Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Possible molecular evolution of biomembranes: from single-chain to double-chain lipids

We have studied a possible evolution process permitting a 'primitive' membrane to evolve towards a membrane structure with an outer wall, similar to that of bacteria. We have investigated whether a polysaccharide bearing hydrophobic phytyl or cholesteryl chains coats giant vesicles made of single- or double-chain lipids. Phytyl-pullulan 5b was found to bind to the surface of vesicles made of either single- or double-chain lipids. In contrast, cholesteryl-pullulan 5a only coated the surface of vesicles made of double-chain lipids. These results indicate that there must be a close match between the size and shape of membrane constituents and the hydrophobic molecules to be inserted. This process could, thus, provide a selection mechanism of lipid-membrane constituents during the course of biomembrane evolution. The presence of the above 'hydrophobized' polysaccharides on the surface of different giant vesicles was identified by lectin binding. Both concanavalin A and annexin V were shown by fluorescence microscopy to bind spontaneously to vesicles made of double-chain lipids. Our experiments exemplify that self-organization of amphiphiles into closed vesicles in aqueous solution automatically leads to the coating of vesicles by 'hydrophobized' polysaccharides, which then permit lectin binding. This is a possible mechanism for the evolution of primitive membranes towards 'proto-cells'.     

Reductive genome evolution from the mother of Rickettsia

The Rickettsia genus is a group of obligate intracellular α-proteobacteria representing a paradigm of reductive evolution. Here, we investigate the evolutionary processes that shaped the genomes of the genus. The reconstruction of ancestral genomes indicates that their last common ancestor contained more genes, but already possessed most traits associated with cellular parasitism. The differences in gene repertoires across modern Rickettsia are mainly the result of differential gene losses from the ancestor. We demonstrate using computer simulation that the propensity of loss was variable across genes during this process. We also analyzed the ratio of nonsynonymous to synonymous changes (Ka/Ks) calculated as an average over large sets of genes to assay the strength of selection acting on the genomes of Rickettsia, Anaplasmataceae, and free-living γ-proteobacteria. As a general trend, Ka/Ks were found to decrease with increasing divergence between genomes. The high Ka/Ks for closely related genomes are probably due to a lag in the removal of slightly deleterious nonsynonymous mutations by natural selection. Interestingly, we also observed a decrease of the rate of gene loss with increasing divergence, suggesting a similar lag in the removal of slightly deleterious pseudogene alleles. For larger divergence (Ks > 0.2), Ka/Ks converge toward similar values indicating that the levels of selection are roughly equivalent between intracellular α-proteobacteria and their free-living relatives. This contrasts with the view that obligate endocellular microorganisms tend to evolve faster as a consequence of reduced effectiveness of selection, and suggests a major role of enhanced background mutation rates on the fast protein divergence in the obligate intracellular α-proteobacteria.     

Comparative proteomics analyses reveal a potential biomarker for the detection of vancomycin-intermediate Staphylococcus aureus strains

Vancomycin-intermediate Staphylococcus aureus (VISA) strains tend to develop during glycopeptide treatment of infections caused by methicillin-resistant S. aureus (MRSA). Rapid and effective detection methods for VISA strains are lacking, and mechanisms of resistance are unclear. Here, global comparative proteomic approaches have been used to identify potential biomarkers of intermediate vancomycin resistance. With the use of high-resolution two-dimensional gels and iTRAQ mass tagging, numerous proteins were found to be differentially expressed between clinical MRSA and VISA isolates of the same multilocus sequence type. One of these, the predicted lytic transglycosylase SAV2095 (SceD-like protein), was selected for further study based on both its high level of induction in Mu50 and its predicted role in modeling the cell wall, which is the target of vancomycin. Relative SAV2095 mRNA expression levels were compared between 25 MRSA and VISA/heterogeneous VISA clinical isolates by real-time RT-PCR. The SAV2095 mRNA was significantly induced in all VISA isolates relative to all MRSA strains ( p < 0.001), and significant induction of SAV2095 was also seen for several potential heterogeneous VISA strains that appear vancomycin-sensitive by standard minimum inhibitory concentration-determining methods. Furthermore, strains selected in vitro for increasing levels of resistance from four unrelated clinical MRSA isolates displayed concomitant increases in levels of SAV2095 expression. Together, these results suggest that SAV2095 expression level could serve as a molecular diagnostic marker for the rapid detection of VISA.     

A retroviral strategy that efficiently creates chromosomal deletions in mammalian cells

Chromosomal deletions, as a genetic tool for functional genomics, remain underexploited for vertebrate stem cells mostly because presently available methods are too labor-intensive. To address this, we developed and validated a set of complementary retroviruses that creates a wide range of nested chromosomal deletions. When applied to mouse embryonic stem cells (ESCs), this retrovirus-based method yielded deletions ranging from 6 kb to 23 Mb (average 2.9 Mb), with an efficiency of 64% for drug-selected clones. Notably, several of the engineered ESC clones, mostly those with large deletions, showed major alteration in cell fate. In comparison to other methods that have also exploited retroviruses for chromosomal engineering, this modified strategy is more efficient and versatile because it bypasses the need for homologous recombination, and thus can be exploited for rapid and extensive functional screens in embryonic and adult stem cells.     

Reporting data from high-throughput screening of small-molecule libraries

Publications reporting results of small-molecule screens are becoming more common as academic researchers increasingly make use of high-throughput screening (HTS) facilities. However, no standards have been formally established for reporting small-molecule screening data, and often key information important for the evaluation and interpretation of results is omitted in published HTS protocols. Here, we propose concise guidelines for reporting small-molecule HTS data.     

Use of cell cultures in vitro to assess the uptake of long dsRNA in plant cells

In Vitro Cellular & Developmental Biology - Plant - While RNAi approaches based on transgenic plants overexpressing double-stranded RNAs (dsRNAs) have yielded a number of genetically modified...     

Correction to: Preliminary insights into the population characteristics and distribution of reef (Mobula alfredi) and oceanic (M. birostris) manta rays in French Polynesia

Coral Reefs - This erratum has been initiated as many corrections which were submitted during proofing were overlooked by vendor.     

Exogenous 24-Epibrassinolide Alleviates Effects of Salt Stress on Chloroplasts and Photosynthesis in Robinia pseudoacacia L. Seedlings

Journal of Plant Growth Regulation - The brassinosteroids (BRs) constitute a recently defined class of plant hormone that can enhance the resistance of plants to multiple stresses. However, the...