A sleep-inducing peptide from the venom of the Indian cone snail Conus araneosus

The marine snail Conus araneosus has unusual significance due to its confined distribution to coastal regions of southeast India and Sri Lanka. Due to its relative scarceness, this species has been poorly studied. In this work, we characterized the venom of C. araneosus to identify new venom peptides. We identified 14 novel compounds. We determined amino acid sequences from chemically-modified and unmodified crude venom using liquid chromatography-electrospray ionization mass spectrometry and matrix assisted laser desorption ionization time-of-flight mass spectrometry. Ten sequences showed six Cys residues arranged in a pattern that is most commonly associated with the M-superfamily of conotoxins. Four other sequences had four Cys residues in a pattern that is most commonly associated with the T-superfamily of conotoxins. The post-translationally modified residue (pyroglutamate) was determined at the N-terminus of two sequences, ar3h and ar3i respectively. In addition, two sequences, ar3g and ar3h were C-terminally amidated. At a dose of 2 nmol, peptide ar3j elicited sleep when injected intraperitoneally into mice. To our knowledge, this is the first report of a peptide from a molluscivorous cone snail with sleep-inducing effects in mice. The novel peptides characterized herein extend the repertoire of unique peptides derived from cone snails and may add value to the therapeutic promise of conotoxins. (C) 2015 Elsevier Ltd. All rights reserved.

Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

De novo mass spectrometric sequencing of two Conus peptides, Vi1359 and Vi1361, from the vermivorous cone snail Conus virgo, found off the southern Indian coast, is presented. The peptides, whose masses differ only by 2 Da, possess two disulfide bonds and an amidated C-terminus. Simple chemical modifications and enzymatic cleavage coupled with matrix assisted laser desorption ionization (MALDI) mass spectrometric analysis aided in establishing the sequences of Vi1359, ZCCITIPECCRI-NH2, and Vi1361, ZCCPTMPECCRI-NH2, Which differ only at residues 4 and 6 (Z = pyroglutamic acid). The presence of the pyroglutamyl residue at the N-terminus was unambiguously identified by chemical hydrolysis of the cyclic amide, followed by esterification. The presence of Ile residues in both the peptides was confirmed from high-energy collision induced dissociation (CID) studies, using the observation Of W-n- and d(n)-ions as a diagnostic. Differential cysteine labeling, in conjunction with MALDI-MS/MS, permitted establishment of disulfide connectivity in both peptides as Cys2-Cys9 and Cys3-Cys10. The cysteine pattern clearly reveals that the peptides belong to the class of T-superfamily conotoxins, in particular the T-1 superfamily.

Lipopeptides from the Banyan Endophyte, Bacillus subtilis K1: Mass Spectrometric Characterization of a Library of Fengycins

Mass spectrometric analysis of a banyan endophyte, Bacillus subtilis K1, extract showing broad spectrum antifungal activity revealed a complex mixture of lipopeptides, iturins, surfactins, and fengycins. Fractionation by reversed-phase high performance liquid chromatography (HPLC) facilitated a detailed analysis of fengycin microheterogeneity. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric studies permitted the identification of several new fengycin variants. Four major sites of heterogeneity are identified: (1) N-terminus beta-hydroxy fatty acid moiety, where chain length variation and the presence of unsaturation occur, (2) position 6 (Ala/Val/Ile/Leu), (3) position 10 (Val/Ile) within the macrocyclic ring, and (4) Gln to Glu replacement at position 8, resulting in fengycin variants that differ in mass by 1 Da. Diagnostic fragment ions provide a quick method for localizing the sites of variation in the macrocycle or the linear segment. Subsequent establishment of the sequences is achieved by MS/MS analysis of linear fengycin species produced by hydrolysis of the macrocyclic lactone. Unsaturation in the fatty acid chain and the presence of linear precursors in the B. subtilis K1 extract are also established by mass spectrometry. The anomalous distribution of intensities within isotopic multiplets is a diagnostic for Gln/Glu replacements. High resolution mass spectrometry facilitates the identification of fengycin species differing by 1 Da by localizing the variable position (Gln(8)/Glu(8)) in the fengycin variants.

Primary Structural Documentation of the Major Urinary Protein of the Indian Commensal Rat (Rattus rattus) Using a Proteomics Platform

Purpose: A number of proteome studies have been performed recently to identify pheromone-related protein expression and their molecular function using genetically modified rodents' urine. However, no such studies have used Indian commensal rodents; interestingly, in a previous investigation, we confirmed the presence of volatile molecules in commensal rodents urine and these molecules seem to be actively involved in pheromonal communication. Therefore, the present study aims to identify the major urinary protein [MUP] present in commensal rat urine, which will help us to understand the protein's expression pattern and intrinsic properties among the rodents globally. Experimental Design: Initially, the total urinary proteins were separated by 1-D and 2-D electrophoresis and then subsequently analyzed by Matrix Assisted Laser Desorption Ionization-Time of Flight and Mass Spectrometer (MALDI-TOF/MS). Furthermore, they were then fragmented with the aid of a Tandem Mass Spectrometer (TOF/TOF) and the identified sequences aligned and confirmed using similarity with the deduced primary structures of members of the lipocalin superfamily.Results: The SDS-PAGE protein profiles showed distinct proteins with molecular masses of 15, 22.4, 25, 28, 42, 50, 55, 68, and 91 kDa. Of these proteins, the 22.4 kDa protein was considered to be target candidate. When 2D electrophoresis was carried out, about similar to 50 spots were detected with different masses and various pI ranges. The 22.4 kDa protein was found to have a pI of about 4.9. This 22.4 kDa protein spot was digested and subjected to mass spectrometry; it was identified as rat MUP. The fragmented peptides from the rat MUP at 935, 1026, 1192, and 1303 m/z were further fragmented with the aid of MS/MS and generated de novo sequence and this confirmed this protein to be the MUP present in the urine of commensal rats.Conclusion: The present investigation confirms the presence of MUP with a molecular mass of 22.4 kDa in the urine of commensal rats. This protein may be involved in the binding of volatile molecules and opens up a discussion about how volatile and non-volatile molecules in the commensal rats' urine may contribute chemo-communication.

SEC-MALDI-TOF mass spectral characterization of a hyperbranched polyether prepared via melt transetherification

An AB(2) monomer, 1-(2-hydroxyethoxy)-3,5-bis-(methoxymethyl)-2,4,6-trimethylbenzene, was synthesized from mesitol and melt-polycondensed in the presence of an acid catalyst via a transetherification process at 145-150 degreesC to yield a soluble, moderately high molecular weight hyperbranched polyether. The degree of branching in the polymer was calculated to be 0.78 by a comparison of its NMR spectrum with that of an appropriately designed model compound. The weight-average molecular weight of the hyperbranched polymer was determined to be 64,600 (weight-average molecular weight/number-average molecular weight = 5.2) by size exclusion chromatography (SEC) in CHCl3, with polystyrene standards. The origin of the broad molecular weight distribution, which could either be intrinsic to such hyperbranched structures or be due to structural heterogeneity, was further probed by the fractionation of the samples by SEC and by the subjection of each fraction to matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. The mass spectral analysis suggested the presence of two primary types of species: one corresponding to the simple branched structure and the other to macrocyclics. Interestingly, from the relative intensities of the two peaks, it was apparent that cyclization became favorable at higher conversions in the melt transetherification process. (C) 2002 Wiley Periodicals, Inc.

Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase{triangledown}

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (–)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Mass Spectrometry-Based Systems Approach for Identification of Inhibitors of Plasmodium falciparum Fatty Acid Synthase{triangledown}

The emergence of strains of Plasmodium falciparum resistant to the commonly used antimalarials warrants the development of new antimalarial agents. The discovery of type II fatty acid synthase (FAS) in Plasmodium distinct from the FAS in its human host (type I FAS) opened up new avenues for the development of novel antimalarials. The process of fatty acid synthesis takes place by iterative elongation of butyryl-acyl carrier protein (butyryl-ACP) by two carbon units, with the successive action of four enzymes constituting the elongation module of FAS until the desired acyl length is obtained. The study of the fatty acid synthesis machinery of the parasite inside the red blood cell culture has always been a challenging task. Here, we report the in vitro reconstitution of the elongation module of the FAS of malaria parasite involving all four enzymes, FabB/F (β-ketoacyl-ACP synthase), FabG (β-ketoacyl-ACP reductase), FabZ (β-ketoacyl-ACP dehydratase), and FabI (enoyl-ACP reductase), and its analysis by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS). That this in vitro systems approach completely mimics the in vivo machinery is confirmed by the distribution of acyl products. Using known inhibitors of the enzymes of the elongation module, cerulenin, triclosan, NAS-21/91, and (–)-catechin gallate, we demonstrate that accumulation of intermediates resulting from the inhibition of any of the enzymes can be unambiguously followed by MALDI-TOF MS. Thus, this work not only offers a powerful tool for easier and faster throughput screening of inhibitors but also allows for the study of the biochemical properties of the FAS pathway of the malaria parasite.

Biodegradation of acrylamide by acrylamidase from Stenotrophomonas acidaminiphila MSU12 and analysis of degradation products by MALDI-TOF and HPLC

The present study examines an improved detoxification and rapid biological degradation of toxic pollutant acrylamide using a bacterium. The acrylamide degrading bacterium was isolated from the soil followed by its screening to know the acrylamide degrading capability. The minimal medium containing acrylamide (30 mM) served as a sole source of carbon and nitrogen for their active growth. The optimization of three different factors was analyzed by using Response Surface Methodology (RSM). The bacteria actively degraded the acrylamide at a temperature of 32 degrees C, with a maximum growth at 30 mM substrate (acrylamide) concentration at a pH of 7.2. The acrylamidase activity and degradation of acrylamide was determined by High Performance Liquid Chromatography (HPLC) and Matrix Assisted Laser Desorption and Ionization Time of Flight mass spectrometer (MALDI-TOF). Based on 168 rRNA analysis the selected strain was identified as Gram negative bacilli Stenotrophomonas acidaminiphila MSU12. The acrylamidase was isolated from bacterial extract and was purified by HPLC, whose mass spectrum showed a molecular mass of 38 kDa. (C) 2014 Elsevier Ltd. All rights reserved.

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.

Numerical modeling of heat and mass transfer in laser surface alloying: Non-equilibrium solidification effects

A transient macroscopic model is developed for studying heat and mass transfer in a single-pass laser surface alloying process, with particular emphasis on non-equilibrium solidification considerations. The solution for species concentration distribution requires suitable treatment of non-equilibrium mass transfer conditions. In this context, microscopic features pertaining to non-equilibrium effects on account of solutal undercooling are incorporated through the formulation of a modified partition-coefficient. The effective partition-coefficient is numerically modeled by Means of a number of macroscopically observable parameters related to the solidifying domain. The numerical model is so developed that the modifications on account of non-equilibrium solidification considerations can be conveniently implemented in existing numerical codes based on equilibrium solidification considerations.

Desorption from concentrated polymer solutions in good and poor solvents

The objective of the study was to investigate the effects of the nature of solvent and polymer concentration on the mass-transfer coefficients in desorption of solvents and to develop a correlation to predict them. Desorption was experimentally studied in a Lewis cell with concentrated binary solutions of polymer in good and poor solvents. The range of parameters covered are polymer weight fraction between 0.25 and 0.6, Reynolds number between 3 and 100; Schmidt number between 1.4 X lo6 and 2.5 X lo8, and Sherwood number between 3.5 X lo2 and 1.2 X lo4. Desorption from moderately concentrated solutions (polymer weight fraction -0.25) is gas-phase controlled. Studies with more concentrated solutions showed that the effects of solvent and concentration were such that corrections due to concentration-dependent diffusivity and viscosity as well as high flux had to be applied to the mass-transfer coefficients before they could be correlated.

Anatomy of Higgs mass in supersymmetric inverse seesaw models

We compute the one loop corrections to the CP-even Higgs mass matrix in the supersymmetric inverse seesaw model to single out the different cases where the radiative corrections from the neutrino sector could become important. It is found that there could be a significant enhancement in the Higgs mass even for Dirac neutrino masses of O(30) GeV if the left-handed sneutrino soft mass is comparable or larger than the right-handed neutrino mass. In the case where right-handed neutrino masses are significantly larger than the supersymmetry breaking scale, the corrections can utmost account to an upward shift of 3 GeV. For very heavy multi TeV sneutrinos, the corrections replicate the stop corrections at 1-loop. We further show that general gauge mediation with inverse seesaw model naturally accommodates a 125 GeV Higgs with TeV scale stops. (C) 2014 The Authors. Published by Elsevier B.V.

The partition of unity finite element method for elastic wave propagation in Reissner-Mindlin plates

This paper reports a numerical method for modelling the elastic wave propagation in plates. The method is based on the partition of unity approach, in which the approximate spectral properties of the infinite dimensional system are embedded within the space of a conventional finite element method through a consistent technique of waveform enrichment. The technique is general, such that it can be applied to the Lagrangian family of finite elements with specific waveform enrichment schemes, depending on the dominant modes of wave propagation in the physical system. A four-noded element for the Reissner-indlin plate is derived in this paper, which is free of shear locking. Such a locking-free property is achieved by removing the transverse displacement degrees of freedom from the element nodal variables and by recovering the same through a line integral and a weak constraint in the frequency domain. As a result, the frequency-dependent stiffness matrix and the mass matrix are obtained, which capture the higher frequency response with even coarse meshes, accurately. The steps involved in the numerical implementation of such element are discussed in details. Numerical studies on the performance of the proposed element are reported by considering a number of cases, which show very good accuracy and low computational cost. Copyright (C)006 John Wiley & Sons, Ltd.

Type I seesaw mechanism for quasi-degenerate neutrinos

We discuss symmetries and scenarios leading to quasi-degenerate neutrinos in type I seesaw models. The existence of degeneracy in the present approach is not linked to any specific structure for the Dirac neutrino Yukawa coupling matrix y(D) and holds in general. Basic input is the application of the minimal flavour violation principle to the leptonic sector. Generalizing this principle, we assume that the structure of the right-handed neutrino mass matrix is determined by y(D) and the charged lepton Yukawa coupling matrix y(l) in an effective theory invariant under specific groups G(F) contained in the full symmetry group of the kinetic energy terms. G(F) invariance also leads to specific structure for the departure from degeneracy. The neutrino mass matrix (with degenerate mass m(0)) resulting after seesaw mechanism has a simple form Mv approximate to m(0)(I - py(l)y(l)(T)) in one particular scenario based on supersymmetry. This form is shown tolead to correct description of neutrino masses and mixing angles. The thermal leptogenesis after inclusion of flavour effects can account for the observed baryon asymmetry of the universe within the present scenario. Rates for lepton flavour violating processes can occur at observable levels in the supersymmetric version of the scenario. (c) 2010 Elsevier B.V. All rights reserved.

Flavored co-annihilations

Neutralino dark matter in supersymmetric models is revisited in the presence of flavor violation in the soft supersymmetry breaking sector. We focus on flavor violation the sleptonic sector and study the implications for the co-annihilation regions. Flavor is introduced by a single (mu) over tilde (R) - (T) over tilde (R) insertion in the slepton mass matrix. Limits on insertion from BR(tau -> mu + gamma) are weak in some regions of the parameter space where happen within the amplitudes. We look for overlaps in parameter space where the co-annihilation condition as well as the cancellations within the amplitudes occur. mSUGRA, such overlap regions are not existent, whereas they are present in models non-universal Higgs boundary conditions (NUHM). The effect of flavor violation is fold: (a) it shifts the co-annihilation regions towards lighter neutralino masses (b) co-annihilation cross sections would be modified with the inclusion of flavor violating which can contribute significantly. Even if flavor violation is within the presently limits, this is sufficient to modify the thermally averaged cross-sections by about 15)% in mSUGRA and (20{30)% in NUHM, depending on the parameter space. In overlap regions, the flavor violating cross sections become comparable and in some even dominant to the flavor conserving ones. A comparative study of the channels is for mSUGRA and NUHM cases.