C2 domain is responsible for targeting rice phosphoinositide specific phospholipase C

Phosphoinositide-specific phospholipase C (PLC) is involved in Ca2+ mediated signalling events that lead to altered cellular status. Using various sequence-analysis methods, we identified two conserved motifs in known PLC sequences. The identified motifs are located in the C2 domain of plant PLCs and are not found in any other protein. These motifs are specifically found in the Ca2+ binding loops and form adjoining beta strands. Further, we identified certain conserved residues that are highly distinct from corresponding residues of animal PLCs. The motifs reported here could be used to annotate plant-specific phospholipase C sequences. Furthermore, we demonstrated that the C2 domain alone is capable of targeting PLC to the membrane in response to a Ca2+ signal. We also showed that the binding event results from a change in the hydrophobicity of the C2 domain upon Ca2+ binding. Bioinformatic analyses revealed that all PLCs from Arabidopsis and rice lack a transmembrane domain, myristoylation and GPI-anchor protein modifications. Our bioinformatic study indicates that plant PLCs are located in the cytoplasm, the nucleus and the mitochondria. Our results suggest that there are no distinct isoforms of plant PLCs, as have been proposed to exist in the soluble and membrane associated fractions. The same isoform could potentially be present in both subcellular fractions, depending on the calcium level of the cytosol. Overall, these data suggest that the C2 domain of PLC plays a vital role in calcium signalling.

Regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C through the transcriptional repression of upstream activating sequence inositol containing genes

The regulation of phospholipid biosynthesis in Saccharomyces cerevisiae through cis-acting upstream activating sequence inositol (UAS(ino)) and trans-acting elements, such as the INO2-INO4 complex and OPI1 by inositol supplementation in growth is thoroughly studied. In this study, we provide evidence for the regulation of lipid biosynthesis by phosphatidylinositol-specific phospholipase C (PLC) through UAS(ino) and the trans-acting elements. Gene expression analysis and radiolabelling experiments demonstrated that the overexpression of rice PLC in yeast cells altered phospholipid biosynthesis at the levels of transcriptional and enzyme activity. This is the first report implicating PLC in the direct regulation of lipid biosynthesis. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Sensitive Liquid Chromatography-Tandem Mass Spectrometry Method for the Determination of Olanzapine in Human Urine

A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method was developed to determine olanzapine (OLZ) in human urine. After solid-phase extraction with SPE cartridge, the urine sample was analysed on a C-18 column (Symmetry 3.5 mu m, 50 x 4.6 mm i.d) interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of ammonium acetate (pH 7.8)-acetonitrile (10:90, v/v). The method was linear over a concentration range of 1-100 ngml(-1). The lower limit of quantitation was 1 ngml(-1). The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was < 11.5 %. The accuracy determined at three concentrations (8.0, 50.0 and 85.0 ngml(-1) OLZ) was within +/- 1.21 % in terms of relative errors.

Quantum corrections to screening at strong coupling

We compute a certain class of corrections to (specific) screening lengths in strongly coupled non-abelian plasmas using the AdS/CFT correspondence. In this holographic framework, these corrections arise from various higher curvature interactions modifying the leading Einstein gravity action. The changes in the screening lengths are perturbative in inverse powers of the `t Hooft coupling or of the number of colors, as can be made precise in the context where the dual gauge theory is superconformal. We also compare the results of these holographic calculations to lattice results for the analogous screening lengths in QCD. In particular, we apply these results within the program of making quantitative comparisons between the strongly coupled quark-gluon plasma and holographic descriptions of conformal field theory. (c) 2012 Elsevier B.V. All rights reserved.

Role of Specific Cations and Water Entropy on the Stability of Branched DNA Motif Structures

DNA three-way junctions (TWJs) are important intermediates in various cellular processes and are the simplest of a family of branched nucleic acids being considered as scaffolds for biomolecular nanotechnology. Branched nucleic acids are stabilized by divalent cations such as Mg2+, presumably due to condensation and neutralization of the negatively charged DNA backbone. However, electrostatic screening effects point to more complex solvation dynamics and a large role of interfacial waters in thermodynamic stability. Here, we report extensive computer simulations in explicit water and salt on a model TWJ and use free energy calculations to quantify the role of ionic character and strength on stability. We find that enthalpic stabilization of the first and second hydration shells by Mg2+ accounts for 1/3 and all of the free energy gain in 50% and pure MgCl2 solutions, respectively. The more distorted DNA molecule is actually destabilized in pure MgCl2 compared to pure NaCl. Notably, the first shell, interfacial waters have very low translational and rotational entropy (i.e., mobility) compared to the bulk, an entropic loss that is overcompensated by increased enthalpy from additional electrostatic interactions with Mg2+. In contrast, the second hydration shell has anomalously high entropy as it is trapped between an immobile and bulklike layer. The nonmonotonic entropic signature and long-range perturbations of the hydration shells to Mg2+ may have implications in the molecular recognition of these motifs. For example, we find that low salt stabilizes the parallel configuration of the three-way junction, whereas at normal salt we find antiparallel configurations deduced from the NMR. We use the 2PT analysis to follow the thermodynamics of this transition and find that the free energy barrier is dominated by entropic effects that result from the decreased surface area of the antiparallel form which has a smaller number of low entropy waters in the first monolayer.

Orthogonal biofunctionalization of magnetic nanoparticles via ``clickable'' poly(ethylene glycol) silanes: a ``universal ligand'' strategy to design stealth and target-specific nanocarriers

The present work demonstrates a novel strategy to synthesize orthogonally bio-engineered magnetonanohybrids (MNPs) through the design of versatile, biocompatible linkers whose structure includes: (i) a robust anchor to bind with metal-oxide surfaces; (ii) tailored surface groups to act as spacers and (iii) a general method to implement orthogonal functionalizations of the substrate via ``click chemistry''. Ligands that possess the synthetic generality of features (i)-(iii) are categorized as ``universal ligands''. Herein, we report the synthesis of a novel, azido-terminated poly(ethylene glycol) (PEG) silane that can easily self-assemble on MNPs through hetero-condensation between surface hydroxyl groups and the silane end of the ligand, and simultaneously provide multiple clickable sites for high density, chemoselective bio-conjugation. To establish the universal-ligand-strategy, we clicked alkyl-functionalized folate onto the surface of PEGylated MNPs. By further integrating a near-infrared fluorescent (NIRF) marker (Alexa-Fluor 647) with MNPs, we demonstrated their folate-receptor mediated internalization inside cancer cells and subsequent translocation into lysosomes and mitochondria. Ex vivo NIRF imaging established that the azido-PEG-silane developed in course of the study can effectively reduce the sequestration of MNPs by macrophage organs (viz. liver and spleen). These folate-PEG-MNPs were not only stealth and noncytotoxic but their dual optical and magnetic properties aided in tracking their whereabouts through combined magnetic resonance and optical imaging. Together, these results provided a strong motivation for the future use of the ``universal ligand'' strategy towards development of ``smart'' nanohybrids for theragnostic applications.

Site-specific N-Linked Glycosylation of Receptor Guanylyl Cyclase C Regulates Ligand Binding, Ligand-mediated Activation and Interaction with Vesicular Integral Membrane Protein 36, VIP36

Guanylyl cyclase C (GC-C) is a multidomain, membrane-associated receptor guanylyl cyclase. GC-C is primarily expressed in the gastrointestinal tract, where it mediates fluid-ion homeostasis, intestinal inflammation, and cell proliferation in a cGMP-dependent manner, following activation by its ligands guanylin, uroguanylin, or the heat-stable enterotoxin peptide (ST). GC-C is also expressed in neurons, where it plays a role in satiation and attention deficiency/hyperactive behavior. GC-C is glycosylated in the extracellular domain, and differentially glycosylated forms that are resident in the endoplasmic reticulum (130 kDa) and the plasma membrane (145 kDa) bind the ST peptide with equal affinity. When glycosylation of human GC-C was prevented, either by pharmacological intervention or by mutation of all of the 10 predicted glycosylation sites, ST binding and surface localization was abolished. Systematic mutagenesis of each of the 10 sites of glycosylation in GC-C, either singly or in combination, identified two sites that were critical for ligand binding and two that regulated ST-mediated activation. We also show that GC-C is the first identified receptor client of the lectin chaperone vesicular integral membrane protein, VIP36. Interaction with VIP36 is dependent on glycosylation at the same sites that allow GC-C to fold and bind ligand. Because glycosylation of proteins is altered in many diseases and in a tissue-dependent manner, the activity and/or glycan-mediated interactions of GC-C may have a crucial role to play in its functions in different cell types.

Experiments and zero D modeling studies using specific Wiebe coefficients for producer gas as fuel in spark-ignited engines

The paper addresses experiments and modeling studies on the use of producer gas, a bio-derived low energy content fuel in a spark-ignited engine. Producer gas, generated in situ, has thermo-physical properties different from those of fossil fuel(s). Experiments on naturally aspirated and turbo-charged engine operation and subsequent analysis of the cylinder pressure traces reveal significant differences in the heat release pattern within the cylinder compared with a typical fossil fuel. The heat release patterns for gasoline and producer gas compare well in the initial 50% but beyond this, producer gas combustion tends to be sluggish leading to an overall increase in the combustion duration. This is rather unexpected considering that producer gas with nearly 20% hydrogen has higher flame speeds than gasoline. The influence of hydrogen on the initial flame kernel development period and the combustion duration and hence on the overall heat release pattern is addressed. The significant deviations in the heat release profiles between conventional fuels and producer gas necessitates the estimation of producer gas-specific Wiebe coefficients. The experimental heat release profiles are used for estimating the Wiebe coefficients. Experimental evidence of lower fuel conversion efficiency based on the chemical and thermal analysis of the engine exhaust gas is used to arrive at the Wiebe coefficients. The efficiency factor a is found to be 2.4 while the shape factor m is estimated at 0.7 for 2% to 90% burn duration. The standard Wiebe coefficients for conventional fuels and fuel-specific coefficients for producer gas are used in a zero D model to predict the performance of a 6-cylinder gas engine under naturally aspirated and turbo-charged conditions. While simulation results with standard Wiebe coefficients result in excessive deviations from the experimental results, excellent match is observed when producer gas-specific coefficients are used. Predictions using the same coefficients on a 3-cylinder gas engine having different geometry and compression ratio(s) indicate close match with the experimental traces highlighting the versatility of the coefficients.

Estimation of design basis earthquake using region-specific M-max, for the NPP site at Kalpakkam, Tamil Nadu, India

The objective of the paper is to estimate Safe Shutdown Earthquake (SSE) and Operating/Design Basis Earthquake (OBE/DBE) for the Nuclear Power Plant (NPP) site located at Kalpakkam, Tamil Nadu, India. The NPP is located at 12.558 degrees N, 80.175 degrees E and a 500 km circular area around NPP site is considered as `seismic study area' based on past regional earthquake damage distribution. The geology, seismicity and seismotectonics of the study area are studied and the seismotectonic map is prepared showing the seismic sources and the past earthquakes. Earthquake data gathered from many literatures are homogenized and declustered to form a complete earthquake catalogue for the seismic study area. The conventional maximum magnitude of each source is estimated considering the maximum observed magnitude (M-max(obs)) and/or the addition of 0.3 to 0.5 to M-max(obs). In this study maximum earthquake magnitude has been estimated by establishing a region's rupture character based on source length and associated M-max(obs). A final source-specific M-max is selected from the three M-max values by following the logical criteria. To estimate hazard at the NPP site, ten Ground-Motion Prediction Equations (GMPEs) valid for the study area are considered. These GMPEs are ranked based on Log-Likelihood (LLH) values. Top five GMPEs are considered to estimate the peak ground acceleration (PGA) for the site. Maximum PGA is obtained from three faults and named as vulnerable sources to decide the magnitudes of OBE and SSE. The average and normalized site specific response spectrum is prepared considering three vulnerable sources and further used to establish site-specific design spectrum at NPP site.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

Determination of size-independent specific fracture energy of concrete mixes by two methods

The RILEM work-of-fracture method for measuring the specific fracture energy of concrete from notched three-point bend specimens is still the most common method used throughout the world, despite the fact that the specific fracture energy so measured is known to vary with the size and shape of the test specimen. The reasons for this variation have also been known for nearly two decades, and two methods have been proposed in the literature to correct the measured size-dependent specific fracture energy (G(f)) in order to obtain a size-independent value (G(F)). It has also been proved recently, on the basis of a limited set of results on a single concrete mix with a compressive strength of 37 MPa, that when the size-dependent G(f) measured by the RILEM method is corrected following either of these two methods, the resulting specific fracture energy G(F) is very nearly the same and independent of the size of the specimen. In this paper, we will provide further evidence in support of this important conclusion using extensive independent test results of three different concrete mixes ranging in compressive strength from 57 to 122 MPa. (c) 2013 Elsevier Ltd. All rights reserved.

Rheometry of dense granular materials: the crucial effects of gravity and confining walls

We describe here the rheological response of dense, slowly deforming granular materials to shear in a cylindrical Couette cell. All components of the stress on the outer cylinder are measured pointwise as a function of the depth, for different methods of construction of the bed that presumably lead to distinct fabrics. The static stress profiles for the different construction protocols are different, but a stress profile that is independent of construction history emerges when the granular column is sheared for sufficient time, in accord with the predictions of plasticity theories. However the qualitative features of the the stress profile under shear differs radically from the predictions of plasticity theories and data reported in earlier studies. We discuss a hypothesis for the anomalous stress profiles that was proposed recently by us, and the ways in which further experiments may to conducted to verify it.

Rheometry of granular materials in cylindrical Couette cells: Anomalous stress caused by gravity and shear

The cylindrical Couette device is commonly employed to study the rheology of fluids, but seldom used for dense granular materials. Plasticity theories used for granular flows predict a stress field that is independent of the shear rate, but otherwise similar to that in fluids. In this paper we report detailed measurements of the stress as a function of depth, and show that the stress profile differs fundamentally from that of fluids, from the predictions of plasticity theories, and from intuitive expectation. In the static state, a part of the weight of the material is transferred to the walls by a downward vertical shear stress, bringing about the well-known Janssen saturation of the stress in vertical columns. When the material is sheared, the vertical shear stress changes sign, and the magnitudes of all components of the stress rise rapidly with depth. These qualitative features are preserved over a range of the Couette gap and shear rate, for smooth and rough walls and two model granular materials. To explain the anomalous rheological response, we consider some hypotheses that seem plausibleapriori, but showthat none survive after careful analysis of the experimental observations. We argue that the anomalous stress is due to an anisotropic fabric caused by the combined actions of gravity, shear, and frictional walls, for which we present indirect evidence from our experiments. A general theoretical framework for anisotropic plasticity is then presented. The detailed mechanics of how an anisotropic fabric is brought about by the above-mentioned factors is not clear, and promises to be a challenging problem for future investigations. (C) 2013 AIP Publishing LLC.

Bilinear tension softening diagrams of concrete mixes corresponding to their size-independent specific fracture energy

The study of the fracture behaviour of concrete structures using the fictitious crack model requires two fracture properties of the concrete mix, namely, the size-independent specific fracture energy G(F). and the corresponding tension softening relation sigma(w) between the residual stress carrying capacity sigma and the crack opening w in the fracture process zone ahead of a real crack. In this paper, bi-linear tension softening diagrams of three different concrete mixes, ranging in compressive strength from 57 to 122 MPa whose size-independent specific fracture energy has been previously determined, have been constructed in an inverse manner based on the concept of a non-linear hinge from the load-crack mouth opening plots of notched three-point bend beams. (C) 2013 Elsevier Ltd. All rights reserved.

Multi-task learning using shared and task specific information

Multi-task learning solves multiple related learning problems simultaneously by sharing some common structure for improved generalization performance of each task. We propose a novel approach to multi-task learning which captures task similarity through a shared basis vector set. The variability across tasks is captured through task specific basis vector set. We use sparse support vector machine (SVM) algorithm to select the basis vector sets for the tasks. The approach results in a sparse model where the prediction is done using very few examples. The effectiveness of our approach is demonstrated through experiments on synthetic and real multi-task datasets.