) was added to the PSS solution (both KRB and 0 [Ca2+]e KRB). mApoE-PA-LIP were dissolved at a final concentration of 0.01mg/ml  in PSS (both KRB and 0 [Ca2+]e KRB).iAstro KRB solution  in PSS for 30 min at 37 °C away from light. The coverslip, after being washed in PSS, was disposed in a low-profile chamber and maintained in physiological solution at 37 °C for the entire duration of the experiments. Fura-2 fluorescence ratio  was observed by wide-field fluorescence time lapse Nikon Eclipse FN1 upright microscope  equipped with a 60X Nikon objective . For experiments with astrocytes, we used 40X Nikon objective . The excitation filters were mounted on a filter wheel . The fluorescent signal was collected by means of a Coolsnap Photometrics CCD camera through a bandpass 510-nm filter.2+ levels monitored by measuring, for each ROI, the ratio (340/380) of the mean fluorescence. For the entire duration of the experiment, ratio measurements were performed and plotted online every 1200 ms with 800 ms exposure time. Duration and area values were measured using Origin tools per each response in different conditions.By using MetaFluor  software, we measured and plotted online, every 1200 ms, the fluorescence from 8–12 regions of interest (ROI) inside each loaded cell; each ROI was identified by a number. Changes in intracellular CaCholesterol (chol), phosphatidic acid (PA), sphyngomielin, and 1,2-distearoyl-sn-glycero-3- phospho-ethanolamine-N [maleimide(polyethyleneglycol)-2000]  were from Avanti Polar Lipids Inc . Adenosine 5′-triphosphate disodium salt hydrate (ATP) and cyclopiazonic acid (CPA—1 mM stock in dimethyl sulfoxide—DMSO) were obtained from Sigma Aldrich (C1530–5MG).2+ binding.mApoE peptide  was synthetized by Karebay Biochem . Fura-2 acetoxymethyl ester (Fura2/AM - 1 mM stock in DMSO) was obtained from Thermo Fisher. This indicator has an emission peak at 505 nm and changes its excitation peak from 340 to 380 nm in response to CaT test for unpaired observations with Gaussian distributions and by the Mann-Whitney non-parametric test with non-Gaussian distributions. Differences were considered significant at *p value < 0.05, **p value < 0.01, and ***p value < 0.001.All data have been collected from hCMEC/D3 and iAstro-WT. The 1st spike amplitude evoked by ATP was measured considering the value from the baseline before and after the stimulus trigger. Area under the curve (A.U.C) was obtained using Origin Integration function. Statistical analysis was performed using Microsoft Office Excel. Pooled data were given as mean ± SE and statistical significance was evaluated by the Student’s The total lipid recovery for NL after extrusion was about 70%. The different lipid components of the mixtures were recovered with equal efficiency and always reflected the proportion in the starting mixture. The yield of NL coupling with mApoE was 70 ± 9%. Final preparations of mApoE-PA-LIP had a diameter of 122.7 ± 4.85 nm with a PDI of 0.1 ± 0.02.2+ “add-back” protocol is a widely employed protocol to monitor both endogenous Ca2+ release and SOCE (store-operated Ca2+ entry) in non-excitable cells [2+]e condition to evaluate passive ER (endoplasmic reticulum) Ca2+ egression through leakage channels. Subsequently, restitution of extracellular Ca2+ induces a second increase in [Ca2+]i which is due to Ca2+ entry through open store-operated Ca2+ channels. As reported in Fig. 2+]i, which reflected the depletion of the ER Ca2+ pool, followed by massive SOCE activation arising after extracellular Ca2+ restitution. These data are consistent with those recently described in hCMEC/D3 cells i by stimulating P2Y2 receptors in hCMEC/D3 cells [n = 87) of the duration of 5 min increased the Ca2+ dynamics evoked by a short (30 sec) ATP pulse in comparison to control conditions (n = 139)  in comparison to controls (n = 16) also in the absence of extracellular Ca2+ (0 [Ca2+]e)  of the duration of the ATP-evoked Ca2+ waves was found in presence of mApoE-PA-LIP in comparison to controls    in comparison to controls (mean A.U.C ± SE 25.44 ± 2.82)  in comparison to controls   Fig. -Bb.Fig. 2+ signals in hippocampal astrocytes e) increased ATP-evoked Ca2+ waves in comparison to control  in comparison to control . Under this condition, also the A.U.C value increased (1.71 ± 0.07) in comparison to control (A.U.C ± SE 1± 0.08)  Fig. .Fig. 6(A2+ waves by stimulating phospholipase Cβ (PLCβ), thereby inducing InsP3-dependent Ca2+ release from the ER. In order to confirm that the ER represents the main endogenous Ca2+ store targeted by ATP, we then evaluated the Ca2+ response to ATP in presence of CPA in PSS and in 0 [Ca2+]e both in hCMEC/D3 cells  and cholesterol (Chol), as a well-tolerated valuable new nanotechnological tools for AD therapy , in ligh2+ dynamics within two main cellular constituents of the NVU.Starting from these evidences, we assessed mApoE-PA-LIP activities on hCMEC/D3 as an in vitro human BBB model and on cultured astrocytes in order to evaluate mApoE-PA-LIP ability of modulating the intracellular Ca2+ waves triggered by extracellular ATP in cultured hCMEC/D3 and astrocytes.Our results proved that mApoE-PA-LIP actively modulate the intracellular Ca2+. These results could be basically related to the increased mobilization of Ca2+ from the intracellular stores induced by mApoE-PA-LIP mediated by the activation of the metabotropic purinergic receptors. Due to this “additional” intracellular calcium mobilization, the number of cells reaching the threshold of the ATP-evoked Ca2+ wave increased. Indeed, a trigger stimulus of 50 and 100 μM ATP increased the duration and the A.U.C of the Ca2+ wave when both hCMEC/D3 and astrocytes were pre-treated with mApoE-PA-LIP at the final concentration of 0.01mg/ml for 5 min. Interestingly, the pre-treatment with mApoE-LIP without PA functionalization failed to increase both the duration and the A.U.C of the intracellular Ca2+ wave triggered by ATP. mApoE-PA-LIP increased the ATP-evoked intracellular Ca2+ waves in cultured hCMEC/D3 and astrocytes even under 0 [Ca2+]e conditions, thus indicating that the increased intracellular Ca2+ wave triggered by ATP is mainly due to endogenous Ca2+ release from ER. Indeed, when SERCA activity was blocked by CPA, the extracellular application of ATP failed to trigger any intracellular Ca2+ waves. These data are consistent with previous results by Bintig and colleagues [2+ signaling in hCMEC/D3 cells acts via G-protein-coupled P2Y2 receptor subtype that triggers Ca2+ wave by means of InsP3-dependent ER Ca2+ release.The percentage of responding hCMEC/D3 cells increased after a pre-treatment with mApoE-PA-LIP both in standard PSS solution as well as in absence of extracellular Calleagues , who dem2+ signaling toolkit is remodeled after dynamic changes of astroglial morphology and function during AD [The astrocytic Cauring AD , 9. TherCerebrovascular pathology is considered the major risk factor for clinically diagnosed AD-type dementia . FunctioIn addition, PA could accumulate and form microdomains highly negatively charged, which potentially serve as membrane retention sites for several key proteins for exocytosis, such as the SNARE protein syntaxin-1 , or othe2+ release from ER in the context of AD [2+, modulates Ca2+ dynamics evoked by ATP when SERCA is active. In agreement with our findings related to mApoE-PA-LIP activities on intracellular Ca2+ waves, a recent paper by Krajnak and Dahl [2+ unbalance, could exert neuroprotective effects and improves memory and cognition in AD model mice. Clearly, a better understanding of how dysregulation of neuronal Ca2+ handling contributes to neurodegeneration and neuroprotection in AD is needed as Ca2+ signaling modulators are targets of great interest as potential AD therapeutics [Recent evidence has revealed new insights into potential role of enhanced Caxt of AD . Our resand Dahl  providesapeutics .2+ wave triggered by ATP when both hCMEC/D3 and iAstro-WT were pre-treated with mApoE-PA-LIP would at the end increase these neuroprotective effects.Occurrence of AD symptoms is sometimes preceded by pathological changes in the brain vascular system, including accumulation of Aβ in the walls of blood vessels and lowering of CBF . The act2+ waves both in hCMEC/D3 cells and astrocytes. Indeed, P2Y receptors, due to their subcellular expression, acting on voltage-gated membrane channels, are able to inhibit neurotransmitter release, modulate dendritic integration, facilitate neuronal excitability, or affect other various neuronal functions such as synaptic plasticity or gene expression [The oxidative stress may also be counteracted via the purinergic signaling. Indeed, ADP activates P2Y13 receptors, leading to the increased activity of heme-oxygenase, which has a cytoprotective activity. Such modulating activity may be advantageous in AD  and mApopression . FurtherThe here outlined results could give additional support to promote mApoE-PA-LIP as putative therapeutic tool for AD treatment. Indeed, targeting the neurovascular unit in AD instead of a classical neuron-centric approach in the development of neuroprotective drugs may result in improved clinical outcomes.


However, a comprehensive summary and explanation for evolutionary and functional synergies in Ca2+ signaling remains elusive in green plants. We review mechanisms of Ca2+ membrane transporters and intracellular Ca2+ sensors with evolutionary imprinting and structural clues. These may provide molecular and bioinformatics insights for the functional analysis of some non-model species in the evolutionarily important green plant lineages. We summarize the chronological order, spatial location, and characteristics of Ca2+ functional proteins. Furthermore, we highlight the integral functions of calcium-signaling components in various nodes of the Ca2+ signaling pathway through conserved or variant evolutionary processes. These ultimately bridge the Ca2+ cascade reactions into regulatory networks, particularly in the hormonal signaling pathways. In summary, this review provides new perspectives towards a better understanding of the evolution, interaction and integration of Ca2+ signaling components in green plants, which is likely to benefit future research in agriculture, evolutionary biology, ecology and the environment.Adaptation to unfavorable abiotic stresses is one of the key processes in the evolution of plants. Calcium (Ca This review focuses on the dynamic properties of Ca2+ signaling and its complex molecular network from environmental interaction, cellular function and evolutionary perspectives. Readers are also directed to existing excellent reviews for a more systematic overview of calcium signaling i increase 1 (AtOSCA1) encodes a plasma membrane calcium-permeable channel, which is responsible for the hyperosmolality-induced transient elevation in Ca2+ . Mo. Mo2+ seignaling . Similarignaling ,33. Over2+ signals; SOS3 cooperates with SOS2/CIPK24 to induce phosphorylation and activation of SOS1/NHX7, a plasma membrane Na+/H+ transporter . Ca. Ca2+-pe process . Ca2+-ATley root  and wateley root . AtCAX1  vacuole ,89. Howe2+ is subsequently decoded in the targeted cells to induce appropriate responses depending on the types and levels of abiotic stresses, where calcium sensors play vital roles in this process. Calcium sensors are divided into three groups: sensor relays , sensor protein kinases , and bimolecular sensor responders , Ca2+-CaM-dependent kinases (CCaMKs), and CIPKs  are activated or enhanced by binding with specific CaMs and there are CaMKs that harbor a CaM-binding domain in some plant species  100,101101. Somebidopsis . Calmodubidopsis ,104. Thebidopsis ,104,105.ey shoot . Taken t2+ concentration through the translocation of an autoinhibitory domain and a conformational change resulting from the interaction between Ca2+ and the binding domain , a serine/threonine protein kinase domain (PKD), an autoinhibitory junction (AJ), and an N-terminal variable domain (NTD) . CDPKs ag domain  . I. I2+ bin)-domain ,125. The)-domain . In addi)-domain . CBL-CIP)-domain . Thus, t)-domain ,127, whipression ,124. In pression ,129. In 2+-binding proteins including the annexins (ANN) . T. T2+ “met stress . Moreoved plants . These f2+ signaling systems fulfill the role of signal transduction in organisms ranging from aquatic unicellular algae to terrestrial multi-cellular higher plants | > 1. The differentially expressed genes (DEG) of the calcium-related gene family present with drought treatments and light conditions are more numerous, at 137 and 132, respectively (Nicotiana tabacum [Arabidopsis and transgenic barley [The major plant response to abiotic stresses is mediated through gene expression patterns, leading to reconfiguration of the proteome and resulting in conferred stress tolerance. Among them, the proteins of Caectively , where tectively . Meanwhi tabacum . Screenic barley . Neverthc barley ,169. Simc barley ,171. SimArabidopsis [Certainly, several reported genes are not listed in the five modules due to different experimental condition settings and regulatory mechanisms; for example, CDPK23 is involved in the osmotic stress exerted by drought and salt stresses in bidopsis , and CaMbidopsis ,172. The2+ signaling events has been refined in plant cells during their adaptation and interaction with the environment. Ca2+ channels are involved in the influx of Ca2+ and the regulation of Ca2+ gradients, Ca2+ sensors decode Ca2+ signatures to decipher the nature of the stimulus, and the signals are then transduced through the appropriate stress hormone pathways to produce a series of physiological responses (2+ spatiotemporal patterns coordinated by Ca2+-permeable channels, and the subsequent positional-directional interaction of downstream sensors, remain questionable. Specifically, the majority of channels do not possess strict selectivity for Ca2+, which suggests that other regulatory pathways may have made some concessions for Ca2+ flux [2+ waves as mobile signals precisely integrates activities throughout plants, whose mechanisms, nevertheless, lack systematic descriptions [2+ sensors exhibit functional redundancy, and the absence of a single sensor does not elicit significant changes in response to abiotic stress. Collectively, Ca2+ is implicated in crosstalk between multiple signaling pathways, coupled with phytohormone transduction, forming an intricate signal network. However, these above-mentioned questions, as challenges for scientists, still need urgent intensive research.Calcium has emerged as a ubiquitous second messenger between abiotic stresses and physiological responses. A large and sensitive network of Caesponses  [9,12,17a2+ flux . Remarkariptions . In para2+ signaling displays fundamental differences to other protein evolutions in general; Ca2+ signaling components encompass a wide evolutionary diversity, with either conservation or variation, which are both required for developing the capability to adapt to complex and changing environments [2+-sensing proteins may carry the imprints of evolutionary adaptation to abiotic stresses or break evolutionary bottlenecks in various ways. With the increasing number of sequenced green plant genomes, significant progress has been made towards our understanding of this evolutionary fundamental process, which may provide the molecular and bioinformatics background for their functional analysis in some non-model species in the evolutionarily important green plant lineages. Consequently, despite the considerable advancements in the recent research of Ca2+ signaling system in plants, comprehensive analysis from signal recognition to adversity response requires further exploration. We propose future research work in exploring Ca2+ signaling functional genes from the model plants Arabidopsis and rice to lay the foundation for improving crop abiotic stress tolerance. It is also worth investigating the evolution and functional differentiation of Ca2+-related gene families in abiotic stresses using the model moss Physcomitrella patens and other early divergent plant lineages. Finally, we should build the regulatory network of Ca2+-related proteins for individual cell types and integrate the potential molecular interactions between different Ca2+ response modules for different abiotic stresses.From an evolutionary perspective, the functional exertion of calcium-related genes is likely to have certain dosage and complementary effects. Caronments ,108. Fur


Methods: We utilized emergency department data on acute urticaria-related visits managed by the California Office of Statewide Planning and Operations for 201 zip codes located in southern central California  collected during the years 2016 and 2017. Census data from the same zip codes were considered as a population at risk. Socioeconomic and environmental parameters using CalEnviroScreen  database for the zip codes were evaluated as risk factors. Results: The incidence rate of acute urticaria in San Joaquin Valley during 2016–2017 was 1.56/1000 persons . Multivariate Poisson analysis revealed that zip codes with high population density (RR = 2.81), high percentage of farm workers (RR = 1.49), and the composite of those with high and medium percentage of poverty and those with high and medium percentage of non-white residents (RR = 1.59) increased the likelihood of the occurrence of acute urticaria. Conclusion: High population density, farm work, poverty and minority status is associated with a high risk of having acute urticaria. At least 15–20% of the population in the world suffers from urticaria. Allergy triggers contribute to the development of urticaria. Not much is known about the demographic and environmental risk factors that contribute to the occurrence of acute urticaria.  Urticaria is a common health outcome. Around 15–20% of the population in the world suffers from urticaria . The couStudies focusing on urticaria are limited . Women hKnowledge of specific triggers or causes for the presentation of urticaria is much more developed. Potential triggers include environmental allergens such as pollen, spores originating from mold, dust mites, animal hair , food prSan Joaquin Valley, the southern part of the Central Valley of California, USA has one of the worst air quality outcomes in the United States . AgriculThe purpose of this study was to identify environmental and sociodemographic risk factors contributing to the visits for the treatment of acute urticaria at emergency departments (ED) of the medical facilities located in the southern Central Valley counties. The study was approved by the institution’s review board for the protection of human subjects. This study was a retrospective cross-sectional analysis of emergency department visits for acute urticaria. Health data were collected from the Office of Statewide Planning and Development (OSHPD) in California . To mainICD-10 CodeDiagnosisL50.0 Allergic UrticariaL50.1Idiopathic UrticariaL50.2Cold and Heat UrticariaL50.3Dermatographic UrticariaL50.4Vibratory UrticariaL50.5Cholinergic UrticariaL50.6Contact UrticariaL50.8Chronic or Recurrent UrticariaL50.9Unspecified UrticariaThe incidence rate of urticaria-related emergency department visits was the primary outcome of interest. According to the American Academy of Allergy Asthma and Immunology , the diaIn this study, these codes except L50.4 and L50.8 were used to extract urticaria-related ED events from OSHPD administered data. Allergic urticaria includes acute allergic reaction resulting in urticaria, allergic medicamentosa, and urticaria resulting from the use of a particular food or drug . IdiopatIn multivariate analysis (Poisson regression), the count of urticaria-related events per zip code was the primary outcome assessed. Zip codes vary in population size; therefore, the population at risk for contracting urticaria varies across zip codes. Population count estimates were used to adjust for the population at risk (offset) within each zip code. This method adjusts the model and allows for urticaria events to be treated as the numerator and the population at risk as the denominator so that regression coefficients can be interpreted as rate ratios. We used patient’s zip code of residence to merge hospital data with the US Census data.3), we used a natural log of the values of population density. High unevenness in the values of variables can affect the model fit and estimations. The percentage of agricultural workers and population density were treated continuously in analysis. We utilized additional data sources to identify the effect of covariates on ED visits for the management of acute urticaria. The percentage of agricultural workers and rurality of a zip code were the two primary covariates of interest. To estimate the percentage of agricultural workers in each zip code, we used 2011–2015 US Census estimates of agriculture, forestry, fishing, and hunting using the American Community Survey. In the 2012–2016 US Census data, population density is estimated by dividing the total population per zip code. Because the average value of population density per zip code was much higher , compared to the average values of other covariates . Therefore, we combined the two variables and used the dichotomous composite in the final multivariate model. This process is referred to as forming a composite to avoid the multicolinearity effect [For other covariates, additional measures were retrieved from the CalEnvironScreen v1.0 tool, Office of Environmental Health Hazard Assessment, Sacramento, USA which aggregated data to the zip code level from various data sources including the US Census and the California Air Resources Board, among others . Adminisy effect . Multicor and unadjusted Poisson models were conducted to test bivariate associations between each of the predictor variables considered in the analysis and the outcome variable. If the predictor variable was not previously explored in the literature or was not significant, it was not included in the final adjusted model. White’s test of heteroscedasticity demonstrated that an ordinary least squares model was a poor fit for these data  because of a violation of the assumption of homogeneity of error variance. Therefore, ordinary least squares regression was not used in the final model although it was used in a fully adjusted model to investigate collinearity across predictor variables. Statistical analysis was conducted in the R data analysis system [To fit the discrete nature of the outcome variable, Poisson-based regression was used. Pearson’s s system .n = 3,237,113). As reflected in n = 99) of missing data were removed and we successfully merged 14,417 cases with zip codes, and these were used for the analysis. There were 14,417 acute urticaria-related emergency department visits from 2016 to 2017 in the San Joaquin Valley composing 0.4% of all ED visits , multiplied by 1000. The number of people at risk being the entire population of the selected counties. Overall, the San Joaquin Valley had an incidence rate of 1.56 per 1000 persons from 2016–2017. Women had a higher rate (1.9 per 1000 persons) of acute urticaria-related ED visits compared to those among men (1.5 per 1000 persons). Children less than five years of age had the highest rate of ED visits (5.5 per 1000 persons) followed by those among youth with age less than nineteen years (3.4 per 1000 persons). Black/African American (2.0 per 1000 persons) and Hispanic/Latino (2.2 per 1000 persons) residents had nearly twice the rate of ED visits in comparison to the rate of ED visits among white residents (1.2 per 1000 persons). p < 0.05 and Pearson’s r = 0.249, p < 0.05, respectively). Poverty and non-white concentrations were positively associated with the rate of urticaria ED visits . Diesel exhaust PM was positively associated with urticaria ED visits .p < 0.05) where one percent increase in population density resulted in 2.81 times the risk of acquiring acute urticaria We found that the percentage of agricultural workers was positively associated with ED visits  where a one percent increase in the percentage of agricultural workers was associated with a 1.49 times increase in the rate of urticaria ED visits. Diesel exhaust PM was significant and positively associated with urticaria-related ED visits  where a one unit increase in diesel PM was associated with a 6% increase in urticaria-related ED visits. The composite of the percentage of those living in low poverty areas and areas with a high percentage of the white population was negatively associated with urticaria-related ED visits . In other words, zip codes (n = 61) with low levels of poverty and low levels of non-white individuals have about a 37% lower risk of acute urticaria-related ED visits compared to all other zip codes included in the analysis (n = 140).In As shown in As shown in San Joaquin valley presents a unique environment. The valley has a semiarid climate with very hot and dry summers and mild winters. As America’s breadbasket, agriculture and related industries are the most common among the industries in the valley. Exposures resulting from agricultural operations such as dust and pesticides add to the pollution in addition to the pollutants from other natural and manmade sources including frequent bushfires in California. Allergens from air pollution have contributed to an increase in asthma outcome in the Central Valley ,20. HoweIn our study, we found that working in agriculture increased the risk of acute urticaria. Multiple studies have reported contrary evidence. In a Finnish study based on an analysis of national level physician-reported data, the authors found that agricultural workers were the most common occupational group with contact urticaria and that cow dander was the most commonly found cause of contact urticaria . In one In our study, we found that living in an urban setting increased the risk of having acute urticaria. A Polish survey reported no effect of urban lifestyle on the first episode of urticaria . Our resWe showed that the individuals living in zip codes that have high and medium percentage of poverty level had a higher risk of urticaria-related ED visits compared to the individuals living in zip codes that have a low percentage of poverty level. As shown in In our study, individuals living in zip codes with a high and medium percentage of non-white neighborhoods had a higher risk of urticaria-related ED visits compared to the individuals living in zip codes with low percentage of non-white neighborhoods. As shown in In our study, we found that being less than 18 years of age increased the risk of developing acute urticaria. In a Polish population survey, the authors reported that the mean age for the first episode of acute urticaria was 15 years . An AustIn our study, we found that the rate ratio was nearly 3:4 for male to female. Others reported similar results. Salvarates et al.  reportedIn our study, we found a positive association between diesel particulate matter and increased incidence of ED visits for acute urticaria. According to the International Agency for Research on Cancer, diesel exhaust is a probable carcinogen (group 2A) . ParticuTo our knowledge, no other study has addressed the urticaria situation in the Central Valley of California; the unique environment that might be contributing to the triggers for different allergic conditions. Another major strength of this study was the application of combined ED visit data and the census data using the American Community Survey resulting in estimates that are more generalizable compared to the situation where analysis restricted to ED visit data only. The possibility of self-reporting bias was also eliminated because of the use of ED data.The study had some limitations. The outcome variable was the ED visits count for acute urticaria and the visits could have been first time or recurrent for the individual patient. We did not account for the specific allergic triggers that could exacerbate urticaria episodes other than diesel particulates. Further efforts should focus on explaining the cause-and-effect relationship for the triggers that could be unique to the Central Valley. As opposed to a two-year data in our study, a five-year or more data can present opportunities to understand the trends of acute urticaria. In this population-based study based in the Central Valley of California, we found that high and medium percentage of poverty and high and medium percentage of non-white population, greater percentage of agricultural workers, young age (less than 18), and higher concentration of diesel exhaust were associated with a high risk of acute urticaria. In particular, the magnitude of the risk of acute urticaria was highest among zip codes with high population density, high percentage of agricultural workers and those containing high and medium percentage of individuals living in poverty and had high and medium percentage of racial minorities. Furthermore, it is possible that most agricultural workers lived in zip codes with high and medium percentage of poverty and the greater to medium percentage of racial minorities. With the unique situation of farming practices (production of diverse commodities) and workforce  in California compared to the other regions in the United States, it is important to understand the needs of farm workers as they may have these environmental and social challenges that could put them at risk for acute urticaria. Further studies should explore more about agricultural worker living conditions, access to care and economic situation, and should also emphasize designing intervention to reduce acute urticaria outcomes targeted for other communities such as those living in urban and suburban neighborhoods with high social and economic inequalities.