Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Automatic compilation of MATLAB programs for synergistic execution on heterogeneous processors

MATLAB is an array language, initially popular for rapid prototyping, but is now being increasingly used to develop production code for numerical and scientific applications. Typical MATLAB programs have abundant data parallelism. These programs also have control flow dominated scalar regions that have an impact on the program's execution time. Today's computer systems have tremendous computing power in the form of traditional CPU cores and throughput oriented accelerators such as graphics processing units(GPUs). Thus, an approach that maps the control flow dominated regions to the CPU and the data parallel regions to the GPU can significantly improve program performance. In this paper, we present the design and implementation of MEGHA, a compiler that automatically compiles MATLAB programs to enable synergistic execution on heterogeneous processors. Our solution is fully automated and does not require programmer input for identifying data parallel regions. We propose a set of compiler optimizations tailored for MATLAB. Our compiler identifies data parallel regions of the program and composes them into kernels. The problem of combining statements into kernels is formulated as a constrained graph clustering problem. Heuristics are presented to map identified kernels to either the CPU or GPU so that kernel execution on the CPU and the GPU happens synergistically and the amount of data transfer needed is minimized. In order to ensure required data movement for dependencies across basic blocks, we propose a data flow analysis and edge splitting strategy. Thus our compiler automatically handles composition of kernels, mapping of kernels to CPU and GPU, scheduling and insertion of required data transfer. The proposed compiler was implemented and experimental evaluation using a set of MATLAB benchmarks shows that our approach achieves a geometric mean speedup of 19.8X for data parallel benchmarks over native execution of MATLAB.

Control of switching surges due to energization of 765kV UHV transmission line

Reduction of switching surge over voltages allows an economic design of UHV transmission system with reduced insulation. The various means of switching surge over voltage control with pre-insertion resistors/closing resistors, shunt re-actors and controlled switching are illustrated. The switching surge over voltages during the energization of series compensated line are compared with uncompensated line. An Electromagnetic transients program has been developed for studying the effect of various means of control of switching transients during 765kV UHV transmission line energization. This paper presents the studies carried out on switching surges control in 765kV UHV transmission line energization.

Tunability of electronic states in ultrathin gold nanowires

The electronic state in ultrathin gold nanowires is tuned by careful engineering of the device architecture via a chemical methodology. The electrons are localized to an insulating state (showing variable range hopping transport) by simply bringing them close to the substrate, while the insertion of an interlayer leads to a Tomonaga Luttinger liquid state.

Multivalent glycoliposomes and micelles to study carbohydrate-protein and carbohydrate-carbohydrate interactions

This tutorial review describes multivalent carbohydrate-protein and carbohydrate-carbohydrate interaction studies that utilize self-assembled aggregates of thermodynamically stable liposomes and micelles. Strategies to prepare multivalent glycoliposomes and micelles include: (i) insertion of synthetic glycolipids into matrix lipids; (ii) preparation of glycolipids that aggregate to liposomes and micelles and (iii) modification of the hydrophilic surfaces with desired sugars. Several design strategies have been developed in order to obtain constituent glycolipids, having multivalent sugar moieties and their subsequent interactions with proteins were assessed in relation to the type of linkers that connect the hydrophilic and lipophilic segments. Lipophilic segments other than alkyl chains have also been developed. Polymer based glycoliposomes and micelles form an emphasis. Further, glycoliposomes facilitate studies of carbohydrate-carbohydrate interactions. An overview of the various types of glycoliposomes and micelles used to study carbohydrate-protein and carbohydrate-carbohydrate recognition phenomena is presented.

Effect of signal peptide on stability and folding of escherichia coli thioredoxin

The signal peptide plays a key role in targeting and membrane insertion of secretory and membrane proteins in both prokaryotes and eukaryotes. In E. coli, recombinant proteins can be targeted to the periplasmic space by fusing naturally occurring signal sequences to their N-terminus. The model protein thioredoxin was fused at its N-terminus with malE and pelB signal sequences. While WT and the pelB fusion are soluble when expressed, the malE fusion was targeted to inclusion bodies and was refolded in vitro to yield a monomeric product with identical secondary structure to WT thioredoxin. The purified recombinant proteins were studied with respect to their thermodynamic stability, aggregation propensity and activity, and compared with wild type thioredoxin, without a signal sequence. The presence of signal sequences leads to thermodynamic destabilization, reduces the activity and increases the aggregation propensity, with malE having much larger effects than pelB. These studies show that besides acting as address labels, signal sequences can modulate protein stability and aggregation in a sequence dependent manner.

Recent advances in muffler acoustics

Exhaust noise in engines has always been a major source of automotive noise. Challenges for muffler design have been constraints on size, back pressure, and, of course, the cost. Designing for sufficient insertion loss at the engine firing frequency and the first few harmonics has been the biggest challenge. Most advances in the design of efficient mufflers have resulted from linear plane wave theory, making use of the transfer matrix method. This review paper deals with evaluating approximate source characteristics required for prediction of the unmuffled intake and exhaust noise, making use of the electroacoustical analogies. In the last few years, significant advances have been made in the analysis of variable area perforated ducts, transverse plane wave analysis of short elliptical as well as circular chambers, double-tuned expansion chambers and concentric tube resonators, catalytic converters, diesel particulate filters, air cleaners, etc. The development of long strand fibrous materials that can be used in hot exhaust systems without binders has led to the use of combination mufflers in exhaust systems. Breakthroughs have been achieved in the prediction and control of breakout noise from the elliptical and circular muffler shell as well as the end plates of typical mufflers. Diesel particulate filters and inlet air cleaners have also been modeled acoustically. Some of these recent advances are the subject of this review paper.

Synthon modularity in Cocrystals of 4-Bromobenzamide with n-Alkanedicarboxylic acids: type I and type ll Halogen center dot center dot center dot Halogen interactions

A Cambridge Structural Database (CSD) analysis on halogen center dot center dot center dot halogen contacts (X...X) in organic crystals has been carried out to review the classification criteria for type I, type II, and quasi type I/II halogen interactions. Trends observed in previous CSD analyses of the phenomenon are reinforced in the present study. The manner in which these interactions are manifested in cocrystals of 4-bromobenzamide and dicarboxylic acid is examined. The design strategy for these cocrystals uses synthon theory and follows from an understanding of the crystal structures of gamma-hydroquinone and a previously studied set of 4-hydroxybenzamide dicarboxylic acid cocrystals, making use of Br/OH isostructurality. All cocrystals are obtained by clean insertion of dicarboxylic acids between 4-bromobenzamide molecules. The strategy is deliberate and the prediction of synthons done well in advance, as evidenced from the robustness of the acid-amide heterosynthons in all nine crystal structures, with no aberrant structures in the crystallization experiments. Formation of the acid-amide synthon in these cocrystals is identified with IR spectroscopy. The packing in these cocrystals can be distinguished in terms of whether the Br...Br interactions are type I or II. Eight sets of dimorphs were retrieved from the CSD, wherein the basis of the polymorphism is that one crystal has a type I Br...Br interaction, while the other has a type II interaction.

C-13-H-1 dipolar couplings for probing rod-like hydrogen bonded mesogens

Hydrogen bonding is the most important non-covalent interaction utilised in building supramolecular assemblies and is preferred often as a means of construction of molecular, oligomeric as well as polymeric materials that show liquid crystalline properties. In this work, a pyridine based nematogenic acceptor has been synthesized and mixed with non-mesogenic 4-methoxy benzoic acid to get a hydrogen bonded mesogen. The existence of hydrogen bonding between the pyridyl unit and the carboxylic acid was established using FT-IR spectroscopy from the observation of characteristic stretching vibrations of unionized type at 2425 and 1927 cm(-1). The mesogenic acceptor and the complex have been investigated using C-13 NMR in solution, solid and liquid crystalline states. Together with the 2D separated local field NMR experiments, the studies confirm the molecular structure in the mesophase and yield the local orientational order parameters. It is observed that the insertion of 4-methoxy benzoic acid not only enhances the mesophase stability but also induces a smectic phase due to an increase in the core length of the hydrogen bonded mesogen.

Structural Insights into Saccharomyces cerevisiae Msh4-Msh5 Complex Function Using Homology Modeling

The Msh4-Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4-Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs (Msh2, Msh3, Msh6). Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4-Msh5 complex has hindered an understanding of the structural aspects of Msh4-Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4-Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4-Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4-Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4-Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.

Synthesis, characterisation and application of an exfoliated graphite-diamond composite electrode in the electrochemical degradation of trichloroethylene

A composite electrode made up of exfoliated graphite (EG) and diamond was prepared for the electrochemical oxidation of trichloroethylene (TCE). The SEM images of the EG-diamond material showed that diamond powders were dispersed on the surface of EG materials. The N-2 adsorption-desorption isotherm of EG-diamond material resulted in a poor adsorption capability due to the insertion of diamond powders into the porous matrix of EG. Raman spectroscopy revealed the presence of characteristic sp(3) bands of diamond confirming good interaction of diamond with EG. Electrochemical characterisation of EG-diamond in 0.1 M Na2SO4 resulted in an enhanced working potential window. The EG-diamond electrode was employed for the electrochemical oxidation of trichloroethylene (0.2 mM) in a Na2SO4 supporting electrolyte. The EG-diamond, in comparison to the pristine EG electrode, exhibited a higher removal efficiency of 94% (EG was 57%) and faster degradation kinetics of 25.3 x 10(-3) min(-1) showing pseudo first order kinetic behaviour. Under the optimised conditions, 73% total organic content (TOC) removal was achieved after 4 h of electrolysis. The degradation of TCE was also monitored with gas chromatography-mass spectrometry. Dichloroacetic acid (DCAA) was identified as a major intermediate product during the electrochemical oxidation of TCE. The electrochemical degradation of TCE at the EG-diamond electrode represents a cost effective method due to the ease of preparation of EG-diamond composite material without the necessity of diamond activation which is normally achieved through doping.

CAVVA: Computational Affective Video-in-Video Advertising

Advertising is ubiquitous in the online community and more so in the ever-growing and popular online video delivery websites (e. g., YouTube). Video advertising is becoming increasingly popular on these websites. In addition to the existing pre-roll/post-roll advertising and contextual advertising, this paper proposes an in-stream video advertising strategy-Computational Affective Video-in-Video Advertising (CAVVA). Humans being emotional creatures are driven by emotions as well as rational thought. We believe that emotions play a major role in influencing the buying behavior of users and hence propose a video advertising strategy which takes into account the emotional impact of the videos as well as advertisements. Given a video and a set of advertisements, we identify candidate advertisement insertion points (step 1) and also identify the suitable advertisements (step 2) according to theories from marketing and consumer psychology. We formulate this two part problem as a single optimization function in a non-linear 0-1 integer programming framework and provide a genetic algorithm based solution. We evaluate CAVVA using a subjective user-study and eye-tracking experiment. Through these experiments, we demonstrate that CAVVA achieves a good balance between the following seemingly conflicting goals of (a) minimizing the user disturbance because of advertisement insertion while (b) enhancing the user engagement with the advertising content. We compare our method with existing advertising strategies and show that CAVVA can enhance the user's experience and also help increase the monetization potential of the advertising content.

Evaluation of the Genotoxic and Antigenotoxic Potential of the Alkaloid Punarnavine from Boerhaavia diffusa

Boerhaavia diffusa is a traditional herbal medicine extensively used in the Ayurveda and Unani forms of medicine in India and many parts of the world. Different parts of the plant are used as an appetizer, alexiteric, eye tonic, for flushing out the renal system, and to treat blood pressure. This study was conducted to evaluate the in vivo genotoxic and/or antigenotoxic potential of punarnavine, a separated alkaloid from the root of B. diffusa using toxicity studies (OECD guideline 474, 1997). The genotoxic and antigenotoxic potential of punarnavine was assayed using the comet assay on lymphocytes, liver, spleen, brain, and bone marrow as well as using the micronucleus test in bone marrow cells including the in vitro chromosomal aberration test. The results demonstrated that none of the tested doses of punarnavine showed genotoxic effects by the comet assay, or clastogenic effects in the micronucleus test. On the other hand, for all cells evaluated, the three tested doses of punarnavine promoted inhibition of DNA damage induced by cyclophosphamide. Based on these results, we concluded that punarnavine, an alkaloid from the Boerhaavia diffusa root, has no genotoxic or clastogenic effects in our experimental conditions. However, it caused a significant decrease in DNA damage induced by cyclophosphamide. It is suggested that the antigenotoxic properties of this alkaloid may be of great pharmacological importance and beneficial for cancer prevention.

In Vivo Genotoxicity Assessment of Titanium Dioxide Nanoparticles by Allium cepa Root Tip Assay at High Exposure Concentrations

The industrial production and commercial applications of titanium dioxide nanoparticles have increased considerably in recent times, which has increased the probability of environmental contamination with these agents and their adverse effects on living systems. This study was designed to assess the genotoxicity potential of TiO2 NPs at high exposure concentrations, its bio-uptake, and the oxidative stress it generated, a recognised cause of genotoxicity. Allium cepa root tips were treated with TiO2 NP dispersions at four different concentrations (12.5, 25, 50, 100 mu g/mL). A dose dependant decrease in the mitotic index (69 to 21) and an increase in the number of distinctive chromosomal aberrations were observed. Optical, fluorescence and confocal laser scanning microscopy revealed chromosomal aberrations, including chromosomal breaks and sticky, multipolar, and laggard chromosomes, and micronucleus formation. The chromosomal aberrations and DNA damage were also validated by the comet assay. The bio-uptake of TiO2 in particulate form was the key cause of reactive oxygen species generation, which in turn was probably the cause of the DNA aberrations and genotoxicity observed in this study.

MazF-induced Growth Inhibition and Persister Generation in Escherichia coli

Background: MazEF is a chromosomally encoded bacterial toxin-antitoxin system whose cellular role is controversial. Results: Expression of chromosomal MazF inhibits cell killing by multiple antibiotics in a Lon and ClpP dependent manner. Conclusion: MazF is involved in reversible growth inhibition and bacterial drug tolerance. Significance: Inactive, active-site toxin mutants yield functional insights by selectively activating the corresponding WT toxin in vivo. Toxin-antitoxin systems are ubiquitous in nature and present on the chromosomes of both bacteria and archaea. MazEF is a type II toxin-antitoxin system present on the chromosome of Escherichia coli and other bacteria. Whether MazEF is involved in programmed cell death or reversible growth inhibition and bacterial persistence is a matter of debate. In the present work the role of MazF in bacterial physiology was studied by using an inactive, active-site mutant of MazF, E24A, to activate WT MazF expression from its own promoter. The ectopic expression of E24A MazF in a strain containing WT mazEF resulted in reversible growth arrest. Normal growth resumed on inhibiting the expression of E24A MazF. MazF-mediated growth arrest resulted in an increase in survival of bacterial cells during antibiotic stress. This was studied by activation of mazEF either by overexpression of an inactive, active-site mutant or pre-exposure to a sublethal dose of antibiotic. The MazF-mediated persistence phenotype was found to be independent of RecA and dependent on the presence of the ClpP and Lon proteases. This study confirms the role of MazEF in reversible growth inhibition and persistence.