Pou4f2‐GFP knock‐in mouse line: A model for studying retinal ganglion cell development

Pou4f2 acts as a key node in the comprehensive and step‐wise gene regulatory network (GRN) and regulates the development of retinal ganglion cells (RGCs). Accordingly, deletion of Pou4f2 results in RGC axon defects and apoptosis. To investigate the GRN involved in RGC regeneration, we generated a mouse line with a POU4F2‐green fluorescent protein (GFP) fusion protein expressed in RGCs. Co‐localization of POU4F2 and GFP in the retina and brain of Pou4f2‐GFP/+ heterozygote mice was confirmed using immunofluorescence analysis. Compared with those in wild‐type mice, the expression patterns of POU4F2 and POU4F1 and the co‐expression patterns of ISL1 and POU4F2 were unaffected in Pou4f2‐GFP/GFP homozygote mice. Moreover, the quantification of RGCs showed no significant difference between Pou4f2‐GFP/GFP homozygote and wild‐type mice. These results demonstrated that the development of RGCs in Pou4f2‐GFP/GFP homozygote mice was the same as in wild‐type mice. Thus, the present Pou4f2‐GFP knock‐in mouse line is a useful tool for further studies on the differentiation and regeneration of RGCs.     

Chemiluminescence analysis of antioxidant capacity for serum albumin isolated from healthy or uremic volunteers

Regular hemodialysis treatment induces an elevation in oxidative stress in patients with end‐stage renal failure, resulting in oxidative damage of the most abundant serum protein, albumin. Oxidation of serum albumin causes depletion of albumin reactive thiols, leading to oxidative modification of serum albumin. The aim of this study was to screen the antioxidant capacity of albumins isolated from uremic patients (HD‐ALB) or healthy volunteers (N‐ALB). From high‐performance liquid chromatography spectra, we observed that one uremic solute binds to HD‐ALB via the formation of disulfide bonds between HD‐ALB and the uremic solute. Furthermore, we found using chemiluminescent analysis that the antioxidant capacities for N‐ALB to scavenge reactive oxygen species including singlet oxygen, hypochlorite and hydrogen peroxide were higher than HD‐ALB. Our results suggest that protein‐bound uremic solute binds to albumin via formation of disulfide bonds, resulting in the depletion of albumin reactive thiols. The depletion of albumin reactive thiols leads to a reduced antioxidant capacity of HD‐ALB, implying postmodification of albumin. This situation may reduce the antioxidant capacity of albumin and increase oxidative stress, resulting in increase in complications related to oxidative damage in uremic patients. Copyright © 2016 John Wiley & Sons, Ltd.     

Construction of photodynamic‐effect immunofluorescence probes by a complex of quantum dots,immunoglobulin G and chlorin e6 and their application in HepG2 cell killing

In this study, tri‐functional immunofluorescent probes (Ce6–IgG–QDs) based on covalent combinations of quantum dots (QDs), immunoglobulin G (IgG) and chlorin e6 (Ce6) were developed and their photodynamic ability to induce the death of cancer cells was demonstrated. Strategically, one type of second‐generation photosensitizer, Ce6, was first coupled with anti‐IgG antibody using the EDC/NHS cross‐linking method to construct the photosensitive immunoconjugate Ce6–IgG. Then, a complex of Ce6–IgG–QDs immunofluorescent probes was obtained in succession by covalently coupling Ce6–IgG to water soluble CdTe QDs. The as‐manufactured Ce6–IgG–QDs maintained the bio‐activities of both the antigen–antibody‐based tumour targeting effects of IgG and the photodynamic‐related anticancer activities of Ce6. By way of polyclonal antibody interaction with rabbit anti‐human epidermal growth factor receptor (anti‐EGFR antibody, N‐terminus), Ce6–IgG–QDs were labelled indirectly onto the surface of human hepatocarcinoma (HepG2) cells in cell recognition and killing experiments. The results indicated that the Ce6–IgG–QDs probes have excellent tumour cell selectivity and higher photosensitivity in photodynamic therapy (PDT) compared with Ce6 alone, due to their antibody‐based specific recognition and location of HepG2 cells and the photodynamic effects of Ce6 killed cells based on efficient fluorescence resonance energy transfer between QDs and Ce6. Copyright © 2015 John Wiley & Sons, Ltd.     

南方水稻黑条矮缩病对褐飞虱和白背飞虱体内三种保护酶活性的影响

为探寻植物感染病毒病后对刺吸性害虫体内生化酶活性的影响,本文研究了感染南方水稻矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的水稻对褐飞虱Nilaparvata lugens和白背飞虱Sogatella furcifera成虫及若虫体内三种保护酶活性的影响。结果表明,取食染病水稻的白背飞虱和褐飞虱成虫及若虫体内超氧化物歧化酶(superoxide dismutase,SOD)、过氧化物酶(peroxidase,POD)和过氧化氢酶(catalase,CAT)的活性均随取食时间的延长而增加。在带毒水稻上取食12 h后,白背飞虱成虫、褐飞虱成虫、若虫体内SOD活性与对照比差异不显著外,其他均显著高于对照;取食24 h后,白背飞虱若虫体内SOD、POD活性和褐飞虱若虫SOD活性虽高于对照但未达显著水平;取食5 d后,白背飞虱若虫、褐飞虱成虫、若虫POD活性未达显著外,其他均显著高于对照。以上研究结果可为进一步研究植物-病毒-寄主三种之间的关系提供参考。     

基于16S rDNA与Cytb序列的疏广蜡蝉属三个近似种的分子鉴定

本文通过PCR扩增首次获得了疏广蜡蝉属Euricania Melichar,1898 3个近似种:透明疏广蜡蝉E.clara Kato,1932、长刺疏广蜡蝉E.longa Xu,LiangJiang,2006和短刺疏广蜡蝉E.brevicula Xu,LiangJiang,2006的16S r DNA序列和Cytb序列;使用MEGA 6.0软件,分析了其序列组成及变异,计算了遗传距离,以宽广蜡蝉属Pochazia AmyotServille,1843的圆纹宽广蜡蝉P.guttifera Walker,1851和眼斑宽广蜡蝉P.discreta Melichar,1898为外群,利用邻接法(NJ)和最大简约法(MP)构建了系统发育树。结果显示:透明疏广蜡蝉、长刺疏广蜡蝉和短刺疏广蜡蝉的16S r DNA序列长度分别为414 bp,404 bp,435 bp,而Cytb基因序列长度分别为492 bp,468 bp,472 bp,三者的序列比对、碱基组成成分存在差异;2属5种广翅蜡蝉16S r DNA基因的种间遗传距离为0.008-0.098,属间遗传距离为0.122-0.197,而Cytb基因的种间遗传距离为0.038-0.055,属间遗传距离为0.181-0.188,表明16S r DNA和Cytb基因序列可作为DNA条形码候选基因片段,用于广翅蜡蝉近似属、种的分子鉴定。系统发育树的拓扑结构显示,3个近似种的亲缘关系基本一致,各自聚为一支,且置信值均达96%以上。本文亦结合了透明疏广蜡蝉、长刺疏广蜡蝉和短刺疏广蜡蝉形态学特征的差异对其进行了讨论和分析。     

Functional TRPV2 and TRPV4 channels in human cardiac c‐kit+ progenitor cells

The cellular physiology and biology of human cardiac c‐kit⁺ progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c‐kit⁺ progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c‐kit⁺ cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca²⁺ (Ca²⁺_i), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α‐phorbol 12‐13‐dicaprinate induced Ca²⁺_i oscillations, which can be inhibited by the TRPV4 blocker RN‐1734. The alteration of Ca²⁺_i by probenecid or 4α‐phorbol 12‐13‐dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c‐kit⁺ progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c‐kit⁺ progenitor cells.     

MOLECULAR CHARACTERIZATION OF AUTOPHAGY‐RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS

Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry‐SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl‐HP cells and partially co‐localized with BmATG6‐GFP, but it almost has no significant co‐localization with GFP‐HaATG8. Then, the Western blot analysis demonstrated that GFP‐SeATG5 conjugated with ATG12. Moreover, real‐time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.     

EXPRESSION AND EFFECTS OF MUTANT Bombyx mori ACETYLCHOLINESTRASE1 IN BmN CELLS

The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP‐ or CB‐resistant strains have been generated. In this study, wild‐type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21‐fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.     

Phylogeny of Impatiens (Balsaminaceae): integrating molecular and morphological evidence into a new classification

Impatiens L. is one of the largest angiosperm genera, containing over 1000 species, and is notorious for its taxonomic difficulty. Here, we present, to our knowledge, the most comprehensive phylogenetic analysis of the genus to date based on a total evidence approach. Forty‐six morphological characters, mainly obtained from our own investigations, are combined with sequence data from three genetic regions, including nuclear ribosomal ITS and plastid atpB‐rbcL and trnL‐F. We include 150 Impatiens species representing all clades recovered by previous phylogenetic analyses as well as three outgroups. Maximum‐parsimony and Bayesian inference methods were used to infer phylogenetic relationships. Our analyses concur with previous studies, but in most cases provide stronger support. Impatiens splits into two major clades. For the first time, we report that species with three‐colpate pollen and four carpels form a monophyletic group (clade I). Within clade II, seven well‐supported subclades are recognized. Within this phylogenetic framework, character evolution is reconstructed, and diagnostic morphological characters for different clades and subclades are identified and discussed. Based on both morphological and molecular evidence, a new classification outline is presented, in which Impatiens is divided into two subgenera, subgen. Clavicarpa and subgen. Impatiens; the latter is further subdivided into seven sections.