PocketAnnotate: towards site-based function annotation

A computational pipeline PocketAnnotate for functional annotation of proteins at the level of binding sites has been proposed in this study. The pipeline integrates three in-house algorithms for site-based function annotation: PocketDepth, for prediction of binding sites in protein structures; PocketMatch, for rapid comparison of binding sites and PocketAlign, to obtain detailed alignment between pair of binding sites. A novel scheme has been developed to rapidly generate a database of non-redundant binding sites. For a given input protein structure, putative ligand-binding sites are identified, matched in real time against the database and the query substructure aligned with the promising hits, to obtain a set of possible ligands that the given protein could bind to. The input can be either whole protein structures or merely the substructures corresponding to possible binding sites. Structure-based function annotation at the level of binding sites thus achieved could prove very useful for cases where no obvious functional inference can be obtained based purely on sequence or fold-level analyses. An attempt has also been made to analyse proteins of no known function from Protein Data Bank. PocketAnnotate would be a valuable tool for the scientific community and contribute towards structure-based functional inference. The web server can be freely accessed at http://proline.biochem.iisc.ernet.in/pocketannotate/.

A novel gas flow sensing application using piezoelectric ZnO thin films deposited on Phynox alloy

We report on the novel flow sensing application of piezoelectric ZnO thin film deposited on Phynox alloy sensing element. Characterization of piezoelectric ZnO films deposited on Phynox (Elgiloy) substrate at different RF powers is discussed. ZnO films deposited at RF power of 100W were found to have fine c-axis orientation, possesses excellent surface morphology with lower rms surface roughness of 1.87 nm and maximum d(31) coefficient value 4.7 pm V-1. The thin cantilever strip of Phynox alloy with ZnO film as a sensing layer for flow sensing has been tested for flow rates ranging from 2 to 18 L min(-1). A detailed theoretical analysis of the experimental set-up showing the relationship between output voltage and force at a particular flow rate has been discussed. The sensitivity of now sensing element is similar to 18 mV/(L min(-1)) and typical response time is of the order of 20 m s. The sensing element is calibrated using in-house developed testing set-up. (C) 2012 Elsevier B.V. All rights reserved.

Precision and long-term stability of clumped-isotope analysis of CO2 using a small-sector isotope ratio mass spectrometer

RATIONALE The ratio of the measured abundance of 13C18O bonding CO2 to its stochastic abundance, prescribed by the delta 13C and delta 18O values from a carbonate mineral, is sensitive to its growth temperature. Recently, clumped-isotope thermometry, which uses this ratio, has been adopted as a new tool to elucidate paleotemperatures quantitatively. METHODS Clumped isotopes in CO2 were measured with a small-sector isotope ratio mass spectrometer. CO2 samples digested from several kinds of calcium carbonates by phosphoric acid at 25 degrees C were purified using both cryogenic and gas-chromatographic separations, and their isotopic composition (delta 13C, delta 18O, Delta 47, Delta 48 and Delta 49 values) were then determined using a dual-inlet Delta XP mass spectrometer. RESULTS The internal precisions of the single gas Delta 47 measurements were 0.005 and 0.02 parts per thousand (1 SE) for the optimum and the routine analytical conditions, respectively, which are comparable with those obtained using a MAT 253 mass spectrometer. The long-term variations in the Delta 47 values for the in-house working standard and the heated CO2 gases since 2007 were close to the routine, single gas uncertainty while showing seasonal-like periodicities with a decreasing trend. Unlike the MAT 253, the Delta XP did not show any significant relationship between the Delta 47 and delta 47 values. CONCLUSIONS The Delta XP gave results that were approximately as precise as those of the MAT 253 for clumped-isotope analysis. The temporal stability of the Delta XP seemed to be lower, although an advantage of the Delta XP was that no dependency of delta 47 on Delta 47 was found. Copyright (c) 2012 John Wiley & Sons, Ltd.

Advanced multifunctional inorganic nanostructured oxides for controlled release and sensing

ADVANCED MULTIFUNCTIONAL INORGANIC NANOSTRUCTURED OXIDES FOR CONTROLLED RELEASE AND SENSING. We demonstrate here certain examples of multifunctional nanostructured oxidematerials for biotechnological and environmental applications.Various in-house synthesized homogeneous nanostructured viz.mesoporous and nanotubes silica and titania have been employed for controlled drug delivery and electrochemical biosensing applications. Confinement of macromolecules such as proteins studied via electrochemical, thermal and spectroscopic methods showed no detrimental effect on native protein structure and function, thus suggesting effective utility of oxide nanostructures as bio-encapsulators. Multi-functionalitywas demonstrated via employing similar nanostructures for sensing organic water pollutants e.g. textile dyes.

Rice LHS1/OsMADS1 Controls Floret Meristem Specification by Coordinated Regulation of Transcription Factors and Hormone Signaling Pathways

SEPALLATA (SEP) MADS box transcription factors mediate floral development in association with other regulators. Mutants in five rice (Oryza sativa) SEP genes suggest both redundant and unique functions in panicle branching and floret development. LEAFY HULL STERILE1/OsMADS1, from a grass-specific subgroup of LOFSEP genes, is required for specifying a single floret on the spikelet meristem and for floret organ development, but its downstream mechanisms are unknown. Here, key pathways and directly modulated targets of OsMADS1 were deduced from expression analysis after its knockdown and induction in developing florets and by studying its chromatin occupancy at downstream genes. The negative regulation of OsMADS34, another LOFSEP gene, and activation of OsMADS55, a SHORT VEGETATIVE PHASE-like floret meristem identity gene, show its role in facilitating the spikelet-to-floret meristem transition. Direct regulation of other transcription factor genes like OsHB4 (a class III homeodomain Leu zipper member), OsBLH1 (a BEL1-like homeodomain member), OsKANADI2, OsKANADI4, and OsETTIN2 show its role in meristem maintenance, determinacy, and lateral organ development. We found that the OsMADS1 targets OsETTIN1 and OsETTIN2 redundantly ensure carpel differentiation. The multiple effects of OsMADS1 in promoting auxin transport, signaling, and auxin-dependent expression and its direct repression of three cytokinin A-type response regulators show its role in balancing meristem growth, lateral organ differentiation, and determinacy. Overall, we show that OsMADS1 integrates transcriptional and signaling pathways to promote rice floret specification and development.

An experimental set-up for carbon isotopic analysis of atmospheric CO2 and an example of ecosystem response during solar eclipse 2010

We present here, an experimental set-up developed for the first time in India for the determination of mixing ratio and carbon isotopic ratio of air-CO2. The set-up includes traps for collection and extraction of CO2 from air samples using cryogenic procedures, followed by the measurement of CO2 mixing ratio using an MKS Baratron gauge and analysis of isotopic ratios using the dual inlet peripheral of a high sensitivity isotope ratio mass spectrometer (IRMS) MAT 253. The internal reproducibility (precision) for the PC measurement is established based on repeat analyses of CO2 +/- 0.03 parts per thousand. The set-up is calibrated with international carbonate and air-CO2 standards. An in-house air-CO2 mixture, `OASIS AIRMIX' is prepared mixing CO2 from a high purity cylinder with O-2 and N-2 and an aliquot of this mixture is routinely analyzed together with the air samples. The external reproducibility for the measurement of the CO2 mixing ratio and carbon isotopic ratios are +/- 7 (n = 169) mu mol.mol(-1) and +/- 0.05 (n = 169) parts per thousand based on the mean of the difference between two aliquots of reference air mixture analyzed during daily operation carried out during November 2009-December 2011. The correction due to the isobaric interference of N2O on air-CO2 samples is determined separately by analyzing mixture of CO2 (of known isotopic composition) and N2O in varying proportions. A +0.2 parts per thousand correction in the delta C-13 value for a N2O concentration of 329 ppb is determined. As an application, we present results from an experiment conducted during solar eclipse of 2010. The isotopic ratio in CO2 and the carbon dioxide mixing ratio in the air samples collected during the event are different from neighbouring samples, suggesting the role of atmospheric inversion in trapping the emitted CO2 from the urban atmosphere during the eclipse.

Common recognition principles across diverse sequence and structural families of sialic acid binding proteins

Sialic acids form a large family of 9-carbon monosaccharides and are integral components of glycoconjugates. They are known to bind to a wide range of receptors belonging to diverse sequence families and fold classes and are key mediators in a plethora of cellular processes. Thus, it is of great interest to understand the features that give rise to such a recognition capability. Structural analyses using a non-redundant data set of known sialic acid binding proteins was carried out, which included exhaustive binding site comparisons and site alignments using in-house algorithms, followed by clustering and tree computation, which has led to derivation of sialic acid recognition principles. Although the proteins in the data set belong to several sequence and structure families, their binding sites could be grouped into only six types. Structural comparison of the binding sites indicates that all sites contain one or more different combinations of key structural features over a common scaffold. The six binding site types thus serve as structural motifs for recognizing sialic acid. Scanning the motifs against a non-redundant set of binding sites from PDB indicated the motifs to be specific for sialic acid recognition. Knowledge of determinants obtained from this study will be useful for detecting function in unknown proteins. As an example analysis, a genome-wide scan for the motifs in structures of Mycobacterium tuberculosis proteome identified 17 hits that contain combinations of the features, suggesting a possible function of sialic acid binding by these proteins.

Analytical model for stress-strain response of plastic waste mixed soil

Recycling plastic water bottles has become one of the major challenges world wide. The present study provides an approach for the use of plastic waste as reinforcement material in soil, which can be used for ground improvement, subbases, and subgrade preparation in road construction. The experimental results are presented in the form of stress-strain-pore water pressure response and compression paths. On the basis of experimental test results, it is observed that the strength of soil is improved and compressibility reduced significantly with the addition of a small percentage of plastic waste to the soil. In this paper, an analytical model is proposed to evaluate the response of plastic waste mixed soil. It is noted that the model captures the stress-strain and pore water pressure response of all percentages of plastic waste adequately. The paper also provides a comparative study of failure stress obtained from different published models and the proposed model, which are compared with experimental results. The improvement in strength attributable to the inclusion of plastic waste can be advantageously used in ground improvement projects.

Effect of post-deposition annealing on transverse piezoelectric coefficient and vibration sensing performance of ZnO thin films

The present experimental study investigates the influence of post-deposition annealing on the transverse piezoelectric coefficient (d(31)) value of ZnO thin films deposited on a flexible metal alloy substrate, and its relationship with the vibration sensing performance. Highly c-axis oriented and crystalline ZnO thin films were deposited on flexible Phynox alloy substrate via radio frequency (RF) reactive magnetron sputtering. ZnO thin film samples were annealed at different temperatures ranging from 100 degrees C to 500 degrees C, resulting in the temperature of 300 degrees C determined as the optimum annealing temperature. The crystallinity, morphology, microstructure, and rms surface roughness of annealed ZnO thin films were systematically investigated by X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), and Atomic Force Microscopy (AFM), respectively. The piezoelectric d(31) coefficient value was measured by 4-point bending method. ZnO thin film annealed at 300 degrees C was highly c-axis oriented, crystalline, possesses fine surface morphology with uniformity in the grain size. This film showed higher d(31) coefficient value of 7.2 pm V-1. A suitable in-house designed and developed experimental set-up, for evaluating the vibration sensing performance of annealed ZnO thin films is discussed. As expected the ZnO thin film annealed at 300 degrees C showed relatively better result for vibration sensing studies. It generates comparatively higher peak output voltage of 147 mV, due to improved structural and morphological properties, and higher piezoelectric d(31) coefficient value. (C) 2014 Elsevier B. V. All rights reserved.

PLIC: protein-ligand interaction clusters

Most of the biological processes are governed through specific protein-ligand interactions. Discerning different components that contribute toward a favorable protein-ligand interaction could contribute significantly toward better understanding protein function, rationalizing drug design and obtaining design principles for protein engineering. The Protein Data Bank (PDB) currently hosts the structure of similar to 68 000 protein-ligand complexes. Although several databases exist that classify proteins according to sequence and structure, a mere handful of them annotate and classify protein-ligand interactions and provide information on different attributes of molecular recognition. In this study, an exhaustive comparison of all the biologically relevant ligand-binding sites (84 846 sites) has been conducted using PocketMatch: a rapid, parallel, in-house algorithm. PocketMatch quantifies the similarity between binding sites based on structural descriptors and residue attributes. A similarity network was constructed using binding sites whose PocketMatch scores exceeded a high similarity threshold (0.80). The binding site similarity network was clustered into discrete sets of similar sites using the Markov clustering (MCL) algorithm. Furthermore, various computational tools have been used to study different attributes of interactions within the individual clusters. The attributes can be roughly divided into (i) binding site characteristics including pocket shape, nature of residues and interaction profiles with different kinds of atomic probes, (ii) atomic contacts consisting of various types of polar, hydrophobic and aromatic contacts along with binding site water molecules that could play crucial roles in protein-ligand interactions and (iii) binding energetics involved in interactions derived from scoring functions developed for docking. For each ligand-binding site in each protein in the PDB, site similarity information, clusters they belong to and description of site attributes are provided as a relational database-protein-ligand interaction clusters (PLIC).

DEFINING alpha-HELIX GEOMETRY BY C-alpha ATOM TRACE vs (phi-psi) TORSION ANGLES: A COMPARATIVE ANALYSIS

A regular secondary structure is described by a well defined set of values for the backbone dihedral angles (phi,psi and omega) in a polypeptide chain. However in real protein structures small local variations give rise to distortions from the ideal structures, which can lead to considerable variation in higher order organization. Protein structure analysis and accurate assignment of various structural elements, especially their terminii, are important first step in protein structure prediction and design. Various algorithms are available for assigning secondary structure elements in proteins but some lacunae still exist. In this study, results of a recently developed in-house program ASSP have been compared with those from STRIDE, in identification of alpha-helical regions in both globular and membrane proteins. It is found that, while a combination of hydrogen bond patterns and backbone torsional angles (phi-psi) are generally used to define secondary structure elements, the geometry of the C-alpha atom trace by itself is sufficient to define the parameters of helical structures in proteins. It is also possible to differentiate the various helical structures by their C-alpha trace and identify the deviations occurring both at mid-positions as well as at the terminii of alpha-helices, which often lead to occurrence of 3(10) and pi-helical fragments in both globular and membrane proteins.

Experimental Investigation on Switching Characteristics of High-Current Insulated Gate Bipolar Transistors at Low Currents

Insulated gate bipolar transistors (IGBTs) are used in high-power voltage-source converters rated up to hundreds of kilowatts or even a few megawatts. Knowledge of device switching characteristics is required for reliable design and operation of the converters. Switching characteristics are studied widely at high current levels, and corresponding data are available in datasheets. But the devices in a converter also switch low currents close to the zero crossings of the line currents. Further, the switching behaviour under these conditions could significantly influence the output waveform quality including zero crossover distortion. Hence, the switching characteristics of high-current IGBTs (300-A and 75-A IGBT modules) at low load current magnitudes are investigated experimentally in this paper. The collector current, gate-emitter voltage and collector-emitter voltage are measured at various low values of current (less than 10% of the device rated current). A specially designed in-house constructed coaxial current transformer (CCT) is used for device current measurement without increasing the loop inductance in the power circuit. Experimental results show that the device voltage rise time increases significantly during turn-off transitions at low currents.

De novo inference of protein function from coarse-grained dynamics

Inference of molecular function of proteins is the fundamental task in the quest for understanding cellular processes. The task is getting increasingly difficult with thousands of new proteins discovered each day. The difficulty arises primarily due to lack of high-throughput experimental technique for assessing protein molecular function, a lacunae that computational approaches are trying hard to fill. The latter too faces a major bottleneck in absence of clear evidence based on evolutionary information. Here we propose a de novo approach to annotate protein molecular function through structural dynamics match for a pair of segments from two dissimilar proteins, which may share even <10% sequence identity. To screen these matches, corresponding 1 mu s coarse-grained (CG) molecular dynamics trajectories were used to compute normalized root-mean-square-fluctuation graphs and select mobile segments, which were, thereafter, matched for all pairs using unweighted three-dimensional autocorrelation vectors. Our in-house custom-built forcefield (FF), extensively validated against dynamics information obtained from experimental nuclear magnetic resonance data, was specifically used to generate the CG dynamics trajectories. The test for correspondence of dynamics-signature of protein segments and function revealed 87% true positive rate and 93.5% true negative rate, on a dataset of 60 experimentally validated proteins, including moonlighting proteins and those with novel functional motifs. A random test against 315 unique fold/function proteins for a negative test gave >99% true recall. A blind prediction on a novel protein appears consistent with additional evidences retrieved therein. This is the first proof-of-principle of generalized use of structural dynamics for inferring protein molecular function leveraging our custom-made CG FF, useful to all. (C) 2014 Wiley Periodicals, Inc.

Matrix-Assisted Refolding, Purification and Activity Assessment Using a `Form Invariant' Assay for Matrix Metalloproteinase 2 (MMP2)

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed `form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

Experimental Investigation on Switching Characteristics of High-Current Insulated Gate Bipolar Transistors at Low Currents

Insulated gate bipolar transistors (IGBTs) are used in high-power voltage-source converters rated up to hundreds of kilowatts or even a few megawatts. Knowledge of device switching characteristics is required for reliable design and operation of the converters. Switching characteristics are studied widely at high current levels, and corresponding data are available in datasheets. But the devices in a converter also switch low currents close to the zero crossings of the line currents. Further, the switching behaviour under these conditions could significantly influence the output waveform quality including zero crossover distortion. Hence, the switching characteristics of high-current IGBTs (300-A and 75-A IGBT modules) at low load current magnitudes are investigated experimentally in this paper. The collector current, gate-emitter voltage and collector-emitter voltage are measured at various low values of current (less than 10% of the device rated current). A specially designed in-house constructed coaxial current transformer (CCT) is used for device current measurement without increasing the loop inductance in the power circuit. Experimental results show that the device voltage rise time increases significantly during turn-off transitions at low currents.