NMRof peptides

Developments and applications of NMR spectroscopy especially with biomolecules has taken big strides over the decades. This review gives a brief overview of peptide analysis by NMR as carried out in the author’s laboratory. A brief introduction to peptide biomolecules and NMR useful parameters are discussed in the beginning. This is followed by diagnostics features observed in NMR for identification of secondary structures. It further goes on to show how a three dimensional structure could be obtained by all-important NOE and hydrogen bond information. Use of heteronuclear experiments, which could be done at natural abundance is also highlighted in getting more details of peptide structures.Applications using Solid state NMR at natural abundance in connecting peptide solution and x-ray structures is demonstrated with couple of examples.

Molecular Identification and Phylogeny of Bactrocera Species (Diptera: Tephritidae)

Fruit flies that belong to the genus Bactrocera (Diptera: Tephritidae) are major invasive pests of agricultural crops in Asia and Australia. Increased transboundary movement of agricultural produce has resulted in the chance introduction of many invasive species that include Bactrocera mainly as immature stages. Therefore quick and accurate species diagnosis is important at the port of entry, where morphological identification has a limited role, as it requires the presence of adult specimens and the availability of a specialist. Unfortunately when only immature stages are present, a lacunae in their taxonomy impedes accurate species diagnosis. At this juncture, molecular species diagnostics based on COX-I have become handy, because diagnosis is not limited by developmental stages. Yet another method of quick and accurate species diagnosis for Bactrocera spp. is based on the development of species-specific markers. This study evaluated the utility of COX-I for the quick and accurate species diagnosis of eggs, larvae, pupae and adults of B. zonata Saunders, B. tau Walker, and B. dorsalis Hendel. Furthermore the utility of species-specific markers in differentiating B. zonata (500bp) and B. tau (220bp) was shown. Phylogenetic relationships among five subgenera, viz., Austrodacus, Bactrocera, Daculus, Notodacus and Zeugodacus have been resolved employing the 5' region of COX-I (1490-2198); where COX-I sequences for B. dorsalis Hendel, B. tau Walker, B. correcta Bezzi and B. zonata Saunders from India were compared with other NCBI-GenBank accessions. Phylogenetic analysis employing Maximum Parsimony (MP) and Bayesian phylogenetic approach (BP) showed that the subgenus Bactrocera is monophyletic.

Language identification using parallel sub-word recognition

Parallel sub-word recognition (PSWR) is a new model that has been proposed for language identification (LID) which does not need elaborate phonetic labeling of the speech data in a foreign language. The new approach performs a front-end tokenization in terms of sub-word units which are designed by automatic segmentation, segment clustering and segment HMM modeling. We develop PSWR based LID in a framework similar to the parallel phone recognition (PPR) approach in the literature. This includes a front-end tokenizer and a back-end language model, for each language to be identified. Considering various combinations of the statistical evaluation scores, it is found that PSWR can perform as well as PPR, even with broad acoustic sub-word tokenization, thus making it an efficient alternative to the PPR system.

Robust Approach for Identification of Bad Data in State Estimation Using SLP Technique

This paper proposes a new approach for solving the state estimation problem. The approach is aimed at producing a robust estimator that rejects bad data, even if they are associated with leverage-point measurements. This is achieved by solving a sequence of Linear Programming (LP) problems. Optimization is carried via a new algorithm which is a combination of “upper bound optimization technique" and “an improved algorithm for discrete linear approximation". In this formulation of the LP problem, in addition to the constraints corresponding to the measurement set, constraints corresponding to bounds of state variables are also involved, which enables the LP problem more efficient in rejecting bad data, even if they are associated with leverage-point measurements. Results of the proposed estimator on IEEE 39-bus system and a 24-bus EHV equivalent system of the southern Indian grid are presented for illustrative purpose.

Higher topoisomerase 2 alpha gene transcript levels predict better prognosis in GBM patients receiving temozolomide chemotherapy: identification of temozolomide as a TOP2A inhibitor

The search for molecular markers which predict response to chemotherapy is an important aspect of current neuro-oncology research. MGMT promoter methylation is the only proved marker of glioblastoma. The purpose of this study was to assess the effect of topoisomerase expression on glioblastoma survival and study the mechanisms involved. The transcript levels of all isoforms of the topoisomerase family in all grades of diffuse astrocytoma were assessed. A prospective study of patients with glioblastoma treated by a uniform treatment procedure was performed with the objective of correlating outcome with gene expression. The ability of TOP2A enzyme to relax the super coiled plasmid DNA in the presence of temozolomide was evaluated to assess its effect on TOP2A. The temozolomide cyctotoxicity of TOP2A-silenced U251 cells was assessed. The transcript levels of TOP2A, TOP2B, and TOP3A are upregulated significantly in GBM in comparison with lower grades of astrocytoma and normal brain samples. mRNA levels of TOP2A correlated significantly with survival of the patients. Higher TOP2A transcript levels in GBM patients predicted better prognosis (P = 0.043; HR = 0.889). Interestingly, we noted that temozolomide inhibited TOP2A activity in in-vitro enzyme assays. We also noted that siRNA knock down of TOP2A rendered a glioma cell line resistant to temozolomide chemotherapy. We demonstrated for the first time that temozolomide is also a TOP2A inhibitor and established that TOP2A transcript levels determine the chemosensitivity of glioblastoma to temozolomide therapy. Very high levels of TOP2A are a good prognostic indicator in GBM patients receiving temozolomide chemotherapy.

Isomeric identification of methylated naphthalenes using gas phase infrared spectroscopy

We report gas phase mid-infrared spectra of 1- and 2- methyl naphthalenes at 0.2 cm(-1) resolution. Assignment of observed bands have been made using scaled quantum mechanical (SQM) calculations where the force fields rather the frequencies are scaled to find a close fit between observed and calculated bands. The structure of the molecules has been optimized using B3LYP level of theory in conjunction with standard 6-311G** basis set to obtain the harmonic frequencies. Using the force constants in Cartesian coordinates from the Gaussian output, scaled force field calculations are carried out using a modified version of the UMAT program in the QCPE package. Potential energy distributions of the normal modes obtained from such calculations helped us assign the observed bands and identify the unique features of the spectra of 1- and 2-MNs which are important for their isomeric identification.

Prediction of inter-laminar stresses in composite honeycomb sandwich panels under mechanical loading using Variational Asymptotic Method

This work focuses on the formulation of an asymptotically correct theory for symmetric composite honeycomb sandwich plate structures. In these panels, transverse stresses tremendously influence design. The conventional 2-D finite elements cannot predict the thickness-wise distributions of transverse shear or normal stresses and 3-D displacements. Unfortunately, the use of the more accurate three-dimensional finite elements is computationally prohibitive. The development of the present theory is based on the Variational Asymptotic Method (VAM). Its unique features are the identification and utilization of additional small parameters associated with the anisotropy and non-homogeneity of composite sandwich plate structures. These parameters are ratios of smallness of the thickness of both facial layers to that of the core and smallness of 3-D stiffness coefficients of the core to that of the face sheets. Finally, anisotropy in the core and face sheets is addressed by the small parameters within the 3-D stiffness matrices. Numerical results are illustrated for several sample problems. The 3-D responses recovered using VAM-based model are obtained in a much more computationally efficient manner than, and are in agreement with, those of available 3-D elasticity solutions and 3-D FE solutions of MSC NASTRAN. (c) 2012 Elsevier Ltd. All rights reserved.

Effect of magnetic field on Mott's variable-range hopping parameters in multiwall carbon nanotube mat

We report the temperature and magnetic field dependence of the conductivity of multiwall carbon nanotube mat in the temperature range 1.4-150 K and in magnetic fields up to 10 T. It is observed that charge transport in this system is governed by Mott's variable-range hopping of three-dimensional type in the higher temperature range and two-dimensional type in the lower temperature range. Mott's various parameters, such as localization length, hopping length, hopping energy and density of states at the Fermi level are deduced from the variable-range hopping fit. The resistance of the sample decreases with the magnetic field applied in the direction of tube axis of the nanotubes. The magnetic field gives rise to delocalization of states with the well-known consequence of a decrease in Mott's T-0 parameter in variable-range hopping. The application of magnetic field lowers the crossover temperature at which three-dimensional variable-range hopping turns to two-dimensional variable-range hopping. The conductivity on the lower temperature side is governed by the weak localization giving rise to positive magnetoconductance. Finally, a magnetic field-temperature diagram is proposed showing different regions for different kinds of transport mechanism.

Extent of Structural Asymmetry in Homodimeric Proteins: Prevalence and Relevance

Most homodimeric proteins have symmetric structure. Although symmetry is known to confer structural and functional advantage, asymmetric organization is also observed. Using a non-redundant dataset of 223 high-resolution crystal structures of biologically relevant homodimers, we address questions on the prevalence and significance of asymmetry. We used two measures to quantify global and interface asymmetry, and assess the correlation of several molecular and structural parameters with asymmetry. We have identified rare cases (11/223) of biologically relevant homodimers with pronounced global asymmetry. Asymmetry serves as a means to bring about 2:1 binding between the homodimer and another molecule; it also enables cellular signalling arising from asymmetric macromolecular ligands such as DNA. Analysis of these cases reveals two possible mechanisms by which possible infinite array formation is prevented. In case of homodimers associating via non-topologically equivalent surfaces in their tertiary structures, ligand-dependent mechanisms are used. For stable dimers binding via large surfaces, ligand-dependent structural change regulates polymerisation/depolymerisation; for unstable dimers binding via smaller surfaces that are not evolutionarily well conserved, dimerisation occurs only in the presence of the ligand. In case of homodimers associating via interaction surfaces with parts of the surfaces topologically equivalent in the tertiary structures, steric hindrance serves as the preventive mechanism of infinite array. We also find that homodimers exhibiting grossly symmetric organization rarely exhibit either perfect local symmetry or high local asymmetry. Binding of small ligands at the interface does not cause any significant variation in interface asymmetry. However, identification of biologically relevant interface asymmetry in grossly symmetric homodimers is confounded by the presence of similar small magnitude changes caused due to artefacts of crystallisation. Our study provides new insights regarding accommodation of asymmetry in homodimers.

Crowd Sourcing a New Paradigm for Interactome Driven Drug Target Identification in Mycobacterium tuberculosis

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative `Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed `3interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/ mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2(-/-) mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnf alpha and Ifn gamma in sera are not significantly affected, Nos2(-/-) mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.