Previous studies have reported that CAX activity is affected by an N-terminal autoinhibitory region. CAXs may be present as heterodimers in plant cells, and this phenomenon necessitates further study. In this study, we demonstrate that there is an interaction between CAX4 and CAX1 as determined by the use of a yeast two-hybrid system and a bimolecular fluorescence complementation assay. More specifically, the N-terminal of CAX4 interacts with CAX1. We further observed the over-expression and either a single or double mutant of CAX1 and CAX4 in response to abiotic stress in Arabidopsis. These results suggest that CAX1 and CAX4 can interact to form a heterodimer, and the N-terminal regions of CAX4 play important roles in vivo; this may provide a foundation for a deep study of CAX4 function in the future.High-capacity tonoplast cation/H This auof CAX1) . Our preto yeast . These fArabidopsis thaliana genome appears to contain six CAX genes, designated AtCAX1–AtCAX6 [CAX1 is most closely related to CAX3 at the amino acid level and, together with CAX4, belong to type I-A CAX family genes [CAX4 has been partially characterized biochemically through heterologous expression in yeast and tobacco to determine its cation transport properties [AtCAX4 is expressed in the root apex and in lateral root primordia, and the level of CAX4 mRNA was reported to increase in response to Mn2+, Na+, and Ni+ treatments [CAX4 display symptoms consistent with increased vacuolar sequestration of Ca2+ and Cd2+. High levels of expression of CAX4 in an Arabidopsis cax1 mutant line with weak vacuolar Ca2+/H+ antiport activity results in a 29% increase in Ca2+/H+ antiport activity. A cax4 loss-of-function mutant and CAX4 RNA interference (CAX4 RNAi) lines display altered root growth and development in response to Cd2+, Mn2+, and auxin [+ antiporter that plays an important functional role in root growth under abiotic stress conditions [The 1–AtCAX6 . Members1–AtCAX6 ,24,25,26ly genes ,11,27. Coperties . Recent eatments ,28. Thisnd auxin . These fnditions .CAX deletions are constructed, the resulting plants display severe growth defects, implying the importance and compensatory function of CAXs [CAX1 and CAX3 in yeast results in the production of functional transporters with different biochemical properties than CAX1 alone [In-depth genetic studies of CAX family members have provided new information that suggests that CAXs play overlapping roles in many cell functions. For example, plants that have lost a single CAX transport function are almost indistinguishable from wild-type plants under normal growth conditions . However of CAXs ,25. In aX1 alone . Native X1 alone . PreviouX1 alone . In someX1 alone  conducteX1 alone . Thus, C2+/H+ exchangers CAX4 and CAX1. Using a yeast two-hybrid system and BiFC assay, we demonstrate that there is an interaction between full-length CAX4 and CAX1. More specifically, the N-terminus of CAX4 interacts with CAX1. Then, we analyzed the responses of CAX1 and CAX4 to abiotic stress in plants using over-expression or the use of single and double mutants of CAX1 and CAX4. Our results suggest that CAX1 and CAX4 may form a heterodimer in plants in response to abiotic stress.In the present study, we have analyzed the interaction between the Arabidopsis Ca2+ homeostasis [2+ ion balance in plants. Results indicated that AtCAX4 and AtCAX1 do interact with each other. Then, full-length AtCAX1 and AtCAX4 or N-truncated AtCAX1 (∆NAtCAX1 or AtCAX1-C), C-truncated AtCAX1 (∆CAtCAX1 or AtCAX1-N), N-truncated AtCAX4 (∆NAtCAX4 or AtCAX4-C), and C-truncated AtCAX4 (∆CAtCAX4 or AtCAX4-N) were cotransformed into Y2HGold strains to confirm the interaction. The obtained results demonstrated that full-length AtCAX1 and AtCAX4 produce blue colonies, thus confirming their ability to interact with each other (AtCAX1 (AtCAX1-N) or C-terminal sequences of AtCAX1 (AtCAX1-C), and the N-terminal sequence of AtCAX4 (AtCAX4-N) were present. However, the presence of only C-terminal sequences of AtCAX1 (AtCAX1-C) and the C-terminal sequence of AtCAX4 (AtCAX4-C) did not produce blue colonies  or C-terminal sequences of AtCAX1-nGFP (AtCAX1-C) and the N-terminal sequence of AtCAX4-cGFP (AtCAX4-N) were expressed together in onion cells D. These 2 and peaked at 12 h or 6 h after treatment in leaf and root tissues, respectively, and then declined. In contrast, AtCAX4 mRNA expression gradually increased in both leaf and root tissues, peaking at 24 h  analysis conducted on various tissues indicated that AtCAX1 and AtCAX4 are expressed in all Arabidopsis plant organs, with the expression level of AtCAX1 being higher in silique and leaf tissues, while AtCAX4 was expressed more highly in root tissues A. The le at 24 h D,E. ThesA T-DNA insertion located in the third exon of AtCAX1 and eight introns in AtCAX4  was confatcax1, atcax4, and atcax1/atcax4 double mutants. WT plants also grew better than the transgenic lines on salt-amended media. In contrast, the growth rates of atcax1, atcax4, atcax1/atcax4, and WT did not obviously differ when seeds were germinated and grown on a medium that was not amended with NaCl   medium supplemented with different concentrations of NaCl A, assessith NaCl A. Moreov 135 mM) A. The senditions B. OveralAtCAX1 and AtCAX4 transgenic plants to salt and ion stress was further assessed in vivo to determine the effect of the AtCAX1 and AtCAX4 interaction on abiotic stress tolerance. Results indicated that the sensitivity of atcax1 and atcax4 mutants to NaCl stress increased with NaCl concentration, while the tolerance of plants overexpressing AtCAX1 or AtCAX4 to salt stress was significantly enhanced. This was mainly manifested in significantly longer root length in transgenic plants relative to WT plants, while atcax1 and atcax4 mutants had the shortest roots  are involved as a second messenger in many physiological processes, where they play a regulatory role in transducing cell signals involved in the growth and stress response of plants. The CAX family is thought to play an important role in regulating intracellular Ca2+ and the balance of other cations.Calcium ions , relative to WT and single mutants, when planted in soil. When seeds were germinated and grown on a culture medium amended with the different levels of Ca2+, the atcax1/atcax3 double mutant exhibited a higher sensitivity to Ca2+, exhibiting poorer growth, producing shorter plants, and a greater level of leaf tip necrosis, relative to WT and single mutant plants. Notably, the growth rate of the atcax1/atcax3 double mutant on a medium containing 15 mM MgCl2 was better than the control and the atcax3 single mutant, while the poor growth rate of the atcax1/atcax3 double mutation was improved by MgCl2 [AtCAX3 and AtCAX4 in Arabidopsis thaliana was significantly increased in atcax1 inserted mutant plants [2+/H+ transport activity. The interaction of AtCAX1  and AtCAX4  was detected in the Y2H system used in the current study. The Ca2+ transport capacity of full-length AtCAX1 can be specifically activated by the CAX-interacting protein, CXIP4 [+/H+ exchanger isoform 1 (NHE1) and the cyanobacterial Na+/H+ antiporter [There are six CAX members in C assays . Previouby MgCl2 . In addit plants . These sn, CXIP4 . The C-ttiporter ,35,36. OAtCAX1 and AtCAX4 in Arabidopsis in response to different salt and ion treatments. In addition to the effect of NaCl, we also assessed the effect of Ca2+ treatments on the expression of CAX1 and CAX4. Results indicated that CAX1 and CAX4 expression was induced to different degrees in roots and leaves  and CAX4 RNA interference (CAX4 RNAi) lines were reported to exhibit altered root growth and development in response to Cd2+, Mn2+, and auxin [CAX4 is most highly expressed in roots  or AtCAX4 (∆NAtCAX4 or AtCAX4-C) and C-terminal truncated 36 aa sequences of AtCAX1 (∆CAtCAX1 or AtCAX1-N) or AtCAX4 (∆CAtCAX4 or AtCAX4-N) were amplified using the primers listed in Saccharomyces cerevisiae strain in the Y2HGold and cultured on SD media lacking leucine and tryptophan (SD/-Leu/-Trp) and SD media lacking leucine, tryptophan, histidine, and adenine and containing Aureobasidin A (AbA) (SD/–Leu/–Trp/–His/–Ade+AbA), as described in [HIS3, ADE2, AUR1-C, and MEL1), which were activated by a positive interaction between interacting CAX proteins.Y2H experiments were performed with the Matchmaker™Gold Yeast Two-Hybrid System as described by the manufacturer . Full-length ribed in . The intAtCAX1 and AtCAX4, as well as the N-terminal truncated of AtCAX1 (∆NAtCAX1 or AtCAX1-C), and C-terminal truncated sequences of AtCAX1 (∆CAtCAX1 or AtCAX1-N) or AtCAX4 (∆CAtCAX4 or AtCAX4-N) were amplified using the primers listed in The vector for BiFC was constructed by replacing the GFP in pBS-35S-GFP with the N-terminus (154 aa) or C-terminus (80 aa) of the mVenus plasmid, yielding pBS-35S: VN154 (nGFP) and pBS-35S:VC80 (cGFP), respectively . Full-leArabidopsis thaliana ecotype Columbia-0 (Col-0) was used in this study as wild-type (WT), T-DNA insertion mutants, and to create transgenic plants. The AtCAX1 and AtCAX4 T-DNA insertion mutant SALK-108310 (atcax1) and SALK-201217C (atcax4) were obtained from the Arabidopsis Biological Resource Center (ABRC: http://www.arabidopsis.org/). The mutant lines atcax1 and atcax4 were crossed to generate double mutants (atcax1/atcax4), and homozygous double mutant plants were screened from the resulting F2 progeny by PCR. Homozygous mutant plants were identified using the gene-specific primers and T-DNA left border-specific primers listed in Arabidopsis  transgenic plants were generated: 35S::AtCAX1/Col-0 (AtCAX1OE), and 35S::AtCAX1/Col-0 (AtCAX4OE). The full-length coding sequence of AtCAX1 and AtCAX4 were separately amplified and individually ligated into the pBI121 vector under the control of the 35S promoter to generate transgenic OEAtCAX1 and OEAtCAX4 constructs. The resulting plasmids were introduced into wild-type Arabidopsis thaliana, ecotype Columbia plants using a floral dip infiltration method for Agrobacterium tumefaciens-mediated transformation [The following ormation . Homozygatcax1, atcax4, atcax1/atcax4 mutants, OE,AtCAX1 and OEAtCAX4 were surface sterilized and placed on 1/2 Murashige and Skoog (MS) medium supplemented with different concentrations of NaCl (125 and 135 mM), or CaCl2 (50 and 65 mM). After 14 days, seedling phenotypes were photographed, and the root length of the seedlings were measured. The experiment was repeated three times. Significant statistical differences between treatment means were determined at p < 0.05 using a Student’s t-tests.For the stress tolerance assay, 30 seeds of Col-0, AtCAX1-RT-FW and AtCAX1-RT-RV were used for AtCAX1, AtCAX4-RT-FW and AtCAX4-RT-RV were used for AtCAX4, while Actin-FW and Actin-RV were used to amplify the Actin gene .Total RNA was isolated using a RNeasy plant Mini kit  and treated with RNasefree DNaseI . First-strand cDNA was synthesized using SuperScript III reverse transcriptase . Pairs of gene-specific primer pairs, tin gene . TranscrArabidopsis lines was separated on a 1% (m/v) agarose–formaldehyde gels and transferred to Hybond N+ membranes. Hybridizations were carried out at 50 °C using AtCAX1 and AtCAX4 probes labeled with digoxigenin . Signals were detected with CDP-Star using a luminescent image analyzer . The single lines were named #1, #2, and #3, respectively.For northern blotting analysis, total RNA (10 µg) obtained from transgenic Arabidopsis seeds were surface sterilized and placed on solid half MS medium. After 2 days of stratification at 4 °C, the plates were moved to a 22 °C incubator for propagation. The seedlings were transferred from the plates to a 1:1 mixture of soil and vermiculite and grown to maturity at 22 °C. The plants were grown under a 16-hlight/8-h-dark cycle in a growth chamber. Roots, stems, leaves, panicle, and siliques of two-month-old plants were sampled and used in the RT-qPCR analysis. A second batch of seedlings was pre-cultured for 2 weeks on 1/2 solid medium and then treated with different concentrations of NaCl (125 and 135 mM) and CaCl2 (50 and 65 mM). Shoots and roots were sampled after 0, 6, 12, and 24 h after treatment and used in the RT-qPCR analyses.Gene expression was quantified by RT-qPCR. AtCAX4 with AtCAX1, CAX family members in Arabidopsis, was demonstrated using a yeast two-hybrid system and a bimolecular fluorescence complementation assay. This is the first study to report the interaction between CAX1 and CAX4 in plants. Further experiments demonstrated that the AtCAX1 and AtCAX4 expression is induced in roots and leaves in response to abiotic stress (NaCl and Ca2+). Functional analysis of AtCAX1 and AtCAX4 in Arabidopsis demonstrated that atcax1, atcax4 single mutants, and atcax1/atcax4 double mutants were more sensitive to abiotic stress than WT plants. In contrast, the overexpression of AtCAX1 or AtCAX4 in Arabidopsis improves abiotic stress tolerance. AtCAX1 and AtCAX4 may form a heterodimer in plants in response to abiotic stress.In the present study, the interaction between the N-terminal region of


Secondary acute myeloid leukemia (sAML) is a high-risk AML evolving from heterogenous prior hematological disorders. Compared to de novo AML, sAML has even worse responses to current therapy and thus is associated with lower remission rates, inferior overall survival (OS) and higher relapse rates. Many efforts have been devoted to improving the overall but with limited success, and novel strategy is thus highly needed. Recent research has identified that CLL1 is highly expressed on AML leukemia stem cells and blasts cells but not on normal hematopoietic stem cells. In this case report, we treated a secondary AML patient with anti -CLL1 CAR-T therapy and achieved morphological, immunophenotypic and molecular complete remission for over 10 months. Although only one successful case is presented here, the anti-CLL1 CAR T-cells should be considered as another treatment option for secondary AML in the future. The prognosis of patients with acute myeloid leukemia (AML) has been improving in the last decades through the intensification of combination chemotherapy and hematopoietic stem cell transplantation. However, the 5-year survival rate of patients with AML varies from 33.3–79.5% worldwide, as documented in the recent CONCORD-2 study , with a de novo AML, sAML has worse response to current therapy, and thus is associated with lower remission rates, inferior overall survival (OS), and higher relapse rates (Secondary AML (sAML) is considered high-risk as it evolves from heterogenous prior hematological disorders such as myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), and other malignancies exposed to cytotoxic agents or radiation therapy . Comparese rates . Many efse rates .ex vivo, primary human AML cells, and human AML patient-derived xenograft mice and monkey models in vivo; however, direct evidence regarding responses in patients with AML has not been reported.Chimeric antigen receptor (CAR) T cells have emerged as a highly effective therapy for relapsed/refractory hematological malignancies –6; howevIn this case report, we treated a patient with secondary AML using anti-CLL1 CAR-T cells therapy and achieved morphological, immunophenotypic, and molecular complete remission for over a 10-month period. Although only one successful case is presented here, the anti-CLL1 CAR T-cells should be considered as another treatment option for secondary AML in the future.+CD38+CD10+CD19+CD123+ immunophenotype. Initially, she was enrolled into the GD-2008-ALL clinical trial (NCT00846703) and then achieved complete remission (CR) after one course of induction therapy, while her minimal residual disease (MRD) evaluated by flow cytometry remained above 0.01% during all treatment phases (+CD38+CD10dimCD19dimCD123+CD33+CLL1+) and not from the B cell lineage  relapse. Bone marrow (BM) aspiration was performed every 2 weeks from July 2018, and blast percentage gradually increased from initial 7.5% to 19.5% within 1 month of observation . Furthert phases . Unfortut phases . We then lineage . Moreovemutation . Resultsmutation , which c2/d and fludarabine 30 mg/m2/d for 4 days) before CAR T-cell transfer to enhance in vivo expansion of CAR T-cells, after which she received a single dose  infusion over 10 min through a peripherally inserted central venous catheter on 11 September 2018. Subsequently, she experienced Grade I-II cytokine release syndrome (CRS) manifested by a temperature ≥38°C; at this time, she developed transient hypotension requiring fluid resuscitation under the CAR T-cell therapy management consensus guidelines  containing a CLL1-specific single chain variable fragment, in combination with a fourth-generation CAR lentiviral vector (4SCAR) carrying T cell costimulatory signals for CD28-CD27-CD3z . The 4SCidelines , 16. Seridelines . After t therapy . Upon co therapy . However therapy , during  therapy . Even wiIn this reported case, the main finding was the achievement of a surprisingly long-term complete remission with anti-CLL1 CAR T-cells therapy. Only grade I-II cytokine release syndrome was observed and successfully managed.Anthracyclines- and cytarabine-based conventional chemotherapy are the main treatments for AML patients; however, both have significant toxicities. In addition, the overall prognosis for patients with this treatment has remained stagnant in the last two decades , 19. WitIn contrast to chemotherapy and new FDA-approved targeting agents, cellular CAR-T cell therapy and cytotoxic T lymphocytes can minimize systemic cytotoxicity and morbidity while generating maximal anti-tumor activity . HoweverIn conclusion, our prolonged survival observation in this patient with sAML showed that anti-CLL1 CAR-T might be of great potential in AML treatment. Interestingly, Liu F et al. has reported the efficacy of combination CLL1-CD33 CAR-T cells in a 6-year-old girl with sAML transformed from prior faconi anemia and juvenile myelomonocytic leukemia . Given tThe studies involving human participants were reviewed and approved by GIMI-IRB-16001. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.HZ, W-TG, and L-JC performed the research, analyzed the data, and wrote the paper. W-GH and P-FW included the patients and collected clinical data. Z-YL contributed to clinical nursing. All authors critically revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In terms of redox effects, δ-tocopherol was the only isomer that evoked a significant change in the HyPer signal at 5 μM. By mimicking Ca2+ entry with ionomycin and monensin, a decline in the HyPer signal was induced as well. Finally, by silencing calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), an intracellular Ca2+ chelator, none of the isomers were able to induce redox changes. Altogether, our data indicate that an elevation in cytoplasmic Ca2+ is necessary for the development of a tocopherol-induced antioxidant impact on the cytoplasm of Caco-2 cells reported by HyPer biosensor.Most of the biological impacts of Vitamin E, including the redox effects, have been raised from studies with α-tocopherol only, despite the fact that tocopherol-containing foods carry mixed tocopherol isomers. Here, we investigated the cellular mechanisms involved in the immediate antioxidant responses evoked by α-, γ- and δ-tocopherol in Caco-2 cells. In order to track the cytosolic redox impact, we performed imaging on cells expressing HyPer, a fluorescent redox biosensor, while cytosolic calcium fluctuations were monitored by means of Fura-2 dye and imaging. With this approach, we could observe fast cellular responses evoked by the addition of α-, γ- and δ-tocopherol at concentrations as low as 2.5 μM. Each isomer induced rapid and consistent increases in cytosolic calcium with fast kinetics, which were affected by chelation of extracellular Ca Vitamin E is a term that groups tocopherols and tocotrienols. However, the concept has evolved to refer mainly to α-tocopherol, likely due to its high relative abundance in plasma and blood cells . This noOn the other hand, the relative abundance of tocopherol isomers in food is quite diverse. Using electroanalytical techniques, Robledo et al. compared the content of tocopherol isomers in edible oils such as corn, soybean and canola, among others. Between them, γ-tocopherol represented more than 65% of the total Vitamin E content . InteresThe antioxidant properties of tocopherol isomers determined by chemical-based assays have been well known since 1996 . Particu2+ and redox fluctuations induced by α-, γ- and δ-tocopherol isomers and α-tocotrienol in living Caco-2 cells. Both phenomena were tracked with a temporal resolution of seconds in order to gain information about the first cellular components affected by the tocopherol/tocotrienol arrival to the plasma membrane and if they participate in the redox properties of these compounds. Our findings indicate that upon exposure to isomers at concentrations as low as 2.5 μM, cytosolic Ca2+ levels experienced a fast increase, showing different kinetic patterns among the isomers. By chelating the extracellular Ca2+, the cytoplasmic Ca2+ elevations induced by all isomers were suppressed, indicating that the calcium response was dependent on Ca2+ entry. Similarly, differential redox responses were induced upon isomer exposure in HyPer-expressing Caco-2 cells with higher impact of δ-tocopherol at low concentrations than any of the other isomers. Finally, by mimicking an elevation in cytosolic Ca2+ with ionophores and avoiding Ca2+ responses with an intracellular calcium chelator, we unveiled a fast connection between calcium signalling and redox adjustment in living Caco-2 cells. In summary, our data show that the immediate antioxidant effect of tocopherol isomers is mediated by calcium signalling, which in turn is dependent on the opening of Ca2+ permeability at the plasma membrane of living Caco-2 cells.In this work, we followed the cytosolic CaReagents and salts for buffer preparation were obtained from Sigma-Aldrich . Media culture and supplements were purchased from Life Technologies . Ionophores like ionomycin and monensin were acquired from Tocris . Fura-2 AM and SBFI-AM dyes, along with pluronic acid, were acquired from Thermo Fisher Scientific . Tocopherols  were purchased from Sigma-Aldrich , α-tocotrienol was purchased from Cayman Chemicals Company . Stock solutions were prepared in absolute ethanol at 50 mM. The 30% hydrogen peroxide solution and absolute ethanol used in this work were from Merck .TM, colorectal adenocarcinoma) were purchased from the American Type Culture Collection  and cultured in Minimum Essential Medium supplemented with 10% fetal bovine serum. In general terms, cells were maintained under a humidified atmosphere with 5% CO2/air, and culture medium was renewed every 2 or 3 days. When 70–80% confluence was reached, cultures were expanded to other plaques or seeded on glass coverslips  to be further imaged. Cells were used at passages 20–40.Caco-2 cells  supplemented with 5 mM glucose (KRH-glc). Dyes were prepared in 0.01% pluronic acid in DMSO solution; 5 μM of the selected dye was used to load the cells for 30 min at room temperature in KRH-glc. After that time, cells were rinsed three times with KRH-glc and left for another 20 min to allow complete de-esterification of the acetoxymethyl ester (AM) motif from the remanent intracellular dye.For imaging experiments, the coverslips were mounted in an open recording chamber, and media were replaced by Krebs Ringer HEPES (KRH) buffer  equipped with 40× oil objectives . A xenon lamp was coupled to the monochromator device , which allowed dual excitation at 340 and 380 nm for Fura-2 and SBFI, respectively. Emission over 520 nm was collected by a long-pass filter, fluorescence was digitalized by a cooled charge-coupled device (CCD) camera  and image analysis was performed using free micromanager software  . Data saThe HyPer biosensor was introduced into the cytoplasm of Caco-2 cells by infection with adenovirus at 1:100 dilution. Adeno-particle production has been explained in detail by our group . BrieflyTwo days after infection, HyPer was homogeneously expressed in the cytosol with no signs of cytotoxicity, and cells were healthy and full-adhered to the glass surface, ready for imaging. As described for calcium imaging, coverslips containing HyPer-expressing Caco-2 cells were mounted in an open recording chamber with KRH-glc buffer. The HyPer biosensor was excited at 420 and 490 nm, whereas the emitted light was collected with a long-pass filter over 520 nm. For every recording, an initial lapse of 20 min was done to ensure a reliable and stable baseline, which functions as the basal value.t-tests were executed only for comparisons of before-after experimental designs, whereas comparisons of multiple groups or repeated measurements were both evaluated by ANOVA with Bonferroni post hoc analysis for parametric data or Dunn’s method for nonparametric data. A p-value less than 0.05 was considered statistically significant. In order to classify cells that elicited a positive calcium response to tocopherol exposure, which we called responding cells, we defined a threshold of 3 standard deviations (SD) over baseline value to consider an increase in the cytosolic calcium level; this analysis was performed by comparing mean values and SD obtained from 3-min bins before tocopherol exposure and two sequential bins after tocopherol exposures (3 or 6 min). A similar analysis was performed with HyPer data as well. Ca2+, Na+ and redox cellular responses elicited by tocopherol/tocotrienol isomers took place on cells with the same passage and the same day. Throughout the manuscript, data are expressed as mean ± standard error. Paired Student’s 2+ indicator, we detected rapid increases in Ca2+ upon exposure to α-, γ- and δ-tocopherol at concentrations as low as 2.5 μM  of Caco-2 cells were responsive to α-tocopherol during the first 3 min of exposure, which augmented to 86% in the next 3 min of exposure. Under the same analysis, the percentage of cells responding to γ-tocopherol was more stable; 60% and 52% of 79 imaged cells were quantified in the first 3 min and the next 3 min of exposure, respectively. δ-tocopherol, on the other hand, showed 68% of cells responding during the first 3 min, which evolved to 97% for the next 3 min of recording (31 single-cell recordings). From this real-time Ca2+ imaging, we presented the differential acute effects on cytosolic Ca2+ evoked by tocopherol isomers, in which δ-tocopherol-induced Ca2+ increases developed more gradually than those induced by the other isomers. In addition to tocopherols, we also evaluated the capacity of α-tocotrienol to induce cytosolic Ca2+ increases. Exposure to 5 μM α-tocotrienol did not evoke significant changes in the cytoplasmic Ca2+ levels; however, at 50 μM, some cells (4 out of 17) experienced Ca2+ augments -N,N,N′,N′-tetraacetic acid (EGTA), a calcium chelator. Under Ca2+ free medium, the number of tocopherol-responding cells and the magnitude of calcium responses diminished significantly. It is noteworthy to point out that in the absence of extracellular Ca2+, the immediate peak evoked either by α- or γ-tocopherol disappeared, suggesting that this fast component of the calcium response has an extracellular source. On the other hand, Ca2+ responses evoked by δ-tocopherol, which lacked this early peak, elicited a slow development, an observation that was noticeable even in the absence of extracellular Ca2+, indicating that δ-tocopherol may trigger a calcium mobilization from intracellular sources  was necessary to mobilize cytosolic calcium in primary rat aortic smooth muscle and HEK293 cells [2+ increase that developed rapidly and transiently. However, the authors did not explore the contribution of extracellular calcium, assuming that most of the calcium effect was due to protein G-coupled receptor signalling. Our experiments indicate that the fast cytoplasmic calcium responses induced by α- and γ-tocopherols (50 μM) were abolished by the removal of extracellular Ca2+, suggesting an interaction of these isomers with other entities capable of permeating this divalent ion through the plasma membrane of Caco-2 cells. TRPC5, a member of the transient receptor potential (TRP) family, is a candidate for connecting calcium signalling with the extracellular presence of antioxidant compounds, since this channel presents an extracellular pair of cysteines able to form a disulfide bond; this structural feature works as a redox switch that controls the subcellular traffic, stabilization and activity of this channel [2+ in Caco-2 cells, a general phenomenon that has the potential to promote the intestinal barrier function by stimulating mucin production, as demonstrated in HT-29 cell line [2+-activated cationic channel [2+ might also improve the thickness and stagnancy of secreted mucus by participating in the secretion of the calcium-activated chloride channel regulator I, a very abundant metalloprotease in intestinal cells. Once at the extracellular space, this metalloprotease is able to process the inner mucus layer, modifying its physicochemical properties [2+ have unveiled an interaction between tocopherols and molecular entities at the plasma membrane that conduct calcium responses, which in turn affect the redox homeostasis of intestinal cells.All three tocopherol isomers evaluated induced calcium responses within seconds to minutes, albeit with some variability through the recordings. In Caco-2 cells, tocopherol uptake is mainly mediated by cholesterol transport carriers such as Scavenger Receptors class B type I (SR-BI)  and Niem93 cells . Interes channel . DTT and channel . In addi channel . All theell line , or stim channel . Increasoperties . Our exp2+ entry in terms of seconds, suggesting that the disulfide bond reduction observed in the biosensor occurred by a Ca2+-dependent cytoplasmic mechanism and not by a direct antioxidant action of tocopherols. Such calcium-coupled redox events have been observed in HEK293 cells by simultaneous tracking of cytoplasmic calcium and H2O2 after a short exposure to thapsigargin (2 min). This treatment induced a transient cytoplasmic calcium elevation that decreased gradually with time. HyPer signal, on the other hand, exhibited an initial small increase, to later, in approximately 10 min, stabilize at a new level with lower values than those observed at the baseline. These results indicate that the redox tone in the cytoplasm of HEK293 reaches a more reduced level, triggered initially by the thapsigargin-evoked Ca2+ increase [Regarding the acute redox effects elicited by tocopherol molecules recorded in this work, we consider the plasma membrane as the first cellular structure where tocopherol must interact. Ultrastructural studies on artificial bilayers indicate that the hydroxyl group located at the chromanol ring of α-tocopherol molecules faces the aqueous interface in both extra- and intracellular compartments, suggesting that tocopherols can exert their antioxidant feature not only restricted to the central lipid environment but also connecting the soluble components from the extra- and intracellular environment of living cells . Howeverincrease . In primincrease . Here, w2O2 for 40 min [2O2 (24 h) with some differences in the level of lipid peroxidation induced by H2O2. Certainly, the longer the exposure with tocopherol/tocotrienol, the more chances the compounds have to interact with cell components. In osteocyte-like cells, 2 h of treatment with δ-tocotrienol (25 μM) was effective to protect cells against death induced by 3 h of tert-butyl hydroperoxide (250 μM), a protection that was sensitive to the pharmacological inhibition of Nrf2 as well as PI3K signalling [We have exposed differential acute effects of tocopherol isomers on the cytoplasmic redox status, a phenomenon that requires the mobilization of calcium, suggesting that tocopherols, beyond their redox properties as ROS scavengers, have a redox impact by interacting with the cellular machinery of human intestinal cells. In other cellular models, such tocopherol as tocotrienol isomers has been studied regarding its capacity to protect cells from an oxidative challenge. For instance, 24 h preincubation with α-tocopherol or α-tocotrienol showed no differences in avoiding ROS generation in SH-SH5Y cells subjected to 1 μM Hr 40 min . In ceregnalling , supportThe tocopherol concentrations used in this work were between 2.5 to 50 μM, a range frequently found in plasma of healthy subjects  or subjeTocopherol isomers evoked fast calcium responses in Caco-2 cells with different kinetics.δ-tocopherol was the only isomer capable of inducing a redox change in HyPer biosensor at 5 µM.2+, but not in Na+, was necessary and sufficient to induce a reduction in the HyPer biosensor for all the isomers evaluated.An increase in cytosolic Ca2+ increase induced by both ionophores, ionomycin and monensin, was enough to generate a redox change in HyPer.A massive cytosolic Ca


The structural changes in HF RV myocytes correlate with more frequent spontaneous Ca2+ release activity than in control RV myocytes. This is accompanied by hyperactivated L-type Ca2+ channels (LTCCs) located specifically in the T-tubules of HF RV myocytes. The increased open probability of tubular LTCCs and Ca2+ sparks activation is linked to protein kinase A-mediated channel phosphorylation that occurs locally in T-tubules. Thus, our approach revealed that alterations in RV myocytes in heart failure are specifically localized in microdomains. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. These data will foster better understanding of mechanisms of heart failure and it could promote an optimized treatment of patients.Right ventricle (RV) dysfunction is an independent predictor of patient survival in heart failure (HF). However, the mechanisms of RV progression towards failing are not well understood. We studied cellular mechanisms of RV remodelling in a rat model of left ventricle myocardial infarction (MI)-caused HF. RV myocytes from HF rats show significant cellular hypertrophy accompanied with a disruption of transverse-axial tubular network and surface flattening. Functionally these cells exhibit higher contractility with lower Ca The increased arrhythmogenic propensity after MI is responsible for more than 50% of deaths among patients with HF2. In recent years, right ventricle (RV) dysfunction (reduced ejection fraction) has been shown to be relatively frequent in patients with HF4. RV dysfunction in HF is thought to arise from several factors, including ischemia/infarct of the RV, septal dyssynergia, pulmonary hypertension, neurohormonal activation, and inflammation6. Moreover, patients with left ventricle (LV) systolic dysfunction and HF who develop RV failure have worse prognosis and their average survival is usually less than two years9. Thus, elucidating why the RV function is declined in the settings of LV disorder has a particular importance in understanding the mechanisms of HF.Heart failure (HF) caused by myocardial infarction (MI) is a major cause of hospitalization and mortality worldwide2+-signalling microdomains disrupts excitation–contraction coupling, suppressing cardiomyocyte contractility10, and promotes the development of arrhythmogenic triggers at the whole heart level11. Ventricular myocytes have a well-organized transverse-axial tubular (TAT) network, which brings L-type Ca2+ channels (LTCCs) in close proximity to clusters of ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum, allowing for a synchronous contraction of the cell. Disruption of TAT network and flattening of sarcolemma membrane in HF can provoke a deranged intracellular Ca2+ handling and malignant arrhythmias13. Specifically, in failing LV myocytes (LVMs) LTCC relocate from their predominant localization in T-tubules to the surface membrane (crest) where they can become hyperphosphorylated11. TAT network loss in failing LVMs promotes “orphaning” of RyR2 and facilitates the dyssynchronization of sarcoplasmic reticulum Ca2+ release16.Pathophysiological remodelling in HF occurs at multiple levels. Recent studies of LV dysfunction in HF showed that the progressive disorganization of Ca2+ handling in failing RVMs. We showed that 16 weeks after the left ventricle coronary arterial ligation caused HF, RVMs become hypertrophied with a higher contractility, and showed significant structural changes including a less regular TAT network and a flatter cell surface topography. While the distribution of functional LTCCs in RVMs was preserved in HF, the open probability (Po) of LTCCs was dramatically increased in T-tubules of failing myocytes. The localized increase in LTCC Po was accompanied by an enhanced frequency and a size of spontaneous Ca2+ sparks observed in failing RVMs resulting in elevated local Ca2+ wave frequency. Our findings may indicate the development of compensatory, though potentially arrhythmogenic, RV remodelling in the setting of LV failure. This work provides new insights into nanoscale level of RVMs remodelling in HF.In this work, we used a combination of structural and functional studies to probe the microdomain-specific alteration in local Ca17. However, using directional analysis of T-tubule alignment18, we did not find any changes in T-tubule fraction oriented transversally and axially in control versus MI RVMs  and propagated Ca2+ waves. The frequency of local Ca2+ waves attributed to a local cluster of Ca2+ sparks was 80% higher in MI than in control RVM  of LTCC at T-tubule and crest was analysed at the activation step of -6.7 mV. In control RVMs, the Po of LTCC from T-tubules was significantly lower as compared to the Po of LTCC from crest  Act 1986 Amendment Regulations 2012, incorporating the EU Directive 2010/63/EU, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Approval for this work was obtained from the Animal Welfare and Ethics Review Board (AWERB) of Imperial College London.10. Rats were kept for 16 weeks after MI surgery to allow development of heart failure condition. RVMs were isolated using a standard enzymatic protocol as described previously40, full details of myocyte isolation can be found in supplementary materials.MI was performed in rats by left anterior descending coronary artery ligation19. Z-groove ratio was calculated as a total Z-groove length observed on the image normalized by the predicted value19. TAT network was visualized in live myocytes via Di-8-ANEPPS membrane staining. Analysis of TAT network was performed as described before18. Detailed description of the analysis can be found in supplementary materials.Surface topography of live myocytes was visualized by scanning ion conductance microscopy (SICM), which uses a glass nanopipette as a sensitive probe 11. The pipette was then positioned to a desired location and lowered until a contact with the membrane was established and a high resistance seal achieved. Single LTCC recordings were performed using cell-attached patch-clamp as described in detail previously11. Full details of the protocols and solutions can be found in supplementary material.After visualization of the myocyte surface topography, the scanning pipette was clipped according to the established protocol41. Cells were paced at 0.5 Hz and sarcomere shortening and calcium transient were recorded for multiple cells selected. The data was analyzed using Transient Analyses Tool Software .Cardiomyocytes attached to laminin were loaded with 1uM of Fura-2AM. CytoCypher system setup and operation has been previously described2+ optical mapping in cells loaded with the Ca2+-sensitive fluorescent dye Fluo-4AM was performed via CMOS camera ULTIMA-L  at 500 fps, 1.5–2 μm/pixel, connected to an inverted Nikon Eclipse Ti microscope42. All spontaneous Ca2+ releases were divided into local (occupying a portion of the cell surface and attributed to discrete clusters of Ca2+ sparks) and propagated waves, that propagate though the whole surface. Single Ca2+ sparks were monitored using line-mode of the confocal microscope10. The analysis of Ca2+ sparks images was performed using SparkMaster plugin in FIJI with a determination criterion of 4.2. Full details of can be found in supplementary material.CaAll graphs were produced and statistical analysis was performed using GraphPad Prism 6 . Normality was tested using the Kolmogorov–Smirnov test. Statistical differences were assessed with Student t-test, Mann–Whitney test, one-way analysis of variance (ANOVA) and Kruskal–Wallis test. All data are expressed as mean ± standard error of mean (S.E.M.). A value of P < 0.05 was considered statistically significant.Supplementary Information.


MCU activity is regulated by several intrinsic proteins including MICU1, MICU2, and EMRE. While significant details about the role of MICU1, MICU2, and EMRE in MCU function have emerged recently, a key challenge for the future experiments is to investigate how these regulatory proteins modulate mitochondrial Ca2+ influx through MCU in intact cells under pathophysiological conditions. This is further complicated by the fact that several variables affecting MCU function change dynamically as cell functions. To overcome this void, we develop a data-driven model that closely replicates the behavior of MCU under a wide range of cytosolic Ca2+ ([Ca2+]C), [Ca2+]M, and mitochondrial membrane potential values in WT, MICU1 knockout (KO), and MICU2 KO cells at the single mitochondrion and whole-cell levels. The model is extended to investigate how MICU1 or MICU2 KO affect mitochondrial function. Moreover, we show how Ca2+ buffering proteins, the separation between mitochondrion and Ca2+-releasing stores, and the duration of opening of Ca2+-releasing channels affect mitochondrial function under different conditions. Finally, we demonstrate an easy extension of the model to single channel function of MCU.Mitochondrial Ca Below this critical concentration, Ca2+ uptake through MCU is essentially negligible. MICU1 is also responsible for the observed highly cooperative activation of MCU activity where the gatekeeping is cooperatively relieved when [Ca2+]C exceeds 1.3 μM [2+]C threshold for the gatekeeping and reduce the gain of cooperative activation of MCU activity [2+]M is approximately 400 nM [2+]M drops below or rises above 400 nM  values in WT, MICU1 KO, and MICU2 KO cells; (2) combine the observations at the cell culture (whole-cell) and single mitoplasts levels into a single mathematical framework; (3) investigate how the Ca2+ signaling in the microdomain between the endoplasmic reticulum (ER) and mitochondrion compares in WT, MICU1 KO, and MICU2 KO mitoplasts by modeling the interaction between the ER-bound inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) channel and MCU; (4) how does MICU1 or MICU2 KO affect mitochondrial function both in terms of Ca2+ uptake and ATP production; and (5) investigate the effect of Ca2+ buffers, width of the microdomain, and open duration of IP3R on mitochondrial Ca2+ uptake and ATP production in WT, MICU1 KO, and MICU2 KO cells.In this paper, we (1) develop a comprehensive data-driven model that reproduces the key observations about the function of MCU under a range of [CaThe experimental data used in this paper is adapted from Riley et al.  and VaisOP) on [Ca2+]C, [Ca2+]M, and Δψ, combining a wide range of observations from patch clamp electrophysiology of individual mitoplasts and fluorescence microscopy of mitochondrial networks in cell cultures. The model assumes Ca2+ sensors on both sides of the IMM. On the cytosolic side, both MICU1 and MICU2 can bind up to two Ca2+ (both MICU1 and MICU2 have two EF hands each and each EF hand can bind one Ca2+) (2+]C regime (0–1.3 μM), whereas MICU2 interacts with MICU1 to increase the [Ca2+]C threshold for MCU activation and reduces the gain of cooperative activation of MCU activity. Thus, the channel is open when Ca2+ is bound to both EF hands of MICU1 and MICU2. On the matrix side, MCU activity is regulated by coupled inhibitory and activating Ca2+ sensors. At intermediate [Ca2+]M (50–800 nM), Ca2+ binds to the inhibitory sensor that closes the channel . At high [Ca2+]M, Ca2+ binds to the activating senor, relieving the gatekeeping on the matrix side of the IMM. Our best fit criterion (BIC score) warrants the binding of three Ca2+ to the activating sensor in addition to the one Ca2+ that is bound in state 41C to open the channel again (state 44O). More recently, it was shown that the affinities of activating and inhibitory sensors on the matrix side are regulated by another Ca2+ sensor termed “flux sensor” C)X([Ca2+]M)Y with occupancy parameters 00C to that state  as a function of [Ca2+]C and [Ca2+]M is given by2+ uptake rate for each experiment was determined by fitting an exponential function to [Ca2+]C beginning from Ca2+ addition until a new steady-state is reached 300 s later to obtain parameters A (extent of uptake) and τ (time constant). The instantaneous rate of uptake at t = 0 was taken as equal to A/τ. In these experiments, only mitochondrial Ca2+ uptake through MCU was active, whereas all other Ca2+ pathways were either pharmacologically blocked or were inactive  ,35. In oeader to ,35 for aeriments . We remasee Ref.  for deta2+ buffers, equilibrating Ca2+ concentration in bath solution and cytoplasm. Ca2+ uptake into the ER and mitochondrial efflux were inhibited by applying 2 μM thapsigargin (Sarco/ER Ca2+ ATPase (SERCA) blocker) and 20 μM CGP37157 (Na+/Ca2+ exchange blocker) at 100 and 400 s, respectively. Similarly, no inositol 1,4,5-trisphosphate (IP3) was used in these experiments, implying no Ca2+ efflux from the ER through IP3 receptor (IP3R) channels. After [Ca2+]C reached a steady-state at 700 s, MCU-mediated Ca2+ uptake was initiated by adding boluses of CaCl2 to achieve increases in [Ca2+]C between 100 nM and 10 μM. All these considerations lead to the following rate equations for [Ca2+]C and [Ca2+]M, respectively.StimV represents Ca2+ stimulus (bolus of CaCl2). The value of StimV is set so that the peak [Ca2+]C given by the model matches the observed value. Parameters m =f 3 × 10−4 and δ = 0.067 are the Ca2+ buffering of mitochondria and the ratio of mitochondrial to cytosolic volume [2+]M. We add LeakV to the rate equation for [Ca2+]C to incorporate the effect of potential Ca2+ leak from the ER to the cytoplasm:2+ concentration in the ER.In the cell culture experiments, HEK293 cells were treated with 0.004% digitonin at 50 s after the beginning of the experiment to permeabilize plasma membrane in intracellular-like medium lacking free Cac volume ,37. As wM), mitochondrial ADP concentration ([ADP]M), Δψ, and cytosolic ADP concentration ([ADP]C). The rate equations, relevant fluxes (with the exception of MCUV and NaCaV), and parameters are adopted from Ref. i = 11 × 103 μM, F, R, and T = 310.16 oK is the cytosolic Na+ concentration, Faraday’s constant, gas constant, and temperature, respectively. Note that Δ0ψ = 91 mV does not represent the resting MMP. It was originally selected so that Δψ = Δ0ψ, i.e., under uncoupled conditions M in WT, MICU1, and MICU2 KO conditions. The full model used for mitochondrial function at the whole-cell level consists of Equations (7) (with Equations (3) and (8)) and To assess how MICU1 or MICU2 KO affect mitochondrial function, we extend the model to incorporate the dynamics of mitochondrial NADH concentration ([NADH]rom Ref.  and are  (VNaCa) .(7)d[Canditions . As we d2+ uptake rate as a function of [Ca2+]C observed in cell cultures using fluorescence microscopy on WT , MICU1 KO , and MICU2 KO  cells are shown in 2+]C regime (0–1.3 μM), the cooperative opening once the gatekeeping is relieved, relief of gatekeeping and reduced cooperative activation in MICU1 KO cells, and reduced threshold for gatekeeping and increased gain of cooperative activation in MICU2 KO cells. We remark that our fitting criterion searches for the parameters that result in the best fit to all observations simultaneously. For example, in case of MICU1 KO experiments, we do not restrict our search to states that are associated with MICU1 only. In other words, we make no a priori assumption about the independent binding of Ca2+ to the MICU1 EF hands. This is motivated by the complex structure of MCU C and [Ca2+]M for the fit. However, the experiments on cell cultures report the uptake rate as a function of [Ca2+]C, whereas the values of [Ca2+]M at the [Ca2+]C used are not known. In one set of experiments, Vias et al. [2+]C and [Ca2+]M simultaneously (without measuring MCU activity)  observed at the single mitoplast level [2+]C and [Ca2+]M are known in the patch clamp experiments, we choose the first set of parameters . We usest level  . However, we found that such change is not supported as the model predicts higher Ca2+ uptake rate in WT cells  observed in patch clamp experiments, which is equal to 206, 237, and 217 pA/pF in case of WT, MICU1 KO, and MICU2 KO mitoplasts, respectively. The model fits a range of observations about individual mitoplasts. The theoretical curves mirror the inverted bell-shaped behavior of IMCU/Cm as a function of [Ca2+]M and the lack of inhibition in the MICU1 KO and MICU2 KO mitoplasts  to the single mitoplast model is straightforward. The only parameter that changes while fitting to the single mitoplast data is that toplasts C. We alstoplasts D. The di2+]M with a minimum (a maximum suppression of 75%) at 0.4 μM at [Ca2+]C = 1 mM in WT mitoplasts  shifts to even lower [Ca2+]M values and eventually disappears as we decrease [Ca2+]C  is another key variable influencing current through MCU by providing a driving force for Caare open ,43. 2+]C, [Ca2+]M, and Δψ constitutes a robust signaling mechanism, controlling mitochondrial function. For example, raising [Ca2+]C by a few μM increases [Ca2+]M by a few tens of μM [2+]C is raised by 5 μM in patch clamp experiments on WT mitoplasts  gets replaced by the maximum uptake rate observed at the whole-cell level (600 nM/s). The interplay between C in the cell culture experiments (2+]C (>7 μM), the higher MCUV causes [Ca2+]C in MICU2 KO to drop significantly faster than both WT and MICU1 KO cells  given by the model is an order of magnitude lower than that observed experimentally  and the relative volume of mitochondria with respect to cytosol (δ) both play key roles in the free [Ca2+]M. We repeat the simulation in 2+ bolus of different strengths and record the peak [Ca2+]C during the simulation and [Ca2+]M 300 s after the Ca2+ bolus is added to the cytoplasm. As shown in 2+ increases dramatically as we increase mf  or decrease δ . Using the simultaneously measured values of [Ca2+]C and [Ca2+]M from Ref. [mf ~ 0.09 results in a close correspondence between [Ca2+]C and [Ca2+]M when δ = 0.067 is considered. The mf value giving the correct correspondence between model and observations would decrease  as we decrease δ. The peak M) and ATP ([ATP]M) change as mitochondria buffer Ca2+ in WT, MICU1 KO, and MICU2 KO cells. A key parameter missing in our whole-cell Ca2+ model is the maximum flux through Na+/Ca2+ exchanger ). Although we do not have direct access to this parameter in our experimental results, we use the observed resting value of [Ca2+]M to estimate NaCa,max.V Knowing that the Ca2+ fluxes through MCU and Na+/Ca2+ exchanger balance each other out in resting conditions M given by the model in resting state matches the observed value  when compared to WT cells (0.958 nM/s). In simulations shown in nditions , and theed value . The valexchange , our mod2+ to the cytoplasm, raising [Ca2+]C from its resting value of 0.1 μM to 1.5 μM to see how key mitochondrial variables respond to the [Ca2+]C rise in the three conditions. Typical time traces of [Ca2+]M, Δψ, [NADH]M, and [ATP]M are shown in 2+]M  results from the Ca2+ and Na+ influx through MCU and Na+/Ca2+ exchanger, respectively. The small depolarization is followed by a relatively large hyperpolarization , mainly due to the increase in [NADH]M that in turn results from the stimulation of the pyruvate dehydrogenase reaction. While the values of [NADH]M, Δψ (more hyperpolarized), and [ATP]M in resting conditions are larger, the relative change in all these variables in response to the Ca2+ bolus is smaller in MICU1 KO cells as compared to both WT and MICU2 KO cells. The relative smaller increase in [ATP]M in response to [Ca2+]C rise is consistent with the observations by Bhosale et al. M  and slig in 2+]M  when com 2+]M . First,ments in  investigMCU/Cm) observed in the patch-clamp experiments is equal to 206, 237, and 217 pA/pF in the case of WT, MICU1 KO, and MICU2 KO mitoplasts respectively at Δψ = −160 mV rest = 100 nM, and CaD = 223 μm2/s C at rest, and diffusion coefficient of Ca2+. λ is given byIn intact cells, mitochondrial Carodomain . We consequation ,47:(11)r23 μm2/s  is the cTB is the total concentration of Ca2+ binding protein with dK = 2.0 μM M given by the model to the observed values  [3R channel is allowed to open for 100 ms. Typical time-traces of [Ca2+]M, Δψ, [NADH]M, and [ATP]M are shown in 2+ uptake leads to the depolarization of IMM at the single mitoplast level  . After rst level —in line WT cells . Finally [NADH]M . Furtherrvations . 2+]M, Δψ, [NADH]M, and [ATP]M from the model as we increase the width of the microdomain in WT, MICU1 KO, and MICU2 KO cells are shown in 2+]M (A), Δψ (B), [NADH]M (C), and [ATP]M (D) in WT and MICU2 KO cells decrease significantly as we increase the width of the microdomain. This occurs because the mitochondrion is exposed to lower extra-mitochondrial Ca2+ concentration as we increase the width of the microdomain (longer distance between the IP3R and MCU). However, the decrease in the maximum values of the four variables is not as dramatic in the MICU1 KO mitoplast, even if we consider the microdomain to be a few hundred nanometers wide. Furthermore, the change in [ATP]M in MICU1 KO cells is significantly lower in addition to the fact that [ATP]M decreases in response to [Ca2+]C rise . Csordás et al. found that the average spatial width of the microdomain is ~24 nm in RBL-2H3 cells . However3R channel is open and analyze the changes in the mitochondrial variables as [Ca2+]mic rises. As is clear from 2+]M  is in the range where the reaction is almost saturated already and any further increase in [Ca2+]M would lead to little change in this reaction. The second contribution to [NADH]M in the model comes from the malate-aspartate shuttle (MAS) M in WT and MICU2 KO mitoplasts results in a significant increase in [ATP]M which increases further as the open duration of the channel increases  and MICU1 KO mitochondria where we observe an increasingly positive and negative change in ATP, respectively. These results are in line with reports claiming that the human loss-of-function mutations in MICU1 results in early-onset neuromuscular weakness, impaired cognition, extrapyramidal motor disorder, and perinatal lethality C and [Ca2+]M values using the method previously developed [2+]C (44 state in particular increases significantly as we increase [Ca2+]M from 0.4 μM (As a toy example, we used the double-exponential behavior of open dwell-time histogram observed in . Specifieveloped ,76,77. Ced [2+]C B,C. Furtm 0.4 μM B to 1 μMm 0.4 μM C. 2+ overload either due to MICU1 mutations [+/Ca2+ exchangers [2+ overload was shown to accelerate memory decline, amyloidosis, and tau pathology by promoting the production of reactive oxygen species, metabolic dysfunction, and upregulation of β secretase expression, and neurodegeneration [2+ overload was proven sufficient to impede Alzheimer’s disease-associated pathology and memory loss [2+ uptake in Alzheimer’s disease and many other diseases where mitochondrial Ca2+ overload is believed to play a major role [Mitochondrial Cautations ,29,30,31utations , or impachangers  plays a changers ,73. Mitoneration ,73. Furtory loss ,73. A dajor role ,79. Our


CBFB-MYH11 fusion is associated with a favorable prognosis. The presence of a KIT mutation modifies it to an intermediate prognosis. Additionally, inv(16) can cooperate with other genetic aberrations to further increase cell proliferation. Coexistence of inv(16) and t is extremely rare (20 cases). We present a case of a 55-year-old male with elevated white blood cell count. Bone marrow evaluation and flow cytometry analysis were compatible with AML with monocytic features. Cytogenetic studies revealed two-related clones, a minor clone with inv(16) and a major clone with concurrent inv(16) and t rearrangements. Fluorescent in situ hybridization studies confirmed these rearrangements. Molecular analysis detected a p190 BCR-ABL1 transcript protein. KIT mutations were negative. The patient was initially treated with standard induction regimen; 7 daily doses of cytarabine from day 1–day 7, 3 daily doses of daunorubicin from day 1–day 3, and 1 dose of Mylotarg (gemtuzumab ozogamicin) on day 1. The detection of t led to the addition of daily doses of dasatinib (tyrosine kinase inhibitor) from day 7 onwards. The patient achieved complete remission on day 45. During his treatment course, he acquired disseminated Fusarium infection. Day 180 bone marrow evaluation revealed florid relapse with 64% blasts. Cytogenetic study showed clonal evolution of the inv(16) clone with no evidence of the t subclone. Eventually, bone marrow transplantation was contraindicated, and the patient was transferred to palliative care. Literature review revealed that AML with co-occurrence of CBFB-MYH11 and BCR-ABL1 gene rearrangements was involved by only a small number of cases with de novo and therapy-related AML. Most cases were in myeloid blast crisis of chronic myeloid leukemia (CML). Treatment and prognosis among the de novo AML cases varied and majority of them achieved clinical remission. In contrast, these cytogenetic abnormalities in the blast phase of CML had a poor prognosis. As the prognosis and management of AML is dependent upon the underlying genetic characteristics of the neoplasm, it is imperative to include clinical outcome with such rare combinations of genetic alterations.Acute myeloid leukemia (AML) with inv(16)(p13.1q22) resulting in  Acute myeloid leukemia (AML) is one of the most commonly encountered types of leukemias in adults. AML is characterized by clonal expansion of undifferentiated myeloid blasts and consequentially results in impaired hematopoiesis and bone marrow failure. It is a heterogenous disease clinically, morphologically, and genetically. Cytogenetic aberrations and gene mutations play critical roles in the pathogenesis of AML. There are many driver mutations, and the disease can evolve to accumulate more competing clones.RUNX1-RUNX1T1 and inv(16)/t/CBFB-MYH11 and are classified as AML with recurrent genetic abnormalities under the World Health Organization (WHO) classification [MYH11) at 16p13 and the core binding factor beta subunit gene (CBFB) at 16q12. This gene fusion results in chimeric proteins that suppress transactivation mediated by CBF and lead to impaired hematopoietic differentiation [Eo [KIT mutation is present [Core binding factor (CBF) leukemias include t8;21)//RUNX1-RUfication . This abBCR-ABL1 is one such example. BCR-ABL1 translocation, also known as the Philadelphia (Ph) chromosome, results from a reciprocal translocation between chromosomes 9 and 22: t. This translocation is necessary in defining chronic myeloid leukemia (CML) and is also responsible for a subset of B cell acute lymphoblastic leukemia (B-ALL) and less commonly in de novo AML [BCR-ABL1 fusion (b2a2 or b3a2) in CML and blast phase of CML results in a 210 kDa product (Major breakpoint), whereas in B-ALL, the main fusion form (e1a2) results in a 190 kDa product (minor breakpoint) [In addition, genetic alterations can also activate signal transduction pathways and confer a proliferation advantage on hematopoietic cells. tq3; q11.2. akpoint) .BCR-ABL1 rearrangements, presenting as de novo AML. Our case also utilized the 190 kDa BCR-ABL1 fusion protein. With only 20 cases of AML described in literature with similar coexisting inv(16) and BCR-ABL1 rearrangements, there is a greater need to report such cases.We report a case with combined occurrence of inv(16) and 9/L), thrombocytopenia (platelets—25 × 109/L), and anemia (hemoglobin—7.6 g/dL). Review of the peripheral blood smear revealed 57% circulating blasts, and flow cytometry was compatible with AML with monocytic features. Myeloblasts comprised 15% of cells and showed expression of CD13, CD33, CD117, CD34, HLA-DR, partial CD123, minimal CD64, CD38, and partial cMPO; the blasts were negative for CD56, CD15, CD11b/C, CD14, TdT, and B and T cell markers. There were around 8% immature monocytic cells present which expressed dim CD45, CD13, CD117, CD33, CD15, partial CD11b, and partial CD64 and were negative for CD34. There were also 14% mature monocytic cells.A 55-year-old male presented with worsening fatigue. Upon evaluation, he had leukocytosis  with decreased erythropoiesis, decreased megakaryopoiesis, increased eosinophils, 19% monocytes, and 24% blasts and promonocytes. Morphologically, the blasts appeared large, with irregular and convoluted nuclear contours, dispersed chromatin, and small amounts of basophilic cytoplasm. They were admixed with an increased number of promonocytes that had delicately folded nuclei, dispersed chromatin, inconspicuous nucleoli, and finely granulated cytoplasm. Importantly, there were increased eosinophils that had increased and coarse basophilic granules . Flow cyCBFB rearrangement and BCR/ABL1 translocation in 94.0∼95.0% of the interphase nuclei  141 gene panel that was obtained based on the first bone marrow reported only two mutations of unknown significance in TET2 (p.L1322R) and P2RY2 (p.V77A). There were no pathogenic variants detected for FLT3 exon 14 , FLT3 exon 20 (tyrosine kinase domain), NPM1 exon 11, CEBPA, and KIT exon 17 p.D816V at a minimum allelic fraction of 1.0%.A qualitative reverse transcriptase polymerase chain reaction (RT-PCR) was positive for an e1a2 2) from day 1 to day 7, three daily doses of daunorubicin (90 mg/m2) from day 1 to day 3, and a single dose of Mylotarg  on day 1. The detection of p190 BCR-ABL1 transcript at day 5 led to the addition of daily doses of 100 mg of dasatinib  from day 7 onwards. Intrathecal cytarabine therapy was omitted due to a low number of platelets. The patient was pancytopenic and needed multiple red blood cells and platelets transfusions. On day 14, peripheral blood showed severe pancytopenia and bone marrow showed a hypocellular (<5%) bone marrow with decreased trilineage hematopoiesis and 1% blasts. Although flow cytometry evaluation revealed a negative immunophenotypic study, FISH studies were positive for t and inv(16) in 25.1–27.1% of the interphase cells. Conventional cytogenetics did not yield enough metaphases for a complete study. Dasatinib was stopped on day 21 due to persistent leukopenia. G-colony stimulating factor was given once on day 21 and on day 23. Notably on day 20, the patient's clinical course was complicated by a skin ulceration in his toes that later resulted in disseminated Fusarium infection , and the patient was eventually discharged on day 60.The patient was initially treated with a 7 + 3 cytarabine-based induction regimen: seven daily doses of cytarabine . He was later enrolled on a clinical trial for an alternative oral-based antifungal medication therapy. His fungal infection was reported to have improved with decreased pulmonary infiltrates and a healed foot wound. The patient returned on day 180 for bone marrow transplant evaluation. His peripheral blood smear was remarkable for pancytopenia and 9% circulating blasts. His bone marrow showed persistent AML with 64% blasts and blast equivalents involving a mildly hypercellular bone marrow (50–60%) with decreased trilineage hematopoiesis. The corresponding flow cytometry on the bone marrow specimen revealed 30% of aberrant myeloid blasts that were positive for CD45 (dim), CD34, CD117, HLA-DR, CD13, CD33, and CD38. Chromosome study showed complete resolution of the t subclone (negative by FISH and RT-PCR); however, the inv(16) clone resurfaced with other additional abnormalities significant for clonal evolution , t/RUNX1-RUNX1T1, t/PML-RARA, inv(3), 5q deletion, and NPM1 mutation [BCR-ABL1 (p190 form) and inv(16) abnormalities.An inv16) abnormality is found in 5–8% of younger patients with AML, while the Ph chromosome is a rare event in AML with a reported incidence ranging from 0.5% to 3% [6 abnormamutation . Our casBCR-ABL1), but the inv(16) clone resurfaced with other additional abnormalities significant for clonal evolution. This finding substantiates the efficacy of a TKI inhibitor in cases of Ph-positive AML.The Ph chromosome was observed as a subclone in our patient suggestive for “real AML” as Bacher et al. described in his case series . In caseBCR-ABL1 rearrangements are compared in BCR-ABL1 fusion by FISH or RT-PCR after 3 weeks of therapy. Salem et al. included one patient with de novo AML and one with initial AML with CBFB rearrangement but subsequently acquired a BCR-ABL1 translocation [Similar de novo and therapy-related AML cases with concurrent inv(16) and location . The forlocation . Miura eBCR-ABL1 translocation was in combination with inv(3) where the patient did not respond to initial treatment and died [We also know that the Ph chromosome can pair up with other abnormalities in AML. Han JY and team presented an AML case with a known prognostically adverse aberration inv(3) and monosomy 7 with Ph chromosome as a secondary abnormality at diagnosis . The patand died . The othBCR-ABL1 cytogenetic abnormalities and seven of which were CML cases [BCR-ABL1-positive acute lymphoblastic leukemia than AML [Interestingly, cases with CML in the blast phase that acquire an inv16) aberration have a poor prognosis. Salem et al. studied ten patients with combined inv(16) and ML cases . In the ML cases . Analyzi aberratithan AML , 18. HowCBFB rearrangement in AML with no additional mutation is known to have a favorable prognosis. A study with 201 de novo AML patients indicated that the 5-year survival probability was 43% for CBFB-MYH11 rearrangements [BCR-ABL1 abnormalities. Our case and the study by Salem et al. did not detect any pathogenic variants. This indicates that these co-occurring abnormalities act independently of other commonly seen mutations in myeloid neoplasms. Our patient had initially shown a favorable response to treatment and achieved CR by day 45 postdiagnosis. He later developed disseminated Fusarium infection, which compromised his leukemic treatment. Incidence of Fusarium spp. infection in patients with acute leukemia is 0.06% and the survival rate is 4% [BCR-ABL1 genetic abnormalities confers an intermediate prognosis.Due to the rarity of this disease, the prognosis is difficult to determine. ngements . Ph-posiBCR-ABL1 fusion transcript surveillance can be used to establish a standard of care for such Ph-positive AML patients with additional rearrangements [De novo AML patients with combined inv(16) and t cytogenetic abnormalities seem to benefit from intensive chemotherapy and targeted TKIs. Decisions about HSCT in intermediate-risk AML were less clear-cut in the past, and nowadays, most patients are considered for HSCT in their first CR . Patientngements .Molecularly defined genetic abnormalities in AML have a significant impact on a patient's management and prognosis. AML presenting with both inv(16) and t abnormalities confers an intermediate prognosis. The pathognomic effect of the occurrence of these abnormalities acts independent of other common molecular mutations seen in myeloid neoplasms. These cases are rare and need to be documented and studied further to formulate better management strategies. Such cases may benefit from intensive chemotherapy and TKIs as a bridge to stem cell transplantation. Comorbidities and infectious diseases arising from an immunocompromised state must be taken into consideration when planning treatment regimens to prevent doing more harm than benefit to the patient.


Recently, whole exome sequencing for acute myeloid leukemia (AML) has been performed by a next-generation sequencer in several studies. It has been revealed that a few gene mutations are identified per AML patient. Some of these mutations are actionable mutations that affect the response to an approved targeted treatment that is available for off-label treatment or that is available in clinical trials. The era of precision medicine for AML has arrived, and it is extremely important to detect actionable mutations relevant to treatment decision-making. However, the percentage of actionable mutations found in AML is about 50% at present, and therapeutic development is also needed for AML patients without actionable mutations. In contrast, the newly approved drugs are less toxic than conventional intensive chemotherapy and can be combined with low-intensity treatments. These combination therapies can contribute to the improvement of prognosis, especially in elderly AML patients who account for more than half of all AML patients. Thus, the treatment strategy for leukemia is changing drastically and showing rapid progress. In this review, we present the latest information regarding the recent development of treatment for AML. Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy of hematopoietic stem cells. Conventionally, the prognosis of AML is determined based on chromosomal abnormalities and fusion genes. Intensive chemotherapy, such as 7 days of cytarabine + 3 days of anthracycline (7 + 3) or high-dose cytarabine, are standard treatments for younger AML patients, and the indication of transplantation is considered based on the prognosis of AML. In contrast, more than half of the patients with newly diagnosed AML are elderly  and inelFLT3 (28%), NPM1 (27%), DNMT3A (26%), and IDH1/2 (20%) mutations are observed in 20% to 30% of cases, but the frequency of more than 10 other types of mutations is less than 10% [Recently, whole exome sequencing for AML has been performed by a next-generation sequencer in several studies ,5. It wa DNMT3A 2%, and IDHowever, the percentage of actionable mutations found in AML is about 50% at present [Thus, the treatment strategies for leukemia are drastically changing with the rapid development of new drugs. In this review, we provide the latest information regarding the recent developments in AML treatment, including small molecule drugs targeting mutant genes, small molecule drugs targeting signal pathways, drugs targeting epigenetic regulation, antibody therapy, immune checkpoint inhibitors, and adoptive therapy .Abbreviation; AML, acute myeloid leukemia; ADC, antibody drug conjugate; α-KG, a-ketoglutarate; BAK, BCL-2 antagonist/killer; BAX, BCL-2-associated X protein; BCL2, B-cell leukemia/lymphoma 2; BET, bromodomain and extra-terminal motif; BiTE, bispecific T cell engagers; BRD, bromodomain; CAR, chimeric antigen receptor; CDK, cyclin dependent kinase; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; DART, dual affinity retargeting molecules; DNMT, DNA methyltransferase; DOT1L, disruptor of telomeric silencing-1-like histone methyltransferase; FLT3, FMS-like tyrosine kinase 3; GLI, glioma-associated oncogene; IDH, isocitrate dehydrogenases; IL15R, interleukin 15 receptor; LSD1, lysine-specific histone demethylase 1; MCL1, myeloid cell leukemia 1; NAE, NEDD8-activating enzyme; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; PTCH, Patched-1; SMO, smoothened; TriKE, trispecific killer cell engagers; 2HG, R-2-hydroxyglutarateFLT3 mutations are found in approximately 30% of patients with AML, and two types of mutations in the FLT3 gene are well-known. FLT3 internal tandem duplications (ITDs) of the juxtamembrane domain occurs in around 25% of AML patients [FLT3-ITD is a poor prognostic factor based on the FLT3-ITD to wild-type (WT) allelic ratio [FLT3-ITD to WT allelic ratio  is associated with poor prognosis, but a low FLT3-ITD to WT allelic ratio  is not associated with poor prognosis [FMS-like tyrosine kinase 3 (FLT3) belongs to a cytokine receptor CD135) tyrosine kinase family and regulates the proliferation and differentiation of early hematopoietic stem and progenitor cells . FLT3 mupatients , and poipatients . FLT3-IT5 tyrosinic ratio ,15,16. Mrognosis .KIT, PDGFR, and VEGFR as multikinase inhibitors [First-generation FLT3 tyrosine kinase inhibitors (TKIs), such as midostaurin and sorafenib, were not designed to target FLT3 kinase . Howeverhibitors .p = 0.009) and event-free survival (EFS)  compared with a placebo with intensive chemotherapy in a randomized placebo-controlled phase III trial in 717 patients (<60 years old) with newly diagnosed FLT3-mutated AML (FLT3-ITD and FLT3-TKD) (RATIFY) [high, ≥0.7; FLT3-ITDlow, <0.7; or FLT3-TKD) [Midostaurin with intensive chemotherapy prolonged the overall survival (OS)  0.78; 95% confidence interval (CI), 0.63–0.96; one-sided (RATIFY) . In thisLT3-TKD) . AccordiFLT3-ITD-positive AML [p = 0.0135) as maintenance therapy post-allogeneic stem cell transplantation for FLT3-ITD-positive AML [FLT3-ITD-positive relapsed or refractory AML [Sorafenib is another first-generation multi-kinase inhibitor and was approved for several solid tumors. Although the addition of sorafenib to intensive chemotherapy did not show clinical benefits in a phase II trial , severaltive AML ,22,23. Itory AML . FLT3-ITD inhibitor, but the inhibitory activity against FLT3-TKD is low [D835FLT3-mutated AML [CRi 47%)) in FLT3-ITD-positive relapsed/refractory (R/R) AML patients [n = 367), quizartinib showed a survival benefit versus (vs.) salvage chemotherapy , with a manageable safety profile in R/R FLT3-ITD-positive AML patients [FLT3-ITD in Japan but not the USA in 2019.Second-generation FLT3 TKIs such as quizartinib, crenolanib, and gilteritinib have a more selective inhibition of FLT3 than first-generation FLT3 TKIs. Quizartinib is designed to target FLT3 and a highly selective D is low ,26. In cated AML . Notableated AML ,29. In apatients . There wpatients . In a phpatients . QuizartFLT3-ITD and FLT3-TKD, including D835 [FLT3-ITD-positive R/R AML patients treated with crenolanib as a single agent, there was an ORR of 50%  11%) among 18 patients who had not received prior FLT3 inhibitors and 31%  among 36 patients who had received prior FLT3 inhibitors [FLT3-mutated (ITD and TKD) AML patients (<60 years) [FLT3 mutation is ongoing (NCT03258931).Crenolanib shows inhibitory activity against both ing D835 . In a phhibitors . Crenola0 years) . A phaseFLT3 TKI; it also inhibits AXL, which is another receptor tyrosine kinase that promotes proliferation and activates AML cells [FLT3 ITD or TKD mutation in a phase II trial including 252 R/R AML patients [p < 0.001) compared with salvage chemotherapy in the phase III ADMIRAL trial including 247 R/R FLT3-mutated AML patients [FLT3-mutated AML has been initiated (NCT03836209). Moreover, gilteritinib is currently being studied as an upfront treatment vs. midostaurin in combination with intensive chemotherapy and as a maintenance therapy following induction/consolidation treatment in first remission (NCT02236013 and NCT 02927262). Gilteritinib is also being studied as a maintenance therapy following allogeneic stem cell transplantation for patients with FLT3-ITD-positive AML in the phase III setting (NCT02997202).Gilteritinib is a highly selective ML cells ,35. Giltpatients . Gilteripatients . Based oIDH1 or IDH2 mutations occur in 15% to 20% of AML patients, and are more prevalent in AML patients with a normal karyotype [Isocitrate dehydrogenases (IDH) are enzymes that catalyze the oxidative decarboxylation of isocitrate to a-ketoglutarate (α-KG) . The IDHaryotype .R140Q- and R172K-mutated IDH2 [IDH2 mutant AML [IDH2 mutations in 2017. Enasidenib combined with intensive chemotherapy achieved a cCR (CRi or CRp) rate of 72% in an open-label, multicenter, phase I study including 89 patients with newly diagnosed AML with an IDH2 mutation [IDH2-mutated AML is ongoing (NCT03839771).Enasidenib inhibits both ted IDH2 . In a phtant AML . The medtant AML . The FDAmutation . CurrentIDH1 mutation inhibitor. Ivosidenib showed an ORR of 41%  as a single agent in a phase I dose-escalation and dose-expansion study including 258 R/R AML patients with the IDH1 mutation [IDH1 mutation in patients who are at least 75 years old or who are unfit for intensive chemotherapy on 2 May, 2019. In the frontline setting, ivosidenib (500 mg daily) in combination with intensive chemotherapy showed clinical efficacy (cCR rate of 80%) in a phase I trial of 60 newly diagnosed AML patients with an IDH1 mutation [Ivosidenib is a selective mutation . The medmutation .IDH1 mutation (NCT03839771 and NCT03173248).Ivosidenib in combinational therapy (intensive or low intensive chemotherapy) is currently being studied in randomized phase III trials investigating previously untreated AML patients with an TP53 has been observed in more than 50% of human cancers, whereas only 5–10% of AML cases have TP53 mutations [TP53 mutations is higher in therapy-related AML patients (~30%) and in elderly AML patients with a complex karyotype (~70%) [TP53 mutation is an independent indicator of poor outcomes [TP53 mutation [TP53 [TP53-mutated myeloid neoplasms, including oligoblastic AML (20–30% myeloblasts) (NCT03072043). The preliminary results of this study showed a CR rate of 50% (n = 8). Arsenic trioxide/Trisenox/ATO is one of the therapeutic agents that configure the standard treatment of acute promyelocytic leukemia [TP53 via the 26S proteasome pathway and also activates wild-type TP53, leading tumor cells to apoptosis [TP53 mutations has a better relapse-free survival and complete response compared to using decitabine and cytarabine (NCT03381781). Furthermore, the cholesterol-lowering drugs named statins (atorvastatin/lipitor) can induce the degradation of abnormal TP53 proteins and inhibit tumor growth in TP53-mutated tumor cells [TP53 in TP53 mutants and TP53 wild-type malignancies, including AML, is ongoing (NCT03560882).The tumor suppressor gene utations ,46,47. Te (~70%) ,49. The outcomes . Intensimutation . APR-246on [TP53 . A phasepoptosis ,55. A muor cells . A pilotn = 145) [B-cell leukemia/lymphoma 2 (BCL-2), which is one of the BCL-2 family proteins that also include BCL-XL and MCL-1, promotes cell survival. BCL-2 regulates the mitochondrial apoptotic pathway and plays an important role in the chemoresistance and survival of AML blasts ,58,59. Vn = 145) . The CR/BCL2, MYC, and Cyclin-D1) involved in leukemia cell survival and proliferation [n = 115) that showed the efficacy of glasdegib + LDAC compared with LDAC alone ) in newly diagnosed AML patients unfit for intensive chemotherapy [n = 720) is ongoing (NCT03416179). The other SMO inhibitors (vismodegib and sonidegib) are also being currently investigated in early phase trials .The activation of the hedgehog (HH) signaling pathway is known to be involved in leukemia cell survival and drug resistance. Hedgehog, a secreted protein, activates smoothened (SMO) by binding to the PTCH (Patched-1) receptor. Activated SMO initiates GLI (glioma-associated oncogene) protein activation and increased HH target-genes , which is a novel inhibitor of NAE, impairs NEDD8-regulated cullin-RING-type ligase action by inhibiting NEDD8, and causes antiproliferative effects . A phaseMYC and MCL-1, and this has been observed in AML [MCL-1-dependent R/R AML is ongoing (NCT02520011). Outside of that, there is an ongoing phase I study which was initiated to explore alvocidib and standard 7 + 3 chemotherapy in patients with newly diagnosed AML (NCT03298984). The other CDK9 inhibitors (BAY 1143572 and TG02) are also being investigated in early phase trials .A cyclin-dependent kinase 9 (CDK9) is a member of the CDK family which controls cell-cycle progression and gene transcription. Dysregulation in the CDK9 pathway activates the mRNA transcription of target genes including d in AML . Alvocidd in AML ,72 and nd in AML ,74,75. OThe system that controls gene expression by chromosomal changes but not changes in the DNA base sequence is called epigenetics. Chromosomal changes refer to chemical modifications, such as the methylation of DNA in nucleosomes, histone acetylation and methylation, and chromatin modification. The Cancer Genome Atlas Research Network reported that, of 200 AML patients, DNA methylation-related genetic mutations occurred in 44% and chromatin modification-related genetic mutations occurred in 30% .Azacitidine or decitabine, which was the first class of epigenetic drug used as DNA-hypomethylating agents, was approved by the FDA in the 2000s. They inhibit DNA methyltransferase, which suppresses tumor suppressor genes by the methylation of the CpG island in the promoter region. These drugs have been used for the treatment of high-grade myelodysplastic syndrome and AML with a low blast count ,77,78. IIn addition to DNA-hypomethylating agents, chromatin modulators have recently been developed as an epigenetic therapy for AML. The three key targets to inhibit are the function of the epigenetic writers (disruptor of telomeric silencing 1-like histone methyltransferase (DOT1L)), epigenetic erasers (lysine-specific histone demethylase 1 (LSD1)), and epigenetic readers ) in AML patients.MLL rearrangements or MLL partial tandem duplication, and only one patient out of the 43 AML patients achieved CR.DOT1L is a lysine methyltransferase that methylates a specific amino acid on histone H3K79 and activates a cancer-promoting gene via an MLL fusion protein (MLL-AF9) . PinometLSD is a lysine demethylase that catalyzes the demethylation of dimethyl and monomethyl forms of H3K4 and regulates gene expression epigenetically. LSD1, one of the LSD isozymes, is involved in the proliferation of various cancer cells ,83. NumeBET inhibitors suppress cancer cell growth by inhibiting the binding of bromodomain proteins to histones with acetylation modification. BET inhibitors induce the apoptosis of MLL-fusion leukemia cells . Some clMonoclonal antibodies (MoAb) play an important role in cancer treatment, and it is believed that treatment strategies using MoAb are reasonable for leukemia because of the accessibility of malignant cells in the blood and bone marrow. Surface antigens targeted by MoAb are limited for leukemia, because most of the suitable antigens on AML for MoAb are also found on healthy myeloid precursors, which can easily result in severe cytopenia. Most MoAbs target CD33 or CD123 in clinical studies of AML. MoAb conjugated with a toxic agent  has been developed, because unconjugated MoAb is ineffective for AML. Furthermore, MoAbs that bind to both immune cells and leukemia cells have been developed as a novel approach. These MoAbs bring cytotoxic T cells (by binding to CD3) in proximity with leukemia cells (by binding to a specific leukemia antigen) and T cell activation and leukemia cell destruction. This includes bispecific T cell engagers (BiTEs), bispecific/trispecific killer cell engagers designed to target CD16 on NK cells (BiKE/TriKE), or dual-affinity retargeting (DART) molecules.CD33 is a myeloid differentiation antigen and is expressed in about 90% of leukemic cells ,86. Gemtn = 131), vadastuximab talirine as monotherapy showed clinical activity (cCR 28% (CR 11% + CRi 17%); 95% CI, 9.5–53.5%, 5 of 18 patients) at the recommended monotherapy dose of 40 µg/kg [n = 42), vadastuximab talirine and intensive chemotherapy (7 + 3 chemotherapy) showed a promising efficacy, with a 78% CR and CRi rate [225Ac-lintuzumab (Actimab-A) is a radioimmunoconjugate composed of 225Ac linked to a humanized anti-CD33 monoclonal antibody that is currently being studied in untreated older AML patients unfit for intensive chemotherapy. In a phase II study, 225Ac-lintuzumab showed a 56% CRR (CRp 22% and CRi 34%) in nine patients [Another ADC targeting CD33 is vadastuximab talirine (SGN-CD33A) using a pyrrolobenzodiazepine dimer. In a phase I trial NCT01902329) , have been developed in AML. BiTE is an antibody drug that combines an antibody against a tumor cell surface antigen and an antibody against an antigen expressed on immune cells, such as CD3. BiTE is already being developed as a treatment for patients with B-cell acute lymphoblastic leukemia (ALL). Blinatumomab, which has dual specificity for CD19 and CD3, showed a high response and relapse-free survival in R/R CD19-positive ALL patients ,100,101.DART is a dual-affinity re-targeting molecule that incorporates two single-chain variable fragments (scFv) stabilized by a C-terminal disulfide bridge, while BiTE is a small molecule comprising two scFvs linked in tandem ,103,104.Similar to BiTE and DART antibodies, bi- and tri-specific killer engagers  against tumor antigens to activate NK cell cytotoxicity have also been developed. GTB-3550 (161533) is a CD16/IL-15/CD33 TriKE antibody. A phase I/II clinical trial of GTB-3550 is currently underway in CD33-expressing myeloid malignancies, including AML patients (NCT03214666).Immune checkpoint inhibitors have been already approved for several solid tumors by the FDA. In hematologic malignancies, immune checkpoint inhibitors have shown effectiveness in Hodgkin lymphoma and have been approved . Severaln = 28), including 12 patients with relapsed AML after allogeneic stem cell transplantation [CTLA-4 (CD152) is a protein receptor on T-cells which downregulates immune responses. CTLA-4 acts as an off switch by competing with the costimulatory receptor CD28 for CD80 and CD86 on the surface of antigen-presenting cells, and inhibits T-cell maturation and differentiation . In AML,antation . Severaln = 14) to evaluate the efficacy of nivolumab maintenance in high-risk AML patients in CR after induction and consolidation chemotherapy, the 6- and 12-month rates of CR duration were 79% and 71%, respectively (NCT02532231) [n = 51), a CR/CRi rate of 18% (6/35) with a median OS of 9.3 months (1.8-14.3 months) was shown in R/R AML patients with poor risk features  who received nivolumab (anti-PD-1 antibody) and azacitidine approximately every 4–5 weeks indefinitely [PD-1 is a cell surface molecule that inhibits T-cell proliferation, cytokine production, and cytolytic function by binding to its ligands PD-L1 or PD-L2 on the surface of antigen-presenting cells . In a pi2532231) . In a phfinitely . CurrentCD47 is a cell transmembrane protein that inhibits phagocytosis by interacting with signal regulatory protein-α on antigen-presenting cells . The uprChimeric antigen receptor (CAR) consists of an extracellular domain generated by joining the heavy and light chain variable regions of a monoclonal antibody with a linker to form an scFv molecule. CAR T-cells are genetically engineered to express CARs on autologous T-cells by a retro-, adeno-, or lentiviral vector carrying the CAR gene, and are infused back into the patient. CAR T-cells combine the antibody in its antigen on the surface of target cells and show tumor-lytic activity.In the setting of B-ALL and non-Hodgkin’s lymphoma, many clinical trials to evaluate the efficacy of CAR T-cell therapy have been conducted and have shown remarkable clinical activities ,113,114.Although there are some challenges, several early phase trials of CAR T-cell therapy targeting AML-associated antigens such as CD33, CD38, CD56, and CD123 are currently ongoing .A lot of clinical trials evaluating the efficacy of promising investigational drugs in AML are ongoing , and morDuring treatments such as small molecule drugs targeting mutant genes, leukemia cells acquire secondary resistance  ,124,125.Not only upon new diagnoses but also in relapse or refractory periods, genome profiling analyses should be conducted to detect the secondary resistance of leukemia cells and select an optimal second line treatment in AML patients. Molecular therapy includes small molecule drugs targeting mutant genes detectable by a genome profiling test.


Although azelaic acid is effective for treatment of acne and rosacea, the biological activity of azelaic acid and the effect of its combination therapy with minoxidil were not elucidated with regard to hair growth.Gli1, Gli2, and Axin2 mRNA levels was evaluated using real-time polymerase chain reaction (PCR) analysis. We further investigated the protective effects of azelaic acid against ultraviolet B (UVB) irradiation in cultured bulb and bulge cells by determining catalase activity. An irradiation dose of 20 mJ/cm2 UVB for 4 sec was chosen.In this study, mouse vibrissae follicles were dissected on day 10 after depilation. Then, the bulb and bulge cells of the hair follicle were treated with minoxidil and azelaic acid for 10 days to evaluate Sonic hedgehog (Shh) protein expression. Moreover, bulge and bulb cells of the hair follicles were cultivated and the expression of Gli1 and Gli2 expression in the bulge cells and 100 µ minoxidil caused Gli1 and Axin2 overexpression in the bulb region of the hair follicle. Moreover, minoxidil (100 µM) alone and in combination with azelaic acid (25 mM) led to Shh protein overexpression in the hair follicles in vitro and in organ culture. The results showed that catalase activity significantly (p<0.05) increased in the bulge cells after exposure to 2.5 mM and 25 mM azelaic acid. Meanwhile, treatment of the bulb cells with azelaic acid (2.5 and 25 mM) did not cause significant changes in catalase activity. We also found that azelaic acid (25 mM) alone upregulated Our results indicated a potential role for azelaic acid in the protection of bulge cells from UVB damage and its combination with minoxidil may activate hair growth through overexpression of Shh protein. Hair follicle undergoes cyclic morphogenetic changes of anagen, catagen, and telogen, throughout life  residing in the bulge region are maintained by activation of Wnt and sonic hedgehog (Shh) signaling pathways. Shh directs patterning and cellular differentiation during embryonic development of the skin and hair follicle. Moreover, Shh is involved in controlling anagen stage of hair follicle during postnatal life  affects about 30-50% of men and 13-37% of women by the age of 50 years old . Minoxidil was dissolved in HCl and azelaic acid was dissolved in culture medium.AnimalsMale BALB/c mice (6-8 weeks old) were obtained from Razi Vaccine and Serum Research Institute . The animals were kept under a 12:12 hr light/dark cycles and they had free access to food and water. The experimental procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of Ardabil University of Medical sciences (IR.ARUMS.REC.1394.50).Isolation of bulb and bulge regionsAll mice upper lip hairs were depilated to synchronize the hair follicles in anagen. Ten days later, the whisker pads were collected. Briefly, after anesthesia induction using dimethyl ether, anagen stage-follicles were dissected from the whisker pads under a stereomicroscope . The vibrissa hair follicles lifted out and treated with minoxidil (100 μM), azelaic acid (25 mM) alone and azelaic (25 mM)/minoxidil (100 µM). Then, the bulge regions were isolated from the hair follicles. Bulb region was also isolated by cutting a piece of around 2–2.5 mm in length from the bottom of the hair follicle . An irradiation dose of 20 mJ/cm2 UVB was chosen based on previous results . Prior to the irradiation, the medium was replaced by Determination of catalase activityThe bulb and bulge regions were isolated and the cells were cultivated in DMEM: F12 supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. The groups were: (i) control (untreated bulb and bulge cells); (ii) Sham (untreated bulb and bulge cells+UV irradiation); (iii) UV+2.5 mM azelaic acid; (iv) UV+25 mM azelaic acid; (v) 2.5 mM azelaic acid+UV; (vi) 25 mM azelaic acid+UV. centrifugal force (RCF), aliquots of the obtained supernatant were subjected to three freeze-thaw cycles at -80°C. Next, 50 μl supernatant was diluted 500 times with PBS and 2 μl of that was added to 1 ml hydrogen peroxide (30 mM). The absorption was monitored by a spectrophotometer  at λ=240 nm every 15 sec for 2 min. Assays were performed in triplicate.Catalase activity was measured according to Aebi method . Briefly, the bulb and bulge cells were washed and suspended in 0.1 M Tris-HCl (pH 7.5) for 20 min on ice. Then, the cells were sonicated for two 30-sec bursts. After 10 min of centrifugation at 14000 relative ImmunoassayImmunohistochemistry was performed as described previously  analysis2O 4 μl). The primer sequences and products lengths are shown in B2M) gene was applied as the housekeeping gene for normalization of the real-time data. The 2−ΔΔCt formula was used to calculate relative changes in gene expression . The concentration of total mRNA was measured using a NanoDrop Spectrophotometer . cDNA was synthesized from total RNA using a cDNA Synthesis Kit . Reactions were performed in a final volume of 20 μl  – ΔCt (control))ΔΔCt = . p values<0.05 were regarded as statistically significant. Graphs were plotted using Sigma Plot software .Azelaic acid induced catalase activity in bulge cells To evaluate the short-term sensitivity of hair follicle to UVB exposure, we irradiated the bulge and bulb regions of the hair follicle with a single dose of UVB. When azelaic acid (2.5 mM) was added into culture medium before UVB irradiation or when added promptly after irradiation, it significantly increased catalase activity in the bulge cells . The incMinoxidil (100 µM) and its combination withazelaic acid (25 mM) increased the expression of Shh proteinTo elucidate whether the treated hair follicles undergo anagen phase, we tested the expression of Shh protein using immunoassay. Our results showed that Shh protein expression was increased in 100 µM minoxidil-treated cells compared to the control cells (untreated) on day 10 post-treatment. In addition, as shown in Effects of minoxidil, azelaic acid and the combination treatments on Gli1, Gli2, and Axin2 expressionGli1, Gli2, and Axin2 mRNA expression in the bulb and bulge regions of the hair follicles. The fold changes of Gli1 and Gli2 mRNA levels were 194.81 and 10.82 in the azelaic acid-treated bulge cells, respectively. In addition, 100 µM minoxidil-treated bulb cells showed a significant increase in Gli1 (6.026-fold change) and Axin2 (5.15-fold change) mRNA level , minoxidil (100 µM) and azelaic acid (25 mM)/minoxidil (100 µM) on NA level . In contGli1 and Gli2 genes and an increase in catalase activity in the bulge cells. Indeed, its combination with minoxidil also elevated Shh protein expression in the hair follicles and combination therapy had a higher effect over minoxidil alone in terms of increased Shh expression level. Similar to our results, previous studies showed that Shh/Gli1/Gli2 activation may directly promote telogen to anagen transition in the hair follicle (St-Jacques et al., 1998γ-independent effects in the hair follicle. Moreover, direct upregulation of Gli2 shown here and by other studies (Mill et al., 2003Gli2 is a transcriptional partner for β-catenin, that acts in synergy to induce anagen promotion and hair growth morphogenesis (Reddy et al., 2001In our results, the effectiveness of azelaic acid alone in inducing hair growth, was shown with respect to up-regulation of . β-catenin, Shh, noggin, and STAT3 are key factors that trigger the anagen phase (Botchkarev and Kishimoto, 2003Several signals were previously shown to participate in the hair follicle cyclic changesFurthermore, we showed that exposure of azelaic acid to bulge cells protected them from UVB damage and caused a significant increase in the catalase activity in the bulge area. Hair loss, like acne, is strongly linked to inflammatory factors and DHT (Knussmann et al., 1992Axin2 mRNA level was significantly up-regulated in the bulb cells after 100 µM minoxidil treatment. Axin2 and PPARγ participate in a negative feedback loop to degenerate and inhibit β-catenin (Lecarpentier and Vallée, 2016β-catenin in the cytoplasm protects the activation of target genes such as cyclin D and c-myc (Tetsu and McCormick, 1999Finally, we found that β-catenin and Shh (Dastan et al., 2016Axin2 gene inhibits Wnt signaling in the bulb cells in a negative feedback mechanism after anagen initiation (Lim et al., 2016β-catenin and up-regulates the expression of Axin2, Lef-1, and EP2 (Kwack et al., 2011Gli1 and Axin2.Our previous work revealed that minoxidil promotes anagen phases through up-regulation of Shh signaling regulates dermal papilla fibroblasts and matrix keratinocytes interaction during embryonic follicle morphogenesis and postnatal hair follicles cycling (Callahan and Oro, 2001Gli1 and Gli2 genes and it can protect bulge cells from UVB damage. Our results suggest that azelaic acid combination with minoxidil promotes hair growth via induction of Shh protein expression in the hair follicles. Altogether, this evidence provides valuable insights into the role of azelaic acid in the hair follicles growth. In conclusion, we showed that azelaic acid is a regulator of