Reactive Pulsed Laser Deposition of titanium nitride thin film: Optimization of process parameters using Secondary Ion Mass Spectrometry

Reactive Pulsed Laser Deposition is a single step process wherein the ablated elemental metal reacts with a low pressure ambient gas to form a compound. We report here a Secondary Ion Mass Spectrometry based analytical methodology to conduct minimum number of experiments to arrive at optimal process parameters to obtain high quality TiN thin film. Quality of these films was confirmed by electron microscopic analysis. This methodology can be extended for optimization of other process parameters and materials. (C) 2009 Elsevier B.V. All rights reserved.

Quantitation and characterization of glutathionyl haemoglobin as an oxidative stress marker in chronic renal failure by mass spectrometry

Objectives: Glutathionyl haemoglobin (GS-Hb) belonging to the class of glutathionylated proteins has been investigated as a possible marker of oxidative stress in different chronic diseases. The purpose of this study was to examine whether glutathionyl haemoglobin can serve as an oxidative stress marker in non-diabetic chronic renal failure patients on different renal replacement therapies (RRT) through its quantitation, and characterization of the specific binding site of glutathione in haemoglobin molecule by mass spectrometric analysis. Design and methods: The study group consisted of non-diabetic chronic renal failure patients on renal replacement therapy (RRT): hemodialysis (HD), continuous ambulatory peritoneal dialysis (CAPD) and renal allograft transplant (Txp) patients. Haemoglobin samples of these subjects were analyzed by liquid chromatography electrospray ionization mass spectrometry for GS-Hb quantitation. Characterization of GS-Hb was done by tandem mass spectrometry. Levels of erythrocyte glutathione (GSH) and lipid peroxidation (as thiobarbituric acid reacting substances) were measured spectrophotometrically, while glycated baernoglobin (HbA1c) was measured by HPLC. Results: GS-Hb levels were markedly elevated in the dialysis group and marginally in the transplant group as compared to the controls. GS-Hb levels correlated positively with lipid peroxidation and negatively with the erythrocyte glutathione levels in RRT groups indicating enhanced oxidative stress. De novo sequencing of the chymotryptic fragment of GS-Hb established that glutathione is attached to Cys-93 of the beta globin chain. Mass spectrometric quantitation of total glycated haemoglobin showed good agreement with HbA1c estimation by conventional HPLC method. Conclusions: Glutathionyl haemoglobin can serve as a clinical marker of oxidative stress in chronic debilitating therapies like RRT. Mass spectrometry provides a reliable analytical tool for quantitation and residue level characterization of different post-translational modifications of haemoglobin. (c) 2007 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Proteomics and mass spectrometric studies reveal planktonic growth of Mycobacterium smegmatis in biofilm cultures in the absence of rpoZ

Mycobacterium smegmatis is known to form biofilms and many cell surface molecules like core glycopeptidolipids and short-chain mycolates appear to play important role in the process. However, the involvement of the cell surface molecules in mycobacteria towards complete maturation of biofilms is still not clear. This work demonstrates the importance of the glycopeptidolipid species with hydroxylated alkyl chain and the epoxylated mycolic acids, during the process of biofilm development. In our previous study, we reported the impairment of biofilm formation in rpoZ-deleted M. smegmatis, where rpoZ codes for the ω subunit of RNA polymerase (R. Mathew, R. Mukherjee, R. Balachandar, D. Chatterji, Microbiology 152 (2006) 1741). Here we report the occurrence of planktonic growth in a mc2155 strain which is devoid of rpoZ gene. This strain is deficient in selective incorporation of the hydroxylated glycopeptidolipids and the epoxy mycolates to their respective locations in the cell wall. Hence it forms a mutant biofilm defective in maturation, wherein the cells undertake various alternative metabolic pathways to survive in an environment where oxygen, the terminal electron acceptor, is limiting.

Vibrational phase relaxation of O–H stretch in bulk water: Role of large amplitude angular jumps and negative cross-correlations among the forces on the O–H bond

The ultrafast vibrational phase relaxation of O–H stretch in bulk water is investigated in molecular dynamics simulations. The dephasing time (T2) of the O–H stretch in bulk water calculated from the frequency fluctuation time correlation function (Cω(t)) is in the range of 70–80 femtosecond (fs), which is comparable to the characteristic timescale obtained from the vibrational echo peak shift measurements using infrared photon echo [W.P. de Boeij, M.S. Pshenichnikov, D.A. Wiersma, Ann. Rev. Phys. Chem. 49 (1998) 99]. The ultrafast decay of Cω(t) is found to be responsible for the ultrashort T2 in bulk water. Careful analysis reveals the following two interesting reasons for the ultrafast decay of Cω(t). (A) The large amplitude angular jumps of water molecules (within 30–40 fs time duration) provide a large scale contribution to the mean square vibrational frequency fluctuation and gives rise to the rapid spectral diffusion on 100 fs time scale. (B) The projected force, due to all the atoms of the solvent molecules on the oxygen (FO(t)) and hydrogen (FH(t)) atom of the O–H bond exhibit a large negative cross-correlation (NCC). We further find that this NCC is partly responsible for a weak, non-Arrhenius temperature dependence of the dephasing rate.

Non-segmented negative sense RNA viruses as vectors for vaccine development

This article intends to cover two aspects of non-segmented negative sense RNA viruses. In the initial section, the strategy employed by these viruses to replicate their genomes is discussed. This would help in understanding the later section in which the use of these viruses as vaccine vectors has been discussed. For the description of the replication strategy which encompasses virus genome transcription and genome replication carried out by the same RNA dependent RNA polymerase complex, a member of the prototype rhabdovirus family - Chandipura virus has been chosen as an example to illustrate the complex nature of the two processes and their regulation. In the discussion on these viruses serving as vectors for carrying vaccine antigen genes, emphasis has been laid on describing the progress made in using the attenuated viruses as vectors and a description of the systems in which the efficiency of immune responses has been tested.

Detection of alpha(2u)-globulin and its bound putative pheromones in the preputial gland of the Indian commensal rat (Rattus rattus) using mass spectrometry

The role of pheromones and pheromone-binding proteins in the laboratory rat has been extensively investigated. However, we have previously reported that the preputial gland of the Indian commensal rat produces a variety of pheromonal molecules and preputial glands would seem to be the predominant source for pheromonal communication. The presence of pheromone-binding proteins has not yet been identified in the preputial gland of the Indian commensal rat; therefore, the experiments were designed to unravel the alpha(2u)-globulin (alpha 2u) and its bound volatiles in the commensal rat. Total preputial glandular proteins were first fractionated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently analyzed by mass spectrometry. Further, we purified alpha 2u and screened for the presence of bound pheromonal molecules with the aid of gas chromatography/mass spectrometry (GC/MS). A novel alpha 2u was identified with a high score and this protein has not been previously described as present in the preputial gland of Indian commensal rats.This novel alpha 2u was then characterized by tandem mass spectrometry (MS/MS). Peptides with m/z values of 969, 1192, 1303 and 1876 were further fragmented with the aid of MS/MS and generated de novo sequences which provided additional evidence for the presence of alpha 2u in the preputial gland. Finally, we identified the presence of farnesol 1 and 2 bound to alpha 2u. The present investigation confirms the presence of alpha 2u (18.54 kDa) in the preputial gland of the Indian commensal rat and identifies farnesol 1 and 2 as probably involved in chemo-communication by the Indian commensal rat.Copyright (C) 2010 John Wiley & Sons, Ltd.

Negative nuclear Overhauser effects as probes of macromolecular structure

Nuclear Overhauser effects (NOE) and circular dichroism (CD) techniques have been used to probe @-turn conformations in acyclic and cyclic peptides containingPro-Xsequences. The model peptides studied are of the type Piv-Pro-X-NHMe (X = Aib, D-Ala, Gly, Val, and Leu) and Boc-Cys-Pro-X-C s NHMe (X = Aib, L-Ala, D-Ala, Gly, and Leu). In the acyclic series, observation of NOES between Pro C"H and X-NH, together with solvent and temperature dependence of NH chemical shifts, establishes a 4 - 1 hydrogen bond stabilized type I1 @-turn in the Gly, D-Ala, and Aib peptides, in CDC13 and (CD3)2S0. A positive n-r* CD band at -225-230 nm appears to be characteristic of this structure. For the acyclic Pro-Leu peptide the observation of NOE's for both Pro and Leu C"H resonances on saturation of Leu NH is compatible with a type V bend or consecutive y-turn conformation. In the cyclic disulfide series the Pro-Aib and Pro-D-Ala peptides favor type I1 @-turns, whereas all other peptides adopt type I (111) conformations. All the cyclic disulfides exhibit an intense negative CD band at -228-230 nm. The results suggest thatgeneralcorrelations between CD spectral type and specific 0-turn conformations may not be obtained. Evidence for solvent-dependent structural changes in the Pro-Aib sequence in both cyclic and acyclic peptides is presented.

Simultaneous observation of positive and negative nuclear Overhauser effects in oligopeptides due to segmental motio

Nuclear Overhauser effect (NOE) studies of the symmetrical cystine peptides (Formula: see text) (n = 1-3) in dimethylsulfoxide, have resulted in the simultaneous observation of both positive and negative NOEs. Positive NOEs are observed on the Trp C2H and C4H protons of the indole ring upon irradiation of Trp C alpha H and C beta H2 resonances in the peptides where n = 1 and 2. Negative NOEs are observed between backbone NH and C alpha H protons. The magnitudes of the observed NOEs are sensitive to changes in molecular size and solvent viscosity. The results demonstrate that NOEs may be a useful probe of sidechain segmental motion in oligopeptides.

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.

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.

Large latitudinal gradients and temporal heterogeneity in aerosol black carbon and its mass mixing ratio over southern and northern oceans observed during a trans-continental cruise experiment

Extensive, and collocated measurements of the mass concentrations (M-B) of aerosol black carbon (BC) and (M-T) of composite aerosols were made over the Arabian Sea, tropical Indian Ocean and the Southern Ocean during a trans-continental cruise experiment. Our investigations show that MB remains extremely low(<50 ng m(-3)) and remarkably steady (in space and time) in the Southern Ocean (20 degrees S to 56 degrees S). In contrast, large latitudinal gradients exist north of similar to 20 degrees S; M-B increasing exponentially to reach as high as 2000 ng m(-3) in the Arabian Sea (similar to 8 degrees N). Interestingly, the share of BC showed a distinctly different latitudinal variation, with a peak close to the equator and decreasing on either side. Large fluctuations were seen in M-T over Southern Ocean associated with enhanced production of sea-salt aerosols in response to sea-surface wind speed. These spatio-temporal changes in M-B and its mixing ratio have important implications to regional and global climate.

Approximate methods for step function response of undamped non-linear spring mass systems with arbitrary hardening type spring characteristic

The possibility of applying two approximate methods for determining the salient features of response of undamped non-linear spring mass systems subjected to a step input, is examined. The results obtained on the basis of these approximate methods are compared with the exact results that are available for some particular types of spring characteristics. The extension of the approximate methods for non-linear systems with general polynomial restoring force characteristics is indicated.

Effect of static deflection on natural frequency of non-linear spring mass system by direct linearization method

An exact expression for the frequency of a non-linear cubic spring mass system is obtained considering the effect of static deflection. An alternative expression for the approximate frequency is also obtained by the direct linearization procedure; it is shown that this is very accurate as compared with the exact method. This approximate frequency equation is used to explain a “dual behaviour” of the frequency amplitude curves.

Response spectrum of non-linear spring mass system subjected to transient disturbances

The transient response spectrum of a cubic spring mass system subjected to a step function input is obtained. An approximate method is adopted where non-linear restoring force characteristic is replaced by two linear segments, so that the mean square error between them is a minimum. The effect of viscous damping on the peak response is also discussed for various values of the damping constant and the non-linearity restoring force parameter.