[Objective] To compare next generation sequencing (NGS) library construction technology between probe hybridization capture and amplicon methods, and analyze the influencing factors of HLA genotyping resolution level and its prospects in clinical applications. [Methods] A total of 207 clinical samples with known typing results and samples from the proficiency testing plan were selected. The conformity rate of HLA genotyping results, allele coverage and typing data analysis indicators were confirmed, and the effects of two library construction methods on the level of HLA genotyping discrimination were compared. [Results] The concordance rate of 207 samples with the feedback results of PT or prior well-characterized HLA genotypes was 100%. Among them, 91 samples were captured using hybridization probe capture method. Compared with the original amplicon method, the hybridization probe capture method can distinguish the alleles of DRB1 and DPB1 that cannot be determined in 13 samples. The allelic imbalance of DRB1, DPA1, and DQB1 loci in 6 samples was resolved. Three samples were found to have missed detection of alleles at the DQA1 and DQB1 loci. [Conclusion] The performance indicators of hybridization probe capture and amplicon performance confirmation meet the requirements of clinical detection of HLA genotyping, which provides an experimental method and basis for clinical application.

BACKGROUND:Previous research by the research team found that domestically produced porous tantalum is beneficial for early adhesion and proliferation of MG63 cells,and can be used as a scaffold material for bone tissue engineering. OBJECTIVE:To investigate the effect of domestic porous tantalum modified by osteogenic induction factor slow-release system on the adhesion,proliferation,and differentiation of MG63 cells. METHODS:Osteogenic induction factor slow-release system was constructed by adding 15%volume fraction of osteogenic factor solution to poly(lactic-co-glycolic-acid)gel.The passage 3 MG63 cells were inoculated on a porous tantalum surface(control group),porous tantalum surface coated with poly(lactic-co-glycolic-acid)copolymer gel(gel group),and porous tantalum surface coated with osteoblastic induction factor slow-release system(slow-release system group),and co-cultured for 5 days.The surface cytoskeleton of the material was observed by phalloidine staining.Cell proliferation was detected by flow cytometry.Western blot assay and RT-qPCR were used to detect the protein and mRNA expressions of type Ⅰ collagen,osteopontin,and RUNX-2 on the surface cells of the material. RESULTS AND CONCLUSION:(1)Phalloidine staining showed that MG63 cells adhered to and grew on the surface and inside of the three groups of porous tantalum,and the matrix secreted by the cells covered the surface of the material.(2)Flow cytometry showed that the cell proliferation in the slow-release system group was faster than that in the control group and the gel group(P<0.05).(3)Western blot assay and RT-qPCR showed that the protein and mRNA expressions of type Ⅰ collagen,osteopontin,and RUNX-2 in the slow-release system group were higher than those in the control group and gel group(P<0.05).(4)The results showed that the domestic porous tantalum modified by the osteogenic induction factor slow-release system was beneficial to the adhesion,proliferation,and differentiation of MG63 osteoblasts.

Objective:The incidence of early-onset colorectal cancer (EOCRC) is increasing globally; however, the molecular characteristics and prognosis of sporadic EOCRC are unclear. In this systematic review and meta-analysis, we aimed to investigate the incidence of gene mutations and their association with cancer survival in sporadic EOCRC, focusing on six common gene mutations ( TP53, BRAF, KRAS, NRAS, PTEN, and APC). Methods:Ovid Embase and Ovid Medline electronic databases were searched for studies involving patients with sporadic EOCRC (i.e., diagnosed with colorectal cancer before the age of 50 years and with no evidence of hereditary syndromes predisposing to colorectal cancer). The included articles were evaluated using quality assessment tools. Meta-analysis was performed using random-effects and fixed-effects models. Cochran's Q statistic and the I2 index were used to assess heterogeneity. The incidence of the six common gene mutations listed above in sporadic EOCRC and their association with cancer survival were evaluated.Results:(1) Incidence of specific gene mutations in sporadic EOCRC. A total of 34 articles were included in this meta-analysis. The incidence of APC gene mutation was 36% (from 13 articles, 95%CI: 19%-55%, P=0.043); of KRAS gene mutation 30% (from 26 articles, 95%CI: 24%-35%, P=0.190); of BRAF gene mutation 7% (from 18 articles, 95%CI: 5%-11%, P=0.422); of NRAS gene mutation 4% (from five articles, 95%CI: 3%-5%, P=0.586); of PTEN gene mutation 6% (from six articles, 95%CI: 4%-10%, P=0.968); and of TP53 gene mutation 59% (from 13 articles, 95%CI: 49%-68%, P=0.164). (2) Association between gene mutations and survival in sporadic EOCRC . A total of six articles were included in this meta-analysis. Compared with wild-type BRAF, mutant BRAF was significantly associated with increased overall mortality risk in patients with EOCRC (pooled HR=2.85, 95%CI: 1.45-5.60, P=0.002). Subgroup analysis showed that the incidence of BRAF gene mutation was higher in Eastern than in Western countries, whereas the incidence of TP53, KRAS, NRAS, and APC gene mutations was lower. There was no significant difference in the incidence of PTEN gene mutation between different regions. Conclusion:Compared with colorectal cancer occurring in the general population, the incidence of APC and KRAS mutations is lower in EOCRC, whereas the incidence of TP53 mutation remains consistent. BRAF mutation is associated with increased overall mortality risk in patients with EOCRC.

Objective:The incidence of early-onset colorectal cancer (EOCRC) is increasing globally; however, the molecular characteristics and prognosis of sporadic EOCRC are unclear. In this systematic review and meta-analysis, we aimed to investigate the incidence of gene mutations and their association with cancer survival in sporadic EOCRC, focusing on six common gene mutations ( TP53, BRAF, KRAS, NRAS, PTEN, and APC). Methods:Ovid Embase and Ovid Medline electronic databases were searched for studies involving patients with sporadic EOCRC (i.e., diagnosed with colorectal cancer before the age of 50 years and with no evidence of hereditary syndromes predisposing to colorectal cancer). The included articles were evaluated using quality assessment tools. Meta-analysis was performed using random-effects and fixed-effects models. Cochran's Q statistic and the I2 index were used to assess heterogeneity. The incidence of the six common gene mutations listed above in sporadic EOCRC and their association with cancer survival were evaluated.Results:(1) Incidence of specific gene mutations in sporadic EOCRC. A total of 34 articles were included in this meta-analysis. The incidence of APC gene mutation was 36% (from 13 articles, 95%CI: 19%-55%, P=0.043); of KRAS gene mutation 30% (from 26 articles, 95%CI: 24%-35%, P=0.190); of BRAF gene mutation 7% (from 18 articles, 95%CI: 5%-11%, P=0.422); of NRAS gene mutation 4% (from five articles, 95%CI: 3%-5%, P=0.586); of PTEN gene mutation 6% (from six articles, 95%CI: 4%-10%, P=0.968); and of TP53 gene mutation 59% (from 13 articles, 95%CI: 49%-68%, P=0.164). (2) Association between gene mutations and survival in sporadic EOCRC . A total of six articles were included in this meta-analysis. Compared with wild-type BRAF, mutant BRAF was significantly associated with increased overall mortality risk in patients with EOCRC (pooled HR=2.85, 95%CI: 1.45-5.60, P=0.002). Subgroup analysis showed that the incidence of BRAF gene mutation was higher in Eastern than in Western countries, whereas the incidence of TP53, KRAS, NRAS, and APC gene mutations was lower. There was no significant difference in the incidence of PTEN gene mutation between different regions. Conclusion:Compared with colorectal cancer occurring in the general population, the incidence of APC and KRAS mutations is lower in EOCRC, whereas the incidence of TP53 mutation remains consistent. BRAF mutation is associated with increased overall mortality risk in patients with EOCRC.

Objective:To establish a linkage prediction model for human leukocyte antigen (HLA) DPA1-DPB1 and validate it by using clinical data and follow-up data from unrelated allogeneic hematopoietic stem cell transplantation donors and recipients, and to explore the clinical application value of the prediction model in transplantation prognosis.Methods:This is a retrospective study. Leveraging the artificial neural network algorithm of NetMHCⅡpan and the DPA1-DPB1 haplotype linkage database of the Chinese population established in our previous research, and incorporating the amino acid FASTA data of DPA1-DPB1 of all known sequences newly published by the Latest International Immunogenetics/Human Leukocyte Antigens, 47 DPA1-DPB1 linkage models were established. Employing next-generation sequencing technology based on the hybridization capture library construction method, HLA genotyping tests for HLA-A, -B, -C, DRB1, DQB1, DQA1, DRB3/4/5, DPB1, and DPA1 (9 loci) were performed on 250 donor-recipients pairs who underwent unrelated-donor hematopoietic stem cell transplantation in the Department of Hematology of the First Affiliated Hospital of Soochow University between January 2016 and September 2021. HLA typing data and clinical information of transplant donors and recipients were retrospectively analyzed to assess and predict the impact of permissive and non-permissive linkage mismatches of DPA1-DPB1 on transplantation prognosis. The Kaplan-Meier method with the log-rank test was applied to compare the survival curves of overall survival (OS) rates between different groups. Additionally, a competing risks model was utilized to compare the cumulative incidence of grade Ⅱ-Ⅳ acute graft-versus-host disease and non-relapse mortality (NRM) across groups. The area under the receiver operating characteristic curve was employed to compare the predictive performance of the established prediction model with that of the T-cell epitope (TCE) model.Results:According to the different hydrophilic and hydrophobic properties of amino acids, the DPA1-DPB1 linkage model is categorized into types Ⅰ-Ⅳ: type I consists of 6 hydrophobic types at P1-P8 plus hydrophilic type at P9; type Ⅱ includes 17 hydrophobic types; type Ⅲ comprises 9 amphiphilic types; and type Ⅳ consists of 15 hydrophilic types. According to the prediction model, DPA1-matched and DPB1-mismatched donor-recipient cases were classed into P1-matched or P1-mismatched groups. Compared with fully matched DPA1 and DPB1 cases, P1-mismatched patients had a 2-year OS rate of 75% (12/16) versus 96.2%(25/26) (χ2=4.13, P=0.04), and a NRM rate of 4/16 versus 0 (χ2=7.05, P<0.01). However, there was no statistically significant difference in the 2-year OS and NRM rates compared to DPA1 and DPB1 cases ( P>0.05). The prediction model established in this study demonstrated a larger area under the receiver operating characteristic curve for predicting the 2-year OS rate compared with the DPB1 TCE model ( Z=0.71, P=0.48). In donor-recipient cases where both DPA1 and DPB1 were mismatched, the 2-year OS rates decreased and the NRM increased in both P1-matched and P1-mismatched cases compared with fully matched DPA1 and DPB1. Moreover, P1-mismatched patients had a worse prognosis compared to P1-matched patients. Conclusion:The DPA1-DPB1 linkage prediction model established based on high-throughput next-generation sequencing technology can be used to predict the impact of HLA-DP mismatches on OS and NRM in transplantation, and the prediction performance is superior to the TCE model.

ObjectiveTo explore the effect and possible mechanism of Shenqi Pills （肾气丸） on cognitive impairment and hippocampal glucose energy metabolism in type 2 diabetes mellitus （T2DM）. MethodsSixty C57BL/6 mice were randomly divided into control group， model group， rosiglitazone group and Shenqi Pills low-， medium- and high-dose groups， with 10 mice in each group. T2DM model was induced by a high-fat diet combined with intraperitoneal injection of streptozotocin in all the groups except for the control group. After successful modeling， the high-， medium-， and low-dose Shenqi Pills groups were given 2.08， 1.04， and 0.52 g/（kg·d） of Shenqi Pills granules by gavage respectively， while the rosiglitazone group was given 3 mg/（kg·d） of rosiglitazone tablets by gavage， and the control group and model group were gavaged with 10 ml/（kg·d） of distilled water， all for 8 consecutive weeks. The body weight and fasting blood glucose （FBG） level were recorded every two weeks. The Morris water maze test was performed on the 8th week of medication. After 8-week medication， oral glucose tolerance test （OGTT） and fasting insulin level were measured， hippocampal glucose energy metabolism-related products were quantitatively detected by liquid chromatography tandem mass spectrometry， and KEGG annotation analysis was performed. ResultsCompared to those measured at the same timepoints in the control group， the body mass on week 6 and 8， as well as the FBG level on week 2， 4， 6 and 8 in the model group increased； the blood glucose level at 0， 30， 60 and 120 minutes of the OGTT test increased， while fasting insulin level after 8-week medication decreased. The escape latency of the model group was significantly prolonged on the 3rd and 4th days， and the escape latency time increased， while the total swimming distance， platform quadrant residence time and the number of platform crossings decreased （P＜0.05 or P＜0.01）. Compared to those measured at the same timepoints in the model group， the body mass on week 6 in the low-dose Shenqi Pills group， on week 6 and 8 in the medium- and high-dose groups， and on week 8 in the rosiglitazone group were significantly reduced； the FBG levels in all the Shenqi Pills groups and rosiglitazone group on week 6 and 8 decreased， while fasting insulin levels increased. In the OGTT test， blood glucose in the medium-dose group of Shenqi Pills at all timepoints decreased； in the Morris water maze test， the escape latency period of the medium- and high-dose Shenqi Pills groups was shortened on the 3rd and 4th days， while the escape latency time was reduced， and the total swimming distance， platform quadrant residence time， and number of platform crossings increased in the medium-dose Shenqi Pills group （P＜0.05 or P＜0.01）.The medium-dose Shenqi Pills showed best effect， therefore it was selected for the targeted quantitative detection of metabolites. The medium-dose Shenqi Pills group could regulate the disorder of glucose metabolism in the hippocampus of T2DM mice， and 13 differential metabolites were found，up-regulating α-ketoglutarate and 3-phosphoglyceric acid， and down-regulating fumaric acid， glutamatic acid， lactatic acid， inosine， malic acid， adenine， fructose 1,6-diphosphate and others. KEGG annotation of differential metabolites suggested that Shenqi Pills was closely related to the regulation of glucose metabolism disorder and insulin resistance in the hippocampus region of T2DM model mice， as well as neurodegenerative diseases and ABC transport， hypoxia-inducible factor 1 （HIF）， forkhead transcription factor （FoXO） and cyclic adenosine monophosphate （cAMP） signaling pathways. ConclusionShenqi Pills can improve learning and memory abilities and cognitive impairment in T2DM mice， and may act its role by regulating glucose energy metabolism in the hippocampus of T2DM.

Objective:Establish the decision threshold value of mean fluorescence intensity of anti-human leukocyte antigen(HLA)antibody through statistical analyzing the results of international proficiency testing(PT)organized by American Society for Histocompatibility and Immunogenetics(ASHI).Methods:Single antigen reagent and liquid chip(Luminex)technique were used to detect anti-HLA antibody. A retrospective analysis of the HLA antibody PT results of 55 quality control samples from 11 times organized by ASHI from 2012 to 2019 was reviewed.Results:Among 79 kinds of HLA-I antibodies, 21, 43 and 15 types of HLA-A, B and Cw antibodies were detected respectively, while among 44 kinds of HLA-Ⅱ antibodies, 18, 7 and 19 types of HLA-DRB1, DQB1 and DPB1 antibodies were detected respectively. After analyzing the MFI detection value of different specific antibodies in each PT samples at our laboratory and the coincidence rate of the negative / positive results judged by ASHI through summarizing the results of multicenter participating in the same period, MFI values of HLA antibody were arranged from high to low into the intervals of possible saturation value, positive decision value, positive judgment threshold value, suspicious positive reference value and suspicious negative reference value , according to the coincidence rate of 95%, 90%, 80%, 79%~50% and <50%.Thus, the decision limit value table of HLA specific antibody at our laboratory was established. And 42 kinds of HLA antibody types were detected with complete data.When the MFI values of various HLA-I or HLA-Ⅱ antibodies are found to be 80% or more in the table, it can be used to judge the detection of HLA antibodies. When HLA antibody MFI value reaches the positive decision value, it may have a certain guiding significance for clinical diagnosis and treatment. And when antibody MFI value reaches the saturation value and lies in the suspicious positive or suspicious negative reference threshold, it just suggests that the clinical need for dynamic follow-up of anti-HLA antibody detection.Conclusions:The decision limit value of MFI of laboratory HLA antibody is established based on the international PT experimental results, which is of reference value for the interpretation of experimental results and clinical diagnosis and treatment. A transplant ation center should pay attention to the quality control of comparison test between laboratories in the detection of HLA antibodies.

Objective: To analyze family-based haplotype frequencies of HLA-A, -B, -C, -DRB1 and -DQB1 genes and their clinical significance. Methods: The data of HLA genotyping in 3568 families undergoing related haploidentical transplantation between 2012 and 2017 at the First Affiliated Hospital of Soochow University were retrospectively evaluated. The HLA genotyping was performed by PCR amplification with sequence-based typing (PCR-SBT) and sequence-specific oligonucleotide probe (PCR-SSOP) methods. The family genetic analysis and haplotype frequencies were also investigated. Results: All the families were divided into 3 groups, including group1 of 1 422 entire families; group2 of 1 310 patients and either of their parents or one of their children; group3 of 836 patients and their HLA≥5/10 matched sibling donors. In the haplotypes with frequencies greater than 0.1% in group1+ group2, the frequency of A*11∶01-B*40∶01-C*03∶04-DRB1*11∶01-DQB1*03∶01, A*02∶07-B*51∶01-C*14∶02-DRB1*09:01-DQB1*03∶03 were significantly different between group1 and group2 (P=0.029, 0.033) . The frequency of A*11∶01-B*46∶01-C*01∶02∶01G-DRB1*09∶01-DQB1*03∶03 was significantly different between group1 and group3 (P=0.035) . The frequency of A*02∶01-B*40∶01-C*07∶02-DRB1*09∶01-DQB1*03∶03 was significantly different between group1 and group2 (P=0.034) , or group1 and group3 (P=0.034) . The frequency of A*24∶02-B*13∶01-C*03∶04-DRB1*12∶02-DQB1*03:01 was significantly different between group2 and group3 (P=0.046) . Conclusion In this study, we summarize the prevalence of haplotype frequencies in terms of HLA-A, -B, -C, -DRB1 and-DQB1. Based on the database of family haplotype analysis, patients and donor candidates are sorted with matched HLA genotype while unmatched HLA haplotype. Even in patients without entire family information, HLA haplotype analysis assists in choosing the optimal related or unrelated donors.

Mitochondrial diseases are maternally inherited heterogeneous disorders that are primarily caused by mitochondrial DNA (mtDNA) mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruction of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells (MiPSCs) that harbored a high proportion of m.3243A>G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes (MELAS). We engineered mitochondrial-targeted transcription activator-like effector nucleases (mitoTALENs) and successfully eliminated the m.3243A>G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Furthermore, we successfully achieved reduction in the human m.3243A>G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.
Animals ; DNA, Mitochondrial ; genetics ; Humans ; Induced Pluripotent Stem Cells ; cytology ; metabolism ; MELAS Syndrome ; genetics ; Male ; Mice ; Microsatellite Repeats ; genetics ; Mitochondria ; genetics ; metabolism ; Mutation ; genetics

AIM: To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS: The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS: Compared with control group, incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein, and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels, increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation, and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells, and reduced Akt protein phosphorylation and NO production.CONCLUSION: Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.