Holistic differential analysis of embryo-induced alterations in the proteome of bovine endometrium in the preattachment period

During the peri-implantation period, molecular signaling between embryo and endometrium (layer of tissue lining the uterus lumen) is supposed to be crucial for the maintenance of pregnancy. To investigate embryo-induced alterations in the proteome of bovine endometrium in the preattachment period (day 18), we used monozygotic cattle twins (generated by embryo splitting) as a model eliminating genetic variability as a source for proteome differences. One of the twins was pregnant after the transfer of two in vitro produced blastocysts, while the corresponding twin received a sham-transfer and served as a nonpregnant control. The two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of the endometrium samples of three twin pairs (pregnant/nonpregnant) revealed four proteins with significantly higher abundance (p < 10(-9)) in each sample derived from the pregnant animals: Rho GDP dissociation inhibitor beta; 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD); soluble NADP(+)-dependent isocitrate dehydrogenase 1; and acyl-CoA-binding protein. To verify the accuracy of the 2-D DIGE quantification, the abundances of 20 alpha-HSD were quantified by a targeted cleavable isotope-coded affinity tag (ICAT) approach. The mass spectrometry-based ICAT quantification matched perfectly the results obtained by 2-D DIGE quantification, demonstrating the accuracy of our data. These results demonstrate that our model (monozygotic twins) in combination with the appropriate analytical tools is particularly suitable for the detection of the proteins involved in the embryo-maternal interactions.     

圈养獐分娩的初步观察

2004年的5~7月,在浙江省嵊州市河麂种源繁育基地对34只獐(Hydropotes inermis)的分娩活动进行了观察。结果表明,母獐通常选择安全的地点进行分娩。獐集中分娩的时间为6月3日到6月19日,占分娩母獐总数的73.53%(n=34),并且分娩多在白天进行。产程(从胎膜露出到胎盘娩出)平均为(302.20±15.27)min(n=15)。胎儿娩出的姿势多为前躯前置(前足、头先露出),占83.78%;少数为后躯前置(后足先露出),占16.22%。幼仔首次吮乳时间在单胎和多胎间无显著差异,平均为出生后(44.97±2.73)min(n=35)。幼仔出生后首次平均站立时间在双胞胎、三胞胎和四胞胎分别为(32.25±2.49)min(n=16)(、29.42±2.52)min(n=12)和(65.00±7.39)min(n=6)。每胎幼仔数越多,初生幼仔的平均体重越轻。     

家蚕核型多角体病毒同源重复区hr5的结构和功能

家蚕核型多角体病毒(BmNPV)和首蓿尺蠖核型多角体病毒(AcMNPV)是宿主不同的同类昆虫杆状病毒.AcMNPV有5个同源重复区,hr5具较强增强子功能,最近被发现可能参与病毒DNA复制.Maeda等曾将BmNPV hrs图谱定位,最近又报道了BmNPV hr s的结构,但功能研究至今未见报道.与Maeda等同时,从野生型BmNPV基因组中克隆了hr 5区,进行了全序列测定,与Maeda等的报道有一定差别.功能分析证实,含BmNPV hr 5的质粒在辅助病毒存在下,不仅在宿主细胞(BmN)中能进行复制,而且在非宿主细胞 Sf 21中亦能进行复制.此外,不仅完整的BmNPV hr 5(含 8个重复单位),而且部分序列(含6个重复单位)亦显示上述功能.以上研究结果,并对hr 5在病毒 DNA复制过程中可能有的功能进行了探讨.     

Influence of Nutrient Concentrations on MPN Quantification and Enrichment of Nitrate-Reducing Fe(II)-Oxidizing and Fe(III)-Reducing Bacteria from Littoral Freshwater Lake Sediments

The application of culture-dependent studies to quantify Fe-metabolizing microorganisms from the environment is a necessity, as there are so far no universal functional marker genes for application in culture-independent studies. Media composition can vary between studies, therefore, we determined the effects of three different growth media on the quantification (MPNs) and identity (via cloning and sequencing of dominant DGGE bands) of nitrate-reducing Fe(II)-oxidizers and lactate- or acetate-oxidizing Fe(III)-reducers from a lacustrine sediment: low sulphate freshwater medium (FWM), sterile filtered bicarbonate-buffered lake water (BLW) and a mixture of both (MIX). We consistently found fewer cells in the BLW than in the FWM and the MIX. The DGGE banding patterns of the microbial communities enriched in different media types clustered together according to the e^? donor and acceptor couples and not according to the medium used. Thus, although the medium composition significantly influenced the quantification and thereby conclusions on the abundance and potential significance of the targeted group within the ecosystem, biodiversity assessments through enrichment cultures were less influenced by the medium, but instead were affected by the type and concentration of the e^? donor/acceptor.     

ICU鲍曼不动杆菌感染的流行状况及耐药性分析

目的对ICU住院患者鲍曼不动杆菌感染的流行状况及耐药情况进行调查,为临床合理选用抗菌药物提供参考。方法对2012年7月至2013年12月ICU住院患者送检的693份各种临床标本进行病原菌的分离鉴定,对分离出的鲍曼不动杆菌采用K-B法做体外药敏试验。结果 693份临床标本共分离出110株鲍曼不动杆菌,其中多重耐药菌株有103株（93.6%）,感染的标本来源以痰液最为常见（86.4%）。鲍曼不动杆菌对常用抗菌药物耐药广泛,对氨苄西林、头孢唑啉100%耐药,对哌拉西林、头孢曲松、复方新诺明、氨曲南、环丙沙星、头孢吡肟、哌拉西林/他唑巴坦及庆大霉素等多种抗菌药物的耐药率大于80.0%。结论 ICU患者鲍曼不动杆菌感染多由多重耐药菌株引起,感染部位以下呼吸道为主,其对抗菌药物耐药情况相当严峻,加强耐药性监测及合理使用抗菌药物,对减少耐药菌株的产生具有重要意义。     

Rac and phosphatidylinositol 3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL 2H3 mast cells

FcepsilonRI signaling in rat basophilic leukemia cells depends on phosphatidylinositol 3-kinase (PI3-kinase) and the small GTPase Rac. Here, we studied the functional relationship among PI3-kinase, its effector protein kinase B (PKB), and Rac using inhibitors of PI3-kinase and toxins inhibiting Rac. Wortmannin, an inhibitor of PI3-kinase, blocked FcepsilonRI-mediated tyrosine phosphorylation of phospholipase Cgamma, inositol phosphate formation, calcium mobilization, and secretion of hexosaminidase. Similarly, Clostridium difficile toxin B, which inactivates all Rho GTPases including Rho, Rac and Cdc42, and Clostridium sordellii lethal toxin, which inhibits Rac (possibly Cdc42) but not Rho, blocked these responses. Stimulation of the FcepsilonRI receptor induced a rapid increase in the GTP-bound form of Rac. Whereas toxin B inhibited the Rac activation, PI3-kinase inhibitors (wortmannin and LY294002) had no effect on activation of Rac. In line with this, wortmannin had no effect on tyrosine phosphorylation of the guanine nucleotide exchange factor Vav. Wortmannin, toxin B, and lethal toxin inhibited phosphorylation of PKB on Ser(473). Similarly, translocation of the pleckstrin homology domain of PKB tagged with the green fluorescent protein to the membrane, which was induced by activation of the FcepsilonRI receptor, was blocked by inhibitors of PI3-kinase and Rac inactivation. Our results indicate that in rat basophilic leukemia cells Rac and PI3-kinase regulate PKB and suggest that Rac is functionally located upstream and/or parallel of PI3-kinase/PKB in FcepsilonRI signaling.     

Parallel Exploitation of Diverse Host Nutrients Enhances Salmonella Virulence

Pathogen access to host nutrients in infected tissues is fundamental for pathogen growth and virulence, disease progression, and infection control. However, our understanding of this crucial process is still rather limited because of experimental and conceptual challenges. Here, we used proteomics, microbial genetics, competitive infections, and computational approaches to obtain a comprehensive overview of Salmonella nutrition and growth in a mouse typhoid fever model. The data revealed that Salmonella accessed an unexpectedly diverse set of at least 31 different host nutrients in infected tissues but the individual nutrients were available in only scarce amounts. Salmonella adapted to this situation by expressing versatile catabolic pathways to simultaneously exploit multiple host nutrients. A genome-scale computational model of Salmonella in vivo metabolism based on these data was fully consistent with independent large-scale experimental data on Salmonella enzyme quantities, and correctly predicted 92% of 738 reported experimental mutant virulence phenotypes, suggesting that our analysis provided a comprehensive overview of host nutrient supply, Salmonella metabolism, and Salmonella growth during infection. Comparison of metabolic networks of other pathogens suggested that complex host/pathogen nutritional interfaces are a common feature underlying many infectious diseases.     

食管穿孔外科治疗22例临床分析

目的:探讨食管穿孔的诊断、临床特征与外科治疗方法.方法:回顾分析1999年-2009年我院收治的食管穿孔22例.结果:本组病例4例保守治疗,2例行颈部脓肿切开引流,8例行单纯食管穿孔修补术,3例行纵隔及胸腔脓肿清除术并引流,1例行食管下段切除、胃代食管弓上吻合术,2例行食管修补并肺叶切除,2例行空肠双管造瘘加纵隔胸腔引流,2例因经济原因放弃治疗出院.治愈19例,治愈率86.3%,死亡1例,死亡率4.5%.结论:早期诊断和及时采取正确的处理措施是提高本病治愈率,降低死亡率的关键.     

Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes

Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.