Aligning the proteome and genome of the silkworm, Bombyx mori

A technology of mass spectrometry (MS) was used in this study for the large-scale proteomic identification and verification of protein-encoding genes present in the silkworm (Bombyx mori) genome. Peptide sequences identified by MS were compared with those from an open reading frame (ORF) library of the B. mori genome and a cDNA library, to validate the coding attributes of ORFs. Two databases were created. The first was based on a 9× draft sequence of the silkworm genome and contained 14,632 putative proteins. The second was based on a B. mori pupal cDNA library containing 3,187 putative proteins of at least 30 amino acid residues in length. A total of 81,000 peptide sequences with a threshold score of 60% were generated by the MS/MS analysis, and 55,400 of these were chosen for a sequence alignment. By searching these two databases, 6,649 and 250 proteins were matched, which accounted for approximately 45.4% and 7.8% of the peptide sequences and putative proteins, respectively. Further analyses carried out by several bioinformatic tools suggested that the matches included proteins with predicted transmembrane domains (1,393) and preproteins with a signal peptide (976). These results provide a fundamental understanding of the expression and function of silkworm proteins.     

Cliques in mitotic spindle network bring kinetochore‐associated complexes to form dependence pathway

The mitotic spindle is an essential molecular machine for chromosome segregation during mitosis. Achieving a better understanding of its organization at the topological level remains a daunting task. To determine the functional connections among 137 mitotic spindle proteins, a protein–protein interaction network among queries was constructed. Many hub proteins, which connect more than one query and serve as highly plausible candidates for expanding the mitotic spindle proteome, are ranked by conventional degree centrality and a new subnetwork specificity score. Evaluation of the ranking results by literature reviews and empirical verification of SEPT6, a novel top‐ranked hub, suggests that the subnetwork specificity score could enrich for putative spindle‐related proteins. Topological analysis of this expanded network shows the presence of 30 3‐cliques and six 4‐cliques (fully connected subgraphs) that, respectively, reside in eight kinetochore‐associated complexes, of which seven are evolution conserved. Notably, these complexes strikingly form dependence pathways for the assembly of the kinetochore complex. These analyses indicate the feasibility of using network topology, i.e. cliques, to uncover novel pathways to accelerate our understanding of potential biological processes.     

A simple and sensitive HPLC–ESI-MS/MS method for the determination of andrographolide in human plasma

A liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) method for the determination of andrographolide in human plasma was established. Dehydroandrographolide was used as the internal standard (I.S.). The plasma samples were deproteinized with methanol and separated on a Hanbon C₁₈ column with a mobile phase of methanol–water (70:30, v/v). HPLC–ESI-MS/MS was performed in the selected ion monitoring (SIM) mode using target ions at [M?H₂O–H]^?, m/z 331.1 for andrographolide and [M?H]^?, m/z 331.1 for the I.S. Calibration curve was linear over the range of 1.0–150.0 ng/mL. The chromatographic separation was achieved in less than 6.5 min. The lower limits of quantification (LLOQ) was 1.0 ng/mL. The intra and inter-run precisions were less than 6.95 and 7.22%, respectively. The method was successfully applied to determine the plasma concentrations of andrographolide in Chinese volunteers.     

A platform to standardize,store, and visualize proteomics experimental data

With the development of functional genomics research, large-scale proteomics studies are now widespread, presenting significant challenges for data storage, exchange, and analysis. Here we present the Integrated Proteomics Exploring Database （IPED） as a platform for managing proteomics experimental data （both process and result data）. IPED is based on the schema of the Proteome Experimental Data Repository （PEDRo）, and complies with the General Proteomics Standard （GPS） drafted by the Proteomics Standards Committee of the Human Proteome Organization. In our work, we developed three components for the IPED platform： the IPED client editor, IPED server software, and IPED web interface. The client editor collects experimental data and generates an extensible markup language （XML） data file compliant with PEDRo and GPS; the server software parses the XML data file and loads information into a core database; and the web interface displays experimental results, to provide a convenient graphic representation of data. Given software convenience and data abundance, IPED is a powerful platform for data exchange and presents an important resource for the proteomics community. In its current release, IPED is available at http：//www. biosino.org/iped2.     

空肠弯曲菌fliG基因敲除突变株动力、体外趋化和小鼠定植能力的改变

空肠弯曲菌( Campylobacter jejuni) 是人类细菌性胃肠炎的病原体。趋化是细菌向合适的寄生部位定向运动, 也是空肠弯曲菌在宿主空肠黏膜表面实现定植的关键起始步骤, 该趋化运动由趋化相关二元信号系统( che-TCS) 以MCPs→Ches→Flis/Mots 方式调控。FliG是Fli 蛋白家族成员, 一些病原菌的FliG被证明是鞭毛马达蛋白, 也是细菌鞭毛马达中开关复合体的必需组分, 但空肠弯曲菌FliG在细菌趋化中的作用未明。本研究中, 我们根据同源重组原理, 构建空肠弯曲菌NCTC11168 株fliG基因敲除( fliG^- ) 突变株, 然后对fliG^- 突变株的动力、趋化和定植能力进行测定。动力实验和体外趋化实验结果证实, fliG^- 突变株在半固体琼脂平板上的菌落直径、在硬琼脂平板上对0. 2 mol/L 脱氧胆酸钠( SDC) 的趋化聚集环直径均明显小于野生株( P <0. 05) 。与野生株比较, 黏附于BALB/c-ByJ小鼠空肠黏膜表面以及空肠内容物中的fliG^- 突变株数量也明显减少( P <0. 05) 。实验结果表明, fliG是空肠弯曲菌鞭毛动力以及细菌感染时向空肠黏膜趋化运动的必需基因。     

高效液相色谱法测定妇血荣胶囊中芍药苷的含量

目的:建立妇血荣胶囊中芍药苷含量的HPLC检测方法.方法:采用甲醇提取样品;色谱条件:Alltech ODS柱(5μm,250mm×4.6 mm);流动相为乙腈-水(16:84,v/v);检测波长为230 nm.结果:线性范围为24.3～218.7 μg.mL-1(r=0.9999),平均回收率为101.21%,RSD为0.80%.结论:高效液相色谱法简单易行,准确,灵敏度高,适用于妇血荣胶囊中芍药苷的含量测定.     

Multilocus variable-number tandem repeat analysis for molecular typing and phylogenetic analysis of Shigella flexneri

Background

Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.

Results

Five of six strains invaded HeLa and T84 cells in a range of 13.3%–20.9% and 5.8%–17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.

Conclusion

Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.     

Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD

Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.