Mitochondria, the power house of cells, are important organelles in eukaryotic cells. Having their own unique and complete DNA (mtDNA) and genetic system, mitochondria play an essential role in cellular energy metabolism, intracel?lular signaling and apoptotic pathways, as well as many other biological functions, which are closely related with cellular met?abolic network. A disruption of mitochondrial genes can therefore result in mitochondrial dysfunction and human diseases, thus they have been widely used in molecular biology, development biology, genetics, forensic identification and clinical diag?nosis. Consequently, sequencing mitochondrial genome has shown great significance in mitochondrial structure and function research. In this review, research progress in mitochondrial genome sequencing method is summarized, mainly focusing on Sanger sequencing, long-PCR and next-generation sequencing. Also rolling circle amplification and indirect sequencing of mtDNA are reviewed. The ambiguities caused by numts in indirect sequencing are mentioned and resolved.

Objective To investigate the expression profile of mRNAs in brain samples collected from pronuclear transfer (PNT) mice. Methods Female CD-1 mice were superovulated, and zygotes were collected after mating with adult male mice. Zygotes with two pronuclei were selected for pronuclear transfer manipulation, and then the reconstructed zygotes were transferred into the oviduct of pseudopregnant female mice. The infant mice obtained from pronuclear transfer were called PNT group, while the embryoes that were not performed pronuclear transfer was regarded as control group. Total RNA were extracted from brain samples of both PNT and control mice, and cDNA were labeled with fluorescent dye. Genes that were differentially expressed were identified using the Agilent mouse mRNA array. Gene ontology analysis and pathway analysis were also completed. Results Compared with control group, 392 mRNAs were expressed differentially, which showed more than 2.0 times variation and statistical significance, accounting for 1.7% of all mRNAs. Among those 366 mRNAs were up-regulated and 26 mRNAs were down-regulated. Eleven mRNAs came to 4.0 times variation in total. Gene ontology analysis indicated that differentially expressed genes were significantly enriched in alternative mRNA splicing, small GTPase mediated signal transduction, regulation of insulin receptor signaling pathway, hydrolase activity, transmembrane transporter activity and pyrophosphatase activity. Significant enriched pathway terms contained ion channel transport, fatty acid metabolism, butanoate metabolism, triacylglycerol and ketone body metabolism. Conclusion Pronuclear transfer might influence some key metabolism process in mouse brain.

Objective To explore the expression and function of miR-125b, miR-30b and miR-424 in endometrial cells. Methods Human endometrial samples were obtained in natural cycles and stimulating cycles. Endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) were isolated and confirmed by immunofluorescence. The expressions of miR-125b, miR-30b and miR-424 were detected by real-time PCR. Results The expression levels of miR-125b, miR-30b and miR-424 were higher in proliferative phase in ESCs than those in EECs. And in EECs, the expression levels of miR-125b, miR-30b and miR-424 were significantly up-regulated in secretory phase than in proliferative phase, while it was stable in ESCs. In addition, the expressions of miR-125b in EECs and miR-30b were increased in ESCs in women with elevated progesterone on the day of HCG administration than those of the control. The target genes of miR-125b, miR-30b and miR-424 mainly participated in cell migration and motion, cell-cell adherens junction and Wnt signaling pathway. Conclusion miR-125b, miR-30b and miR-424 were differently expressed in endometrial cells in different phases, and may participate in regulation of endometrial receptivity.

Objective: To investigate the response to neoadjuvant chemotherapy (NAC) among different molecular subtypes of breast cancers using molecular classification with Ki-67 (ER+ PR+ HER2+ Ki-67) or without Ki-67 (ER+ PR+ HER2). Methods: One hundred and twenty-seven cases of invasive breast cancer confirmed by core needle biopsy before NAC were collected from January 2007 to December 2009 and diagnosed at West China Hospital, Sichuan University. The cases were classified into different molecular subtypes using molecular classifications with or without Ki-67. Their clinical and pathological response to NAC was evaluated and compared. Results: The different subtypes using both molecular classifications showed significant difference in clinical response(with Ki-67: χ²=22.40, P<0.01; without Ki-67: χ²=9.202, P=0.027)but not pathological(P>0.05) response to NAC. By multivariate analysis, Ki-67 was predictive for a clinical complete response (P=0.041) and clinical overall response (P<0.01); also Ki-67 was the only clinicopathological factor predictive of pathological response(P=0.041). Conclusion The molecular classification with Ki-67 is better to predict breast cancers responsiveness to NAC than the molecular classification without Ki-67.

Objective: To analyze and evaluate the comprehensiveness and reliability of the content related to comprehensive sexuality education in 16 subject curriculum standards in the current compulsory education system, and the feasibility and effectiveness of comprehensive sexuality education teaching practice. Methods: Based on the textbook analysis model, a curriculum standard analysis model was established, and the interpretive structure model was used to analyze and evaluate the content related to comprehensive sexual education in the curriculum standards of 16 subjects in the current compulsory education system, including Ethics and Rule of Law, Chinese, History, Mathematics, English, Japanese, Russian, Geography, Science, Physics, Chemistry, Biology, Information Technology, Physical Education and Health, Arts, Labor. Results: There were 7 subject curriculum standards including Ethics and Rule of Law, Chinese, English, Science, Biology, Information Technology, Physical Education and Health reflecting 5 core concepts and 12 themes of comprehensive sexuality education, and the most frequent core concept was Violence and Staying Safe and the Human Body and Development,as well as the most frequent theme was "Puberty". History, Mathematics, Japanese, Russian, Geography, Physics, Chemistry, Art and Labor didn t include content related to comprehensive sexuality education. The content related to comprehensive sexuality education in the curriculum standard of compulsory education presented three characteristics:it was closely related to the subject content, partial content was consistent with the teaching goal of the subject, and the content depth increased with the growth of grade. The comprehensiveness and reliability of comprehensive sexual education in the current compulsory education curriculum standards of China needed to be improved. Conclusion The comprehensiveness, reliability, feasibility and effectiveness of teaching practice of comprehensive sexuality education in 16 subject curriculum standards in the current compulsory education stage need to be improved.

Objective:To investigate the effects of erythropoietin producing hepatocyte receptor A2 (EphA2) gene silencing on the growth, migration and invasion of human ovarian cancer HeyA8-MDR cells.Methods:After stable screening by plasmid and liposome mediated transfection, the expression of EphA2 protein was detected by Western blot. The effects of EphA2 gene silencing on the biological characters of HeyA8-MDR cells of ovarian cancer were observed by CCK-8 and Transwell chamber invasion assay.Results:Compared with HeyA8-MDR (negative control group) and HeyA8-MDR+ blank control group, the expression level of EphA2 protein in HeyA8-MDR+ experimental group was significantly decreased ( P<0.01). Cell proliferation, migration and invasion were inhibited ( P<0.05). Conclusions:EphA2 gene silencing can inhibit the growth, migration and invasion of HeyA8-MDR cells of ovarian cancer, providing a new idea for the treatment of ovarian cancer.

Objectives:To investigate the clinical impacts of chronic total occlusion (CTO) in acute non-ST segment elevation myocardial infarction (NSTEMI) patients underwent primary percutaneous coronary intervention (PCI).Methods:A total of 2 271 acute NSTEMI patients underwent primary PCI from China Acute Myocardial Infarction Registry were enrolled in this study and divided into the CTO group and the non-CTO group according to the angiography. The primary endpoint was in-hospital mortality and mortality during a 2-year follow-up. The secondary endpoint was major adverse cardiovascular events (MACE) including revascularization, death, re-myocardial infarction, heart failure readmission, stroke and major bleeding.Results:Thirteen-point four percent of the total acute NSTEMI patients had concurrent CTO. In-hospital mortality (3.6% vs. 1.4%, P<0.01) and 2-year mortality (9.0% vs. 5.1%, P<0.01) were significantly higher in the CTO group than those in the non-CTO group, respectively. Multiple regression analyses showed that chronic obstructive pulmonary disease ( HR 7.28, 95% CI 1.50-35.35, P=0.01) was an independent risk factor of in-hospital mortality, and advanced age ( HR 1.04, 95% CI 1.01-1.07, P<0.01), and low levels of ejection fraction ( HR 0.95, 95% CI 0.93-0.98, P<0.01) were independent risk factors of 2-year mortality. CTO ( HR1.67, 95% CI 1.10-2.54, P=0.02) was an independent risk factor of revascularization, but not a risk factor of mortality. Conclusions:Although acute NSTEMI patients concurrent with CTO had higher mortality, CTO was only an independent risk factor of revascularization, but not of mortality. Advanced age and low levels of ejection fraction were independent risk factors of long-term death among acute NSTEMI patients.

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 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.

Atrial fibrillation (AF) is one of the most common arrhythmias, which does great harm to patients. Effective methods were urgently required to prevent the recurrence of AF. Four methods were used to analyze RR sequence in this paper, and differences between Pre-AF (preceding an episode of AF) and Normal period (far away from episodes of AF) were analyzed to find discriminative criterion. These methods are: power spectral analysis, approximate entropy (ApEn) and sample entropy (SpEn) analysis, recurrence analysis and time series symbolization. The RR sequence data used in this research were downloaded from the Paroxysmal Atrial Fibrillation Prediction Database. Supporting vector machine (SVM) classification was used to evaluate the methods by calculating sensitivity, specificity and accuracy rate. The results showed that the comprehensive utilization of recurrence analysis parameters reached the highest accuracy rate (95%); power spectrum analysis took second place (90%); while the results of entropy analyses and time sequence symbolization were not satisfactory, whose accuracy were both only 70%. In conclusion, the recurrence analysis and power spectrum could be adopted to evaluate the atrial chaotic state effectively, thus having certain reference value for prediction of AF recurrence.
Atrial Fibrillation ; diagnosis ; Entropy ; Heart Atria ; physiopathology ; Humans ; Recurrence ; Sensitivity and Specificity ; Support Vector Machine