Bayesian Variable Selection with Joint Modeling of Categorical and Survival Outcomes: An Application to Individualizing Chemotherapy Treatment in Advanced Colorectal Cancer

Summary Colorectal cancer is the second leading cause of cancer related deaths in the United States, with more than 130,000 new cases of colorectal cancer diagnosed each year. Clinical studies have shown that genetic alterations lead to different responses to the same treatment, despite the morphologic similarities of tumors. A molecular test prior to treatment could help in determining an optimal treatment for a patient with regard to both toxicity and efficacy. This article introduces a statistical method appropriate for predicting and comparing multiple endpoints given different treatment options and molecular profiles of an individual. A latent variable‐based multivariate regression model with structured variance covariance matrix is considered here. The latent variables account for the correlated nature of multiple endpoints and accommodate the fact that some clinical endpoints are categorical variables and others are censored variables. The mixture normal hierarchical structure admits a natural variable selection rule. Inference was conducted using the posterior distribution sampling Markov chain Monte Carlo method. We analyzed the finite‐sample properties of the proposed method using simulation studies. The application to the advanced colorectal cancer study revealed associations between multiple endpoints and particular biomarkers, demonstrating the potential of individualizing treatment based on genetic profiles.     

Impact of DNA gyrase inhibition by antisense ribozymes on rec A in <Emphasis Type="Italic">E. coli</Emphasis>

The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluroquinolones.     

Modeling and experimental studies on intermittent starch feeding and citrate addition in simultaneous saccharification and fermentation of starch to flavor compounds

Simultaneous saccharification and fermentation (SSF) is a combined process of saccharification of a renewable bioresource and fermentation process to produce products, such as lactic acid and ethanol. Recently, SSF has been extensively used to convert various sources of cellulose and starch into fermentative products. Here, we present a study on production of buttery flavors, namely diacetyl and acetoin, by growing Lactobacillus rhamnosus on a starch medium containing the enzyme glucoamylase. We further develop a structured kinetics for the SSF process, which includes enzyme and growth kinetics. The model was used to simulate the effect of pH and temperature on the SSF process so as to obtain optimum operating conditions. The model was experimentally verified by conducting SSF using an initial starch concentration of 100 g/L. The study demonstrated that the developed kinetic was able to suggest strategies for improved productivities. The developed model was able to accurately predict the enhanced productivity of flavors in a three stage process with intermittent addition of starch. Experimental and simulations demonstrated that citrate addition can also lead to enhanced productivity of flavors. The developed optimal model for SSF was able to capture the dynamics of SSF in batch mode as well as in a three stage process. The structured kinetics was also able to quantify the effect of multiple substrates present in the medium. The study demonstrated that structured kinetic models can be used in the future for design and optimization of SSF as a batch or a fed-batch process. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Evaluation of RAPD-PCR and protein profile analysis to differentiate <Emphasis Type="Italic">Vibrio harveyi</Emphasis> strains prevalent along the southwest coast of India

Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index (D) value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.     

An analysis of acetylcholinesterase sequence for predicting mechanisms of its non-catalytic actions

The enzyme acetylcholinesterase (AChE) which belongs to the family of alpha/beta hydrolases is well known for hydrolyzing the neurotransmitter acetylcholine (ACh). In addition to its catalytic function, AChE appears to play a significant non-catalytic role in development, regeneration and modulation of properties of neurons. However the mechanisms underlying these important actions of AChE are unknown. It prompted the analysis of the sequence of fetal bovine serum AChE to seek clue(s) for the mechanisms of AChE non-catalytic actions. The searches for motifs, finger prints and domains demonstrated the presence of a highly conserved carboxylesterase type B signature in AChE from slime molds to human. Interestingly, the presence of finger prints for a metabotropic glutamate receptor, gap junction protein connexin, a calcium binding motif, several phosphorylation sites, and a motif similar to nicotinic acetylcholine receptor were also found in the sequence of AChE suggesting a role for AChE in cell signalling. The similarity search using gapped BLAST retrieved previously known similar proteins, namely neurotactin, neuroligin and thyroglobulin. When aligned neurotactin, neuroligin and bovine AChE showed the presence of three stretches of consensus, one of which is carboxylesterase B signature. The identity of the other two are not known. Amino acids in these stretches in AChE may be involved in possible growth factor like action and awaits experimental verification. Thus sequence analysis can be very useful for biologists while searching for a possible mechanism of action of a protein. Further, these results underscore the significance of performing both local and global searches to identify possible functional domains as well as consensus sequence.     

Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.     

The 1.59 Å resolution structure of the minor pseudopilin EpsH of Vibrio cholerae reveals a long flexible loop

The type II secretion complex exports folded proteins from the periplasm to the extracellular milieu. It is used by the pathogenic bacterium Vibrio cholerae to export several proteins, including its major virulence factor, cholera toxin. The pseudopilus is an essential component of the type II secretion system and likely acts as a piston to push the folded proteins across the outer membrane through the secretin pore. The pseudopilus is composed of the major pseudopilin, EpsG, and four minor pseudopilins, EpsH, EpsI, EpsJ and EpsK. We determined the x-ray crystal structure of the head domain of EpsH at 1.59 Å resolution using molecular replacement with the previously reported EpsH structure, 2qv8, as the template. Three additional N-terminal amino acids present in our construct prevent an artifactual conformation of residues 160–166, present in one of the two monomers of the 2qv8 structure. Additional crystal contacts stabilize a long flexible loop comprised of residues 104–135 that is more disordered in the 2qv8 structure but is partially observed in our structure in very different positions for the two EpsH monomers in the asymmetric unit. In one of the conformations the loop is highly extended. Modeling suggests the highly charged loop is capable of contacting EpsG and possibly secreted protein substrates, suggesting a role in specificity of pseudopilus assembly or secretion function.     

Development and characterization of microsatellite markers in Indian sesame (Sesamum indicum L.)

The genetic characterization of Indian sesame cultivars and related wild species was analysed using 102 simple sequence repeat (SSR; microsatellite) markers. Of these, 62 were novel sesame-specific microsatellites isolated in the course of the present investigation by constructing genomic libraries. Characterization of the 68 sesame accessions and three related wild species using 72 polymorphic SSR primers resulted in the detection of 170 alleles. The number of alleles ranged from two to four with an average of 2.5 alleles per locus. Polymorphic information content of the markers ranged from 0.43 to 0.88 with an average of 0.66. UPGMA cluster analysis grouped all the accessions into two major clusters with a genetic similarity ranging from 0.40 to 0.91. A moderate to high level of genetic variability was observed. The three wild accessions used in the study formed separate clades and distant genetic relationships were observed between the cultivar lines and wild species. Differentiation of genotypes according to geographical region was not observed. Analysis of molecular variance (AMOVA) analysis revealed that a high percentage of variation was within populations (87.1 %). An overall F _st of 0.11 among the populations indicated low population differentiation. The SSR markers developed will be useful for further genetic analysis, linkage mapping and selection of parents in future breeding programmes.     

Growth optimization of an ectomycorrhizal fungus with respect to pH and temperature in vitro, using Design of Experiments

The effect of external factors like pH of the medium, temperature and incubation period on the growth of the ectomycorrhizal fungus, Pisolithus tinctorius was studied as a function of the total fungal biomass produced, extracellular protein content and residual pH of the medium. The effect of the three parameters on the three responses was found to be significant and interdependent. Therefore, an attempt was made to optimize the growth of the fungus under varying conditions of pH, temperature and incubation period using the Box-Behnken Design of Experiments which is a second order model involving Response Surface Methodology and a second order quadratic equation. With this design expert, the optimum conditions of the three parameters that favoured the maximum growth of the fungus in vitro were found. The experimental values corroborated with the predicted values got from the model with the correlation coefficients 0.9349 for biomass, 0.9913 for protein and 0.9959 for final pH.