CASVM: web server for SVM-based prediction of caspase substrates cleavage sites

Caspases belong to a unique class of cysteine proteases which function as critical effectors of apoptosis, inflammation and other important cellular processes. Caspases cleave substrates at specific tetrapeptide sites after a highly conserved aspartic acid residue. Prediction of such cleavage sites will complement structural and functional studies on substrates cleavage as well as discovery of new substrates. We have recently developed a support vector machines (SVM) method to address this issue. Our algorithm achieved an accuracy ranging from 81.25 to 97.92%, making it one of the best methods currently available. CASVM is the web server implementation of our SVM algorithms, written in Perl and hosted on a Linux platform. The server can be used for predicting non-canonical caspase substrate cleavage sites. We have also included a relational database containing experimentally verified caspase substrates retrievable using accession IDs, keywords or sequence similarity. AVAILABILITY: http://www.casbase.org/casvm/index.html     

Exome Sequencing of Phenotypic Extremes Identifies CAV2 and TMC6 as Interacting Modifiers of Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

Discovery of rare or low frequency variants in exome or genome data that are associated with complex traits often will require use of very large sample sizes to achieve adequate statistical power. For a fixed sample size, sequencing of individuals sampled from the tails of a phenotype distribution (i.e., extreme phenotypes design) maximizes power and this approach was recently validated empirically with the discovery of variants in DCTN4 that influence the natural history of P. aeruginosa airway infection in persons with cystic fibrosis (CF; MIM219700). The increasing availability of large exome/genome sequence datasets that serve as proxies for population-based controls affords the opportunity to test an alternative, potentially more powerful and generalizable strategy, in which the frequency of rare variants in a single extreme phenotypic group is compared to a control group (i.e., extreme phenotype vs. control population design). As proof-of-principle, we applied this approach to search for variants associated with risk for age-of-onset of chronic P. aeruginosa airway infection among individuals with CF and identified variants in CAV2 and TMC6 that were significantly associated with group status. These results were validated using a large, prospective, longitudinal CF cohort and confirmed a significant association of a variant in CAV2 with increased age-of-onset of P. aeruginosa airway infection (hazard ratio = 0.48, 95% CI=[0.32, 0.88]) and variants in TMC6 with diminished age-of-onset of P. aeruginosa airway infection (HR = 5.4, 95% CI=[2.2, 13.5]) A strong interaction between CAV2 and TMC6 variants was observed (HR=12.1, 95% CI=[3.8, 39]) for children with the deleterious TMC6 variant and without the CAV2 protective variant. Neither gene showed a significant association using an extreme phenotypes design, and conditions for which the power of an extreme phenotype vs. control population design was greater than that for the extreme phenotypes design were explored.     

HMMConverter 1.0: a toolbox for hidden Markov models

Hidden Markov models (HMMs) and their variants are widely used in Bioinformatics applications that analyze and compare biological sequences. Designing a novel application requires the insight of a human expert to define the model''s architecture. The implementation of prediction algorithms and algorithms to train the model''s parameters, however, can be a time-consuming and error-prone task. We here present HMMConverter, a software package for setting up probabilistic HMMs, pair-HMMs as well as generalized HMMs and pair-HMMs. The user defines the model itself and the algorithms to be used via an XML file which is then directly translated into efficient C++ code. The software package provides linear-memory prediction algorithms, such as the Hirschberg algorithm, banding and the integration of prior probabilities and is the first to present computationally efficient linear-memory algorithms for automatic parameter training. Users of HMMConverter can thus set up complex applications with a minimum of effort and also perform parameter training and data analyses for large data sets.     

A novel selective growth medium-PCR assay to isolate and detect Sphingomonas in environmental samples

Sphingomonas species can be found ubiquitously in the environment and can be frequently found in surface biofilms. Some Sphingomonas strains are well known for metabolizing complex organic pollutants but some are opportunistic human pathogens. Despite the importance of the Sphingomonas species, a reliable system to isolate this group of bacteria from the environment has not been developed. In this study, a combined streptomycin-piperacillin selective growth medium/polymerase chain reaction (PCR) detection approach is developed to isolate and identify the Sphingomonas bacteria. A total of 72 known Sphingomonas strains (including 21 different Sphingomonas species type strains) and 14 non-Sphingomonas species were tested using a new Sphingomonas-specific growth medium containing 100 and 50 µg/ml streptomycin and piperacillin, respectively. All the Sphingomonas strains showed positive growth on the selective medium and no growth was shown by the non-Sphingomonas species. In addition, two sets of PCR primers targeting the serine palmitoyltransferase gene (spt), a crucial sphingolipid biosynthesis gene, were developed. With the exception of the Sphingomonas subarctica type strain, 71 of the 72 known Sphingomonas samples were amplified positively by either one or both of the spt-specific primers. None of the non-Sphingomonas bacteria were amplified by the spt primers. To verify the effectiveness of this novel approach for use in environmental screening applications the Sphingomonas selective medium was used to isolate 165 potential Sphingomonas isolates, including 101 yellow, 4 orange and 58 unpigmented isolates, from the influent water and biofilm samples of a pulp and paper mill in Northwestern Ontario. Screening of these isolates with the two Sphingomonas spt-PCR primer sets showed that 98% of the yellow isolates and 100% of the orange isolates were positive to the spt-PCR test. None of the unpigmented isolates was positive to the spt-PCR assay. The 16S rDNA of 17% of the spt + ve and − ve isolates were sequenced and analyzed. All of the yellow and orange pigmented isolates were Sphingomonas while none of the unpigmented isolates were Sphingomonas. REP-PCR was performed on 79 Sphingomonas samples randomly selected from the paper mill and hospital isolates and showed that a diverse group of Sphingomonas can be grown or isolated by our Sphingomonas selective growth medium. Therefore, by using the streptomycin-piperacillin selective growth medium in combination with the colour pigmentation and the positive spt-PCR reactions of the isolates, a diverse population of Sphingomonas strains can be isolated and identified from complex microbial communities with high accuracy.     

The discovery of novel tartrate-based TNF-α converting enzyme (TACE) inhibitors

A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP’s. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.     

The Effect of a DNA Damaging Agent on Embryonic Cell Cycles of the Cnidarian Hydractinia echinata

The onset of gastrulation at the Mid-Blastula Transition can accompany profound changes in embryonic cell cycles including the introduction of gap phases and the transition from maternal to zygotic control. Studies in Xenopus and Drosophila embryos have also found that cell cycles respond to DNA damage differently before and after MBT (or its equivalent, MZT, in Drosophila). DNA checkpoints are absent in Xenopus cleavage cycles but are acquired during MBT. Drosophila cleavage nuclei enter an abortive mitosis in the presence of DNA damage whereas post-MZT cells delay the entry into mitosis. Despite attributes that render them workhorses of embryonic cell cycle studies, Xenopus and Drosophila are hardly representative of diverse animal forms that exist. To investigate developmental changes in DNA damage responses in a distant phylum, I studied the effect of an alkylating agent, Methyl Methanesulfonate (MMS), on embryos of Hydractinia echinata. Hydractinia embryos are found to differ from Xenopus embryos in the ability to respond to a DNA damaging agent in early cleavage but are similar to Xenopus and Drosophila embryos in acquiring stronger DNA damage responses and greater resistance to killing by MMS after the onset of gastrulation. This represents the first study of DNA damage responses in the phylum Cnidaria.