Abnormal Rab11‐Rab8‐vesicles cluster in enterocytes of patients with microvillus inclusion disease

Microvillus inclusion disease (MVID) is a congenital enteropathy characterized by accumulation of vesiculo‐tubular endomembranes in the subapical cytoplasm of enterocytes, historically termed “secretory granules.” However, neither their identity nor pathophysiological significance is well defined. Using immunoelectron microscopy and tomography, we studied biopsies from MVID patients (3× Myosin 5b mutations and 1× Syntaxin3 mutation) and compared them to controls and genome‐edited CaCo2 cell models, harboring relevant mutations. Duodenal biopsies from 2 patients with novel Myosin 5b mutations and typical clinical symptoms showed unusual ultrastructural phenotypes: aberrant subapical vesicles and tubules were prominent in the enterocytes, though other histological hallmarks of MVID were almost absent (ectopic intra‐/intercellular microvilli, brush border atrophy). We identified these enigmatic vesiculo‐tubular organelles as Rab11‐Rab8‐positive recycling compartments of altered size, shape and location harboring the apical SNARE Syntaxin3, apical transporters sodium‐hydrogen exchanger 3 (NHE3) and cystic fibrosis transmembrane conductance regulator. Our data strongly indicate that in MVID disrupted trafficking between cargo vesicles and the apical plasma membrane is the primary cause of a defect of epithelial polarity and subsequent facultative loss of brush border integrity, leading to malabsorption. Furthermore, they support the notion that mislocalization of transporters, such as NHE3 substantially contributes to the reported sodium loss diarrhea.      

Effect of siRNAs on HSV-1 plaque formation and relative expression levels of RR mRNA

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.     

Asymbiotic Seed Germination and in vitro Seedling Rapid Development of Paphiopedilum spicerianum

In this paper, we studied the asymbiotic seed germination and seedling development of Paphiopedilum spicerianum, a wild plant with extremely small populations (PSESP) in China. By analogizing the influences of different media, seed maturity, pretreatments and lights on the seed germination, the results showed that the best germination condition is using the seeds at 270 days after pollination, pretreating with 1% NaOCl for 40 min, on the 1/4MS+10% coconut water medium under 24 hours darkness for 4 weeks. For seedling formation, 30g·L-1 Hyponex No 1 medium with 10mg·L-1 α naphthlene acetic acid, 05g·L-1 activated charcoal and 10% banana homogenate was the most effective. For seedling development, the same medium used for seedling formation with supplemented 10g·L-1 6 benzyladenine was most suitable.     

灰飞虱高带毒（RSV）群体酵母双杂交cDNA文库的构建

为了研究灰飞虱Laodelphax striatellus Fallén与水稻条纹病毒（rice stripe virus, RSV）互作机制, 本研究构建了灰飞虱高带毒群体酵母双杂交cDNA文库。以实验室筛选的灰飞虱高带毒群体为材料, 分离纯化mRNA, 反转录合成双链cDNA, 并连接三框型接头, 层析柱分级纯化。采用同源重组反应制备三框型cDNA入门文库, 再通过同源重组将入门文库转移到Gateway兼容载体pGADT7-DEST上, 构建获得酵母双杂交cDNA文库。检测结果表明： 文库库容量为3.68×10⁷ cfu, 扩增文库滴度为2.62×10¹⁰ cfu/mL; 文库重组率大于95%, cDNA插入片段平均长度>1 kb, 达到了标准cDNA文库的要求。灰飞虱高带毒群体酵母双杂交cDNA文库的构建为开展昆虫介体与水稻条纹病毒互作机制的研究奠定了基础。     

Production and preliminary characterization of a recombinant triheme cytochrome c7 from Geobacter sulfurreducens in Escherichia coli

Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c₇ from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c₇ and expressed it in Escherichiacoli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c₇ determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c₇ of known structure.     

An expert system to estimate the pesticide contamination of small streams using benthic macroinvertebrates as bioindicators II. The knowledge base of LIMPACT

The development and the evaluation of a biological indicator system for pesticide pollution in streams are presented. For small headwater streams with an agricultural catchment area, the expert system LIMPACT estimates the pesticide contamination according to the four classes: Not Detected (ND), Low (L), Moderate (M) and High (H) contamination without any specification of the chemical agents. The input parameters are the abundance data of benthic macroinvertebrate taxa within four time frames in a year (March/April, May/June, July/August, September/October) and nine basic water-quality and morphological parameters. The heuristic knowledge base was developed with the shell-kit D3 and contains 921 diagnostic rules with scores either to establish or to de-establish a diagnosis. The 418 rules had less than three symptoms, and only 47 rules had more than four symptoms in their rule condition. We differentiate between positive indicator (PI) taxa, which indicate contamination by high abundance values and positive abundance dynamics, and negative indicator (NI) taxa, a high abundance of which rules out contamination and indicates an uncontaminated site. We analysed 39 taxa and found 13 positive and 24 negative indicators. The database comprises 157 investigations per stream and year with rainfall event-controlled pesticide sampling and repeated benthic sampling as described in Part 1 [Ecol. Indicators, this issue]. For the evaluation of LIMPACT, we used the same cases. The correct class for the 157 investigations per stream and year is established by LIMPACT in 66.7–85.5% of the cases, with better results for uncontaminated sites. The overall alpha error probability (false positive) is 9.6% while the beta error probability (false negative) varied between 0 and 8% depending on the contamination class. If each stream is considered only once in the system (n=104), the correct diagnosis is established by LIMPACT in 51.9–88.6% of the cases. In most of the remaining cases no diagnosis is established instead of a wrong one.     

供肥对小麦间作蚕豆群体产量及根系的调控

利用田间,池栽,盆栽等方法,对不同N,P水平下小麦间作蚕豆群体根系的时空分布及产量效应进行了研究。结果表明,供P对小麦-蚕豆间作系统有明显的增产作用,与不供P相比,田间试验中供P处理间作小麦和蚕豆分别提高了48.39%和16.69%,池栽试验中群体产量增产幅度为20.07%-43.14%。间作蚕豆经济产量增幅为58.46%-78.78%,小麦间作蚕豆多作系统2种作物根密度生长高峰期交错出现,小麦的峰值出现早于蚕豆,其中,小麦和蚕豆根干重最大值分别出现在抽穗期和成熟期,而根长的最大值分别出现在拔节期和成熟期,这在一定程度上减轻了共生期2种作物对水肥的竞争,也是此群体增产增效的原因之一,施P处理间作小麦的根重,根长和根表面积分别比不施P处理提高54.33%,48.88%和47.00%;施N处理间作小麦的根重,根长和根表面积分别比不施N的增加15.25%,11.61%和11.46%。间作小麦根重的57.61%和蚕豆的69.20%分布在0-30cm土层,随施P水平的提高,总根干重及根密度都趋于增加,且深层土壤中根系增加明显。     

Modular estuarine mesocosm validation: ecotoxicological assessment of direct effects with the model compound endosulfan

This study represents the first in a series of validation experiments for the modular estuarine mesocosm testing direct pesticide effects. Endosulfan, an agricultural insecticide, was selected as a model contaminant for studying direct toxic effects as well as uptake of this model contaminant by estuarine biota. The grass shrimp, Palaemonetes pugio, and the mummichog, Fundulus heteroclitus, showed significant mortality (96 h LC₅₀: 0.12 and 2.2 μg/l, respectively) to endosulfan as predicted by laboratory bioassays. There was no effect on fiddler crabs and eastern oysters. The mesocosm was also useful in demonstrating the bioconcentration of endosulfan by eastern oysters (bioconcentration factor [BCF]=375) similar to results reported in field studies. This study illustrates the modular estuarine mesocosms' ability to detect direct effects of pesticide exposure and the uptake of a pesticide by estuarine fauna.     

普通大鵟胃容物和食茧分析

对青海省玛多县境内的普通大鵟(Buteo hemilasius)的14个胃容物和118个食茧中的猎物逐个分析鉴定,共分为3种类型：①高原鼠兔(Ochotona curzoniae),②青海田鼠(Lasiopodomys fuscus）,③小型鸟类,分别计算各类猎物出现率(Pi)和生物量比率(bi)。高原鼠免、青海田鼠、小型鸟类在胃容物中,pi分别为0．28、0．68和0．04;bi则分别为0．59、0．40和0．02;食茧中,pi分别为0．70、0．27和0．02,bi则分别为0．89、0．10和0．01。χ^2检验表明取食个体数和生物量在胃容物和食茧之间差异显著,但均说明高原鼠兔和青海田鼠等小型哺乳类是普通大鵟的主要食物来源。     

酪蛋白小肽对幼龄草鱼生长和饲料利用的影响

本实验为酪蛋白小肽在幼龄草鱼营养生理上的意义研究。 2 2 5尾 (3 76± 0 16g)草鱼分为 3组 ,每组 75尾鱼 ,每 2 5尾鱼饲于 0 2 5m3 的水族箱中 ,每组设 3个平行水族箱。对照组添加 0 5 %酪蛋白 ,试验组 1、2日粮中分别添加 0 5 %酶解酪蛋白 (80 %以上为小肽 ,平均链长 2 98) ,0 5 %酸解酪蛋白 (80 %以上为游离氨基酸 )。试验期 5 6d ,水温为 2 7 6± 3 1℃。结果表明 :添加 0 5 %酶解酪蛋白组草鱼相对生长率、蛋白效率比、饲料效率比和血浆中镁含量与小肽总量均显著高于添加 0 5 %酪蛋白组和10 5 %酸解酪蛋白组 (P <0 0 5 )。草鱼日粮中添加一定比例的小肽可提高饲料表观消化率和蛋白消化率 ,增加血液循环中生物活性肽的含量 ,减少肝胰脏和肠系膜脂肪储积 ,提高机体对日粮中氨基酸的利用率 ,从而增加体内氮沉积 ,提高生长速度。