Functional characterization of UvrD helicases from Haemophilus influenzae and Helicobacter pylori

Haemophilus influenzae and Helicobacter pylori are major bacterial pathogens that face high levels of genotoxic stress within their host. UvrD, a ubiquitous bacterial helicase that plays important roles in multiple DNA metabolic pathways, is essential for genome stability and might, therefore, be crucial in bacterial physiology and pathogenesis. In this study, the functional characterization of UvrD helicase from Haemophilus influenzae and Helicobacter pylori is reported. UvrD from Haemophilus influenzae (HiUvrD) and Helicobacter pylori (HpUvrD) exhibit strong single-stranded DNA-specific ATPase and 3'5' helicase activities. Mutation of highly conserved arginine (R288) in HiUvrD and glutamate (E206) in HpUvrD abrogated their activities. Both the proteins were able to bind and unwind a variety of DNA structures including duplexes with strand discontinuities and branches, three- and four-way junctions that underpin their role in DNA replication, repair and recombination. HiUvrD required a minimum of 12 nucleotides, whereas HpUvrD preferred 20 or more nucleotides of 3'-single-stranded DNA tail for efficient unwinding of duplex DNA. Interestingly, HpUvrD was able to hydrolyze and utilize GTP for its helicase activity although not as effectively as ATP, which has not been reported to date for UvrD characterized from other organisms. HiUvrD and HpUvrD were found to exist predominantly as monomers in solution together with multimeric forms. Noticeably, deletion of distal C-terminal 48 amino acid residues disrupted the oligomerization of HiUvrD, whereas deletion of 63 amino acids from C-terminus of HpUvrD had no effect on its oligomerization. This study presents the characteristic features and comparative analysis of Haemophilus influenzae and Helicobacter pylori UvrD, and constitutes the basis for understanding the role of UvrD in the biology and virulence of these pathogens.

Elasto-plastic analysis of jointed rocks using discrete continuum and equivalent continuum approaches

Results from elasto-plastic numerical simulations of jointed rocks using both the equivalent continuum and discrete continuum approaches are presented, and are compared with experimental measurements. Initially triaxial compression tests on different types of rocks with wide variation in the uniaxial compressive strength are simulated using both the approaches and the results are compared. The applicability and relative merits and limitations of both the approaches for the simulation of jointed rocks are discussed. It is observed that both the approaches are reasonably good in predicting the real response. However, the equivalent continuum approach has predicted somewhat higher stiffness values at low strains. Considering the modelling effort involved in case of discrete continuum approach, for problems with complex geometry, it is suggested that a proper equivalent continuum model can be used, without compromising much on the accuracy of the results. Then the numerical analysis of a tunnel in Japan is taken up using the continuum approach. The deformations predicted are compared well against the field measurements and the predictions from discontinuum analysis. (C) 2012 Elsevier Ltd. All rights reserved.

Control of a Duct Flow Network for Wind Display in a Virtual Environment

Control of flow in duct networks has a myriad of applications ranging from heating, ventilation, and air-conditioning to blood flow networks. The system considered here provides vent velocity inputs to a novel 3-D wind display device called the TreadPort Active Wind Tunnel. An error-based robust decentralized sliding-mode control method with nominal feedforward terms is developed for individual ducts while considering cross coupling between ducts and model uncertainty as external disturbances in the output. This approach is important due to limited measurements, geometric complexities, and turbulent flow conditions. Methods for resolving challenges such as turbulence, electrical noise, valve actuator design, and sensor placement are presented. The efficacy of the controller and the importance of feedforward terms are demonstrated with simulations based upon an experimentally validated lumped parameter model and experiments on the physical system. Results show significant improvement over traditional control methods and validate prior assertions regarding the importance of decentralized control in practice.

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.

Clinical phenotype and the lack of mutations in the CHRNG, CHRND, and CHRNA1 genes in two Indian families with Escobar syndrome

The objective of this study was to report the clinical phenotype and genetic analysis of two Indian families with Escobar syndrome (ES). The diagnosis of ES in both families was made on the basis of published clinical features. Blood samples were collected from members of both families and used in genomic DNA isolation. The entire coding regions and intron-exon junctions of the ES gene CHRNG (cholinergic receptor, nicotinic, gamma), and two other related genes, CHRND and CHRNA1, were amplified and sequenced to search for mutations in both families. Both families show a typical form of ES. Sequencing of the entire coding regions including the intron-exon junctions of the three genes did not yield any mutations in these families. In conclusion, it is possible that the mutations in these genes are located in the promoter or deep intronic regions that we failed to identify or the ES in these families is caused by mutations in a different gene. The lack of mutations in CHRNG has also been reported in several families, suggesting the possibility of at least one more gene for this syndrome. Clin Dysmorphol 22:54-58 (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Evidence for the role of Mycobacteriumtuberculosis RecG helicase in DNA repair and recombination

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacteriumtuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M.tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichiacoli recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions.

Mutation analysis of the SLC4A11 gene in Indian families with congenital hereditary endothelial dystrophy 2 and a review of the literature

Purpose: Congenital hereditary endothelial dystrophy 2 (CHED2) is an autosomal recessive disorder caused by mutations in the solute carrier family 4, sodium borate transporter, member 11 (SLC4A11) gene. The purpose of this study was to identify the genetic cause of CHED2 in six Indian families and catalog all known mutations in the SLC4A11 gene. Methods: Peripheral blood samples were collected from individuals of the families with CHED2 and used in genomic DNA isolation. PCR primers were used to amplify the entire coding region including intron-exon junctions of SLC4A11. Amplicons were subsequently sequenced to identify the mutations. Results: DNA sequence analysis of the six families identified four novel (viz., p.Thr262Ile, p.Gly417Arg, p.Cys611Arg, and p.His724Asp) mutations and one known p.Arg869His homozygous mutation in the SLC4A11 gene. The mutation p.Gly417Arg was identified in two families. Conclusions: This study increases the mutation spectrum of the SLC4A11 gene. A review of the literature showed that the total number of mutations in the SLC4A11 gene described to date is 78. Most of the mutations are missense, followed by insertions-deletions. The present study will be helpful in genetic diagnosis of the families reported here.

Unsteady separation and vortex shedding from a laminar separation bubble over a bluff body

Boundary layers are subject to favorable and adverse pressure gradients because of both the temporal and spatial components of the pressure gradient. The adverse pressure gradient may cause the flow to separate. In a closed loop unsteady tunnel we have studied the initiation of separation in unsteady flow past a constriction (bluff body) in a channel. We have proposed two important scalings for the time when boundary layer separates. One is based on the local pressure gradient and the other is a convective time scale based on boundary layer parameters. The flow visualization using a dye injection technique shows the flow structure past the body. Nondimensional shedding frequency (Strouhal number) is calculated based on boundary layer and momentum thicknesses. Strouhal number based on the momentum thickness shows a close agreement with that for flat plate and circular cylinder.

High speed digital holographic interferometry for hypersonic flow visualization

Optical imaging techniques have played a major role in understanding the flow dynamics of varieties of fluid flows, particularly in the study of hypersonic flows. Schlieren and shadowgraph techniques have been the flow diagnostic tools for the investigation of compressible flows since more than a century. However these techniques provide only the qualitative information about the flow field. Other optical techniques such as holographic interferometry and laser induced fluorescence (LIF) have been used extensively for extracting quantitative information about the high speed flows. In this paper we present the application of digital holographic interferometry (DHI) technique integrated with short duration hypersonic shock tunnel facility having 1 ms test time, for quantitative flow visualization. Dynamics of the flow fields in hypersonic/supersonic speeds around different test models is visualized with DHI using a high-speed digital camera (0.2 million fps). These visualization results are compared with schlieren visualization and CFD simulation results. Fringe analysis is carried out to estimate the density of the flow field.

Engineered spin-valve type magnetoresistance in Fe3O4-CoFe2O4 core-shell nanoparticles

Naturally occurring spin-valve-type magnetoresistance (SVMR), recently observed in Sr2FeMoO6 samples, suggests the possibility of decoupling the maximal resistance from the coercivity of the sample. Here we present the evidence that SVMR can be engineered in specifically designed and fabricated core-shell nanoparticle systems, realized here in terms of soft magnetic Fe3O4 as the core and hard magnetic insulator CoFe2O4 as the shell materials. We show that this provides a magnetically switchable tunnel barrier that controls the magnetoresistance of the system, instead of the magnetic properties of the magnetic grain material, Fe3O4, and thus establishing the feasibility of engineered SVMR structures. (C) 2013 AIP Publishing LLC.

Experimental Investigation of Heat Fluxes in the Vicinity of Protuberances on a Flat Plate at Hypersonic Speeds

Heat transfer rates measured in front and to the side of a protrusion on an aluminum flat plate subjected to hypersonic flow at zero angle of attack are presented for two flow enthalpies of approximately 2 MJ/kg and 4.5 MJ/kg. Experiments were conducted in the hypersonic shock tunnel (HST2) and free piston driven HST3 at a freestream Mach number of 8. Heat transfer data was obtained for different geometries of the protrusion of a height of 4 mm, which is approximately the local boundary layer thickness. Comparatively high rates of heat transfer were obtained at regions of flow circulation in the separated region, with the hottest spot generally appearing in front of the protuberance. Experimental values showed moderate agreement with existing empirical correlations at higher enthalpy but not at all for the lower enthalpy condition, although the correlations were coined at enthalpy values nearer to the lower value. Schlieren visualization was also done to investigate the flow structures qualitatively.

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.

Stability of long unsupported twin circular tunnels in soils

The stability of two long unsupported circular parallel tunnels aligned horizontally in fully cohesive and cohesive-frictional soils has been determined. An upper bound limit analysis in combination with finite elements and linear programming is employed to perform the analysis. For different clear spacing (S) between the tunnels, the stability of tunnels is expressed in terms of a non-dimensional stability number (gamma H-max/c); where H is tunnel cover, c refers to soil cohesion, and gamma(max) is maximum unit weight of soil mass which the tunnels can bear without any collapse. The variation of the stability number with tunnels' spacing has been established for different combinations of H/D, m and phi; where D refers to diameter of each tunnel, phi is the internal friction angle of soil and m accounts for the rate at which the cohesion increases linearly with depth. The stability number reduces continuously with a decrease in the spacing between the tunnels. The optimum spacing (S-opt) between the two tunnels required to eliminate the interference effect increases with (i) an increase in H/D and (ii) a decrease in the values of both m and phi. The value of S-opt lies approximately in a range of 1.5D-3.5D with H/D = 1 and 7D-12D with H/D = 7. The results from the analysis compare reasonably well with the different solutions reported in literature. (C) 2013 Elsevier Ltd. All rights reserved.

Numerical and experimental investigation and optimization of an open wheeled race car cooling air duct

In this study the cooling performance due to air flow and aerodynamics of the Formula Student open wheeled race car has been investigated and optimized with the help of CFD simulations and experimental validation. The race car in context previously suffered from overheating problems. Flow analysis was carried out based on the detailed race car 3D model (NITK Racing 2012 formula student race car). Wind tunnel experiments were carried out on the same. The results obtained from the computer simulations are compared with experimental results obtained from wind tunnel testing of the full car. Through this study it was possible to locate the problem areas and hence choose the best configuration for the cooling duct. The CFD analysis helped in calculating the mass flow rate, pressure and velocity distribution for different velocities of the car which is then used to determine the heat dissipated by the radiator. Area of flow separation could be visualized and made sure smooth airflow into the radiator core area. This significantly increased the cooling performance of the car with reduction in drag.

RNA-dependent RNA polymerase of Japanese encephalitis virus binds the initiator nucleotide GTP to form a mechanistically important pre-initiation state

Flaviviral RNA-dependent RNA polymerases (RdRps) initiate replication of the single-stranded RNA genome in the absence of a primer. The template sequence 5'-CU-3' at the 3'-end of the flaviviral genome is highly conserved. Surprisingly, flaviviral RdRps require high concentrations of the second incoming nucleotide GTP to catalyze de novo template-dependent RNA synthesis. We show that GTP stimulates de novo RNA synthesis by RdRp from Japanese encephalitis virus (jRdRp) also. Crystal structures of jRdRp complexed with GTP and ATP provide a basis for specific recognition of GTP. Comparison of the jRdRp(GTP) structure with other viral RdRp-GTP structures shows that GTP binds jRdRp in a novel conformation. Apo-jRdRp structure suggests that the conserved motif F of jRdRp occupies multiple conformations in absence of GTP. Motif F becomes ordered on GTP binding and occludes the nucleotide triphosphate entry tunnel. Mutational analysis of key residues that interact with GTP evinces that the jRdRp(GTP) structure represents a novel pre-initiation state. Also, binding studies show that GTP binding reduces affinity of RdRp for RNA, but the presence of the catalytic Mn2+ ion abolishes this inhibition. Collectively, these observations suggest that the observed pre-initiation state may serve as a check-point to prevent erroneous template-independent RNA synthesis by jRdRp during initiation.