Rationalization and prediction of drug resistant mutations in targets for clinical anti-tubercular drugs

Resistance to therapy limits the effectiveness of drug treatment in many diseases. Drug resistance can be considered as a successful outcome of the bacterial struggle to survive in the hostile environment of a drug-exposed cell. An important mechanism by which bacteria acquire drug resistance is through mutations in the drug target. Drug resistant strains (multi-drug resistant and extensively drug resistant) of Mycobacterium tuberculosis are being identified at alarming rates, increasing the global burden of tuberculosis. An understanding of the nature of mutations in different drug targets and how they achieve resistance is therefore important. An objective of this study is to first decipher sequence as well as structural bases for the observed resistance in known drug resistant mutants and then to predict positions in each target that are more prone to acquiring drug resistant mutations. A curated database containing hundreds of mutations in the 38 drug targets of nine major clinical drugs, associated with resistance is studied here. Mutations have been classified into those that occur in the binding site itself, those that occur in residues interacting with the binding site and those that occur in outer zones. Structural models of the wild type and mutant forms of the target proteins have been analysed to seek explanations for reduction in drug binding. Stability analysis of an entire array of 19 mutations at each of the residues for each target has been computed using structural models. Conservation indices of individual residues, binding sites and whole proteins are computed based on sequence conservation analysis of the target proteins. The analyses lead to insights about which positions in the polypeptide chain have a higher propensity to acquire drug resistant mutations. Thus critical insights can be obtained about the effect of mutations on drug binding, in terms of which amino acid positions and therefore which interactions should not be heavily relied upon, which in turn can be translated into guidelines for modifying the existing drugs as well as for designing new drugs. The methodology can serve as a general framework to study drug resistant mutants in other micro-organisms as well.

Generation and analysis of drought stressed subtracted expressed sequence tags from safflower (Carthamus tinctorius L.)

Drought is the most crucial environmental factor that limits productivity of many crop plants. Exploring novel genes and gene combinations is of primary importance in plant drought tolerance research. Stress tolerant genotypes/species are known to express novel stress responsive genes with unique functional significance. Hence, identification and characterization of stress responsive genes from these tolerant species might be a reliable option to engineer the drought tolerance. Safflower has been found to be a relatively drought tolerant crop and thus, it has been the choice of study to characterize the genes expressed under drought stress. In the present study, we have evaluated differential drought tolerance of two cultivars of safflower namely, A1 and Nira using selective physiological marker traits and we have identified cultivar A1 as relatively drought tolerant. To identify the drought responsive genes, we have constructed a stress subtracted cDNA library from cultivar A1 following subtractive hybridization. Analysis of similar to 1,300 cDNA clones resulted in the identification of 667 unique drought responsive ESTs. Protein homology search revealed that 521 (78 %) out of 667 ESTs showed significant similarity to known sequences in the database and majority of them previously identified as drought stress-related genes and were found to be involved in a variety of cellular functions ranging from stress perception to cellular protection. Remaining 146 (22 %) ESTs were not homologous to known sequences in the database and therefore, they were considered to be unique and novel drought responsive genes of safflower. Since safflower is a stress-adapted oil-seed crop this observation has great relevance. In addition, to validate the differential expression of the identified genes, expression profiles of selected clones were analyzed using dot blot (reverse northern), and northern blot analysis. We showed that these clones were differentially expressed under different abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance are discussed in our study.

The defect sequence for contractive tuples

We introduce the defect sequence for a contractive tuple of Hilbert space operators and investigate its properties. The defect sequence is a sequence of numbers, called defect dimensions associated with a contractive tuple. We show that there are upper bounds for the defect dimensions. The tuples for which these upper bounds are obtained, are called maximal contractive tuples. The upper bounds are different in the non-commutative and in the commutative case. We show that the creation operators on the full Fock space and the coordinate multipliers on the Drury-Arveson space are maximal. We also study pure tuples and see how the defect dimensions play a role in their irreducibility. (C) 2012 Elsevier Inc. All rights reserved.

Structural and molecular basis of interaction of HCV non-structural protein 5A with human casein kinase 1 alpha and PKR

Background: Interaction of non-structural protein 5A (NS5A) of Hepatitis C virus (HCV) with human kinases namely, casein kinase 1 alpha (ck1 alpha) and protein kinase R (PKR) have different functional implications such as regulation of viral replication and evasion of interferon induced immune response respectively. Understanding the structural and molecular basis of interactions of the viral protein with two different human kinases can be useful in developing strategies for treatment against HCV. Results: Serine 232 of NS5A is known to be phosphorylated by human ck1 alpha. A structural model of NS5A peptide containing phosphoacceptor residue Serine 232 bound to ck1 alpha has been generated using the known 3-D structures of kinase-peptide complexes. The substrate interacting residues in ck1 alpha has been identified from the model and these are found to be conserved well in the ck1 family. ck1 alpha - substrate peptide complex has also been used to understand the structural basis of association between ck1 alpha and its other viral stress induced substrate, tumour suppressor p53 transactivation domain which has a crystal structure available. Interaction of NS5A with another human kinase PKR is primarily genotype specific. NS5A from genotype 1b has been shown to interact and inhibit PKR whereas NS5A from genotype 2a/3a are unable to bind and inhibit PKR efficiently. This is one of the main reasons for the varied response to interferon therapy in HCV patients across different genotypes. Using PKR crystal structure, sequence alignment and evolutionary trace analysis some of the critical residues responsible for the interaction of NS5A 1b with PKR have been identified. Conclusions: The substrate interacting residues in ck1 alpha have been identified using the structural model of kinase substrate peptide. The PKR interacting NS5A 1b residues have also been predicted using PKR crystal structure, NS5A sequence analysis along with known experimental results. Functional significance and nature of interaction of interferon sensitivity determining region and variable region 3 of NS5A in different genotypes with PKR which was experimentally shown are also supported by the findings of evolutionary trace analysis. Designing inhibitors to prevent this interaction could enable the HCV genotype 1 infected patients respond well to interferon therapy.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Translocation and encapsulation of siRNA inside carbon nanotubes

We report spontaneous translocation of small interfering RNA (siRNA) inside carbon nanotubes (CNTs) of various diameters and chirality using all atom molecular dynamics simulations with explicit solvent. We use umbrella sampling method to calculate the free energy landscape of the siRNA entry and translocation event. Free energy profiles show that siRNA gains free energy while translocating inside CNT, and barrier for siRNA exit from CNT ranges from 40 to 110 kcal/mol depending on CNT chirality and salt concentration. The translocation time tau decreases with the increase of CNT diameter with a critical diameter of 24 angstrom for the translocation. In contrast, double strand DNA of the same sequence does not translocate inside CNT due to large free energy barrier for the translocation. This study helps in understanding the nucleic acid transport through nanopores at microscopic level and may help designing carbon nanotube based sensor for siRNA. (C) 2013 American Institute of Physics. http://dx.doi.org/10.1063/1.4773302]

InSt: an integrated steering framework for critical weather applications

Online remote visualization and steering of critical weather applications like cyclone tracking are essential for effective and timely analysis by geographically distributed climate science community. A steering framework for controlling the high-performance simulations of critical weather events needs to take into account both the steering inputs of the scientists and the criticality needs of the application including minimum progress rate of simulations and continuous visualization of significant events. In this work, we have developed an integrated user-driven and automated steering framework InSt for simulations, online remote visualization, and analysis for critical weather applications. InSt provides the user control over various application parameters including region of interest, resolution of simulation, and frequency of data for visualization. Unlike existing efforts, our framework considers both the steering inputs and the criticality of the application, namely, the minimum progress rate needed for the application, and various resource constraints including storage space and network bandwidth to decide the best possible parameter values for simulations and visualization.

Parametric study of the behavior of double toggle switching mechanisms

The paper identified and characterized a special multi-degree of freedom toggle behavior, called double toggle, observed in a typical MCCB switching mechanism. For an idealized system, the condition of toggle sequence is derived geometrically. The existing tools available in a multi-body dynamics package are used for exploring the dynamic behavior of such systems parametrically. The double toggle mechanism is found to make the system insensitive to the operator's behavior; however, the system is vulnerable under extreme usage. The linkage kinematics and stopper locations are found to have dominant role on the behavior of the system. It is revealed that the operating time is immune to the inertial property of the input link and sensitive to that of the output link. Novel designs exploiting this observation, in terms of spring and toggle placements, to enhance switching performance have also been reported in the paper. Detailed study revealed that strategic placement of the spring helps in selective alteration of system performance. Thus, the study establishes the critical importance of the kinematic design of MCCB over the dynamic parameters. (C) 2013 Elsevier Ltd. All rights reserved.

The curvature of the liquid-vapor reduced pressure curve and its relation with the critical region

For most fluids, there exist a maximum and a minimum in the curvature of the reduced vapor pressure curve, p(r) = p(r)(T-r) (with p(r) = p/p(c) and T-r = T/T-c, p(c) and T-c being the pressure and temperature at the critical point). By analyzing National Institute of Standards and Technology (NIST) data on the liquid-vapor coexistence curve for 105 fluids, we find that the maximum occurs in the reduced temperature range 0.5 <= T-r <= 0.8 while the minimum occurs in the reduced temperature range 0.980 <= T-r <= 0.995. Vapor pressure equations for which d(2)p(r)/dT(r)(2) diverges at the critical point present a minimum in their curvature. Therefore, the point of minimum curvature can be used as a marker for the critical region. By using the well-known Ambrose-Walton (AW) vapor pressure equation we obtain the reduced temperatures of the maximum and minimum curvature in terms of the Pitzer acentric factor. The AW predictions are checked against those obtained from NIST data. (C) 2013 Elsevier Ltd. All rights reserved.

Improved Detection of Remote Homologues Using Cascade PSI-BLAST: Influence of Neighbouring Protein Families on Sequence Coverage

Background: Development of sensitive sequence search procedures for the detection of distant relationships between proteins at superfamily/fold level is still a big challenge. The intermediate sequence search approach is the most frequently employed manner of identifying remote homologues effectively. In this study, examination of serine proteases of prolyl oligopeptidase, rhomboid and subtilisin protein families were carried out using plant serine proteases as queries from two genomes including A. thaliana and O. sativa and 13 other families of unrelated folds to identify the distant homologues which could not be obtained using PSI-BLAST. Methodology/Principal Findings: We have proposed to start with multiple queries of classical serine protease members to identify remote homologues in families, using a rigorous approach like Cascade PSI-BLAST. We found that classical sequence based approaches, like PSI-BLAST, showed very low sequence coverage in identifying plant serine proteases. The algorithm was applied on enriched sequence database of homologous domains and we obtained overall average coverage of 88% at family, 77% at superfamily or fold level along with specificity of similar to 100% and Mathew's correlation coefficient of 0.91. Similar approach was also implemented on 13 other protein families representing every structural class in SCOP database. Further investigation with statistical tests, like jackknifing, helped us to better understand the influence of neighbouring protein families. Conclusions/Significance: Our study suggests that employment of multiple queries of a family for the Cascade PSI-BLAST searches is useful for predicting distant relationships effectively even at superfamily level. We have proposed a generalized strategy to cover all the distant members of a particular family using multiple query sequences. Our findings reveal that prior selection of sequences as query and the presence of neighbouring families can be important for covering the search space effectively in minimal computational time. This study also provides an understanding of the `bridging' role of related families.

Diverse functions of restriction-modification systems in addition to cellular defense

Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

Queuing of concurrent movement plans by Basal Ganglia

How the brain converts parallel representations of movement goals into sequential movements is not known. We tested the role of basal ganglia (BG) in the temporal control of movement sequences by a convergent approach involving inactivation of the BG by muscimol injections into the caudate nucleus of monkeys and assessing behavior of Parkinson's disease patients, performing a modified double-step saccade task. We tested a critical prediction of a class of competitive queuing models that explains serial behavior as the outcome of a selection of concurrently activated goals. In congruence with these models, we found that inactivation or impairment of the BG unmasked the parallel nature of goal representations such that a significantly greater extent of averaged saccades, curved saccades, and saccade sequence errors were observed. These results suggest that the BG perform a form of competitive queuing, holding the second movement plan in abeyance while the first movement is being executed, allowing the proper temporal control of movement sequences.

Critical investigation on hydrogen bonding by Fourier transform infrared spectroscopy in hydrogenated amorphous silicon thin films

Fourier Transform Infrared (FTIR) spectroscopic analysis has been carried out on the hydrogenated amorphous silicon (a-Si:H) thin films deposited by DC, pulsed DC (PDC) and RF sputtering process to get insight regarding the total hydrogen concentration (C-H) in the films, configuration of hydrogen bonding, density of the films (decided by the vacancy and void incorporation) and the microstructure factor (R*) which varies with the type of sputtering carried out at the same processing conditions. The hydrogen incorporation is found to be more in RF sputter deposited films as compared to PDC and DC sputter deposited films. All the films were broadly divided into two regions namely vacancy dominated and void dominated regions. At low hydrogen dilutions the films are vacancy dominated and at high hydrogen dilutions they are void dominated. This demarcation is at C-H = 23 at.% H for RF, C-H = 18 at.% H for PDC and C-H = 14 at.% H for DC sputter deposited films. The microstructure structure factor R* is found to be as low as 0.029 for DC sputter deposited films at low C-H. For a given C-H, DC sputter deposited films have low R* as compared to PDC and RF sputter deposited films. Signature of dihydride incorporation is found to be more in DC sputter deposited films at low C-H.

Multiwalled-carbon-nanotube-induced miscibility in near-critical PS/PVME blends: assessment through concentration fluctuations and segmental relaxation

The effects of multiwalled carbon nanotubes (MWNTs) on the concentration fluctuations, interfacial driven elasticity, phase morphology, and local segmental dynamics of chains for near-critical compositions of polystyrene/poly(vinyl to methyl ether) (PS/PVME) blends were systematically investigated using dynamic shear rheology and dielectric spectroscopy. The contribution of the correlation length (xi) of the concentration fluctuations to the evolving stresses was monitored in situ to probe the different stages of demixing in the blends. The classical upturn in the dynamic moduli was taken as the rheological demixing temperature (T-rheo), which was also observed to be in close agreement with those obtained using concentration fluctuation variance, <(delta phi)(2)>, versus temperature curves. Further, Fredrickson and Larson's approach involving the mean-field approximation and the double-reptation self-concentration (DRSC) model was employed to evaluate the spinodal decomposition temperature (T-s). Interestingly, the values of both T-rheo and T-s shifted upward in the blends in the presence of MWNTs, manifesting in molecular-level miscibility. These phenomenal changes were further observed to be a function of the concentration of MWNTs. The evolution of morphology as a function of temperature was studied using polarized optical microscopy (POM). It was observed that PVME, which evolved as an interconnected network during the early stages of demixing, coarsened into a matrix-droplet morphology in the late stages. The preferential wetting of PVME onto MWNTs as a result of physicochemical interactions retained the interconnected network of PVME for longer time scales, as supported by POM and atomic force microscopy (AFM) images. Microscopic heterogeneity in macroscopically miscible systems was studied by dielectric relaxation spectroscopy. The slowing of segmental relaxations in PVME was observed in the presence of both ``frozen'' PS and MWNTs interestingly at temperatures much below the calorimetric glass transition temperature (T-g). This phenomenon was observed to be local rather than global and was addressed by monitoring the evolution of the relaxation spectra near and above the demixing temperature.

The sequence and structure of snake gourd (Trichosanthes anguina) seed lectin, a three-chain nontoxic homologue of type II RIPs

The sequence and structure of snake gourd seed lectin (SGSL), a nontoxic homologue of type II ribosome-inactivating proteins (RIPs), have been determined by mass spectrometry and X-ray crystallography, respectively. As in type II RIPs, the molecule consists of a lectin chain made up of two beta-trefoil domains. The catalytic chain, which is connected through a disulfide bridge to the lectin chain in type II RIPs, is cleaved into two in SGSL. However, the integrity of the three-dimensional structure of the catalytic component of the molecule is preserved. This is the first time that a three-chain RIP or RIP homologue has been observed. A thorough examination of the sequence and structure of the protein and of its interactions with the bound methyl-alpha-galactose indicate that the nontoxicity of SGSL results from a combination of changes in the catalytic and the carbohydrate-binding sites. Detailed analyses of the sequences of type II RIPs of known structure and their homologues with unknown structure provide valuable insights into the evolution of this class of proteins. They also indicate some variability in carbohydrate-binding sites, which appears to contribute to the different levels of toxicity exhibited by lectins from various sources.