BCL2 transcription by activating STAT3. The BCL2 inhibitor venetoclax can enhance the chemotherapy sensitivity of AML cells to sorafenib.Drug resistance is the main cause of sorafenib treatment failure in clinical acute myeloid leukemia (AML) patients, but the mechanism is currently not fully clear. In this study, we analyzed the genetic characteristics of sorafenib-resistant AML cell subclusters using single-cell and bulk transcription data and found that sorafenib-resistant AML cells can promote Sorafenib, a kinase inhibitor, has shown promising therapeutic efficacy in a subset of patients with acute myeloid leukemia (AML). However, despite its clinical effectiveness, sorafenib resistance is frequently observed in clinical settings, and the mechanisms underlying this resistance as well as effective strategies to overcome it remain unclear. We examined both single-cell and bulk transcription data in sorafenib-resistant and control AML patients and integrated a sorafenib resistance gene signature to predict the sensitivity of AML cells and the clinical outcomes of AML patients undergoing sorafenib therapy. In addition, our drug sensitivity analysis of scRNA-seq data using deconvolution methods showed that venetoclax was effective in targeting sorafenib-resistant AML cells. Mechanistically, sorafenib was found to activate the JAK-STAT3 pathway and upregulate BCL2 expression in sorafenib-resistant AML cells. This upregulation of BCL2 expression rendered the cells vulnerable to the BCL2 inhibitor venetoclax. In conclusion, we developed a platform to predict sorafenib resistance and clinical outcomes in AML patients after therapy. Our findings suggest that the combination of sorafenib and venetoclax could be an effective therapeutic strategy for AML treatment. Acute myeloid leukemia (AML) is the most prevalent form of leukemia in adults, comprising 80% of all adult leukemia cases . The incAlthough sorafenib demonstrates therapeutic efficacy in AML treatment , sorafen2.The THP-1 , U937 , HL-60 , MOLM13 , K562 , and Jurkat  cell lines were cultivated in RPMI 1640  supplemented with 10% fetal bovine serum , 1% penicillin–streptomycin , 1% HEPES  and 2 mM glutamine . All cells were maintained in a humidified incubator at 37 °C with 5% CO5 cells were seeded into a 48-well plate and treated with sorafenib  and/or venetoclax  where indicated. Cell growth curves were plotted using flow cytometry to count viable cells, and 7-aminoactinomycin D  was used to exclude dead cells. For apoptosis analysis, 1 × 106 cells were seeded into a 24-well plate and treated with SF and/or Vene where indicated. AnnexinV  and 7-AAD were used to stain the cells. FACS assay was conducted using an analyzer , and the data were analyzed using FlowJo software (FlowJo 10.8.1).To perform proliferation analysis, 1 × 10An equal number (1 million) of cells were lysed using RIPA buffer  containing 1 mM PMSF . The protein extracts were fractionated using 10% SDS-PAGE and transferred to a PVDF membrane . Following blocking with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20  for 1 h at room temperature, the membranes were incubated with primary antibodies for BCL2 , cleaved caspase 3 , p-STAT3 , and β-actin  overnight at 4 °C, as indicated. The membranes were then incubated with secondary antibodies  for 1 h at room temperature. The Western blots were detected either using X-ray film or a digital imaging system (Odyssey Fc). Protein levels were quantified using densitometric intensity.t-distributed stochastic neighbor embedding (tSNE). Graph-based cell clustering was performed using the FindClusters function in Seurat with the appropriate resolution. To remove the batch effect of each scRNA-seq sample, the Harmony R package (version 0.1.1) was utilized.Fresh primary AML cells were isolated using Ficoll  gradient centrifugation from leftover BM aspirates of clinical routine sampling. Patient characteristics for primary samples used are provided in To forecast the cellular constitution of sorafenib-resistant cells in single-cell data, BayesPrism deconvolution was executed utilizing bulk RNA-seq samples obtained from sorafenib-treated AML cells (GSE104594) ,17. The To establish a correlation between patient prognosis and significant genes in sorafenib-resistant clusters, we conducted univariate Cox regression analysis on 553 AML patients from the GEO database (GSE37642). We employed the least absolute shrinkage and selection operator (LASSO) regression analysis to compute the coefficient. Finally, we computed the risk score of individual patients utilizing the following formula: risk score = coefficient1 × expression of gene1 + … + coefficientN × expression of geneN. Based on the median risk scores, patients were categorized into a high-risk (HR) or low-risk (LR) cohort. To assess overall survival (OS) between the HR and LR in each cohort, we employed the Kaplan–Meier analysis. To evaluate the precision of the prognostic model, we utilized ROC curves. We validated our model with 140 samples from the LAML project in the Cancer Genome Atlas (TCGA) database using the same model.We obtained ex vivo drug sensitivity area under the curve (AUC) scores for acute myeloid leukemia (AML) from BEAT-AML and Lee et al. ,19. PearRNA-seq data for THP1, MV4-11, HL60, U937, MOLM13, K562, Jurkat cell lines, and primary patient cells were obtained from the GEO database . The raw count matrices were normalized by applying log2 transformation. The ComBat function in the sva R package (version 3.46.0) was utilized to eliminate batch effects.t-tests were employed for comparison between two groups , while one-way ANOVA followed by Dunnett’s tests were used for multiple comparisons . Statistical significance was established at p < 0.05.The data are presented as means ± standard deviation (s.d.). Student’s SOX4, BCL2, and previously reported LSC markers such as CD82 and CD99 were significantly higher in SF-res than in the SF-sens group, indicating that SF-res cells possess stronger stemness . Our analysis revealed that the OS of patients in the HR cohort was significantly lower than that of patients in the LR cohort. We validated the resistance genes using ROC curves, where the area under the ROC curve (AUC) was positively associated with prognostic accuracy. The AUCs for the 1-year, 3-year, and 5-year patient survival rates were 0.684, 0.721, and 0.722, respectively , respectively. The AUC values of TC and VC were both greater than 0.65, which confirmed that the 22 sorafenib resistance genes were effective in predicting the prognosis of AML patients.We further validated our prognostic model using the 140 patients in the TCGA database as the validation cohort (VC). The risk score distribution  and survp = 0.041), confirming that the SF-res-7 score can effectively predict the response of patients to different chemotherapy drugs and thus the patient prognosis.To further scrutinize genes linked with sorafenib resistance, we employed our deconvolution method to deduce the abundance of cell types associated with SF sensitivity from 430 AML patient samples gathered in the Leucegene cohorts . The hieTo investigate the correlation between sorafenib resistance genes and the susceptibility of leukemia cells to sorafenib, and to validate the predictive efficacy of SF-res-7 and SF-res-22 prognostic models on cell line sensitivity to sorafenib, we examined the RNA-seq outcomes of various leukemia cells from the GEO database. The prognostic model scored distinct leukemia cell lines, with higher scores reflecting greater resistance to sorafenib. Our findings revealed heterogeneous sorafenib sensitivity across different leukemia cell lines, with the highest resistance scores observed in U937, HL60, Jurkat, and FLT3-ITD mutation patients with sorafenib resistance and the lowest scores in MV4-11, MOLM13, K562, and THP-1 . AdditioNo effective treatment schemes have been clinically applied for AML patients who are resistant to sorafenib. Therefore, it is crucial to find novel targeted drugs to overcome sorafenib resistance. The BEAT-AML database provides information on gene expression signatures and the corresponding sensitivity of AML samples to clinically available drugs ex vivo . To analTo ascertain the correlation between SF-res-7 and the drug sensitivity of AML cells, we analyzed the sensitivity of 33 drugs from BEAT-AML c and 72 The analysis of the drug sensitivity database revealed that venetoclax effectively targeted the sorafenib-resistant population a. To ascBCL2 was higher in the sorafenib-resistant group (SF-res group) than in the SF-unrelated and SF-sens groups , which activate the proliferation and survival signal pathway, account for the sorafenib resistance ,24,25. FBy analyzing the drug sensitivity of the sorafenib-resistant cell group, we found that it exhibited greater sensitivity to lenalidomide, VX.745, azacitidine, and venetoclax. Lenalidomide is an immunomodulatory drug used in the treatment of chronic lymphocytic leukemia (CLL) and multiple myeloma ,31. LenaVenetoclax has been reported to restore the sensitivity of TKI-resistant leukemia cells by inhibiting the MAPK pathway or downregulating BIM expressions ,40. MostBCL2 transcription, and achieve drug resistance. Targeting the activation of BCL2 in drug-resistant AML cells can effectively enhance the sorafenib sensitivity of AML cells.In summary, we analyzed the gene expression characteristics of sorafenib-resistant AML cells in detail and found that its highly expressed gene set can predict sorafenib sensitivity and prognosis in AML patients. In addition, sorafenib-resistant AML cells activate STAT3, promote


TP53 mutations, still have dismal outcomes despite these recent advances, underscoring an ongoing unmet need and opportunity for translational advances. In this review, we will discuss recent updates in the classification and risk stratification of AML, explore the literature regarding low-intensity and novel oral combination therapies, and briefly highlight investigative agents currently in early clinical development for high-risk disease subtypes.Acute myeloid leukemia (AML) is the most common acute leukemia in adults. While survival for younger patients over the last several decades has improved nearly sixfold with the optimization of intensive induction chemotherapy and allogeneic stem cell transplantation , this effect has been largely mitigated in older and less fit patients as well as those with adverse-risk disease characteristics. However, the last 10 years has been marked by major advances in the molecular profiling of AML characterized by a deeper understanding of disease pathobiology and therapeutic vulnerabilities. In this regard, the classification of AML subtypes has recently evolved from a morphologic to a molecular and genetic basis, reflected by recent updates from the World Health Organization and the new International Consensus Classification system. After years of stagnation in new drug approvals for AML, there has been a rapid expansion of the armamentarium against this disease since 2017. Low-intensity induction therapy with hypomethylating agents and venetoclax has substantially improved outcomes, including in those previously considered to have a poor prognosis. Furthermore, targeted oral therapies against driver mutations in AML have been added to the repertoire. But with an accelerated increase in treatment options, several questions arise such as how to best sequence therapy, how to combine therapies, and if there is a role for maintenance therapy in those who achieve remission and cannot undergo alloHSCT. Moreover, certain subtypes of AML, such as those with  Acute myeloid leukemia (AML) is the most common acute leukemia in adults. AML is thought to arise from somatically acquired mutations, which is a fairly ubiquitous process during human aging –11. HoweWhile optimization of intensive chemotherapy and better supportive care over the years has improved survival in AML, this benefit is largely confined to younger patients and those without adverse-risk cytogenetics. Moreover, the only potentially curative strategy for those with intermediate- or adverse-risk disease is allogeneic hematopoietic stem cell transplantation  , which iBCR::ABL1 fusions and CEBPA mutations. Cases of AML without a defining genetic alteration are made based on differentiation patterns and still require a blast count of at least 20%. Similarly, the ICC uses a 10% blast cutoff for most molecularly defined subtypes of AML, again with the exception of BCR::ABL1 to avoid confusion with the diagnosis of chronic myeloid leukemia (CML). Moreover, both the WHO and ICC define cytogenetic and genetic changes almost always associated with antecedent MDS  domain mutations in CEBPA confer a better prognosis with a distinct gene expression profile ), and FLT3 . We will briefly review the landmark studies regarding these agents and discuss the emerging roles for approved therapies  of the catalytic domain of isocitrate dehydrogenase. The prognostic implications of IDH1/IDH2 mutations are not entirely clear. Some reports have suggested that IDH2 mutations are associated with better outcomes while IDH1 mutations confer worse outcomes [NPM1 [, in AML , 79. Mutoutcomes , 81, thoes [NPM1 , 81.R140 or IDH2R172 mutations (mean age 67 years) were treated with ENA [R140 mutations had 2-HG reductions greater than 90% regardless of response, while 2-HG levels in those with IDH2R172 mutations correlated with response . CR/CRi/CR with incomplete platelet recovery (CRp) was observed in 29% of patients. Median OS for all patients was 8.8 months but extended to 22.9 months in patients achieving CR. While reduction in mutant IDH2 VAF was not required for response, follow-up data demonstrated that clearance of IDH2-mutated clones was associated with 100% CR [NRAS or MAPK pathway were suggested to contribute to treatment resistance [IDH2-mutant R/R AML were randomized to ENA or conventional care [DNMT3A co-mutations has been shown to be associated with CR, and no deleterious effects of RAS signaling pathway co-mutations were observed; however, the presence of ≥ 4 co-mutations decreased the overall response rates (ORR) significantly (27.3% compared to 47.1% with < 4 co-mutations) [IDH2 mutations, the NCCN guidelines provide a recommendation to consider frontline ENA use for patients older than 60 years who are not candidates for intensive remission induction [In a phase 1/2 study, patients with R/R AML harboring IDH2with ENA , 83. Patsistance . In a phnal care ; patienttations) . Thus, anduction .IDH1-mutant R/R AML [IDH1 mutations (median age 76.5 years) received IVO in the frontline setting [IDH1 mutant clone clearance was reported in 64.3% of patients who achieved CRc and was not observed in any patients without CRc. These two studies ultimately led to the approval of IVO in both R/R AML with IDH1 mutations and ND AML with IDH1 mutations in patients who are ineligible for standard chemotherapy.In a phase 1 study, DiNardo et al. assessed the use of IVO in  R/R AML . The med setting . The comIDH1-mutant R/R AML who were naïve to IDH1 inhibitors [IDH1 mutations, though this is solely based on preclinical data [On December 1, 2022, the FDA approved another IDH1 inhibitor, OLU , for usehibitors . Patienthibitors . While tcal data  with no IDH1/IDH2 mutations [IDH1/2 mutations [ASXL1, NRAS, U2AF1, and TP53 were associated with worse response rates, while DNMT3A co-mutations were associated with marginally improved CR/CRi/CRp. When assayed by digital polymerase chain reaction (dPCR), 39% of patients treated with IVO cleared IDH1-mutant clones and 23% of patients treated with ENA cleared IDH2-mutant clones. Approximately half of the patients receiving IVO or ENA ultimately proceeded to alloHSCT. The use of IVO or ENA in combination with induction therapy was tolerable and ultimately did not significantly impact the time to recovery of the absolute neutrophil count or platelet count. An unanswered question that will require further investigation is the role of maintenance IVO or ENA after alloHSCT, particularly in patients who are unable to clear their mutant clone prior to transplantation. In patients eligible for intensive chemotherapy, the improved response rates compared to historical controls with the addition of IVO or ENA [Recent studies have evaluated the use of IVO or ENA in combination with other frontline therapies in ND AML. A phase 1 study assessed the use of IVO or ENA in combination with 7 + 3  in patients with utations . Patientutations . In an uutations , the aututations . Co-mutaO or ENA , 95 provO or ENA .IDH1/2 mutations have been shown to be exquisitely sensitive to the combination of AZA/VEN with response rates similar to or higher than those achieved with standard induction. Therefore, it is reasonable to consider low-intensity therapy in this patient population without compromising outcomes. In a pooled analysis of from VIALE-A [IDH1/2 mutations achieved CR/CRi rates of 79% with AZA/VEN compared with 11% with AZA alone [IDH2 mutations compared to IDH1.The majority of  VIALE-A  and a ph VIALE-A , the patZA alone . Median IDH1-mutant AML ineligible for intensive chemotherapy to receive AZA/IVO or AZA/placebo [FLT3, KIT, NRAS, KRAS, PTPN11) and TP53 mutations were more likely to respond to AZA/IVO, and follow-up data suggest that relapse appears to preferentially occur with the acquisition of secondary high-risk mutations, independent of IDH1 [IDH1-mutant AML. While the rate of differentiation syndrome in these patients approaches 20%, the rate of cytopenias compared to AZA/VEN is significantly less. Therefore, when thinking about the various options for patients with IDH1 mutations, toxicity, quality of life, and sequencing of treatment should be considered.The combination of AZA with IVO has recently emerged as an alternative non-intensive treatment option . The AGI/placebo . At a me of IDH1 . The finIDH2 mutations ineligible for intensive chemotherapy [The combination of AZA/ENA has also been studied in patients with R/R  and ND Aotherapy , 101. InIDH1/2-mutant AML. The lack of survival advantage with AZA/ENA compared to AZA monotherapy [IDH2 mutations with the caveat that the median OS of patients with AZA in this study was significantly longer than reported for patients with IDH2 mutations in the AZA/VEN studies [IDH1 mutations, CR rates appear to be better with AZA/VEN [IDH1/2 mutations who are ineligible for intensive chemotherapy, thereby preserving IVO, OLU, or ENA in the case of R/R disease. However, AZA/IVO could be considered in patients who are at high risk of complications with the myelosuppression of AZA/VEN. While ENA and IVO monotherapy are both NCCN-recommended options for frontline therapy [IDH1 mutations [A major question remains as to whether therapies such as AZA/IVO or AZA/ENA would outperform AZA/VEN for ND otherapy  would su studies . For pat AZA/VEN  than wit AZA/VEN , though  therapy , only IVutations , patientFLT3 encodes a type 3 RTK (FMS-like tyrosine kinase 3) and is widely expressed on AML blasts [FLT3 are seen in about 25–32% of cases of ND AML with 25% harboring ITDs and 7–10% harboring TKD mutations [FLT3-targeting TKIs (henceforth referred to as FLT3i) have suggested that AR of FLT3-ITD and the presence of NPM1 co-mutations variably affect outcomes [FLT3-ITD AML irrespective of AR or presence of NPM1 mutation, and these patients should be considered for alloHSCT in first remission (CR1) if eligible [FLT3-mutant AML [L blasts . Mutatioutations , 105. Proutcomes –108. Neveligible , 110; theligible . FLT3i celigible , which aeligible , 113 wittant AML . Newer atant AML –117.FLT3 mutations (TKD or ITD). CR rates were similar between both groups, though median EFS and OS were significantly improved with MIDO . OS was durable with 51.4% of patients in the MIDO group surviving at 4 years. More patients underwent alloHSCT in CR1 with MIDO compared to placebo ; notably, follow-up studies after the publication of the RATIFY trial demonstrated deeper molecular remission with the addition of FLT3i to induction therapy [The RATIFY trial  was a ra therapy , 120, peFLT3-mutant AML [Recent findings from a phase 2 study have also established the efficacy of adding to MIDO to induction, HiDAC consolidation, and maintenance in patients up to the age of 70 with ND tant AML , 122. Ittant AML , 121–123tant AML  noted a FLT3 mutations. The ADMIRAL study was a phase 3 randomized control trial of patients with R/R AML and FLT3-mutations who received GILT or salvage chemotherapy [FLT3-ITD and FLT3-TKD groups that received GILT were 9.3 months and 8 months, respectively. An important aspect of this study was the efficacy in both FLT3-ITD and FLT3-TKD populations, as the latter has been demonstrated to confer secondary resistance to type II FLT3i [GILT is the only FDA-approved FLT3i for use in R/R AML with otherapy . Similarotherapy . CRc ratII FLT3i .FLT3-ITD had a CR/CRi rate of 63% with AZA/VEN and a median OS of 9.9 months, while those with FLT3-TKD had a CR/CRi rate of 77% and a median OS of 19.2 months [FLT3 mutations, approximately 36% had NPM1 mutations in each of the AZA/VEN and AZA groups. Of those with concurrent FLT3-ITD and NPM1 mutation, AZA/VEN conferred a CR/CRi rate of 70% and a median OS of 9.1 months; patients with FLT3-ITD and wild-type NPM1 had a median OS of 10.6 months. This study has two important takeaways for older patients with mutated FLT3. First, the rate of FLT3 mutations in this population was lower, and the patients were older than typical FLT3-driven AML seen in younger patients, possibly suggesting different disease kinetics and biology. Second, while CR/CRi rates were worse for patients with FLT3-ITD mutations with wild-type NPM1 compared to those with mutated NPM1, the overall survival did not differ significantly, suggesting that NPM1 status has an unclear prognostic value for patients treated with HMA/VEN.Given the increasing use of low-intensity regimens in AML, pooled data from VIALE-A  and a ph2 months . Of patiFLT3-ITD [FLT3-ITD [FLT3-ITD AML or in R/R AML with FLT3-ITD [FLT3-wild-type or FLT3-mutant  and FLT3-mutant (dose expansion) R/R AML [FLT3 mutations had received other prior FLT3i. Patients with FLT3-ITD had CR/CRi/CRp rates of 43%, while those with FLT3-TKD has rates of 33%, and response rates were slightly better in those who were FLT3i-naïve. Median OS was 10 months for all FLT3-mutated patients, though there was a significant improvement in those who had undergone alloHSCT after VEN/GILT (not reached) compared to those who did not receive alloHSCT (6.3 months).Trials combining GILT  or MIDO FLT3-ITD . Data deFLT3-ITD  have ledFLT3-ITD , though FLT3-ITD –134. A p R/R AML . The medFLT3-mutant AML and all adult patients with R/R FLT3-mutant AML. In ND AML, the CRc rate was 92% with high rates of 91% MRD negativity in responders by PCR. In patients with R/R AML, CRc rates were 63% with MRD negativity by PCR in all patients who responded. At a median follow-up of 14.5 months, the median OS was not reached in ND patients (2-year OS estimated at 80%); the median OS in R/R patients was 6.8 months. Approximately one-third of patients underwent alloHSCT in either group. These results compare favorably to other reports of FLT3i/HMA in the ND setting [Furthermore, a phase 2 trial evaluated the use of triplet therapy (DAC/VEN/FLT3i) in older patients with ND  setting , 136, th setting .FLT3-mutant AML [FLT3-ITD and randomized them to QUIZ or placebo in addition to induction therapy with 7 + 3. Patients who achieved CR/CRi received up to 4 cycles of HiDAC with QUIZ or placebo and/or alloHSCT followed by up to 3 years of maintenance therapy with QUIZ or placebo. CR/CRi rates were 71.6% and 64.9% in the QUIZ and placebo arms, respectively, with DOR of 38.6 months and 12.4 months, respectively. Median OS and RFS were 31.9 months versus 15.1 months and 39.3 months versus 13.6 months in the QUIZ and placebo arms, respectively. AlloHSCT was performed in CR1 at similar rates between both arms; when censored for alloHSCT, OS trended toward a benefit with QUIZ over placebo. Moreover, an updated report from the study found that QUIZ conferred a deeper molecular remission compared to the placebo arm, perhaps underscoring the durability of benefit [FLT3-ITD mutation.Although not approved, two other FLT3i deserve mention given recent reports of their efficacy in AML. QUIZ is a second-generation type I FLT3i that can achieve significant marrow remissions in R/R tant AML , 137–139tant AML . Due to tant AML  enrolled benefit . Althoug benefit  had alreFLT-mutant ND AML [FLT3 mutations were 75% ITD, 18% TKD, and 7% both ITD and TKD. CR/CRi rates above 80% were reported across several subgroups including those with FLT3-ITD mutations or concomitant FLT3/DNMT3A/NPM1 mutations. MRD-negative CR/CRi was achieved in 94% of evaluable patients, and 50% of patients underwent alloHSCT. Median OS has not been reached at a median follow-up of 45 months. Furthermore, translational studies found that no FLT3 mutant clones were found at relapse in patients who completed protocol therapy.Lastly, a type I FLT3i emerging in clinical discussion is crenolanib (CREN). Long-term data were recently reported regarding the use of CREN in combination with 7 + 3 in adult patients with t ND AML . CREN maFLT3-mutant AML, intensive induction chemotherapy plus MIDO remains a standard of care for eligible patients. However, the formal release of data from QuANTUM-FIRST is awaiting, and ongoing trials will assess other frontline combinations with QUIZ , CREN (NCT03258931), and GILT , including head-to-head comparisons against MIDO. If QUIZ is approved for ND AML, it should be emphasized that its use would be limited to patients with FLT3-ITD, while those with TKD mutations should still receive MIDO. For patients who are ineligible for intensive chemotherapy, AZA/VEN is effective for those with FLT3-TKD mutations; unfortunately, better frontline options for those with FLT3-ITD are currently limited. Nonetheless, GILT remains a very active FLT3i in the relapsed setting, and early data from doublet and triplet FLT3i combinations are encouraging in ND and R/R AML [In considering the role of FLT3i in ND  R/R AML . SeveralFLT3-ITD after alloHSCT [FLT3-ITD [FLT3-mutant AML, it is recommended for use in patients with FLT3-ITD who achieve remission after alloHSCT [FLT3 mutations and has completed enrollment.While the historical focus of induction therapy in AML is to achieve remission and proceed with transplantation in eligible patients, the emergence of tolerable oral therapies posits the role of maintenance therapy, particularly in patients with a higher risk of relapse, such as those with pre-transplantation MRD positivity. There are limited data to support the use of targeted therapies as maintenance after transplant except for SORA. Although SORA has limited efficacy in the frontline setting , 145, thalloHSCT . The invalloHSCT . These rFLT3-ITD ; thus, wFLT3-ITD. The QUAZAR AML-001 study [For patients in remission after induction therapy but unfit for transplant, maintenance options have been limited in the absence of 01 study  randomiz01 study  or with 01 study . Combini01 study . FurtherNPM1 or IDH2 mutation with MRD negativity at cessation. Consequently, in this specific population, MRD negativity may be a reasonable impetus to interrupt treatment, though prospective studies formally evaluating this question are warranted.With several options for low-intensity maintenance therapies, the question remains as to whether treatment needs to be indefinite. A retrospective study reported their experience with discontinuing HMA/VEN or LDAC/VEN in transplant-ineligible patients older than 65 years who achieved CRc after receiving either combination for at least 12 months in the frontline setting . PatientFLT3-ITD versus FLT3-TKD) and co-mutational patterns  have important prognostic and treatment implications. Although TP53-mutant myeloid neoplasms remain one of the largest unmet needs in care, it is encouraging that detailed mechanistic studies may open the door to the development of further targeted therapies. Moreover, with the adoption of genomic data into routine care for patients with AML [The aforementioned studies emphasize an increasingly nuanced approach to treatment decision-making in AML as molecular data such as types of mutations  was presented, which assessed eprenetapopt/AZA as post-alloHSCT maintenance in patients with TP53-mutant MDS and AML [Eprenetapopt (APR-246) is a small-molecular inhibitor originally thought to work through covalent modification of mutated p53, which restores wild-type-like p53 conformation, thus functionally reactivating it . However and AML , 160. In and AML . Patient and AML . An addiTP53 mutations were noted in 27% of patients. Common adverse events included anemia, neutropenia, thrombocytopenia, and infusion reactions. In transfusion-dependent MDS and AML patients, 58% and 64% were able to achieve transfusion independence; moreover, CR/CRi rate was 56% in AML patients. The median duration of response was not reached in MDS, AML, or TP53-mutant AML subpopulations. Preliminary data from a phase 1b/2 study evaluating the combination of magrolimab/AZA/VEN (NCT04435691) noted CR/CRi rates of 63% and 86% in patients with ND AML with or without TP53 mutations, respectively, conferring 1-year OS rates of 53% and 83%, respectively [TP53-mutant group, three phase 3 trials opened which are comparing magrolimab/AZA versus AZA in patients with untreated intermediate- to very high-risk MDS (NCT04313881), magrolimab/AZA versus AZA/VEN or intensive chemotherapy in patients with untreated TP53-mutant AML (NCT04778397), and magrolimab/AZA/VEN versus AZA/VEN in patients with untreated AML who are ineligible for standard intensive chemotherapy (NCT05079230). A phase 2 study of magrolimab with various anti-leukemic therapies in patients with untreated AML is enrolling as well (NCT04778410). An additional combination study of another anti-CD47 antibody, lemzoparlimab, had opened for patients with a higher-risk MDS or AML ineligible for intensive chemotherapy (NCT04912063) but was recently stopped.CD47 is a heavily glycosylated cell surface protein and is expressed by virtually all cells in the body, including those that do not express integrin, such as red blood cells . It provectively . In patiHOXA/B cluster genes and MEIS1, which are critical regulators of hematopoietic stem cell self-renewal and differentiation [N-methyltransferase 2A (KMT2A or MLL) and/or NPM1 mutations [NPM1-mutation using the menin-MLL inhibitor KO-539 (NCT04067336) or menin inhibitor SNDX-5613  which are currently enrolling patients or opening soon. Early data from NCT04067336 and NCT04065399 suggest similar CR/CRh rates (25–30%) and MRD negativity rates in responding patients (75–78%) with either KO-539 or SNDX-5613, respectively [NPM1-mutated AML, in whom differentiation syndrome was not observed.Several types of AML have overexpression of ntiation –171. Theutations –174. KMTutations –177. Whiutations , 179. Coutations , 181. Thectively –184. ToxTP53 alterations responded [TP53-mutated, which is related to a distinct immune microenvironment compared to non-TP53-mutated AML. An ongoing phase 1b/2 study is also evaluating the combination of pivekimab sunirine (IMGN632), a CD123-targeting antibody–drug conjugate, with AZA/VEN [FLT3-ITD, with minimal additive myelosuppression beyond that of AZA/VEN. In regard to checkpoint inhibition, findings from the unpublished REMAIN trial did not demonstrate a PFS or OS benefit with the use of nivolumab maintenance in patients with CR/CRi who are ineligible for alloHSCT [In addition to the aforementioned therapies, several other pathways have been identified as possible therapeutic vulnerabilities in AML and are emerging in early clinical development , 186. Soesponded . There hesponded  with recesponded . The maj AZA/VEN . PatientalloHSCT . NeverthTP53 or RAS-pathway mutations [AML is a very complex and heterogeneous disease as evidenced by the expansion of genetic and cytogenetic qualifiers in the updated WHO  and ICC utations , and cliutations –196.IDH1/2 and FLT3 mutations, progress has unfortunately been slow for those with the highest risk forms of AML, such as TP53-mutant disease. Nevertheless, the incorporation of data such as co-mutational burden and MRD analysis will hopefully allow us to better define patients at the highest risk of relapse [We are entering a unique era of precision oncology whereby molecularly informed data can be exploited to tailor treatments based on disease pathobiology. But while options have rapidly increased for patients  relapse –199 and  relapse –202. PerThe multitude of active studies addressing these questions will lend nuance to clinical practice and contribute to improved outcomes for patients.


In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca2+ sensors. With them, Ca2+ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca2+ levels , to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca2+ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca2+ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca2+ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca2+-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca2+ or calmodulin levels negatively affect the efficiency of the process.Free calcium (Ca One of the most important is its role as signaling molecule in both plant and animal cells , being stored principally at the endoplasmic reticulum, nucleus, cell wall and vacuoles , where the first embryogenic suspension cultures were reported more than six decades ago , a calmodulin (CaM) residue fused to the specific binding peptide M13, and an acceptor yellow fluorescent protein (YFP). Free Ca2+ binds CaM and induces a conformational change that favors the acceptor YPF excitation by the donor CPF emission  cv. Nantese 5 from Semillas Batlle S.A. were used for transformations, induction of somatic embryogenesis and modulation of Ca2+ homeostasis. Transgenic seedlings developed from carrot transformation expressing the PM-YC3.6-LTI6b cameleon construct were used for Ca2+ visualization by confocal microscopy. Carrot seeds were surface-sterilized in two consecutive cycles of 15 minutes in 70% ethanol + 0.1% triton solution, 15 minutes in 50% bleach solution and three rinses in distilled sterile water. Seeds were kept in solid MS2% medium  was transformed with the YC3.6-Bar and PM-YC3.6-LTI6b plasmids respectively following provider instructions. Transformed cells were plated in YM solid medium supplemented with 50 mg/L rifampicin and appropriate bacterial selective agents: 50 mg/L kanamycin for YC3.6-Bar and 100 mg/L spectinomycin and 100 mg/L streptomycin for PM-YC3.6-LTI6b.Two membrane . Both caetail in . EscheriAgrobacterium tumefaciens suspensions prepared in 4.6 g/L MS medium at a final OD600 of 0.2. Incubation was performed in petri dishes. During the process, small wounds were performed in the explants with a scalpel to facilitate the penetration of bacteria in the explants. Then, explants were deposited in sterile paper to remove the excess of bacterial inoculum. Explants were placed in Co-culture D medium and incubated for 3 days at 25°C in darkness. After that, explants were transferred to MI medium  to the explants. In both cases, after inoculation, explants were incubated in Co-culture E medium Table 2 in vitro-growing transgenic seedlings, were cultured in solid Embryogenesis Induction Medium -N,N,N′,N′-tetraacetic acid (EGTA) and N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) were prepared following the guidelines provided by the manufacturer. Embryogenic cell cultures from liquid MS2% flasks, containing mostly proembryogenic masses and cell clumps, were inoculated into 3-cm culture dishes. To allow for comparisons among stages and concentrations for each chemical, equal aliquots (1 mL) of the same embryogenic cell culture were inoculated, so the initial amounts of proembryogenic masses/cell clumps were the same in all cases. Then the different chemicals, at different concentration (as described in Results), were applied during the first three days, during the first seven days, or continuously (for 30 days). Control plates were prepared with the proper concentrations of the solvent used to dilute the corresponding compound. At least three different repeats were performed for each chemical, concentration and exposure time applied. In all cases, plates were kept at 25°C in darkness and no agitation. For the cultures with three and seven days of exposure, experimental plates with the chemical (and their corresponding control) were centrifuged  to wash the compound. The proportion of embryos was calculated 30 days after culture initiation as described for somatic embryogenesis cultures.For the chemical treatments, stocks of CaClTo inhibit callose deposition, we applied 2-deoxy-D-glucose to two different types of cultures, wild-type carrot calli disaggregated in liquid EIM, where predominantly cell clumps can be found, and one-week-old liquid EIM cultures filtered and resuspended in liquid MS2%, where proembryogenic masses can predominantly be found. One mL aliquots of each culture were poured in 3-cm sterile culture dishes and cultured for a week without (controls) and with 2-deoxy-D-glucose at 0.1, 1 and 5 mM. Incubations were performed in a rotary shaker at low speed and at room temperature. At least three different repeats were performed for each 2-deoxy-D-glucose concentration and exposure time applied. The proportion of embryos was calculated 30 days after culture initiation as described for somatic embryogenesis cultures. For visualization in the confocal microscope, control and 2-deoxy-D-glucose samples were collected in tubes, centrifuged  and stained with 1% aniline blue for 10 min. Then, samples were washed three times with PBS 1x. Approximately 20 µL of sample were mounted in a glass slide, covered with a coverslip and observed in the confocal microscope.Carrot samples stained with aniline blue were observed in a Zeiss 780 Axio Observer confocal laser scanning microscope at an excitation wavelength of 405 nm. Emission was recorded at 450 nm. Cell cultures from PCR-positive carrot plants were imaged using a Zeiss LSM880 laser scanning confocal microscope. Samples were excited at 440 nm (for CFP excitation) and 514 nm (for YFP excitation) and emission was recorded between 400 and 600 nm.2+ levels were defined as very low, low, moderate, high or very high according to the colorimetric scale based on the FRET ratio images. Very low Ca2+ levels corresponded to dark blue colors, low levels to light blue, moderate to green-yellow, high to orange-red and very high levels to purple-white colors. Image analysis was performed using the FIJI software  supplemented with 0.3% agarose. Mounted samples were covered with liquid half-strength MS medium (pH 5.7) and observed in a Leica SP8-FLIMan confocal laser scanning microscope using an immersion objective. CFP was excited at 448 nm and emission was recorded between 460 and 493 nm, YFP was excited at 514 nm and recorded between 525 and 560 nm. FRET imaging was performed exciting at 448 nm and recording the emission between 525 and 560 nm. For imaging, the FRET ratio was defined as the ratio between YFP and CFP emissions (480/535 nm). Image treatment and calculation of fluorescence emission ratios were performed as previously described . For allsoftware .Data were analyzed using the ANOVA test. Groups of significance were established according to the least significant difference (LSD) test (p ≤ 0.05). All the analyses were performed with the Statgraphics software.cameleon carrot lines, we first tested two carrot transformation protocols, namely D  to the culture medium (2 produced significantly more embryos (3-fold) than the control. In these conditions, addition of increased CaCl2 levels also reduced the occurrence of disorganized calli and accelerated embryo growth, producing larger and elongated embryos , the shapes, sizes and positions of callose-positive (2+-positive cells (2+ levels could stimulate callose deposition to isolate embryogenic cells (Brassica napus microspore embryogenesis, the embryogenic competence of microspores was shown associated to their high Ca2+ levels and their parallel ability to form an osmoprotective, isolating callose layer through the activation of Ca2+-dependent callose synthases (2+ levels and callose deposition, needed to isolate different cell types in different species as a previous requisite to reprogram them towards in vitro embryogenesis.We showed that a callose layer is created around specific cells of cell clumps and small proembryogenic masses which share a number of features with the embryo-precursor cells that present high Caic cells . In lineic cells . In Brasynthases . Togethe2+ in both zygotic and somatic embryogenesis is widely acknowledged (2+ homeostasis during the process. While some reports proposed that Ca2+ levels can be altered to increase the embryo induction rate (2+ in this process, we used different approaches to modulate intracellular Ca2+ levels. Increasing cytoplasmic Ca2+ availability during the first days of culture by adding extra CaCl2 to the culture medium or by using ionophore A23187 to open plasma membrane channels where through extracellular Ca2+ can enter the cytoplasm, led to higher percentages of embryo induction, accelerated embryo growth and lower callus occurrence. However, there seemed to be a limit for the direct relationship between Ca2+ and induction of embryogenesis, since a continuous increase of Ca2+ levels did not make any significant difference. This is in line with previous reports suggesting that excessive Ca2+ influx may have detrimental consequences on somatic embryogenesis (2+ levels were reduced by chelating Ca2+ with EGTA, the percentage of embryos decreased in a dose-dependent manner. Thus, there is a range of Ca2+ concentrations where somatic embryogenesis can be modulated by altering Ca2+ homeostasis. A possible explanation for this could be that increased extracellular Ca2+ levels would create an even higher Ca2+ gradient (the cytoplasmic resting Ca2+ concentration revolves around 100 nM; (2+ influx in more cells, thereby triggering the 2+ signatureembryogenic Ca in more cells. An alternative explanation would be that higher extracellular Ca2+ levels would make the plasma membrane less permeable. Although counterintuitive in principle, this effect has been documented for some cell types (2 was similar to that of ionophore A23187, a Ca2+ channel that increases Ca2+ influx (2+ concentrations to the culture medium increases Ca2+ influx in more cells, which in turn increases somatic embryogenesis.The role of Caowledged , but in ion rate , other sion rate . To gainogenesis . Convers 100 nM;  that woull types . A calci+ influx . Thus, i2+ levels by adding both CaCl2 and ionophore A23187 had positive effects only when applied during the first days of culture, when cell clumps are converting into proembryogenic masses and early embryos. The application of increased Ca2+ levels for longer times or continuously did not have any significant effect in embryo yield, which implies that the observed positive effect of increased Ca2+ levels during the first stages is compensated by a detrimental effect during later stages of embryo development in order to produce no significant final changes. In turn, a reduction of intracellular Ca2+ by chelation with EGTA produced similarly detrimental effects when applied during 3 days, 7 days or continuously, which indicates that the detrimental effect seems to be exerted principally during the first stage of embryo induction. Thus, increasing the intracellular Ca2+ levels during the inductive stage, when the first embryogenic cells differentiate from cell clumps/proembryogenic masses, increases the percentage of cells effectively induced to embryogenesis, and decreasing the levels has the opposite effect. However, once embryos are induced, during the embryo development stages, any alteration of Ca2+ homeostasis would have detrimental effects on embryo production. This is indicating that Ca2+ has a key role during the induction of somatic embryogenesis from carrot cells, and that Ca2+ homeostasis at this stage is plastic and can be altered to modulate the induction rate, but only during this stage. Once embryos are induced, Ca2+ homeostasis must be strictly maintained in order to allow for an efficient embryo maturation, since fully functional, differentiated embryos are able to autonomously regulate their Ca2+ levels in their different organs and any external interference may be detrimental.It is interesting to note that increasing CaThe original contributions presented in the study are included in the article/Conceptualization: JMSS. Funding Acquisition: JMSS, RM. Investigation: AC-S, PA, RM. Methodology: AC-S, PA, RM. Data curation: AC-S, PA, RM. Formal analysis: AC-S, RM. Project Administration: JMSS. Supervision: JMSS, RM. Writing – Original Draft Preparation: AC-S, JMSS, RM. Writing – Review & Editing: AC-S, JMSS, RM. All authors contributed to the article and approved the submitted version.


Seasonal parameters, especially temperature and humidity, are reasonably speculated to affect livestock production performance. Among farmed animals, rabbits in particular are sensitive to extreme environmental temperature and humidity. This is not conducive to the performance of rabbit production and the improvement of product quality. Thus, a better understanding of the effects of the seasons on rabbits’ physiology is fundamental to ensuring rabbit welfare, production efficiency, and fur quality improvement. Our study proved that the skinning season has a significant influence on the fur quality and hair follicle traits of Rex rabbits. The skin quality of rabbits slaughtered in winter was the best. Seasons may regulate hair follicle development via the Wnt10b/β-catenin, TGFβ-BMP, IGF1, and EGF signaling pathways in Rex rabbits.p < 0.05). Moreover, the coat length was shorter in summer than in spring, autumn, and winter (p < 0.05). The shoulder fat weight, perirenal fat weight, and perigastric fat weight of the Rex rabbits in winter were higher than those in summer (p < 0.05). Furthermore, the leptin levels in serum were higher in winter than in summer in the Rex rabbits (p < 0.05). In terms of serum biochemistry, the glucose levels were higher in autumn and winter than in spring and summer (p < 0.05). The cholesterol, high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) in summer had higher values than in winter in the Rex rabbits (p < 0.05). In winter, the expression of the Wnt10b, catenin beta 1 (CTNNB1), glycogen synthase kinase 3 beta (GSK3β), insulin like growth factor I (IGF-I), Type I insulin-like growth factor receptor (IGF-IR), and epidermal growth factor (EGF) genes was higher (p < 0.05), and the expression of the dickkopf-1 (DDK1), transforming growth factor beta 1 (TGFβ-1), bone morphogenetic protein 2 (BMP2), and bone morphogenetic protein 4 (BMP4) genes was lower than in summer (p < 0.05). In summer, the heat shock 70 kDa protein (HSP70) expression and CTNNB1 protein phosphorylation levels in skin tissue were higher than in spring, autumn, and winter (p < 0.05). In winter, Wnt10b protein expression was higher (p < 0.05), and GSK-3β protein phosphorylation levels were lower than in spring, autumn, and winter (p < 0.05). These results show that the skinning season can affect the production performance and hair follicle development of Rex rabbits. Compared with other seasons, the quality of skin from rabbits slaughtered in winter is better. Seasons may regulate hair follicle development via the Wnt10b/β-catenin, TGFβ-BMP, IGF1, and EGF signaling pathways in Rex rabbits.This experiment was conducted to study the effects of different skinning seasons on the fur quality and hair follicle development of Rex rabbits. A total of 80,150-day-old Rex rabbits were slaughtered on 15 July 2022 (summer), 15 October 2022 (autumn), 15 January 2023 (winter), and 15 April 2023 (spring) in Shandong Province . The results show that the skin weight, skin area, skin thickness, and hair follicle density of the Rex rabbits (at 150 days of age) were lower in summer than in winter ( The season is an important factor affecting the growth and development of many animals, especially fur animals. Hair length and density features could affect the transfer of heat from the domestic animal’s skin to the environment . The envThe experiment was conducted in early January 2022, early April 2022, early July 2022, and early October 2022, and the conception and birth of Rex rabbits were ensured. Both female and young rabbits were fed the same diet formula in Tai’an City, Shandong Province, which belongs to a warm temperate continental subhumid monsoon climate area with four distinct seasons; it is suitable for an analysis of cold seasons and summer, synchronous light and temperature, and rain and heat in the same season. Spring is dry and windy (average daily temperature: 0∼9 °C), summer is hot and rainy (average daily temperature: 22∼31 °C), autumn is sunny and cool (average daily temperature: 18∼28 °C), and winter is cold and has some snow (average daily temperature: 5∼14 °C). The rabbits were reared until five months of age. In spring, summer, autumn, and winter, the rabbits had the same feeding conditions, and all of them were raised in simple outdoor cement cages with access to feed and drinking water, natural lighting, and natural ventilation; there were no cooling or insulation measures in summer and winter. The raw material composition and nutritional level of the basic diet are shown in g for 10 min after coagulation. Upper serum was collected, transferred to a sterile tube, and stored at −20 °C for the determination of blood indicators. Then, the rabbits were sacrificed via cervical dislocation. Shoulder fat, perirenal fat, and perigastric fat were carefully stripped and weighed. Mediodorsal skin samples of each rabbit sample were divided into two parts. One part was frozen in liquid nitrogen and stored in a −80 °C refrigerator for gene and protein expression detection. The other part was fixed in 4% paraformaldehyde and stored at 4 °C for subsequent hematoxylin–eosin (HE) staining for a hair follicle density analysis.The animals were fasted for 24h before the end of the experiment. For Rex rabbits at 150 days of age, 5 mL blood samples were placed into sterile vacuum coagulation vessels and centrifuged at 3000× At 150 days of age, the Rex rabbit skin was collected; the adhesive tissue and fat was removed, weighed, and cut in the middle of the abdomen, and then the length and width of the skin were measured using a soft ruler. The length of the skin was measured from the neck to the tail root, and the width was recorded from the narrowest part of the middle abdomen; the skin area was also calculated. The thickness of the skin was measured using vernier calipers, and the length of the coat was measured using a straightedge. The hair follicle density was observed with paraffin section and HE staining. After HE staining, photos were taken under ordinary light conditions with a microscope , and Image-Pro Plus 6.0 analysis software was used to calculate the primary hair follicle density, secondary hair follicle density, and total hair follicle density, and the ratio of the secondary hair follicle density to the primary hair follicle density was calculated.A rabbit insulin ELISA Kit (CSB-E06992Rb), a rabbit insulin-like growth factor 1 (IGF-1) ELISA Kit (CSB-E06994Rb), and a rabbit leptin ELISA Kit (CSB-E06971Rb) were used for the detection of serum hormone contents . Serum glucose, total protein, cholesterol, triglyceride, high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) were determined using a sequential multiple analyzer .Total RNA extraction and real-time PCR were performed as described previously . The seqThe methods for total protein extraction and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) were performed according to Liu et al. . Total pp < 0.05 was considered to be a significant difference.SPSS 26.0 statistical software was used for a one-way ANOVA analysis of the data, and Duncan’s test was used for multiple comparisons. The results are presented as the mean value and standard error of means (SEMs), and p < 0.05). Additionally, the coat length of the Rex rabbits in summer was shorter than that in the other seasons .The effects of different skinning seasons on the fur quality of Rex rabbits are shown in p < 0.05). However, the primary hair follicle density and the ratio of the secondary hair follicle density to the primary hair follicle density did not change significantly . Compared with summer, the shoulder fat weight, perirenal fat weight, and perigastric fat in the rabbits skinned in winter increased by 28.81%, 62.90%, and 41.54%, respectively.The fat deposition of the Rex rabbits is presented in p < 0.05), and there was no significant difference between spring, summer, autumn, and winter skinning in terms of serum hormones (insulin and IGF-I) (p > 0.05). For serum biochemistry, the glucose levels in the Rex rabbits skinned in autumn and winter were higher than in those skinned in spring and summer (p < 0.05). The cholesterol, HDL, and LDL in the Rex rabbits skinned in summer at 150 days of age were higher than in those skinned in winter (p < 0.05).The serum hormones and biochemical indices of the Rex rabbits are presented in Wnt10b, CTNNB1, GSK3β, IGF-I, IGF-IR, and EGF genes in the skin tissue of the Rex rabbits at 150 days of age was higher for winter skinning than for summer skinning (p < 0.05), and the expression of the DDK1, TGFβ-1, BMP2, and BMP4 genes was lower for winter skinning than for summer skinning (p < 0.05).The effects of the different skinning seasons on the gene expression of hair follicle development in the Rex rabbits are shown in p < 0.05; p < 0.05; p < 0.05; The effects of different skinning seasons on the protein expression (HSP70 and Wnt10b) and protein phosphorylation (CTNNB1 and GSK-3β) levels of hair follicle development in the Rex rabbits at 150 days of age are shown in Global warming and abnormally high ambient temperatures have adversely affected the livestock sector in recent decades . In ChinRabbits with fur have poor heat dissipation mechanisms and are more easily affected by heat stress, and this reduces their performance and the economic efficiency of farms. The quality of fur is mainly based on the hair density, hair length, skin area, and skin thickness, and the density of the hair follicle is a particularly important indicator for evaluating the quality of Rex rabbits’ skin. The skin in summer is generally thin and breaks easily, and the wool can also fall off easily, which affects the rabbit’s skin quality. Compared with winter, the present results show that skinning in summer decreased the skin weight, skin area, skin thickness , and haiWnt10b, CTNNB1, and GSK3β gene expression was higher than in summer, and the DDK1 gene expression was lower than in summer  signaling pathway plays an important role in controlling the growth and development of animal skin hair follicles . BMP sign summer , and then summer C, indicaIGF-I, IGF-IR, and EGF in the rabbits skinned in summer was lower than that in the rabbits skinned in winter (IGF1 has a positive effect on hair follicle development; it is expressed in both the hair follicles and dermal fibroblasts of rats, participates in apoptosis, and promotes the proliferation of dermal cells . In addin winter , which sThe skinning season can affect the production performance and hair follicle development of Rex rabbits. The skin quality of Rex rabbits slaughtered in winter is better than in the other seasons. Seasons may regulate hair follicle development via the Wnt10b/β-catenin, TGFβ-BMP, IGF1, and EGF signaling pathways in Rex rabbits.


Ca2+ exerts a pivotal role in not just regulating oocyte activation and driving fertilisation, but also in influencing the quality of embryogenesis. In humans, a failure of Ca2+ release, or defects in related mechanisms, have been reported to result in infertility. Furthermore, mutations in the PLCζ gene and abnormalities in sperm PLCζ protein and RNA, have been strongly associated with forms of male infertility where oocyte activation is deficient. Concurrently, specific patterns and profiles of PLCζ in human sperm have been linked to parameters of semen quality, suggesting the potential for PLCζ as a powerful target for both therapeutics and diagnostics of human fertility. However, further to PLCζ and given the strong role played by Ca2+ in fertilisation, targets down- and up-stream of this process may also present a significantly similar level of promise. Herein, we systematically summarise recent advancements and controversies in the field to update expanding clinical associations between Ca2+-release, PLCζ, oocyte activation and human fertility. We discuss how such associations may potentially underlie defective embryogenesis and recurrent implantation failure following fertility treatments, alongside potential diagnostic and therapeutic avenues presented by oocyte activation for the diagnosis and treatment of human infertility.Oocyte activation, a fundamental event during mammalian fertilisation, is initiated by concerted intracellular patterns of calcium (Ca This results in a signal transduction cascade, which will convert the oocyte into a diploid zygote, through a series of collective processes termed oocyte activation (OA). OA involves well-defined morphological and biochemical endpoints that occur following sperm-oocyte interaction, such as the resumption of meiosis II, prevention of polyspermy, and cortical granule exocytosis. These endpoints can vary in duration; some will require minutes, and some will require hours after the interaction, but calcium levels are critical for all of them . Prior t[2+ ions .2+ at OA in mammalian and non-mammalian species [2+ release at fertilisation can exert effects upon postnatal growth and the weight of mice offspring [2+ release is a crucial component of OA, occurring in a dramatic wave-like manner, starting from the point of fusion to all over the oocyte. Generally, this increase in intracellular Ca2+ release seems mediated in an inositol 1,4,5-triphosphate (IP3) receptor-dependent manner and from Ca2+ stores, predominantly being the endoplasmic reticulum (ER). While in some species, such as Xenopus and sea urchins, there is a single wave of Ca2+ release, in others, such as mammals, this release pattern occurs in an oscillatory manner, the amplitude and frequency of which vary in duration, amplitude, and frequency depending on the species. Generally, Ca2+ oscillations are a linear result of IP3 activation [3 peaks preceding Ca2+ release in oocytes during fertilisation [2+ ions could also initiate oocyte activation following microinjection in oocytes [3 receptors in hamster and mouse oocytes inhibited Ca2+ oscillations and oocyte activation [Indeed, many experiments have demonstrated the importance of Ca species , with fuffspring . Intracetivation . Many exlisation , while o oocytes ,9. The dtivation ,11,12,132+ levels in the oocyte are kept relatively low compared to the extracellular environment, making the cytosol a favourable site for minor Ca2+ variation, as a response to extracellular or intracellular Ca2+ signalling [2+ oscillation signals the start as a result of the disturbance of cytosolic Ca2+ equilibrium. This disturbance occurs as a response to stimulatory signals that act on Ca2+ receptors which are found either in the cytoplasmic membrane or ER (Ca2 store) and will result in an increase in cytoplasmic Ca2+ [2+ stores, where a sudden increase of Ca2+ will induce more Ca2+ release through a series of events as positive feedback [2+ waves is inositol trisphosphate receptors (IP3Rs) which are found abundantly as calcium-releasing channels on ER in the cytosol [3 resulting from various cascades produced by extracellular stimulants will bind to IP3Rs, causing a conformational change to Ca2+ channels found on the cell membrane leading to Ca2+ influx into the cell [2+ oscillations are initiated that are critical for OA and the completion of meiosis II [2+ oscillations will activate Ca2+ calmodulin-dependent kinase II (CaMKII) which then will activate an anaphase-promoting complex (APC) [2 oscillation has also proven to play a major role in other developmental stages at the genome level and nuclear signalling level [Normally, basal cytosolic Cagnalling . The Ca2mic Ca2+ . Coordinfeedback , a proce cytosol . IP3 resthe cell . Followiiosis II . Ca2+ osex (APC) . The actex (APC) . The degex (APC) ), althoung level ,21,23,242+ oscillation occur in the temporal order, unlike non-mammalian cells where all of the events occur simultaneously after being exposed to single Ca2+ transients. Therefore, mammalian cells are more reactive to the frequency, duration, and amplitude of Ca2+ release. Along with the accuracy of the oscillations, the mature oocyte that is coordinated by important organelles, such as ER and mitochondria, are essential to maintain the periodical increase in Ca2+ [In mammalian oocytes, all of the events following Ca in Ca2+ .2+ is pumped against a concentration gradient from the cytoplasm into the ER by the plasma membrane pump sarco-ER Ca2+-ATPase (SERCA). Three genes in mammals (ATP2A1–3) are responsible for producing three different isoforms of SERCA (SERCA1–3), and through alternative splicing, they produce 11 SERCA isoforms. Each one of these isoforms has a location, developmental expression, and most importantly, a unique affinity and sensitivity to Ca2+. Like any Ca2+ ATPase, SERCA’s functional structure is trans-membranal, containing three cytoplasmic domains (phosphorylation and nucleotide-binding domains in addition to the actuator) and ten membrane-spanning helices. The transmembrane domain contains two Ca2+-binding sites, making SERCA capable of transporting two Ca2+ ions per ATP. Any general plasma membrane ATPase inhibitor, such as orthovanadate, is able to inhibit an undetermined SERCA isoform or a specific one, such as thapsigargin [Casigargin .2+ oscillation during fertilisation in animal models, such as Xenopus, frogs, and mice. Interestingly, thapsigargin treatment significantly reduced the magnitude and duration of the first Ca2+ peak and oscillation persistence. During oocyte maturation, SERCA2B protein levels remain constant but are redistributed spatially from diffuse patterns to cortical clusters mimicking ER redistribution. This arrangement will allow SERCA to pump closer to IP3 receptors. This would be necessary for depletion that follows fertilisation, as it will facilitate the refilling of Ca2+ stores in ER [Many studies have illustrated the influence of SERCA2 isoforms in sustaining Caes in ER .2+ intracellular homeostasis. Alteration in cellular homeostasis depends on the change of Ca2+ concentration, for example, if the concentration of Ca2+ entering the mitochondria decreases more than it should, this will cause a bioenergy disaster. Moreover, if the concentration of Ca2+ in the cell increases, causing apoptosis because the abnormal Ca2+ concentration disrupts the oxidative phosphorylation and can open the transition pore in mitochondria, this will cause mitochondrial dysfunction [Prior to oocyte and mitochondria maturation, the granulosa cells and cumulus provide the cell with energy. Following ovulation, the mitochondria start to activate and become the main source of energy in the mature oocyte . Furtherfunction .2+ oscillations have been proven for a long time in many studies as an important step of OA, but the exact mechanism that results in the oscillation, specifically in relation to gamete fusion, remains unclear. Few hypotheses have been suggested [Cauggested .2+ into the oocyte right after sperm fusion would lead to Ca2+-induced Ca2+ release, allowing Ca2+-influx into the oocyte. However, this model was not successful on other animal models, such as mice and ascidians. Moreover, experiments emphasize the importance of the IP3 pathway to release and maintain calcium in OA [Based on the sea urchin model, it was suggested that the infusion of a considerable amount of Caum in OA .2+ release by injecting the hydrolysis-resistant GTP analogue GTP-γS, in sea urchins and frog eggs. However, resultant patterns of Ca2+ release were not comparable to that at fertilisation, specifically in mammalian cells. Moreover, the direct injection of sperm into the oocyte cytosol using intracytoplasmic sperm injection (ICSI) can undergo successful fertilisation and embryogenesis, without any such membrane-membrane interactions [2+ oscillations and the production of considerable IP3 levels [The basic theory underlying this model suggests that OA would result from the interaction between a specific sperm-ligand and oocyte-receptor, activating a phospholipase C (PLC) inside the oocyte. However, such assertions were supported by indirect evidence and the experiments involved overexpressed G-protein linked receptors which might be responsible for activating PLC-β, as a response to gamete interactions and the corresponding application of ligands. Some experiments showed Caractions , creatin3 levels ,30,31,322+ oscillations seen at fertilisation, while also producing the subsequent events of OA [3-mediated nature of Ca2+ release in mammalian oocytes, it would be suitable to consider that a phosphoinositide (PI)-specific PLC-associated pathway is simulated [2+ release at fertilisation is not stimulated by Ca2+ injection , nor does injection of IP3 or stimulating G-proteins  [2+ release and OA. Indeed, given the specifications underlying the signalling mechanisms underlying OA, most opinions suggest a PLC-mediated mechanism is the essential factor to initiate the IP3 pathway for OA [This model suggests that a soluble sperm factor is released into the oocyte during or immediately after gamete fusion, which in turn is responsible for OA. Injection of sperm cytosolic extracts into the eggs/oocytes of sea urchins, mice, humans, pigs, and cows triggered the characteristic series of Cats of OA ,34,35. Oimulated . Indeed,isation) ,36. Mosty for OA ,32.Xenopus eggs [2+ [2+ oscillations, while the suggested signalling pathway associated with PAWP seemingly has no relevance to OA [A number of factors and proteins have been proposed to be the sperm factor, including the post-acrosomal WW-domain binding protein (PAWP), where its implied function in OA is through a yes-associated protein (YAP) to activate PLC-γ, similar to what happens in pus eggs ,38. The eggs [2+ . Microince to OA . A furthce to OA ,42 (like2+ oscillations that are seen in OA that are attributed to be the function of the “sperm factor” is believed to be the direct result of Ca2+ release via (IP3-mediated reactions [2) into IP3 and diacylglycerol (DAG) [2+ from intracellular stores [2 hydrolysis. PLCs also comprise EF-hands, which are the Ca2+ binding structures in the enzyme; a pleckstrin homology (PH) domain that is generally used for targeting the enzyme substrates; and a C2 domain, which is also essential in Ca2+ activity [2+ release following microinjection into oocytes [The series of Caeactions ,43,44,45ol (DAG) ,46,47,48ol (DAG) ,50,51,52r stores ,46,47,48activity ,54,55. A oocytes .2+ release at OA was first identified using mouse expressed sequence tag (EST) databases to describe a novel, testis-specific PLC, termed PLCzeta (PLCζ), a ~74 kDa protein in mice, its immunodepletion from sperm extracts suppressed Ca2+ release at OA [2+ release similar to those at natural fertilisation [2+ release and OA corresponded to the same range as the amount of PLCζ found in a single sperm, estimated to be ~40 fg, which was also found to be the level at which PLCζ is most effective [2+ oscillations which are seen during fertilisation [The specific PLC isozyme responsible for Case at OA . Recombilisation ,58,59. Tffective ,58,59,60lisation .2 in the oocyte, yielding IP3, which targets the IP3R on Ca2+ stores, such as the endoplasmic receptors, to release intracellular Ca2+ [2+ release before OA, with such mice yielding a decreased number of offspring [2+ release from intracellular stores via hydrolysation of PIP2 into DAG and IP3 [2+ sensing ability and the distribution of the protein mainly in sperm and testes [The suggested PLCζ mechanism of action is that PLCζ targets the cytoplasmic vesicle-bound PIPlar Ca2+ ,62,63. Rffspring . PLCζ, l and IP3 ,43,44,45d testes , PLCζ had testes ,64,65,662+ sensitivity and binding [2+ sensitivity but rather Ca2+ oscillatory activity [PLCζ is currently the smallest known PLC isoform (ranging in size from 70–75 kDa) ,45,54,57 binding , suggestactivity ,72. Thisactivity .2+ oscillations for an extended period of time [2+ release [2+ sensitivity, distinguishing it from other PLC isoforms, but also perhaps exerts a role in nuclear translocation during fertilisation and binding to PIP2 [2+ sensitivity [2+ levels after gamete fusion [2+ sensitivity of the altered PLCζ but did not affect the enzymatic function [2 interaction following the deletion of EF-hands [PLCζ is also involved in nuclear sequestration activity that directs the protein to act in a cell cycle-dependent manner ,74,75. I of time ,77. This of time ,78,79,80 release ,78. The  to PIP2 ,79,81. Isitivity , allowine fusion ,53. Trunfunction ,53,72. Afunction , illustrEF-hands ,67.2+ oscillations are also an important part of fertilisation, which varies between species, extending from minutes to hours in terms of duration [2+ oscillation frequency and amplitude [2 hydrolysation occurred in the cytoplasm, corresponding to PLCζ localisation in the cytoplasm near the nuclear envelope rather than the plasma membrane [2 did not significantly affect Ca2+ oscillations at fertilisation [2 in vesicles led to the inhibition of Ca2+ oscillations [2 hydrolysis from intracellular vesicles, rather than the plasma membrane, is the primary source of the cytosolic Ca2+ oscillations in oocytes induced by PLCζ [The duration and frequency of PLCζ-induced Caduration ,63,82,83mplitude ,84, thismplitude ,54,85. Tmplitude ,75,86. Pmplitude ,81,87,88membrane ,89. Furtlisation . Interesllations . This co2+ oscillations and an increased amount of polyspermy. However, ICSI of such KO sperm was unable to successfully elicit Ca2+ release [2+ release at fertilisation, in addition to (and perhaps independently of) PLCζ (discussed later in this review).Intriguingly, transgenic mice where PLCζ was knocked out (KO) in two different studies indicated that KO male mice were able to have offspring but with a remarkably decreased amount than usual following in vitro fertilisation (IVF) alongside abnormal and delayed Ca release ,64,65,91PLCζ has been found in the sperm of many different species and it typically localizes to different subcellular areas of the sperm head. For instance, it has been shown that PLCζ in mice is localized in the sperm’s head post-acrosomal region . HoweverRecent efforts, using specific antibodies and optimised protocols, specific patterns of PLCζ localisation in human sperm were identified including equatorial, equatorial + acrosomal, and a uniformly dispersed pattern, with a further pattern in the tail and the mid-peace of the sperm . The equ2+ responses in oocytes [While PLCζ has been found to be localized at the sperm tail, it is still unclear whether these results are accurate . However oocytes . Therefo oocytes . The pot oocytes . HencefoAn interesting assertion was made by Aarabi et al. , who sug2+ signalling (and involved downstream pathways) in clinical conditions [2+ release was reduced. This shows that defects or absence of PLCζ may lead to the failure of fertilisation [Similar to defective PLCs and abnormal Canditions ,103, defnditions . The higlisation . Indeed,lisation . Sperm flisation . Furtherlisation , either lisation ,105,106.2+ release following injection into mouse oocytes [2+ oscillations were rescued and OA/fertilisation was able to proceed [2+ release, resulting in failed OA in mouse oocytes, in stark comparison with oocytes injected with wild type PLCζ cRNA [PLCζ levels may also be associated with specific sperm structures, as globozoospermic sperm with acrosomal buds selected from a population of sperm exhibiting a complete round-headed globozoospermic morphology could be used to achieve successful fertilisation without fertility treatment, also corresponding to an acrosomal pattern of PLCζ localisation . Moreove oocytes . However proceed . Inferti proceed ,69,108. LCζ cRNA . NumerouLCζ cRNA ,108.2+ release [2+ oscillations. Single Ca2+ oscillations in some forms of AOA are not ideal for future successful development in humans and mice [AOA is a potential treatment for male-related infertility that aims to mimic physiological Ca release ,110. AOA release . AOA wiland mice ,1102+ through migration of lipid bilayer-charged proteins and pore formation within the membrane [2+ increase in the oocyte [2+ concentration. However, the downside of such a method is the formation of excess reactive oxygen species (ROS), in addition to physical damage to the oocyte [The electrical method has been tested in bovine and human oocytes , aiming membrane . This ree oocyte ,114,115.e oocyte . Interese oocyte , and thu2+ via intracellular store disruption or manual elevations of Ca2+. This could be accomplished by piercing the oocyte, leading to increased Ca2+ influx, or direct microinjection of Ca2+ into the oocyte. Perhaps more invasively, another mechanism involves a physical ER membrane disruption and mitochondrial redistribution, or (more popularly) manual oocyte membrane disruption followed by vigorous oocyte cytoplasm disruption to increase the Ca2+ load. While of course significantly physically disruptive, such mechanisms would perhaps enhance closer contacts between sperm and intracellular membranes, further enhancing the chances of successful OA [Mechanical activation is the result of a mechanical disruption of the oocyte, resulting in a ‘manual’ release of Cassful OA ,117,118.2+ ionophores’ that diffuse into the oocyte and enhance Ca2+ permeability, Ca2+ influx, and release of intracellular stored Ca2+ [2+ [2+ transients, which include thimerosal, phorbol esters, or strontium chloride (SrCl2) [2 efficacy in human oocytes is still debatable [2+ and transports them through biological membranes. Ionomycin has a similar action but is more potent and is specific to Ca2+, and it stimulates gene expressions [Chemical methods of activation stimuli utilise lipid-soluble chemicals termed ‘Cared Ca2+ . Such iored Ca2+ ,119, andred Ca2+  which alCa2+ [2+ ,119. The (SrCl2) ,119. SrCebatable . Ionomycressions .2+ ionophores after ICSI and not with it. Patients’ characteristics also play role in determining the success of ICSI and Ca2+ ionophores. Indeed, the effect in humans is not consistent; with some studies and meta-analyses indicating that the effect of Ca2+ ionophores in the case of sperm morphological abnormalities is negative, while other studies indicate positive results in cases with <30% successful fertilisation rates in previous ICSI cycles [It is more effective to deliver CaI cycles . FurtherI cycles .2+ release following AOA methods differs from physiological release, specifically in the frequency and amplitude of Ca2+ release [Some cases of successful OA after ICSI have been reported. However, ionophore treatment may hold cytotoxic, teratogenic, or even mutagenic effects for the embryo. For instance, the abnormal calcium-induced signal may have poor outcomes on epigenetic processes. Furthermore, current protocols may not be effective for all patients receiving this treatment . The tra release . However release ,123,124, release . Indeed, release , and did release ,128,129. release , particu release .Interestingly, however, chromosomal abnormality and defective embryogenesis following AOA could be overcome by supplementation of AOA media with granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine involved in human preimplantation embryo development . Indeed,2+ transient would result in cyclin B degradation and reduction of CDK1 activity, promoting meiotic resumption [2+ transient would only result in a temporary alleviation of arrest, with cyclin B resynthesis followed by the resumption of CDK1 activity and re-arrest of the oocyte cell cycle [2+ ionophore treatments seem more effective upon in vitro-aged oocytes following ovulation, perhaps due to a decline in cyclin B levels [A single Casumption , which mll cycle . To thisll cycle ,133,134.B levels ,133,134,B levels  recently2+ oscillations following injection of recombinant PLCζ RNA or protein, with both modes leading to successful OA and subsequent embryogenesis to the blastocyst stage at rates comparable to those achieved by IVF [In recent years, numerous studies have successfully demonstrated the induction of Cad by IVF ,120.2+ oscillations and a low rate of blastocyst development [2+ waves. Other disadvantages with RNA injections is the chance that the dose of injected RNA is small or not enough for translation into PLCζ needed for Ca2+ influx or release from ER, or it may lead to abnormal Ca2+ release [2+ oscillations, which is important in proper embryogenesis. Furthermore, the average half-life of mRNA molecules is 9 h, making it difficult to exist in cells beyond that time. Further, mRNA lacks the ability to integrate into the host genome, thus generating induced pluripotent stem (iPS) cells [To this degree, production of active and pure recombinant PLCζ protein is another option for rescuing OA in case of ICSI failure, and other similar male infertility conditions . The useelopment . Indeed, release  followin release . SurprisS) cells .Thus, while the utility of recombinant PLCζ represents a potential therapeutic option for OAD patients, perhaps even for a wider range of patients where fertilisation occurs, but embryogenesis is poor , reliabl2+ release, known as the mouse oocyte activation test (MOAT). However, given that human PLCζ is significantly more potent in activity compared with mouse PLCζ in mouse oocytes, even minimal human PLCζ could result in high frequency and amplitude Ca2+ oscillations, and would thus perhaps not represent a method to examine cases of reduced PLCζ, but cases of outright absence [Considering the large body of work examining PLCζ levels and localisation patterns in mammalian, and particularly human, sperm, it is prudent to consider PLCζ as a powerful diagnostic indicator of sperm fertility not just in cases of OAD, but also perhaps a larger range of male factor conditions and cases of poor/abortive embryogenesis ,110. Ind absence .Thus far, sperm PLCζ protein has predominantly been examined using immunocytological analyses ,156,157.Given the considerable amount of data present in the literature pertaining to PLCζ, the importance of this enzyme is apparent for potential therapeutic/diagnostic applications. However, several issues remain regarding its clinical utilisation, related to both technical aspects, but also perhaps pertaining to the incomplete picture regarding the role of PLCζ in OA. Indeed, the independent PLCζ KO studies, while supporting the importance of PLCζ at OA, also suggest that alternative contributory mechanisms may also be present ,64,65,912+, suggesting that actin could act as an intracellular buffer to store and release [A. aranciacus at the optimum period of fertilisation, induced an increase in Ca2+ and depolarization of plasma membrane after activation [A particular example of this is actin, perhaps the most conserved and abundant family of proteins in eukaryotic cells, may possess specific roles in the oocyte cortex development and fertilisation . Indeed, release ,162,163.tivation ,165,166.tivation .Drosophila, actin was found to be smoothly distributed before OA, the onset of which resulted in actin spreading out, with a relaxed actin cytoskeleton required for initiation and propagation of Ca2+ release, which in turn leads to a reorganization of actin in a wavelike manner [2+ release patterns [2+ levels, while detachment and cortical translocation of actin is a prerequisite for normal cellular cleavage, indicating an important role for Ca2+-dependent actin reorganisation [2+ release [In e manner . Drugs ppatterns . Followinisation ,171,172. release ,174,1752+ could be viewed as one where actin acts as a buffer to store and release Ca2+ [2+ oscillations. This is a non-invasive and safe diagnostic method and can also be related to the development potential of forming zygotes. This test can be used after the injection of PLCζ cRNA into the human oocyte. PIV was used in humans for imaging post-microinjecting with PLCζ cRNA in oocytes that failed ICSI. These oocytes were donated by patients and microinjected with PLCζ cRNA with a mixture of substances using a micropipette needle with a brief electrical pulse. The first Ca2+ spike was delayed by 15–20 s with the use of PLCζ cRNA compared to normal sperm injection. This correlates with the translation of PLCζ protein. The cytoplasmic movement follows Ca2+ oscillation pattern, the higher the Ca2+ peak, the slower the movement [2+ oscillations by confirming cytoplasmic movement, which could be used as a diagnostic predictor of OA efficacy and thus embryogenesis [Considering that the relationship between actin and Caase Ca2+ ,162,163,ase Ca2+ . To thismovement ,177. Thimovement . In summ2+ cytosolic concentration when ER stores are depleted. The major components of the SOCE are sarco-ER Ca2+-ATPase (SERCA), Ca2+ release-activated Ca2+ channel protein 1 (ORAI1), stromal interaction molecule-1 (STIM1), and other membrane channels. Targeting these proteins may produce Ca2+ oscillations without PLCζ. STIM1 senses Ca2+ stored in the ER, and with the help of a sterile alpha motif domain, STIM1 polymerizes to the plasma membrane yielding to the protein-protein interaction with ORAI, which will result in extracellular Ca2+ influx. Any mutation in STIM1 leads to a persistent influx of Ca2+ regardless of ER status. CaMKII and mitogen-activated protein kinase (MAPK) are proteins responsible for progression in MII and pronuclei formation, any modulation in their function can affect OA, making them a potential therapeutic option. These systems are believed to have a role in spontaneous oocyte activation (SOA) [Store-operated calcium entry (SOCE) is a system that maintains Caon (SOA) .2+. One proposed mechanism is the elevation of LH which can initiate Ca2+ release. However, oocyte collection without any hormonal stimulation also revealed SOA, excluding LH as a possible cause. Another theory explains SOA due to problems in cell cycle regulators that arrest oocytes at MII, c-mos KO mice showing SOA can support this hypothesis. Some patients showed a repeated incidence of SOA highlighting the possibility of a genetic cause [2+-ATPases or Ca2+-dependent proteins. MAPK early decrease in addition to activation of spindle assembly checkpoint proteins may have an input in SOA [SOA is a phenomenon where the oocyte decides to exit MII, enter anaphase II and form a single pronucleus without any interaction with sperm. This could perhaps be explained by changes in cell cycle regulators, post-ovulatory oocyte aging, and temperature changes during oocyte harvesting. Such a concept is the extreme opposite to infertility resulting from failure of sperm to activate oocytes through PLCζ and Caic cause . Perhapst in SOA .2+ ATPase 1 (PMCA1) protein support Ca2+ efflux at fertilisation and the proper growth, weight, and body composition of the ensuing offspring, is indicated in mice oocytes. PMCA1, along with other proteins, such as SERCA2B, functions in decreasing cytoplasmic Ca2+ levels following each Ca2+ transient. Furthermore, two Ca2+ influx channels, TRPM7 and Cav3.2, increase cytosolic calcium [2+ and Mg2+ to control Ca2+ influx [2+ oscillations [2+ influx, such as TRPM7 and CaV3.2, can maintain Ca2+ oscillations [2+ channel [2+ release in sea urchins [3-dependent Ca2+ release is an essential component of OA for at least mammalian species, others utilise alternative or additional pathways [2+ release via perhaps the ryanodine receptor in sea urchin fertilisation [Drosophila, induce OA before gamete fusion, mediated via extracellular Ca2+ in response to a physical compression of egg plasma membrane TRP channels during ovulation [2+ is still IP3 receptor-mediated [Further to such aspects, plasma membrane Ca calcium . TRPM7 s+ influx  and leadllations . Modulat channel ,180, whi urchins . While Ipathways . For exalisation ,183, whilisation ,185,186.vulation ,188  in the extracellular milieu [2+ and Mg2+ to control Ca2+ influx [2+:Ca2+ in culture media may exert a role in AOA, as decreasing the Ca2+:Mg2+ ratio increased Ca2+ release within the oocyte [Drosophila egg activation, osmotic pressure generated by the uptake of external fluid drives the initiation of Ca2+ release. This mechanism is regulated by conserved osmoregulatory channels, aquaporins, and DEGenerin/Epithelial Na+ channels, utilising transient receptor potential M channels to transport Ca2+ across the plasma membrane into the egg [In other species, Cand worms . Other solvement ,190, prer milieu ,190. As + influx . Indeed,e oocyte . Indeed,e oocyte . Further the egg .2+ related to OA are the intracellular levels of Zn2+, levels of which increase before fertilisation, while after fertilisation, Zn2+ levels decrease, correlating to the release of meiotic arrest [2+ leads to cell cycle promotion in oocytes, whilst also regulating the function of CDC25, which in turn regulates maturation-promoting factor (MPF) [2+ chelators to trigger a resumption of MII in human oocytes. This concept can be used to treat fertility due to the failure of OA with Ca2+ [2+ chelating agent, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), enabled the effective completion of MII and blastocyst development in pigs, but to a lower extent compared to other Ca2+ ionophores [2+ with heavy metals led to the activation of the oocyte and miotic resumption without changes in Ca2+ levels. Indeed, TPEN affects Zn2+ levels without altering intracellular Ca2+. In mice, TPEN resulted in blastocysts with lower inner cell mass and trophectoderm cell quantity. The effect of TPEN use in humans is not well established and does not seem entirely effective [Perhaps the most intriguing non-Cac arrest . The cheor (MPF) ,193, earor (MPF) ,193,194,ith Ca2+ ,193. A Znophores . The absffective .2+ into the extracellular milieu in a series of events called the ‘zinc spark’ [2+ transient. Similar observations have been made in human, bovine, porcine, and primate systems [2+) that this Zn2+ spark is highly conserved  [2+ depletion at fertilisation occurs in Drosophila [2+ spark, indicating Zn2+ accumulation is required during meiotic maturation [2+ spark dynamics indicated that zygotes successfully able to reach the blastocyst stage released more Zn2+ compared to those unable to develop [2+ could represent a diagnostic marker of embryogenic capacity in mouse zygotes [Imaging experiments indicated that mouse OA triggered transient ejection of Znc spark’ ,195,196, systems ,198, sugmammals) ,200. Altosophila  and zebrosophila . Immaturturation ,204. Ana develop , suggest zygotes .2+ release, the indispensable involvement of the sperm factor PLCζ, and the utilisation of both these players in a therapeutic and diagnostic context. However, research has yet again demonstrated that there is yet much more to be elucidated, particularly regarding the role of seemingly disparate, yet utterly interdependent actors, such as Zn2+, modulators of Ca2+ homeostasis, and the mechanisms of actin cytoskeleton dynamics. The discussion surrounding OA has traditionally revolved around the intracellular Ca2+ release and PLCζ . While these aspects are without a doubt integral to the process, it is increasingly clear that just these by themselves do not constitute the ‘end sum game’ OA. Indeed, as our understanding of several intra- and extracellular aspects surrounding OA increases, it becomes clear that OA (including intracellular Ca2+ release and PLCζ) need to be viewed as part of a much larger, interconnected, and vastly more complex overview. Indeed, much promise is present for the therapeutic and diagnostic targeting of such players, although much more work is yet required to fulfil this potential.Given the complexities underlying OA at fertilisation, it is astounding that much work has been accomplished, establishing the mechanisms underlying Ca


We first overviewed the PAK1 activation mechanism. We then discussed recent updated results showing the action mechanisms of PAK1 signalling on Cav1.2/Cav1.3 (ICaL)-mediated Ca2+ entry, ryanodine receptor type 2-mediated sarcoplasmic reticulum (SR) Ca2+ release, transcriptional regulation of SR Ca2+-ATPase 2a, Na+/Ca2+ exchangers, and Ca2+/calmodulin-dependent protein kinase II. Finally, we proposed a new and exciting route for developing a PAK1-based therapeutic strategy for cardiac arrhythmias.Evidence accumulated over the past decade suggests that p21-activated kinase 1 (PAK1) is a critical cardiac-protective signalling molecule. The present article provides an updated review of recent findings regarding the role of PAK1 in maintaining normal cardiac electrophysiological function through its regulation of membrane and CaThis article is part of the theme issue ‘The heartbeat: its molecular basis and physiological mechanisms’. Approximately 1 00 000 people a year die of VTs in the UK, of which about 50% of them are HF patients. Despite a large number of basic and clinical investigations, current pharmacotherapy is still inadequate, necessitating novel therapeutic approaches . This dysregulated Ca2+ release could stimulate the propagating waves of spontaneous sarcoplasmic Ca2+ release, therefore, decoupling M- and C-clock activity. Moreover, this initial C-clock dysregulation may temporarily elevate electrogenic Na+/Ca2+ exchange current as the cytosolic [Ca2+] rises. This altered Na+/Ca2+ exchange current may then drive delayed after-depolarizations and disrupt ectopic excitation from M-clock –172-term529) (a) . Specifi529) (a) .Figure b). Structurally, inactive PAK1 was shown to be a homodimer with an antiparallel conformation, where the C-lobe of the kinase domain of a monomer interacts with the N-terminal inhibitory switch region of the other, thereby stabilizing in the inactive state i and dysregulated SR Ca2+, especially under β-adrenergic stress conditions, were observed in cardiomyocyte-specific PAK1 knock-out (ckoPAK1). These results demonstrate that the regulation of PAK1 activity in cardiomyocytes plays a critical role in maintaining Ca2+ homeostasis.Recent studies have revealed the regulation of Capathways –35. Over (a)2+/calmodulin-dependent protein kinase II (CaMKII) and the phosphatases (PP1 and PP2A). In addition, RyR2 hyperphosphorylation by PKA or by CaMKII are the major causes of Ca2+-dependent arrhythmia such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and VTs associated with HF i transient amplitude, a reduced rate of [Ca2+]i decay and an increased SR Ca2+ content were observed after an ISO treatment i, leading to spontaneous Ca2+ released. However, the Ca2+ overload in PAKcko VMs could be attenuated by the administration of a reverse mode blocker (KB-R7943) of the Na+/Ca2+ exchanger (NCX) or a Rac1 inhibitor (NSC23766). Suppressed PAK1 activity in PAK1cko VMs or VMs treated with the PAK1 inhibitor (IPA3) were shown to promote greater cellular reactive oxygen species (ROS) production than those of the control group. Voltage clamp recordings also demonstrated higher NCX activity in PAKcko VMs which indicated greater ROS production through NOX channel activity. Overall, these studies suggest that PAK1 is an important negative regulator of NOX2 and thereby attenuates ROS production i.Abnormal automaticity is responsible for both the initiation and maintenance of a major type of VTs, referred as ‘triggered ventricular tachyarrhythmia', that results from aberrant Cac stress ,36,55,56[2+ leak . RyR2 ca[2+ leak ,58. Many[2+ leak ,60. Ther[2+ leak ,61–63. Mdent VTs . In addident VTs . This gi2+ release should attenuate Ca2+-dependent VTs [2+ release via cardiac RyR2 provides an additional avenue for anti-arrhythmic therapy. This may be a promising target to potentially increase survival in patients with acquired heart diseases.Factors that prevent abnormal diastolic SR Cadent VTs . Current (b)CaL) mediated Ca2+ entry, RyR2-mediated SR Ca2+ release, transcriptional regulation of SERCA2a, NCX (class IVd) and CaMKII. Class VII actions: it has longer-term modifications of arrhythmic substrate including electrophysiological and/or structural remodelling for primary or secondary prevention, reducing susceptibility to or progression of established atrial and ventricular arrhythmias, particularly in the presence of cardiac failure and hypertension.Given the critical role of PAK1 in regulating in both M-clock and C-clock in cardiac myocytes, we anticipate that PAK1 can be a feasible target for developing a novel class of anti-arrhythmic drugs. 2+ transient decay constant (τCa) [CaL and phospholamban phosphorylation both probably through PP2A activation. We also showed that a bioactive peptide derived from the PAK1 auto-inhibitory region increases PAK1 activity and counteracts Ang II-induced pathological hypertrophy and ventricular arrhythmias [Previous studies have provided strong evidence for supporting such hypothesis. For example, overexpression of constitutively active PAK1 altered Cant (τCa) , and prohythmias .2+ overload by attenuating NCX activity in a NOX2-dependent manner in atrial myocytes from a canine model of ventricular tachypacing-induced atrial fibrillation [Moreover, previous research has also indicated that FTY720 (Fingolimod), an immuno-modulatory agent, activates PAK1 and prevents I/R injury-induced arrhythmias via activated PAK1/Akt signalling pathways in studies of rat heart subjected to I/R. In addition, FTY720 can induce nitric oxide (NO) production via the PI3K/Akt/eNOS signalling pathway, which is also reported to be important for cardiac protection . FTY720 illation .2+ responsiveness and improves diastolic dysfunction and partially reverses atrial remodelling in a mouse model treated with a hypertrophic cardiomyopathy-linked mutation in tropomyosin [et al. have demonstrated that the modulation of S1PR through FTY720 led to reduced myofilament-Ca2+-responsiveness and improved diastolic function in hypertrophic cardiomyopathy might be owing to decreased oxidative modification of myofilament proteins via downregulation of NOX2 [Recent studies have also demonstrated that FTY720 reduces myofilament Capomyosin . Ryba et of NOX2 . Further of NOX2 . These c. 7There are a large amount of studies for the PAK1 enzyme function in regulating neurodevelopment, neuroplasticity as well as the metastasis and cell motility of cancer cells –71. For 2+ homoeostasis to protect against cardiac arrhythmias. These findings promote future development of PAK1 activators as a potential novel therapeutic strategy for both prophylactic and therapeutic measures involving cardiac arrhythmia. Since PAK1 is involved in the pathogenic and oncogenic effects, as well as PAK inhibitors, which may be developed and deployed in cancer therapy, anti-viral infection, including COVID-19, and other diseases, critical questions are raised to discuss how to balance the cardiac-protective role of PAK1 activation and the anti-tumour effect of PAK1 inhibition in drug discovery research [in vitro and in vivo cancer models, suggesting a potential therapeutic role in cancer patients [Increasing remarkable studies have demonstrated the critical role of PAK1 in the heart from its regulation in the membrane clock and Caresearch –76. Intepatients . Therefo