AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mu...AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall survival.RESULTS: Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 10.7% vs 20.5;Ⅰ/Ⅱ vs Ⅳ, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (Ⅰ/Ⅱ vs Ⅲ, 12.9% vs 16.9%;Ⅰ /Ⅱ vs Ⅳ, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 0% vs 12%;Ⅰ/Ⅱ vs Ⅳ, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. CONCLUSION: Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of BRCA1 and M展开更多
Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis...Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis （PGD） for spinal muscular atrophy （SMA）, an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild- type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses （MARSALA） is a new method allowing embryo selection by a one-step next-generation sequencing （NGS） procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion （carriers） from the wild-type （normal） ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos （homozygous deletion）, which can be regarded as probands for linkage analysis, in case that the affected family member is absent, In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.展开更多
(Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will ...(Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will achieve therapeutic benefits and enable a better understanding of the decisive mechanisms of cell death or survival in response to DNA damaging agents. ) In present paper, we sought to characterize the cell cycle checkpoint profiles in U937-ASPI3K, a U937 cell mutant that was previously established with endogenous ATM knock-out phenotype. Syn- chronized U937-ASPI3K was exposed to 137Cs irradiation, G1, S. G2/M cell cycle checkpoint pro- files were evaluated by determining cell cycle kinetics, p53/p21 protein, cyclin dependent kinase 2 (CDK2) and p34CDC2 kinase activity in response to irradiation. U937-ASPI3K exhibited multiple defects in cell cycle checkpoints as defined by failing to arrest cells upon irradiation. The accumulation of cellular p53/p21 protein and inhibition of CDK kinase was also abolished in U937-ASPI3K. It was concluded that the stable expression of anti-sense PI3K cDNA fragment completely abolished multiple cell cycle checkpoints in U937-ASPI3K, and hence U937-ASPI3K with an AT-like phenotype could serves as a valuable model system for investigating the signal transduction pathway in responding to DNA damaging-based cancer therapy.展开更多
Background： Caffeine suppresses ataxia telangiectasia and Rad3 related and ataxia telangiectasia mutated （ATM） activities; ATM is the major kinase for DNA damage detection. This study aimed to investigate the effec...Background： Caffeine suppresses ataxia telangiectasia and Rad3 related and ataxia telangiectasia mutated （ATM） activities; ATM is the major kinase for DNA damage detection. This study aimed to investigate the effects of caffeine on DNA damage responses in cells from the bladder cancer cell line RT4 those were exposed to ionizing radiation （IR）. Methods： Immunofluorescent staining was performed to investigate changes in the proteins involved in DNA damage responses with or without caffeine. A mouse xenograft model was used to study the effects of caffeine on the DNA damage responses. Western blotting was used to investigate the effects of caffeine pretreatment on the ATM-Chk2-p53-Puma axis, while real-time polymerase chain reaction （RT-PCR） assessed changes in messenger RNA levels of p53 and downstream targets responding to IR. Finally, terminal deoxynucleotidyl transferase-dUTP nick end labeling assay. Western blotting and colony formation assay were used to measure the effects of caffeine on radiation-related apoptosis. All of the data were analyzed with a two-tailed Student＇s t-test. Results： lmmunofluorescent staining showed that caffeine pretreatment profoundly suppressed the formation ofyH2AXand p53-binding protein 1 foci in RT4 cells in response to irradiation. Cellular and animal experiments suggested that this suppression was mediated by suppression of the ATM-Chk2-p53-Puma DNA damage-signaling axis. RT-PCR indicated caffeine also attenuated transactivation of p53 and p53-inducible genes. The colony formation assay revealed that caffeine displayed radioprotective effects on RT4 cells in response to low-dose radiation compared to the radiosensitization effects on T24 cells. Conclusion： Caffeine may inhibit IR-related apoptosis of bladder cancer RT4 cells by suppressing activation of the ATM-Chk2-p53-Puma axis.展开更多
Induction and mobilization of transposable elements （TEs） following DNA damage or other stresses has been reported in prokaryotes and eukaryotes. Recently it was discovered that eukaryotic TEs are frequently associa...Induction and mobilization of transposable elements （TEs） following DNA damage or other stresses has been reported in prokaryotes and eukaryotes. Recently it was discovered that eukaryotic TEs are frequently associated with long non-coding RNAs （IncRNAs）, many of which are also upregulated by stress. Yet, it is unknown whether DNA damage-induced transcriptional activation of TEs and IncRNAs occurs sporadically or is a synchronized, genome-wide response. Here we investigated the transcriptome of Arabidopsis wild- type （WT） and ataxia telangiectasia mutated （atm） mutant plants 3 h after induction of DNA damage. In WT, expression of 5.2% of the protein-coding genes is 〉 2-fold changed, whereas in atm plants, only 2.6% of these genes are regulated, and the response of genes associated with DNA repair, replication, and cell cy- cle is largely lost. In contrast, only less than 0.6% of TEs and IncRNAs respond to DNA damage in WT plants, and the regulation of 〉95% of them is ATM-dependent. The ATM-downstream factors BRCA1, DRM1, JMJ30, AGO2, and the ATM-independent AGO4 participate in the regulation of individual TEs and IncRNAs. Remarkably, protein-coding genes located adjacent to DNA damage-responsive TEs and IncRNAs are frequently coexpressed, which is consistent with the hypothesis that TEs and IncRNAs located close to genes commonly function as controlling elements.展开更多
Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). A...Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP) based detection procedures.展开更多
Glucagon-like peptide-1 （GLP-1） and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases. Its short half-life has limited...Glucagon-like peptide-1 （GLP-1） and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases. Its short half-life has limited the application of GLP-1 in the clinic. We generated a mutated form of human GLP-1 （mGLP-1） using site-directed mutagenesis and gene recombination techniques, and found that these modifications significantly prolonged the biological half-life of GLP-1 compared with native GLP-1 （nGLP-1）. This study investigated the role of mGLP-1 on inducing PC12 cell differentiation, mGLP-1 induced PC12 cell differentiation with neurite outgrowth and increased the expression of growth-associated protein-43 and neuronal class III I^-tubulin, and significantly increased cyclic adenosine monophosphate level. No significant difference was found between mGLP-1 and nGLP-I. The results indicate that mGLP-1 activates the GLP-1 receptor, induces PC12 cell differentiation, and has neurotrophic effects.展开更多
Gastric cancer(GC) is a highly heterogeneous disease with multiple cellular types and poor prognosis.However, the cellular evolution and molecular basis of GC at the individual intra-tumor level has not been well demo...Gastric cancer(GC) is a highly heterogeneous disease with multiple cellular types and poor prognosis.However, the cellular evolution and molecular basis of GC at the individual intra-tumor level has not been well demonstrated. We performed single-cell whole exome sequencing to detect somatic singlenucleotide variants(SNVs) and significantly mutated genes(SMGs) among 34 tumor cells and 9 normal cells from a patient with GC. The Complete Prediction for Protein Conformation(CPPC) approach directly predicting the folding conformation of the protein 3D structure with Protein Folding Shape Code, combined with functional experiments were used to confirm the characterization of mutated SMGs in GC cells. We identified 201 somatic SNVs, including 117 non-synonymous mutations in GC cells. Further analysis identified 24 significant mutated genes(SMGs) in single cells, for which a single amino acid change might affect protein conformation. Among them, two genes(CDC27 and FLG) that were mutated only in single cells but not in the corresponding tumor tissue, were recurrently present in another GC tissue cohort, and may play a potential role to promote carcinogenesis, as confirmed by functional characterization. Our findings showed a mutational landscape of GC at intra-tumor level for the first time and provided opportunities for understanding the heterogeneity and individualized target therapy for this disease.展开更多
Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.W...Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.展开更多
Four pairs of HBV surface antigen genes, in which the preS region was partially deleted,were constructed by the polymerase chain reaction (PCR). The comparison of the levels of the expression in mammalian cells of the...Four pairs of HBV surface antigen genes, in which the preS region was partially deleted,were constructed by the polymerase chain reaction (PCR). The comparison of the levels of the expression in mammalian cells of these genes and the ones constructed before, and the properties of these gene products showed that the missing of a part of the preS region did not affect the overall spatial structure of the S region and the surface localization of the preS region. The removal of the preS1 retention sequence (a. a. 2-19) alleviated significantly the shelter of the major antigenic determinants in the S region by the preS sequence. It was found that the long preS region seriously impaired the secretion of the surface antigen proteins from mammalian cells. In addition to the previously reported preSl retention sequence, the preSl sequence (a.a. 48-65) may also inhibit the secretion of the surface antigen proteins, whereas the preS2 region exerts no major influence on the retention of the large surface antigen protein. One of the expressed surface antigen proteins, in which the preSl sequence (a.a. 21 - 47) and the S region were directly fused, deserves further study and may be developed into a new HBV vaccine which contains the preSl binding site for hepatocyte receptors due to its stability, fine secretability and strong preS1 antigenicity.展开更多
Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogen...Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogenesis and prognosis of cancer.However,the underlying mechanism remains unclear.The bioinformatic analysis predicted a potential antisense transcript ATM-antisense(AS)from the opposite strand of the ATM gene.The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.Methods:Single strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus.qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples.Luciferase reporter gene assays,biological mass spectrometry,ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression.Immunofluorescence and host-cell reactivation(HCR)assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair.Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.Results:The ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter.The reduced ATM-AS level led to the abnormal downregulation of ATM expression,and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro.The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples,and the patient prognosis.Conclusion:The present study demonstrated that ATM-AS,an antisense transcript located within the ATM gene body,is an essential positive regulator of ATM expression,and functions by mediating the binding of KAT5 to the ATM promoter.These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in bre展开更多
In modern scenarios,Industry 4.0 entails invention with various advanced technology,and blockchain is one among them.Blockchains are incorporated to enhance privacy,data transparency aswell as security for both large ...In modern scenarios,Industry 4.0 entails invention with various advanced technology,and blockchain is one among them.Blockchains are incorporated to enhance privacy,data transparency aswell as security for both large and small scale enterprises.Industry 4.0 is considered as a new synthesis fabrication technique that permits the manufacturers to attain their target effectively.However,because numerous devices and machines are involved,data security and privacy are always concerns.To achieve intelligence in Industry 4.0,blockchain technologies can overcome potential cybersecurity constraints.Nowadays,the blockchain and internet of things(IoT)are gaining more attention because of their favorable outcome in several applications.Though they generate massive data that need to be effectively optimized and in this research work,deep learning-based techniques are employed for this.This paper proposes a novel mutated leader sine cosine algorithm-based deep convolutional neural network(MLSC-DCNN)in order to attain a secure and optimized IoT blockchain for Industry 4.0.Here,an MLSC is hybridized using a mutated leader and sine cosine algorithm to enhance the weight function and minimize the loss factor of DCNN.Finally,the experimentation is carried out for various simulation measures.The comparative analysis is made for Best Tip Selection Method(BTSM),Smart Block-Software Defined Networking(SDN),and the proposed approach.The evaluation results show that the proposed approach attains better performances than BTSM and SDN.展开更多
Variations in Caladium humboldtii cv. ＇Phraya Savet＇ from in vitro culture were observed. Callus and small shoots were induced from unexpanded leaf segments cultured on modified MS medium supplemented with 2.69 p.M ...Variations in Caladium humboldtii cv. ＇Phraya Savet＇ from in vitro culture were observed. Callus and small shoots were induced from unexpanded leaf segments cultured on modified MS medium supplemented with 2.69 p.M l-Naphthalene acetic acid （NAA） and 17.76 μM N6-Benzyladenine （BA）. Shoots were transferred onto modified MS medium supplemented with 8.88 μM BA for shoot multiplication. Subsequently, roots were induced on MS without growth regulator. The regenerated plantlets were vigorously grown in glasshouse conditions. From 4 morphological groups （leaf color ratio, leaf color, petiole and leaf pattern）, the regenerated ＇Phraya Savet＇ caladium plants were divided into 6 types. The occurrence of variants was 52%. Most of the mutated plants were observed from only leaf color ratio （green ： white） and leaf shape. The most significance for commercial value from mutated clones was round leaf obtaining 20%. Characteristics of new clones from somatic hybridization between C humboldtii cv. ＇Phraya Savet＇ and C. bicolor cv. ＇Suvarnabhum＇ using thin cell layer technique from in vitro calli were investigated. Each thin cell layer of induced callus, about 0.5 - 1 mm thick, from both caladiums was alternately laid on the top of each other for 8 layers. Subsequently, the combination of thin cell layers was cultured and the regenerated plantlets were grown in glasshouse conditions. From 3 morphological groups （leaf pattern, leaf color and petiole）, the regenerated caladium plants were found dissimilarly to both original caladiums at 85 percent with 8 types of different characters. Somatic hybridization between C. humboldtii cv. ＇Phraya Savet＇ and C. bicolor cv. ＇Suvarnabhum＇ gave rise to a number of most hybrids with all conserving C. bicolor characters.展开更多
Objective To compare the immunophenotypic and clinical characteristics between NPM1mutated acute myeloid leukemia(AML)(NPM1m+AML)and unmutated AML(NPM1m-AML)not otherwise characterized(NOS)
DNA double-strand break(DSB)is generally regarded as the most lethal of all DNA lesions after radiation.Ku80,DNA-PK catalytic subunit(DNA-PKcs)and ataxia telangiectasia mutated(ATM)proteins are major DSB repair protei...DNA double-strand break(DSB)is generally regarded as the most lethal of all DNA lesions after radiation.Ku80,DNA-PK catalytic subunit(DNA-PKcs)and ataxia telangiectasia mutated(ATM)proteins are major DSB repair proteins.In this study,survival fraction at 2Gy(SF2)values of eight human tumor cell lines(including four human cervical carcinoma cell lines HeLa,SiHa,C33A,Caski,three human breast carcinoma cell lines MCF-7,MDA-MB-231,MDA-MB-453,and one human lung carcinoma cell line A549)were acquired by clone formation assay,and western blot was applied to detect the expressions of Ku80,DNA-PKcs and ATM protein.The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis.We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference.The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2(r=0.723,P=0.043),but Ku80 and ATM expression had no correlation with SF2(P>0.05).Thesefindings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.展开更多
The COVID-19 caused by SARS-CoV-2 has resulted in millions of people being infected and thousands of deaths globally since November 2019.To date,no unique therapeutic agent has been developed to slow the progression o...The COVID-19 caused by SARS-CoV-2 has resulted in millions of people being infected and thousands of deaths globally since November 2019.To date,no unique therapeutic agent has been developed to slow the progression of this pandemic.Despite possessing antiviral traits the potential of bacteriocins to combat SARS-CoV-2 infection has not been fully investigated.This review summarizes the mechanisms by which bacteriocins can be manipulated and implemented as effective virus entry blockers with infection suppression potential properties to highly transmissible viruses through comprehensive immune modulations that are potentially effective against COVID-19.These antimicrobial peptides have been suggested as effective antiviral therapeutics and therapeutic supplements to prevent rapid virus transmission.This review also provides a new insight into the cellular and molecular alterations which have made SARS-CoV-2 self-modified with diversified infection patterns.In addition,the possible applications of antimicrobial peptides through both natural and induced mechanisms in infection prevention perspectives on changeable virulence cases are comprehensively analyzed.Specific attention is given to the antiviral mechanisms of the molecules along with their integrative use with synthetic biology and nanosensor technology for rapid detection.Novel bacteriocin based therapeutics with cutting-edge technologies might be potential substitutes for existing time-consuming and expensive approaches to fight this newly emerged global threat.展开更多
Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway....Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.展开更多
文摘AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall survival.RESULTS: Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 10.7% vs 20.5;Ⅰ/Ⅱ vs Ⅳ, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (Ⅰ/Ⅱ vs Ⅲ, 12.9% vs 16.9%;Ⅰ /Ⅱ vs Ⅳ, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 0% vs 12%;Ⅰ/Ⅱ vs Ⅳ, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. CONCLUSION: Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of BRCA1 and M
基金supported by the National Natural Science Foundation of China (Nos. 31522034, 31571544 and 31230047)the National High Technology Research and Development Program (No. 2015AA020407)+1 种基金Beijing Municipal Science and Technology Commission (No. D151100002415004)Research Fund of National Health and Family Planning Commission of China (No. 201402004)
文摘Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis （PGD） for spinal muscular atrophy （SMA）, an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild- type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses （MARSALA） is a new method allowing embryo selection by a one-step next-generation sequencing （NGS） procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion （carriers） from the wild-type （normal） ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos （homozygous deletion）, which can be regarded as probands for linkage analysis, in case that the affected family member is absent, In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.
基金This project was supported by the National Natural ScienceFoundation of China (No. 39800149)
文摘(Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will achieve therapeutic benefits and enable a better understanding of the decisive mechanisms of cell death or survival in response to DNA damaging agents. ) In present paper, we sought to characterize the cell cycle checkpoint profiles in U937-ASPI3K, a U937 cell mutant that was previously established with endogenous ATM knock-out phenotype. Syn- chronized U937-ASPI3K was exposed to 137Cs irradiation, G1, S. G2/M cell cycle checkpoint pro- files were evaluated by determining cell cycle kinetics, p53/p21 protein, cyclin dependent kinase 2 (CDK2) and p34CDC2 kinase activity in response to irradiation. U937-ASPI3K exhibited multiple defects in cell cycle checkpoints as defined by failing to arrest cells upon irradiation. The accumulation of cellular p53/p21 protein and inhibition of CDK kinase was also abolished in U937-ASPI3K. It was concluded that the stable expression of anti-sense PI3K cDNA fragment completely abolished multiple cell cycle checkpoints in U937-ASPI3K, and hence U937-ASPI3K with an AT-like phenotype could serves as a valuable model system for investigating the signal transduction pathway in responding to DNA damaging-based cancer therapy.
基金This work was supported by grants from Zhejiang Provincial Natural Science Foundation （No. LY 13 H05000 l）, and National Natural Science Foundation of China （No. 81500532）.
文摘Background： Caffeine suppresses ataxia telangiectasia and Rad3 related and ataxia telangiectasia mutated （ATM） activities; ATM is the major kinase for DNA damage detection. This study aimed to investigate the effects of caffeine on DNA damage responses in cells from the bladder cancer cell line RT4 those were exposed to ionizing radiation （IR）. Methods： Immunofluorescent staining was performed to investigate changes in the proteins involved in DNA damage responses with or without caffeine. A mouse xenograft model was used to study the effects of caffeine on the DNA damage responses. Western blotting was used to investigate the effects of caffeine pretreatment on the ATM-Chk2-p53-Puma axis, while real-time polymerase chain reaction （RT-PCR） assessed changes in messenger RNA levels of p53 and downstream targets responding to IR. Finally, terminal deoxynucleotidyl transferase-dUTP nick end labeling assay. Western blotting and colony formation assay were used to measure the effects of caffeine on radiation-related apoptosis. All of the data were analyzed with a two-tailed Student＇s t-test. Results： lmmunofluorescent staining showed that caffeine pretreatment profoundly suppressed the formation ofyH2AXand p53-binding protein 1 foci in RT4 cells in response to irradiation. Cellular and animal experiments suggested that this suppression was mediated by suppression of the ATM-Chk2-p53-Puma DNA damage-signaling axis. RT-PCR indicated caffeine also attenuated transactivation of p53 and p53-inducible genes. The colony formation assay revealed that caffeine displayed radioprotective effects on RT4 cells in response to low-dose radiation compared to the radiosensitization effects on T24 cells. Conclusion： Caffeine may inhibit IR-related apoptosis of bladder cancer RT4 cells by suppressing activation of the ATM-Chk2-p53-Puma axis.
文摘Induction and mobilization of transposable elements （TEs） following DNA damage or other stresses has been reported in prokaryotes and eukaryotes. Recently it was discovered that eukaryotic TEs are frequently associated with long non-coding RNAs （IncRNAs）, many of which are also upregulated by stress. Yet, it is unknown whether DNA damage-induced transcriptional activation of TEs and IncRNAs occurs sporadically or is a synchronized, genome-wide response. Here we investigated the transcriptome of Arabidopsis wild- type （WT） and ataxia telangiectasia mutated （atm） mutant plants 3 h after induction of DNA damage. In WT, expression of 5.2% of the protein-coding genes is 〉 2-fold changed, whereas in atm plants, only 2.6% of these genes are regulated, and the response of genes associated with DNA repair, replication, and cell cy- cle is largely lost. In contrast, only less than 0.6% of TEs and IncRNAs respond to DNA damage in WT plants, and the regulation of 〉95% of them is ATM-dependent. The ATM-downstream factors BRCA1, DRM1, JMJ30, AGO2, and the ATM-independent AGO4 participate in the regulation of individual TEs and IncRNAs. Remarkably, protein-coding genes located adjacent to DNA damage-responsive TEs and IncRNAs are frequently coexpressed, which is consistent with the hypothesis that TEs and IncRNAs located close to genes commonly function as controlling elements.
文摘Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP) based detection procedures.
基金the National Natural Science Foundation of China,No.81070876the Shanghai Science and Technology Commission,No.10JC1411200the Fundamental Research Funds for the Central Universities
文摘Glucagon-like peptide-1 （GLP-1） and its long-acting analogues have neuroprotective and neurotrophic properties and are emerging as potential treatments for neurodegenerative diseases. Its short half-life has limited the application of GLP-1 in the clinic. We generated a mutated form of human GLP-1 （mGLP-1） using site-directed mutagenesis and gene recombination techniques, and found that these modifications significantly prolonged the biological half-life of GLP-1 compared with native GLP-1 （nGLP-1）. This study investigated the role of mGLP-1 on inducing PC12 cell differentiation, mGLP-1 induced PC12 cell differentiation with neurite outgrowth and increased the expression of growth-associated protein-43 and neuronal class III I^-tubulin, and significantly increased cyclic adenosine monophosphate level. No significant difference was found between mGLP-1 and nGLP-I. The results indicate that mGLP-1 activates the GLP-1 receptor, induces PC12 cell differentiation, and has neurotrophic effects.
基金supported by the National Key Research and Development Program of China (2017YFC1308900)Beijing Municipal Commission of Health and Family Planning Project (PXM2018_026279_000005)+1 种基金National High-tech R&D Program of China (2012AA02A203, No.2012AA02A504)Beijing talent fund
文摘Gastric cancer(GC) is a highly heterogeneous disease with multiple cellular types and poor prognosis.However, the cellular evolution and molecular basis of GC at the individual intra-tumor level has not been well demonstrated. We performed single-cell whole exome sequencing to detect somatic singlenucleotide variants(SNVs) and significantly mutated genes(SMGs) among 34 tumor cells and 9 normal cells from a patient with GC. The Complete Prediction for Protein Conformation(CPPC) approach directly predicting the folding conformation of the protein 3D structure with Protein Folding Shape Code, combined with functional experiments were used to confirm the characterization of mutated SMGs in GC cells. We identified 201 somatic SNVs, including 117 non-synonymous mutations in GC cells. Further analysis identified 24 significant mutated genes(SMGs) in single cells, for which a single amino acid change might affect protein conformation. Among them, two genes(CDC27 and FLG) that were mutated only in single cells but not in the corresponding tumor tissue, were recurrently present in another GC tissue cohort, and may play a potential role to promote carcinogenesis, as confirmed by functional characterization. Our findings showed a mutational landscape of GC at intra-tumor level for the first time and provided opportunities for understanding the heterogeneity and individualized target therapy for this disease.
基金supported by grants from the National Natural Science Foundation of China (No. 30900631)Hubei Provincial Department of Education (No. Q20102101 andD20082406)
文摘Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.
基金Project supported in part by National 863 Research Grant of China and Public Health Service Grants from the National Institutes of Health of the United States.
文摘Four pairs of HBV surface antigen genes, in which the preS region was partially deleted,were constructed by the polymerase chain reaction (PCR). The comparison of the levels of the expression in mammalian cells of these genes and the ones constructed before, and the properties of these gene products showed that the missing of a part of the preS region did not affect the overall spatial structure of the S region and the surface localization of the preS region. The removal of the preS1 retention sequence (a. a. 2-19) alleviated significantly the shelter of the major antigenic determinants in the S region by the preS sequence. It was found that the long preS region seriously impaired the secretion of the surface antigen proteins from mammalian cells. In addition to the previously reported preSl retention sequence, the preSl sequence (a.a. 48-65) may also inhibit the secretion of the surface antigen proteins, whereas the preS2 region exerts no major influence on the retention of the large surface antigen protein. One of the expressed surface antigen proteins, in which the preSl sequence (a.a. 21 - 47) and the S region were directly fused, deserves further study and may be developed into a new HBV vaccine which contains the preSl binding site for hepatocyte receptors due to its stability, fine secretability and strong preS1 antigenicity.
基金supported by the National Natural Science Foundation of China(No.81802670 and No.82072580).
文摘Objective:The ataxia telangiectasia mutated(ATM)gene is a master regulator in cellular DNA damage response.The dysregulation of ATM expression is frequent in breast cancer,and is known to be involved in the carcinogenesis and prognosis of cancer.However,the underlying mechanism remains unclear.The bioinformatic analysis predicted a potential antisense transcript ATM-antisense(AS)from the opposite strand of the ATM gene.The purpose of this study was to identify ATM-AS and investigate the possible effect of ATM-AS on the ATM gene regulation.Methods:Single strand-specific RT-PCR was performed to verify the predicted antisense transcript ATM-AS within the ATM gene locus.qRT-PCR and Western blotting were used to detect the expression levels of ATM-AS and ATM in normal and breast cancer cell lines as well as in tissue samples.Luciferase reporter gene assays,biological mass spectrometry,ChIP-qPCR and RIP were used to explore the function of ATM-AS in regulating the ATM expression.Immunofluorescence and host-cell reactivation(HCR)assay were performed to evaluate the biological significance of ATM-AS in ATM-mediated DNA damage repair.Breast cancer tissue samples were used for evaluating the correlation of the ATM-AS level with the ATM expression as well as prognosis of the patients.Results:The ATM-AS significantly upregulated the ATM gene activity by recruiting KAT5 histone acetyltransferase to the gene promoter.The reduced ATM-AS level led to the abnormal downregulation of ATM expression,and impaired the ATM-mediated DNA damage repair in normal breast cells in vitro.The ATM-AS level was positively correlated with the ATM expression in the examined breast cancer tissue samples,and the patient prognosis.Conclusion:The present study demonstrated that ATM-AS,an antisense transcript located within the ATM gene body,is an essential positive regulator of ATM expression,and functions by mediating the binding of KAT5 to the ATM promoter.These findings uncover the novel mechanism underlying the dysregulation of the ATM gene in bre
基金The authors extend their appreciation to King Saud University for funding this work through Researchers Supporting Project Number(RSP2022R499)King Saud University,Riyadh,Saudi Arabia.
文摘In modern scenarios,Industry 4.0 entails invention with various advanced technology,and blockchain is one among them.Blockchains are incorporated to enhance privacy,data transparency aswell as security for both large and small scale enterprises.Industry 4.0 is considered as a new synthesis fabrication technique that permits the manufacturers to attain their target effectively.However,because numerous devices and machines are involved,data security and privacy are always concerns.To achieve intelligence in Industry 4.0,blockchain technologies can overcome potential cybersecurity constraints.Nowadays,the blockchain and internet of things(IoT)are gaining more attention because of their favorable outcome in several applications.Though they generate massive data that need to be effectively optimized and in this research work,deep learning-based techniques are employed for this.This paper proposes a novel mutated leader sine cosine algorithm-based deep convolutional neural network(MLSC-DCNN)in order to attain a secure and optimized IoT blockchain for Industry 4.0.Here,an MLSC is hybridized using a mutated leader and sine cosine algorithm to enhance the weight function and minimize the loss factor of DCNN.Finally,the experimentation is carried out for various simulation measures.The comparative analysis is made for Best Tip Selection Method(BTSM),Smart Block-Software Defined Networking(SDN),and the proposed approach.The evaluation results show that the proposed approach attains better performances than BTSM and SDN.
文摘Variations in Caladium humboldtii cv. ＇Phraya Savet＇ from in vitro culture were observed. Callus and small shoots were induced from unexpanded leaf segments cultured on modified MS medium supplemented with 2.69 p.M l-Naphthalene acetic acid （NAA） and 17.76 μM N6-Benzyladenine （BA）. Shoots were transferred onto modified MS medium supplemented with 8.88 μM BA for shoot multiplication. Subsequently, roots were induced on MS without growth regulator. The regenerated plantlets were vigorously grown in glasshouse conditions. From 4 morphological groups （leaf color ratio, leaf color, petiole and leaf pattern）, the regenerated ＇Phraya Savet＇ caladium plants were divided into 6 types. The occurrence of variants was 52%. Most of the mutated plants were observed from only leaf color ratio （green ： white） and leaf shape. The most significance for commercial value from mutated clones was round leaf obtaining 20%. Characteristics of new clones from somatic hybridization between C humboldtii cv. ＇Phraya Savet＇ and C. bicolor cv. ＇Suvarnabhum＇ using thin cell layer technique from in vitro calli were investigated. Each thin cell layer of induced callus, about 0.5 - 1 mm thick, from both caladiums was alternately laid on the top of each other for 8 layers. Subsequently, the combination of thin cell layers was cultured and the regenerated plantlets were grown in glasshouse conditions. From 3 morphological groups （leaf pattern, leaf color and petiole）, the regenerated caladium plants were found dissimilarly to both original caladiums at 85 percent with 8 types of different characters. Somatic hybridization between C. humboldtii cv. ＇Phraya Savet＇ and C. bicolor cv. ＇Suvarnabhum＇ gave rise to a number of most hybrids with all conserving C. bicolor characters.
文摘Objective To compare the immunophenotypic and clinical characteristics between NPM1mutated acute myeloid leukemia(AML)(NPM1m+AML)and unmutated AML(NPM1m-AML)not otherwise characterized(NOS)
基金supported by the National Natural Science Foundation of China(Grant No.30672426).
文摘DNA double-strand break(DSB)is generally regarded as the most lethal of all DNA lesions after radiation.Ku80,DNA-PK catalytic subunit(DNA-PKcs)and ataxia telangiectasia mutated(ATM)proteins are major DSB repair proteins.In this study,survival fraction at 2Gy(SF2)values of eight human tumor cell lines(including four human cervical carcinoma cell lines HeLa,SiHa,C33A,Caski,three human breast carcinoma cell lines MCF-7,MDA-MB-231,MDA-MB-453,and one human lung carcinoma cell line A549)were acquired by clone formation assay,and western blot was applied to detect the expressions of Ku80,DNA-PKcs and ATM protein.The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis.We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference.The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2(r=0.723,P=0.043),but Ku80 and ATM expression had no correlation with SF2(P>0.05).Thesefindings suggest that the expression level of DNA-PKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.
基金the authority of Research Publication Guardians(RPG,Government License No.05-060-06021)for providing logistic support and suggestions which were important to the successful completion of this review。
文摘The COVID-19 caused by SARS-CoV-2 has resulted in millions of people being infected and thousands of deaths globally since November 2019.To date,no unique therapeutic agent has been developed to slow the progression of this pandemic.Despite possessing antiviral traits the potential of bacteriocins to combat SARS-CoV-2 infection has not been fully investigated.This review summarizes the mechanisms by which bacteriocins can be manipulated and implemented as effective virus entry blockers with infection suppression potential properties to highly transmissible viruses through comprehensive immune modulations that are potentially effective against COVID-19.These antimicrobial peptides have been suggested as effective antiviral therapeutics and therapeutic supplements to prevent rapid virus transmission.This review also provides a new insight into the cellular and molecular alterations which have made SARS-CoV-2 self-modified with diversified infection patterns.In addition,the possible applications of antimicrobial peptides through both natural and induced mechanisms in infection prevention perspectives on changeable virulence cases are comprehensively analyzed.Specific attention is given to the antiviral mechanisms of the molecules along with their integrative use with synthetic biology and nanosensor technology for rapid detection.Novel bacteriocin based therapeutics with cutting-edge technologies might be potential substitutes for existing time-consuming and expensive approaches to fight this newly emerged global threat.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.81672743 and 81974464)Beijing Tianjin Hebei Basic Research Cooperation Project(Grant No.19JCZDJC64500(Z))+4 种基金Shenzhen Basic Research Project(Grant No.JCYJ20160331114230843)Tianjin Municipal Health Commission(Grant Nos.2015KR11 and 2013KG134)Tianjin Municipal Science and Technology Bureau(Grant No.18JCYBJC27800)US NIH grant RO 1 CAI33093,the Alabama Innovation Fund of the United Statesthe Tianjin Medical University Cancer Institute and Hospital Innovation Fund(Grant No.1803)。
文摘Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.
