Growing evidence indicates that RNA methylation plays a fundamental role in epigenetic regulation, which is associated with the tumorigenesis and drug resistance. Among them, acute myeloid leukemia (AML), as the top acute leukemia for adults, is a deadly disease threatening human health. Although N7-methylguanosine (m7G) has been identified as an important regulatory modification, its distribution has still remained elusive.The present study aimed to explore the long non-coding RNA (lncRNA) functional profile of m7G in AML and drug-resistant AML cells. The transcriptome-wide m7G methylation of lncRNA was analyzed in AML and drug-resistant AML cells. RNA MeRIP-seq was performed to identify m7G peaks on lncRNA and differences in m7G distribution between AML and drug-resistant AML cells. The Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to predict the possible roles and m7G-associated pathway.Using m7G peak sequencing, it was found that a sequence motif was necessary for m7G methylation in drug-resistant AML lncRNA. Unsupervised hierarchical cluster analysis confirmed that lncRNA m7G methylation occurred more frequently in drug-resistant AML cells than in AML cells. RNA sequencing demonstrated that more genes were upregulated by methylation in drug-resistant AML cells, while methylation downregulated more genes in AML cells. The GO and KEGG pathway enrichment analyses revealed that genes having a significant correlation with m7G sites in lncRNA were involved in drug-resistant AML signaling pathways.Significant differences in the levels and patterns of m7G methylation between drug-resistant AML cells and AML cells were revealed. Furthermore, the cellular functions potentially influenced by m7G in drug-resistant AML cells were predicted, providing evidence implicating m7G-mediated lncRNA epigenetic regulation in the progression of drug resistance in AML. These findings highlight the involvement of m7G in the development of drug resistance in AML. Acute myeloid leukemia (AML) is a type of blood cancer that originates in the bone marrow and is characterized by the uncontrolled growth of myeloid cells. Drug resistance in AML remains a significant challenge to effective treatment, with several patients relapsing after initial therapy. Understanding the mechanisms underlying drug resistance is therefore an essential step towards developing novel therapies . EmerginDysregulated m7G methylation of specific lncRNAs has been associated with altered gene expression patterns, dysfunctions in cellular signaling pathways, and the development of drug-resistant phenotypes in AML cells. Aberrant m7G methylation of lncRNAs has been found to be associated with the modulation of drug efflux pumps, drug metabolism pathways, DNA repair mechanisms, and anti-apoptotic processes, contributing to the resistance of AML cells to therapeutic interventions . TargetiAs a post-transcriptional modification, RNA methylation plays a fundamental role in epigenetic regulation. To date, over 100 RNA methylations have been reported . ExampleNotably, m7G is a widely recognized RNA modification on mRNA caps and inside rRNAs, tRNAs and mRNAs. DespiteLncRNAs are RNA molecules with over 200 nucleotides but no or limited protein-coding potential. Indeed, LncRNAs serve as key gene expression regulators in multiple pathological and physiological processes, including tumorigenesis, cell differentiation, and inflammation . NowadayAccording to the American Cancer Society, around 20,000 cases of AML were diagnosed in 2020in the United States. Despite some breakthroughs in AML diagnosis and treatment over the recent decades, relapsed and refractory nature of AML remains significant owing to its heterogeneity and malignancy . NeverthIn the present study, the influence of m7G methylation on lncRNAs in AML-resistant cells was investigated. Specifically, m7G was globally mapped via RNA MeRIP-seq in AML-resistant and non-resistant cells, while distribution and prevalence in the two cell types and cellular compartments were determined and compared with each other. The results demonstrated that the m7G modification degree was significantly higher in AML-resistant cells than that in non-resistant cells. This could be reflected by intra-cell consistency, as well as inter-cells differences, involving all chromosomes. This study could facilitate the understanding of epigenetic regulation of lncRNAs in AML by m7G, as well as new therapies for AML.2 at 37 °C.HL60/MX2 and HL60 cells were acquired from the American Type Culture Collection and cultured in RPMI-1640 containing 10% thermally deactivated fetal calf serum, 1% (v/v) penicillin streptomycin in a humidified atmosphere containing 5% COIn the present study, m7G peaks were acquired from each sample by using the MeRIP-seq technique. RIP was executed by a commercial service provider. High-quality reads were aligned to the UCSC HG19 by utilizing hisat2 (ver. 2.0.4). Then, fragments per kilobase of exon per million fragments mapped values from transcript-level LncRNAs were obtained by utilizing cuffdiff (ver. 2.2.1) under guidance by the gtf files. Additionally, the P-value and fold-change of the two groups were determined to screen for lncRNAs that were expressed differentially.Sequencing and visualization of the libraries developed were achieved by using a sequencer (Illumina NovaSeq 6000). The mapping quality is reflected by ‘Q30’: a read above 30 was considered to be reliable. Meanwhile, library mapping was achieved by STAR (ver. 2.5.1b). Sequencing datasets were aligned to UCSC HG19 by utilizing Hisat2 (ver. 2.0.4). Methylation peaks of the two cells were identified by using MACS, while their differences were determined by using DiffReps.The GO and KEGG pathway enrichment analyses were carried out using the DAVID online tool to clarify the properties of the above-mentioned genes. The GO analysis aimed to analyze MFs (molecular functions), CCs (cellular components), and BPs  of these genes based on differentially methylated genes. P < 0.05 was set to be the critical level of significant GO terms. Additionally, the KEGG pathway analysis –25 was cm7G methylation over transcriptome in the two cells was investigated by RNA MeRIP-seq of lncRNA. As observed, a total of 7,049 and 6,170 m7G peaks were observed in HL-60 cells and HL60/MX2 cells, respectively  was determined to be the most conservative motif in HL-60 cells and its E-value was 1.1e-009 , 5’UTR region (10.2% in HL-60 cells vs. 13.3% in HL60/MX2 cells), stopC (11.5% in HL-60 cells vs. 11.1% in HL60/MX2 cells) and startC (8.2% in HL-60 cells vs. 11.9% in HL60/MX2 cells) were also determined  in gastric cancer, Hodgkin, non-Hodgkin’s lymphoma, and breast cancer –34. LocaAs a potential critical layer of biological regulation, lncRNAs are aberrantly expressed in AML by transcription factors, DNA methylation or histone modifications, as well as by post-transcriptional modifications. Dysregulated lncRNAs may alter activity and expression of mRNAs, proteins and miRNAs, leading to epigenetic alterations in AML. For instance, AC026150.8, which is upregulated in AML, is correlated with poor prognosis, and its overexpression is positively related to drug resistance of AML cells, and confirms its scaffolding effect combined with splicing factors . WT1 wasThe present study utilized RNA MeRIP-seq to investigate m7G methylation over the transcriptome in human AML-resistant cells. The analysis revealed a total of 7,049 m7G peaks in HL-60 cells and 6,170 m7G peaks in HL60/MX2 cells. These peaks were associated with annotated genes, with 6,491 genes mapped in HL-60 cells and 5,757 genes mapped in HL60/MX2 cells. The study identified specific m7G motifs in the two cell types. In HL-60 cells, the most conservative motif was determined to be URUUUY , while in HL60/MX2 cells, the most conservative motif was YMCAG . The presence of these motifs indicated significant differences in the sequences of m7G methylation between the two cell groups. The density of m7G peaks in lncRNAs was also analyzed based on the cell type. The results showed that approximately 50% of lncRNAs in HL-60 cells and 45.9% of lncRNAs in HL60/MX2 cells contained m7G peaks in the CDS region. The percentages varied in other regions, such as 3’UTR, 5’UTR, stop codon (stopC), and start codon (startC), suggesting differential distribution patterns of m7G methylation in lncRNAs between the two cell types. Furthermore, the study analyzed the sources of methylated lncRNAs and found that most of the methylated lncRNAs were derived from exon sense overlapping regions, followed by intergenic regions. The distributions of lncRNA methylation sites across chromosomes were also examined, revealing differences between the two groups. The X chromosome and chromosome 2 exhibited notable differences in the distribution of lncRNA methylation sites. The differential expression of lncRNAs based on m7G methylation was assessed, and it was observed that 301 lncRNAs were upregulated and 355 lncRNAs were downregulated in HL60/MX2 cells compared to HL-60 cells. The study also provided a list of the top 10 upregulated and downregulated lncRNAs. Functional and pathway enrichment analyses were performed to investigate the biological classifications of m7G genes in lncRNAs of HL60/MX2 cells. GO analysis revealed enriched BPs and MFs that were associated with the up-methylated and down-methylated m7G genes. The up-methylated genes were enriched in cellular metabolic processes, protein binding, and ion binding, while the down-methylated genes were related to nitrogen compound metabolic processes and metal ion binding, among others. The KEGG pathway analysis demonstrated that the lncRNAs with up-methylated m7G in HL60/MX2 cells were enriched in pathways, such as the fanconi anemia pathway, other types of O-glycan biosynthesis, and non-homologous end-joining. On the other hand, the lncRNAs with down-methylated m7G were enriched in pathways including Fc gamma R-mediated phagocytosis, B cell receptor signaling pathway, and osteoclast differentiation.Uptake of amino acid, catabolism and homeostate levels are elevated in LSCs (leukemia stem cells). LSCs that are obtained from naive AML patients are dependent on the metabolism of amino acids for OP (oxidative phosphorylation) and survival . LSCs isAdditionally, it was noted that certain genes, which exhibited low expression levels in AML cells, demonstrated high expression in AML-resistant cells accompanied by the elevated levels of methylation. However, the precise role of this methylation in AML resistance necessitates further investigation. The present study provided a new perspective for the future study on the role of m7G methylation in drug resistance of AML cells to discover new therapeutic targets.In conclusion, the present study explored the regulatory mechanisms of m7G methylation in AML resistance. The findings shed light on the identification of differentially methylated lncRNAs, specific m7G motifs, and their distribution patterns in AML-resistant cells compared with AML cells. The results suggest a potential role for m7G methylation in modulating gene expression in AML-resistant cells. However, it is important to note that further investigation is warranted to fully understand the epigenetic alterations associated with AML resistance. Future research in this area may provide valuable insights into the underlying mechanisms and potential therapeutic targets for overcoming AML resistance.


During the last decade, the underlying pathogenic mechanisms of acute myeloid leukemia (AML) have been the subject of extensive study which has considerably increased our understanding of the disease. However, both resistance to chemotherapy and disease relapse remain the principal obstacles to successful treatment. Because of acute and chronic undesirable effects frequently associated with conventional cytotoxic chemotherapy, consolidation chemotherapy is not feasible, especially for elderly patients, which has attracted a growing body of research to attempt to tackle this problem. Immunotherapies for acute myeloid leukemia, including immune checkpoint inhibitors, monoclonal antibodies, dendritic cell (DC) vaccines, together with T-cell therapy based on engineered antigen receptor have been developed recently. Our review presents the recent progress in immunotherapy for the treatment of AML and discusses effective therapies that have the most potential and major challenges. Acute myeloid leukemia (AML) is a form of leukemia that mostly affects the adult population with most cases occurring in this age group and is diagnosed less frequently in children. First-line chemotherapy has been the mainstay of treatment during the last four decades . DespiteImmune surveillance is pivotal in suppressing tumor growth and maturation. The immune system can identify the antigens specific to the tumor cells. Nevertheless, the tumor microenvironment fosters immunosuppressive activities and antigen loss that would eventually lead to immune evasion. On the one hand, T-cell surface checkpoint regulators such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death protein 1 (PD-1) have been demonstrated to contribute, at least in part, to these immunosuppressive activities . EnhanceImmune checkpoint inhibitors are capable of activating tumor-specific immune cells that are suppressed in the tumor microenvironment (TME). For hematological malignancies such as AML with a maturation and progression pattern different from solid tumors, the mechanisms underlying the activity or inactivity of the immune system may strikingly differ from previous descriptions for solid tumors. Despite the presence of mutual immune evasion pathways, hematological tumors have their unique tolerance mechanisms . MoreoveIn the present review, recent advances with regard to immunotherapies for AML will be discussed, various challenges in the field will be looked at, and emerging strategies that may optimize treatment efficacy will be presented.+ T-cell activation to initiate a strong antitumor response , pembrolizumab (Keytruda®), atezolizumab (Tecentrip®), avelumab (Bavencio®), durvalumab (Imfinzi®), and cemiplimab (Libtayo®). Immune checkpoint inhibitors (ICIs) confer advantages for solid tumors that are yet to be attained for AML   . Anothery or IHC . The comy or IHC . In addiy or IHC . Moreovey or IHC  and avely or IHC  with ency or IHC . Given ty or IHC , as welly or IHC . Neverthy or IHC .In addition to CTLA-4 and PD-1/PD-L1, numbers of clinical trials of TIM-3 inhibitors including sabatolimab and TQB2618 for AML are recruiting patients . Similarly, TIM-3 inhibitors combined with demethylation drugs (azacitidine or decitabine) is used as the treatment strategy for AML, and one of the clinical trial adopts the combination of spartalizumab (PD-1 inhibitor) and sabatolimab (NCT03066648), which indicates that TIM3 inhibitors will occupy the market in the field of AML in the future.Recent studies have identified numerous candidate AML-associated antigens that can be targeted, including CD33 and CD123. By selectively targeting AML specific antigens, monoclonal antibodies have emerged as effective therapeutic agents that can reduce morbidity and mortality. During treatment of AML, monoclonal antibodies can exert their therapeutic effects through selective drug delivery, cell-mediated and complement-mediated mechanisms, and innate immune enhancement. Besides, antibody-drug conjugation combining chemotherapeutic agents or radioactive particles with monoclonal antibodies is also being evaluated to specifically target cells expressing CD33 or CD123. Despite that the immune system is not activated, they can induce cell injuries which may initiate the innate immune system and activate immune defense .FLT3-ITD or NPM1 mutations are more prevalent in groups with higher CD33 expression level  targeting CD33 in 2000 to treat AML  and was ADC linked with pyrrolobenzodiazepine (PBD) dimer is a new strategy for treatment of various cancers . SGN-CD3Lintuzumab is a humanized anti-CD33 antibody with a modest single-agent activity against AML. To improve the efficacy, lintuzumab was conjugated to radioactive elements such actinium-225 and bismuth-213. The radio immune conjugates are undergoing clinical investigation in older patients newly diagnosed with AML for whom standard induction treatment is deemed unfit. Actinium-225-lintuzumab was feasible with myelosuppression and no cases of VOD . SequentCD123 is a cytokine receptor widely overexpressed in multiple hematologic cancer cells, especially in leukemic stem cells (LSCs). AML cells with FLT3-ITD or NPM1 mutations are correlated with significantly increased levels of CD123 as compared to AML cells with wild-type FLT3 or NPM1 . OverexpCSL360 is a chimeric monoclonal antibody directed against CD123 and is believed to inhibit proliferation of AML cells . Howeverin vitro and in mouse models , and flotetuzumab , a dual-affinity retargeting (DART) molecule.Bispecific T-cell-engaging (BiTE) antibodies are composed of dual variable regions that simultaneously bind to CD3 on cytotoxic T lymphocytes and tumor cell antigens on malignant cells, activating the effector function, accompanied by release of cytokines and destruction of tumor cells. By means of this strategy, virtually all T lymphocytes can be recruited and directed against leukemic blasts irrespective of its specificity. The preclinical trial proved that CD3/CD33-directed antibody constructs might be utilized as an alternative treatment in AML . AMG 330e models . A clinitory AML . AdditioCancer vaccines represent a groundbreaking therapeutic approach that employs immune effector cells to eradicate malignant cells. Upon activation of immune signaling, presentation of tumor associated antigens is increased, breaking the tolerance to tumor, and at the same time, immune regulation against autoimmunity is maintained. Dendritic cell (DC)-based vaccines offer the potential of a non-toxic and effective immunotherapeutic strategy. The four most common tumor types can be targeted include melanoma, prostate cancer, glioblastoma and renal cell carcinoma . The priAML cells have been shown to express the Wilms’ tumor 1 (WT1) protein and upregulation of this protein is a predictor of poor prognosis in AML and MDS patients . The speTelomerase is a ribonucleoprotein enzyme that positively expressed in most malignant tumor cells and is critical for increasing proliferative potential of cancer cells . TelomerAn individualized cancer vaccine strategy was developed by Jacalyn Rosenblatt et al. in which fusion hybrids of autologous dendritic cells and patient-derived AML cells are used to generate antileukemic responses . In thisin vitro  can be cultured in vitro . The sucin vitro . A phasein vitro . On the in vitro  or immunin vitro . Future CARs, unlike TCRs which interact with HLA molecules, do not need antigen processing or presentation by the HLA peptidome , and theCD19-specific CAR-T cells represent a well-established CAR-T cell therapy with restrictive expression pattern and favorable safety outcome. The promise of anti-CD19 CAR-T cells was immense in ALL, achieving CR in over 90% of B-ALL patients . ConsideIn a phase I study, four patients infused with anti-LeY CAR-T cells were examined to determine the safety and clinical efficacy of adoptive transfer of T cells. The CAR T cells sustained efficacy for up to 10 months post infusion, confirming the feasibility and safety of anti-LeY CAR-T cells in patients with high-risk AML, and demonstrating long-term efficacy . CLL-1 iNKG2D ligands are selectively expressed on monoblastic cells in AML, but absent or weakly expressed on myeloblastic cells and chemotherapy-resistant leukemic stem cells in AML, which avoid NK-mediated killing and cause immune evasion . TherefoThe safety and lymphodepleting conditioning chemotherapy in participants with AML/MDS or relapsed/refractory multiple myeloma were assessed in a phase I trial (NCT02203825) with dose escalation design . A NKG2DCD123 is a poor prognostic antigen marker found on AML cells and chemo-resistant leukemic stem cells. Anti-CD123 CAR-T therapy was able to eradicate AML blasts in experiments . Gene-edAn early phase I pilot study sponsored by the University of Pennsylvania (NCT02623582) sought to evaluate autologous T lymphocytes containing anti-CD123 linked to TCR/4-1BB domains in AML patients . Five adin vivo NSG mice model experimenting with AML xenotransplantation has reported significant reduction of leukemic burden and prolonged survival  (NCT03126864). The results found that CAR-T cell production was only possible in cohorts with increased lymphocyte levels and decreased blood cells, but along with systemic inflammatory syndrome and neurotoxic effects -1 has achieved remarkable clinical benefits in the treatment of solid tumors . TCR-T cin vivo TCR-T cells should be the urgent problems of TCR-T immunotherapy.In several clinical trials, WT1-specific TCR-T cells has shown strong clinical responses with tolerated safety for AML patients . Moreover, numerous clinical trials are underway to evaluate other antigen-specific TCR-T cells in AML. Recently, Kang et al. reviewed the update and challenges of TCR-T for AML. TCR-T exhibits a promising tool for AML patients according to the benefit efficacy of clinical trials. However, the toxicity produced in the treatment process and the persistence of In this review, we summarized the recent advances for AML immunotherapies and noted the representative emerging strategies. The immunotherapy as a treatment option for AML patients with relapse has shown great promise. However, several limitations to the AML immunotherapy are limited its application in AML. Immune evasion remains a clinical challenge in effectively implementing immunotherapeutic strategies. AML is a complex disorder associated with distinctive genetic features and accumulation of genetic aberrations. The lower level of mutation load in AML would lead to higher chance of immune escape of the immune system and less neoantigen epitopes when compared to melanoma and other types of solid cancers such as lung cancer. Considering reduced mutation load, a core issue around AML requiring further exploration is the extent of immunogenicity. Therefore, clinical success of immune therapy in AML will depend on at least the following efforts: exploiting immune escape mechanism and identifying specific targets.via genetically modified AML models reconstructing the disease. Moreover, integrated data on antigens coupled with analytical platforms to screen out truly AML-specific antigen markers are required. Up till now, transcriptome analyses are mainly used for screening candidate AML targets assuming a direct link between mRNA abundance and protein expression. This complex correlation cannot represent the complete set of proteins expressed by the cell, due to numerous factors such as variations in drug metabolism and mechanisms of post-transcriptional regulation. Therefore, integrative analysis of RNA transcripts and proteins should be performed to complement mRNA or protein expression analysis. In addition, coupling advanced proteomics analytical approaches with plasma membrane proteome enrichment will facilitate direct assessment of surface proteins. In summary, because of the heterogeneous nature of AML, each patient may present distinct cytogenetic aberrations and chromosomal rearrangements that require biomarker-specific individualized treatment. Therefore, tailored treatments combining different forms of immunotherapy with chemotherapy and autologous or allogeneic SCT hold immense promise.AML has a distinct pattern of development and progression, and there is a paucity of data on the mechanisms of immune activation or tolerance. The mechanisms underlying antitumor immune responses are well understood and the strategies of immune evasion employed by solid tumors have been studied extensively. Accumulating evidence suggests that certain immune evasion mechanisms are exclusive to AML despite the fact that PD1/L1, among other immune escape pathways, are shared with solid malignancies. Although currently encountering various obstacles, the progress in exploring the mechanisms by which immune surveillance and evasion take place in AML can be attained


To profile immunological changes in healthy and acute myeloid leukemic (AML) patients’ ex vivo cell cultures, we correlated the cell biological data with the profiles of cell culture supernatant-derived VOCs. DC/DCleu from leukemic or healthy whole blood (WB) were generated without (Control) or with immunomodulatory Kit M (Granulocyte macrophage-colony-stimulating-factor (GM-CSF) + prostaglandin E1 (PGE1)) in dendritic cell cultures (DC culture). Kit-pretreated/not pretreated WB was used to stimulate T cell-enriched immunoreactive cells in mixed lymphocyte cultures (MLC culture). Leukemia-specific adaptive and innate immune cells were detected with a degranulation assay (Deg) and an intracellular cytokine assay (InCyt). Anti-leukemic cytotoxicity was explored with a cytotoxicity fluorolysis assay (CTX). VOCs collected from serum or DC- and MLC culture supernatants (with vs. without Kit M pretreatment and before vs. after culture) were measured using eNose. Compared to the Control (without treatment), Kit M-pretreated leukemic and healthy WB gave rise to higher frequencies of mature (leukemia-derived) DC subtypes of activated and (memory) T cells after MLC. Moreover, antigen (leukemia)-specific cells of several lines (innate and adaptive immunity cells) were induced, giving rise to blast-lysing cells. The eNose could significantly distinguish between healthy and leukemic patients’ serum, DC and MLC culture supernatant-derived volatile phases and could significantly separate several supernatant -derived VOCs within subgroups . Interestingly, the eNose could indicate a Kit M- and culture-associated effect. The eNose may be a prospective option for the deduction of a VOC-based profiling strategy using serum or cell culture supernatants and could be a useful diagnostic tool to recognize or qualify AML disease.Volatile organic compounds (VOCs) reflect the metabolism in healthy and pathological conditions, and can be collected easily in a noninvasive manner. They are directly measured using electronical nose (eNose), and may qualify as a systemic tool to monitor biomarkers related to disease. Myeloid leukemic blasts can be transformed into leukemia-derived dendritic cells (DC AML is a clonal stem cell disorder of the hematopoiesis, which comes with uncontrolled proliferation of myeloid progenitor cells (blasts) . Cytocheleu, leukemia-derived DC; without induction of blast proliferation) from AML patients’ WB in the presence of different combinations of response modifiers (Kits) . Its 32 sensors have a variation of coefficient of 2–10%. The addition of all 32 signals and the determination of the precision of the total signal finally resulted in a mean coefficient of variation of 4.64%. Digitally sensed VOCs using an electronic nose have the potential to become a rapid, immediate, and non-invasive diagnostic tool. With the advent of inexpensive, environmentally friendly, and biocompatible sensor systems, health monitoring using VOCs may transform laborious or invasive procedures that currently can only be used in facilities specialized for this purpose into a technology that can be used anywhere and anytime by individuals.An electronic nose such as the one used here utilizes a standardized approach. An important requirement is that its sensors work correctly, and the 32 sensors of the eNose indicate correct reference ranges. Moreover, sensors must be trained with a training set before the start of measurements and must be regularly calibrated. In contrast to other methods, such as ion mobility spectrometry or mass spectrometry, identification of the individual component in a mixture is usually not possible here or is only possible under very restricted conditions . Consequmat secrete different exosomes compared to immature DCs . Exosomes from mature DCs (compared to immature DCs) can be enriched with MHC class II, B7.2, and ICAM-1, and depleted in MFG-E8 [Analyses of different cellular/humoral, soluble factors ,22,23,24n MFG-E8 .p = −0.0158) (A culture effect could be detected by the eNose in leukemic MLC culture supernatant-derived VOCs. VOCs collected before culture were significantly different compared to after culture (leukemic MLC: Δ−0.0158) . These VWith respect to culture effects, we found significant differences in the VOC profiles of healthy DC culture supernatants (independent of the addition of Kit M), whereas the culture effects of AML samples in the same settings were not different. These findings might be explained by the different compositions of DC culture supernatants in healthy vs. AML DC culture supernatants; healthy samples contain higher frequencies of ‘healthy cells’, and AML samples contain high frequencies of blasts. After culture, different releases of VOCs in the different settings might explain the good differentiation of healthy DC supernatant VOCs, but not AML DC culture supernatant VOCs.With respect to Kit M-mediated effects, we found significant differences in the VOC profiles of healthy DC culture supernatants when comparing Kit M-pretreated vs. non-pretreated samples, whereas the culture effects of AML samples in the same settings were not different. These findings might be explained by different DC compositions in healthy vs. AML DC culture supernatants; healthy samples yield higher frequencies of mature monocyte-derived DCs, and AML samples yield (in addition) leukemia-derived DCs and blasts, which may proliferate/differentiate and produce leukemia-associated VOCs.Moreover, AML patients’ DC supernatants might contain traces of drug (derivates) after chemotherapy and antibiotic/antimycotic therapy, which could be responsible for alternated VOC profiles compared to healthy DC culture supernatant-derived VOCs.With respect to culture effects, we found no significant differences in the VOC profiles of healthy MLC supernatants (independent of Kit M addition), whereas the culture effects of AML samples in the same settings were significantly different. These findings might be explained by different MLC-related supernatants in healthy vs. AML MLC supernatants .With respect to Kit M-mediated effects, we did not find significant differences in the VOC profiles of healthy MLC supernatants when comparing Kit M-pretreated vs. non-pretreated samples, whereas the culture effects of AML samples in the same settings were significantly different. These findings might be explained by the different compositions of immune cells in AML vs. healthy cells; the activation of leukemia-specific immune reactive cells in AML cases might yield significantly different VOCs under the influence of Kit M vs. Control.We could deduce the following culture and Kit M-related effects detectable by VOC profiles in healthy and AML cultures:This means that culture alone gives rise to other changes in healthy and AML VOC profiles; the presence of Kit M changes the setting and produces different results in AML and healthy VOC samples. We have shown that the role and metabolic influences of drugs of different cellular compositions with respect to qualitative/quantitative differences in VOC release profiles are complex, although our chosen technology yielded differences in different settings. Other strategies or more standardized settings might contribute to more refined VOC-based monitoring strategies in the future. According to our results, eNose analyses might then yield a prospective option for deriving a VOC-based disease profiling strategy using serum or cell culture supernatants from patients with leukemia. Due to rapid sample collection and analysis, the present study shows good reproducibility of data. It could therefore be recommended to include VOC analyses as an additional component to monitor the course of disease, and potentially to guide therapy-related decisions.


TP53 gene encodes the p53 protein, which plays a diverse role in responding to various cellular stresses. p53 consists of several functional domains, including a tetramerization domain for forming heterotetramers and a DNA-binding domain for bindings to p53-responsive elements. The most common mutations in acute myeloid leukemia (AML) occur in the DNA-binding domain of p53. TP53 mutations are associated with a very poor prognosis and define a unique disease subgroup within AML. TP53-mutated AML often shows limited response to conventional chemotherapy and even allogeneic hematopoietic stem cell transplantation. There is an urgent need for novel treatment approaches for patients with TP53-mutated AML, given the frustrating treatment outcomes associated with this condition. Incorporating targeted agents and immunologic therapies into future treatment regimens may offer promising options for patients with TP53-mutated AML.The TP53 is the most commonly mutated gene in human cancers and was the first tumor suppressor gene to be discovered in the history of medical science. Mutations in the TP53 gene occur at various genetic locations and exhibit significant heterogeneity among patients. Mutations occurring primarily within the DNA-binding domain of TP53 result in the loss of the p53 protein’s DNA-binding capability. However, a complex phenotypic landscape often combines gain-of-function, dominant negative, or altered specificity features. This complexity poses a significant challenge in developing an effective treatment strategy, which eradicates TP53-mutated cancer clones. This review summarizes the current understanding of TP53 mutations in AML and their implications. TP53 mutation in AML: In patients with acute myeloid leukemia (AML), six hotspot mutations  within the DNA-binding domain are common. TP53 mutations are frequently associated with a complex karyotype and subgroups of therapy-related or secondary AML. The presence of TP53 mutation is considered as a poor prognostic factor. TP53-mutated AML is even classified as a distinct subgroup of AML by itself, as TP53-mutated AML exhibits a significantly distinct landscape in terms of co-mutation and gene expression profiles compared with wildtype (WT)-TP53 AML. Clinical Implications: To better predict the prognosis in cancer patients with different TP53 mutations, several predictive scoring systems have been proposed based on screening experiments, to assess the aggressiveness of TP53-mutated cancer cells. Among those scoring systems, a relative fitness score (RFS) could be applied to AML patients with TP53 mutations in terms of overall survival (OS) and event-free survival (EFS). The current standard treatment, which includes cytotoxic chemotherapy and allogeneic hematopoietic stem cell transplantation, is largely ineffective for patients with TP53-mutated AML. Consequently, most patients with TP53-mutated AML succumb to leukemia within several months, despite active anticancer treatment. Decitabine, a hypomethylating agent, is known to be relatively effective in patients with AML. Numerous trials are ongoing to investigate the effects of novel drugs combined with hypomethylating agents, TP53-targeting agents or immunologic agents. Conclusions: Developing an effective treatment strategy for TP53-mutated AML through innovative and multidisciplinary research is an urgent task. Directly targeting mutated TP53 holds promise as an approach to combating TP53-mutated AML, and recent developments in immunologic agents for AML offer hope in this field.Introduction:  TP53 gene located on chromosome 17p13.1 encodes p53 protein, a tumor suppressor protein which is 393 amino acids long and functions as a tetrameric transcription factor capable of regulating the expression of a huge set of target genes involved in the responses to cellular stress, including cell cycle arrest, DNA repair, apoptosis, and metabolism. TP53 had once been thought to be an oncogene because mutant cDNA clones were capable of inducing cell transformation. Wild-type (WT) TP53 was eventually classified as a tumor suppressor gene based on an experimental demonstration of its capacity for inhibiting the oncogenic transformation of cells in vitro [TP53 can be activated by various cellular stresses, including DNA damage, hypoxia, replicative stress, and oncogene expression. It primarily functions as a transcription factor, mediating various transcriptional regulations, depending on the type of cellular stress and cell type. For instance, minor DNA damage can result in cell cycle arrest and activate DNA repair mechanisms, whereas significant DNA damage may lead to senescence or apoptosis [TP53 gene or indirectly as a result of alterations in genes whose products interact with p53 in about half of cancers. TP53 is the most commonly mutated gene in human cancer cells; the amino-acid-changing mutation in the DNA-binding domain (DBD) of the TP53 gene is frequently observed in various types of cancer, including colon, breast, lung, bladder, brain, pancreas, and stomach, as well as leukemia [The human poptosis . p53 is leukemia .The p53 protein is composed of an N-terminal transactivation motif, a DNA-binding domain, and a tetramerization domain . The traTP53 gene. Each p53 isoform is regulated by small interfering RNA in an isoform-specific manner, providing the wide range of regulatory mechanisms of p53 [p21/WAF and Bcl-XL. Aside from its role in cellular stress response, p53 plays a vital role in multiple metabolic regulations. p53 is involved in the synthesis and storage of fatty acids through the targeting of SREBP1 and ABCAs, enhances gluconeogenesis and glycolysis by binding SIRT6 and GLUTs, and regulates amino acid metabolism by targeting SLC7As and PRODH [DNMT3A and DNMT3B [p53 dimers are generated co-translationally on the polyribosomes, and then two dimers gather and form a tetramer, which is an active form of p53 in cytosol or on DNA-binding sites . p53 hass of p53 . Althougs of p53 . As a res of p53 . In respnd PRODH . Furtherd DNMT3B . Additiod DNMT3B  , which are called the “six hotspot mutations”. Although these hotspot mutations are the most common, they comprise only around one-quarter of all TP53 mutations in human cancers, because the TP53 missense mutations have a remarkably broad spectrum and extreme diversity over various tumor types. A specific amino acid change due to mutations in the DBD of TP53 causes the loss of DNA-binding potential, and TP53 mutants at the six hotspots can be classified as a loss-of-function mutation. [TP53 mutants can have the capability of abrogating WT TP53-induced transactivation, which is called “dominant-negative potential”, since the p53 heterotetramer consisting of the WT TP53 dimer and TP53 mutant dimer is generated in tumor cells harboring the TP53 mutation and WT TP53 as heterozygotes [TP53 mutants with the loss-of-function characteristic also have a gain-of-function activity. This feature was proved by a knock-in mouse model showing a more invasive and metastatic phenotype in Trp53 mutants compared with Trp53−/− or Trp53+/− mice [TP53 mutations can exhibit different transactivating activities. The p.S121F TP53 mutant is not capable of inducing the CDKN1A gene, but it is capable of transactivating other target genes such as BAX, BBC3, and TNFRSF10B, and inducing apoptosis [TP53 mutations investigated in their work, TP53 mutations can be classified as follows [TP53 mutations in terms of binding affinity toward variable target response elements might be a “hand” playing many keys with variable intensity on the piano, which was proposed by Resnick and Inga [The most common type of utation. ,20. Cancozygotes . Interes+/− mice ,23,24. Spoptosis ,26. Somepoptosis ,30. Base follows : (1) losand Inga ,32.TP53 mutations on the clinical outcomes of patients with TP53-mutation-harbored cancers. Poeta et al. classified TP53 mutation as disruptive and non-disruptive mutations, based on the location of the mutation and the predicted amino acid alterations [TP53 mutation had clinical significance in terms of overall survival in patients with head and neck squamous cell carcinoma. Neskey et al. proposed an Evolutionary Action Score of TP53 (EAp53) using a computational model based on the phenotype–genotype relationship of TP53 mutations in the DBD. Scores ranging from 0 to 100 were calculated and outcomes of head and neck cancers with high and low EAp53 scores were compared [TP53-mutated head and neck cancers, and for consistent application to patients with AML. Eran Kotler et al. tested the functional impact of nearly every kind of TP53 mutation in the DBD in a colon cancer cell line using a synthetically designed library. They performed extensive experiments to measure the proliferative capacity of each TP53 variant relative to that of the WT p53-coding sequence (synonymous TP53 mutation), and proposed a prediction model called “relative fitness score” [TP53 mutations were applied to patients with AML [TP53-mutated AML. Variables of disruption (yes/no) and EAp53 (<75 or ≥75) did not prove to be significant predictive factors for OS and EFS. However, RFS (≤−0.135 or >−0.135) emerged as a significant predictive factor for OS  and EFS  in multivariate analysis. Although this last prediction model can be applied to patients with TP53-mutated AML, it was not derived from a tissue-specific approach targeting TP53-mutated cell types, but rather from a mutation-specific approach. Consequently, we anticipate future prediction models based on findings from TP53 mutations in AML cells.There have been several suggestions regarding how to determine the impact of different erations . They decompared . These ts score” . In a rewith AML . In thisTP53 mutations are found in less than 10% of patients with newly diagnosed AML; however, they increase remarkably by up to 50% or more in the setting of therapy-related AML [TP53 mutation can emerge after irradiation or cytotoxic stress [TP53 mutations represent the primary mutational event in chemotherapy- or radiation therapy-induced MDS/AML. However, intriguingly, pre-existing TP53 mutations exhibit resistance to chemotherapy or irradiation, and selectively expand after treatment. It is not the case that cytotoxic stress does not directly induce TP53 mutations [TP53-mutated hematopoietic clones have the highest probability of developing AML within a median of 4.9 years following the detection of TP53 mutation in the blood [TP53-mutated HSCs represent only a small fraction of the total hematopoietic stem/progenitor pool, they keep expanding upon their survival advantage and preferentially proliferate through selective survival pressure during chemotherapy and allogeneic hematopoietic stem cell transplantation [TP53 is one of the genes found in age-related clonal hematopoiesis [TP53 exerts a more deterministic influence compared with other genes such as DNMT3A, TET2, and ASXL1 when it comes to leukemic transformation. Further, with aging, HSCs gradually lose their self-renewal capacity and shift toward myeloid lineage with decreases in lymphopoiesis [DNMT3A, TET2, RUNX1, and EZH2, are suggested to have originated in HSCs, based on observations that these mutations have been detected in T-cells in patients with AML, which was called “lympho-myeloid clonal hematopoiesis” [TP53-mutated leukemia, both HSCs and HPCs are thought to be cellular origins [TP53 mutation has been associated with LMPP-like LSC dominant phenotype in patients with newly diagnosed AML in terms of LSC immunophenotype [TP53 mutation over time, and these preleukemic HSCs with multiple mutations co-exist with leukemic bulk at initial diagnosis, persist during intensive chemotherapy, and become sources of relapse in some cases [DNMT3A, TET2, and ASXL1) preludes fully transformed leukemic cells, with additional mutation on signaling or transcriptional genes like RAS, FLT3, NPM1, or CEBPA, is widely accepted [TP53 mutation is enriched in patients with hematologic malignancies without DTA  mutation [TP53 mutation is somewhat different from that caused by DTA mutations. Rather, TP53 mutations commonly co-occur with cytogenetic abnormalities, including deletion of chromosomes 5, 7, and 17 or complex abnormalities of >3 [TP53-mutated AML might involve multiple hits accelerated by a deficient DNA repair mechanism resulting from TP53 mutation rather than epigenetic dysregulation due to DTA mutations. Due to this distinct genetic feature, one of the recent classification systems classifies TP53-mutated AML as a separate disease entity [ated AML . TP53 muc stress . A longiutations . Healthyhe blood . Even thantation . Althougopoiesis , it appeopoiesis  and erytopoiesis . The celpoiesis” ,46. In thenotype . Additiome cases ,49,50. Caccepted . Howevermutation , which ses of >3 .The leuke entity . TransfoTP53-mutated AML. Epigenetic genes such as DNMT3A, ASXL1, and TET2 or RAS/MAPK signaling genes including NF1, KRAS/NRAS, and PTPN11 or transcription factors like RUNX1 or genes involving RNA splicing such as SRSF2 are frequently mutated in the same leukemic clone or sub-clones. SRSF2, RUNX1 and ASXL1 frequently occur in patients with newly diagnosed AML with one TP53 mutation, whereas KRAS/NRAS, PTPN11, and RUNX1 commonly occur in ≥2 TP53 mutations [TP53-mutated AML and TP53-WT AML demonstrated that FLT3, NPM1, and RAS gene mutations are not common in TP53-mutated AML compared with TP53-WT AML, whereas other gene mutations are not different between the two groups [TP53 mutation is also associated with chromosome-level alterations such as complex karyotype, aneuploidy including del(5q), −5, −7, and del (3p) rather than single nucleotide variation. The frequency of TP53 mutation in MDS/AML with a complex karyotype is reported to be up to 83% [TP53 mutation in MDS/AML is closely linked to the presence of a complex karyotype. Both of these abnormalities are associated with prior therapeutic interventions that induce DNA damage. However, it remains unclear how TP53 mutation contributes to the development of a complex karyotype in hematopoietic stem and progenitor cells. Chromothripsis events are thought to be a cause of complex karyotypes in TP53-mutated AML [TP53-mutated AML compared with other AMLs [TP53-mutated AML. Immune infiltration in the bone marrow microenvironment is characteristic of TP53-mutated AML [TP53-mutated AML are stronger than in healthy donors [TP53-mutated AML might be partly a result of increased tumor mutation burden, considering the observation of increased tumor mutation burden in various cancer types with TP53 mutation [TP53-mutated AML cells [TP53-mutated AML at all [TP53-mutated AML [Some co-occurring mutations are detected in patients with utations . Anothero groups . Howeverp to 83% . The higated AML . There aher AMLs . Severalated AML . IFN-α ay donors . Increasmutation . A previML cells . HoweverL at all ,65. Neveated AML ,66  is associated with better survival in TP53-mutated AML [TP53 mutation. In contrast, treatment outcome is independent of mutated TP53 VAF when treated with a prolonged schedule (10 days) of decitabine [TP53 mutation showed a remarkably higher response rate than patients without TP53 mutation (100% (21/21) vs. 41% (32/78), p < 0.001) [TP53-mutated leukemic clones disappeared as a result of decitabine treatment in many cases. It remains unclear why hypomethylating agents are effective in TP53-mutated AML. However, a plausible explanation for this phenomenon is that hypomethylating agents like decitabine may reverse an unbalanced DNA methylation in TP53-mutated AML cells because normal p53 suppresses DNA methylation by up-regulating TET1/2 components of the demethylation machinery [TP53-mutated AML. However, this unrealistic CR rate was not represented in other studies where a combination with 10-day decitabine improved the outcomes of TP53-mutated AML. In a randomized phase II study to investigate the addition of bortezomib, a proteasome inhibitor on 10-day decitabine showed a poor response in patients with TP53-mutated AML [TP53-mutated AML. Two studies investigated the benefits of venetoclax in patients with TP53-mutated AML. In the first study, a prospective phase II trial, investigators tested 10-day decitabine plus venetoclax 400 mg daily followed by 5-day decitabine plus venetoclax after achieving remission in newly diagnosed AML. The overall response rate and complete remission rate were 89% and 77%, respectively, in WT-TP53 AML, whereas they were 66% and 57%, respectively, in TP53-mutated AML. Survival outcomes were less favorable regarding response rate, with a 60-day mortality of 26% vs. 4% and overall survival of 5.2 and 19.4 months in TP53-mutated and WT-TP53 AML, respectively [TP53-mutated AML. In this study, investigators could not identify a significant difference between the venetoclax-based regimen and the non-venetoclax-based regimen in terms of OS  and relapse-free survival  [TP53-mutated and TP53-nonmutated AML, despite treatment being heterogeneous and retrospective.0 months ,84. TP53s of AML . TP53-mup to 70% . Low TP5ated AML . A standcitabine . A corne< 0.001) . Furtherachinery . Followiated AML . A substectively . In the  = 0.43) . The firTP53 mutation was unsatisfactory  [TP53-mutated MDS and ORR 33% and CR 17% in TP53-mutated AML) [TP53-mutated AML/MDS. In this study, median relapse-free survival and overall survival were 12.5 and 20.6 months, respectively [TP53-mutated MDS/AML. APR-246 was also tested as a triple combination therapy with azacytidine and venetoclax in TP53-mutated AML. Complete remission and CR/CRi/MLFS rate in this phase I study were 38% and 59%, respectively. This result indirectly supports the previous finding that the addition of venetoclax is not beneficial in TP53-mutated AML, though we consider that an interpretation of treatment outcomes from a phase I study should be cautious [TP53-mutated AML [TP53-mutated malignancy in a clinical trial context. Several methods to indirectly target mutated TP53 beyond TP53 reactivators have also been studied in TP53-mutated solid cancers. One method constitutes the enhanced degradation of mutated p53 by protein degraders, including heat shock protein 90 inhibitor [TP53-mutated AML in the near future.Considering data obtained so far from drugs which are currently clinically available or soon to be available, there are two paths worth placing our hopes in. The first one is mutated-p53-targeted therapy, which is called p53 reactivators. APR-246 (eprenetapopt) is a prodrug converting to MQ, which binds to a specific thiol group in the p53 DNA-binding domain. Mutant p53 can be refolded to its wildtype configuration and reactivated through MQ binding ,90. In tted AML)  and anotted AML) . Doubletectively . This recautious . COTI-2,cautious . Arsenicated AML . Howevernhibitor , whereasnhibitor ,95. ThesTP53-mutated AML could be effectively treated using novel agents with immunologic mechanisms. Flotetuzumab, a CD123 x CD3 bispecific dual-affinity retargeting antibody (DART) molecule, demonstrated a promising efficacy in relapsed/refractory TP53-mutated AML, showing a 47% complete remission rate and a 10.3-month OS [TP53-mutated AML. In a phase I/II study, the CR/CRi rate in patients with TP53-mutated AML was 100% (7/7) [TP53-mutated AML by immunologic mechanism comes from the current clinical practice. A recent retrospective study extracted independent predictive factors for prolonged OS in a cohort of patients with TP53-mutated AML treated with allogeneic stem cell transplantation [TP53-mutated AML treated with allogeneic HSCT compared with TP53-mutated AML not treated with allogeneic HSCT (median OS 33.7 months vs. 7.0 months), but the Kaplan–Meyer graph in this article strongly suggested that even allogeneic HSCT is not curative for TP53-mutated AML [TP53-mutation. A certain hotspot for TP53 mutation is known to be the immune-cell-stimulating neoantigen, and this can be effectively targeted by T-cells engaging bispecific antibodies [TP53-mutated AML.The second method uses immunologic agents. There is a suggestion that month OS . Magroli0% (7/7) . Severalantation . In thisated AML . Anothertibodies . AlthougTP53-mutated AML is a unique subtype of AML with a very poor prognosis. TP53-mutated AML is highly enriched in therapy-related or secondary AML with a complex karyotype. This type of TP53 mutation is heterogeneous, but the most common types of TP53 mutation in AML are mainly missense mutations in the DNA-binding domain, which are not only characterized by loss of function in view of loss of effective DNA sensing of the target sequence and resultant physiologic regulatory function and/or dominant-negative effect, but are also often characterized by gain of function in terms of gain of mutated oncogenic p53 proteins contributing to the malignant phenotype. The current standard treatment strategies for AML make it difficult to induce durable remission and to cure TP53-muated AML. Five-day or ten-day decitabine could be the preferred options for TP53-mutated AML. Standard therapy including high-dose cytarabine could be considered as an initial induction therapy for some patients with treatment-naive TP53-mutated AML with a low TP53 mutation burden. Allogeneic HCT should be considered in transplant-eligible patients with TP53-mutated AML, despite the low probability of long-term survival. The incorporation of TP53 reactivators and immunologic agents including CD123-targeted DART and CD47-targeting immune-checkpoint inhibitor is now being prepared for running, at the starting line, after completing early-phase clinical trials.


FUT2-encoded fucosyltransferase (Se enzyme) and FUT3-encoded fucosyltransferase (Le enzyme). In Japanese populations, c.385A>T in FUT2 and a fusion gene between FUT2 and its pseudogene SEC1P are the cause of most Se enzyme-deficient alleles (wSe and fusse), and c.59T>G and c.314C>T in FUT3 are tag SNPs for almost all nonfunctional FUT3 alleles . In this study, we first conducted a single-probe fluorescence melting curve analysis (FMCA) to determine c.385A>T and fusse using a pair of primers that collectively amplify FUT2, sefus, and SEC1P. Then, to estimate Lewis blood group status, a triplex FMCA was performed with a c.385A>T and fusse assay system by adding primers and probes to detect c.59T>G and c.314C>T in FUT3. We also validated these methods by analyzing the genotypes of 96 selected Japanese people whose FUT2 and FUT3 genotypes were already determined. The single-probe FMCA was able to identify six genotype combinations: 385A/A, 385T/T, fusse/fusse, 385A/T, 385A/fusse, and 385T/fusse. In addition, the triplex FMCA successfully identified both FUT2 and FUT3 genotypes, although the resolutions of the analysis of c.385A>T and fusse were somewhat reduced compared to that of the analysis of FUT2 alone. The estimation of the secretor status and Lewis blood group status using the form of FMCA used in this study may be useful for large-scale association studies in Japanese populations.Lewis blood group status is determined by two fucosyltransferase activities: those of  This is because the activity of the Se enzyme encoded by wSe is very low but detectable due to a single nucleotide polymorphism (SNP), c.385A>T  [FUT3 allele (Le) have Le(a-b+) red cells in their secretors, Le(a+b-) in their non-secretors, and Le(a+b+) in their weak secretors. On the other hand, Lewis-negative individuals, homozygotes for the nonfunctional FUT3 alleles (le), all have Le(a-b-) red cells [The expression of Lewis blood group antigens, Lewis a (Le enzyme) ,2,3. Seced cells ,14,15.se alleles resulting from non-allelic homologous recombination and several population-specific SNPs in FUT2 and FUT3 have been identified [fusse, which results from an unequal crossover between FUT2 and its pseudogene SEC1P , determining these polymorphisms could provide an accurate estimate of secretor status and Lewis blood group status.To date, five entified ,17,18,19ne SEC1P A, is pre 19q13.3 ,21. In about 50% ,22,23. Fulations . In JapaFUT3 to estimate le allele frequency [Fluorescence melting curve analysis (FMCA) is a simple, robust, and rapid closed-tube post-PCR method for detecting SNPs that analyzes the difference between the melting curve profiles of the fluorescence-labeled probe and PCR amplicon ,26. In arequency .fusse using a single probe. A triplex FMCA was then performed to estimate Lewis blood group status in Japanese people, adding primers and probes to detect c.59T>G and c.314C>T of FUT3 to c.385A>T and fusse assay system.In this study, we performed a FMCA that simultaneously determined the frequency of c.385A>T and FUT2 haplotypes had been determined previously using established methods such as the allele specific PCR and/or DNA sequencing [The genomic DNAs of 96 Japanese people from Fukuoka whose quencing  were useFUT2 and SEC1P genes were numbered as described previously [FUT2, fusse, and SEC1P. The forward primer, 5′-TGGCAGAACTACCACCTGAA-3′, matches exactly 337–356 bp of FUT2 and 379–398 bp of SEC1P, and is the same primer that previously detected c.385A>T by an unlabeled probe HRM analysis [FUT2 and of 456–475 bp of SEC1P, and is the same primer that previously detected fusse by a TaqMan probe assay and HRM analysis [SEC1P, and 369–393 bp of FUT2, but differs by one base from the wild-type  and fusse, and differs by two bases from the 385T allele. The asymmetric PCR reaction mixture with a final volume of 10 µL contained 5 µL of TaKaRa Taq HS Perfect Mix containing modified Taq DNA polymerase, which has neither 5′-3′ exonuclease nor 3′-5′ exonuclease activities , 50 nM of the forward primer, 500 nM of the reverse primer, 200 nM of the SEC1P-FUT2 probe, and 2–20 ng of genomic DNA. The PCR was conducted on LightCycler 480 Instrument II  with the following thermal conditions: 45 cycles of denaturation at 95 °C for 5 s, and annealing/extension at 60 °C for 15 s.The nucleotide positions of the eviously . All prianalysis . The revanalysis ,28. The FUT3 to estimate le allele frequency [fusse of the FUT2 assay system to detect c.59T>G and c.314C>T, excluding c.484A>G. However, since a HEX-labeled probe was used to detectc.385A>T and fusse, a FAM-labeled probe was used instead of a HEX-labeled probe to detect c.59T>G in this study. The primer and probe concentrations for c.59T>G and c.314C>T were as follows: 50 nM of each forward primer, 250 nM of each reverse primer, 50 nM of a probe for c.59T>G, and 100 nM of a probe for c.314C>T. The primer and probe concentrations for c.385A>T and fusse and the thermal conditions of the asymmetric PCR were the same as described above.Recently, we conducted a triplex FMCA for the genotyping of three tag SNPs, c.59T>G, c.314C>T, and c.484A>G, in requency . HoweverThe PCR products were then heated to 95 °C for 1 min and cooled to 40 °C for 1 min, and fluorescence data were acquired using the VIC/HEX/Yellow 555 filter (excitation–emission: 533–580 nm) and/or the FAM filter (465–510 nm) and/or the Cy5/Cy5.5 filter (618–660 nm) during heating from 50 to 80 °C at a 0.1 °C/s ramp rate. Melting curve genotyping and melting temperature (Tm) analyses were carried out using the LightCycler 480 gene scanning software.fusse with a single probe using a primer set that collectively amplified 97 bp amplicons of FUT2, fusse, and SEC1P. As shown in SEC1P amplicon because the probe sequence exactly matches that of SEC1P. On the other hand, an intermediate Tm value around 68 °C was observed for the 385A allele amplicon because the probe sequence differed by one base, and the lowest Tm value around 62 °C was observed for the 385T allele amplicon because the probe sequence differed by two bases. In addition, in fusse, the nucleotides corresponding to the positions of 375 bp and 385 bp in FUT2 (or 419 bp and 429 bp in SEC1P) were both “A”, and therefore, the Tm value for the fusse amplicon was the same (around 68 °C) as that for the 385A amplicon  that hybridized with the probe  showing only one melting peak at around 68 °C, and homozygotes of 385T allele (385T/T) showing two melting peaks at around 73 °C and 62 °C were completely separated by the default settings of the LightCycler 480 gene scanning software . On the other hand, homozygotes of the 385A allele (385A/A) and heterozygotes of 385A/fusse with two melting peaks at around 73 °C and 68 °C, or heterozygotes of c.385A>T (385A/T) and heterozygotes of 385T/fusse with three melting peaks at around 73 °C, 68 °C, and 62 °C, were classified into the same group in the default settings. However, it was possible to separate 385A/A from 385A/fusse and 385A/T from 385T/fusse by changing the settings to normal sensitivity with a score threshold of 0.85 and a resolution threshold of 0.00  are relatively lower than that corresponding to SEC1P for subjects without fusse .We then analyzed 96 Japanese people whose quencing . Homozyg of 0.00 B. The reut sefus A,C.wSe/628se, determined by a Sanger sequencing analysis, was misclassified as wSe/Se in this FMCA because the nucleotide at position 385 of 628se was an “A”, the other results were completely in accordance with previous ones [FUT2 genotyping results of conducting a FMCA of 96 Japanese people were as follows: 23 were 385A/A, 38 were 385A/T, 22 were 385T/T, 9 were 385A/fusse, 2 were 385A/fusse, and one was fusse/fusse. In addition, the repeatability the results was confirmed because the results of two independent assays were identical.Although one weak secretor with the genotype FUT3 and c.385A>T and adding the fusse assay system. The melting curve genotyping results for c.385A>T and fusse from the triplex FMCA were similar to those from the single-probe FMCA with the default settings; however, unlike the single-probe FMCA, some melting peaks were divided into unknown groups . The FUT3 genotyping results of conducting a FMCA of 96 Japanese people were as follows: 35 were 59T/T, 43 were 59T/G, and 18 were 59G/G, while 93 were 314C/C and three were 314C/T.We then attempted a triplex FMCA that could estimate the Lewis blood group status of the Japanese people by adding primers and probes that detect c.59T>G and c.314C>T in eviously B,C. The FUT2 and FUT3 genotypes, secretor status, and Lewis blood group status of the 96 Japanese subjects estimated by the present triplex FMCA. Thus, by the FMCA, 60 of the 96 Japanese subjects were estimated to be Lewis-positive secretors with a Lewis phenotype of Le(a-b+); 16 were Lewis-positive weak secretors of Le(a+b+), one was a Lewis-positive non-secretor of Le(a+b-), and 19 were Lewis-negative subjects of Le(a-b-). In addition, 11 of the 19 people with a phenotype of Le(a-b-) were estimated to be secretors and 8 were estimated to be weak secretors.. Incidentally, conventional serological Lewis phenotyping is somewhat difficult because it depends largely on the strength and specificity of the anti-Lea and anti-Leb antibodies used and the skill of the observer [FUT2 of the same subjects used in this study, we misdiagnosed the serological Lewis phenotype Le(a+b+) as Le(a+b-) [b antibody used. Thus, we classified Le(a+b-) subjects as being of the Se enzyme-deficient phenotype, which includes both weak secretors and non-secretors. In any case, with the exception of one subject (as mentioned above), the previous Lewis phenotyping results were also compatible with the estimate of the Lewis phenotype made by the FMCA.observer . In factLe(a+b-) . This mafusse individually, including high-resolution melting (HRM) analysis and a TaqMan (hydrolysis probe) assay [fusse by an asymmetric PCR using a single probe. The probe-based FMCA showed significant Tm change (about 5–6 °C) between a wild type (385A) allele and/or fusse, and between a mutant type (385T) allele and SEC1P. Thus, this single-probe assay accurately determined 385A>T substitution. fusse was found almost solely in the Japanese population with a frequency of 5–9% [fusse heterozygotes  in this study. As described previously, an artificial recombinant of SEC1P and FUT2 was generated during PCR amplification when a relatively small fragment specific to the fusse sequence was amplified [SEC1P and FUT2, we selected primers that had amplified fusse and SEC1P in the previous studies [fusse, SEC1P, and FUT2 in the present study. The present method has an advantage over the previous methods, which could only detect c.385A>T or fusse, in that it can simultaneously detect fusse and the c.385A>T of FUT2 in a single assay.Several real-time PCR based methods were developed to identify c.385A>T or e) assay ,27,28,30 of 5–9% , and thu studies ,28, and fusse allele contains the 3′ region of the wild-type FUT2 sequence, it was previously misidentified as a functional 385A allele when c.385A>T was genotyped using primers that specifically amplified the FUT2 sequence surrounding 385A>T [fusse signal was also the same as that of the 385A allele signal, but since a chromosome with fusse lacks SEC1P, it appeared that the zygosity of fusse could be determined by the peak height of the SEC1P signal. Namely, fusse/fusse lacked the SEC1P signal and fusse heterozygotes had relatively lower peak SEC1P signal height. The relatively lower peak SEC1P signal height of in the fusse heterozygotes could not be detected by the software of the real-time PCR instrument used with the default settings for melting curve genotyping. According to the instrument manual , ‘score’ is an index of the similarity between the melting curves of a sample and the melting curve of the standard that is most similar to the sample, and ‘resolution’ is the index of the dissimilarity between the melting curve of the sample and the melting curve of the second most similar standard. Therefore, subtle differences in the relatively lower peak SEC1P signal height in the fusse heterozygotes could have been detected by increasing the score threshold and decreasing the resolution threshold from the default settings.Because the g 385A>T ,28. In tfusse of FUT2 and the c.59T>G and c.314C>T of FUT3. Because the resolution was somewhat lower for the triplex FMCA than for the single-probe FMCA, it was difficult to automatically separate the six genotype combinations using the software. Nevertheless, it was possible to first separate them into four groups, fusse/fusse, 385T/T, 385A/A plus 385A/fusse, and 385A/T plus 385T/fusse, by the default settings, and then 385A/A was further separated from 385A/fusse and 385A/T from 385T/fusse by manually observing the relative peak SEC1P signal heights. Therefore, we could estimate not only secretor status but also Lewis blood group status in a single assay.In addition, we developed a triplex FMCA prodcedure that could simultaneously detect the c.385A>T and FUT2 has been observed in African and Oceanian populations [FUT2 allele with this SNP would be determined as SEC1P in the present method. However, in other populations, including the Japanese population, this SNP seems to be rarely observed. Therefore, such a misdiagnosis is unlikely to occur in the genotyping of Japanese subjects. A limitation of the present method is that it cannot detect rare known se alleles such as 571se and 628se and rare unknown se and le alleles. In fact, we misdiagnosed one 628se allele as a functional allele by the present method. In addition, this method is useful almost exclusively for Japanese populations.The c.375A>G  of ulations ,32. The se alleles. However, compared to Sanger sequencing, for the whole coding region of FUT2, the present FMCA method is simple, cost-effective, and rapid, making it suitable for high-throughput analysis [se alleles generated by non-allelic homologous recombination such as fusse by Sanger sequencing for the whole coding region of FUT2. Therefore, the probe-based FMCA procedure used in this study may have some advantages over Sanger sequencing.Sanger sequencing, the golden standard for the determination of SNPs, can detect these rare analysis . In addiThe association between phenotypic polymorphisms of blood types, as represented by the ABO blood group, and specific diseases has been analyzed in the past by case-control studies, but few reports have shown an association of those with specific symptoms such as the presence of duodenal ulcers . On the FUT2 genotyping could determine the secretory status of Lewis-negative subjects. A recent study suggested that red cells with Lewis a phenotype displayed strongly reduced SARS-CoV2-susceptibility [FUT2 and FUT3 genotyping is a useful alternative method for phenotyping, and the FMCA method used here appears to be a valid and feasible method for large-scale association studies of both secretor status and Lewis blood group status in Japanese populations.Furthermore, while serological Lewis phenotyping could not determine the secretory status of Lewis-negative subjects, i.e., those without Le(a-b-), tibility . However


However, the detailed mechanisms of glutamate-evoked Ca2+ signals are not entirely clear. Here, we used a ratiometric Ca2+ and Na+ imaging technique to investigate glutamate-evoked Ca2+ responses. The comparison of Ca2+ responses to glutamate (100 μM) and high (20 mM) K+ solution indicated slower Ca2+ clearance, along with rebound Ca2+ suppression for glutamate-evoked Ca2+ transients. Increasing the length of exposure time in glutamate, but not in 20 mM K+, slowed Ca2+ clearance and increased rebound Ca2+ suppression, a result correlated with glutamate-induced Na+ loads. The rebound Ca2+ suppression was abolished by ouabain, monensin, Na+-free solution, or nimodipine, suggesting an origin of activated Na+/K+-ATPase (NKA) by glutamate-induced Na+ loads. Ouabain or Na+-free solution also slowed Ca2+ clearance, apparently by retarding Na+/Ca2+-exchanger (NCX)-mediated Ca2+ extrusion. Together, our results indicated the involvement of glutamate-induced Na+ loads, NKA, and NCX in shaping the Ca2+ response to glutamate. Nevertheless, in the absence of external Na+ (NMDG substituted), Ca2+ clearance was still slower for the Ca2+ response to glutamate than for 20 mM K+, suggesting participation of additional Ca2+ handlers to the slower Ca2+ clearance under this condition.Glutamate mediates photic entrainment of the central clock in the suprachiasmatic nucleus (SCN) by evoking intracellular Ca The Ca2+ response to high (20 mM) K+, which did not induce Na+ loads, was also investigated in the same cells for comparison. Our results indicate the involvement of glutamate-induced Na+ loads, NKA, and NCX in shaping the Ca2+ response to glutamate.In the rat SCN cells, the Naincrease ,11. The activity  . In 2+ imaging with Fura2-AM was used to determine [Ca2+]i in cells in reduced SCN slice preparations. 2+ responses to 100 μM glutamate. The experiments were performed by first applying 20 (mM) K+ solution for 20 s, followed by two consecutive 20 s applications of 100 μM glutamate to elicit Ca2+ responses (left panels). For the experiment shown in 2+ response that rapidly reached a plateau, followed by rebound Ca2+ suppression (marked by arrowheads) after washing out the glutamate, and the second application of glutamate 5 min later elicited an almost identical response (right panel). For the experiment shown in 2+ response that exhibited a spike-like waveform during the 20-s application, and the second application of glutamate 5 min later failed to elicit a Ca2+ increase, but instead produced a small Ca2+ suppression (right panel). The mechanism for the variable, transient Ca2+ response to glutamate is not clear (see Discussion). Only the tonic glutamate responses with a rapid Ca2+ increase were included in the following experiments.Ratiometric Ca2+ response to 20 mM K+ and 100 μM glutamate (an average of 14 cells). 2+ response induced by 20 K+ and glutamate, with the center plot showing the average Ca2+ response (n = 14 cells) and the surrounding plots showing the Ca2+ responses from eight different SCN cells. Superimposition of the Ca2+ responses to glutamate (black traces) and 20 K+ (grey traces) revealed major differences, particularly in their Ca2+ clearance kinetics. While the amplitude of the Ca2+ increase may be comparable between the Ca2+ response to 20 K+ and 100 μM glutamate, the rate of Ca2+ clearance was slower (marked by the arrow), along with the occurrence of rebound Ca2+ suppression (marked by arrowhead), for the glutamate-evoked Ca2+ transient (center plot). The slower Ca2+ clearance and rebound Ca2+ suppression after glutamate washout suggest more complicated mechanisms for clearing the Ca2+ evoked by glutamate than by 20 K+-induced depolarization i of 20 mM K+ and 100 μM glutamate  and 0.0125 ± 0.0006   B.+]i in the first minute of glutamate application . Superimposition of the Ca2+ responses at the end of glutamate application indicates a more prolonged Ca2+ decay (marked by the arrow) and a larger rebound Ca2+ suppression (marked by the arrowhead) for the 60 s response (blue trace) . 2+ half-decay time (t1/2) and rebound Ca2+ suppression, as measured by the area under the curve (AUC). On average, an increase in the glutamate exposure time from 20 to 60 s increased the t1/2 values from 13.1 ± 0.3 s  to 19.7 ± 0.8 s  (n = 100) to 2.25 ± 0.09   B. Figure t-test) G and the t-test) H. Altoge  to 5.10 ± 0.21 s . The result indicates a lack of effect of increasing 20 mM K+ exposure time on the clearance of Ca2+ or the t1/2 value.For comparison, the same cells were also used to determine their Casolution D. Superikinetics E,F. Figu+ is controlled by Na+/K+-ATPase (NKA) [2+]i [2+ response to glutamate. 2+]i, and at a concentration of 10 μM, it evoked an initial increase during the first 10~15 min, followed by a decrease to the different levels shown above, near or even below the basal level, and then an increase again to various levels close to or above the levels of initial increase (see 2+ level was higher than that before ouabain treatment (2+ clearance and eliminated rebound Ca2+ suppression (marked by the arrowhead), which could be better visualized by superimposing the normalized Ca2+ transients in the control (black trace) and in ouabain (red trace)  compared to that in the control (black trace).Since intracellular Nase (NKA)  and coulA) [2+]i , we firsreatment A. The red trace) B. For co20 mM K+ C,D. The 2+ clearance for the 20 K+-evoked Ca2+ transient confirms our previous observation [2+ extrusion as a result of ouabain-induced Na+ loads. In contrast, for the glutamate-evoked Ca2+ transient, ouabain should additionally inhibit the extrusion of Na+ loaded by glutamate and thereby augment the effects of glutamate-induced Na+ loads on slowing Ca2+ clearance and eliciting rebound Ca2+ suppression as well. The result of ouabain-induced slower Ca2+ clearance is consistent with this view. In contrast, the result of the ouabain-induced elimination of rebound Ca2+ suppression instead suggests that glutamate-induced Na+ loads activate NKA to mediate rebound Ca2+ suppression. In other words, our results suggest that the Na+ loaded by glutamate shapes the Ca2+ response by inhibiting NCX-mediated Ca2+ extrusion and also activating NKA to elicit rebound Ca2+ suppression.The result of the ouabain-induced slowing of Caervation , and cou+ and NKA activation, we determined the effect on the Ca2+ response to glutamate of 10 μM monensin, which increases Na+ to activate NKA, hyperpolarize the resting membrane potentials [2+ clearance for 20 K+-evoked Ca2+ transients [2+ decay and yet completely eliminated rebound Ca2+ suppression (marked by the arrowhead), as shown by superimposing the normalized Ca2+ transients in the control (black trace), monensin (red trace), and after washout (grey trace) , slightly decreased (n = 2), or had no effect (n = 2) on the t1/2 values for the Ca2+ response to glutamate, but always increased the t1/2 values for the Ca2+ response to 20 mM K+.To further investigate the involvement of Natentials , and sloansients . Figure y trace) B. The in20 mM K+ C,D. Cons1/2 value (or the much slowed Ca2+ clearance) for the Ca2+ response to 20 K+ is apparently mediated by the large Na+ loads induced by 10 μM monensin [1/2 value for glutamate Ca2+ response and yet eliminated rebound Ca2+ suppression. A simple explanation is that in the presence of large Na+ loads induced by monensin, NKA may be maximally activated to promote the extrusion of Na+ loaded by glutamate and become refractory to further stimulation by glutamate-induced Na+ loads to elicit rebound Ca2+ suppression (see Discussion).The large increase in the tmonensin , which n2+ suppression, we investigated the effect of the L-type Ca2+ channel blocker nimodipine on the Ca2+ response to glutamate (2+ transient (an average of 16 cells). 2+ transients in the control (black trace), in nimodipine (red trace), and after washout (grey trace). Apart from lowering the basal Ca2+, nimodipine slowed the rate of Ca2+ increase to slightly reduce the amplitude, had no effect on t1/2, and abolished the rebound Ca2+ suppression (marked by the arrowhead). The result suggests that the rebound Ca2+ suppression is mediated by the inhibition of nimodipine-sensitive Ca2+ influx. For comparison, the same cells were also used to determine the effect of nimodipine on the Ca2+ response to 20 mM K+  solution on the Ca2+ response to glutamate. 2+ transients in the control (black trace), in Na+-free solution (red trace), and after washout (grey trace) to indicate the reversible effect of Na+-free solution on slowing the Ca2+ decay and eliminating the rebound Ca2+ suppression (marked by arrowheads). For comparison, the same cells were also used to determine the effect of Na+-free solution on the Ca2+ response to 20 mM K+  and to 20 mM K+ (blue trace)  and 9.9 ± 0.2 s  . The result suggests the contribution of additional Ca2+ handlers to the slower Ca2+ clearance for glutamate response, at least under the condition of a Na+-free solution.The slowing of Cae trace) E. On ave t-test) F. In oth+ loads, NKA, and NCX in shaping the Ca2+ response to glutamate. Compared to the Ca2+ response to 20 mM K+, the glutamate-evoked Ca2+ response shows slower Ca2+ clearance, along with a rebound Ca2+ suppression, both being associated with glutamate-induced Na+ loads. The increase in intracellular Na+ induced by glutamate appears to inhibit NCX activity to slow Ca2+ extrusion and also enhance NKA activity to mediate nimodipine-sensitive rebound Ca2+ suppression. Nevertheless, there remain unidentified mechanisms responsible for the slower Ca2+ clearance for glutamate-evoked Ca2+ responses under the condition of occurring in a Na+-free solution.This study demonstrates the involvement of glutamate-induced Na2+ responses can generally be discerned into two different types: a reproducible, tonic Ca2+ response and a variable, transient Ca2+ response , with the activation of the former receptors inhibiting the activation of the latter in a time-dependent manner. Further work is needed to better resolve this issue.We show that the glutamate-evoked Caresponse . The ton kainate . Furthercultures . The var studies ,6,7,9. T studies . Our pre2+ response to 100 μM glutamate and 20 mM K+ solution, we found major differences in the kinetics of Ca2+ clearance, namely, slower Ca2+ clearance followed by rebound Ca2+ suppression for the glutamate-evoked Ca2+ response  and the rebound Ca2+ suppression   , ouabaintituted) , and by channels . Monensipression . The res neurons ,15.+ loads slow Ca2+ clearance, apparently by inhibiting NCX-mediated Ca2+ extrusion, an idea consistent with the result of slower Ca2+ decay in Na+-free solution to block NCX activity , it is tempting to speculate that the monensin-activated NKA may promote extrusion of Na+ to the extent that it actually speeds the clearance of Ca2+. Taken together, our results indicate a critical role of glutamate-evoked Na+ loads, coupled with NKA activation and NCX inhibition, to mediate slower Ca2+ clearance and rebound Ca2+ suppression for the Ca2+ response to glutamate.On the other hand, glutamate-induced Naactivity . Neverth+ (NMDG+ substituted), the Ca2+ response to glutamate still has a slower Ca2+ clearance than 20 mM K+  solution. Further investigations are needed to answer this question. Nonetheless, is should be kept in mind that in the absence of external Na+ (NMDG substituted), the SCN neurons are hyperpolarized to ~–80 mV [+-dependent action potentials. As such, the Ca2+ handlers involved in the Ca2+ response elicited by glutamate might differ between the Na+-free solution and the physiological condition.Our results also show that in the absence of external Na20 mM K+ . This re ~–80 mV  and cann2+ suppression remains to be determined. The rebound Ca2+ suppression after glutamate washout, however, resembles rebound Ca2+ suppression after the washout of K+-free solution : 125 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, and 10 glucose. A coronal slice (200–300 μm) containing the SCN and the optic chiasm was cut with a DSK microslicer DTK-1000 , and was then incubated at room temperature (22–25 °C) in the incubation solution, which contained (in mM): 140 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 10 glucose, 10 HEPES, pH 7.4, bubbled with 100% O2.All experiments were carried out according to procedures approved by the Institutional Animal Care and Use Committee of Chang Gung University. Sprague–Dawley rats (18–24 days old) were kept in a temperature-controlled room under a 12:12 light:dark cycle (light on 0700–1900 h). Hypothalamic brain slices and reduced SCN preparations were formulated as described previously ,11. An a2+ and Na+ imaging, a reduced SCN preparation was obtained by excising a small piece of tissue (circa one-ninth the size of SCN) from the medial SCN using a fine needle , followed by further trimming down to 4–10 smaller pieces with a short strip of razor blade. The reduced preparation  [+-sensitive fluorescent indicator sodium-binding benzofuran isophthalate (SBFI-AM) [+ and 0.5 Hz for Ca2+. Ca2+ and Na+ levels in regions of interest (ROI) over the soma were spatially averaged and presented by fluorescence ratios (F340/F380) after background subtraction. Data were analyzed and plotted with custom-made programs written in Visual Basic 6.0 and the commercial software GraphPad PRISM 8.0.1 . Data were given as means ± SEM and analyzed with paired t-test.Ratiometric fluorescence imaging was carried out as described previously ,11. Fluoura2-AM)  and the SBFI-AM) . The red+ solution was prepared with equal molar substitution of K+ for Na+. Na+-free solutions were prepared with a total replacement of extracellular Na+ with N-methyl-D-glucamine (NMDG+). All solutions were adjusted to pH 7.4 before use.Stock solutions of nimodipine (20 mM in DMSO) and monensin (10 mM in 100% ethanol) were stored at –20 °C, and were diluted at least 1000 times to reach the desired final concentrations. Nimodipine was purchased from Tocris Cookson , and ouabain and monensin were obtained from Sigma-Aldrich . A 20 mM K