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.

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.

Skeletal Muscle Degeneration and Regeneration in Mice and Flies

Many aspects of skeletal muscle biology are remarkably similar between mammals and tiny insects, and experimental models of mice and flies (Drosophila) provide powerful tools to understand factors controlling the growth, maintenance, degeneration (atrophy and necrosis), and regeneration of normal and diseased muscles, with potential applications to the human condition. This review compares the limb muscles of mice and the indirect flight muscles of flies, with respect to the mechanisms of adult myofiber formation, homeostasis, atrophy, hypertrophy, and the response to muscle degeneration, with some comment on myogenic precursor cells and common gene regulatory pathways. There is a striking similarity between the species for events related to muscle atrophy and hypertrophy, without contribution of any myoblast fusion. Since the flight muscles of adult flies lack a population of reserve myogenic cells (equivalent to satellite cells), this indicates that such cells are not required for maintenance of normal muscle function. However, since satellite cells are essential in postnatal mammals for myogenesis and regeneration in response to myofiber necrosis, the extent to which such regeneration might be possible in flight muscles of adult flies remains unclear. Common cellular and molecular pathways for both species are outlined related to neuromuscular disorders and to age-related loss of skeletal muscle mass and function (sarcopenia). The commonality of events related to skeletal muscles in these disparate species (with vast differences in size, growth duration, longevity, and muscle activities) emphasizes the combined value and power of these experimental animal models.

Skeletal Muscle Degeneration and Regeneration in Mice and Flies

Many aspects of skeletal muscle biology are remarkably similar between mammals and tiny insects, and experimental models of mice and flies (Drosophila) provide powerful tools to understand factors controlling the growth, maintenance, degeneration (atrophy and necrosis), and regeneration of normal and diseased muscles, with potential applications to the human condition. This review compares the limb muscles of mice and the indirect flight muscles of flies, with respect to the mechanisms of adult myofiber formation, homeostasis, atrophy, hypertrophy, and the response to muscle degeneration, with some comment on myogenic precursor cells and common gene regulatory pathways. There is a striking similarity between the species for events related to muscle atrophy and hypertrophy, without contribution of any myoblast fusion. Since the flight muscles of adult flies lack a population of reserve myogenic cells (equivalent to satellite cells), this indicates that such cells are not required for maintenance of normal muscle function. However, since satellite cells are essential in postnatal mammals for myogenesis and regeneration in response to myofiber necrosis, the extent to which such regeneration might be possible in flight muscles of adult flies remains unclear. Common cellular and molecular pathways for both species are outlined related to neuromuscular disorders and to age-related loss of skeletal muscle mass and function (sarcopenia). The commonality of events related to skeletal muscles in these disparate species (with vast differences in size, growth duration, longevity, and muscle activities) emphasizes the combined value and power of these experimental animal models.

Microstructure-hardness-fretting wear resistance correlation in ultrafine grained Cu-TiB2-Pb composites

The retention of the desired combination of mechanical/tribological properties in ultrafine grained materials presents important challenges in the field of bulk metallic composites. In order to address this aspect, the present work demonstrates how one can achieve a good combination of hardness and wear resistance in Cu-Pb-TiB2 composites, consolidated by spark plasma sintering at low temperatures ( < 500 degrees C). Transmission electron microscope (TEM) studies reveal ultrafine grains of Cu (100-400 nm) with coarser TiB2 particles (1-2 mu m) along with fine scale Pb dispersoid at triple junctions or at the grain boundaries of Cu. Importantly, a high hardness of around 2.2 GPa and relative density of close to 90% relative density (rho(theo)) have been achieved for Cu-15 wt% TiB2-10 wt% Pb composite. Such property theo, combination has never been reported for any Cu-based nanocomposite, by conventional processing route. In reference to the tribological performance, fretting wear tests were conducted on the sintered nanocomposites and a good combination of steady state COF (0.6-0.7) and wear rate (10-4 mm(3)/N m) were measured. An inverse relationship between wear rate and hardness was recorded and this commensurates well with Archard's relationship of abrasive wear. The formation of a wear-resistant delaminated tribolayer consisting of TiB2 particles and ultrafine oxide debris, (Cu, Fe, Ti)(x)O-y as confirmed from subsurface imaging using focused ion beam microscopy has been identified as the key factors for the low wear rate of these composites. (C) 2014 Elsevier B.V. All rights reserved.

Highly sensitive and rapid detection of acetylcholine using an ITO plate modified with platinum-graphene nanoparticles

Determining the concentrations of acetylcholine (ACh) and choline (Ch) is clinically important. ACh is a neurotransmitter that acts as a key link in the communication between neurons in the spinal cord and in nerve skeletal junctions in vertebrates, and plays an important role in transmitting signals in the brain. A bienzymatic sensor for the detection of ACh was prepared by co-immobilizing choline oxidase (ChO) and acetylcholinesterase (AChE) on graphene matrix/platinum nanoparticles, and then electrodepositing them on an ITO-coated glass plate. Graphene nanoparticles were decorated with platinum nanoparticles and were electrodeposited on a modified ITO-coated glass plate to form a modified electrode. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) studies. The optimum response of the enzyme electrode was obtained at pH 7.0 and 35 degrees C. The response time of this ACh-sensing system was shown to be 4 s. The linear range of responses to ACh was 0.005-700 mu M. This biosensor exhibits excellent anti-interferential abilities and good stability, retaining 50% of its original current even after 4 months. It has been applied for the detection of ACh levels in human serum samples.

Multi-agent Reinforcement Learning for Traffic Signal Control

Optimal control of traffic lights at junctions or traffic signal control (TSC) is essential for reducing the average delay experienced by the road users amidst the rapid increase in the usage of vehicles. In this paper, we formulate the TSC problem as a discounted cost Markov decision process (MDP) and apply multi-agent reinforcement learning (MARL) algorithms to obtain dynamic TSC policies. We model each traffic signal junction as an independent agent. An agent decides the signal duration of its phases in a round-robin (RR) manner using multi-agent Q-learning with either is an element of-greedy or UCB 3] based exploration strategies. It updates its Q-factors based on the cost feedback signal received from its neighbouring agents. This feedback signal can be easily constructed and is shown to be effective in minimizing the average delay of the vehicles in the network. We show through simulations over VISSIM that our algorithms perform significantly better than both the standard fixed signal timing (FST) algorithm and the saturation balancing (SAT) algorithm 15] over two real road networks.

Vacancy mediated clipping of multi-layered graphene: A precursor for 1, 2 and 3D carbon structures

Using first principles calculations, we show that the overlapping defects in bi-layer graphene (both AA-and AB-stacked) interact forming inter-layer covalent bonds, giving rise to two-dimensional (2D) clipped structures, without explicit use of functional groups. These clipped structures can be transformed into one-dimensional (1D) double wall nanotubes (DWCNT) or multi-layered three dimensional (3D) bulk structures. These clipped structures show good mechanical strength due to covalent bonding between multi-layers. Clipping also provides a unique way to simultaneously harness the conductivity of both walls of a double wall nanotube through covalently bonded scattering junctions. With additional conducting channels and improved mechanical stability, these clipped structures can lead to a myriad of applications in novel devices. (C) 2015 Elsevier Ltd. All rights reserved.

Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

Edge states, spin transport, and impurity-induced local density of states in spin-orbit coupled graphene

We study graphene, which has both spin-orbit coupling (SOC), taken to be of the Kane-Mele form, and a Zeeman field induced due to proximity to a ferromagnetic material. We show that a zigzag interface of graphene having SOC with its pristine counterpart hosts robust chiral edge modes in spite of the gapless nature of the pristine graphene; such modes do not occur for armchair interfaces. Next we study the change in the local density of states (LDOS) due to the presence of an impurity in graphene with SOC and Zeeman field, and demonstrate that the Fourier transform of the LDOS close to the Dirac points can act as a measure of the strength of the spin-orbit coupling; in addition, for a specific distribution of impurity atoms, the LDOS is controlled by a destructive interference effect of graphene electrons which is a direct consequence of their Dirac nature. Finally, we study transport across junctions, which separates spin-orbit coupled graphene with Kane-Mele and Rashba terms from pristine graphene both in the presence and absence of a Zeeman field. We demonstrate that such junctions are generally spin active, namely, they can rotate the spin so that an incident electron that is spin polarized along some direction has a finite probability of being transmitted with the opposite spin. This leads to a finite, electrically controllable, spin current in such graphene junctions. We discuss possible experiments that can probe our theoretical predictions.

Flow Investigation of Linear Cluster Plug Nozzle

Computational and experimental tools have been used to understand the linear cluster plug nozzle flowfield for a range of pressure ratios. The experimental cluster configuration is arrived at from a linear plug nozzle by introducing splitter plates in the primary nozzle, and computational analysis of corresponding geometry is also carried out. The flow development on the plug surface has been analyzed for two different cluster module spacings. The interactions between the cluster module jets is a complex one with a three-dimensional shock structure because of the differential end condition the shock experiences on the plug wall and freejet boundary. A prominent streamwise vorticity resulting from curvature of the shock is also seen along the length of the plug downstream of the module junctions. The out-of-phase wave interactions occurring along the module centerline and the splitter plate centerline, resulting in a wavy surface-limiting streamline pattern, particularly at lower pressure ratios, is explained.

In Situ Synthesis Of Nanocrystalline Intermetallic Layer During Surface Plastic Deformation Of Zirconium

By means of a surface plastic deformation method a nanocrystalline (NC) intermetallic compound was in situ synthesized on the surface layer of bulk zirconium (Zr). Hardened steel shots (composition: 1.0C, 1.5Cr, base Fe in wt.%) were used to conduct repetitive and multidirectional peening on the surface layer of Zr. The microstructure evolution of the surface layer was investigated by X-ray diffraction and scanning and transmission electron microscopy observations. The NC intermetallic layer of about 25 gm thick was observed and confirmed by concentration profiles of Zr, Fe and Cr, and was found to consist of the Fe100-xCrx compound with an average grain size of 22 nm. The NC surface layer exhibited an extremely high average hardness of 10.2 GPa. The Zr base immediately next to the compound/Zr interface has a grain size of similar to 250 nm, and a hardness of similar to 3.4 GPa. The Fe100-xCrx layer was found to securely adhere to the Zr base. (c) 2007 Elsevier B.V All rights reserved.