烟草祖先种及重要野生种PDR1基因的生物信息学分析

植物的多向耐药性(Pleiotropic drug resistance,PDR)转运蛋白隶属于ATP结合盒(ATP-binding cassette,ABC)转运蛋白家族的G亚家族。该类蛋白参与植物多种初生、次生代谢物的跨膜运输,在植物的生长发育、响应逆境胁迫、解毒过程中起着重要作用。近年的研究还表明,该蛋白与多种植物对真菌病原的抗性有关。本文以烟草祖先种和重要野生种为研究对象,以拟南芥PDR12蛋白、烟草的PDR1基因为探针,通过搜索同源序列,从数据库中提取烟草及祖先种、重要野生种中的PDR基因、蛋白序列,并通过生物信息学分析手段对其编码蛋白的理化特性、氨基酸序列特征、蛋白结构、功能、启动子序列及表达调控特征进行了分析,并以本氏烟(Nicotiana benthamiana)NbPDR1为代表,预测了该基因启动子的顺式作用元件,以普通烟草(Nicotiana tabacum)NtPDR1基因为例,预测了其基因编辑位点。结果表明,这些PDR基因相似性高,均编码跨膜蛋白,具有典型的跨膜结构域和核酸结合结构域。该基因启动子区有多个与病原真菌诱导、干旱等逆境胁迫响应相关的元件,暗示该基因可能是抗真菌病害、响应逆境胁迫的广谱抗性基因。最后,本文预测的基因编辑位点,为烟草PDR基因的功能研究、基因编辑及抗病育种提供理论基础。

Establishment and Application of a Multiplex PCR System for the Detection of Blast Resistance Genes Pi-ta and Pi-b in Rice

Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.

Genetic diversity of the hepatitis C virus: Impact and issues inthe antiviral therapy

The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The re- sulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the pre- dictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic suc- cess. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antivi- ral therapy in patients infected by the same HCV geno- type. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Inter- feron pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non- structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.

Efflux pump gene hefA of Helicobacter pylori plays an important role in multidrug resistance

AIM: To determine whether efflux systems contribute to multidrug resistance of H pylori. METHODS: A chloramphenicol-induced multidrug resistance model of six susceptible H pylori strains (5 isolates and H pylori NCTC11637) was developed. Multidrug-resistant (MDR) strains were selected and the minimal inhibitory concentration (MIC) of eryth-romycin, metronidazole, penicillin G, tetracycline, and ciprofloxacin in multidrug resistant strains and their parent strains was determined by agar dilution tests. The level of mRNA expression of hefA was assessed by fluorescence real-time quantitative PCR. A H pylori LZ1026 knockout mutant (ΔH pylori LZ1026) for (puta-tive) efflux protein was constructed by inserting the kanamycin resistance cassette from pEGFP-N2 into hefA, and its susceptibility profiles to 10 antibiotics were evaluated. RESULTS: The MIC of six multidrug-resistant strains (including 5 clinical isolates and H pylori NCTC11637) increased signifi cantly (≥ 4-fold) compared with their parent strains. The expression level of hefA gene was significantly higher in the MDR strains than in their parent strains (P = 0.033). A H pylori LZ1026 mutant was successfully constructed and the ΔH pylori LZ1026 was more susceptible to four of the 10 antibiotics. All the 20 strains displayed transcripts for hefA that con-fi rmed the in vitro expression of these genes.CONCLUSION: The efflux pump gene hefA plays an important role in multidrug resistance of H pylori.

PLASMA RESISTIN LEVELS AND SINGLE-NUCLEOTIDE POLYMORPHISMS IN RESISTIN GENE 5 FLANKING REGION IN PATIENTS WITH STROKE

Objective To analyze the role of resistin in insulin resistance (IR) through investigating the variation of plasma resistin levels and single-nucleotide polymorphisms (SNPs) in resistin gene 5’ flanking region in stroke patients.Methods In 103 atherothrombotic cerebral infarction (ACI) patients, 85 lacunar infarction (LI) patients, 70 intracerebral hemorrhage (ICH) patients, and 86 healthy controls, plasma resistin and insulin levels were measured by ELISA , SNPs in resistin gene 5’ flanking region were detected by PCR and direct DNA sequencing. The subjects’ body height and weight, the body mass index, quantitative insulin sensitivity check index (QUICKI), blood pressure, and the concentration of fasting plasma glucose, triglyceride, total cholesterol, creatinine, low-density lipoprotein, and high-density lipoprotein were also determined. Results QUICKI was significantly lower in the ACI and ICH patients (0.316±0.037 and 0.309±0.032, respectively) than that in the controls (0.342±0.043, P<0.001), while plasma resistin level was significantly higher in the ACI and ICH patients (6.36±3.79 and 7.15±4.27 ng/mL, respectively) than that in the controls (5.28±2.56 ng/mL, P<0.05), but such difference was not observed in the LI patients compared with controls. There was a statistically negative correlation between plasma resistin level with QUICKI (r=-0.228, P<0.001). The distributions of allele and genotype frequencies of resistin gene -420C>G and -537A>C SNPs were not significantly different among the different groups, and those SNPs were not correlated with other clinical and biochemical parameters.Conclusions Plasma resistin is associated with stroke by participating in the development of IR. The SNPs in resistin gene 5’ flanking region has no impact on the plasma resistin level.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to chronic hepatitis B

AIM： To assess the three polymorphJsm regions within cytotoxic T-lymphocyte antigen 4 （CTLA-4） gene, a C/T base exchange in the promoter region-318 （CTLA-4 -318C/T）, an A/G substitution in the exon 1 position 49 （CTLA-4 49A/G）, a T/C substitution in 1172 （CTLA-4 -1172T/C） in patients with chronic hepatitis B. METHODS： Fifty-one patients with chronic hepatitis B virus infection and 150 healthy subjects were recruited sequentially as they presented to the hepatic clinic. Classification of chronic hepatitis B virus （HBV）-infected patients was as asymptomatic carrier state （26 patients） and chronic hepatitis B （25 patients）. Genomic DNA was isolated from anti-coagulated peripheral blood Bully coat using Miller＇s salting-out method. The presence of the CTLA-4 gene polymorphisms was determined using polymerase chain reaction amplification refractory mutation system （ARMS）. RESULTS： We observed a significant association between -318 genotypes frequency （T＋C-, T＋C＋, T-C＋） and susceptibility to chronic hepatitis B （P=0.012, OR=0.49, 95%CI： 0.206-1.162）. However, we did not observe a significant association for ＋49 genotype frequency （T＋C＋, T＋C- T-C＋） and -1172 genotype frequency （C＋T＋, T＋C- C＋T-） and state of disease. CONCLUSION： Our results suggest that CTLA-4 gene polymorphisms may partially be involved in the susceptibility to chronic hepatitis B.

IMPROVING P-gp EXPRESSION IN HUMAN MONONUCLEAR CELLS IN VITRO TRANSFECTED BY MULTIDRUG RESISTANCE-1 mRNA

Objective To evaluate the expression and function activity of P-glycoprotein (P-gp) in human mononuclear cells (MNCs) in vitro transfected by multidrug resistance-1(MDR1) mRNA. Methods Two MDR1 cDNA vectors, pT7TS_MDR1 and pGEM5Zf(+)_MDR1, were constructed and transcripted in vitro. Vector pGEM5Zf(+)_MDR1 only contained the coding region of mdr1 cDNA, and pT7TS_MDR1 also included Xeponus β-globin 5’ and 3’ untranslated region. MNCs were prepared from peripheral blood of parvicellular lung cancer patient. The two human mdr1 mRNAs were then transferred into human MNCs in vitro by DOTAP. And the expression efficiency and pump function of P-gp were measured with flow cytometry. Results Expression of P-gp significantly elevated in both transferred cells compared with untransferred cells (P < 0.01). And pT7TS_MDR1 showed higher capability in elevating the expression of P-gp than pGEM5Zf(+)_MDR1 (P < 0.01). The P-gp function was elevated in both pT7TS_MDR1 and pGEM5Zf(+)_MDR1 groups. The survival ratio of MNCs in erythrocyte-lysis-solution (ELS, 86.07%) and lymphocyte-isolation-solution (LIS, 83.67%) had no significant difference. The CD34+ cells content of the MNCs used for transfection was 2.65% and 1.01% in ELS and LIS group, respectively (P < 0.01).Conclusions It is a feasible approach to improve P-gp expression in human MNCs by transfection of MDR-1 mRNA. And the ELS may be more suitable for purifing MNCs for mRNA transfection than LIS.

Crohn's disease genotypes of patients in remission vs relapses after infliximab discontinuation

AIM： To investigate genetic differences between Crohn＇s disease （CD） patients with a sustained remission vs relapsers after discontinuing infliximab while in cortico- steroid-free remission. METHODS： Forty-eight CD patients received infliximab and were in full corticosteroid-free clinical remission but then discontinued infliximab for reasons other than a loss of response, were identified by review of an electronic database and charts. Infliximab-associated remis- sion was defined as corticosteroid-free plus normaliza- tion of clinical disease activity [CD activity index （CDAI） 〈 150] during follow-up visits based on physician global assessments. A CD relapse （loss of infliximab-induced remission） was clinically defined as a physician visit for symptoms of disease activity （CDAI 〉 220） and a thera- peutic intervention with CD medication（s）, or a hospital- ization with complications related to active CD. Genetic analyses were performed on samples from 14 patients （n = 6 who had a sustained long term remission after stopping infliximab, n -- 8 who rapidly relapsed after stopping infliximab）. Nucleotide-binding oligomerization domain 2 （NOD2）/caspase activation recruitment do- main 15 （CARD15） polymorphisms （R702W, G908R and L1007fs） and the inflammatory bowel disease 5 （IBDS） polymorphisms （IGR2060a1 and IGR3081a1） were ana- lyzed in each group. RESULTS： Five single nucleotide polymorphisms of IBD5 and NOD2/CARD15 genes were successfully analyzed for all 14 subjects. There was no signifcant increase in frequency of the NOD2/CARD15 polymor- phisms （R702W, G908R and L1007fs） and the IBD5 polymorphisms （IGR2060al and IGR3081a1） in either group of patients; those whose disease relapsed rap- idly or those who remained in sustained long term remission following the discontinuation of infliximab. Nearly a third of patients in full clinical remission who stopped infliximab for reasons other than loss of re- sponse remained in sustained clinical remission, while two-thirds relapsed rapidly. There was a marked dif- ference in the duration of clinical remission following discontinuance of infliximab between the two groups. The patients who lost remission did so after 1.0 years 4- 0.6 years, while those still in remission were at the time of this study, 8.1 years 4- 2.6 years post-discon- tinuation of infliximab, P 〈 0.001. The 8 patients who had lost remission after discontinuing infiiximab had a mean number of 5 infusions （range 3-7）, with a mean treatment time of 7.2 mo （range 1.5 mo-15 mo）. The mean duration of time from the last infusion of inflix- imab to the time of loss of remission was 382 d （range 20 d-701 d）. The 6 patients who remained in remission after discontinuing infliximab had a mean number of 6 infusions （range 3-12）, with a mean treatment dura- tion of 12 mo （range 3.6 mo-32 too） （P = 0.45 relative to those who lost remission）. CONCLUSION： There are no IBD5 or NOD2/CARD15 mutations that predict which patients might have sus- tained remission and which will relapse rapidly after stopping infliximab.

polymorphism Analysis of Resistance Genes in Chinese Populations with HIV-1 Infection

Objective: To analyze the genotypes of CCR5 △ 32,CCR2b-64I and SDF 1-3 A and mutation frequencies of allelicgenes in Chinese populations infected with HIV-1. Methods: Genome DNA from peripheral blood mononuclearcells (PBMCs) of 78 HIV-1 infectors was amplified bypolymerase chain reaction (PCR). CCR5, CCR2b and SDF1gene fragments were obtained from restrictive fragmentlength polymorphism (RFLP) and/or CCR△32, CCR5m303,CCR2b-64I and SDF1-3' A allelic genes' mutationalfrequencies were sequenced directly from PCR products. Results: None of CCR5△32, CCR5m303 gene mutationwere found in 78 subjects with HIV-1 infection. The allelicgene mutation frequencies of CCR2b-64I and SDF1-3'Acorresponding to 14.9-34.0% and 17.6-38.2% of 95% CI, were22.79% and 26.92% respectively. Their colony distributionconformed to the Hardy-Weinberg equilibrium. Conclusion: The HIV-1 infections found at present are allsusceptible population of CCR5△32 and CCR5m303. Thepolymorphism and frequencies of CCR5△32, CCR5m303,CCR2b-64I and SDF1-3'A alleles from Chinese HIV-1infected population were disclosed in this study for the firsttime, which is of significance for studying the geneticresistance to susceptibility to HIV-1 infection as well as AIDSdisease progression.

Gene polymorphism in IL-1 receptor antagonist affects its production by monocytes in IgA nephropathy and Henoch-Schonlein nephritis

Objective To define the functional significance of IL-1 receptor antagonist (IL-1ra) gene polymorphism and to investigate, the production of IL-1ra by monocytes from individuals with different genotypes of IL-1.Methods The genotype of IL-1ra was detected by polymerase chain reaction (PCR). Peripheral monocytes obtained from patients with immunoglobin A nephropathy (IgAN), Henoch-Schonlein purpura nephritis (HSPN) and normal subjects were matched in sex and age between the IL1RN-2 allele carriers and non-carriers. The secretion of IL-1ra, IL-1α and IL-1β in the supernatant of GM-CSF (10ng/ml) treated and untreated monocytes were measured by ELISA.Results The secretion of IL-1ra by monocytes stimulated with GM-CSF was significantly higher in the IL1RN-2 allele non-carriers than those of carriers both in IgAN (21.55±3.08 vs 13.85±2.24ng/ml, P<0.001) and HSPN (23.72±6.68 vs 12.67±2.24ng/ml, P<0.01) as well as in normal controls (20.29±1.45 vs 10.51±2.3ng/ml, P<0.001). All showed no significant differences in monocyte secretion of IL-1α and IL-1β by GM-CSF stimulation between the IL1RN-2 allele carriers and non-carriers. Conclusions These results indicate that a functional correlation of the IL1RN-2 allele and IL-1ra production is present in patients with IgAN and HSPN. This gene polymorphism control of IL-1ra production may contribute to the variety of clinical responses to inflammatory stimulation in individuals with different genotype of IL-1ra.

Genomic insights into the ESBL and MCR-l-producing ST648 Escherichia coli with multi-drug resistance

Polymyxin acts as an ultimate line of refuge against the severe infections by multidrug-resistant Gram- negative pathogens. This conventional idea is challenged dramatically by the recent discovery of mobile colistin resistance gene （mcr-1） is prevalent in food animals and human beings worldwide. More importantly, the mcr-1 gene was found to be co-localized with other antibiotic resistance genes, raising the possibility that super-bugs with pan-drug resistance are emerging. However, little is reported on the genomes of the mcr-l-positive bacterial host reservoirs. Here we report genome sequencing of three human isolates of the mcr-l-positive Escherichia coli （E15004, E15015 and E15017） and define general features through analyses of bacterial comparative genomics. Fur- ther genomic mining together with sequence typing allowed us to elucidate that the MCR-l-carrying E. coli E15017 belongs to the sequence type ST648 and copro- duces extended-spectrum β-1actamase （ESBL）. Given the fact that ST648 has been known to associate New Delhi metallo-β-1actamase 1 or ESBL, with either our results highlighted the possibility of ST648 as an epidemic clone with multidrug resistances.