Reducing false-positive prediction of minimotifs with a genetic interaction filter

Background

Minimotifs are short contiguous peptide sequences in proteins that have known functions. At its simplest level, the minimotif sequence is present in a source protein and has an activity relationship with a target, most of which are proteins. While many scientists routinely investigate new minimotif functions in proteins, the major web-based discovery tools have a high rate of false-positive prediction. Any new approach that reduces false-positives will be of great help to biologists.

Methods and Findings

We have built three filters that use genetic interactions to reduce false-positive minimotif predictions. The basic filter identifies those minimotifs where the source/target protein pairs have a known genetic interaction. The HomoloGene genetic interaction filter extends these predictions to predicted genetic interactions of orthologous proteins and the node-based filter identifies those minimotifs where proteins that have a genetic interaction with the source or target have a genetic interaction. Each filter was evaluated with a test data set containing thousands of true and false-positives. Based on sensitivity and selectivity performance metrics, the basic filter had the best discrimination for true positives, whereas the node-based filter had the highest sensitivity. We have implemented these genetic interaction filters on the Minimotif Miner 2.3 website. The genetic interaction filter is particularly useful for improving predictions of posttranslational modifications such as phosphorylation and proteolytic cleavage sites.

Conclusions

Genetic interaction data sets can be used to reduce false-positive minimotif predictions. Minimotif prediction in known genetic interactions can help to refine the mechanisms behind the functional connection between genes revealed by genetic experimentation and screens.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

Na,K-ATPase is a target of cigarette smoke and reduced expression predicts poor patient outcome of smokers with lung cancer

Diminished Na,K-ATPase expression has been reported in several carcinomas and has been linked to tumor progression. However, few studies have determined whether Na,K-ATPase function and expression are altered in lung malignancies. Because cigarette smoke (CS) is a major factor underlying lung carcinogenesis and progression, we investigated whether CS affects Na,K-ATPase activity and expression in lung cell lines. Cells exposed to CS in vitro showed a reduction of Na,K-ATPase activity. We detected the presence of reactive oxygen species (ROS) in cells exposed to CS before Na,K-ATPase inhibition, and neutralization of ROS restored Na,K-ATPase activity. We further determined whether Na,K-ATPase expression correlated with increasing grades of lung adenocarcinoma and survival of patients with smoking history. Immunohistochemical analysis of lung adenocarcinoma tissues revealed reduced Na,K-ATPase expression with increasing tumor grade. Using tissue microarray containing lung adenocarcinomas of patients with known smoking status, we found that high expression of Na,K-ATPase correlated with better survival. For the first time, these data demonstrate that CS is associated with loss of Na,K-ATPase function and expression in lung carcinogenesis, which might contribute to disease progression.     

Achieving High Accuracy Prediction of Minimotifs

The low complexity of minimotif patterns results in a high false-positive prediction rate, hampering protein function prediction. A multi-filter algorithm, trained and tested on a linear regression model, support vector machine model, and neural network model, using a large dataset of verified minimotifs, vastly improves minimotif prediction accuracy while generating few false positives. An optimal threshold for the best accuracy reaches an overall accuracy above 90%, while a stringent threshold for the best specificity generates less than 1% false positives or even no false positives and still produces more than 90% true positives for the linear regression and neural network models. The minimotif multi-filter with its excellent accuracy represents the state-of-the-art in minimotif prediction and is expected to be very useful to biologists investigating protein function and how missense mutations cause disease.     

A novel computational and structural analysis of nsSNPs in CFTR gene

Single Nucleotide Polymorphisms (SNPs) are being intensively studied to understand the biological basis of complex traits and diseases. The Genetics of human phenotype variation could be understood by knowing the functions of SNPs. In this study using computational methods, we analyzed the genetic variations that can alter the expression and function of the CFTR gene responsible candidate for causing cystic fibrosis. We applied an evolutionary perspective to screen the SNPs using a sequence homology-based SIFT tool, which suggested that 17 nsSNPs (44%) were found to be deleterious. The structure-based approach PolyPhen server suggested that 26 nsSNPS (66%) may disrupt protein function and structure. The PupaSuite tool predicted the phenotypic effect of SNPs on the structure and function of the affected protein. Structure analysis was carried out with the major mutation that occurred in the native protein coded by CFTR gene, and which is at amino acid position F508C for nsSNP with id (rs1800093). The amino acid residues in the native and mutant modeled protein were further analyzed for solvent accessibility, secondary structure and stabilizing residues to check the stability of the proteins. The SNPs were further subjected to iHAP analysis to identify htSNPs, and we report potential candidates for future studies on CFTR mutations.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Recurrent heat shock impairs the proliferation and differentiation of C2C12 myoblasts

Heat-related illness and injury are becoming a growing safety concern for the farmers, construction workers, miners, firefighters, manufacturing workers, and other outdoor workforces who are exposed to heat stress in their routine lives. A primary response by a cell to an acute heat shock (HS) exposure is the induction of heat-shock proteins (HSPs), which chaperone and facilitate cellular protein folding and remodeling processes. While acute HS is well studied, the effect of repeated bouts of hyperthermia and the sustained production of HSPs in the myoblast-myotube model system of C2C12 cells are poorly characterized. In C2C12 myoblasts, we found that robust HS (43 °C, dose/time) significantly decreased the proliferation by 50% as early as on day 1 and maintained at the same level on days 2 and 3 of HS. This was accompanied by an accumulation of cells at G2 phase with reduced cell number in G1 phase indicating cell cycle arrest. FACS analysis indicates that there was no apparent change in apoptosis (markers) and cell death upon repeated HS. Immunoblot analysis and qPCR demonstrated a significant increase in the baseline expression of HSP25, 70, and 90 (among others) in cells after a single HS (43 °C) for 60 min as a typical HS response. Importantly, the repeated HS for 60 min each on days 2 and 3 maintained the elevated levels of HSPs compared to the control cells. Further, the continuous HS exposure resulted in significant inhibition of the differentiation of C2C12 myocytes to myotubes and only 1/10th of the cells underwent differentiation in HS relative to control. This was associated with significantly higher levels of HSPs and reduced expression of myogenin and Myh2 (P < 0.05), the genes involved in the differentiation process. Finally, the cell migration (scratch) assay indicated that the wound closure was significantly delayed in HS cells relative to the control cells. Overall, these results suggest that a repeated HS may perturb the active process of proliferation, motility, and differentiation processes in an in vitro murine myoblast-myotube model.     

Physiological significance of proline and glycinebetaine: Maintaining photosynthesis during NaCl stress in wheat

Experiments on the physiological significance of accumulation of proline and glycinebetaine (GB) in sustaining photosynthesis during salt stress in wheat in vivo showed that pre-treatment with GB, but not proline, alleviated NaCl-induced stomatal and non-stomatal inhibition of photosynthesis completely. A permeating and non-dissociating osmoticum, 3-orthomethyl-glucopyranose, also alleviated NaCl-induced perturbations of photosynthesis, suggesting that GB may work by maintaining chloroplast volume and not by specific effects on photosynthetic processes.