During heightened vigilance, noradrenergic signaling leads to quasi-synchronous Ca2+ elevations encompassing soma and processes across the brain-wide astroglia network. Distinct from this vigilance-associated global Ca2+ rise are apparently spontaneous fluctuations within spatially restricted microdomains. Over the years, several strategies have been pursued to shed light on the physiological impact of these signals including deletion of endogenous ion channels or receptors and reduction of intracellular Ca2+ through buffering, extrusion or inhibition of release. Some experiments that revealed the most compelling behavioral alterations employed chemogenetic and optogenetic manipulations to modify astroglia Ca2+ signaling. However, there is considerable contrast between these findings and the comparatively modest effects of inhibiting endogenous sources of Ca2+. In this review, we describe the underlying mechanisms of various forms of astroglia Ca2+ signaling as well as the functional consequences of their inhibition. We then discuss how the effects of exogenous astroglia Ca2+ modification combined with our knowledge of physiological mechanisms of astroglia Ca2+ activation could guide further refinement of behavioral paradigms that will help elucidate the natural Ca2+-dependent function of astroglia.Astroglia display a wide range of spontaneous and behavioral state-dependent Ca Astroglia are ubiquitous in the brain and exhibit a range of morphological structure adapted for each brain region. Star-shaped or protoplasmic astrocytes in gray matter possess minimal internal structure and are shaped by the surrounding neuropil, whereas fibrous astrocytes in white matter contain fibrils that assist integration within axon bundles . Special2+. A list of many but not all membrane proteins enabling rise in astroglia intracellular Ca2+ is shown in 3R2 protein or ITPR2 gene) – whether demonstrated or expected; in vivo characterization – a crucial factor given that even in the most conservative ex vivo preparation that is acute brain slice preparation, alterations in astroglia protein expression, morphology and Ca2+ dynamics can occur within less than 2 h of preparation  whole-cell, behavior-associated signals as well as (ii) spatially localized microdomain activity, and (iii) the functional consequences of their inhibition. We then discuss (iv) what effects of exogenous astroglia Ca2+ activation have been identified that could guide further refinement of behavioral paradigms to study the natural function of astroglia.In this review, we describe the various forms of astroglia Ca2+ dynamics directly to behavioral state became possible with the introduction of an approach that allows study of cellular brain activity in head-fixed awake mice on a spherical treadmill using two-photon microscopy -positive neurons, which depends on activation of nicotinic acetylcholine receptors  GCaMP6s in mouse noradrenergic and cholinergic neurons, respectively. Locomotion is accompanied by Ca2+ elevations in both types of terminals in primary visual and auditory cortices  type glutamate receptors , during inhibition of glutamatergic receptors, and during isoflurane general anesthesia. These features suggest that a considerable portion of sparkles resemble intrinsically-generated Ca2+ elevations that have been previously described in cortical astrocytes of anesthetized mice , ryanodine receptors, voltage-dependent Ca2+ channels or Na+-Ca2+ exchangers  visualization of astroglia Ca2+ dynamics in awake organisms during behavior, (2) reduction or abolishment of the behavior-dependence of Ca2+ dynamics through astroglia-specific genetic deletion of a signaling pathway component, and (3) the detection of consequences for circuit activity and/or behavior.The most convincing and complete evidence if and how astroglia Ca2+ elevations. Its importance is further demonstrated by its evolutionary conservation. Sensory stimulation (olfaction) or startling touch of Drosophila larvae triggers release of the Drosophila adrenergic transmitter analogs octopamine and tyramine from Tdc2-positive neurons  channel Water witch (Wtrw). Somatic astrocyte Ca2+ elevation, through an unknown mechanism, potentially causes release of ATP, which following breakdown to adenosine, activates the inhibitory AdoR on dopaminergic neurons, relieving chemotaxis and startle-induced reversal behaviors from dopaminergic inhibition , which suppresses locomotion-induced norepinephrine release, results in considerable loss of Bergmann glia Ca2+ responsiveness that lasts for more than 45 min whereas ataxic motor coordination, present 15 min after onset of exposure to ethanol, is completely recovered 30 min later   beyond individual synapses , specifically the humanized Gq protein-coupled muscarinic receptor type 3-based DREADD (hM3Dq). This finding could either mean that even excessive Ca2+ elevation in Hes5-positive supSDH astrocytes does not modulate extracellular D-serine signaling, or it could suggest that the detection method was not sensitive enough to take advantage of the Adra1a conditional knockout mouse line to imperatively link capsaicin-induced, noradrenergic Ca2+ activation of Hes5-positive supSDH astrocytes to D-serine signaling.An independently developed (supSDH) . Althougmolecule , but seesynapses . It is c1-adrenergic receptor-dependent widespread, global Ca2+ elevation in astrocytes but not in neurons  was specifically deleted from hippocampal CA1 astrocytes. Extracellular L-serine does not follow circadian oscillations, indicating that circadian D-serine fluctuations originate from astrocytes. Remarkably, α7nAChR-dependent D-serine release causes the glycine-binding site of N-methyl D-aspartate receptors (NMDARs) to reach saturation during the active/wakeful phase of the mouse. This finding raises the possibility that transient vigilance-independent D-serine release may be less impactful during the active phase, when NMDAR glycine-binding sites appear to be saturated, than during sleep, and may offer an explanation why several phenotypes arising from astroglia-selective reductions in Ca2+ dynamics have been found linked to the regulation of sleep  receptor GABABR  spontaneously release ATP which activates P2Y1 autoreceptors and together with intense gap junction coupling among ISCs, intracellular Ca2+ rises in clusters of neighboring cells . The Gourine laboratory studied the pressure sensing mechanism in more detail in an in vitro preparation and proposed a model for the initial pressure-dependent ATP release mechanism based on an interaction between TRPV4 ion channels and connexin 43. After finding that P2Y1 autoreceptors appear to serve as signal amplifiers, they deduced that vesicular release of ATP from brainstem astrocytes is the signal that excites central sympathetic centers  settled an earlier controversy about the expression of this receptor in astrocytes . In addievations . It stilin vivo hippocampal CA3-CA1 long-term depression (LTD)  . Cannabion (LTD) . A recenon (LTD) .2+ stores with thapsigargin, while DSE was not. Also, it was inhibited by loading astrocytes with the high-affinity Ca2+ chelator BAPTA or the G-protein inhibitor GDPβS, a procedure leading toward enhanced DSE. While pyramidal neuron depolarization-induced astrocyte Ca2+ elevations were insensitive to inhibition of mGlu1, synaptic potentiation was inhibited, suggesting presynaptic mGlu1 activation downstream of astrocyte Ca2+ elevation is responsible for synaptic potentiation  or excitation (DSE) . An inventiation .in vivo high-frequency stimulation-induced long-term potentiation (LTP). Consistent with the dependence on NMDA receptors, high-frequency stimulation-induced LTP as well as novel object recognition could be rescued in astroglia-specific CB1R knockout mice by substituting with saturating concentration of D-serine  in anesthetized mice  regulator, facilitating endocytosis of GAT, impairing synaptic GABA clearance, and leading to paralysis  or with NMDA receptor inhibition  to wakefulness, there is an enhanced IP3R2-dependent Ca2+ elevation. This finding is consistent with an independent recent astroglia Ca2+ imaging study where intracellular Ca2+ release was attenuated by astroglia-specific gene deletion of stromal interaction molecule 1 (STIM1)  discussed below   . The tra (STIM1) . SurprisEM sleep  when eleEM sleep . This fiagonists  raise thlidation . This fied above  or for ied below . Bojarskrivation . This ste (VIPP) . There t2+ pump and will be discussed below. For actuators that represent the overexpression of an exogenous G protein-coupled receptor that elevates Ca2+, one needs to consider the physiological dynamic range of the expected signal regarding expression level, its subcellular location, and duration/pattern of ligand application. Predictions of the consequences of overexpression of ion channels, some with ion selectivity for which no physiological counterpart is known in astroglia, a cell type with ion buffering function, are most complex. Nevertheless, these are very powerful tools that may inform follow-up studies focused on the physiological role of specific endogenous astroglia signaling pathways.In recent years, astroglia-specific overexpression of exogenous actuators for manipulation and interrogation of astroglia physiology has become very popular . The adv2+ ATPase pump (hPMCA) isoform 2, w/b splice variant with excised cytosolic interaction domains (hPMCA2w/b), resulting in a modified pump that constitutively extrudes cytosolic Ca2+. The model was coined “CalEx” due to its function in Ca2+ extrusion2  due to high dose ethanol sedation, reducing astrocyte Ca2+ dynamics with CalEx has protective, opposite effects. The aggravated sedating effect of enhanced astrocyte Ca2+ could be reversed by inhibiting adenosine A1 receptors  projection is critical for remote memory but not for recent memory. Inhibiting CA1 astrocyte Ca2+ dynamics then disrupts remote memory (3R2 gene deletion impairs remote but not recent memory (The Goshen group recently expressed hM4Dievations . This is signals , and thi signals . One coue memory . These rt memory .q protein-coupled orphan receptor MrgA1, which is naturally only expressed in some nociceptive neurons (2+ activation (2+ elevation is accompanied by extracellular reduction in potassium concentration leading to a temporary hyperexcitability of Purkinje cells, the cerebellar principal neurons (5 in cortical astrocytes of awake mice and found that Arch light-activation causes Ca2+ elevations that, in terms of their spatial restriction, resemble microdomain events, but last tens of seconds (2+ elevation remains elusive. As a consequence of astrocyte Arch activation, extracellular glutamate spikes can be detected accompanying slow oscillatory and more synchronized neuronal activity (The mas-related genes G neurons , was thetivation . When ex neurons ; however seconds . The mecactivity .1-adrenergic receptor-based opsin Opto-Gq (2+ has been demonstrated to be sufficient to induce NMDA receptor-dependent LTP and contextual memory (2+ elevations and hyperactivity (2+ activity, such as the Huntington’s disease mouse model (BR activation, elevates Ca2+ levels in astrocytes in an IP3R2-dependent manner. Yet, the phospholipase C inhibitor U73122 failed to inhibit hM4Di as well as GABABR responses in astrocytes. This suggests the possibility that in general, Gi/o protein-coupled receptors in astroglia couple via the βγ subunit to the IP3R2 (Activation of hM3Dq and the α Opto-Gq  in hippol memory . In contl memory . Activatactivity . Transcractivity . Treatmeactivity . They fuse model . Durkee he IP3R2 .2+ dynamics for the processing of information in the brain. Behavioral state transitions occur constantly, ranging from different sleep states to various levels of vigilance during wakefulness and are disrupted in psychiatric disease. Emerging experimental findings, which are schematized in 2+ signal, what spatial and temporal pattern it assumes, and how it feeds back to behavior. While the precise astroglia Ca2+ signals in unanesthetized animals have not yet been defined in vivo for many of the discussed receptors, it appears that a significant portion of astroglia Ca2+ dynamics are representations of neuromodulator signaling and are primed for a role in informing neural populations about the behavioral and environmental context. Since dependence of astroglia Ca2+ activation on behavioral state relies on variable release of neuromodulator and not on variable intrinsic responsiveness of astroglia, it is possible that exogenous astroglia-specific actuators have led to more robust phenotypes because the limited spatial and temporal precision of this approach allows the recruitment of astroglia Ca2+ activation out of behavioral or environmental context. On the other hand, it is encouraging that several similarities in phenotypes have been independently defined. For example, the importance of astroglia Ca2+ dynamics in memory consolidation allowing remote memory formation has been discovered using the IP3R2 gene deletion model as well as independently using inhibitory DREADD overexpression in hippocampal astrocytes. Similarly, a role for astroglia Ca2+ dynamics in developmental synapse elimination was discovered using the same IP3R2 gene deletion model, and independently it was found that developmental synaptogenic cues can be reactivated by exogenous enhancement of astroglia Ca2+ dynamics in adult mice. It will be important to combine insight from exogenous activation studies with our increasing understanding of the behavioral and environmental context when endogenous astroglia signaling pathways are naturally engaged to revise and customize conventional behavioral paradigms for testing the physiological impact of astroglia Ca2+ dynamics. This will be a prerequisite for recognizing alterations in astroglia Ca2+ dynamics and for understanding how they can contribute to nervous system disease.Considerable progress has been made in our understanding of the role of astroglia CaAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


TLR genes influence the risk of developing hematological malignancies. We aimed to evaluate the effect of TLR2 (rs5743708), TLR4 , TLR9  on AML risk, the relation between investigated SNPs and somatic mutations, clinical features, and the overall survival of adult AML patients. All mentioned SNPs were genotyped in 511 AML cases and 503 healthy controls. DNMT3A (R882), FLT3 , and NPM1 mutations’ status were investigated in AML patients. TLR4 rs4986791 was associated with an increased risk of AML under the dominant model . Variant genotypes of the TLR4 rs4986790 or rs4986791 were associated with the odds of developing AML in the codominant model . The TLR9 rs5743836 variant genotype was associated with the NPM1 mutation (p = 0.002). The investigated SNPs were not associated with the DNMT3A, FLT3 mutations and had no significant contribution to the hazard of death after adjusting for covariates. Our findings suggest that TLR4 rs4986791 is associated with AML susceptibility. The combined variant genotypes of TLR4 rs4986790 and rs4986791 increase AML risk, the TLR9 C-G-A haplotype may represent a promising approach to predict a person’s risk for developing AML.Toll-like receptors (TLRs) have an important role in innate immunity, and single nucleotide polymorphisms (SNPs) of  Acute myeloid leukemia (AML), an aggressive malignant hematologic neoplasm with an incidence that is increasing with age, is characterized by complex pathogenicity that arises as a consequence of the accumulation of genetic abnormalities . AML repIt has been reported that the disruption of the immune system is involved in cancer’s pathogenesis . The innTLR genes had been reported .Median survival time (MeST) was calculated to estimate the time point when 50% of studied AML patients died. Log-rank test was used to compare the distribution of overall survival time across studied SNPs under the dominant genetic model. The univariable and multivariable Cox proportional-hazards models were used in order to evaluate the genetic SNPs effects on risk of death expressed as hazard ratio (HR) with 95% CIs adjusted for known clinical covariates . Further, we transformed the number of variant genotypes into a categorical variable with the following categories: 0–1, 2–3, and 4–5 variant genotypes with frequencies of 168 (16.57%), 748 (73.77%), and 98 (9.66%), respectively. Although we noticed an increased odds of AML for subjects carrying 4–5 variant genotypes, the result did not reach statistical significance .We combined all studied SNPs by the number of variant genotypes . For both the TLR4 rs4986790 and TLR4 rs4986791 gene polymorphisms, we combined both the heterozygous and variant homozygous genotypes as a single group and compared it with the double wild-type genotype. Subjects carrying combined heterozygous and homozygous variant genotype of the two TLR4 SNPs (rs4986790 and rs4986791) had higher odds of AML compared with individuals possessing double wild-type genotypes . We did not find a significant association between the other double combinations of the TLR2, TLR4 and TLR9 variant genotypes and the odds of AML (p > 0.05).We also analyzed the joint effect of double combinations of SNPs variant genotypes and we found no significant effect, except for the genotype combination of variants . The preTLR SNP–SNP interactions using a log-likelihood ratio test under the dominant genetic model, but we did not find a statistically significant interaction effect of any two SNPs.Moreover, we performed all TLR4 rs4986790 SNP did not have a modifying effect on the odds of AML initiated by the TLR4 rs4986791 SNP . Taking into account the fact that the presence of the combined variant genotype of TLR4 rs4986790 and TLR4 rs4986791 was significantly associated with increased odds of AML, we found that these two gene polymorphisms may have a combined effect and no modifying effect on AML susceptibility.The results indicated that the TLR2, TLR4 and TLR9 SNPs and somatic mutations in AML patients (TLR SNPs and odds of AML). The results showed that the TLR9 rs5743836 variant genotype (AG + GG) was significantly negatively associated with NPM1 mutation , a higher relative frequency of the TLR9 rs5743836 variant genotype (AG + GG) being observed in the subgroup without the NPM1 (30.5%) vs. patients with NPM1 mutation (14.4%). We noticed a trend towards statistical significance for the association between the FLT3 ITD mutations and TLR4 rs11536889 SNP variant genotype (GC + CC) , the variant genotype (GC + CC) being observed in 26.6% of the patients without the FLT3 IDT mutation vs. 37% of the patients with the FLT3 IDT mutation. No significant associations were found between the DNMT3A and FLT3 mutations and the other investigated SNPs.We further analyzed the relationship between the studied TLR2 rs5743708 GA genotype had a better overall survival than carriers of the wild genotype (p = 0.012). In the separate univariable Cox proportional-hazards models, we observed that only the TLR2 rs5743708 GA genotype was significantly associated with a decreased HR for death , while a tendency toward statistical significance was noticed for the TLR4 rs4986790 AG + GG variant genotype (p = 0.092) and TLR4 rs4986791 CT genotype (p = 0.070). TLR2 rs5743708 did not remain as an independent predictor for the hazard of death (adjusted p = 0.272) after adjusting for other clinical known covariates  variant genotypes is consistent with that reported by Schnetzke et al. in AML patients from Germany [In this study, we sought to address the question of whether the presence of the polymorphisms of the genes involved in the innate immune system, namely  Germany . One expTLR genes, and aimed to figure out the correlation between these SNPs and the known prognostic factors in AML .Moreover, our study investigated the associations between the prognostic indicators in AML and seven SNPs of TLR2 rs5743708, TLR4 , and TLR9  gene polymorphisms were not associated with AML risk. Our findings are partly similar to those reported by other studies previously [TLR2 rs5743708 SNP was reported in a meta-analysis conducted by Wang et al. [The present study showed that variant genotypes of the g et al. .TLR2 rs5743708, TLR4 rs4986790, and rs4986791 polymorphisms [Aref et al. included in their study 120 AML patients and 100 healthy subjects from Egypt and reported no significant differences in genotype or alleles frequency between patients and healthy controls regarding orphisms .TLR4 rs4986790 and rs4986791 with the risk of development of colorectal cancer was reported by a study that included 268 subjects  [TLR4 rs4986791 and rs11536889, might represent a genetic risk factor for cancer development [TLR4 rs4986791 SNP is associated with an increased risk of AML. Our finding is in contradiction with that reported by Aref et al. [Recently, a significant association of variant alleles of the yptians) . Similarelopment . In agref et al. . One expTLR4 rs4986790 or TLR4 rs4986791 SNPs in the dominant model was positively associated with the odds of developing AML in investigated Caucasian patients.Moreover, we observed that the presence of the double combination of the variant genotype of the TLR4 rs1156889 and TLR9 rs187084 and rs1927911 SNPs were found to be associated with the increased risk of developing cervical cancer among Indian women [TLR9 rs187084 SNP and AML in our cohort of adult patients.The variant genotypes of an women . DespiteTLR9 rs187084 polymorphism was associated with the progression of AML when they investigated a small group of 62 Polish AML patients [TLR9 rs187084 influenced patients’ survival [TLR9 rs187084 influenced the outcome of transplants in AML cases after a myeloablative, non-T-cell-depleted transplant. Furthermore, a reduced relapse rate was observed after transplant in AML patients with the TLR9 gene variant [TLR9 rs187084 SNP, especially with cervical cancer, when they stratified analyses by specific tumor types [Rybka et al. found that the patients . In addisurvival . This is variant . Wan GX or types .TLR9 rs5743836 SNP conferred almost a fourfold increased risk of B-cell Non-Hodgkin lymphomas (B-NHL) in patients from Egypt  [TLR9 rs352140 and rs5743836 SNPs were associated with a decreased risk of development of breast and digestive system cancers [The variant heterozygous genotype of the 16–7.14) . This ob cancers .TLR2 and TLR3 SNPs with the development of chronic lymphoproliferative diseases and considered that these SNPs might represent early additional diagnostic and prognostic criteria [Nazarova et al. suggested a relationship between criteria . HoweverTLR9 was associated with a fivefold increase in AML risk, while C-A-G and T-A-A haplotypes showed an about twofold elevated risk of AML among adult patients.No previous study investigated the impact of haplotypes of the mentioned gene polymorphisms on the AML risk. Our findings revealed that the C-G-A haplotype of TLR9 rs5743836 and rs187084 SNPs haplotype on cancer risk and reported a protective effect of the TLR9 SNPs haplotype for endometrial cancer risk  [Ashton et al. investigated the influence of the = 0.002) .TLR4  SNPs showed no association with increased susceptibility to AML. Contrary to our observation, the haplotype analysis of TLR4 SNPs, (namely AT and GT haplotypes) were associated with increased odds for colorectal cancer  [TLRs’ haplotype is restricted to certain subgroups of patients.In our study, the haplotypes of the ctively) . The disTLR2, TLR4 and TLR9 SNPs on the overall survival in AML cases was studied by the Kaplan–Meier curve. AML patients who had the TLR2 rs5743708 GA genotype had better overall survival. A previous study reported that the homozygous variant genotype of TLR2 rs5743708 polymorphism was significantly associated with the shortest overall survival in Egyptian AML cases [TLR4 and TLR9 gene polymorphisms did not affect the overall survival in our AML patients. These findings are in agreement with those reported by a previous study [TLR SNPs and the hazard of death, we may consider/suppose that the poorer outcome was conferred by other causes.The individual contribution of the ML cases . In addius study . ConsideTLR SNPs and somatic mutations, we observed a significant association between the variant genotype of the TLR9 rs5743836 and NPM1 mutation and also a trend towards statistical significance between the TLR4 rs11536889 SNP and FLT3 ITD mutations. To the best of our knowledge, the impact of the mentioned TLR2, TLR4 and TLR9 variants on Caucasian adult AML cases have not been studied simultaneously. Rybka et al. investigated TLR4  and TLR9  susceptibility to AML in a smaller group of subjects (62 AML cases and 126 controls) [TLR2, TLR4, and TLR9 genes with somatic mutation , patient’s gender, age group, and characteristics of AML cases.Regarding the relationship between investigated ontrols) . To our TLR2, TLR4, and TLR9 gene expression, and CEBPA, IDH1, IDH2, etc., mutation status was not investigated in all cases and heterogeneity of the AML patients included in the study (different AML subtype). Another limitation of this study is that this is an observational study of the association of TLR2, TLR4 and TLR9 polymorphisms, and TLR9 haplotypes with AML risk and the overall survival of AML patients based on a single time-point evaluation. Taking into account the suppression of the immune system in active cancers such as AML, further prospective longitudinal studies with multiple time-point measures  should be conducted in the future to establish if the TLRs of concern are being suppressed by the active cancer.The present study has some limitations: a lack of data regarding Moreover, further research that estimates the effect of gene–gene and gene–environment interactions may eventually provide a better and more comprehensive understanding.TLR4 rs4986791 SNP and AML susceptibility. The combined variant genotypes of TLR4 rs4986790 and TLR4 rs4986791 appear to increase AML risk. In addition, the current findings indicate that the identification of patterns of genetic variations, in the form of the C-G-A haplotype of TLR9 rather than the TLR9 single nucleotide polymorphism, may represent a promising approach to predict the risk for developing acute myeloid leukemia.Our findings suggest a potential association between the


EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1–G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.Deregulation of the  EVI1 is transcribed from the MECOM locus on chromosome 3q26.2, which also encodes the Myelodysplasia Syndrome-associated Protein 1 (MDS1) and the longer splice form MDS1-EVI1 .–37.G2DHEEVI1 downregulation induced by G2DHE deletion, immunophenotyping of PARPi treated inv(3) MUTZ-3 cells showed a loss of CD34 expression along with an increase in myelomonocytic differentiation as measured by CD14 expression , whereas the immature progenitor cells (CD34+/CD14−) were almost completely lost in comparison to the controls /t AML that may render it a therapeutic liability for this disease category. The interplay and additive effect of those TFBS is integral to G2DHE function as both modules are required for maximum enhancer activity. Among the most recurrent and functionally important TFs in reporter studies of G2DHE in vitro were RUNX1, MYB, CEBPA, IKZF1, FLI1, ELK1, and GATA2 itself, which play key roles in hematopoiesis [EVI1 transcription in AML cells by studies of the minimal EVI1 promoter of the MECOM locus, which is the target of the hijacked G2DHE in 3q-rearranged AML [CEBPA has been reported to be negatively regulated by EVI1, we have observed measurably high expression of CEBPA in inv(3)/t cell lines /t cells, and activating potential in reporter experiments [G2DHE allowed targeted chromatin-capture and thereby identification of novel interacting partners. A limitation of ChIP-SICAP is its lack of targeted identification of locus-specific interactions, thus it was not possible to discriminate between TF complexes of the orthotopic and rearranged G2DHE alleles. However, histone ChIP-Seq data point to the generation of a super-enhancer accessible for TFs only on the rearranged 3q21 allele [Our studies provide molecular and functional evidence of the TF composition of opoiesis –41. In lnged AML . Althouges Fig. S, clear beriments . The pre1 allele .ChIP-SICAP was used to identify a panel of CEBPA and RUNX1 interactors. Association of the two hits PARP1 and IKZF1 was confirmed by an in vitro pull-down approach Fig. ; howeverG2DHE impacting EVI1 promoter activity and chromatin co-occupancy with RUNX1 and CEBPA as found in ChIP-SICAP experiments implicate an interaction of IKZF1 with EVI1/GATA2 pathways, which was previously indicated by a study investigating the mutational landscape of EVI1-rearranged AML [EVI1 expression, but no reduced viability of inv(3)/t AML cell lines in response to lenalidomide treatment (data not shown), which causes selective degradation of IKZF1 [The mapping of IKZF1 DNA binding sites in nged AML . We haveof IKZF1 . Notwithof IKZF1 .G2DHE baits RUNX1 and CEBPA in different 3q-rearranged cell lines. Inhibition of PARP1 catalytic activity and PARP1 knockdown led to downregulation of EVI1. RNA-Seq analysis showed that more genes were upregulated than downregulated after PARPi treatment  AML cells, and increased differentiation, which can be partially rescued by overexpression of EVI1. This differentiation pattern was similar to that observed after EVI1 knockdown, BRD4 inhibition, or G2DHE deletion previously [EVI1 knockdown confirmed induction of differentiation gene expression patterns and revealed a subset of coregulated genes. Interestingly, one of the strongest negatively regulated gene both in PARPi and EVI1 knockdown in inv(3) AML cells was PHGDH, a serine biosynthesis enzyme implicated as a critical metabolic regulator necessary for propagation of leukemia cells and other cancers [PARP1 was identified in ChIP-SICAP experiments using both nt Fig. S. Hence, eviously . Compara cancers –47.G2DHE – EVI1 promoter interaction frequency, indicating that the persistent activity of the oncogenic super-enhancer relies on the constitutive presence and chromatin modulation of PARP1. PARP1-mediated chromatin remodeling has been shown to be a facilitator of binding of the pioneer TF Sox2 to intractable enhancers in closed chromatin regions of embryonic stem cells, making PARP1 a requirement for the expression of pluripotency genes and stemness [Previous studies showed that PARP1 can exert its positive gene regulatory function by poly(ADP-ribosyl)ation (PARylation) of histones and the histone-associated protein DEK Fig.  and the stemness . Conversstemness . AltogetSupplemental MaterialFile S1 Supplement ChIP-SICAP resultsFile S2 Supplement TPM values RNA-SeqFile S3 Supplement differential gene expression


Dear Editor,With increased survival of patients with multiple myeloma (MM), therapy-related myelodysplastic syndrome (t-MDS) and t-acute myeloid leukemia (AML) may occur more frequently , 2. We pIn June 2015, a 64-year-old female was diagnosed with IgG kappa (κ) MM. IgG levels were 46g/L, κ-serum-free light chains (SFLC) 75.4mg/L and ß2-microglobulin 8.2mg/L Fig. . Anemia 6/L). BM assessment did not reveal increased PCs or MDS, and serological parameters indicated stable disease.First-line therapy with bortezomib, cyclophosphamide, and dexamethasone was followed by autologous stem cell transplantation and maintenance therapy with lenalidomide Fig. . After 16/L, platelets 12x106/L) and κ-SFLCs increased .Due to frailty at that time, she was ineligible for intensive AML induction therapy. Therefore, treatment with decitabine/venetoclax was started in February 2019. A BM biopsy in March 2019 confirmed CR of the t-AML Fig. , right; ted Fig. . This inIn June 2019, after worsening pancytopenia re-emerged and myeloid blasts were detectable in PB smears, decitabine/venetoclax was re-initiated. The BM biopsy in August 2019 showed persisting (30%) immature myeloid blasts .


Despite recent progress achieved in the management of acute myeloid leukemia (AML), it remains a life-threatening disease with a poor prognosis, particularly in the elderly, having an average 5-year survival of approximately 28%. However, recent evidence suggests that immunotherapy can provide the background for developing personalized targeted therapy to improve the clinical course of AML patients. Our review aimed to assess the immunotherapy effectiveness in AML by discussing the impact of monoclonal antibodies, immune checkpoint inhibitors, chimeric antigen receptor T cells, and vaccines in AML preclinical and clinical studies.Acute myeloid leukemia (AML) is a malignant disease of hematopoietic precursors at the earliest stage of maturation, resulting in a clonalproliferation of myoblasts replacing normal hematopoiesis. AML represents one of the most common types of leukemia, mostly affecting elderly patients. To date, standard chemotherapy protocols are only effective in patients at low risk of relapse and therapy-related mortality. The average 5-year overall survival (OS) is approximately 28%. Allogeneic hematopoietic stem cell transplantation (HSCT) improves prognosis but is limited by donor availability, a relatively young age of patients, and absence of significant comorbidities. Moreover, it is associated with significant morbidity and mortality. However, increasing understanding of AML immunobiology is leading to the development of innovative therapeutic strategies. Immunotherapy is considered an attractive strategy for controlling and eliminating the disease. It can be a real breakthrough in the treatment of leukemia, especially in patients who are not eligible forintensive chemotherapy. In this review, we focused on the progress of immunotherapy in the field of AML by discussing monoclonal antibodies (mAbs), immune checkpoint inhibitors, chimeric antigen receptor T cells (CAR-T cells), and vaccine therapeutic choices. Acute myeloid leukemia (AML) is the leading cause of mortality among all leukemias .It starts in the bone marrow (BM), moves into the blood, and sometimes spreads to other organs including the lymph nodes, liver, spleen, skin, testicles, and central nervous system ,3. AML iIn the past, a one-size-fits-all regimen of either high- or low-intensity chemotherapy combined with HSCT was the only available treatment for AML. Response to this treatment is not always fully satisfactory. Hematological remission is only obtained in about a third of patients. This percentage is higher in patients under the age of 60–65, with success rates approaching 50–60%, while, for older patients, long-term survival beyond 5 years does not exceed 10–20% of cases [The characterization of human tumor antigens , the intIn acute lymphoblastic leukemia (ALL), immunotherapy is extensively utilized, and defined antibody-based approaches are already included in standard protocols . In contAlthough HSCT remains the most effective treatment, AML relapse can still occur. In addition, most AML patients are elderly and therefore are not suitable for this type of treatment. Leukemic stem cells (LSCs), resistant to chemotherapy and radiotherapy, are supposed to be responsible for the minimal residual disease (MRD) that predicts relapse but may not be a contraindication for HSCT , and whoA variety of treatment protocols for AML, based on immune-mediated therapeutic mechanisms, have been developed in recent years. Here, we will provide a critical overview of the most important immunotherapeutic treatments for AML.The current concept of antibody-based therapy began with Porter’s finding which disclosed the basic structure of immunoglobulin (Ig). He showed that papain digestion of a rabbit antibody produced two antigen-binding fragments (Fab) and a third fragment that is easily crystallizable (Fc). The former interfered with the antigen binding of the undigested antibody, while the latter did not . To date2 on day 4 plus a daunorubicin and cytarabine (DA) induction chemotherapy regimen to that of standard DA, patients receiving GO had a higher mortality rate, due to venous occlusive disease (VOD) [CD33 is a sialic acid binding receptor highly expressed on myeloid cells . It is rse (VOD) ,27. Morese (VOD) .2) can be safely associated with induction chemotherapy regimens [2 of GO on day 1 induction. GO was well tolerated; karyotypic analysis indicated a significant interaction with GO and enhanced survival of patients with favorable cytogenetics. The latter was a randomized study involving untreated AML or MDS patients who were given DA or daunorubicin and clofarabine (DC) with or without GO (3 mg/m2) on day 1 of induction therapy. No toxicity was observed in the presence of GO, while there was a better 3-year cumulative incidence of relapse and survival.However, based on preliminary studies showing that implementation of lower doses of GO  was significantly higher in the GO regimen. The GOELAMS AML 20061R study was a phase III clinical trial testing the impact of GO’s association with standard chemotherapy, focusing on patients with an intermediate karyotype. The study included 238 AML patients between 18 and 60 years of age who were randomized to standard DA plus or minus GO 6 mg/m2, maximum dose of 5 mg) on days 1, 4, and 7. GO had no impact on the number of patients achieving CR. However, in the following 2 years, the EFS, OS, and relapse-free survival (RFS) of patients treated with GO were significantly better than without GO. In contrast, hematologic toxicity, and particularly thrombocytopenia, was more common with GO. The study suggested that fractionation of a low dose of GO is safe and improves the clinical course of AML. The ALFA-0701  study waSince the results obtained from randomized phase III trials were not conclusive, a meta-analysis of individual patients included in the five above-described studies was carried out. GO did not increase the CR rate, while it significantly improved 5–6-year OS and significantly decreased the risk of relapse in patients with favorable and intermediate-risk cytogenetics .In 2017, GO was re-approved for newly diagnosed and relapsed/refractory (R/R) AML owing to new data on the clinical efficacy and safety of GO administered according to a fractionated dosing schedule .Up to that date, several clinical trials were carried out to study how to reduce GO’s toxicity. Many of these chose to enrich GO treatment with azacytidine. Azacytidine induced maturation of AML blasts, increased CD33 expression, and enhanced GO uptake by these cells . This trAn alternative approach is represented by the use of SGN-CD33A, a humanized anti-CD33 mAb conjugated to a new pyrrolobenzodiazepine dimer, whose safety and efficacy, alone or in various combinations, were evaluated in several clinical trials. Encouraging results by Sutherland et al. demonstrated that SGN-CD33A is highly active in a broad panel of preclinical AML models .http://businesswire.com/Clinical-Hold-Phase1, accessed on 15 October 2021).Interestingly, SGN-CD33 used as monotherapy shows a favorable antileukemic activity. In a phase I trial (NCT01902329), 14 out of 27 (54%) high-risk older AML patients achieved complete remission (CR) and incomplete blood count recovery (CRi), thereby more than doubling the response rate expected upon standard non-intensive therapies such as HMA or low-dose cytarabine. Furthermore, treatment with SGN-CD33A + HMA in older AML patients (NCT01902329) allowed reaching high rates of remission and protracted myelosuppression, with an encouraging CR/CRi rate of 73% . It remaAnother reliable therapeutic target in the treatment of hematological malignancies is the interleukin 3 (IL-3) receptor α-chain (IL3RA) or CD123. It is strongly expressed on myeloid blasts and LSCs, but also in a small subset of CD56+ monocytes ,38. The https://www.ema.europa.eu/en/medicines/human/EPAR/elzonris#authorisation-details-section, accessed on 8 November 2021), followed by the FDA authorization in December 2018. Despite the limited amount of information about this treatment, under exceptional circumstances, tagraxofusp was approved as an orphan medicine by the European Medicines Agency (EMA) in November 2015 (The first paper showing the description of the production of bispecific antibodies (BsAbs) was published in 1961 . Then, tThe interesting data obtained with bispecific antibodies in ALL have prompted the development of the first bispecific antibody CD33/CD3BiTE, named AMG330 , for AMLThe second BsAb format is represented by DART, in which heavy- and light-chain variable domains are on two separate polypeptides stabilized by a C-terminal disulfide bridge .Flotetuzumab (FLZ), also known as MGD006 or S80880, is currently being investigated as a treatment in AML. This so-called DART simultaneously binds CD3 and CD123, leading T cells to recognize and kill in vitro and in vivo AML cell lines and primary AML blasts expressing CD123 . An ongo® technology, which, in contrast to BiTEs and DARTs, retains its Fc region and its associated effector functions and in vivo stability.Together with AMG330 and Flotetuzumab, JNJ-63709178  and MCLA-117 have also entered clinical trials for AML . The firIn addition to its safety and tolerability, a phase I study (NCT02715011) is evaluating the antitumor activity of JNJ-63709178 in R/R AML patients. Instead, the second one is a new T cell-redirecting antibody targeting CD3 on T cells and C-type lectin-like molecule-1 (CLL1) on leukemic cells . This biLSCs play an important role in promoting high relapse rates and treatment resistance in AML patients. LSCs are capable of self-renewal and, at the same time, continue to generate proliferating progenitors and leukemic blasts . TherefoCLL-1 is one of the most studied targets that is involved in the regulation of key regulatory immune functions. Additionally, it is highly expressed in myeloid cells, AML blasts , and LSCCD44 is a transmembrane glycoprotein mainly expressed on hematopoietic cells. The gene is composed of 20 exons which give rise to a variety of isoforms: the standard isoform (CD44s), of about 85kDa , and theCD47 is a cell surface protein belonging to the Ig superfamily, implicated in multiple cellular processes including protein–protein interactions . It is uRegarding CD123, strongly expressed on LSCs, it has been reported that the murine CD123-specific 7G3mAb mediates ADCC by effector cells with specific effectiveness against leukemic cells. In NOD/SCID mice treated with 7G3, AMLLSC engraftment was deeply reduced, and survival improved. Two types of 7G3 mAbs are available. The first is chimeric (CSL360) and the second is humanized (CSL362), also known as talacotuzumab. The results of a phase I study of CSL360 in 40 patients with R/R or high-risk AML (NCT00401739) did not show a significant impact on the disease .Given the inefficacy of CSL360, CSL362 was modified to bind, with higher affinity, both CD16A on NK cells by its Fc region and CD123 by its variable region, thereby more effectively mediating ADCC. As shown in Leukemia 2020, the use of talacotuzumab as monotherapy was clinically less effective in the high-risk myelodysplastic syndrome (HR-MDS) and AML groups of patients resistant to previous HMA therapy NCT02992860). Significant toxicity compromised the success of the study and determined an early treatment discontinuation and disease progression . Given t0. SignifAmong engineered antibodies, there are those in which the Fc region is modified to enhance their antitumor activity by ADCC, antibody-dependent cellular phagocytosis (ADCP), and CDC. A variety of Fc-engineered mAbs are under investigation as potential effective therapies in hematologic malignancies. Besides CSL362, additional Fc-optimized antibodies are described below, based on evidence of their significant activity against cell surface AML-associated antigens.The Fc-engineered CD33 antibody BI 836858 is one of them. Vasu et al. reported that it can mediate the killing of AML blasts by NK cells, which is further enhanced after decitabine pretreatment of AML blasts . A cliniMEN1112, which is directed against CD157 and has recently entered clinical testing, is also an additional Fc-engineered antibody. Krupka et al. found that CD157 is often expressed in primary AML patient samples. Analysis by flow cytometry of 101 AML patient samples at primary diagnosis or relapse showed that CD157 is expressed in 97% of samples. It is more expressed at relapse than at the onset of the disease. Its persistent expression, from primary diagnosis to relapse, makes it an attractive candidate for targeted therapy at any stage of the disease . An ongoThe antitumor immune response is regulated by the type and the function of inflammatory cells infiltrating the tumor microenvironment. Numerous studies have clearly shown that CD8+ T cell infiltration is associated with a favorable prognosis in many solid tumors ,91. NeveSeveral checkpoint inhibitors have been approved for the treatment of solid tumors including the anti-CTLA-4 antibody ipilimumab and the anti-PD-1antibody pembrolizumab. Today, they have become the standard treatment in metastatic melanoma ,102. A PThe use of anti-PD-1 antibodies shows remarkable success both in Hodgkin and some non-Hodgkin lymphomas ,105,106.PD-1 expressed on “exhausted” Tcells binds its ligand PD-L1 on APC. This link delivers intracellular signals inhibiting lymphocyte proliferation and activation. This pathway is normally used by the immune system to avoid response against self-antigens. In some tumors, upregulation of PD-1 andPD-L1 expression allows them to evade the host immune system. Antibodies against these molecules, such as nivolumab and pembrolizumab, are currently under investigation. As shown in the NCT02532231 phase II study, the use of nivolumab in high-risk AML patients in CR ineligible for SCT allowed reaching CR durations of 6 (79%) and 12 months (71%). Rates of OS were 86% at 12 months and 67% at 18 months . Since PNivolumab in combination with azacytidine was assessed in a phase IB/II trial in relapsed AML patients (NCT02397720); the first 53 patients enrolled were evaluated for response: 11 (21%) achieved CR/Cri, and 7 (14%) had hematologic improvement with an overall response rate of 35% .At present, clinical results are promising, but responding patients are still few, and toxicity is considerable. Hence, there is a need to develop combination strategies to increase clinical benefits and reduce toxicities.An effective therapeutic solution to treat patients that develop resistance to anti-PD-1 therapy could be represented by a combined blockade of PD-1 and other checkpoint inhibitors, including T cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3).TIM-3 is an immunoregulatory protein expressed on LSCs in most types of AML, but not on normal HSCs, which is able to enhance antitumor immunity and suppress tumor growth in several preclinical tumor models . As repoRecent studies revealed that TIM-3 and PD-1 are coexpressed during exhausted T cell differentiation and influence T cell immunotherapy efficacy . It seemAdoptive cell therapy (ACT) with tumor-infiltrating lymphocytes (TILs) or gene-modified T cells expressing novel T cell receptors (TCRs) or chimeric antigen receptors (CARs) is a rapidly emerging immunotherapy approach. Among the different types of ACT, the type that has made the most progress in clinical development is CAR-T cell therapy. It is based on the genetic engineering of a patient’s T lymphocytes to induce the expression of a chimeric receptor to be able to recognize a marker expressed on tumor cells, leading to cancer cell elimination. T lymphocytes are sampled from the patient’s peripheral blood and transduced with viral vectors encoding the desired genes. Genetically engineered lymphocytes are then led to proliferating in vitro before re-infusion into the patient’s blood .Severe immune-mediated adverse events following CAR-T cell infusion have been reported; therefore, unwanted toxicity management is one of the key points in implementing this type of ACT. Good selection of the optimal target antigens is essential to overcome the limitations of this approach. Thus far, CD19 has been the main target antigen for CAR-T cell therapy, and encouraging results have been reported, especially in the treatment of B cell neoplasms . In 2013Nevertheless, several single-chain variable fragment (scFv) antibodies specific for AML antigens have been utilized to produce CAR-T cells for clinical investigation. Among them, the single-chain anti-CD33 (CART-33) seems to be the most relevant. However, severe side effects including neurotoxicity and CRS have been experienced. In addition, in a clinical trial (NCT01864902), CART-33 did not affect the clinical course of refractory AML, leading to progression within nine weeks following CART-33 cell infusion. Thus, the trial has been suspended by the US FDA for two months until the adoption of more severe criteria. For the treatment of R/R AML patients, the authors proposed the use of CART-33 infusions as a short-term problem-solving approach followed by chemotherapy or HSCT .However, only a few clinical studies of CAR-T cell immunotherapy for AML patients are currently being undertaken . This maFollowing HSCT, the powerful graft-versus-leukemia (GVL) effect, mediated by allogeneic donor lymphocytes, has proven to be the most impressive evidence of how the immune system can control AML spread. However, given the low range of specificity of the donor lymphocytes, the recipient may develop graft-versus-host disease (GVHD), which is linked to increased morbidity and mortality . An attrThese data were also confirmed by a phase I study NCT01266083) that investigated the use of a multivalent WT1 peptide vaccine  in adults with AML in the first complete remission . However66083 thaVaccination could also be performed using DCs, potent APCs able to induce strong specific antitumor immune responses. DCs can be obtained from autologous or allogeneic leukapheresis  and by “Clinical trials have shown that DC-based immunotherapy is feasible, safe, and devoid of serious side effects ,137,138.As reported in Van Acker 2019, major efforts are concentrated on the use of DCs derived from autologous peripheral blood monocytes (moDCs)  or autolThe identification of novel therapeutic methods capable of modulating the expression of relevant genes in human diseases is challenging. A major hurdle for successful immunotherapy of AML is the lack of optimal AML antigens to be targeted. In this context, a recently developed area of investigation is epitranscriptomics. This area focuses on the changes occurring in the cells following post-transcriptional RNA modification. More than 170 RNA modifications are known. These modifications impact RNA functionality in collaboration with RNA binding proteins termed writers, readers, and erasers. Writer enzymes “write” the RNA modification. The readers read and interpret them, and the erasers remove them. The changes can affect both messenger RNA (mRNA) and ribosomal RNA (rRNA) as well as transfer RNA (tRNA). One of the most common modifications is N6-methyladenosine (m6A) methylation; it can carry out relevant biological functions that affect hematopoietic malignancies. The writer methyltransferase-like 3 (METTL-3) and its associated methyltransferase-like 14 (METTL-14), and the reader YTH domain-containing family protein 2, are critical for LSC survival. Similar considerations can be applied to the demethylases fat mass and obesity-associated (FTO) protein, and AlkB homolog 5 RNA demethylase. Additionally, N6-methyladenosine (m6A) methylation can produce significant effects in the regulation of innate and adaptive immune responses including tumor immunology and immunotherapy .This scenario opens new perspectives in the treatment of AML. However, the state of the art of current knowledge represents the platform for further investigation toward a better understanding of the role of both them6A and other RNA modifications in the identification of optimal AML antigens for innate and/or adaptive immunotherapy of AML.AML continues to be a severe disease. The identification of novel immunotherapeutics, among them GO and tagraxofusps, represents an additional step ahead in the fight against AML. Therefore, there is a hope to increase the availability of these molecules in a way to reduce the use of chemotherapy to enhance the selectivity of the treatment and reduce the patient side effects. Additionally, these new treatments are easy to handle, providing investigators with the possibility to administer the treatment in an outpatient setting.Although this is only the beginning, these new drugs are showing never-before-seen results, saving patients who would not have had survival chances without them.In conclusion, the latest immunotherapeutic drugs allow more patients to experience a better clinical course of the disease.


Oenothera biennis (DSOB) are a by-product of evening primrose oil production that are currently not effectively used. In this study, α-glucosidase inhibition, aldose reductase inhibition, antioxidant capacity, polyphenol composition, and nutritional value  of DSOB were evaluated using the seeds of Oenothera biennis (SOB) as a reference. DSOB was an excellent inhibitor of α-glucosidase (IC50 = 3.31 μg/mL) and aldose reductase (IC50 = 2.56 μg/mL). DSOB also showed considerable antioxidant capacities . DSOB was a reservoir of polyphenols, and 25 compounds in DSOB were temporarily identified by liquid chromatography coupled with electrospray ionization–quadrupole time of flight–mass spectrometry analysis. Moreover, the carbohydrate, protein, and mineral content of DSOB were increased compared to that of SOB. DSOB contained large amounts of fiber and low levels of sugars, and was rich in calcium and iron. These results imply that DSOB may be a potential functional food ingredient for diabetes, providing excellent economic and environmental benefits.The defatted seeds of  Diabetes mellitus, characterized by abnormal hyperglycemia, affects 463 million patients globally , of whicOenothera biennis (OB), commonly known as evening primrose, is an herbaceous plant of the family Onagraceae [agraceae . In receagraceae . Thus, Oagraceae . The annagraceae . However−)). Moreover, the polyphenol composition and nutritional value of DSOB were analyzed compared to SOB as a reference.SOB is a rich source of not only a valuable oil, containing essential fatty acids, but also of polyphenols, which have shown remarkable bioactivity . MoreoveSaccharomyces cerevisiae, ascorbic acid, acarbose, ammonium sulfate, p-nitrophenyl-α-D-glucopyranoside, Trolox, β-nicotinamide adenine dinucleotide 2-phosphate reduced tetrasodium salt hydrate (NADPH), DL-glyceraldehyde, epalrestat, quercetin, oxalic acid, citric acid, glucose, fructose, galactose, lactose, maltose, sucrose, tocopherols , hydrogen peroxide (H2O2), ABTS, DPPH, Folin and Ciocalteu’s phenol reagent, Griess reagent, and 2,6-di-tert-butyl-4-methylphenol (BHT) were purchased from Sigma-Aldrich . The standards used for the determination of mineral contents, including sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn), were obtained from the Central Iron and Steel Research Institute . ONOO− was obtained from Cayman Chemical . All organic solvents were purchased from Concord Technology . Ultrapure water (18.2 MΩ cm) was prepared using a water purification system . Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich.α-Glucosidase from n-hexane over a period of 48 h to obtain DSOB (788.69 g). Voucher DSOB (WLL-2018-01) and SOB (WLL-2018-02) were deposited at the College of Public Health, Hebei University.SOB was purchased from Yiyuan Chinese Herb Medicine Co. Ltd.  in August 2018. Dry ground SOB (1000.21 g) was defatted twice with Macronutrients , energy, and sugars  were determined using Association of Official Agricultural Chemists methods . Specifiv/v/v, (B) at 0.7 mL/min: 0% B at 0–1.9 min, 0–57% B at 1.9–18.1 min, 57–100% B at 18.1–18.6 min, and 100% B at 18.6–22.3 min. The excitation and emission wavelengths of the fluorescence detector were 340 nm and 450 nm, respectively. Amino acids were identified by comparing the retention times with those of the standards and were further quantified using calibration curves. Moreover, the chemical score (CS), essential amino acid index (EAAI), and biological value (BV) were calculated using the following equations:Proteins extracted from SOB or DSOB were hydrolyzed with hydrochloric acid at 110 °C for 21 h, and then filtered using a 0.22 μm membrane for injection into a high-performance liquid chromatography (HPLC)  instrument equipped with a fluorescence detector . Separation was achieved using a ZORBAX Eclipse-AAA column  at 35 °C. The samples (10 μL) were eluted with sodium dihydrogen phosphate solution  and acetonitrile–methanol–water solution . The digested solutions were then filtered using 0.22 μm membranes. The filtrate was subsequently analyzed using an inductively coupled plasma-atomic emission spectrometer . The response signals were tracked at 766.49 nm (K), 589.59 nm (Na), 324.75 nm (Cu), 317.93 nm (Ca), 279.55 nm (Mg), 259.94 nm (Fe), 257.61 nm (Mn), and 206.20 nm (Zn). Each element was quantitatively analyzed in accordance with its standard calibrated curves. n-hexane (4 mL), and saturated sodium chloride water solution (2 mL). The clear upper layer was collected and filtered for further HPLC analysis. The Eclipse Plus C18 column  was used for separation. Samples were eluted with methanol at 0.7 mL/min and detected at 300 nm. Tocopherols  were confirmed and quantified using standard compounds with calibrated curves.Tocopherols in SOB and DSOB were determined using the UltiMate 3000 HPLC system with a UV detector (UltiMate 3000), as previously described with some modifications . BrieflyOrganic acids  in SOB and DSOB were extracted by meta-phosphoric acid and subsequently analyzed by HPLC-UV (UltiMate 3000) at 215 nm and 210 nm, respectively. Sodium dihydrogen phosphate solution  was used as the mobile phase. The eluent was monitored using an Ultimate AQ-C18 column  that was used for separation at 35 °C. Compounds were identified by comparison with standards and further quantified using calibration curves .v/v) for 30 min. The obtained extracts were concentrated at 37 °C using a rotary evaporator  under vacuum, lyophilized , and stored at −20 °C for the determination of TPC, TFC, polyphenols, and antioxidant capacities. TPC was determined using the Folin–Ciocalteu colorimetric method and expressed as gallic acid equivalents (GAE) [SOB and DSOB were extracted twice by sonication at 30 °C with an ethanol aqueous solution  . TFC wasts (GAE)  and exprv/v, (B) at a flow rate of 0.3 mL/min. The following were used as the optimized gradient chromatography conditions: 5–20% B at 0–2 min, 20–30% B at 2–5 min, 30–40% B at 5–7 min, 40–100% B at 7–10 min. Peak identification was performed in both negative and positive modes, and mass spectra in the m/z range of 50–1000 were obtained. The mass spectrometry conditions were as follows: source temperature, 120 °C; desolvation temperature, 450 °C; gas flow rate, 800 L/h; and nebulizer gas pressure, 6.5 bar. The capillary voltage was set to 2.0 kV. Data acquisition and analysis were performed using MassLynx V4.1 SCN 901 (Waters). The polyphenols were temporarily identified by comparing the literature and searching the library using UNIFI (Waters). The content ratio of polyphenols in DSOB and SOB were calculated by peak areas in TIC.The DSOB and SOB polyphenols were characterized using an LC–ESI–QTOF/MS instrument  for tentative identification. Separation was accomplished at 30 °C using a CORTECS C18 column . SOB or DSOB (4 μL) were eluted with acidified water  and organic solutions . The DPPH, ABTS, HO•, and NO scavenging assays were performed as reported by Kwon et al. [− scavenging assay was carried out as reported by Hazra et al. [− scavenging assays. The antioxidant activities were expressed as the percentage of radical elimination (%) and the sample concentration for 50% inhibition (IC50).The antioxidant capacities of SOB and DSOB were assessed using electron transfer assays (DPPH and ABTS) and ROS scavenging assays , and α-glucosidase solution  were incubated at 37 °C for 30 min. Acabose was used as a positive control. Then, the absorbance of the incubated mixture was measured at 405 nm using a microplate reader . The inhibitory activity was expressed as the percentage inhibition (%) and the IC50.The α-glucosidase inhibition assay was performed as previously reported . Specifi50. The rat lens aldose reductase inhibition assay of SOB and DSOB was performed as described previously . In brieAll animal experimental procedures were conducted in accordance with the guidelines and approval of the Institutional Animal Care and Use Committee (IACUC) of Hebei University (IACUC-20180051). Prior to the experiment, rats were placed in a standardized laboratory environment under a 12 h light/12 h dark cycle at temperature of 20–26 °C and humidity of 40–70%. All animals had access to water and food. t-test or one-way analysis of variance, as required. p < 0.05 was considered significant.All experiments were repeated at least in triplicate. Results are expressed as the mean ± standard deviation. Data were analyzed using SPSS Statistics software version 19.0 . The comparison of mean values was performed using Student’s unpaired 50 of 3.31 μg/mL for α-glucosidase inhibition, which showed no significant difference compared to the IC50 of SOB (3.18 μg/mL). Interestingly, both DSOB and SOB showed dramatically higher α-glucosidase inhibitory capacities than acarbose. Grape seed and green tea are popular functional food ingredients that possess excellent α-glucosidase inhibitory activities. In a previous study, Yilmazer-Musa et al. reported that grape seed has an IC50 of 1.2 μg/mL for α-glucosidase inhibition, green tea has an IC50 of 0.5 μg/mL for α-glucosidase inhibition, and acarbose has an IC50 of 91 μg/mL for α-glucosidase inhibition [Phellinus merrillii has an IC50 of 13.73 μg/mL for α-glucosidase inhibition [α-Glucosidase inhibitors from natural sources are often used as functional foods for intervening in postprandial hyperglycemia in patients with diabetes. Thus, the effect of DSOB on α-glucosidase inhibition was first investigated to assess its potential as a functional food ingredient in diabetes. As shown in hibition . Anotherhibition . These r50 (2.56 μg/mL) than that of SOB (3.44 μg/mL), as shown in Phellinus merrillii has an IC50 of 12.55 μg/mL for aldose reductase inhibition [Recently, the development of naturally derived aldose reductase inhibitors with less toxicity as functional foods has attracted much attention. Our previous research has reported that SOB is a potent aldose reductase inhibitor , but thehibition . Althoug−, DPPH, and ABTS, on the basis of the antioxidant mechanisms of electron transfer and ROS scavenging [−, is formed when NO interacts with superoxide radicals, leading to a series of harmful events [50 = 1.08 μg/mL) and ONOO− (IC50 = 512.49 μg/mL), with no significant difference with SOB. DSOB also showed excellent scavenging capacities for ABTS (IC50 = 1.22 μg/mL) and DPPH (IC50 = 15.62 μg/mL) radicals, with no significant difference compared to SOB. These findings demonstrated that DSOB is a good natural antioxidant.The antioxidant capacities of DSOB were further assessed using five assays, namely, O•, NO, ONOOavenging . HO• is avenging . As showl events . DSOB shIn recent years, an increase in the incidence of diabetes associated with lifestyle has led to a search for effective and safe methods of prophylaxis and/or therapy. The results of epidemiological studies indicate that diabetes may be prevented by enriching diets with plant foods, since these foods are rich in polyphenols with pharmacological activity ,28. OverTo better understand the polyphenol composition of DSOB, we performed LC–ESI–QTOF/MS because it provides high-resolution mass data containing affluent structural information of the analyte. In this work, 25 components in DSOB have been identified, including (+)-catechin, ellagic acid, gallic acid, procyanidins, quercetin, syringic acid, and their derivatives, as listed in The nutritional and energy values of DSOB are presented as dw in The amount and type of carbohydrates to eat is an important consideration for patients with diabetes when planning their diet. As shown in As mentioned previously, SOB is a good source of protein and, interestingly, DSOB has a higher protein content. As shown in As shown in Two organic acids  were identified in DSOB and SOB. As shown in The tocopherol contents  have been determined, as shown in In summary, DSOB was found to be a reservoir of nutrients and polyphenols, and had remarkable inhibitory activity for α-glucosidase and aldose reductase, as well as antioxidant capacities. Moreover, 25 compounds in DSOB were temporarily identified using LC–ESI–QTOF/MS analysis, which were associated with the remarkable bioactivities of DSOB. DSOB also contained a high content of carbohydrates, proteins, and minerals. The fat content in DSOB decreased after EPO extraction from SOB, resulting in low levels of γ-tocopherol and energy. DSOB contained large amounts of fiber and low levels of sugars, providing a low glycemic index. Notably, DSOB was a good source of Ca and Fe, and the anti-nutrient and oxalic acid content was low. These findings imply that DSOB, that is, the waste material generated by SOB after EPO extraction, has potential as a functional food ingredient in diabetes.


Wounding triggers cell fate transition in detached plant tissues and induces de novo root organogenesis. While the hormonal regulation of root organogenesis has been widely studied, the role of early wound signals including ROS and Ca2+ remains largely unknown.Reactive oxygen species (ROS) and calcium ions (Ca2+ are required for de novo root organogenesis, but have different functions in Arabidopsis explants. The inhibition of the ROS and Ca2+ signals delayed root development in detached leaves. Examination of the auxin signaling pathways indicated that ROS and Ca2+ did not affect auxin biosynthesis and transport in explants. Additionally, the expression of key genes related to auxin signals during root organogenesis was not significantly affected by the inhibition of ROS and Ca2+ signals. The addition of auxin partially restored the suppression of root development by the ROS inhibitor; however, auxin supplementation did not affect root organogenesis in Ca2+-depleted explants.We identified that ROS and Ca2+ are key molecules, at least in part of the auxin signals acts downstream of ROS signaling, and Ca2+ acts downstream of auxin during de novo root organogenesis in leaf explants.Our results indicate that, while both ROS and CaThe online version contains supplementary material available at 10.1186/s12870-022-03524-w. De novo root organogenesis is a type of plant regeneration that forms adventitious roots in detached leaves without exogenous hormone supplementation –3. The eYUC genes increases within a few hours after leaf detachment [WUSCHEL-RELATED HOMEOBOX 11 (WOX11) and WOX 12 expression [WOX5 and WOX7 by directly binding to their promoters for cell fate transition from the root founder cells to root primordium cells [Auxin is a key hormone in de novo root organogenesis . Auxin btachment . The YUCpression , 8. WOX12+) are early signaling molecules involved in plant wound responses [rboh mutations [rbohd mutants [2+ is also a long-distance signaling molecule involved in plant wound responses [2+ concentration after wounding [2+ propagation and expression of JA-responsive genes have not been detected in glr3.3 glr3.6 double mutants, indicating the role of Ca2+ in mediating plant wound responses.Reactive oxygen species (ROS) and calcium ions -inhibited primary root growth [2+ is related to auxin signaling pathways during root development, and changes in nuclear Ca2+ signals by nuclear membrane-located ion channels regulate auxin homeostasis, which affects primary root development [2+ also acts as a cofactor of Ca2+-DEPENDENT MODULATOR OF ICR1 (CMI1)- INTERACTOR OF CONSTITUTIVELY ACTIVE ROP 1 (ICR1) interactions, which regulate auxin responses during primary root development [2+ signals have been reported to be involved in lateral and adventitious root development in rice and cucumber [2+ is a general upstream regulator of root development in different plant species. While ROS and Ca2+ play key roles in root development and wound responses, their roles in de novo root organogenesis in detached leaf explants remain largely unknown.In addition to wound responses, ROS and Cat growth . During t growth . Additiot growth . It was t growth , demonstelopment . Ca2+ alelopment . Ca2+ sicucumber , 24, dem2+ are required for de novo root organogenesis in leaf explants. ROS and Ca2+ are not involved in auxin biosynthesis and transport; thus, they do not significantly affect the expression of auxin-responsive genes involved in cell fate transition in explants. Further experiments suggested that ROS are independent of auxin signaling, while Ca2+ acts downstream of the auxin signaling pathways during root development. Our results suggest the distinct role of ROS and Ca2+ in de novo root organogenesis in leaf explants, which will be beneficial in understanding how early wound signals affect root development in detached tissues.In this study, we identified that early wound signals of ROS and CaIn the detached leaves, adventitious roots develop near the wound site without exogenous phytohormone supplementation . As wounTo investigate the role of ROS in de novo root organogenesis, we incubated leaf explants in a phytohormone-free medium containing diphenylene iodonium (DPI), a general inhibitor of ROS-producing RBOH proteins in plants . In NBT YUC2 and YUC4 genes after DPI treatment. DPI did not significantly change the expression of both YUC genes . As reported previously, NAA treatment elevated the rooting rate by up to ~ 100% in mock-treated explants . Notablyesponses . GFP flu2+ is another early signal of plant wound responses. Ca2+ signals rapidly increase after wounding and are transmitted to systemic tissues to induce JA responses [2+ assists ROS production via the activation of RBOH proteins through the function of Ca2+-binding proteins and ion channels [2+ is also involved in de novo root organogenesis in detached leaves. First, we analyzed the relationship between Ca2+ and ROS production in leaf explants by NBT and DAB staining. Again in these experiments, the ROS kinetics in mock-treated leaves were different in compared to those in Figs. 2+ signals by the Ca2+ chelator, ethylene glycol tetraacetic acid (EGTA), did not affect superoxide accumulation  is highly suppressed by glr3.3 glr3.6 mutations in systemic tissues, but not in local tissues [2+ signals would affect auxin-dependent root organogenesis in leaf explants. Taken together, we propose a working model for the role of ROS and Ca2+ in de novo root organogenesis  was obtained from Dr. Chung-Mo Park’s lab at the Seoul National University and it was originally from the Arabidopsis Biological Resource Center . The rbohC (N2259), rbohD (N9555), rbohF (N9557), rbohDF (N9558), glr3.3 (N663463), and glr3.6 (N663316) mutants were obtained from the Nottingham Arabidopsis Stock Centre . The DR5rev:GFP (CS9361) was obtained from the ABRC. The glr3.3 glr3.6 double mutants were obtained from crossbreeding glr3.3 and glr3.6 mutants. S.Y.S. and H.J.L. performed identification of all plant materials. Newly generated plant materials in this study are not deposited in a publicly available herbarium, but they are available from the corresponding author on reasonable request. Seeds were surface-sterilized in 75% (v/v) ethanol with 0.03% (v/v) Triton X-100 and then washed twice using 70% (v/v) ethanol before stratification at 4 °C. After three days, the seeds were transferred to a growth room set at 24 °C with 40–50% humidity under long-day conditions . The seedlings were grown on 1/2 X Murashige and Skoog-agar (MS-agar) plates containing 0.7% (w/v) of plant agar with pH 5.7. White light with an intensity of 100 μmol m−2 s−1 was applied using fluorescent FL40EX-D tubes .WOX11pro:GUS transgenic plants, 4.87-kb upstream sequences of the WOX11 gene were amplified using pWOX11-F (5’-AATGGATCCCTAACTGTTACGATTGAATTCAAACGATA-3’) and pWOX11-R (5’-TTGGCGCGCCTGCTTTGAAGAATATTGATATTA-TCTGGTG-3’). The PCR products were combined into a modified pBA-GUSII vector using BamHI and AscI restriction enzymes.To generate Seedlings were grown for 9 days and first rosette leaves were detached. The explants were incubated on Gamborg B5 basal medium-agar (B5-agar) plates containing 0.7% (w/v) of plant agar with pH 5.7 for up to 14 days to induce de novo root organogenesis. For DPI treatment, explants were incubated on B5-agar plates containing 1 or 10 μM of DPI. For EGTA treatment, explants were incubated on B5-agar plates containing 0.5, 1, or 1.5 mM of EGTA. For NAA treatment, explants were incubated on B5-agar plates containing 0.1 μM of NAA.Seedlings were grown on MS-agar plates for 9 days and first rosette leaves were incubated on B5-agar plates containing 1μM of DPI or 0.5 mM of EGTA for the indicated time periods in figures. For DAB staining, explants were immersed in DAB staining solution containing 40 mg of 3,3'-diaminobenzidine and 20 μL of Tween 20 in 40 mL of distilled water, and then vacuum-infiltrated for 5 min. The seedlings were then incubated for approximately 8 h in the darkness at 24 °C without shaking. After the staining, DAB solutions were removed and then explants were immersed in 70% (v/v) ethanol to remove the chlorophyll. For NBT staining, explants were immersed in NBT staining solution containing 70 mg of nitroblue tetrazolium and 13 mg of sodium azide in 20 mL of 10 mM potassium phosphate buffer (pH 7.8) and vacuum-infiltrated for 2 min. Explants were then incubated for 2 h in the darkness at 24 °C without shaking. The NBT staining solution was then removed and the plant materials were immersed in 70% (v/v) ethanol to remove the chlorophyll. The plant materials were mounted on slide glasses and photographed using a Nikon D5600 digital camera. The ImageJ software was used to measure the DAB and NBT signal intensity.pWOX11:GUS transgenic plants were grown for 9 days on MS-agar plates and first rosette leaves were incubated on B5-agar plates containing 1 μM of DPI or 0.5 mM of EGTA for the indicated time periods in figures. Explants were fixed with 90% cold acetone for 20 min on ice. Explants were then washed two times using rinsing solution containing 50 mM of sodium phosphate pH 7.2, 0.5 mM of K3Fe(CN)6, and 0.5 mM of K4Fe(CN)6. Washed leaves were completely submerged in staining solution containing 2 mM of X-Gluc (Duchefa) in the rinsing solution and incubated at 37 °C for 16 h. Explants were then incubated in 70% ethanol for 24 h to remove the chlorophyll. Stained leaves were mounted on slide glasses and photographed using Nikon D750 digital camera. The ImageJ software was used to measure the GUS signal intensity.The DR5rev:GFP transgenic plants were grown for 9 days on MS-agar plates. First rosette leaves were detached and incubated on B5-agar plates containing 1 μM of DPI or 0.5 mM of EGTA for the indicated time periods in figures. The plant materials were mounted on the slide glasses and abaxial side of the leaves were observed. GFP and chlorophyll fluorescence imaging were performed using an LSM 800 confocal microscope (Carl Zeiss). The excitation and emission wavelengths of GFP fluorescence were 488 and 500–600 nm, respectively. For analysing chlorophyll fluorescence, excitation and emission wavelengths were 561 and 560–700 nm, respectively. Fluorescence images were analyzed using the ZEN 2.5 LITE software. To measure fluorescence intensity of GFP signals, the ImageJ software was used. Three to five biological repeats were analyzed.The UBQ10 as a reference gene.Plants were grown for 9 days on MS-agar plates. First rosette leaves were detached and incubated on B5-agar plates containing 1 μM of DPI or 0.5 mM of EGTA for the indicated time periods in figures. Total RNA was extracted from the explants using the Trizol (Thermo Fisher Scientific) or the RNeasy Plant Mini Kit  according to the manufacturer’s recommendations. First-strand complementary DNA synthesis was performed using AccuPower CycleScript RT PreMix (Bioneer) according to the manufacturer’s protocol. QPCR was performed using TOPreal qPCR 2X PreMIX . Primers used for qPCR were listed in Table St-test were performed using Rstudio and Excel software, respectively.All statistical methods as well as the number of biological replicates in each assay are annotated in the figure legends. To determine statistically significant differences, one-way analysis of variance (ANOVA) with post hoc Tukey’s test and Student’s Additional file 1: Fig. S1. Effects of DPI on root organogenesis in the leaf explants. Fig. S2. Observation of auxin responses using DR5rev:GFP at 0 DAC. Fig. S3. Effects of EGTA on root organogenesis in the leaf explants of rboh mutants. Fig. S4. Proposed working model for the role of ROS and Ca2+ on root organogenesis. Table S1. Primers used in this study.


Acute myeloid leukemia (AML) with t is categorized as favorable-risk AML, but KIT mutations show a significantly poor prognostic impact in such patients. Persistent vulnerability to relapse is a major challenge in the treatment of this subtype of patients. Venetoclax is a BCL-2 selective inhibitor. The venetoclax+HMA strategy is also a notable salvage regimen that achieves good clinical outcomes in the treatment of relapsed or refractory (R/R) AML. However, in our clinical practice, we found that disease progressed rapidly even after venetoclax+azacitidine (AZA) therapy in two relapsed t AML patients with KIT mutations. We report for the first time the therapeutic potential of venetoclax+midostaurin as a new combination therapy for relapsed t AMLs with KIT mutations showing resistance to venetoclax+AZA therapy. Our ex vivo study also showed that midostaurin alone could inhibit proliferation and induce apoptosis of Kasumi-1 cells  and observed a synergistic anti effect when the two drugs were combined. Our study shows that the venetoclax+midostaurin regimen may be a promising treatment option for R/R t AML with KIT mutations. Acute myeloid leukemia (AML) with t8;21)/AML1-ETO belongs to the core-binding factor (CBF) category of leukemia, which constitutes a distinct entity in the World Health Organization 2016 classification and comprises AML with inv(16)p13.1q22) or t/CBFB-MYH11  is an oral BCL-2 selective inhibitor that has activity in a variety of hematologic malignancies . ClinicaIn vitro, midostaurin or its major active metabolites inhibit the activity of KIT (wild type and the D816V mutant), FLT3 , PDGFRα/β, VEGFR2 and members of the serine/threonine kinase protein kinase C family (Midostaurin (PKC412) is an inhibitor of multiple receptor tyrosine kinases (TKs). C family . MidostaC family . We expl4 abl copies), and KIT exon 17 and CSF3R mutations were detected. She was initially treated with a standard “3+7” regimen with idarubicin and cytarabine (IA) as induction with complete remission (CR) followed by four cycles of high-dose cytarabine (HiDAC) consolidation. However, 2 months after the last consolidation, the disease relapsed, and she failed to achieve CR again after salvage chemotherapy with CLAG . BM analysis revealed 71.5% blasts and high AML1-ETO (40412 copies). This patient then received venetoclax+AZA for one cycle. After the treatment, the BM revealed 75% blasts, and the patient presented with symptoms of fatigue, lassitude and bone pain, which suggested disease progression. Then, one cycle of venetoclax+midostaurin chemotherapy  was administered, and the BM showed 4.5% blasts upon finishing the therapy. Then, the patient underwent allo-HSCT   , and AML9/L. No pneumonitis, hepatotoxicity or tumor lysis syndrome occurred. Investigator-assessed AEs were graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (NCI CTCAE version 5.0)  occurred in both patients. Both cases received red blood cell (RBC) (10 U vs. 6 U) and platelet (5 U vs. 6.5 U) transfusions to treat low hemoglobin and platelet levels. G-CSF could be administered when the neutrophil count was below 1.0×10ex vivo study showed that venetoclax and midostaurin alone could inhibit proliferation and induce apoptosis of Kasuim-1 cells (harboring AML1-ETO fusion and KIT mutation) and observed a synergistic anti effect when the two drugs were combined  AML with KIT mutation showing resistance to venetoclax+AZA therapy. Taken together, our study shows that the venetoclax+midostaurin regimen may be a promising treatment option for R/R t AML with KIT mutations.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Soochow University. The patients/participants provided their written informed consent to participate in this study.ZL, JW, and S-SG collected, analyzed data and wrote the manuscript. Q-CQ contributed to methodology and investigation. J-HD, S-SS, X-DS, and C-LW performed research and interpreted data. B-RW performed the experiments. D-PW, H-YQ, and S-LX conceived and designed the study. All authors read and approved the final manuscript.This work was supported by the grants from the National Natural Science Foundation of China (Grant No. 81970138), Translational Research Grant of NCRCH (Grant No. 2020ZKMB05), Jiangsu Province”333”project, Jiangsu Province Medical Youth Talent Program (Grant No. QNRC2016719), and Gusu Key Medical Talent Program (Grant No. GSWS2019007), Social Development-Clinical Frontier Project of Jiangsu Province (Grant No. BE2018652).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.