Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of mycobacterium tuberculosis

Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.

Effects of two-wheelers on saturation flow at signalized intersections in developing countries

Two-wheelers (TW) constitute a major proportion of urban traffic in developing countries and therefore their effect on the saturation flow at signalized intersections could be substantial. This paper attempts to study and analyze the effect of two-wheelers on the saturation flow of signalized intersections by collecting data at a few signalized intersections in Bangalore, India. A strong correlation is observed between the measured saturation flow and the proportion of two-wheeler traffic, which suggest that two-wheelers have significant impact and should be considered in the capacity analysis of signalized intersections. In this paper, the effect of two-wheelers on saturation flow rate is incorporated in a previous model by calibrating and introducing a new adjustment factor for two-wheelers. Results show that saturation flow measured using the modified HCM equation is closer to observed saturation flow values.

Influence of rock depth on seismic site classification for shallow bedrock regions

Seismic site classifications are used to represent site effects for estimating hazard parameters (response spectral ordinates) at the soil surface. Seismic site classifications have generally been carried out using average shear wave velocity and/or standard penetration test n-values of top 30-m soil layers, according to the recommendations of the National Earthquake Hazards Reduction Program (NEHRP) or the International Building Code (IBC). The site classification system in the NEHRP and the IBC is based on the studies carried out in the United States where soil layers extend up to several hundred meters before reaching any distinct soil-bedrock interface and may not be directly applicable to other regions, especially in regions having shallow geological deposits. This paper investigates the influence of rock depth on site classes based on the recommendations of the NEHRP and the IBC. For this study, soil sites having a wide range of average shear wave velocities (or standard penetration test n-values) have been collected from different parts of Australia, China, and India. Shear wave velocities of rock layers underneath soil layers have also been collected at depths from a few meters to 180 m. It is shown that a site classification system based on the top 30-m soil layers often represents stiffer site classes for soil sites having shallow rock depths (rock depths less than 25 m from the soil surface). A new site classification system based on average soil thickness up to engineering bedrock has been proposed herein, which is considered more representative for soil sites in shallow bedrock regions. It has been observed that response spectral ordinates, amplification factors, and site periods estimated using one-dimensional shear wave analysis considering the depth of engineering bedrock are different from those obtained considering top 30-m soil layers.

Mycobacterium tuberculosis mutT1 (Rv2985) and ADPRase (Rv1700) proteins constitute a two-stage mechanism of 8-Oxo-dGTP and 8-Oxo-GTP detoxification and adenosine to cytidine mutation avoidance

Approximately one third of the world population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. A better understanding of the pathogen biology is crucial to develop new tools/strategies to tackle its spread and treatment. In the host macrophages, the pathogen is exposed to reactive oxygen species, known to damage dGTP and GTP to 8-oxo-dGTP and 8-oxo-GTP, respectively. Incorporation of the damaged nucleotides in nucleic acids is detrimental to organisms. MutT proteins, belonging to a class of Nudix hydrolases, hydrolyze 8-oxo-G nucleoside triphosphates/diphosphates to the corresponding nucleoside monophosphates and sanitize the nucleotide pool. Mycobacteria possess several MutT proteins. However, a functional homolog of Escherichia coli MutT has not been identified. Here, we characterized MtuMutT1 and Rv1700 proteins of M. tuberculosis. Unlike other MutT proteins, MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP, and 8-oxo-GTP to 8-oxo-GDP. Rv1700 then converts them to the corresponding nucleoside monophosphates. This observation suggests the presence of a two-stage mechanism of 8-oxo-dGTP/8-oxo-GTP detoxification in mycobacteria. MtuMutT1 converts 8-oxo-dGTP to 8-oxo-dGDP with a K-m of similar to 50 mu M and V-max of similar to 0.9 pmol/min per ng of protein, and Rv1700 converts 8-oxo-dGDP to 8-oxo-dGMP with a K-m of similar to 9.5 mu M and V-max of similar to 0.04 pmol/min per ng of protein. Together, MtuMutT1 and Rv1700 offer maximal rescue to E. coli for its MutT deficiency by decreasing A to C mutations (a hallmark of MutT deficiency). We suggest that the concerted action of MtuMutT1 and Rv1700 plays a crucial role in survival of bacteria against oxidative stress.

Processing of DNA double-stranded breaks and intermediates of recombination and repair by saccharomyces cerevisiae Mre11 and its stimulation by Rad50, Xrs2, and Sae2 proteins

Saccharomyces cerevisiae RAD50, MRE11, and XRS2 genes are essential for telomere length maintenance, cell cycle checkpoint signaling, meiotic recombination, and DNA double-stranded break (DSB) repair via nonhomologous end joining and homologous recombination. The DSB repair pathways that draw upon Mre11-Rad50-Xrs2 subunits are complex, so their mechanistic features remain poorly understood. Moreover, the molecular basis of DSB end resection in yeast mre11-nuclease deficient mutants and Mre11 nuclease-independent activation of ATM in mammals remains unknown and adds a new dimension to many unanswered questions about the mechanism of DSB repair. Here, we demonstrate that S. cerevisiae Mre11 (ScMre11) exhibits higher binding affinity for single-over double-stranded DNA and intermediates of recombination and repair and catalyzes robust unwinding of substrates possessing a 3' single-stranded DNA overhang but not of 5' overhangs or blunt-ended DNA fragments. Additional evidence disclosed that ScMre11 nuclease activity is dispensable for its DNA binding and unwinding activity, thus uncovering the molecular basis underlying DSB end processing in mre11 nuclease deficient mutants. Significantly, Rad50, Xrs2, and Sae2 potentiate the DNA unwinding activity of Mre11, thus underscoring functional interaction among the components of DSB end repair machinery. Our results also show that ScMre11 by itself binds to DSB ends, then promotes end bridging of duplex DNA, and directly interacts with Sae2. We discuss the implications of these results in the context of an alternative mechanism for DSB end processing and the generation of single-stranded DNA for DNA repair and homologous recombination.

Linearization of Drucker-Prager yield criterion for axisymmetric problems: implementation in lower-bound limit analysis

The linearization of the Drucker-Prager yield criterion associated with an axisymmetric problem has been achieved by simulating a sphere with the truncated icosahedron with 32 faces and 60 vertices. On this basis, a numerical formulation has been proposed for solving an axisymmetric stability problem with the usage of the lower-bound limit analysis, finite elements, and linear optimization. To compare the results, the linearization of the Mohr-Coulomb yield criterion, by replacing the three cones with interior polyhedron, as proposed earlier by Pastor and Turgeman for an axisymmetric problem, has also been implemented. The two formulations have been applied for determining the collapse loads for a circular footing resting on a cohesive-friction material with nonzero unit weight. The computational results are found to be quite convincing. (C) 2013 American Society of Civil Engineers.

Modeling the dynamic response of wrap-faced reinforced soil retaining walls

This paper describes the development of a numerical model for simulating the shaking table tests on wrap-faced reinforced soil retaining walls. Some of the physical model tests carried out on reinforced soil retaining walls subjected to dynamic excitation through uniaxial shaking tests are briefly discussed. Models of retaining walls are constructed in a perspex box with geotextile reinforcement using the wraparound technique with dry sand backfill and instrumented with displacement sensors, accelerometers, and soil pressure sensors. Results showed that the displacements decrease with the increase in number of reinforcement layers, whereas acceleration amplifications were not affected significantly. Numerical modeling of these shaking table tests is carried out using the Fast Lagrangian Analysis of Continua program. The numerical model is validated by comparing the results with experiments on physical models. Responses of wrap-faced walls with varying numbers of reinforcement layers are compared. Sensitivity analysis performed on the numerical models showed that the friction and dilation angle of backfill material and stiffness properties of the geotextile-soil interface are the most affecting parameters for the model response.

Bearing capacity of piles in soft clay underlaid by cohesive frictional soil

By using the axisymmetric quasi-lower bound finite-element limit analysis, the bearing capacity factors N-c(p) and N-gamma q(p) have been computed for axially loaded piles, with the shaft embedded in a fully cohesive soil medium and the tip placed over cohesive frictional soil strata. The results were obtained for various combinations of L/D, phi(l), and c(l)/c(u); the subscripts l and u refer to lower and upper soil strata, respectively. The factors N-c(p) and N-gamma q(p) increase continuously with increases in L/D and phi(l); the rate of increase of N-c(p) and N-gamma q(p) with L/D, however, decreases with an increase in L/D. For c(l)/c(u) > 100, the factor N-c(p) hardly depends on L/D.

Human la protein interaction with GCAC near the initiator AUG enhances hepatitis C virus RNA replication by promoting linkage between 5 ` and 3 ` untranslated regions

Human La protein has been implicated in facilitating the internal initiation of translation as well as replication of hepatitis C virus (HCV) RNA. Previously, we demonstrated that La interacts with the HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG within stem-loop IV by its RNA recognition motif (RRM) (residues 112 to 184) and influences HCV translation. In this study, we have deciphered the role of this interaction in HCV replication in a hepatocellular carcinoma cell culture system. We incorporated mutation of the GCAC motif in an HCV monocistronic subgenomic replicon and a pJFH1 construct which altered the binding of La and checked HCV RNA replication by reverse transcriptase PCR (RT-PCR). The mutation drastically affected HCV replication. Furthermore, to address whether the decrease in replication is a consequence of translation inhibition or not, we incorporated the same mutation into a bicistronic replicon and observed a substantial decrease in HCV RNA levels. Interestingly, La overexpression rescued this inhibition of replication. More importantly, we observed that the mutation reduced the association between La and NS5B. The effect of the GCAC mutation on the translation-to-replication switch, which is regulated by the interplay between NS3 and La, was further investigated. Additionally, our analyses of point mutations in the GCAC motif revealed distinct roles of each nucleotide in HCV replication and translation. Finally, we showed that a specific interaction of the GCAC motif with human La protein is crucial for linking 5' and 3' ends of the HCV genome. Taken together, our results demonstrate the mechanism of regulation of HCV replication by interaction of the cis-acting element GCAC within the HCV IRES with human La protein.

Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Diverse functions of restriction-modification systems in addition to cellular defense

Restriction-modification (R-M) systems are ubiquitous and are often considered primitive immune systems in bacteria. Their diversity and prevalence across the prokaryotic kingdom are an indication of their success as a defense mechanism against invading genomes. However, their cellular defense function does not adequately explain the basis for their immaculate specificity in sequence recognition and nonuniform distribution, ranging from none to too many, in diverse species. The present review deals with new developments which provide insights into the roles of these enzymes in other aspects of cellular function. In this review, emphasis is placed on novel hypotheses and various findings that have not yet been dealt with in a critical review. Emerging studies indicate their role in various cellular processes other than host defense, virulence, and even controlling the rate of evolution of the organism. We also discuss how R-M systems could have successfully evolved and be involved in additional cellular portfolios, thereby increasing the relative fitness of their hosts in the population.

Preliminary study of the effects of carbon nanotubes on Cpu cooling

Managing heat produced by computer processors is an important issue today, especially when the size of processors is decreasing rapidly while the number of transistors in the processor is increasing rapidly. This poster describes a preliminary study of the process of adding carbon nanotubes (CNTs) to a standard silicon paste covering a CPU. Measurements were made in two rounds of tests to compare the rate of cool-down with and without CNTs present. The silicon paste acts as an interface between the CPU and the heat sink, increasing the heat transfer rate away from the CPU. To the silicon paste was added 0.05% by weight of CNTs. These were not aligned. A series of K-type thermocouples was used to measure the temperature as a function of time in the vicinity of the CPU, following its shut-off. An Omega data acquisition system was attached to the thermocouples. The CPU temperature was not measured directly because attachment of a thermocouple would have prevented its automatic shut-off A thermocouple in the paste containing the CNTs actually reached a higher temperature than the standard paste, an effect easily explained. But the rate of cooling with the CNTs was about 4.55% better.

Factors influencing stick-slip motion: effect of hardness, crystal structure and surface texture

In the present investigation, efforts were made to study the different frictional responses of materials with varying crystal structure and hardness during sliding against a relatively harder material of different surface textures and roughness. In the experiments, pins were made of pure metals and alloys with significantly different hardness values. Pure metals were selected based on different class of crystal structures, such as face centered cubic (FCC), body centered cubic (BCC), body centered tetragonal (BCT) and hexagonal close packed (HCP) structures. The surface textures with varying roughness were generated on the counterpart plate which was made of H-11 die steel. The experiments were conducted under dry and lubricated conditions using an inclined pin-on-plate sliding tester for various normal loads at ambient environment. In the experiments, it was found that the coefficient of friction is controlled by the surface texture of the harder mating surfaces. Further, two kinds of frictional response, namely steady-state and stick-slip, were observed during sliding. More specifically, stead-state frictional response was observed for the FCC metals, alloys and materials with higher hardness. Stick-slip frictional response was observed for the metals which have limited number of slip systems such as BCT and HCP. In addition, the stick-slip frictional response was dependent on the normal load, lubrication, hardness and surface texture of the counterpart material. However, for a given kind of surface texture, the roughness of the surface affects neither the average coefficient of friction nor the amplitude of stick-slip oscillation significantly.

Topology optimization-based design of a compliant aircraft compliant aircraft wing for morphing leading and trailing edges

In this paper, we present a methodology for designing a compliant aircraft wing, which can morph from a given airfoil shape to another given shape under the actuation of internal forces and can offer sufficient stiffness in both configurations under the respective aerodynamic loads. The least square error in displacements, Fourier descriptors, geometric moments, and moment invariants are studied to compare candidate shapes and to pose the optimization problem. Their relative merits and demerits are discussed in this paper. The `frame finite element ground structure' approach is used for topology optimization and the resulting solutions are converted to continuum solutions. The introduction of a notch-like feature is the key to the success of the design. It not only gives a good match for the target morphed shape for the leading and trailing edges but also minimizes the extension of the flexible skin that is to be put on the airfoil frame. Even though linear small-displacement elastic analysis is used in optimization, the obtained designs are analysed for large displacement behavior. The methodology developed here is not restricted to aircraft wings; it can be used to solve any shape-morphing requirement in flexible structures and compliant mechanisms.