Identification and characterization of doublesex in Bemisia tabaci

Bemisia tabaci (Gennadius) is an important agricultural pest with a worldwide distribution. Although B. tabaci is known to have a unique haplodiploid reproductive strategy, its sex determination mechanism is largely unknown. In this study, we cloned the full‐length sequence of B. tabaci doublesex (Btdsx) and found that Btdsx has 28 splicing isoforms. We found two new splicing isoforms of transformer 2 (Bttra2), which encode two proteins. We also confirmed that both genes lack sex‐specific splicing isoforms. Real‐time quantitative PCR analysis showed that the expression of Btdsx and Bttra2 is higher in males than in females. RNA interference of Bttra2 affected the expression of Btdsx and vice versa. Furthermore, silencing of Bttra2 or Btdsx caused malformation of the male genitalia (anal style). It did not affect the female phenotype, but reduced the expression of vitellogenin gene in females. These results indicate that Btdsx is associated with sex determination in B. tabaci and that Btdsx and Bttra2 affect each other and are important for male genitalia formation. In addition to increasing our understanding of the roles of dsx and tra2 in the sex determination of B. tabaci, the results will be useful for studies of sex determination in other haplodiploid species.     

Molecular characterization of N-methyl-d-aspartate receptor from Bemisia tabaci

The N-methyl-d-aspartate receptors (NMDARs) are ionotropic ligand gated channels that are highly permeable to calcium ions. In insects, NMDARs are associated with glutamatergic neurotransmission governing diverse physiological and biological processes like vitellogenesis and ovarian development. Therefore, NMDAR may act as attractive target for insect pest control. In present study, we performed structural and functional characterization of NMDARs in Bemisia tabaci, a highly invasive crop pest and potent virus vector. We identified that NMDAR consists of three subunits each encoded by single gene in whiteflies which are highly conserved among different insect orders. Expression analysis suggests that subunit 1 (BtNR1) and subunit 2 (BtNR2) are the main functional units. External supplementation of NMDAR ligand or BtNRs silencing was lethal to insects, which suggested that NMDAR function is highly balanced in whiteflies.     

Molecular characterizations of DNA methyltransferase 3 and its roles in temperature tolerance in the whitefly,Bemisia tabaci Mediterranean

The Bemisia tabaci Mediterranean (MED) cryptic species is an invasive pest, distributed worldwide, with high ecological adaptability and thermotolerance. DNA methylation (a reversible chromatin modification) is one possible change that may occur within an organism subjected to environmental stress. To assess the effects of temperature stress on DNA methyltransferase 3 (Dnmt3) in MED, we cloned and sequenced BtDnmt3 and identified its functions in response to high and low temperatures. The full‐length cDNA of BtDnmt3 was 3913 bp, with an open reading frame of 1962 bp, encoding a 73.89 kDa protein. In situ hybridization showed that BtDnmt3 was expressed mainly in the posterior region. BtDnmt3 messenger RNA expression levels were significantly down‐regulated after exposure to heat shock and significantly up‐regulated after exposure to cold shock. Furthermore, after feeding on double‐stranded RNA specific for BtDnmt3, both heat resistance and cold resistance were significantly decreased, suggesting that BtDnmt3 is associated with thermal stress response and indicating a differential response to high‐ and low‐temperature stress in MED. Together, these results highlight a potential role for DNA methylation in thermal resistance, which is a process important to successful invasion and colonization of an alien species in various environments.     

Use of the synergist piperonyl butoxide can slow the development of alpha‐cypermethrin resistance in the whitefly Bemisia tabaci

The development of insecticide resistance in insect pests of crops is a growing threat to sustainable food production, and strategies that slow the development of resistance are therefore urgently required. The insecticide synergist piperonyl butoxide (PBO) inhibits certain insect detoxification systems and so may delay the evolution of metabolic resistance. In the current study we characterized resistance development in the silverleaf whitefly, Bemisia tabaci, after selection with either a neonicotinoid (thiacloprid) or pyrethroid (alpha‐cypermethrin) insecticide alone or in combination with PBO. Resistance development was significantly suppressed (> 60%) in the line selected with alpha‐cypermethrin + PBO compared to the line selected with alpha‐cypermethrin alone. RNA sequencing (RNAseq) analyses revealed an increase in frequency of a knock‐down resistance mutation but no differentially expressed genes were identified that could explain the sensitivity shift. No significant difference was observed in the level of resistance between the thiacloprid and thiacloprid + PBO selected lines, and RNA sequencing (RNAseq) analyses revealed that the cytochrome P450 monooxygenase CYP6CM1, known to metabolize neonicotinoids, was significantly upregulated (>10‐fold) in both lines. The findings of this study demonstrate that PBO used in combination with certain insecticides can suppress the development of resistance in a laboratory setting; however, the mechanism by which PBO supresses resistance development remains unclear.     

Identification,characterization and expression of a defensin‐like antifungal peptide from the whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)

Defensins are a class of small and diverse cysteine‐rich proteins which have broad‐spectrum antimicrobial activities. We identified and characterized a full‐length cDNA encoding a putative defensin‐like peptide from the whitefly Bemisia tabaci by RACE and quantitative real‐time (qRT)‐PCR. The full‐length cDNA, named Btdef, was 388 bp long and contained an open reading frame of 228 bp. The putative mature Btdef had 46 amino acids with a molecular weight of 5.06 kDa. The deduced amino acid sequence showed significant homology with insect defensins from Heliothis virescens (76%) and Galleria mellonella (75%). The predicted mature form of Btdef was expressed as a recombinant peptide in Escherichia coli. Antimicrobial assays of the purified product indicated that Btdef was most active against fungi. qRT‐PCR analyses indicated that Btdef mRNA was constitutively expressed in different tissues of B. tabaci, including fat body, midgut, ovaries and salivary gland, and was induced by fungal infection. Btdef mRNA expression was also significantly altered after feeding on different host plants, indicating that diet affects immune defences in B. tabaci. These results describe for the first time the basic properties of a defensin‐like peptide from B. tabaci that probably plays an important role in the immune response against pathogens.     

Protective functions of intracellular heat-shock protein (HSP) 70-expression in patients with severe sepsis

Intracellular expression of heat-shock-protein 70 (HSP70) arose early in evolutionary development as a tool to protect cellular homeostasis. HSP70 detects proteins that are incorrectly folded or denatured. They form a complex with such proteins which can lead to correct folding, compartmentalization in organelles, or to proteolytic degradation. HSP70 also appears to protect proteins from degeneration. Intracellular HSP70-expression is induced by a wide variety of stimuli including heat, fever, hypoxia, oxygen radicals, endotoxins, cytokines, and heavy metal ions. Pre-emptive induction of HSP70-expression reduces organ dysfunction and mortality in animal models of sepsis.     

Characterization of acetylcholinesterases, and their genes, from the hemipteran species Myzus persicae (Sulzer), Aphis gossypii (Glover), Bemisia tabaci (Gennadius) and Trialeurodes vaporariorum (Westwood)

Gene sequences encoding putative acetylcholinesterases have been reported for four hemipteran insect species. Although acetylcholinesterase insensitivity occurs in insecticide-resistant populations of each of these species, no mutations were detected in the gene sequences from the resistant insects. This, coupled with a series of experiments using novel reversible inhibitors to compare the biochemical characteristics of acetylcholinesterase from a range of insect species, showed that the cloned cDNA fragments are unlikely to encode the hemipteran synaptic acetylcholinesterases, and there is likely to be a second ace locus.     

Transovarial transmission of sugarcane white leaf phytoplasma in the insect vector Matsumuratettix hiroglyphicus (Matsumura)

White leaf is a serious disease of sugarcane caused by phytoplasma. The disease is transmitted to the plant by the leafhopper Matsumuratettix hiroglyphicus (Matsumura). The reservoir of phytoplasma was suspected to be weeds that grow in sugarcane farming areas because they can be infected with phytoplasma and show symptoms similar to sugarcane white leaf. However in previous work we have demonstrated by RFLP and sequencing that this is not the case. Here we have reared M. hiroglyphicus through two generations by feeding them phytoplasma free sugarcane grown from tissue culture. By nested-PCR followed by sequencing, we demonstrated the presence of the phytoplasma in eggs, nymphs and adults of the first and second generations thereby showing transovarial transmission. We have also shown by in situ PCR that phytoplasmas were widely distributed throughout the body of the insect. RFLP and sequencing showed that the same phytoplasma was present in the vector and in the plant. Together, these data point to the leafhopper M. hiroglyphicus as the reservoir of phytoplasma that cause sugarcane white leaf disease.     

人β防御素-2基因的克隆及原核表达载体的构建

目的构建人β-防御素2的融合性原核表达载体，为今后基因工程手段表达纯化及生产人β-防御素-2奠定基础。方法体外基因合成人β-防御素2（HBD-2）的基因片段，并克隆人pMD18T中间载体中，经酶切鉴定后再克隆人pET32a原核表达载体中，与载体中所固有的TrX—A形成融合基因。结果构建的融合性原核表达载体经酶切鉴定无突变发生。结论成功地构建了pET-32a-（HBD-2）。     

肺表面活性蛋白质A的提取纯化和初步应用

为了进一步研究肺表面活性物质（PS）的生理功能和今后建立肺表面活性蛋白质A（SPA）含量测定，本研究用正丁醇和八－辛－吡喃葡萄糖酐离心法在水中提取和纯化了SPA并免疫兔制备了兔抗人抗体，所得SPA经免疫电泳及蛋白印迹分析纯度都较佳,鉴于SPA在国外已用于抗原测定及在合成磷脂中加入SPA可提高对新生儿呼吸窘迫症和成人呼吸窘迫症的疗效，本研究今后建立SPA含量测定，用于临床诊断和治疗奠定了基础。     

免疫透射比浊法多点非线性校准在脂蛋白(a)测定中的应用

目的探讨免疫透射比浊法测定脂蛋白(a)中,采用2种多点非线性校准方法的测定结果,并确定最合适的校准曲线。方法分别采用Y=aX~2+bX+c和Spline2种方法在AU680生化仪上进行脂蛋白(a)校准。分别检测200例来自临床患者的血清标本和4个标准定值血清,200例临床患者血清按照测定值高低分为5个不同梯度水平,分别统计2组数据的均值(x)、标准差(s),相关系数(r),相对误差(v),偏倚(%)。采用SPSS19.0软件对2组数据偏倚进行统计学分析,以P0.05表示差异有统计学意义。结果 200例临床患者数据表明,采用Y=aX~2+bX+c曲线时,0~100、400~600、600~800mg/L组的脂蛋白(a)测定值比Spline曲线所得数值高;100~200mg/L组和200~400mg/L组的脂蛋白(a)测定值相反,Spline曲线所得数值较高。4个标准定值血清的测定结果,Spline曲线所得测定值更接近靶值;而Y=aX~2+bX+c曲线所测定结果中,100~200mg/L组和300~400mg/L组所得值偏低,600~800mg/L组所得值偏高,这与临床患者的血清标本结果一致。结论 Spline曲线更为客观,能更准确地体现免疫透射比浊法测定脂蛋白(a)整个反应过程。     

糖尿病合并脑卒中患者脂蛋白(a)测定及临床分析

目的探讨2型糖尿病合并脑卒中患者脂蛋白(a)水平的变化特点及其意义。方法选择2型糖尿病合并脑卒中患者75例为观察组(58例脑梗死,17例脑出血),单纯2型糖尿病患者35例为对照组,采用免疫透射比浊法测定两组患者的血浆脂蛋白(a)水平,观察其脂蛋白(a)水平及其差异。结果与对照组比较,观察组脂蛋白(a)水平差异有高度统计学意义(P<0.01)。结论测定糖尿病合并脑卒中患者脂蛋白(a)水平对鉴别诊断及危险度分层具有重要的临床意义。     

Characterization of a serum protein with selective affinity for denatured collagen (author's transl)

A protein known as antigelatin factor (AGF) was isolated from human serum by affinity chromatography with immobilized denatured collagen. In biochemical and immunological assays AGF showed specificity to denatured, but not to native collagen of the types I, II and III. A close relationship to Cell Attachment Protein and Cold Insoluble Globulin was found in comparative studies.     

Three different LDL apheresis columns efficiently and equally reduce lipoprotein(a) concentrations in patients with familial hypercholesterolemia and small apolipoprotein(a) particles

Introduction

High levels of lipoprotein(a) [Lp(a)] and apolipoprotein(a) [apo(a)] are associated with cardiovascular disease. In this study we determined apo(a) particle size and compared the Lp(a) reducing efficacy of three different LDL apheresis columns; DL-75, LA-15 and EC-50W in patients with familial hypercholesterolemia (FH).

Results

Average Lp(a) concentration was reduced by 70%, 74% and 75% (all p < 0.0001) for DL-75, LA-15 and EC-50W, respectively. No significant changes in the relative proportion of the isoforms of 14 and 32 K 4 domains were observed after apheresis.

Conclusion

Three different LDL apheresis columns reduced Lp(a) efficiently with preserved ratio between apo(a) isoforms.     

Middle region of FancM interacts with Mhf and Rmi1 in silkworms,a species lacking the Fanconi anaemia (FA) core complex

The Fanconi anaemia (FA) pathway is responsible for interstrand crosslink (ICL) repair. Among the FA core complex components, FANCM is believed to act as a damage sensor for the ICL‐blocked replication fork and also as a molecular platform for FA core complex assembly and interaction with Bloom's syndrome (BS) complex that is thought to play an important role in the processing of DNA structures such as stalled replication forks. In the present study, we found that in silkworms, Bombyx mori, a species lacking the major FA core complex components (FANCA, B, C, E, F, and G), FancM is required for FancD2 monoubiquitination and cell proliferation in the presence of mitomycin C (MMC). Silkworm FancM (BmFancM) was phosphorylated in the middle regions, and the modification was associated with its subcellular localization. In addition, BmFancM interacted with Mhf1, a histone‐fold protein, and Rmi1, a subunit of the BS complex, in the different regions. The interaction region containing at least these two protein‐binding domains played an essential role in FancM‐dependent resistance to MMC. Our results suggest that BmFancM also acts as a platform for recruitment of both the FA protein and the BS protein, although the silkworm genome seems to lose FAAP24, a FancM‐binding partner protein in mammals.