Characterization of integrons and novel cassette arrays in bacteria from clinical isloates in China,2000-2014

Rapid dissemination of antibiotic resistance genes among bacterial isolates is an increasing problem in China. Integron, a conserved DNA sequence, which is carried on episomal genetic structures, plays a very important role in development of antibiotic resistance. This systematic analysis was based on MEDLINE and EMBASE databases. We summarized the distribution and proportion of different types of gene cassette arrays ofintegrons （including class 1, 2, 3 and atypical class 1 integron） from clinical bacteria isolates in China. Fifty-six literatures were included in this study. Most of the strains were Gram-negative bacteria （94.1%, 7,364/7,822） while only 5.9% strains were Gram- positive bacteria. Class 1 integrons were detected in 54.2% （3956/7295） Gram-negative strains, aadA2 was the most popular gene cassette array detected from 60 Gram-positive bacteria while dfrA 17-aadA5 were detected in 426 Gram- negative bacteria. This study identified 12 novel gene cassette arrays which have not been previously found in any species. All the novel gene cassette arrays were detected from Gram-negative bacteria. A regional characteristic of distribution of integrons was presented in this study. The results highlight a need for continuous surveillance of integrons and provide a guide for future research on integron-mediated bacteria resistance.

Transferring a Gene Expression Cassette Lacking the Vector Backbone Sequences of the 1Ax1 High Molecular Weight Glutenin Subunit into Two Chinese Hexaploid Wheat Genotypes

1Ax1 high molecular weight glutenin subunit （HMW-GS） gene expression cassette （GEC） lacking vector backbone sequences together with selectable marker Bar GEC were co-transformed into Chinese hexaploid cultivars Een 1 and Emai 12 to test the feasibility and the efficiency of explant regeneration, transformation frequency and transgene expression comparing with whole vector transformation by the approaches of plasmid extraction and excision, immature embryo isolation, particle co-bombardment, tissue culture, DNA extraction, PCR amplification, southern hybridization, leaf-painting test and SDS-PAGE etc. No significant difference was shown in tissue culture response of the proportion of embryogenic calli, somatic embryogenesis and regeneration frequency between GEC and whole plasmid bombarded embryos, but both regenerated less well than non-bombarded control. Total 56 plantlets that survived PPT selection had insertion of at least the Bar gene, 18 were from the GEC treatment and 38 from the whole plasmid treatment, the escape ratio averaged 0.23. Six independent transplants f230 - f235 with GEC transformation from genotype Emai 12 presented clear PCR amplification bands of Bar and 1Ax1 gene. The transformation and co-transformation frequency were 3.51 and 100% respectively. PCR amplification using a primer-pair specific for ampicillin resistant gene indicated the existence of Amp^R gene in whole vectors but the removal in GECs and transplants. Southern blot of total DNA and PCR products from transgenic plants of 1Ax1 GEC confirmed the integration of the transgene 1Ax1 and the absence of the EcoR Ⅰ recognition site at both ends of the 1Ax1 GEC when integrated. SDS-PAGE showed the expression of 1Ax1 GEC and un-expression of whole plasmid. The length of integrated fragment, the proportion of the gene of interest （GOI） and the selectable marker （MG）, bombardment pressure and genotypes are vital for the expression of a transformed GEC.

ATP-binding cassette transporters in progression and clinical outcome of pancreatic cancer: what is the way forward?

Pancreatic ductal adenocarcinoma(PDAC) is one of the most aggressive diseases and is characterized by high chemoresistance, leading to the lack of effective therapeutic approaches and grim prognosis. Despite increasing understanding of the mechanisms of chemoresistance in cancer and the role of ATPbinding cassette(ABC) transporters in this resistance, the therapeutic potential of their pharmacological inhibition has not been successfully exploited yet. In spite of the discovery of potent pharmacological modulators of ABC transporters, the results obtained in clinical trials have been so far disappointing, with high toxicity levels impairing their successful administration to the patients. Critically, although ABC transporters have been mostly studied for their involvement in development of multidrug resistance(MDR), in recent years the contribution of ABC transporters to cancer initiation and progression has emerged as an important area of research, the understanding of which could significantly influence the development of more specific and efficient therapies. In this review, we explore the role of ABC transporters in the development and progression of malignancies, with focus on PDAC. Their established involvement in development of MDR will be also presented. Moreover, an emerging role for ABC transporters as prognostic tools for patients' survival will be discussed, demonstrating the therapeutic potential of ABC transporters in cancer therapy.

ATP binding cassette C1 (ABCC1/MRP1)-mediated drug efflux contributes to disease progression in T-lineage acute lymphoblastic leukemia

Purpose: In acute lymphoblastic leukemia (ALL), multidrug resistance is often mediated by AT- Pase Binding Cassette (ABC) proteins, which principally involve ABCC1 (multidrug resistance protein 1, MRP1) and ABCB1 (multidrug resistance 1, MDR1). However, direct comparisons between the differential effects of ABCC1 and ABCB1 have been difficult, since identical cell lines with differential expression of these transporters have not been developed. Experimental Design: In this study, we developed and compared the biological profiles of Jurkat cell lines that selectively over-expressed ABCC1 and ABCB1. Vincristine (VCR) plays an important role in the treatment of T-lineage ALL (T-ALL), and is often the first drug given to newly-diagnosed patients. Because of its importance in treatment, we provide descalating, sub-lethal doses of VCR to Jurkat cells, and extended our observations to expression profiling of newly diagnosed patients with T-ALL. Results: We found that VCR-resistant cells over-expressed ABCC1 nearly 30-fold. The calcein AM assay confirmed that VCR-resistant cells actively extruded VCR, and that ABCC1-mediated drug resistance conferred a different spectrum of multidrug resistance than other T-ALL induction agents. siRNA experiments that blocked ABCC1 export confirmed that VCR resistance could be reversed in vitro. Analyses of T-lymphoblasts obtained from 100 newly diagnosed T-ALL patients treated on Children’s Oncology Group Phase III studies 9404 and AALL0434 that induction failure could be could be partially explained by the over-expression of ABCC1 and ABCB1. Conclusions: Taken together, these results suggest that over-expression of ABC transporters plays a contributing role in mediating treatment failure in T-ALL, and underscore the need to employ alternate treatment approaches in patients for whom induction failed or for those with relapsed disease.

Identification of effective siRNA against K-ras in human pancreatic cancer cell line MiaPaCa-2 by siRNA expression cassette

AIM: We shall construct the small interfering RNA (siRNA)expression cassette (SEC) targeting activated K-ras gene sequence, identify more effective siRNA sequence against K-ras gene in human pancreatic cancer cell line MiaPaCa-2 by SEC and reveal the anti-cancer effects of RNA interference (RNAi) and its therapeutic possibilities.WETHODS: Three different sites of SECs were constructed by PCR. K1/siRNA, K2/siRNA and K3/siRNA are located at sites 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of sites 194 and 491, we detected the apoptosis in cells by FACS after they were incubated for 48 h, then we tested the alternation of K-ras gene in MiaPaCa-2 cells by RT-PCR immunofluorescence, respectively.RESULTS: Introduction of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells leads to increased apoptosis, and the number of apoptotic cells is increased compared with control cells. The tests of RT-PCR immunofluorescence show the effects of inhibiting expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also find that the introduction of K3/siRNA has no effect on MiaPaCa-2 cells.CONCLUSION: K1/siRNA and K2/siRNA can inhibit the expression of activated K-fas but K3/siRNA has no effect,demonstrating that K1/siRNA and K2/siRNA are effective sequences against K-ras gene and K3/siRNA are not. We conclude that specific siRNA against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

Class Ⅰ integron with a novel cassette array in an ESBL-producing multidrug-resistant Klebsiella pneumoniae isolate

Objective： To analyze the molecular mechanism of integron mediated mulfi-resistanc, e in an ESBL-producing K. pneumoniae NJ 12 isolate. Methods： Susceptibility test was carried out by Kirby-Bauer method. Class Ⅰ, Ⅱ and Ⅲ integrons were detected by integrase gene PCR with primers that annealed to conserved regions of integron-encoded integrase genes intll, intl2 and intl3. The variable region of integron was amplified by integron PCR with primers that targeted the conserved flanking regions, and the product was sequenced. Six aminoglycoside modifying-enzyme genes, including ant（2＂）-Ⅰ, ant（3＂）- Ⅰ, aac（3）- Ⅰ, aac（3）-Ⅱ, aac （6＇）-Ⅰ, and aac（6＇）-Ⅱ , were detected. Results： K. pneumoniae NJ 12 was resistant to nine antibiotics, including piperacillin, ampicillin, cefuroxime, ceftazidime, cefotaxime, aztreonam, streptomycin, gentamicin and amikacin. This isolate was shown that there was positive with class Ⅰ integron, ant（2＂）- Ⅰ , ant（3＂）- Ⅰ , aac（3）-Ⅱ and aac（6＇）- Ⅰ modifying-enzyme genes. Neither class Ⅱ nor Ⅲ integron was detected; DNA sequencing of the fragment amplified by integron PCR revealed a novel cassette array aadR-cat-blaoxa-10/ aadA1. Conclusion： Class I integron with a novel cassette array in an ESBL-producing multidrug-resistant K. pneumon/ae NJ 12 isolate was reported from Nanjing area of China, with the GenBank accession number DQ141319.

ATP-binding cassette subfamily B member 1 (ABCB1) and subfamily C member 10(ABCC1O) are not primary resistance factors for cabazitaxel

Introduction:ATP-binding cassette subfamily B member 1(ABCB1) and subfamily C member 10(ABCCIO) proteins are efflux transporters that couple the energy derived from ATP hydrolysis to the translocation of toxic substances and chemotherapeutic drugs out of cells.Cabazitaxel is a novel taxane that differs from paclitaxel by its lower affinity for ATP-binding cassette(ABC) transporters.Methods:We determined the effects of cabazitaxel,a novel tubulin-binding taxane,and paclitaxel on paclitaxelresistant,ABCB1-overexpressing KB-C2 and LLC-MDR1-WT cells and paclitaxel-resistant,ABCC10-overexpressing HEK293/ABCC10 cells by calculating the degree of drug resistance and measuring ATPase activity of the ABCB1 transporter.Results:Decreased resistance to cabazitaxel compared with paclitaxel was observed in KB-C2,LLC-MDR1-WT,and HEK293/ABCC10 cells.Moreover,cabazitaxel had low efficacy,whereas paclitaxel had high efficacy in stimulating the ATPase activity of ABCB1,indicating a direct interaction of both drugs with the transporter.Conclusion:ABCB1 and ABCC10 are not primary resistance factors for cabazitaxel compared with paclitaxel,suggesting that cabazitaxel may have a low affinity for these efflux transporters.

Characterization of Class 1 Integron Gene Cassettes among Clinical Bacteria Isolated from One Large Hospital in Northern China

The class 1 integron and complex gene cassettes among different species of clinical isolates in northern China were characterized in this study. 383 clinical isolates were obtained from northern China, and class 1 integrons containing gene cassettes widely distributed among gram negative clinical isolates was observed. We find that the class 1 integron showed positive correlation with multidrug resistance phenotype of gram negative bacteria. In addition, we find that isolates belonged to one species harbored different types of gene cassette arrays, while same types of gene cassette arrays were observed in different species of isolates. The diversity of gene cassette arrays among the isolates indicated the complexity of multidrug resistance in clinical isolates in northern China.

INHIBITION OF ACTIVATED K-RAS GENE BY SIRNA EXPRESSION CASSETTES IN HUMAN PANCREATIC CARCINOMA CELL LINE MIAPACA-2

Objective: To construct the small interfering RNA(siRNA) expression cassettes (SECs) targeting activated K-ras gene sequence and investigate the effects of SECs on K-ras gene in human pancreatic cancer cell line MIAPaCa-2. Methods: Three different sites of SECs were constructed by PCR. The K1/siRNA, K2/siRNA and K3/siRNA were located at the site 194, 491 and 327, respectively. They were transfected into MiaPaCa-2 cells by liposome to inhibit the expression of activated K-ras. In the interfering groups of site 194,491, we observed the cytopathic effect of confluent MiaPaCa-2 cells after they were incubated for 48 hours, and detected the apoptosis in cells by FACS, then we tested the alternation of K-ras gene in confluent MiaPaCa-2 cells by RT-PCR,immunofluorescence and western blot, respectively. Results: Introductions of the K1/siRNA and K2/siRNA against K-ras into MiaPaCa-2 cells led to cytopathic effect, slower proliferation and increased apoptosis, while the appearances of control MiaPaCa-2 cells remained well. The number of apoptotic cells increased compared with control cells. RT-PCR,immunofluorescence and western blot showed the effects of inhibited expression of activated K-ras gene by RNA interference in the K1/siRNA and K2/siRNA groups. We also found that the introduction of K3/siRNA had no effect on MiaPaCa-2 cells. Conclusion: K1/siRNA and K2/siRNA can inhibit the expression of activated K-ras and decrease the growth of MiaPaCa-2 cells, while K3/siRNA has no such effect, demonstrating that the suppression of tumor growth by siRNA is sequence-specific. We conclude that K-ras is involved in maintenance of tumor growth of human pancreatic cancer, and SECs against K-ras expression may be a powerful tool to be used therapeutically against human pancreatic cancer.

Emergence of staphylococcal cassette chromosome mec type Ⅰ with high-level mupirocin resistance among methicillin-resistant Staphylococcus aureus

Objective: To investigate the molecular epidemiology and antimicrobial resistance patterns of methicillin-resistant Staphylococcus aureus(MRSA) among healthcare workers and patients.Methods: MRSA isolates were recovered from nasal swabs collected at a tertiary care hospital of Nepal and confirmed on the basis of Gram staining, conventional biochemical tests, and PCR amplification of mec A gene. PCRs were also used for detection of the different resistance genes and staphylococcal cassette chromosome(SCC) mec types.Antibiotic susceptibility patterns of isolates were assessed by disc diffusion method and minimum inhibitory concentrations were determined by E-test.Results: A total of 29 MRSA were isolated from 536 nasal swabs(5.4%) of health care workers and patients at a tertiary care hospital in Nepal. All isolates were susceptible to amikacin, gentamicin, vancomycin(minimal inhibitory concentrations < 2 mg/m L), tigecycline, tetracycline, nitrofurantoin, rifampicin, quinupristin-dalfopristin, and linezolid. Among the 29 MRSA isolates, resistance to erythromycin(72%), ciprofloxacin(75%), co-trimoxazole(62%), clindamycin(10%), and chloramphenicol(10%) was found, and fifteen isolates(51%)exhibited high-level mupirocin resistance(minimal inhibitory concentrations > 1 024 mg/m L).Fourteen isolates were found harboring the mup A gene and one isolate was found carrying the novel mup B gene. High prevalence(68%) of SCCmec I type was found, followed by SCCmec V(13%) and SCCmec III(3%) among all the MRSA isolates.Conclusions: We found the emergence of SCCmec type I with high-level mupirocin resistance among MRSA in Nepal. Data also suggest that MRSA SCCmec type V strain has spread from the community to the hospital.

ZBM30 suppresses atherosclerosis through up-regulating ATP-binding cassette A1 and G1

Atherosclerosis is the most common cause of cardiovascular diseases, such as myocardial infarction and stroke. The aim of this study was to investigate the effects of a novel compound ZBM30 on atherosclerosis in ApoE- deficient mice and its associated mechanism. ApoE-deficient mice （6 weeks old）, fed an atherogenic high-fat and high cholesterol diet for 8 weeks, were divided into three groups. Two groups were orally administrated ZBM30 （10, 30 nag ~ kg-1） daily for 12 weeks, while the control group was administered saline. Atherosclerotic lesions with en face aortas were evaluated by Sudan IV staining, and lesion areas in aortic sinuses were evaluated by oil red O staining. Necrotic core areas and fibrous cap areas in the lesion were evaluated by henaatoxylin and eosin （HE） staining and Masson＇ s trichronae staining in the aorta sinuses. The effects of ZBM30 on cholesterol accumulation in naacrophages and cholesterol transporters： ATP binding cassette A1 （ABCA1） and ATP binding cassette G1 （AB- CG1） were evaluated by oil red O assay, 3H-cholesterol efflux assay, Western blot, and real-time PCR on macro- phage cell lines： Raw 264.7 and THP-1. Inanauno-fluoresces was used to determine the ABCA1 expression in naac- rophage in aorta sinuses. Luciferase reporters of wild type and mutant types of ABCA1 promoter were constructed to determine the regulatory domain of ZBM30 on ABCA1 promoter. Results showed that, compared with the control group, en face lesions in ZBM30 group （ 10, 30 mg · kg^-1 ） were reduced 54.96 ± 10.06% and 71.50 ± 15.37% respectively, and aorta sinus lesions were reduced 41.85 ± 11.21% and 82.23 ± 8.25% respectively. Necrotic core areas in the ZBM30 group were markedly reduced and fibrous cap areas were not changed. Oil red O staining and 3 H-cholesterol efflux assays on Raw 264.7 cell line revealed that ZBM30 significantly attenuated the cholesterol accumulation in naacrophages by enhancing apolipoprotein AI and HDL mediated cholesterol efflux. Furthermore, ZBM30 induced the protein and naRNA expression of cholesterol transporters such as ABCA1 and ABCG1. Inanauno- fluoresces experiment revealed that ZBM30 induced the ABCA1 expression in naacrophage in the lesion, which is consistent with the results in vitro. Luciferase reporter assay revealed that ZBM30 exerted its effect on ABCA1 via liver X receptor （LXR） binding domain. In conclusion, ZBM30 suppresses atherosclerosis through up-regulating cholesterol efflux via ABCA1 and ABCG1 transporters in ApoE-deficient mice.

Diversity and Distribution of Staphylococcal Chromosomal Cassettes Mec (SCCmec) Types I, II and III in Coagulase-Negative Staphylococcal Strains Isolated from Surfaces and Medico-Technical Materials of the University Hospital of Abomey-Calavi/Sô-Ava

The coagulase-negative staphylococci (CoNS) have long been considered to be low pathogenicity. The possibility of a horizontal transfer of resistance and virulence genes from S. aureus to CoNS could increase the pathogenicity of these bacteria. The objective of this work is to contribute to a better knowledge of the pathogenicity of (CoNS) strains isolated from surfaces and medico-technical materials of the University Hospital of Abomey-Calavi/Sô-Ava. Seventy strains of CoNS isolated from surfaces and medico-technical materials of the University Hospital of Abomey-Calavi were tested for methicillin resistance. The resistance to methicillin was evaluated phenotypically by the resistance of the strains to cefoxitin and then confirmed by the search for the mecA gene using PCR. The genes encoding staphylococcal chromosomal cassette (SCCmec) types I, II and III originally found in S. aureus were tested in CoNS by multiplex PCR using specific primers. All the strains studied showed resistance to methicillin. However, only 28.5% (20/70) carried the mecA gene. SCCmec was identified in only 17.14% (12/70) of these strains. Four strains carried mecA gene as well as one of the three types of SCCmec searched. SCCmec types I, II and III were identified in CoNS strains studied. SCCmec type I was the most frequent chromosomal cassette in mecA+ strains, only or in association with another SCCmec. The study also revealed methicillin-resistant strains carrying SCCmec lacking the mecA gene. Finally, 60% (12/20) of the strains were found to be non-typeable. Our results show that CoNS strains present a high resistance to methicillin and the source of this resistance in the CoNS of our study is not only the mecA gene. There is also a high diversity of SCCmec, justified by a large number of non-typeable CoNS strains. The mecA− SCCmec+ methicillin-resistant strains deserve to be sequenced for further studies.

Inhibiting NF-κB increases cholesterol efflux from THP-1 derived-foam cells treated with AngⅡ via up-regulating the expression of ATP-binding cassette transporter A1

Objective： To study the role of nuclear factor-kappa B（NF- κB） in cholesterol efflux from THP-1 derived-foam cells treated with Angiotensin Ⅱ（Ang Ⅱ ）. Methods：Cultured THP-1 derived-foam cells were treated with Ang Ⅱ or preincubated with tosyl-phenylalanine chloromethyl-ketone（TPCK） NF- κB inhibitor. The levels of activated NF- κB in the cells were examined by sandwich ELISA, Cellular cholesterol content was studied by electron microscopy scanning and zymochemistry via fluorospectrophotometer and cholesterol effiux was detected by scintillation counting technique. ABCA1 mRNA and protein were quantified by RT-PCR and Western blotting. Results：Addition of TPCK to the cells before Ang Ⅱ stimulation attenuated the response of NF- κB p65 nuclear translocation induced by Ang Ⅱ and showed no peak in foam cells group and caused a reduction in cholesterol content and an increase in cholesterol efflux by 24.1%（P〈 0.05） and 41.1%（P〈 0.05） respectively, when compared with Ang Ⅱ group. In accordance, the ABCA1 mRNA and protein were increased by 30% and 19%（P 〈 0.05） respectively, when compared with Ang Ⅱ group. Conclusion：Ang Ⅱ can downregulate ABCA1 in THP-1 derived-foam cells via NF- K B, which leads to less cholesterol effiux and the increase of cholesterol content with the consequence of the promotion of atherosclerosis.

Prevalence of Class 1 Integron, Resistance Gene Cassettes and Antimicrobial Susceptibility Profiles among Isolates of Pseudomonas aeruginosa in Iran

Pseudomonas aeruginosa is one of the most important opportunistic human pathogens worldwide. High prevalence of Multi Drug Resistant P. aeruginosa (MDRPa) in Iran is a serious problem for antimicrobial therapy. Several studies have reported the MDRPa in Europe and Asia. Due to the use of broad-spectrum antibiotics, bacterial resistance is increasing in Iran, located in Middle East. The present cross-sectional study was designed to investigate the prevalence of class1 integron, resistance gene cassettes and antimicrobial susceptibility profiles among isolates of P. aeruginosa in Al-Zahra Hospital, Isfahan City, central part of Iran from Jan-Sep 2014. The aim of this study was to determine the antimicrobial susceptibility, the prevalence of Class1 integron, resistance gene a measuring in Iran. A total of 231 P. aeruginosa isolates were collected from clinical specimens including urine (50.6%), tracheal tube (25.5%), wound (13.4%), blood (6.1%), catheter (2.2%), cerebrospinal fluid (1.7%) and sputum (0.4%) isolates from hospitalized patients (mean age: 50.27 ± 24.12 years).The majority of patients (68%) were male. Isolates were collected from different parts of the hospital as follows: ICU, Internal Medicine, Emergency care, Pediatrics, Nephrology, Transplant Center, General surgery and Infectious. Revealed data show a high rate of MDR P. aeruginosa isolates in the studied area;also, the result signifies the spread of aadA6 among clinical isolates in hospitalized patients.

自然衰老型小鼠海马及肝脏组织中胆固醇转运相关蛋白表达的研究

目的研究自然衰老型小鼠海马及肝脏组织胆固醇转运相关蛋白表达变化,观察衰老对胆固醇转运的影响。方法选取清洁级雄性昆明小鼠1月龄10只,10月龄10只。1月龄小鼠为对照组,10月龄小鼠为老年组,饲养7 d后,采用Y字迷宫检测小鼠行为学及记忆,采用免疫组化检测肝脏脂肪酸合酶(FASN)蛋白表达变化、海马载脂蛋白E(ApoE)蛋白表达变化,采用ELISA法检测总胆固醇、三酰甘油、高密度胆固醇及低密度胆固醇含量变化,采用Western Blot法检测肝脏三磷酸腺苷结合盒转运体A1(ABCA1)、低密度脂蛋白受体(LDLR)蛋白和海马ABCA1蛋白、ApoE蛋白表达变化。结果老年组小鼠出现摄食量减少、懒动及反应较迟钝的表现。老年组小鼠的总进臂次数和交替次数少于对照组,差异有统计学意义(P<0.05)。老年组小鼠的高密度脂蛋白胆固醇含量低于对照组,三酰甘油和低密度脂蛋白胆固醇高于对照组,差异有统计学意义(P<0.05)。老年组小鼠肝脏的FASN、LDLR和ABCA1水平低于对照组,海马组织的ApoE蛋白表达高于对照组,ABCA1蛋白表达低于对照组,差异有统计学意义(P<0.05)。结论老年组小鼠外周及中枢胆固醇代谢出现异常,可能与肝脏及海马ABCA1蛋白、ApoE蛋白、LDLR蛋白表达异常有关。

THE HARDWARE & SOFTWARE DESIGN AND IMPLEMENTATION OF A NEW TYPE INTERFACE FOR THE CASSETTE TAPE RECORDER

This paper analyzes in detail the operating principle and characteristics of hardware and software of a new type interface for the cassette tape recorder,which has been developed by this author for the 8097BH target prototype machine.It discusses the design of the audio cassette tape recording format and some important aspects which must be taken care of in software for reading the cassette tape.The paper also illustrates debugging methods thoroughly.

建立检测猕猴三磷酸腺苷结合盒转运蛋白G2的mRNA相对表达水平的RT-qPCR方法

目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。

荻草谷网蚜ABCG1基因的克隆及表达模式分析

荻草谷网蚜Sitobion miscanthi是严重威胁我国小麦生产安全的迁飞性害虫。蜕皮激素是参与蚜虫翅型分化调控的内激素,在有翅成蚜体内保持高滴度,且诱导后代产生更高比例的无翅蚜,其进出靶细胞需要经过细胞膜上特定蛋白的转运。ATP结合盒转运蛋白家族G亚家族(ATP-binding cassette transporter G, ABCG)中的ABCG1是通过跨膜转运昆虫类固醇、对蜕皮激素信号进行负调控的功能蛋白之一,在蚜虫中尚未见报道。本文克隆了荻草谷网蚜ABCG1(SmisABCG1)基因,并进行了序列比对、系统进化分析以及不同组织部位和发育时期表达模式分析。结果显示,SmisABCG1基因的开放阅读框全长为1 851 bp,编码616个氨基酸,含7个跨膜结构域,符合ABCG蛋白家族典型结构特性,基因登录ID:OP626323。昆虫间ABCG1较保守,该蛋白系统进化关系与各自物种间亲缘关系的远近保持一致。其中,SmisABCG1与来自豌豆蚜、禾谷缢管蚜、棉蚜、花生蚜和雪松长足大蚜等的ABCG1氨基酸序列高度一致(>87%),以上蚜虫聚为一支。与SmisABCG1亲缘关系最近的是豌豆蚜的ABCG1,其次是半翅目的褐飞虱、白背飞虱和灰飞虱,与膜翅目的新疆菜叶蜂、阿里山潜蝇茧蜂以及鞘翅目的赤拟谷盗、蜂箱小甲虫亲缘关系较远。该基因在伪胚胎和成蚜阶段高表达。包含伪胚胎的有翅、无翅成蚜整蚜SmisABCG1的转录水平无显著差异,但其在来自有翅成蚜的伪胚胎中的转录水平高于无翅成蚜伪胚胎,证实无翅成蚜自身的转录水平较高,而有翅成蚜较低。进一步分析显示这一差异主要是无翅蚜胸部显著高表达所导致。基于该蛋白对蜕皮激素负调控,与有翅成蚜转录水平低,但蜕皮激素水平更高相符合。

三磷酸腺苷结合盒转运蛋白G2与痛风病关联的病理机制研究进展

痛风病是指与高尿酸血症、尿酸盐沉积(关节、肾脏)等尿酸代谢紊乱密切相关的进展性代谢疾病,根据其病理进展特点和临床表现,其病程可分为高尿酸血症阶段、尿酸盐沉积阶段、痛风性炎症阶段。三磷酸腺苷结合盒转运蛋白G2(ABCG2)是高通量的尿酸转运体,既参与了机体尿酸外排的肠-肾途径,又与痛风性炎症密切相关。通过切入ABCG2,对痛风病病理机制研究进展进行综述,以期为临床痛风病的深入研究及防治提供新的见解。

冠心病患者ABCG1、ANGPTL2启动子区甲基化与心力衰竭发生的关系研究

目的分析冠状动脉粥样硬化性心脏病(冠心病)患者三磷酸腺苷结合盒转运蛋白G1(ABCG1)、血管生成素样蛋白2(ANGPTL2)启动子区甲基化与心力衰竭(心衰)发生的关系。方法选取2020年3月至2022年3月于湖南省第二人民医院收治的冠心病患者120例。根据是否发生心衰,将患者分为合并心衰组(n=26)和不合并心衰组(n=94)。采用全自动生化仪检测血清总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)。采用心脏超声检查患者左室射血分数(LVEF)、左心室舒张末期内径(LVEDD)、左室收缩末期内径(LVESD)水平。特异性PCR检测ABCG1和ANGPTL2基因甲基化。采用实时荧光定量聚合酶链反应检测ABCG1和ANGPTL2基因mRNA表达水平。Logistic回归分析探讨影响冠心病患者发生心衰的因素。结果两组患者LDL-C、LVEF、LVEDD、LVESD、ABCG1、ANGPTL2基因启动子区甲基化等方面比较,差异具有统计学意义(P<0.05)。LVESD、LVEF、ABCG1甲基化和ANGPTL2甲基化是冠心病患者发生心衰的危险因素。ABCG1甲基化、ANGPLT2甲基化患者中LVESD水平均高于未甲基化患者(P<0.05)。ABCG1甲基化、ANGPLT2甲基化患者中LVEF水平均低于未甲基化患者(P<0.05)。合并组ABCG1、ANGPTL2基因甲基化的患者mRNA的表达量明显低于非甲基化患者(P<0.05)。结论ABCG1和ANGPTL2基因甲基化是冠心病患者发生心衰的危险因素,检测上述两基因甲基化状态可为诊治冠心病心衰提供理论支持。