The property optimization design strategy and the cation characterization method are not only helpful in exploring nonlinear optical materials but also enlightening in material design and selection.The design of new materials with special performances is still a great challenge, especially for the deep-ultraviolet nonlinear optical materials in which it is difficult to balance large bandgaps and strong second harmonic generation responses due to their inverse relationship. Cation variation not only influences the whole structure frameworks but also directly participates in the formation of electronic structures, both of which could lead to the uncontrollability of the properties of the designed materials. Here, a novel approach, aiming at purposeful and foreseeable material designs, is proposed to characterize the role of cations. By the verification of several series of borates, the influences of cation variation on property changes are explored systematically. Accordingly, a feasible strategy of designing deep-ultraviolet nonlinear optical materials by substituting barium for lead has been concluded, which could obviously blue-shift the ultraviolet cutoff edge and maintain the relatively strong second harmonic generation response (more than 2 times of KH Eg > 6.2 eV) is indispensable for DUV transparency, while Eg is inversely proportional to the second harmonic generation (SHG) response (dij) 3−, [B3O6]3−, distorted d0 or d10 transition-metal-centered  polyhedra, fluorooxoborate anionic units [BOnFn4-]n+1)−, and so on ) in different bandgap regions (or ultraviolet [UV] cutoff ranges) are highlighted in Fig. 3TeBO9 (20.0 KDP) ∞ layer, and a [B6O9F6]∞ layer, respectively. It should be noticed that the connection of the halide atoms is also different, where F in the fluorooxoborate unit BO3F connects both with the metal Ba and B atoms, while Br connects only to the metal Ba 3Cl , Pb3B6O13B6O11F2 , Pb2B5O9 is better to characterize the cations but hard to sum arithmetically for composite cations. ESD [V(M2+)] can also be summed arithmetically, but its deviation γ is too large. More importantly, all of the above ESD values are incapable of ion groups, such as [NH4]+, [CH3NH3]+, and so on. Consequently, ESD based on Bader volumes could overcome the above restrictions, where the electron density in the whole space is partitioned by DFT calculation  . Taking the above isostructural substitutions of lead/barium borates as examples, the magnitudes of ESD between lead and barium atoms are compared on the basis of atom radius R(M), cation radius R(M)3 Table . Here, dculation ,66, and 2BO3Br and BaB2O3F2 mentioned above. As the lattice parameters are relaxed in geometric structure optimization, the values of ESD have been calculated from both optimized and experimental (no-optimization) structures. The ESD and crystal volume in Pb2BO3Br are 27.7/28.3 and 143.6/149.0 Å3 without/with optimization, and their deviation γ is 2.2% and 3.7% 3Cl, Pb3B6O11F2, and Pb2B5O9Cl, are 3.54, 3.81, and 3.36 eV, which are lower than their experimental or HSE06 values, 3.97 eV (312 nm), 5.17 eV (240 nm), and 4.41 eV , respectively. Here, the underestimation of bandgaps under GGA-PBE functional is derived from the inherent discontinuous exchange-correlation energy [To assess the influence of cation variation on property change, the optical properties of the selected lead/barium borates are evaluated and analyzed in detail by DFT calculation. Based on generalized gradient approximation (GGA)-Perdew-Burke-Ernzerhof (PBE) functionals, the calculated bandgaps of these lead borates, Pbn energy , and a sn energy , is used2B5O9Cl , were synthesized in experiments, and the UV cutoff edge and SHG intensity were also measured 3Cl, Ba3B6O11F2, and Ba2B5O9Cl, which exhibit 76, 50, and 92 nm blue-shift from the isostructural lead borates. Correspondingly, the SHG responses have changed to 0.5, 3.0, and 0.7 KDP for Ba5(BO3)3Cl, Ba3B6O11F2, and Ba2B5O9Cl from 3.2, 4.0, and 1.2 KDP of Pb2Ba3(BO3)3Cl, Pb3B6O11F2, and Pb2B5O9Cl. The experimental SHG intensities of M2B5O9Cl  are shown in the insets of Fig. 3B6O11F2 and Ba2B5O9Cl have obtained DUV cutoff edges (<200 nm), but only borate fluoride Ba3B6O11F2 maintained relatively strong SHG responses (> 2 KDP). For the last three prototypes, borate halide Pb2BO3Br and fluorooxoborates PbB5O7F3 and PbB2O3F2 exhibit remarkably strong SHG responses, 9.5, 6.0, and 13.0 KDP, but their bandgaps are different, 3.33 eV (372 nm), 5.51 (225 nm), and 5.64 eV (220 nm), respectively. After cation substitutions, the combination property of the former changes slightly, 3.87 eV (320 nm) and 7.8 KDP for Ba2BO3Br. Contrarily, BaB5O7F3 and BaB2O3F2 show a great change in their prototype, 7.92 eV (157 nm) with 2.5 KDP, and 8.24 eV (150 nm) with 4.8 KDP. Furthermore, to analyze the source of the variation in bandgaps among the above metal borates, the total and partial density of states are shown in Figs. 3 groups in a series of fluorooxoborates [5O7F3 and BaB2O3F2 is more successful.By cation substitution in the three lead borate prototypes with moderate SHG responses, the bandgaps of barium borates have increased to 5.26 eV , 6.53 eV , and 6.56 eV  for Ba), 5.51 2 nm, and oborates ,50,59. AEg) and SHG response in the cation substitution from lead to barium. The whole region is divided into 4 sections: I, Eg < 6.2 eV, SHG < 2 KDP; II, Eg ≥ 6.2 eV, SHG < 2 KDP; III, Eg < 6.2 eV, SHG ≥ 2 KDP; IV, Eg ≥ 6.2 eV, SHG ≥ 2 KDP, where 6.2 eV and 2 KDP are DUV and relatively strong SHG response bounds, respectively. Here, before the cation substitution, 5 lead borates in 6 are all located at Section III, indicating strong SHG responses but not DUV cutoff edges. After the cation substitution, these 5 barium borates scatter into 3 sections, 1 (Ba2BO3Br) still in Section III, 1 (Ba5(BO3)3Cl) in Section I, and 3 in Section IV . Although Ba2B5O9Cl shows a DUV bandgap, its SHG response is relatively small for the reason that the SHG intensity of its prototype is not strong. It is found that these lead borates could keep strong SHG responses (≥2 KDP) after cation substitution except Pb2Ba3(BO3)3Cl and Pb2B5O9Cl, whose SHG response is the smallest among the 6 lead borates. What is more important, the bandgaps of all the lead borates have visibly widened, and especially four of them have exceeded the DUV limit  after cation substitution. Therefore, the cation substitution from lead to barium could optimize the combination properties.Figure 3B6O11F2 and Ba2B5O9Cl, lead and barium cations contribute equally to the SHG coefficient. In M(BO3)3Cl , the Wyckoff site cations cause various contributions to the SHG coefficient d14. Namely, in Pb2Ba3(BO3)3Cl, two 8c Pb contribute the most of d14, 29% in 33%, and two 4b Ba and one 4a Ba contribute the relatively small parts, 7% and 0.3% in 33%, respectively. In Ba5(BO3)3Cl, two 8c Ba contribute almost all of the d14, but 4a Ba makes a remarkably negative contribution, −30%, which leads to an obvious decrease of the SHG response from Pb2Ba3(BO3)3Cl to Ba5(BO3)3Cl. For MB5O7F3 , the metal cations contribute about 10% to 15%, and the relatively small contribution mainly derives from the ratio of metal cations to B-O/B-O-F anion groups is only 1:5.Considering the importance of SHG response for NLO materials, exploring the role of cation variation on SHG response is vital to deepen the understanding of the structure–property relationship. The real-space atom-cutting technique is an efficient postprocessing tool to evaluate the contribution of microscopic structural groups to optical properties, such as the SHG coefficient. Table 2BO3Br2 and MB2O3F2 , the cations exert a remarkable contribution to the SHG responses. In addition, in the SHG density map , there are plenty of unoccupied states contributing to the VE and virtual-hole (VH) process and occupied states contributing to the VH process around Pb and Ba. In MB2O3F2 , Sn shows a vitally negative contribution for d33 (−130%), which is the reason that the SHG response of SnB2O3F2 is much lower than that of isostructural PbB2O3F2, in accordance with the results from another group [d22, 73% to 72%.Furthermore, in Mer group , while S2O3F2 to BaB2O3F2, two virtual middle structures, Pb0.75Ba0.25B2O3F2 (Pb0.75Ba0.25) and Pb0.25Ba0.75B2O3F2 (Pb0.25Ba0.75) are introduced. As shown in Fig. 33| and |d22| decrease, as the negative relationship between bandgap and SHG response. Here, we add a trend line with the formula d = a / Eg5, where d and Eg are SHG coefficient and HSE06 bandgap values, and the constant a is calculated by the value of d and Eg. Of course, this formula only considers the bandgap effect and ignore the momentum matrix elements [d22 than Ba (22% to 4% in Pb0.75Ba0.25 and 33% to 7% in Pb0.25Ba0.75), while Pb and Ba show similar contributions to d33 (12% to 9% in Pb0.75Ba0.25 and 13% to 13% in Pb0.25Ba0.75). Interestingly, the contributions of a single Ba atom develop both in d22 and d33 as the proportion of Ba content increases. Therefore, the SHG coefficient trends of PbB2O3F2, Pb0.75Ba0.25, and Pb0.25Ba0.75 do not vary so much, while the SHG coefficients of BaB2O3F2 are much larger than the expected value according to the trend lines.On the other hand, to explore the trends of property changing from PbBelements , so it i2O3F2 obtains a smaller bandgap than PbB2O3F2 for stronger stereochemical activity. Table 2+ contributes the main part of d33 in BaB2O3F2, so the BaB2O3F2 could maintain a large SHG coefficient when obtaining a large bandgap. Furthermore, we also calculate the ESD value of Pb0.75Ba0.25 and Pb0.25Ba0.75 and compared them with PbB2O3F2 and BaB2O3F2. Then, Fig. 2O3F2 . It is important to note that the above conclusion has only been obtained in the case of cation substitution between Pb and Ba in MB2O3F2 structures.From Fig. S10, the location of Pb-6p in conduction band minimum is nearer to the Fermi surface than that of Ba-5d for the stereochemical activity of the lone pairs. Meanwhile, the SnB5(BO3)3Cl, Ba3B6O11F2, Ba2B5O9Cl, Ba2BO3Br, BaB5O7F3, and BaB2O3F2, along with their prototypes of cation substitutions lead borates, we found that ESD is very useful to describe the electron occupied space, and the magnitudes of ESD are similar in the isostructural substitutions. As the aim of chemical substitutions is to develop performances, three cases, one borate fluoride, and two fluorooxoborates, in 6 cation substitutions from lead to barium have achieved the property optimization, enlarging bandgaps to enter the DUV region  from outside DUV as the absence of lone pair in metal cations, and maintaining relatively strong SHG responses (> 2 KDP) for the reason that metal cations show less contribution to SHG responses than the anion frames. The cation substitution between Pb and Ba in MB2O3F2 structures shows the negative relation between ESDs and SHG responses, which indicates the direction of searching for the NLO material with larger SHG responses. In conclusion, by analyzing the cation variations, a cation substitution approach toward DUV NLO materials has been proposed, and two predicted fluorooxoborates BaB5O7F3 and BaB2O3F2 have achieved the balance between large bandgaps and strong responses.Through the investigation of a series of barium borates, Ba2B5O9Cl and Pb2B5O9Cl were prepared by using standard high-temperature solid-state techniques. For Pb2B5O9Cl, a stoichiometric mixture of PbCl2 and H3BO3 was heated at 473 K for 10 h, cooled and ground, and then heated again at 873 K for 48 h. For Ba2B5O9Cl, a stoichiometric mixture of BaCl2·2H2O, BaCO3, Ba(OH)2, and H3BO3 was heated at 573 K for 10 h, cooled and ground, and then heated again at 1153 K for 48 h. The starting materials of PbCl2, BaCl2·2H2O, BaCO3, and H3BO3 were gathered from commercial sources without any purification.Polycrystalline samples of Ba2B5O9Cl and Pb2B5O9Cl were obtained on an automated Bruker D2 X-ray diffractometer equipped with a diffracted beam monochromator set for Cu-Kα radiation (λ = 1.5418 Å) at room temperature in the angular range of 2θ = 10° to 70° with a scan step of 0.01° and a fixed counting time of 0.1 s/step.XRD patterns of BaThe diffuse reflectance spectrum was measured by a Shimadzu SolidSpec-3700DUV spectrophotometer at room temperature from 175 to 2600 nm.4 solid-state laser at 1064 nm [2B5O9Cl and Pb2B5O9Cl were ground and sieved into the following particle size ranges: 38 to 55, 55 to 88, 88 to 105, 105 to 150, 150 to 200 μm. To make relevant comparisons with known SHG materials, the crystalline KDP sample was also ground and sieved into the same particle size ranges.Powder SHG effects were measured by using the Kurtz-Perry method with a Q-switched Nd: YVO 1064 nm . Polycry25p66s2, Pb 5s25p65d106s26p2, B 2s22p1, O 2s22p4, F 2s22p5, Cl 3s23p5, and Br 4s24p5. The cutoff energies of M2Ba3(BO3)3Cl, M3B6O11F2, M2B5O9Cl, M2BO3Br, MB5O7F3 and MB2O3F2  were all no more than 940 eV and the Monkhorst-Pack k-point grids were 3 × 3 × 3, 4 × 3 × 3, 6 × 1 × 4, 4 × 4 × 2, 4 × 4 × 3, and 5 × 5 × 4, respectively.In this paper, the CASTEP package, a plane wave pseudo-potential method , was used = χ/2. Through the result of the band structure from the CASTEP package, the second-order nonlinear susceptibilities at the limit of zero frequency, χijk(2)(0), can be expressed as the sum of the contribution of the virtual-electron processes and the VH processes [When the phase-matching condition is met, the SHG conversion efficiency of an NLO material is significantly determined by the SHG coefficients . Also, trocesses ,71.χijk2i, j, and k are Cartesian components, v (v') and c (c') are valence and conduction bands, and P(ijk) represents full permutation.Here, Phonon spectra, powder XRD patterns, total and partial density of states, SHG density, comparison of typical NLO materials in bandgap and SHG response, structure information of predicted structures, and real-space atom-cutting results of SHG coefficients are shown in the Supplementary Materials.


KMT2C and KMT2D are the most frequently mutated epigenetic genes in human cancers. While KMT2C is identified as a tumor suppressor in acute myeloid leukemia (AML), the role of KMT2D remains unclear in this disease, though its loss promotes B cell lymphoma and various solid cancers. Here, it is reported that KMT2D is downregulated or mutated in AML and its deficiency, through shRNA knockdown or CRISPR/Cas9 editing, accelerates leukemogenesis in mice. Hematopoietic stem and progenitor cells and AML cells with Kmt2d loss have significantly enhanced ribosome biogenesis and consistently, enlarged nucleolus, increased rRNA and protein synthesis rates. Mechanistically, it is found that KMT2D deficiency leads to the activation of the mTOR pathway in both mouse and human AML cells. Kmt2d directly regulates the expression of Ddit4, a negative regulator of the mTOR pathway. Consistent with the abnormal ribosome biogenesis, it is shown that CX‐5461, an inhibitor of RNA polymerase I, significantly restrains the growth of AML with Kmt2d loss in vivo and extends the survival of leukemic mice. These studies validate KMT2D as a de facto tumor suppressor in AML and reveal an unprecedented vulnerability to ribosome biogenesis inhibition. KMT2D is identified as a tumor suppressor gene in acute myeloid leukemia. KMT2D deficiency reduces H3K4 methylation levels and suppresses the expression of DDIT4, the negative regulator in the mTOR signaling pathway, which leads to the activation of the mTOR pathway and enhancement of ribosome biogenesis. KMT2D‐deficient cells are vulnerable to ribosome biogenesis inhibitors such as CX‐5461. Among them, the lysine methyltransferase 2 ) family genes are arguably the most notable in AML. There are five KMT2 family members, KMT2A‐E, given their functions to methylate the lysine residues of histones.KMT2A is the major player in large MLL‐fusion proteins found in AML and acute lymphoblastic leukemia, which are often associated with poor prognosis.MLL‐fusion genes have been demonstrated as driver genes to promote AML genesis in mice. In contrast, KMT2C, located on chromosome 7q36.1, is recurrently deleted in −7/del7q AML. The heterozygous loss of KMT2C could promote AML formation in mice. KMT2C and KMT2D are large scaffold proteins that form the KMT2C/D COMPASS complex (complex of proteins associated with Set1), which contains WDR5, RBBP5, hDPY30, ASH2, KDM6A (UTX), PTIP, PA1, and NCOA6. Besides KMT2C, it is also reported that loss‐of‐function mutations of KDM6A, which happened in AML, promoted myeloid leukemogenesis and contributed to chemotherapy resistance.Epigenetic factors are among the most frequently altered genes in human cancers.1 In aKMT2D, a histone‐3 lysine‐4 methyltransferase, is one of the most frequently mutated genes like KMT2C in human cancers.KMT2D plays critical roles in various biological processes, including regulation of development, differentiation, metabolism, and tumor suppression, while its frequent mutations have been found in developmental diseases, such as Kabuki syndrome and congenital heart disease, and various forms of cancers. In many solid cancers and B‐cell lymphomas, KMT2D deficiency or its loss‐of‐function mutants drive the initiation and development of tumors. Its deficiency induces wide epigenetic alterations that affect multiple cellular functions, including transcription stress, genomic stability, metabolism reprogramming, and response to immune checkpoint blockade.KMT2D is frequently mutated in a cohort of Chinese AML. However, several studies in MLL‐AF9‐ or HOXA9‐driven AML found that Kmt2d deficiency did not drive but suppress leukemia formation, which seems to be the opposite effect of Kmt2d on other cancers like B‐cell lymphoma. Therefore, it is worthwhile to investigate the function and mechanism of KMT2D on leukemia genesis and maintenance.KMT2D in AML and its tumor suppression capacity in other cancers, we hypothesized KMT2D is a tumor suppressor in AML. To test our hypothesis, we studied a mouse AML model where we reduced Kmt2d expression by shRNA knockdown and CRISPR/Cas9 technology. Further, we found that ribosome biogenesis was dramatically increased in Kmt2d‐deficient hematopoietic stem and progenitor cells (HSPCs) and AML cells. Inhibition of rRNA synthesis could significantly prolong the survival of Kmt2d‐deficient AML mice. Altogether, our data indicate that Kmt2d deficiency could promote AML and confer a new vulnerability to ribosome biogenesis inhibitors.Given the frequent, seemly loss‐of‐function mutations of 22.1KMT2D in AML patients, we found that compared to normal control , AML samples (n = 43) contained significantly lower KMT2D expression (GSE48173). A similar observation was obtained from another cohort . A28. AKMKMT2D in leukemogenesis, we developed a transplantation‐based mouse model with RNA interference approach. Both shRNAs effectively repressing Kmt2d (shKmt2d) in mouse cells were cloned and confirmed by quantitative real‐time polymerase chain reaction , together with Trp53 and Nf1 loss, into sub‐lethally irradiated syngeneic wild‐type C57BL/6 recipient mice  developed AML significantly faster with shorter overall survival  counts compared to controls 7 weeks after transplantation, while the former had variably decreased hemoglobin (Hb) and platelet (PLT) levels, indicative of leukemic outgrowth with the suppression of normal hematopoiesis  who had mixed lineage leukemia  and splenomegaly  because of extramedullary hematopoiesis and leukemia infiltration. Hematoxylin and eosin (H&E) staining of the spleen, liver, and bone marrow revealed prominent leukemia with the disruption of normal architecture . About 1.6% (3/190) of TCGA AMLs carry KMT2D mutants or deletions. Interestingly, in the OHSU AML cohort , KMT2D mutations (5/531) are all truncating mutations  targeting the genomic DNA sequence encoding the PHD domain of Kmt2d (sgKmt2d), together with Nf1 sgRNA (sgNf1), were transduced into c‐Kit+ HSPCs from Trp53−/−; Cas9 knock‐in transgenic mice, followed by transplantation into 5.5 Gy irradiated syngeneic wild‐type mice  was also used to prevent potential adverse effects associated with constitutive Cas9 expression.We then analyzed the status of eletions.4 InteTrp53−/−; sgNf1; sgKmt2d; sgCas9 HSPCs (hereafter referred to as TNKC) died of AML at a median survival of 59 days, while none of the control sgScramble recipients developed diseases during the observation period , while a withdraw time of 4 days shuts down the TRE promoter and shKmt2d transcription  images and immunofluorescence staining using an antibody against nucleolar protein fibrillarin, shKmt2d AML cells contained significantly enlarged nucleoli and brighter ribosome biogenesis key molecule fibrillarin staining, compared to control cells , in line with the impact of H3K4 methylation on activating gene expressions  to profile the transcriptomes of s Figure . The upr2.4Kmt2d regulates ribosome biogenesis, we sought to conduct multiomics analyses to identify downstream pathways and targets of Kmt2d. First, digging deeper into RNA‐seq data using GSEA, we found that genes in the HALLMARK_MTORC1_SIGNALING pathway were significantly positively enriched in shKmt2d leukemia cells and HSPCs  technique to investigate the corresponding H3K4me1, H3K4me2, or H3K27ac levels in Kmt2d restored and knockdown AML cells. Consistent with the KMT2D enzymatic activity, Kmt2d‐deficient AML cells had a decrease in differentially modified peak numbers of H3K4me1, H3K4me2, and H3K27ac. Specifically, in comparison with the control group, Kmt2d‐deficient AML cells exhibited 6680, 2592, and 1486 significantly lower H3K4me1, H3K4me2, and H3K27ac modified peaks, respectively  and RNA polymerase III (Pol III) bound regions, which specialize in the transcription of rRNA, were marked with elevated H3K27ac modifications and possessed increased genome accessibility levels in Kmt2d‐deficient AML cells, concordant with increased gene expression and enhanced rRNA concentration  and normal Kmt2d control leukemia cells (TN) side by side. Results showed that Kmt2d‐deficient AML cells were more sensitive to the CX‐5461 treatment , we found that in KMT2D‐mutated cells, cell and nuclear sizes were enlarged . Therefore, KMT2D may also regulate ribosome biogenesis and leukemogenesis through mTOR‐independent mechanisms. Altogether, we illustrated a novel function of KMT2D that could regulate protein translation in addition to its critical role in controlling gene transcription.As an important tumor suppressor gene, er cells.26 HerKmt2d‐deficient AML. The drug experiment not only confirmed the important role of ribosome biogenesis in Kmt2d‐deficiency‐induced AML, but also suggested a potential therapeutic target for KMT2D low‐expression or mutated AML patients. Ribosome biogenesis has emerged as an effective pathway for cancer therapeutics. Apart from chemotherapeutic drugs which exert their cytotoxic effects by perturbation of ribosome biogenesis at various levels, several novel compounds that selectively target ribosome production or function, mainly inhibitors of Pol I transcription, such as CX‐5461, have recently entered clinical trials. CX‐5461 has shown therapeutic potential in hematological malignancies. CX‐5461 treatment could effectively inhibit mouse MLL‐fusion AML progression and human AML cell lines in both Trp53‐dependent and ‐independent mechanisms. Also, combination therapy targeting ribosome biogenesis and mTORC1‐dependent translation synergistically extends survival in MYC‐driven lymphoma. Thus, several phase I clinical trials of CX‐5461 in B‐cell lymphomas and solid cancers have been done or are ongoing . According to these studies, CX‐5461 treatment in patients is safe and holds promise. Further validation of CX‐5461 and other inhibitors with similar functions in human cancer cells with KMT2D mutations or deficiency would pave the way for their potential applications.Furthermore, our work revealed that CX‐5461, a specific RNA polymerase I inhibitor, could effectively reduce tumor burden and significantly prolong the survival of mice bearing l trials.49 CX‐KMT2D could be a tumor suppressor gene in AML. KMT2D regulates the expression of a negative regulator DDIT4 in the mTOR pathway via histone methyltransferase activity, suppresses ribosome biogenesis, and eventually prevents leukemogenesis. CX‐5461, the selective inhibitor of RNA Pol I transcription, has an antileukemia effect on Kmt2d‐deficient AML. Besides, KMT2D mutations and abnormalities are also common in other leukemias. As such, our observations may have implications beyond AML.In summary, we demonstrate that eukemias.55 As 4Trp53−/− mice from The Jackson Laboratory  and Trp53−/− Cas9‐expressing mice obtained by breeding Trp53−/− mice with Cas9 knock‐in mice from The Jackson Laboratory  were served as donor mice (both in C57BL/6 background). Bone marrow cells were freshly isolated from femurs and tibias of 6 to 8 weeks old donor mice and HSPCs were sorted using c‐kit (CD117) Microbeads  and a QuadroMACS separator . For in vivo tumorigenesis, HSPCs were transduced with lentiviruses encoding sgRNAs or retroviruses encoding shRNAs, and 10E+6 transfected HSPCs were injected into 5.5 Gy irradiated C57BL/6 recipient mice  via the tail vein injection. All recipient mice were randomly divided into each group before transplantation and monitored by complete blood count (CBC) and blood smears. Blood count measurement was performed on a HemaVet 950FS Blood Analyzer (RRID:SCR_020016). For secondary transplant experiments, 10E+6 bone marrow leukemia cells were injected into 5.5 Gy irradiated C57BL/6 recipient mice.2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% vol/vol fetal bovine serum (FBS) and penicillin (100 U mL−1)/streptomycin (0.1 mg mL−1). MOLM‐13 (RRID:CVCL_2119) was from DSMZ and cultured at 37 °C with 5% CO2 in RPMI 1640 medium supplemented with 10% FBS and penicillin (100 U mL−1)/streptomycin (0.1 mg mL−1). Mouse HSPCs and AML cells were cultured at 37 °C with 7.5% CO2 in BCM medium [45% IMDM + 45% DMEM + 10% FBS + 50 × 10−6mβ‐mercaptoethanol + penicillin (100 U mL−1)/streptomycin (0.1 mg mL−1)] supplemented with 10% stem cell medium (SCM) including mIL‐3 , mIL‐6 , and mSCF .HEK293T (RRID:CVCL_0063) and NIH 3T3  were from ATCC and cultured at 37 °C with 5% COhttp://splashrna.mskcc.org/). Sequences of shRNAs for Kmt2d are displayed in Table Ren and shNf1 are from published reports. shRNAs were cloned into retroviral vectors including MSCV‐shRNA‐SV40‐GFP, MSCV‐shRNA‐SV40‐mCherry, MSCV‐shRNA‐PGK‐puromycin‐IRES‐GFP, MSCV‐shRNA‐IRES‐rtTA3, and TRE‐dsRed‐shRNA‐PGK‐Venus‐IRES‐neomycin.97 bp oligonucleotides with gene‐specific hairpins were designed by splashRNA ( reports.8 shRNhttps://www.atum.bio/eCommerce/cas9/input). Sequences of sgRNAs for Kmt2d, Nf1, Cas9, Scramble, and KMT2D are displayed in Table KMT2D‐mutated MOLM‐13 cell lines, V2T plasmid which expressed Cas9 cDNA and puromycin was transduced into cells, followed by puromycin  selection, and sgRNA transduction. Mutation validation was performed by the T7E1  assay and primers for the T7 endonuclease I mismatch detection assay are displayed in Table sgRNAs were designed by the ATUM CRISPR gRNA Design tool . Data were analyzed using FlowJo software (RRID:SCR_008520).58 Ant−3m Tris‐HCl (pH 6.8), 2% w/v SDS, 150 × 10−3m NaCl, 1% NP‐40, 40 × 10−3m dithiothreitol) followed by sonication with an ultrasonic cell disruptor. Lysate proteins were separated by 12% SDS‐polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes. The primary antibodies used are displayed in Table 10E+6 cells were harvested and lysed in sodium dodecyl sulfate (SDS) buffer  and cDNA was synthesized using Hiscript III RT SuperMix for qPCR  following the manufacturer's instructions. qRT‐PCR was performed using ChamQ Universal SYBR qPCR Master Mix  on the QuantStudio 3 Real Time PCR System (RRID:SCR_018712). Sequences of qRT‐PCR primers are displayed in Table For H&E staining, mouse organs were fixed overnight in 4% paraformaldehyde, embedded in paraffin blocks, and sectioned. Tissue sections were stained with hematoxylin  for 5 min to identify the cell nucleus and eosin  for 5 min to identify the cytoplasm. For immunofluorescence, cells were deposited on slides by cytocentrifuge , fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X‐100 for 15 min. The antibody of Fibrillarin  was used. After secondary antibodies were added, the slides were mounted by mounting medium with 4′,6‐diamidino‐2‐phenylindole . And images were taken with Zeiss LSM 880 with Airyscan confocal laser scanning microscope (RRID:SCR_020925).−3m 5‐FUrd , then washed in cold phosphate‐buffered saline for subsequent immunofluorescence, performed as described above, using the antibody of BrdU .Cells were incubated for 30 min in medium containing 2 × 10Nascent protein synthesis was detected using Click‐iT Plus OPP Protein Synthesis Assays kit  following the manufacturer's instructions. The intensity of OPP signals was detected by flow cytometry analyses and presented as the mean fluorescence index.−1 CX‐5461  every 3 days via oral gavage. Administration timing can be adjusted according to the physical condition of the mice. The leukemia progression in recipient mice was monitored by CBC, flow cytometry, and blood smears.10E+6 bone marrow leukemia cells were injected into 5.5 Gy irradiated C57BL/6 recipient mice. Drug treatments were initiated 10 days after transplantation. Mice were treated with either vehicle (0.5% CMC‐Na) or 40 mg kgKmt2d) or without doxycycline (Kmt2d‐restored) were harvested for RNA‐seq. HSPCs isolated from bone marrow cells of Trp53−/− mice were transfected with retroviruses encoding shKmt2d or shRen. 48 h later GFP‐positive cells were sorted by the BD FACSAria III cell sorter (RRID:SCR_016695) for RNA‐seq. RNA libraries were prepared using standard Illumina protocols. Transcriptome sequencing was performed by the Illumina NovaSeq 6000 platform. RNA‐seq reads were aligned to the reference mouse genome (mm10) by STAR . Raw counts normalization, significance scores, and log2‐fold change values calculation were performed by DESeq2 (RRID:SCR_015687). Differential expression genes were used for GO analysis with R package clusterProfiler (RRID:SCR_016884). GSEA (RRID:SCR_003199) was performed to identify significantly enriched pathways. The software Integrative Genomics Viewer  and the R packages ggpubr (RRID:SCR_021139), ggplot2 (RRID:SCR_014601), pheatmap (RRID:SCR_016418), Vennerable were used for data visualization.AML cells treated with doxycycline . The primary antibodies used are displayed in Table  standard workflow, Bowtie2 (RRID:SCR_016368) was used for mouse genome (mm10) index construction and 150 bp paired‐end clean data alignments with “–local –very‐sensitive‐local –no‐unal –no‐mixed –no‐discordant –phred33 ‐I 10 ‐X 700” option. The program SAMtools (RRID:SCR_002105) was performed to convert sam files into sorted bam files. Duplicates were removed using MarkDuplicates tools from Picard (RRID:SCR_006525) with “REMOVE_DUPLICATES = true” option. Normalized bw files were generated by DeepTools (RRID:SCR_016366) with “bamCoverage ‐bs = 1 –normalizeUsing BPM” option for further IGV (RRID:SCR_011793) visualization. MACS2 was performed for peak calling with “macs2 callpeak –broad ‐q 1e‐2 ‐f BAMPE ‐g mm –keep‐dup all” option. Genomic distribution of peaks was identified by ChIPseeker (RRID:SCR_021322) with “annotatePeak” option and the TSS region “tssRegion” was set as . “narrowPeak” files were transformed into GrangesList forms and peaks were converted into consensus counts. Data normalization and difference comparison were performed using DESeq2 (RRID:SCR_015687). Peaks with a p‐value cutoff of 0.05  were identified as differential binding or modification regions for downstream analyses.shin Table 63 sta and the transposase was from TruePrep DNA Library Prep Kit V2 for Illumina . The library was sequenced on the Illumina NovaSeq 6000 platform. NGmerge was performed to remove adapters from 150 bp paired‐end raw data with “NGmerge ‐a ‐v ‐n 20” option. The mouse genome (mm10) index construction and follow‐up alignments were performed using Bowtie2 (RRID:SCR_016368) with “–very‐sensitive ‐X2000 ‐x mm10” option. Duplicates were removed using MarkDuplicates tools from Picard (RRID:SCR_006525). The mitochondrial genome contamination was removed and bam files were obtained using SAMtools (RRID:SCR_002105) and awk commands. Normalized bw files were generated using DeepTools (RRID:SCR_016366) with “bamCoverage ‐bs = 1 –normalizeUsing BPM” option for further IGV (RRID:SCR_011793) visualization. The standard workflow of HMMRATAC was utilized for the ATAC‐seq peak calling step. The genomic distribution of accessibility sites was identified by ChIPseeker (RRID:SCR_021322) with “annotatePeak” option and the TSS region “tssRegion” was set as . “gappedPeak” files were transformed into GrangesList forms and peaks were converted into consensus counts. Data normalization and difference comparison were performed using DESeq2 (RRID:SCR_015687). Pol I and Pol III binding regions were cited from GSE145874. Peaks with a p‐value cutoff of 0.05  were identified as differential accessibility regions for downstream analyses.Library preparation was performed as previously described,67 and and OHSU AML cohorts. The co‐occurrence of KMT2D, TP53, and NF1 mutations in leukemia patients was analyzed by GENIE Cohort v11.0‐public datasets . Transcriptome data of normal and AML patients were acquired from high‐throughput sequencing data GSE48173  and microarray data GSE1159 . RPKM of GSE48173 was performed with log2 transformation to obtain the standardized expression value. p value was determined by the Wilcoxon signed‐rank test. To estimate the impact of KMT2D expression on the prognosis of AML patients, patients were divided into KMT2D‐high and KMT2D‐low groups by FPKM in the TCGA‐LAML and Beat AML cohorts. The optimal cut‐point for numerical variables of KMT2D expression was calculated with the maximally selected rank statistics by the survminer (RRID:SCR_021094) package to stratify patients for the survival analysis, set as 20.78 and 7.12 in TCGA‐LAML and Beat AML cohort, respectively. According to the algorithm, 122 and 20 patients in the TCGA‐LAML cohort were divided into KMT2D‐high and KMT2D‐low groups. GSEA (RRID:SCR_003199) was performed to identify significantly enriched pathways between the two groups. The ribosome biogenesis gene signature was determined by GOBP_RIBOSOME_BIOGENESIS. Correlations between KMT2D expression and mean expression levels of genes involved in ribosome biogenesis were visualized by ggpubr (RRID:SCR_021139) and correlation values were determined by Spearman's rank correlation coefficient.Genetic alteration data were analyzed from The Cancer Genome Atlas AML project (TCGA‐LAML)4 and R packages ggpubr and ggplot2 were performed to show the expression and accessibility level differences by box plots. Heatmaps were generated by the R package pheatmap to present differentially expressed genes. Bar plots and dot plots of GO and GSEA enrichment results were drawn by ggplot2. The program DeepTools was utilized to visualize the histone modification levels. Matrix computations of differential modification and accessibility peaks were performed using “ComputeMatrix reference‐point –referencePoint center ‐b 3000 ‐a 3000 –skipZeros” option.p values are indicated in the corresponding figure legends. Statistical significance was determined using GraphPad Prism  and PASW Statistics18 software by unpaired two‐tailed t‐test, Wilcoxon signed‐rank test, Wald test, hypergeometric distribution, or log‐rank test. Statistical significance was defined as p < 0.05. Error bars were shown as SD.Statistical test methods, sample sizes, and All animal study procedures and experiments were reviewed and approved by the Experimental Animal Ethics Committee of the State Key Laboratory of Biotherapy and Cancer Center, Sichuan University  and were in accordance with the 8th edition of the Guide for the Care and Use of Laboratory Animals.The authors declare no conflict of interest.J.X., A.Z., and S.Z. contributed equally to this work. C.C. and Y.L. conceived the project and designed experiments. J.X., S.Z., M.C., L.Z., X.H., J.Z., B.W., X.D., Z.W., L.Q., K.S., S.L., Y.W., Q.Z., and P.L. performed experiments. J.X., A.Z., S.Z., R.P.G., and T.N. analyzed data. J.X., A.Z., X.C., and X.P. performed RNA‐seq, ATAC‐seq, and CUT&Tag analyses. J.X., A.Z., X.C., M.W., C.C., Y.L., and T.N. prepared the manuscript. J.X., A.Z., and S.Z. are co‐first authors. J.X. and S.Z. performed much of the experimental work, and A.Z. performed much of the bioinformatic analyses. All authors read and approved the final manuscript.Supporting InformationClick here for additional data file.


As a second messenger, cGMP activates the cGMP-dependent protein kinase G (PKG), which regulates numerous targets/cellular events. The dysregulation of cGMP signaling is observed in varieties of photoreceptor/retinal degenerative diseases. Abnormally elevated cGMP signaling interferes with various cellular events, which ultimately leads to photoreceptor degeneration. In line with this, strategies to reduce cellular cGMP signaling result in photoreceptor protection in mouse models of retinal degeneration. The potential mechanisms underlying cGMP signaling-induced photoreceptor degeneration involve the activation of PKG and impaired Ca2+ homeostasis/Ca2+ overload, resulting from overactivation of the CNG channels, as well as the subsequent activation of the downstream cellular stress/death pathways. Thus, targeting the cellular cGMP/PKG signaling and the Ca2+-regulating pathways represents a significant strategy for photoreceptor protection in retinal degenerative diseases.Photoreceptors in the retina are highly specialized neurons with photosensitive molecules in the outer segment that transform light into chemical and electrical signals, and these signals are ultimately relayed to the visual cortex in the brain to form vision. Photoreceptors are composed of rods and cones. Rods are responsible for dim light vision, whereas cones are responsible for bright light, color vision, and visual acuity. Photoreceptors undergo progressive degeneration over time in many hereditary and age-related retinal diseases. Despite the remarkable heterogeneity of disease-causing genes, environmental factors, and pathogenesis, the progressive death of rod and cone photoreceptors ultimately leads to loss of vision/blindness. There are currently no treatments available for retinal degeneration. Cyclic guanosine 3′, 5′-monophosphate (cGMP) plays a pivotal role in phototransduction. cGMP governs the cyclic nucleotide-gated (CNG) channels on the plasma membrane of the photoreceptor outer segments, thereby regulating membrane potential and signal transmission. By gating the CNG channels, cGMP regulates cellular Ca The human retina is comprised of light-responsive neuronal cells known as photoreceptors. Light stimulation initiates phototransduction in photoreceptors, which is conveyed to secondary neurons, ganglion cells, and, ultimately, to the visual cortex in the brain to form vision. There are two types of photoreceptors: rods and cones. Rods respond to dim light and enable vision at night, whereas cones react to bright light and are responsible for daylight vision, color vision, and visual acuity ,2,3,4. P2+ and Na+ influx. Upon light stimulation, the light-sensing protein rhodopsin/opsin undergoes a conformational rearrangement, which activates the heterotrimeric G protein transducin. Consequently, the α subunit of transducin activates phosphodiesterase 6 (PDE6), leading to the catalytic acceleration of cGMP hydrolysis, closure of the channels, and the hyperpolarization of the cell i.The cellular level of cGMP is highly regulated in photoreceptors. The biosynthesis of cGMP is catalyzed by the retinal guanylyl cyclases (RetGCs) from the guanosine triphosphate (GTP), and its degradation is achieved by PDE6, which specifically hydrolyzes the 3′5′ cyclic phosphate bond  Figure . The bioGUCY2D (Gucy2e in mouse), and RetGC2 is encoded by GUCY2F (Gucy2f in mouse) i is high, the Ca2+-bound GCAPs inactivate RetGC and inhibit cGMP synthesis , and the Mg2+-bound GCAPs activate RetGCs and stimulate cGMP synthesis . RD3 is a 23-kDa monomer with an elongated four-helix bundle . The in PDE6 is the primary regulator of intracellular cGMP concentration in rod and cone photoreceptors . PDE enze enzyme .2+/Mg2+-GCAP/RetGC complex and PDE6, cellular cGMP level is indirectly controlled by photoreceptor CNG channels via [Ca2+]i  keeps the plasma membrane depolarized and promotes the synaptic glutamate release. As a feedback regulatory mechanism, the elevated [Ca2+]i inhibits cGMP synthesis via the GCAP/RetGC complex. The elevated [Ca2+]i also slightly reduces the affinity of cGMP to the channel [2+ influx/[Ca2+]i, and the subsequent activation of the GCAP/RetGC complex  identified to date. These mutations are mainly associated with autosomal-dominant CRD (adCRD)  mouse harbors a 116-bp insertion between exon 4 and 5, as well as an additional 1-bp deletion in exon 7 in the Pde6c gene, leading to an in-frame shift, introducing premature termination codons, the loss of activity of the enzyme, and the accumulation of cGMP i, cannot decrease the intracellular levels of cGMP in Pde6 mutants. The cellular mechanism(s) underlying this observation is unknown at this time. The observation may suggest that the feedback regulation by the GCAP/RetGC complex is less sensitive to the elevation of [Ca2+]i than to the reduction in [Ca2+]i. It may also imply that the feedback regulation is blunted, for some reason, in the absence of functional PDE6. Nevertheless, the feedback regulation of cGMP production by the GCAP/RetGC complex, in the absence of functional PDE6, merits further investigation.As mentioned above, the disease-causing mutations in CNGA1 and CNGB1 are associated with arRP, accounting for 2–3% of arRP cases i and accumulation of cellular cGMP have been shown in the cones of CNG channel-deficient mice [Cngb1 deficiency [2+]i, which subsequently leads to the overproduction of cGMP via the Ca2+/Mg2+-GCAP/RetGC complex.Mutations in CNG channels are associated with photoreceptor degeneration. Mutations in RP cases ,103,104.s i in CNG channel deficiency harms the ER Ca2+ homeostasis and deteriorates ER function. The elevated cGMP/PKG signaling also activates histone deacetylase (HDAC)/poly-ADP-ribose polymerase (PARP) signaling, leading to DNA condensation/damage and cell death. The elevated cGMP signaling and the subsequent cellular Ca2+ overload triggers the calpain-associated cell death pathway. Moreover, the ER stress, activation of PARP signaling, and activation of calpain signaling induce the mitochondrial insult and release of the apoptosis-inducing factor (AIF) to worsen the death process.There are multiple mechanisms involved in cGMP dysregulation-induced photoreceptor degeneration . The ele2+ storage. ER homeostasis is critical for its function, and impaired ER homeostasis will lead to unfolded protein response (UPR)/ER stress, impaired protein processing/trafficking, and impaired ER-associated protein degradation (ERAD). Under the stress conditions or accumulation of incorrectly folded/unfolded proteins, ER will undergo UPR/ER stress, which triggers cytotoxicity and cell death. ER stress is typically characterized by the activation of the three pathways/arms, including the pancreatic ER kinase (PKR)-like ER kinase (PERK)/eukaryotic-initiation factors 2α (phospho-eIF2α) pathway, the cleaved activating transcription factor 6 (ATF6) pathway, and the inositol-requiring kinase 1 (IRE1) pathway i has been shown in the cones of CNG channel-deficient mice i and the subsequent activation of RetGC. Activated cGMP/PKG signaling induces ER stress and the impairment of ER Ca2+ homeostasis. Either cellular Ca2+ overload or Ca2+ depletion will impair cellular homeostasis, leading to cell stress/death. In PDE6 deficiency, RD3 deficiency, or RetGC overaction, the activated cGMP/PKG signaling, along with Ca2+ overload/calpain activation, plays a significant role in the disease pathogenesis and cell death progression. In CNG channel deficiency or RetGC deficiency, the activated cGMP/PKG signaling, along with cellular Ca2+ depletion, plays a significant role in the disease pathogenesis and cell death progression. The activated cGMP/PKG signaling may also induce HDAC and PARP, leading to cell stress/death directly and via mitochondrial dysfunction/the release of AIF. Thus, targeting cGMP/PKG signaling in photoreceptors and the downstream target cellular organelles/molecules represents a significant approach to slow photoreceptor death in retinal degenerative diseases.Photoreceptor cGMP level is tightly controlled by a well-developed, feedback regulatory system. The Ca


Ca2+ deficiency causes a physiological disorder of tip-burn in Brassiceae crops and is involved in the regulation of cellular Ca2+ homeostasis. Although the functions of Ca2+/H+ exchanger antiporters (CAXs) in mediating transmembrane transport of Ca2+ have been extensively characterized in multiple plant species, the potential roles of BrCAX genes remain unclear in Chinese cabbage. In this study, eight genes of the BrCAX family were genome-widely identified in Chinese cabbage. These BrCAX proteins contained conserved Na_Ca_ex domain and belonged to five members of the CAX family. Molecular evolutionary analysis and sequence alignment revealed the evolutionary conservation of BrCAX family genes. Expression profiling demonstrated that eight BrCAX genes exhibited differential expression in different tissues and under heat stress. Furthermore, Ca2+ deficiency treatment induced the typical symptoms of tip-burn in Chinese cabbage seedlings and a significant decrease in total Ca2+ content in both roots and leaves. The expression changes in BrCAX genes were related to the response to Ca2+ deficiency-induced tip-burn of Chinese cabbage. Specially, BrCAX1-1 and BrCAX1-2 genes were highly expressed gene members of the BrCAX family in the leaves and were significantly differentially expressed under Ca2+ deficiency stress. Moreover, overexpression of BrCAX1-1 and BrCAX1-2 genes in yeast and Chinese cabbage cotyledons exhibited a higher Ca2+ tolerance, indicating the Ca2+ transport capacity of BrCAX1-1 and BrCAX1-2. In addition, suppression expression of BrCAX1-1 and BrCAX1-2 genes reduced cytosolic Ca2+ levels in the root tips of Chinese cabbage. These results provide references for functional studies of BrCAX genes and to investigate the regulatory mechanisms underlying Ca2+ deficiency disorder in Brassiceae vegetables.Calcium (Ca As i of Ca2+ . CAX fam of Ca2+ . The inn of Ca2+ . The imp of Ca2+ . Plant C of Ca2+ ,16.Arabidopsis thaliana are most thoroughly studied in biological functions, such as ion transport, abiotic stress signaling, and Ca2+ homeostasis [Arabidopsis cax mutants indicate the phenotypes of an increased sensitivity to ion toxicity and abiotic stress [Arabidopsis CAX1 and CAX2 transporters have been demonstrated the regulatory roles in cation transport [cax4 loss-of-function mutant suggests the important modulation of CAX4 gene in root growth of Arabidopsis under heavy metal stress conditions [cax1/cax3 double mutation exhibits necrosis of the leaf tips and shoot apex in Arabidopsis and suggests the exchange of cytosolic Ca2+ by CAX1 and CAX3 antiporters [Arabidopsis CAX1 in tobacco increases Ca2+ accumulation in leaves and roots and improves salt stress sensitivity [CAX1 exhibits a higher Ca2+ concentration in tissues, it shows an evident increase in the symptoms of Ca2+ disorders [CAX1 presents the phenotype of Ca2+ disorder in fruit, such as a higher accumulation of total Ca2+ and the increased incidence of fruit necrotic lesions [2+ and incidence of Ca2+ disorder may be the result of abnormal cellular Ca2+ partitioning and distribution [The CAX proteins from eostasis ,17. In aeostasis ,18. Sevec stress ,20,21. Aransport ,23. A canditions . The caxiporters . Overexpsitivity ,27. Remaisorders . In toma lesions ,30. The 2+ deficiency disorder is a physiological disorder, such as black heart in celery [2+ deficiency disorder, including localized Ca2+ deficiency and aberrant Ca2+ homeostasis, which are linked to cellular Ca2+ transcript and CAX activity [Brassica rapa L. ssp. pekinensis), belonging to Brassicaceae family, is an economically important leafy vegetable grown worldwide. Ca2+ deficiency-induced tip-burn mainly occurs in the inner fast-grown leaves of Chinese cabbage, leading to a serious threat to quality and yield [2+ transmembrane transport and tip-burn incidence remains largely unveiled in Chinese cabbage. In this study, to understand the specific expression and putative function of CAX genes in response to environmental stresses and Ca2+ deficiency conditions, eight genes from BrCAX family were genome-widely identified and characterized in Chinese cabbage. The temporal and spatial expression profiles of BrCAX genes were analyzed under different Ca2+ deficiency conditions. Moreover, the Ca2+ transport capacities of BrCAX1-1 and BrCAX1-2 genes were validated by overexpression in yeast and Chinese cabbage cotyledons and transient suppression expression in Chinese cabbage. The results of this study will provide references for functional studies of BrCAXs in regulating Ca2+ transport and Ca2+ deficiency-induced tip-burn in Chinese cabbage and other vegetable crops. Can celery , blossomn celery , bitter n celery , and tipn celery ,35, and n celery ,3,36. Coactivity ,36. Chinnd yield . However2+ contents, keeping the consistent content of other elements, especially N, P, and K. The normal Hoagland contained 4 mM Ca2+ contents. The conditions with 1.5 and 0.75 mM Ca2+ contents were considered Ca2+ deficiency treatments. For gene expression analysis, the tissues, including root, leaf base, and leaf apex, were collected at 28 d after treatment. The symptom of tip-burn was observed every 7 d under treatment. For the determination of total Ca2+ content, the roots and leaves of seedlings were also collected at 28 d after treatment.The Chinese cabbage tip-burn susceptible line ‘10S42′ and resistant line ‘10S230′ were used in this study. Germinated seeds were grown in 1/4 Hoagland nutrient solution under standard growth conditions with 7 d. Subsequently, the seedlings were transferred to different treatments with 4, 1.5, and 0.75 mM CaA. thaliana were downloaded from the Arabidopsis Information Resource (TAIR) database and used as BLASTP query to search against the Brassicaceae database (BRAD). On the other hand, Hidden Markov Model (HMM) file of Na_Ca_ex domain (PF01699) was downloaded from Pfam database and subjected to search against the genomic sequences of Chinese cabbage using HMMER (version 3.2.1) search tool with an E-value cut-off of 1.0. After removing the repeat sequences from the two searching results, candidate sequences were verified by analyzing conserved Na_Ca_ex domain through public databases, including NCBI Conserved Domain Database , Pfam , and SMART . The obtained proteins were named as BrCAXs based on their homologous annotations. The physiological and biochemical characteristics of BrCAX proteins were analyzed using ProtParam tool of ExPASy . Subcellular localization of BrCAX proteins was predicted by online WoLF PSORT program . The exon–intron structures of BrCAX genes were analyzed using the online Gene Structure Display Server  with default parameters.The known sequences of CAX family proteins from CAX family genes from other five Brassica species, including Brassica napus, Brassica oleracea, A. thaliana, Arabidopsis lyrata, and Raphanus sativus, were performed to obtain the CAX homologous protein sequences from BRAD database (B. rapa chromosome information in BRAD database.The genome-wide searches of database . MultiplKa (nonsynonymous substitution rate) and Ks (synonymous substitution rate) were calculated to detect positive selection of CAX homologs. The protein sequences of CAX homologs from six Brassica species were aligned using ClustalX 2.1 software. Alignment results were submitted to PAL2NAL  and translated into coding sequence alignments. The output alignments were used to estimate the values of Ka, Ks, and Ka/Ks ratio using KaKs Calculator 2.0 software with Model Selection (MS) method. The boxplot of pairwise comparisons among CAX homologs was constructed using R program.The Saccharomyces cerevisiae VCX1 (NCBI: CAA98696.1) protein was used for homology modeling of BrCAX proteins. The obtained 3D structures of BrCAX proteins were visualized using PyMOL Viewer 2.5 software. Structural alignment and amino acid residue analysis were also performed.The three-dimensional (3D) protein structures of BrCAXs were predicted using the homology model method by Phyre2 web . The knoBrCAX genes were retrieved from BRAD database. The putative cis-acting elements were predicted using PlantCARE program  and visualized using TBtools 1.105 software.The promoter sequences (the 2.0 kb upstream sequences of the 5′ regulatory regions) of BrCAX genes were analyzed using the available RNA-Seq data of Chinese cabbage ‘Chiifu’ [2 FPKM. In addition, the probe intensity (PI) value of BrCAX gene expressions under 45 °C heat stress was obtained from a previous microarray analysis on Chinese cabbage ‘Chiifu’ [BrCAX genes was analyzed using the values of log2 fold change (PI in heat stress/PI in control). Heat map of gene expression was drawn using TBtools 1.105 software.The tissue-specific expression patterns of ‘Chiifu’ . The FPK‘Chiifu’ . ExpressBrCAX genes in response to Ca2+ deficiency stress was performed by quantitative real-time PCR (qRT-PCR). Total RNA extraction and cDNA synthesis were performed according to the previous reports [EF-1α gene (Bra031602) of B. rapa was used as internal reference gene to normalize expression data. The relative expression was calculated using delta-delta-CT method.Further expression analysis of  reports . The qRThttp://stringdb.org, accessed on 15 March 2023) with the parameter of the minimum required interaction score ≥ 0.7. The interaction relationships were analyzed according to the genome information of Chinese cabbage from BRAD and NCBI databases. The predicted interacting proteins were annotated based on KEGG and NCBI databases.An interaction network of BrCAX proteins was predicted using STRING  and BrCAX1-2 (−36 aa) (labeled BrCAX1-1-N and BrCAX1-2-N) were also obtained from the full-length sequences. The obtained sequences were inserted into the yeast expression vector pDR196 and transferred into the Ca2+ sensitive yeast mutant strain k667 (cnb1::LEU2 pmc1::TRP1 vcx1∆) [2+ tolerance assay of yeast, the prepared yeast suspensions (OD600 = 1.0) were serially diluted  and then spotted onto YPDA/SD-ura medium containing different concentrations of CaCl2 . Meanwhile, the yeast solutions cultured to the exponential phase were transferred to liquid YPDA/SD-ura medium containing 50 mM CaCl2 and further cultured. The OD values were measured every 2 h and then used to construct the growth curves of different yeast strains. The yeast cells from different transformants under 25 mM CaCl2 treatment were also collected for measuring the total Ca2+ content. For Ca2+ fluorescence staining assay, the yeast cells (OD600 = 0.4~0.5) under 50 mM CaCl2 treatment were washed using PBS solution three times and incubated with 15 μM Fluo4-AM for 1 h at 30 °C. A confocal laser scanning microscope  was used to observe the green fluorescence signaling under the excitation and emission wavelengths with 488 nm and 512~520 nm. The ImageJ 1.51 software was used to determine the grey values of green fluorescence in yeast cells.The full-length CDS sequences of thaliana , the N-t1 vcx1∆) . The useBrCAX1-1-N and BrCAX1-2-N sequences were inserted into the pCAMBIA-2300 vector containing green fluorescent protein (GFP) sequence and transferred into Agrobacterium strain GV3101. The infiltration experiments of Chinese cabbage cotyledons were performed according to the previously reported methods with minor modifications [Agrobacterium strain containing the target sequences with 1 mL were infiltrated into the back of cotyledons of Chinese cabbage seedlings (5 d old). After 24 h of infiltrating, the seedlings were sprayed with 50 mM CaCl2 solutions per 12 h and cultured in the dark with 2 d. The cotyledons infiltrated with the empty vector strain and sprayed with equivalent ddH2O were used as the controls. Observation of GFP fluorescence was performed using the confocal laser scanning microscope  and used to validate the successful infiltrating. The infiltrated cotyledons were collected for gene expression analysis as the above described. The phenotype and disease index of cotyledon necrosis were determined as follows: “0” representing no obvious necrosis, “1” representing the necrosis area < 50% of cotyledon area, and “2” representing the necrosis area > 50% of cotyledon area. The disease index was calculated using 30 seedlings per replicate, with three independent replicates.The ications ,44. The BrCAX1-1 and BrCAX1-2 in Chinese cabbage were performed using anti-sense oligonucleotides (AS-ODN) treatment as described previously with minor modifications [http://sfold.wadsworth.org/cgi-bin/soligo.pl, accessed on 5 May 2023)  after incubating with Fluo4-AM, according to the reported method by Li et al. [Gene suppression expressions of ications . The 7 day 2023) . The seei et al. .2+ contents were collected, washed, and dried at 85 °C for 24 h. Then, the samples with 0.1 g dry weight were dissolved in 6 mL HNO3. An atomic absorption spectrophotometer  was used to detect the total Ca2+ content according to the method by Bai et al. [The samples for the measurement of total Cai et al. .p < 0.05. The data were presented as the means ± SEM from at least three independent experiments. One-way ANOVA and Duncan’s post hoc tests in SPSS 24.0 software were used to analyze the data. Statistical significance was defined as BrCAX family genes in Chinese cabbage, the gene sequences of Arabidopsis CAX family were used to perform the genome-wide BLASTP and HMM search against BRAD database. As a result, a total of eight candidate sequences belonging to five genes were obtained and considered as BrCAX family members in Chinese cabbage  of BrCAX proteins were also shown in their crystal structures  containing “GNxxE” motifs were also identified in M3 and M8 regions. The specific N-terminal autoinhibitory region was identified in BrCAX1-1 (37 aa) and BrCAX1-2 (36 aa), which was similar to that in AtCAX1.Alignment of amino acid sequences showed that eight BrCAX proteins shared high similarity to AtCAX and ScVCX1 protein sequences and may have conserved protein functions . Remarka2+ transport and marked in green arrow, and their interacting residues of G102 (M3), N103 (M3), G298 (M8), and S325 (M9) in purple arrow were also found in an active site. In addition, three interacting residues (in red arrow) of E83 (M2), E230 (acidic helix), and D234 (acidic helix) in another active site were reported to respond to the cellular Ca2+ concentration. The identification of these highly conserved residues provides rich information for studying the putative ion transport functions of BrCAX proteins.According to the structural basis of ScVCX1 protein , the conBrCAX family genes, the cis-acting elements in the 2.0 kb region upstream of BrCAXs were predicted using PlantCARE online searching. The promotor regions of these BrCAX genes contained a variety of cis-acting elements, which were classified into four categories: light response, hormone response, biotic and abiotic stress, and plant growth and development (BrCAX genes. ABA responsive element (ABRE) and GA responsive element (MYB) were enriched in these BrCAX genes, with the exception of BrCAX3 without ABRE and BrCAX4 without MYB. Moreover, several elements that were associated with drought stress (MBS), temperature stress (LTR), salinity stress (STRE), and wound stress (WUN-motif) were found in these BrCAX genes. In addition, several cis-acting elements, such as light responsive element (Box 4), MeJA responsive element (MYC), and anaerobic induction related element (ARE), were most abundant in all the BrCAX genes, indicating that the putative roles of BrCAXs may respond to the cis-element-related regulatory processes or stress responses.To understand the regulatory patterns of elopment . A list BrCAX genes in six different tissues  were analyzed using FPKM values . The symptoms of tip-burn were observed in the seedlings under 1.5 and 0.75 mM Ca2+ deficiency conditions (1.5 and 0.75 groups)  A,B. The  groups) C–F, werewo lines G–I. Nota‘10S230′ H, the Ca‘10S230′ I, which BrCAX genes to Ca2+ deficiency stress, the expression levels of BrCAX genes were analyzed in the root, leaf base, and leaf apex from the two lines after Ca2+ deficiency treatments. The results showed that these BrCAX genes were differentially expressed in different tissues and under different Ca2+ deficiency treatments  was performed to investigate their Ca2+ transport capacity. Moreover, the N-terminal autoinhibitory region that affect Ca2+ transport was removed from their full-length gene sequences, and the truncated BrCAX1-1-N and BrCAX1-2-N were then transferred to k667 strain. As expected, under high Ca2+ treatment, BrCAX1-1 and BrCAX1-2 did not promote the growth in yeast, while the BrCAX1-1-N and BrCAX1-2-N significantly recovered the growth in yeast  event in the long evolutionary history [BrCAX gene family were identified from the genome of B. rapa. Compared with A. thaliana and A. lyrata, more members of CAX gene family also exist in other Brassica species, such as B. napus and B. oleracea, which confirmed the widespread existence of duplicated genes throughout the Brassica genus and genome duplications. Additionally, the number of CAX family members varied among different species that may be due to gene loss event. The whole genome duplication is usually followed by the substantial gene loss, which is regarded as the major source of adaptive functional novelty in plants [CAX subfamily display a characteristic of phylogenetic diversity within many species. Phylogenetic analysis of our study showed that the CAX proteins from Brassica species shared phylogenetic characteristics with five distinct gene subgroups. Moreover, analysis of evolutionary selective pressure showed the various values of Ka, Ks, and Ka/Ks ratio among the pairwise comparisons of CAX homologs. The variation of Ks values representing the divergence time suggested that these CAX genes diverged concurrently with the Brassica-specific WGT event. In addition, a higher value of Ka/Ks ratio was reported to indicate the genes that may be more likely to evolve new functions during the long evolutionary time [Ks values and lower Ka/Ks values, indicating the ancient evolutionary division but high evolutionary conservation in CAX1 and CAX2 genes.The expansion of a gene family is always accompanied by the tandem and segmental duplication events in species ,54. It i history ,56, whicn plants ,58. The ary time . In the α-repeat regions that include conserved residues for cation binding [α-repeat and reported to be opposite in topology [2+ binding and transport functions [α-repeat regions in an active site may determine their putative ion transport functions. The crystal structure of ScVCX1 shows an acidic helix containing two central residues of Glu and Asp in another active site, and the acidic helix region underneath α-repeat region locates on the cytosolic side with parallel orientation to membrane [2+ binding. Previous studies have reported that plant CAXs seemed to maintain the structural basis with implications for functional activity in Ca2+ homeostasis and signal transduction [2+ signaling.Although CAX family has multimember divergence, the CAX homologous proteins commonly share high structural conservation ,14. Insiunctions . All thesduction . In thisCAX genes focused on their physiological and molecular biological roles in diverse aspects of plant growth and development and in response to external stresses [BrCAX genes had various expression levels in different tissues, indicating that these BrCAXs may participate in diverse biological processes. BrCAX1-1 and BrCAX1-2 genes had higher expression levels in all the tissues than other genes, implying that the two genes may be the most actively expressed gene members. It is well known that CAX1 acts as a central ion transporter and has unique responses to biotic and abiotic stresses [Arabidopsis cax1 mutant enhanced the resistance against avirulent biotrophic pathogens and the accumulation of defense hormone salicylic acid (SA). Liu et al. [CAX1 gene from Puccinellia tenuiflora could complement active Ca2+ transporters and confer Ba2+ tolerance to yeast. In addition, apple CAX3 protein, a homologous protein to CAX1, was validated to have calcium transport activity in yeast [BrCAX genes, indicating the putative roles of BrCAXs in response to various stress conditions.Extensive research on stresses ,11. In tstresses ,22. Zhanstresses  reportedu et al.  reportedin yeast . In thisCAXs as Ca2+ transporters are involved in the process of Ca2+ deficiency disorders by controlling Ca2+ storage and transport and modulating intracellular Ca2+ homeostasis [sCAX1 gene showed Ca2+ deficiency symptoms in leaf and tuber tissues, which provided insights into the role of sCAX1 in Ca2+ homeostasis [sCAX1 and CAX4 contained significantly more Ca2+ [sCAX1 overexpression induced the mobility of Ca2+ to vacuole and a significant increase in the occurrence of BER [BrCAX1-1-N and BrCAX1-2-N genes improved the tolerance of yeast to high Ca2+ stress and promoted the accumulation of Ca2+ in the vacuole. The BrCAX1-1-N and BrCAX1-2-N overexpressed Chinese cabbage cotyledons exhibited lower index of necrosis and indicated high tolerance to environmental Ca2+ stress. In addition, suppression of BrCAX1-1 and BrCAX1-2 gene expressions reduced the cytosolic Ca2+ levels in the root tips of Chinese cabbage. These findings demonstrated that BrCAX1-1-N and BrCAX1-2-N genes function in Ca2+ transmembrane transport.Notably, the critical roles of eostasis ,62. Overeostasis . In tomaore Ca2+ , and sCAe of BER . In the CAX genes are demonstrated to respond to the change in Ca2+ concentration and the occurrence of Ca2+ deficiency-induced tip-burn in Brassica species [B. oleracea have identified several differentially expressed tip-burn related genes [BoCAX2 and BoCAX5 expressions between tip-burn susceptible and resistant lines [2+ deficiency condition, the expression levels of two BrCAX1 homologous genes in tip-burn resistant line were higher than that in susceptible line [2+ deficiency stress [2+ deficiency treatment induced the occurrence of tip-burn symptoms and a decrease in total Ca2+ content in tip-burn susceptible line ‘10S42′ and resistant line ‘10S230′ of Chinese cabbage. The expression changes of BrCAX genes exhibited obvious difference between the two lines and were responsive to Ca2+ deficiency stresses. Specifically, the expression patterns of BrCAX1-1 and BrCAX1-2 genes depended on different Ca2+ deficiency treatments, implying the possible participation of BrCAX1-1 and BrCAX1-2 in the response process and modulation of tip-burn in Chinese cabbage. Moreover, transformation of Chinese cabbage cotyledons showed that the expressions of BrCAX1-1 and BrCAX1-2 genes were significantly increased after spraying CaCl2 solutions, further suggesting that BrCAX gene expression changes may directly respond to external Ca2+ stress. Notably, Ca2+ is relatively immobile and not easily circulated to growing parts of the plants, resulting in the differences of Ca2+ concentration in different tissues and the occurrence of Ca2+ deficiency-induced tip-burn in leafy vegetables, especially in new leaves and leaf apex [2+content in the leaf of ‘10S42′ was lower than that in ‘10S230′ after Ca2+ deficiency treatment, which provides evidence for the occurrence of more serious symptoms of tip-burn in susceptible line ‘10S42′ of Chinese cabbage. Moreover, these BrCAX genes showed significantly differential expressions between leaf apex and leaf base, which was consistent with the previous studies that the expressions of tip-burn related genes differed in leaf apex and leaf base [CAX2 homologous genes, BrCAX2-1 and BrCAX2-3, were not detected in the present study, which was in agreement with the reported studies that CAX2 may do not play a major physiological role in regulating Ca2+ homeostasis [BrCAX genes provide insight into further functional characterizations of BrCAXs in Chinese cabbage. Furthermore, the transcript levels and expression patterns of  species . Previoued genes  and founnt lines . In Chinble line . In addiy stress ,66. In teaf apex ,52. As eeaf base . In addieostasis . In geneBrCAX family members were identified in Chinese cabbage by genome-wide searching. These BrCAX proteins contained conserved Na_Ca_ex domains and shared close phylogenetic relationships with their homologous proteins. Molecular evolution analysis and structural alignment showed the evolutionary conservation of BrCAX family genes. Further sequence alignment revealed the highly conserved Ca2+ binding residues among BrCAX proteins. Moreover, these BrCAX genes exhibited tissue-specific expression patterns and differential expression under Ca2+ deficiency treatments. Notably, BrCAX1-1 and BrCAX1-2 genes exhibited significantly differential expression in the two different tip-burn sensitive lines of Chinese cabbage under Ca2+ deficiency stress. Moreover, the N-terminal truncated BrCAX1-1 and BrCAX1-2 genes significantly promoted the accumulation of Ca2+ in the vacuole of yeast cells and improved the tolerance of Chinese cabbage cotyledons to environmental high Ca2+, which validated the functions of BrCAX1-1 and BrCAX1-2 in Ca2+ transmembrane transport. These results provide rich information for further functional exploration of BrCAX genes in regulating Ca2+ deficiency response in Brassica species.In this study, eight genes belonging to five


Some mutations impair the gating and thus cause muscle disease. The RyR1 mutation T4706M is linked to a myopathy characterized by muscle weakness. Although, low expression of the T4706M RyR1 protein can explain in part the symptoms, little is known about the function RyR1 channels with this mutation. In order to learn whether this mutation alters channel function in a manner that can account for the observed symptoms, we examined RyR1 channels isolated from mice homozygous for the T4709M (TM) mutation at the single channel level. Ligands, including Ca2+, ATP, Mg2+ and the RyR inhibitor dantrolene were tested. The full conductance of the TM channel was the same as that of wild type (wt) channels and a population of partial open (subconductive) states were not observed. However, two unique sub-populations of TM RyRs were identified. One half of the TM channels exhibited high open probability at low (100 nM) and high (50 μM) cytoplasmic [Ca2+], resulting in Ca2+-insensitive, constitutively high Po channels. The rest of the TM channels exhibited significantly lower activity within the physiologically relevant range of cytoplasmic [Ca2+], compared to wt. TM channels retained normal Mg2+ block, modulation by ATP, and inhibition by dantrolene. Together, these results suggest that the TM mutation results in a combination of primary and secondary RyR1 dysfunctions that contribute to disease pathogenesis.Physiological muscle contraction requires an intact ligand gating mechanism of the ryanodine receptor 1 (RyR1), the Ca When RyR1 channels open under the control of an action potential in the sarcolemma, Ca2+ is released from the SR, which is used to drive muscle contraction. The communication between the sarcolemma and RyR1 is mediated by the dihydropyridine receptor (DHPR) in a voltage dependent manner through an allosteric mechanism, called excitation–contraction coupling (ECC)2. RyR1 channels are regulated by a variety of ligands, including Ca2+, ATP and Mg2+9. In terms of regulation, the most relevant ligand is Ca2+, which activates RyR1 in the micromolar range and allows maximal channel activity at 50 µM6. The role of cytoplasmic Ca2+ in ECC is to open RyR channels that are not directly allosterically coupled to DHPRs, within a process termed Ca2+-induced Ca2+ release11. In this way Ca2+ release amplifies the Ca2+ signal. Mg2+ inhibits RyR1 channels in the millimolar range by competing for the Ca2+ binding site8. ATP is one of the most effective agonists of RyR1, as it significantly activates the channel even in the absence of Ca2+ and can further enhance RyR1 channel open probability even in 50 µM Ca2+9.Ryanodine receptor type 1 (RyR1) is the Ca12. Among these, RyR1-related myopathies are often classified according to their specific histological presentation. Major histopathological groups of RYR1-related myopathies include central core disease (CCD), multiminicore disease, core-rod myopathy, and centronuclear myopathy. The patients suffer from mild, slowly- or non-progressive disabilities due to proximal muscle weakness and fatigue, low muscle tone and slow contraction. Symptoms also include respiratory muscle weakness, scoliosis, orthopedic deformities, pronounced facial weakness and dysmorphism, ptosis, ophthalmoparesis, exertional heat stroke, exertional myalgia and malignant hyperthermia susceptibility14. Malignant hyperthermia is an idiosyncratic reaction to volatile anaesthetics  and succinylcholine. Susceptible patients exhibit muscle contractures all over the body when exposed to therapeutic concentrations of these trigger-drugs. Consequently, the body temperature rapidly increases, acidosis and hyperkalaemia develop, leading to an acute life-threatening condition unless the body is cooled down and dantrolene, an RyR1 inhibitor and muscle relaxant, is immediately administered20.Mutations impairing RyR function or its regulation cause a wide spectrum of muscle disorders, collectively called ryanopathies2+ leak from the SR. Nevertheless, both the Ca2+ content of the SR and the resting myoplasmic Ca2+ concentration remained normal, indicating that the leak was low enough to be compensated by the SR Ca2+ pump. However, the threshold for activation of Ca2+ release by voltage and caffeine was significantly lower, suggesting that the mutations increased the sensitivity of RyR1 to activation by both endogenous  and exogenous (caffeine) activators23. In contrast, RyR1 channels carrying other more severe CCD mutations showed a substantial elevation resting activity, leading to decompensated Ca2+ leak and consequent depletion of Ca2+ from the SR, resulting in attenuated Ca2+ release24. Other mutations located within the RyR1 pore region result in a reduction in Ca2+ RyR1 permeation, and thus, a function uncoupling of excitation from SR Ca2+ release (termed “EC uncoupling”)25. Apparently, the clinical symptoms can be explained by both of these mechanistic alterations in RyR1 function (RyR1 Ca2+ leak and EC uncoupling).At the cellular level, RyR1 channels carrying certain MH causing mutations showed elevated basal activity when expressed in skeletal muscle fibres, indicating that there was a resting Ca2+ leak was shown to be associated with low levels of the regulatory protein FKBP12  bound to the RyR126. FKBP12 stabilizes the closed state of the RyR1 channel and dissociation causes leaky RyR1 channels by promoting subconductive openings. Posttranslational RyR1 modifications, such as PKA mediated hyperphosphorylation, oxidation and S-nitrosylation were reported to dissociate FKBP12, and thus, increase RyR1 Ca2+ leak in certain muscle disorders33. On the other hand, some RyR1-related myopathy mutations were reported to affect RyR1 channel conductance, ligand  regulation or expression of the channel35.In some new cases of RyR1-related myopathies, pathologic SR Ca36, but recently the diagnostic procedure is enhanced by the sequencing of the entire RyR1 gene. Owing to this technical development, the number of newly discovered mutations is increasing39. Both dominant and recessive forms of RyR1-related myopathies have been reported, with the recessive forms being often associated with a more severe clinical presentation41. An example for such a mutation is T4706M. Despite the low incidence rate of these recessive mutations, their medical relevance may be significant, because some heterozygous cases occur together with epigenetic allele silencing of the wild type allele resulting in a pseudo-recessive phenotype, including severe facial and proximal weakness, scoliosis, opthalmoplegia and respiratory impairment43. Homozygous T4709M knock-in RyR1 mice  were generated and examined previously42. Homozygous TM mice exhibit mild muscle weakness, kyphosis, enhanced isoflurane sensitivity and reduced RyR1 expression (~ 30% reduction). Compound heterozygous mice with one TM allele and one frameshift (null) allele express a markedly lower level of RyR1 channels (~ 80% reduction) and exhibit an even more severe  myopathy. Importantly, both models are expected to result in only homotypic TM/TM channels, with a higher level of overall channel expression in muscle of homozygous TM/TM mice42.Diagnosis of myopathies traditionally relies on the histological findings from a muscle biopsy44. Prior studies conducted on recombinant RyR1 channels expressed and purified from Hek293 cells found that the homotypic TM RyR1 channels exhibited a normal low open probability in low Ca2+, but an increased sensitivity to activation by caffeine. However, the function of endogenous homotypic TM channels in skeletal muscle and the degree to which TM channel dysfunction contributes to muscle dysfunction is unclear43. This question of homotypic T4706M RyR’s functionality is particularly important because reactivation of the wt silenced allele using DNA methyltransferase inhibitors appears to be a promising future therapeutic option to improve RyR1 expression, but improvement in muscle function can only be expected if RyR1 function is adequate43.TM mutation is located adjacent to the RyR1 caffeine binding site2+, ATP, and Mg2+) sensitivity, and biophysical and pharmacological properties of single wt and homotypic T4709M RyR1 channels reconstituted in planar lipid bilayers.As homozygous TM/TM mice (TM) express a higher level of homotypic RyR1 channels, purified RyR1 channels from skeletal muscle of wt and homozygous TM mice in order to directly evaluate the impact of the T4706M mutation on RyR1 channel function at the single-channel level. Here, we characterized the unitary conductance, ligand .Phospholipids were obtained from Avanti Polar Lipids, Inc. . All other chemicals, if not specified, were purchased from Sigma-Aldrich .42. Mice were euthanized by CO2 anaesthesia followed by cervical dislocation. Each step of the purification protocol was performed on ice or at 4 °C in the presence of protease inhibitors . The muscle samples pooled from 10 mice and were homogenised in: 100 mM NaCl, 20 mM EGTA, 20 mM Na-HEPES (pH = 7.5). Cell debris was pelleted at 3500 × g, for 35 min by using a laboratory centrifuge. SR microsomes were isolated by using differential centrifugation as follows. Crude microsomes were collected from the supernatant by centrifugation in a Ti45 rotor at 40,000 × g, for 30 min. To remove the actomyosin contamination the pellet was resuspended in: 600 mM KCl, 10 mM K-PIPES, 250 mM sucrose, 1 mM EGTA, 0.9 mM CaCl2 (pH = 7.0) and incubated for 1 h. Then, the microsomes were collected by centrifugation at 109,000 × g for 30 min, and the pellet was resuspended in: 300 mM sucrose, 10 mM K-PIPES (pH = 7.0), snap-frozen in liquid nitrogen, and stored at – 70 °C until the solubilisation step.13 g of skeletal muscle was collected from wild type (wt) and genetically modified homozygous mice carrying the patient-relevant T4709M mutation in RyR1  (n = 10 for each genotype)2, 5 mM AMP, 0.45% phosphatidylcholine, 20 mM Na-PIPES (pH = 7.2) for 2 h. The samples were loaded onto a 10–28% linear sucrose gradient and centrifuged overnight at 90,000 × g in an SW27 rotor. RyR-containing fractions of the gradient were snap-frozen in liquid nitrogen and stored at − 70 °C in small aliquots47.Microsomes were allowed to melt on ice and solubilized in: 1% CHAPS, 1 M NaCl, 100 μM EGTA, 150 μM CaCl6. Images were converted to black and white and were not further processed. Full length gels are presented.SDS-PAGE was used to verify the purified samples. 30 μl of each fraction (~ 400 μl) of the sucrose gradient was loaded in each well of a 10% linear gel. After electrophoresis, gels were stained with Coomassie Blue2, 10 mM HEPES (pH = 7.2). Transmembrane voltages were reference to ground (trans-to-cis), allowing ionic currents of physiological direction through open RyR channels. The free [Ca2+] of the recording medium was adjusted using an EGTA stock solution.Voltage-clamp measurements were performed on purified RyR1 channels incorporated into artificial planar lipid bilayers. Bilayers were formed across an aperture with a diameter of 200 μm drilled in the wall of a Delrin cap , which separated two chambers. The chambers were filled with a recording solution containing: 250 mM KCl, 50 μM CaCl47.The lipid mixture contained phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine  in a ratio of 5:4:1 was dissolved in n-decane at a final concentration of 20 mg/mL. The bilayer currents were recorded in voltage-clamp mode using an AxoPatch 200 amplifier and pCLAMP 6.03  software. The holding potentials were as indicated in the text. The currents were filtered at 1 kHz through an eight-pole low-pass Bessel filter and digitized at 3 kHzo) were determined using the pClamp software suite . Statistical analyses were performed in Origin 7.0  and in Excel . Results were expressed as mean ± standard error (SE). Relative Po data were calculated by normalizing each data point to their own control. Statistical significance of differences was evaluated by using the independent two-sample t-test. Differences were considered significant when p was less than 0.05 (marked by *). The number of observations is displayed in the figure.Open probabilities  RyR1 knock-in mice and RyRs were purified from the tissue using sucrose gradient ultracentrifugation. The resulting samples were verified for RyR1 content using SDS-PAGE. Small volumes of 10 different fractions, collected between the 18 and 21% sucrose concentration range were loaded on a gel and stained with Coomassie blue after electrophoresis. Consistent with prior reports, the RyR1 expression was lower in the case of TM muscle, but no evidence for fragmentation of the protein was found  decreased in only 8 out of 16 TM channels   and Ca2+ sensitive TM RyR1 channels, as well as a typical Ca2+ sensitive wt RyR1 in the presence of various cytoplasmic [Ca2+]s are shown in Fig. 2+ sensitive TM population to wt revealed that the maximal channel activity measured at 50 μM Ca2+ was not significantly different between the two groups (Po = 0.55 ± 0.09 vs. 0.51 ± 0.12). We compared the Ca2+ dependence of the Ca2+ sensitive channels in 5; 1; 0.5; and 0.1 μM cytoplasmic [Ca2+] , but Po values were not significantly different at 100 nM (resting) Ca2+ (p = 0.8)  than that of their Ca2+ sensitive counterparts (0.55 ± 0.09), whereas their average Po did not decrease appreciably when the [Ca2+] was reduced to 500 nM (0.84 ± 0.11). It should be noted that we have not previously observed a similarly high proportion of reluctant channels in prior RyR1 preparations. This unusually high proportion of Ca2+-insensitive, high PO mutant channels would be expected to exhibit a “leaky” phenotype in spite of the absence of a significant proportion of subconduction states as is typically observed following FKBP dissociation. The reasons for the loss of Ca2+ regulation in this subpopulation of TM RyR1 channels is unclear, but could reflect either primary  and/or secondary  consequences of the mutation.As Caels Fig. A, while 3 Ryanodine-binding and single channel bilayer recordings of recombinant TM channels expressed in HEK cells to conclude that TM channels exhibit a normal low Po in low Ca2+ concentrations but an increased sensitivity to activation by caffeine 44. Our results on TM/TM channels isolated from native skeletal muscle tissue are quite different or at least our observation of two different classes of channels  is different. This suggests that it is important to assess the function of RyR1 disease mutant channels purified from native skeletal muscle as their function can be influenced by numerous muscle-specific factors .Kushnir et al. reported that the TM mutation is located adjacent to the caffeine binding site and they used H2+ and ATP sensitivity of wt and TM RyR1s were also tested. In the presence of 50 µM cytoplasmic [Ca2+], Mg2+ was added to the cytosolic face of the channels at increasing concentrations. The concentration-dependent inhibition of RyR1 activity (PO) by cytoplasmic Mg2+ not statistically different between wt and TM channels  produced a similar, significant (~ 60%) reduction in channel activity (PO) for both wt and TM channels , these RyR1 channels most likely represented Ca2+ insensitive “reluctant” channels.As the T4706M mutation is associated with increased susceptibility for malignant hyperthermia susceptibilityels Fig. , demonsto that was not reduced following a reduction in cytoplasmic Ca2+ to physiological levels observed in resting skeletal muscle (e.g. 100 nM), which would be expected to result in a high level of RyR1-dependent SR Ca2+ leak. While the other population of TM RyR1 channels exhibited a normal PO at 100 nM cytoplasmic Ca2+, these channels showed lower responsiveness to moderate elevations in cytoplasmic Ca2+ (0.5–10 μM) that occur during muscle excitation. Our results suggest that Ca2+-insensitive, high PO (i.e. reluctant) channels will enhance resting SR Ca2+ leak and potentially deplete SR Ca2+, which would reduce the driving force for Ca2+-release and contribute to T4706M-linked myopathy. In addition, the reduced Ca2+-sensitivity a subpopulation of TM RyR1 channels may also participate in the pathomechanism of muscle weakness by since these channels would be less activated by cytoplasmic Ca2+ during muscle excitation. We must note that functional dichotomy of homotypic TM channels may reflect either primary conformational or secondary post-translational (e.g. Ca2+-dependent modifications) mechanisms. Although, our description of two functionally distinct classes of RyR1 channels in TM muscle falls short of determining their relative role in disease pathogenesis, these results should be taken into consideration in the development of new therapeutic interventions for RyR1-related myopathy.In this study, we identified two functionally distinct populations of T4706M RyRs. One group exhibited high baseline P