家蝇抗药性相关乙酰胆碱酯酶基因突变的进化起源分析

家蝇是多种人与动物疾病的传播媒介，化学防治是家蝇防治的重要手段之一，但杀虫剂抗性是家蝇化学防治的一大障碍。以往的研究表明，乙酰胆碱酯酶(由Ace基因编码)的某些点突变与家蝇对有机磷和氨基甲酸酯类杀虫剂抗性相关。在我国野外家蝇群中发现了可以导致抗药性的乙酰胆碱酯酶V260L、G342A、G342V和F407Y氨基酸置换，但对其进化起源模式尚缺乏认识。本研究采用基因片段测序的方法，获得了来自北京、山东、上海、广东4个省份的728头家蝇的Ace基因片段序列，从中鉴定出9种不同的Ace单倍型。分子系统树和网络图分析发现了V260L、G342A和F407Y突变的多起源性，4种三位点突变(L-A-Y)的单倍型源自两条不同的进化途径。     

用mRNA RT-PCR差异显示技术克隆拟除虫菊酯抗性家蝇细胞色素P450基因片段

细胞色素P450单加氧酶系是一类广泛分布在生物有机体中的重要酶系,具有多样的功能。细胞色素P450介导的杀虫剂代谢解毒作用的增强是昆虫对杀虫剂产生抗性的普遍机制。为揭示家蝇对拟除虫菊酯类杀虫剂的分子基础,我们利用mRNA差异显示技术(DDRTPCR)从溴氰菊酯抗性品系家蝇Muscadomestica中分离出2个新的基因片段(M1,M2)。序列比对(BlastP)结果表明,M1与黑腹果蝇Drosophilamelanogaster中的Cyp6A9在氨基酸水平上具有54%的相似性,M2分别与果蝇Drosophilasimulans中的Cyp6g1有65%的相似性。M1和M2包含P450的特征序列,并与细胞色素P450具有最高程度的序列相似性,是为细胞色素P450基因片段。     

贵阳市家蝇对拟除虫菊酯类杀虫剂的抗性现状与kdr(L1014F)等位基因频率分析

探讨贵阳市区家蝇对拟除虫菊酯类杀虫剂抗药性水平与kdr等位基因上的LI014F点突变频率相关性,为贵阳市科学合理使用卫生杀虫剂控制家蝇提供科学依据.本研究采用WHO推荐的微量点滴法进行抗性测定.结果显示贵阳市4城区家蝇对氯菊酯和高效氯氰菊酯的LD50分别为0.00620 ～ 0.013339μg/雌蝇、0.02298 ～0.03614 μg/雌蝇,抗性系数分别为3.35 ～7.56、7.38 ～11.62.采用特异性等位基因PCR法测定基因频率,结果显示4城区家蝇种群的L1014F点突变频率在29％ ～37％.相关性分析显示L1014F点突变等位基因频率与高效氯氰菊酯LD50值之间存在直线相关.研究结果表明贵阳市家蝇对氯菊酯、高效氯氰菊酯已经产生了抗药性,击倒抗性是其抗性机理之一.     

家蝇抗药性的分子机制：乙酰胆碱酯酶介导的抗药性

乙酰胆碱酯酶（AchE）是有机磷和氨基甲酸酯杀虫剂的主要作用靶标,该酶氨基酸残基的替换可以降低AchE对这两类杀虫剂的敏感性,从而使昆虫表现出对这两类杀虫剂的抗性。至今在抗性家蝇AchE中鉴定出多个不同位点的点突变,以G262A／V和F327Y突变最为普遍。262和327两位点上的氨基酸残基靠近AchE的活性位点三联体,G262A／V和F327Y替换可以通过改变起催化作用的丝氨酸的定位或通过降低酰基．结合袋的空间来改变酶的活性,在抗性进化过程中起着重要的作用。家蝇AchE点突变的效用是累加,多突变组合往往导致更高水平且更广谱的抗性。从不同抗性家蝇种群中检测出相同的AchE遗传突变,表明这一抗性进化机制的保守性;多种抗性等位基因共存于家蝇种群中,反映了杀虫剂使用的多重性。家蝇乙酰胆碱酯酶抗性等位基因种类分布因地区而不同,但也有重叠。家蝇种群表现出抗性等位基因的演替,推测是杀虫药剂的轮替使用的结果。     

寡核苷酸探针法(PCR-ASO)检测家蝇kdr抗性相关的L1014F点突变

为检测家蝇对拟除虫菊酯击倒抗性kdr等位基因上的L1014F点突变,采用了寡核苷酸探针法(PCR-AlleleSpecificOligonucleotideprobe,PCR-ASO),用于检测该点突变。对L1014位的上下游DNA进行PCR扩增,并将纯化产物平行点样在带正电荷的尼龙膜上,用2条地高辛标记的特异性寡核苷酸探针,1条为敏感型,1条为突变型,与之杂交,根据显影的阳性信号判断待检样品的kdr等位基因型。该方法能区分敏感纯合子、抗性纯合子及杂合子,是检测家蝇kdr抗性的有用工具。     

家蝇对氯菊酯抗性的分子机制研究

家蝇是一种重要的病媒生物,可以传播上百种人畜疾病。化学防治是家蝇控制的重要手段,但杀虫剂的重复和广泛使用可导致家蝇产生抗药性。在我国,不少事例显示家蝇对拟除虫菊酯杀虫剂普遍产生了抗药性,但对家蝇种群抗药性的分子机制还缺乏系统了解。本研究以来源于山东济南的家蝇为材料,在明确其对氯菊酯抗性水平的基础上,分析其抗药性的分子机制。结果显示,野外采集未经杀虫剂汰选实验室济南品系(JN)相对于敏感品系(WHO)表现出对氯菊酯近50倍的抗性,而经氯菊酯汰选7代后的JN-Sel品系获得了比JN更高水平的抗性,抗性倍数超过110倍。核酸检测显示JN和JN-Sel品系家蝇钠离子通道的基因型为1014LL敏感纯合型。通过检测8个细胞色素P450基因的表达水平,发现3个P450基因(CYP6A5v2、CYP6A36和CYP6G4)在抗性品系中的表达量高于敏感品系,其中CYP6G4的表达与氯菊酯抗性成正相关,JN品系CYP6G4的表达量是敏感品系的35倍,而在高抗性的JN-Sel品系中的表达量是敏感品系的70倍。研究结果强烈提示CYP6G4的过量表达是JN和JN-Sel品系对氯菊酯抗性的重要机制。     

家蝇二氯苯醚菊酯抗性机理的初步研究

为了研究家蝇拟除虫菊酯抗性机理,在实验室选育了家蝇二氯苯醚菊酯抗性品系,根据P450基因CYP6D1的保守序列设计合成引物,用PCR方法从敏感品系和抗性品系个体都能扩增出一条约210bp的特异性片段,从片段大小和条带明亮程度上均未显示出二者之间有什么差异,初步表明抗性家蝇P450基因在DNA水平上没有明显的变化。RT-PCR结果多次重复显示抗性品系的扩增带明显比敏感品系的亮,揭示二者RNA的含量可能不同,抗性品系P450基因的转录活性可能升高。这些为在分子水平研究P450基因相关的拟除虫菊酯抗性机理打下基础。     

纯合子Cys329Gly导致的遗传性凝血因子Ⅶ缺陷症家系基因突变分析

目的对1个姨表近亲婚配的遗传性凝血因子Ⅶ(FⅦ)缺陷症家系进行表型和F7基因突变分析,探讨其分子发病机制。方法检测先证者及其家系成员(共4代9人)凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)、血浆FⅦ活性(FⅦ:C)等来明确诊断。PCR扩增先证者全部外显子及其侧翼序列、5′和3′非翻译区及家系成员相应的突变位点区域,PCR产物纯化后直接测序,寻找突变位点,以反向测序验证所发生的突变;使用生物信息学软件(Poly Phen-2和Mutation Taster)预测突变位点对蛋白质功能的影响,利用Py MOL软件构建正常FⅦ蛋白空间模型,进行定点突变观察构型改变。结果先证者PT(36.1s)和FⅦ:C(2%)明显异常,家系中其余8位成员PT均略高于正常对照组和FⅦ:C均略低于正常对照组;基因分析显示先证者F7基因第8外显子存在c.1165 TG纯合型突变,即TGT→GGT(Cys329Gly),8位家系成员的F7基因分析均显示c.1165有杂合型突变;两种生物信息学软件都提示此突变会引起蛋白功能变化,有致病性;F7基因蛋白构型显示突变后329位点与310位点之间二硫键消失,周边静电磁场发生改变。结论该家系F7基因存在Cys329Gly突变,是引起FⅦ缺陷症的主要分子机制,推测先证者纯合Cys329Gly突变基因分别遗传自近亲结婚的杂合子父母。     

黄体生成素β Gly102Ser基因多态性与多囊卵巢综合征的相关性研究

目的探讨黄体生成素β(Luteinizing hormoneβ-subunit,LHβ)Gly102Ser及黄体生成素受体(Luteinizing hormone receptor,LHR)基因多态性与PCOS的相关性。方法应用聚合酶链反应-限制性片段长度多态性(Polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)检测LHβGly102Ser多态性,分析LHβ基因多态性与多囊卵巢综合征(polycystic ovary syndrome,PCOS)的相关性。结果 PCOS组中LHβGly102Ser基因有14例突变型,占3.3%。对照组中3例突变,占1.1%;PCOS组LHβGly102Ser基因突变率显著高于对照组(P=0.006);LHβGly102Ser基因突变型的血清基础LH水平低于对照组(P0.05),但突变组有降低趋势;PCOS组内血清LH2mmol/L的患者,LHβ基因Gly102Ser发生突变的比例显著增加(P=0.003)。结论 PCOS人群中存在LHGly102Ser基因多态性,此多态性与PCOS有明显的相关性。LHβGly102Ser基因多态性可能是PCOS发病的危险因素之一。     

自然种群淡色库蚊钠通道基因全长的克隆及序列分析

本研究采用反转录多聚酶链式反应（ RT-PCR）的方法对野外采集的河北种群淡色库蚊的钠离子通道基因进行了克隆和序列分析。得到的克隆序列与GenBank中公布的序列能够吻合,说明成功克隆出淡色库蚊的钠通道基因全长,为抗性突变的研究奠定了基础。本研究在淡色库蚊钠通道基因上发现了多处氨基酸突变（A32T、 T336A、 S342P、 K535E、 L1035F、 N1595S和F1982L）和7处可变剪接。其中,包括经典的抗性突变L1014F （ L1035F）。但这些突变和可变剪接与蚊虫抗性的关系有待进一步研究。     

家蝇抗性相关P450基因的初步研究

为了在分子水平上研究拟除虫菊酯抗性家蝇的 P4 50基因的特性 ,更好地研究其与抗性相关的机制 ,本研究根据在美洲地区的抗性家蝇品系 ( LPR)克隆的 P4 50基因 MDC YP6D1的保守序列设计合成引物 ,用 PCR方法从北京地区的家蝇中也扩增出 1条约 2 1 0 bp的特异性片段 ,片段长度与原序列对应片段相近 ,说明北京地区的家蝇 P4 50基因与 MDCYP6D1间有很大的同源性。H inf I酶切发现扩增产物上并没有相应的酶切位点 ,说明扩增产物与原序列 MDCYP6D1的相应部分不一样 ,提示这一用 PCR方法在北京地区的家蝇中扩增出的 P4 50基因片段有可能对应一新的 P4 50基因。 PCR-SSCP结果显示扩增产物在一级结构上有一定的多态性 ,这一现象可能与 P4 50基因在昆虫体内的复杂多样性有关     

Insecticide resistance of Dengue vectors in South East Asia: a systematic review

BackgroundThe insecticides used widely has led to resistance in the vector and impose a challenge to vector control operation.ObjectivesThis review aims to analyse the distribution of insecticide resistance of dengue vectors in South East Asia and to describe the mechanism of insecticide resistance.MethodsLiterature search for articles published on 2015 to 2019 from PubMed, Scopus and ProQuest was performed. Total of 37 studies included in the final review from the initial 420 studies.ResultsPyrethroid resistance was concentrated on the west coast of Peninsular Malaysia and Northern Thailand and scattered at Java Island, Indonesia while organophosphate resistance was seen across the Java Island (Indonesia), West Sumatera and North Peninsular Malaysia. Organochlorine resistance was seen in Sabah, Malaysia and scattered distribution in Nusa Tenggara, Indonesia. V1016G, S989P, F1269C gene mutation in Aedes Aegypti were associated with Pyrethroid resistance in Singapore and Indonesia. In Malaysia, over-expressed with monooxygenase P450 genes (CYP9J27, CYP6CB1, CYP9J26 and CYP9M4) Glutathione S-transferases, carboxylesterases commonly associated with pyrethroids resistance in Aedes Aegypti and CYP612 overexpressed in Aedes Albopictus. The genetic mutation in A302S in Aedes Albopictus was associated with organochlorine resistance in Malaysia.ConclusionsRotation of insecticide, integration with synergist and routine assessment of resistance profile are recommended strategies in insecticide resistance management.     

Multiple NADPH-cytochrome P450 reductases from Trypanosoma cruzi suggested role on drug resistance

Cytochrome P450 hemoproteins (CYPs) are involved in the synthesis of endogenous compounds such as steroids, fatty acids and prostaglandins as well as in the activation and detoxification of foreign compounds including therapeutic drugs. Cytochrome P450 reductase (CPR, E.C.1.6.2.4) transfers electrons from NADPH to a number of hemoproteins such as CYPs, cytochrome c, cytochrome b5, and heme oxygenase. This work presents the complete sequences of three non-allelic CPR genes from Trypanosoma cruzi. The encoded proteins named TcCPR-A, TcCPR-B and TcCPR-C have calculated molecular masses of 68.6kDa, 78.4kDa and 71.3kDa, respectively. Deduced amino acid sequences share 11% amino acid identity, possess the conserved binding domains for FMN, FAD and NADPH and differ in the hydrophobic 27-amino acid residues of the N-terminal extension, which is absent in TcCPR-A. Every T. cruzi CPRs, TcCPR-A, TcCPR-B and TcCPR-C, were cloned and expressed in Escherichia coli. All of the recombinant enzymes reduced cytochrome c in a NADPH absolutely dependent manner with low K(m) values for this cofactor. They all were also strongly inhibited by diphenyleneiodonium, a classical flavoenzyme inhibitor. In addition, TcCPRs could support CYP activities when assayed in reconstituted systems containing rat liver microsomes. Polyclonal antiserum rose against the recombinant enzymes TcCPR-A and TcCPR-B demonstrated its presence in every T. cruzi developmental stages, with a remarkable expression of TcCPR-A in cell-cultured trypomastigotes. Overexpression of TcCPR-B in T. cruzi epimastigotes increased its resistance to the typical chemotherapeutic agents Nifurtimox and Benznidazole. We suggest a participation of TcCPR-B in the detoxification metabolism of the parasite.     

Antimicrobial agent resistance in mycobacteria: molecular genetic insights.

The primary theme emerging from molecular genetic work conducted with Mycobacterium tuberculosis and several other mycobacterial species is that resistance is commonly associated with simple nucleotide alterations in target chromosomal genes rather than with acquisition of new genetic elements encoding antibiotic-altering enzymes. Mutations in an 81-bp region of the gene (rpoB) encoding the beta subunit of RNA polymerase account for rifampin resistance in 96% of M. tuberculosis and many Mycobacterium leprae isolates. Streptomycin resistance in about one-half of M. tuberculosis isolates is associated with missense mutations in the rpsL gene coding for ribosomal protein S12 or nucleotide substitutions in the 16S rRNA gene (rrs). Mutations in the katG gene resulting in catalase-peroxidase amino acid alterations nad nucleotide substitutions in the presumed regulatory region of the inhA locus are repeatedly associated with isoniazid-resistant M. tuberculosis isolates. A majority of fluoroquinolone-resistant M. tuberculosis isolates have amino acid substitutions in a region of the DNA gyrase A subunit homologous to a conserved fluoroquinolone resistance-determining region. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. Molecular strategies show considerable promise for rapid detection of mutations associated with antimicrobial resistance. These approaches are now amenable to utilization in an appropriately equipped clinical microbiology laboratory.     

Toll‐like receptor polymorphism in host immune response to infectious diseases: A review

Immunopolymorphism is considered as an important aspect behind the resistance or susceptibility of the host to an infectious disease. Over the years, researchers have explored many genetic factors for their role in immune surveillance against infectious diseases. Polymorphic characters in the gene encoding Toll‐like receptors (TLRs) play profound roles in inducing differential immune responses by the host against parasitic infections. Protein(s) encoded by TLR gene(s) are immensely important due to their ability of recognizing different types of pathogen associated molecular patterns (PAMPs). This study reviews the polymorphic residues present in the nucleotide or in the amino acid sequence of TLRs and their influence on alteration of inflammatory signalling pathways promoting either susceptibility or resistance to major infectious diseases, including tuberculosis, leishmaniasis, malaria and filariasis. Population‐based studies exploring TLR polymorphisms in humans are primarily emphasized to discuss the association of the polymorphic residues with the occurrence and epidemiology of the mentioned infectious diseases. Principal polymorphic residues in TLRs influencing immunity to infection are mostly single nucleotide polymorphisms (SNPs). I602S (TLR1), R677W (TLR2), P554S (TLR3), D299G (TLR4), F616L (TLR5), S249P (TLR6), Q11L (TLR7), M1V (TLR8), G1174A (TLR9) and G1031T (TLR10) are presented as the major influential SNPs in shaping immunity to pathogenic infections. The contribution of these SNPs in the structure‐function relationship of TLRs is yet not clear. Therefore, molecular studies on such polymorphisms can improve our understanding on the genetic basis of the immune response and pave the way for therapeutic intervention in a more feasible way.     

Analysis of Pneumocystis jirovecii DHPS alleles implicated in sulfamethoxazole resistance using an Escherichia coli model system

Pneumocystis jirovecii is a major opportunistic pathogen that causes Pneumocystis pneumonia (PCP). Drug treatment failure has been associated epidemiologically with point mutations in the gene for dihydropteroate synthase which is part of a gene that encodes three covalently linked enzymes involved in folic acid synthesis (FAS). The evaluation of whether mutations found in P. jirovecii FAS lead to sulfa drug resistance is hampered by the lack of a culture system for P. jirovecii as well as the failure of P. jirovecii FAS to complement in a heterologous system. Therefore, we chose to model the P. jirovecii mutations in the Saccharomyces cerevisiae FAS protein (encoded by FOL1) via its expression in Escherichia coli. An optimized drug diffusion assay was used to evaluate the FAS mutants against 15 sulfa drugs. It was established that the single amino acid substitution, P599S, in the (DHPS) domain of FAS led to sulfa drug resistance, whereas the T597A substitution led to increased sensitivity. The presence of both mutations (T597A and P599S) was cooperative and led to increased sulfa drug resistance. Analysis of a novel double mutant, (T597V P599S) was found to have significantly higher sulfa drug resistance than the T597A P599S mutant. These data suggest that further amino acid substitutions may lead to the evolution of higher sulfa drug resistance. Two sulfa drugs (sulfachloropyridazine and sulfathiazole) were identified that had higher inhibitory potential than sulfamethoxazole, which is currently the preferred treatment for PCP.