Computational bacterial genome-wide analysis of phylogenetic profiles reveals potential virulence genes of Streptococcus agalactiae

The phylogenetic profile of a gene is a reflection of its evolutionary history and can be defined as the differential presence or absence of a gene in a set of reference genomes. It has been employed to facilitate the prediction of gene functions. However, the hypothesis that the application of this concept can also facilitate the discovery of bacterial virulence factors has not been fully examined. In this paper, we test this hypothesis and report a computational pipeline designed to identify previously unknown bacterial virulence genes using group B streptococcus (GBS) as an example. Phylogenetic profiles of all GBS genes across 467 bacterial reference genomes were determined by candidate-against-all BLAST searches,which were then used to identify candidate virulence genes by machine learning models. Evaluation experiments with known GBS virulence genes suggested good functional and model consistency in cross-validation analyses (areas under ROC curve, 0.80 and 0.98 respectively). Inspection of the top-10 genes in each of the 15 virulence functional groups revealed at least 15 (of 119) homologous genes implicated in virulence in other human pathogens but previously unrecognized as potential virulence genes in GBS. Among these highly-ranked genes, many encode hypothetical proteins with possible roles in GBS virulence. Thus, our approach has led to the identification of a set of genes potentially affecting the virulence potential of GBS, which are potential candidates for further in vitro and in vivo investigations. This computational pipeline can also be extended to in silico analysis of virulence determinants of other bacterial pathogens.     

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5'' amine modified to allow fixation to the membrane. Primers are 5'' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours).The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.Published applications of mPCR/RLB include detection of antibiotic resistance genes^1,2, typing of methicillin-resistant Staphylococcus aureus^3-5 and Salmonella sp⁶, molecular serotyping of Streptococcus pneumoniae^7,8, Streptococcus agalactiae⁹ and enteroviruses^10,11, identification of Mycobacterium sp¹², detection of genital^13-15 and respiratory tract¹⁶ and other¹⁷ pathogens and detection and identification of mollicutes¹⁸. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.     

The network of global corporate control

The structure of the control network of transnational corporations affects global market competition and financial stability. So far, only small national samples were studied and there was no appropriate methodology to assess control globally. We present the first investigation of the architecture of the international ownership network, along with the computation of the control held by each global player. We find that transnational corporations form a giant bow-tie structure and that a large portion of control flows to a small tightly-knit core of financial institutions. This core can be seen as an economic "super-entity" that raises new important issues both for researchers and policy makers.     

Defining reference sequences for Nocardia species by similarity and clustering analyses of 16S rRNA gene sequence data

Background

The intra- and inter-species genetic diversity of bacteria and the absence of ‘reference’, or the most representative, sequences of individual species present a significant challenge for sequence-based identification. The aims of this study were to determine the utility, and compare the performance of several clustering and classification algorithms to identify the species of 364 sequences of 16S rRNA gene with a defined species in GenBank, and 110 sequences of 16S rRNA gene with no defined species, all within the genus Nocardia.

Methods

A total of 364 16S rRNA gene sequences of Nocardia species were studied. In addition, 110 16S rRNA gene sequences assigned only to the Nocardia genus level at the time of submission to GenBank were used for machine learning classification experiments. Different clustering algorithms were compared with a novel algorithm or the linear mapping (LM) of the distance matrix. Principal Components Analysis was used for the dimensionality reduction and visualization.

Results

The LM algorithm achieved the highest performance and classified the set of 364 16S rRNA sequences into 80 clusters, the majority of which (83.52%) corresponded with the original species. The most representative 16S rRNA sequences for individual Nocardia species have been identified as ‘centroids’ in respective clusters from which the distances to all other sequences were minimized; 110 16S rRNA gene sequences with identifications recorded only at the genus level were classified using machine learning methods. Simple kNN machine learning demonstrated the highest performance and classified Nocardia species sequences with an accuracy of 92.7% and a mean frequency of 0.578.

Conclusion

The identification of centroids of 16S rRNA gene sequence clusters using novel distance matrix clustering enables the identification of the most representative sequences for each individual species of Nocardia and allows the quantitation of inter- and intra-species variability.     

The Hiroshima thermal-neutron discrepancy for 36Cl at large distances. Part II: Natural in situ production as a source

For Hiroshima, a large discrepancy between calculated and measured thermal-neutron fluences had been reported in the past, for distances to the epicenter larger than about 1,000 m. To be more specific, measured (36)Cl concentrations in environmental samples from Hiroshima were too large at these distances, and the ratio of measured to calculated values reached about 70, at a distance of 1,800 m. In an attempt to identify other sources that might also produce (36)Cl in Hiroshima samples, the role of cosmic rays and of neutrons from natural terrestrial sources was investigated. Four reaction mechanisms were taken into account: spallation reactions of the nucleonic (hadronic) component of the cosmic rays on potassium (K) and calcium (Ca) in the sample material, particle emission after nuclear capture of negative muons by K and Ca, reactions of fast-muon induced electromagnetic, and hadronic showers with K and Ca, and neutron capture reactions with (35)Cl in the sample where the neutrons originate from the above three reaction mechanisms and from uranium and thorium decay. These mechanisms are physically described and mathematically quantified. It is shown that among those parameters important for the production of (36)Cl in granite, the chemical composition of the sample, the depth in the quarry where the sample had initially been taken, and the erosion rate at the site of the quarry are most important. Based on these physical, chemical, and geological parameters, (36)Cl concentrations were calculated for different types of granite that are typical for the Hiroshima area. In samples that were of these granite types and that had not been exposed to atomic bomb(A-bomb) neutrons, the (36)Cl concentration was also determined experimentally by means of accelerator mass spectrometry, and good agreement was found with the calculated values. The (36)Cl signal due to natural in situ production was also calculated in granite samples that had been exposed to A-bomb neutrons at distances up to 1,500 m from the hypocenter. It is demonstrated that, for granite samples from Hiroshima exposed to A-bomb neutrons beyond distances of about 1,300 m from the hypocenter, the (36)Cl signal is dominated by natural in situ production.     

Ser1928 is a common site for Cav1.2 phosphorylation by protein kinase C isoforms

Voltage-dependent Ca(2+) channel (Ca(v)1.2, L-type Ca(2+) channel) function is highly regulated by hormones and neurotransmitters in large part through the activation of kinases and phosphatases. Regulation of Ca(v)1.2 by protein kinase C (PKC) is of significant physiologic importance, mediating, in part, the cardiac response to hormonal regulation. Although PKC has been reported to mediate activation and/or inhibition of Ca(v)1.2 function, the molecular mechanisms mediating the response have not been definitively elucidated. We show that PKC forms a macromolecular complex with the alpha(1c) subunit of Ca(v)1.2 through direct interaction with the C terminus. This interaction leads to phosphorylation of the channel in response to activators of PKC. We identify Ser(1928) as the residue that is phosphorylated by PKC in vitro and in vivo. Ser(1928) has been identified previously as the site mediating, in part, the protein kinase A up-regulation of channel activity. Thus, the protein kinase A and PKC signaling pathways converge on the Ca(v)1.2 complex at Ser(1928) to increase channel activity. Our results identify two mechanisms leading to regulation of Ca(v)1.2 activity by PKC: pre-association of the channel with PKC isoforms and phosphorylation of specific sites within the alpha(1c) subunit.     

Quantitative 13C and 2H NMR relaxation studies of the 723-residue enzyme malate synthase G reveal a dynamic binding interface

A detailed understanding of molecular recognition is predicated not only on high-resolution static structures of the free and bound states but also on information about how these structures change with time, that is, molecular dynamics. Here we present a deuterium ((2)H) and carbon ((13)C) NMR relaxation study of methyl side chain dynamics in the 82 kDa enzyme malate synthase G (MSG) that is a promising target for the development of new antibiotic agents. It is shown that excellent agreement between (2)H- and (13)C-derived measures of dynamics is obtained, with correlation coefficients exceeding 0.95. The binding interface formed by MSG and its substrates is found to be highly dynamic in the ligand-free state of the enzyme with rigidification upon binding substrate. This study establishes that detailed, quantitative information about methyl side chain dynamics can be obtained by NMR on proteins with molecular masses on the order of 100 kDa and opens up the possibilities for studies of motion in a large number of important systems.