NEXCADE: Perturbation Analysis for Complex Networks

Recent advances in network theory have led to considerable progress in our understanding of complex real world systems and their behavior in response to external threats or fluctuations. Much of this research has been invigorated by demonstration of the 'robust, yet fragile' nature of cellular and large-scale systems transcending biology, sociology, and ecology, through application of the network theory to diverse interactions observed in nature such as plant-pollinator, seed-dispersal agent and host-parasite relationships. In this work, we report the development of NEXCADE, an automated and interactive program for inducing disturbances into complex systems defined by networks, focusing on the changes in global network topology and connectivity as a function of the perturbation. NEXCADE uses a graph theoretical approach to simulate perturbations in a user-defined manner, singly, in clusters, or sequentially. To demonstrate the promise it holds for broader adoption by the research community, we provide pre-simulated examples from diverse real-world networks including eukaryotic protein-protein interaction networks, fungal biochemical networks, a variety of ecological food webs in nature as well as social networks. NEXCADE not only enables network visualization at every step of the targeted attacks, but also allows risk assessment, i.e. identification of nodes critical for the robustness of the system of interest, in order to devise and implement context-based strategies for restructuring a network, or to achieve resilience against link or node failures. Source code and license for the software, designed to work on a Linux-based operating system (OS) can be downloaded at http://www.nipgr.res.in/nexcade_download.html. In addition, we have developed NEXCADE as an OS-independent online web server freely available to the scientific community without any login requirement at http://www.nipgr.res.in/nexcade.html.     

Organizational and Functional Status of the Y-linked Genes and Loci in the Infertile Patients Having Normal Spermiogram

Male fertility is an orchestrated interplay of loci on the Y chromosome with a number of genes from across the other chromosomes. In this context, micro-deletions in the Y chromosome have been correlated with spermatogenic failure often leading to infertility. However, causes of infertility in the patients with the normal spermiogram have remained unclear and therefore pose another level of challenge. In the present study, we analyzed 64 STSs, studied different Y-linked genes and loci and conducted single nucleotide variant (SNV) analyses in 31 infertile males with normal spermiogram along with 67 normal fertile males (NFMs) to gain an insight into the organization of their Y chromosome. Further, employing quantitative real-time PCR (qPCR), we studied copy number variation of DYZ1 arrays and three genes and mutational status of SRY by direct sequence analyses. STS analyses of the AZFa, b and c regions in these patients showed known and new mutations. Further, copies of DAZ and BPY2 in the patients were found to be affected [Formula: see text] compared to those in NFMs. All the patients had normal copy number of the SRY however its sequence analysis (in silico) showed mutations in eight patients. In four of these eight patients, SRY mutations resulted into truncated proteins. Similarly, DYZ1 analysis showed micro-deletions and it's much reduced copy number [Formula: see text] as compared to those in NFMs. Present study in males with unexplained infertility revealed deletions similar to those observed in oligospermic and azoospermic patients. Thus, there are some common but still unknown factors underlying infertility in these patients irrespective of their spermatogenic status. This work is envisaged to augment DNA diagnosis, proving beneficial in the context of in vitro fertilization (IVF) and genetic counselling.     

Enhanced Expression of Rabies Virus Surface G-Protein in Escherichia coli using SUMO Fusion

Fusion systems are known to increase the expression of difficult to express recombinant proteins in soluble form to facilitate their purification. Rabies glycoprotein was also tough to express at sufficient level in soluble form in both E. coli and plant. The present work was aimed to over-express and purify this membrane protein from soluble extract of E. coli. Fusion of Small Ubiqutin like Modifier (SUMO) with rabies glycoprotein increased ~1.5 fold higher expression and ~3.0 fold solubility in comparison to non-fused in E. coli. The SUMO fusion also simplified the purification process. Previously engineered rabies glycoprotein gene in tobacco plants provides complete protection to mice, but the expression was very low for purification. Our finding demonstrated that the SUMO-fusion was useful for enhancing expression and solubility of the membrane protein and again proves to be a good alternative technology for applications in biomedical and pharmaceutical research.     

Gene expression profiling through microarray analysis in Arabidopsis thaliana colonized by Pseudomonas putida MTCC5279, a plant growth promoting rhizobacterium

Plant growth promotion is a multigenic process under the influence of many factors; therefore an understanding of these processes and the functions regulated may have profound implications. Present study reports microarray analysis of Arabidopsis thaliana plants inoculated with Pseudomonas putida MTCC5279 (MTCC5279) which resulted in significant increase in growth traits as compared with non-inoculated control. The gene expression changes, represented by oligonucleotide array (24652 genes) have been studied to gain insight into MTCC5279 assisted plant growth promotion in Arabidopsis thaliana. MTCC5279 induced upregulated Arabidopsis thaliana genes were found to be involved in maintenance of genome integrity (At5g20850), growth hormone (At3g23890 and At4g36110), amino acid synthesis (At5g63890), abcissic acid (ABA) signaling and ethylene suppression (At2g29090, At5g17850), Ca⁺² dependent signaling (At3g57530) and induction of induced systemic resistance (At2g46370, At2g44840). The genes At3g32920 and At2g15890 which are suggested to act early in petal, stamen and embryonic development are among the downregulated genes. We report for the first time MTCC5279 assisted repression of At3g32920, a putative DNA repair protein involved in recombination and DNA strand transfer in a process of rapid meiotic and mitotic division.     

Prevalence of methylenetetrahydrofolate reductase C677T polymorphism in eastern Uttar Pradesh

AIM:

This study was aimed to evaluate the 5, 10-methylenetetrahydrofolate reductase (MTHFR) C677T mutation in eastern Uttar Pradesh population.

MATERIALS AND METHODS:

Polymerase chain reaction (PCR) using specific primers followed by amplicon digestion by Hinf I restriction enzyme was used for MTHFR C677T polymorphism analysis. Total 250 subjects were analyzed.

RESULTS:

The CC genotype was found in 192 subjects, followed by CT in 56 subjects and TT in 2 subject. Genotype frequencies of CC, CT and TT were 0.768, 0.224 and 0.008, respectively. The frequency of C allele was found to be 0.88 and that of T allele was 0.12.

CONCLUSION:

It is evident from the results of the present study that the percentage of homozygous genotype (CC) is highest in the target population.     

Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment.     

Metabolic pathway analysis and molecular docking analysis for identification of putative drug targets in Toxoplasma gondii: novel approach

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that can infect a wide range of warm-blooded animals including humans. In humans and other intermediate hosts, toxoplasma develops into chronic infection that cannot be eliminated by host's immune response or by currently used drugs. In most cases, chronic infections are largely asymptomatic unless the host becomes immune compromised. Thus, toxoplasma is a global health problem and the situation has become more precarious due to the advent of HIV infections and poor toleration of drugs used to treat toxoplasma infection, having severe side effects and also resistance have been developed to the current generation of drugs. The emergence of these drug resistant varieties of T. gondii has led to a search for novel drug targets. We have performed a comparative analysis of metabolic pathways of the host Homo sapiens and the pathogen T. gondii. The enzymes in the unique pathways of T. gondii, which do not show similarity to any protein from the host, represent attractive potential drug targets. We have listed out 11 such potential drug targets which are playing some important work in more than one pathway. Out of these, one important target is Glutamate dehydrogenase enzyme; it plays crucial part in oxidation reduction, metabolic process and amino acid metabolic process. As this is also present in the targets of tropical diseases of TDR (Tropical disease related Drug) target database and no PDB and MODBASE 3D structural model is available, homology models for Glutamate dehydrogenase enzyme were generated using MODELLER9v6. The model was further explored for the molecular dynamics simulation study with GROMACS, virtual screening and docking studies with suitable inhibitors against the NCI diversity subset molecules from ZINC database, by using AutoDock-Vina. The best ten docking solutions were selected (ZINC01690699, ZINC17465979, ZINC17465983, ZINC18141294_03, ZINC05462670, ZINC01572309, ZINC18055497_01, ZINC18141294, ZINC05462674 and ZINC13152284_01). Further the Complexes were analyzed through LIGPLOT. On the basis of Complex scoring and binding ability it is deciphered that these NCI diversity set II compounds, specifically ZINC01690699 (as it has minimum energy score and one of the highest number of interactions with the active site residue), could be promising inhibitors for T. gondii using Glutamate dehydrogenase as Drug target.     

Crystal structure of the novel complex formed between zinc alpha2-glycoprotein (ZAG) and prolactin-inducible protein (PIP) from human seminal plasma

This is the first report on the formation of a complex between zinc α2-glycoprotein (ZAG) and prolactin-inducible protein (PIP). The complex was purified from human seminal plasma and crystallized using 20% polyethylene glycol 9000 and 5% hexaethylene glycol. The structure of the complex has been determined using X-ray crystallographic method and refined to an R_cryst of 0.199 (R_free = 0.239). The structure of ZAG is broadly similar to the structure of serum ZAG. The scaffolding of PIP consists of seven β-strands that are organized in the form of two antiparallel β-pleated sheets, resulting in the formation of a sandwiched β-sheet. The amino acid sequence of PIP contains one potential N-glycosylation site at Asn77, and the same is found glycosylated with four sugar residues. The structure of the complex shows that the β-structure of PIP is ideally aligned with the β-structure of domain α3 of ZAG to form a long interface between two proteins. The proximal β-strands at the long interface are arranged in an antiparallel manner. There are 12 hydrogen bonds and three salt bridges between ZAG and PIP. At the two ends of vertical interface, two salt bridges are formed between pairs of Lys41-Asp233 and Lys68-Glu229. On the perpendicular interface involving α1-α2 domains of ZAG and a loop of PIP, another salt bridge is formed. The internal space at the corner of the L-shaped structure is filled with solvent molecules including a carbonate ion. The overall buried area in the complex is approximately 914 Å², which is considerably higher than the 660 Å² reported for the class I major histocompatibility complex structures.     

Specific detection and quantification of culturable and non-culturable mycobacteria in metalworking fluids by fluorescence-based methods

Aims: To optimize and evaluate fluorescence microscopy assays for specific assessment of mycobacteria and co‐contaminants, including culturable and non‐culturable sub‐populations, in metalworking fluids (MWF). Methods and Results: Auramine‐O‐rhodamine (AR) staining and LIVE/DEAD BacLight? Bacterial Viability staining (L/D staining) were adapted and evaluated for detection/quantification and differentiation (viable vs non‐viable) of the MWF‐associated mycobacteria and the background bacterial flora, respectively. The AR staining method was found to be specific to MWF mycobacteria with a minimum detection limit of 10 cells ml^?1 and was comparable to the QPCR in quantification efficiency in MWF matrix. The L/D staining‐based microscopy allowed differential quantification of viable vs non‐viable cells. In general, a 3‐log difference was observed between the L/D microscopy count and culture count accounting for the presence of non‐culturable fraction in the bacterial population in in‐use MWF. Conclusions: The optimized AR staining‐ and the L/D staining‐based microscopy methods have the potential for rapid, specific and differential assessment (viable vs non‐viable) of MWF‐associated mycobacteria and co‐contaminants in field MWF. Significance and Impact of the study: Early detection of MWF mycobacteria by rapid, low‐cost, less‐skill intensive and culture‐independent fluorescence‐based microscopy methods will facilitate timely intervention to protect the machine workers from occupational hazards.