Direct control of type IIA topoisomerase activity by a chromosomally encoded regulatory protein

Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coil gyrase, a type HA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holo enzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state.

Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

Depression biased non-Hebbian spike-timing-dependent synaptic plasticity in the rat subiculum

The subiculum is a structure that forms a bridge between the hippocampus and the entorhinal cortex (EC), and plays a major role in the memory consolidation process. Here, we demonstrate spike-timing-dependent plasticity (STDP) at the proximal excitatory inputs on the subicular pyramidal neurons of juvenile rat. Causal (positive) pairing of a single EPSP with a single back-propagating action potential (bAP) after a time interval of 10 ms (+10 ms) failed to induce plasticity. However, increasing the number of bAPs in a burst to three, at two different frequencies of 50 Hz (bAP burst) and 150 Hz, induced long-term depression (LTD) after a time interval of +10 ms in both the regular-firing (RF), and the weak burst firing (WBF) neurons. The LTD amplitude decreased with increasing time interval between the EPSP and the bAP burst. Reversing the order of the pairing of the EPSP and the bAP burst induced LTP at a time interval of -10 ms. This finding is in contrast with reports at other synapses, wherein prebefore postsynaptic (causal) pairing induced LTP and vice versa. Our results reaffirm the earlier observations that the relative timing of the pre- and postsynaptic activities can lead to multiple types of plasticity profiles. The induction of timing-dependent LTD (t-LTD) was dependent on postsynaptic calcium change via NMDA receptors in the WBF neurons, while it was independent of postsynaptic calcium change, but required active L-type calcium channels in the RF neurons. Thus the mechanism of synaptic plasticity may vary within a hippocampal subfield depending on the postsynaptic neuron involved. This study also reports a novel mechanism of LTD induction, where L-type calcium channels are involved in a presynaptically induced synaptic plasticity. The findings may have strong implications in the memory consolidation process owing to the central role of the subiculum and LTD in this process.

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers that drives fusion of viral and cellular membranes. While it is expected that native Env is a good immunogen, its metastability results in exposure of immunodominant nonneutralizing epitopes. In the present study, we stabilize native Env trimers by incorporation of a number of different mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The stabilized constructs described here can be incorporated into DNA vaccine candidates.

Phthalate mediated hydrogelation of a pyrene based system: a novel scaffold for shape-persistent, self-healing luminescent soft material

Two-component super-hydrogelation triggered by the acid-base interaction of a L-histidine appended pyrenyl derivative (PyHis) and phthalic acid (PA) was reported. The use of isomeric isophthalic or terephthalic acid or other comparable acids in place of PA does not lead to salt formation and therefore hydrogelation is not observed. Excimer formation of the pyrenyl unit has not been detected although the PyHis : PA = 1: 1 system undergoes extensive self-assembly in aqueous solution. The synergistic effect of intermolecular H-bonding forces, pi-pi stacking, electrostatic interactions, etc. is found to be responsible for robust hydrogel formation. Development of chiral supramotecular assemblies has been verified through circular dichroism spectroscopy. Morphological investigations involving the PyHis : PA = 1: 1 system show vesicular nano-structures with a definite bilayer width at relatively low concentrations. The latter fuses to construct coiled-coil left-handed helical fibers upon increase in the concentrations of the gelators. The intertwining of the resultant helical fibers eventually results in hydrogel formation. The probable bilayer packing in the self-assembled structures has been probed using X-ray diffraction (XRD) studies and lanthanide sensitization, which suggests that the polar imidazolium hydrogen phthalate unit of the gelator forms the head group and faces the hydrophilic water environment while the hydrophobic pyrenyl units sit inside the hydrophobic core of the bilayer. The hydrogel exhibits multi-stimuli responsiveness including thixotropic behavior. In addition, shape-persistent as well as rapid self-healing behaviour of the hydrogel was established. Furthermore load-bearing characteristics of the hydrogel have also been demonstrated.

On the quality of hydrogenated amorphous silicon deposited by sputtering

Amorphous hydrogenated silicon (a-Si:H) is well-known material in the global semiconductor industry. The quality of the a-Si:H films is generally decided by silicon and hydrogen bonding configuration (Si-H-x, x=1,2) and hydrogen concentration (C-H). These quality aspects are correlated with the plasma parameters like ion density (N-i) and electron temperature (T-e) of DC, Pulsed DC (PDC) and RF plasmas during the sputter-deposition of a-Si:H thin films. It was found that the N-i and T-e play a major role in deciding Si-H-x bonding configuration and the C-H value in a-Si:H films. We observed a trend in the variation of Si-H and Si-H-2 bonding configurations, and C-H in the films deposited by DC, Pulsed DC and RF reactive sputtering techniques. Ion density and electron energy are higher in RF plasma followed by PDC and DC plasma. Electrons with two different energies were observed in all the plasmas. At a particular hydrogen partial pressure, RF deposited films have higher C-H followed by PDC and then DC deposited films. The maximum energy that can be acquired by the ions was found to be higher in RF plasma. Floating potential (V-f) is more negative in DC plasma, whereas, plasma potential (V-p) is found to be more positive in RF plasma. (C) 2014 Elsevier Ltd. All rights reserved.

Room temperature ferromagnetic and ferroelectric properties of Bi1-xCaxMnO3 thin films

Bi1-xCaxMnO3 (BCMO) thin films with x = 0, 0.1, 0.2, 0.3 and 0.4 are successfully deposited on the n-type Si (100) substrate at two different temperatures of 400 degrees C and 800 degrees C using RF magnetron sputtering. The stoichiometry of the films and oxidation state of the elements have been described by X-ray photoelectron spectroscopy analysis. Dielectric measurement depicts the insulating property of BCMO films. Magnetic and ferroelectric studies confirm the significant enhancement in spin orientation as well as electric polarization at room temperature due to incorporation of Ca2+ ions into BiMnO3 films. The BCMO (x = 0.2) film grown at 400 degrees C shows better magnetization (M-sat) and polarization (P-s) with the measured values of 869 emu / cc and 6.6 mu(C)/cm(2) respectively than the values of the other prepared films. Thus the realization of room temperature ferromagnetic and ferroelectric ordering in Ca2+ ions substituted BMO films makes potentially interesting for spintronic device applications. (C) 2014 Author(s).

Application of Qualitative Imaging Methods to Electrical Performance-Aware Package Board Design

Package-board co-design plays a crucial role in determining the performance of high-speed systems. Although there exist several commercial solutions for electromagnetic analysis and verification, lack of Computer Aided Design (CAD) tools for SI aware design and synthesis lead to longer design cycles and non-optimal package-board interconnect geometries. In this work, the functional similarities between package-board design and radio-frequency (RF) imaging are explored. Consequently, qualitative methods common to the imaging community, like Tikhonov Regularization (TR) and Landweber method are applied to solve multi-objective, multi-variable package design problems. In addition, a new hierarchical iterative piecewise linear algorithm is developed as a wrapper over LBP for an efficient solution in the design space.

Thick PECVD Germanium films for MEMS Application

Plasma enhanced chemical vapour deposition (PECVD) of thick germanium (Ge) films (similar to 1 mu m) on silicon dioxide (SiO2) at low temperatures is described. A diborane pretreatment on SiO2 films is done to seed the Ge growth, followed by the deposition of thick Ge films using germane (GeH4) and argon (Ar). Further, the effect of hydrogen (H-2) dilution on the deposition rate is also investigated. The film thickness and morphology is characterized using SEM. Use of high RF power and substrate temperature show increased deposition rate. EDS analysis indicates that these films contain 97-98 atomic percentage of Ge. A recipe for anisotropic dry etching of the deposited Ge films with 10nm/ min etch rate is also suggested.

Raman Spectroscopic Studies on Screening of Myopathies

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld1 (up1), heldup(2) (hdp(2)), myosin heavy chain7 (Mhc7), actin88F(KM88) (Act88F(KM88)), upheld101 (up101), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and beta-sheet and/or random coil protein content along with a decrease in a-helix protein. Interestingly, the 12th day samples of up1 and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up1 and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up101 and hdp2 (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up1). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.

Space-Time Coded Spatial Modulated Physical Layer Network Coding for Two-Way Relaying

Using the spatial modulation approach, where only one transmit antenna is active at a time, we propose two transmission schemes for two-way relay channel using physical layer network coding with space time coding using coordinate interleaved orthogonal designs (CIODs). It is shown that using two uncorrelated transmit antennas at the nodes, but using only one RF transmit chain and space-time coding across these antennas can give a better performance without using any extra resources and without increasing the hardware implementation cost and complexity. In the first transmission scheme, two antennas are used only at the relay, adaptive network coding (ANC) is employed at the relay and the relay transmits a CIOD space time block code (STBC). This gives a better performance compared to an existing ANC scheme for two-way relay channel which uses one antenna each at all the three nodes. It is shown that for this scheme at high SNR the average end-to-end symbol error probability (SEP) is upper bounded by twice the SEP of a point-to-point fading channel. In the second transmission scheme, two transmit antennas are used at all the three nodes, CIOD STBCs are transmitted in multiple access and broadcast phases. This scheme provides a diversity order of two for the average end-to-end SEP with an increased decoding complexity of O(M-3) for an arbitrary signal set and O(M-2 root M) for square QAM signal set. Simulation results show that the proposed schemes performs better than the existing ANC schemes under perfect and imperfect channel state information.

Sensitivity characteristics of Ag doped BaTiO3-CuO mixed oxide as Carbon-dioxide S ensor

Ag doped BaTiO3-CuO mixed oxide thin films are evaluated for their carbon-dioxide sensing characteristics. The metal oxide films of different thicknesses are deposited on oxidized p type Si < 100 > substrate by RF Sputtering. Sensing characteristics for different CO2 concentration, (300 ppm - 1000 ppm) are obtained for different operating temperatures, (100 degrees C - 400 degrees C). Optimum temperature for maximum sensitivity is found to be 250 degrees C. The effect of annealing on sensing properties is also evaluated. The unannealed films give better sensitivity than that of annealed films. Response time and recovery time are also calculated.

Effect of radio frequency power and thickness on the electrochemical properties of Li2-x MnO3-y thin films

In the present work, Li2-x MnO3-y (LMO) thin films have been deposited by radio frequency (RF) reactive magnetron sputtering using acid-treated Li2MnO3 powder target. Systematic investigations have been carried out to study the effect of RF power on the physicochemical properties of LMO thin films deposited on platinized silicon substrates. X-ray diffraction, electron microscopy, surface chemical analysis and electrochemical studies were carried out for the LMO films after post deposition annealing treatment at 500 A degrees C for 1 h in air ambience. Galvanostatic charge discharge studies carried out using the LMO thin film electrodes, delivered a highest discharge capacity of 139 mu Ah mu m(-1) cm(-2) in the potential window 2.0-3.5 V vs. Li/Li+ at 100 W RF power and lowest discharge capacity of 80 mu Ah mu m(-1) cm(-2) at 75 W RF power. Thereafter, the physicochemical properties of LMO films deposited using optimized RF power 100 W on stainless steel substrates has been studied in the thickness range of 70 to 300 nm as a case study. From the galvanostatic charge discharge experiments, a stable discharge capacity of 68 mu Ah mu m(-1) cm(-2) was achieved in the potential window 2.0-4.2 V vs. Li/Li+ tested up to 30 cycles. As the thickness increased, the specific discharge capacity started reducing with higher magnitude of capacity fading.

Hybrid Elements for Modelling Squeeze Film Effects Coupled with Structural Interactions in Vibratory MEMS Devices

We present a hybrid finite element based methodology to solve the coupled fluid structure problem of squeeze film effects in vibratory MEMS devices, such as gyroscopes, RF switches, and 2D resonators. The aforementioned devices often have a thin plate like structure vibrating normally to a fixed substrate, and are generally not perfectly vacuum packed. This results in a thin air film being trapped between the vibrating plate and the fixed substrate which behaves like a squeeze film offering both stiffness and damping. For accurate modelling of such devices the squeeze film effects must be incorporated. Extensive literature is available on squeeze film modelling, however only a few studies address the coupled fluid elasticity problem. The majority of the studies that account for the plate elasticity coupled with the fluid equation, either use approximate mode shapes for the plate or use iterative solution strategies. In an earlier work we presented a single step coupled methodology using only one type of displacement based element to solve the coupled problem. The displacement based finite element models suffer from locking issues when it comes to modelling very thin structures with the lateral dimensions much larger than the plate thickness as is typical in MEMS devices with squeeze film effects. In this work we present another coupled formulation where we have used hybrid elements to model the structural domain. The numerical results show a huge improvement in convergence and accuracy with coarse hybrid mesh as compared to displacement based formulations. We further compare our numerical results with experimental data from literature and find them to be in good accordance.

Synthesis and Characterization of Au@Pt Nanoparticles with Ultrathin Platinum Overlayers

Gold-core platinum-shell (Au@Pt) nanoparticles with ultrathin platinum overlayers, ranging from submonolayer to two monolayers of platinum atoms, were prepared at room-temperature using a scalable, wet-chemical synthesis route. The synthesis involved the reduction of chloroauric acid with tannic acid to form 5 nm (nominal dia.) gold nanoparticles followed by addition of desired amount of chloroplatinic acid and hydrazine to form platinum overlayers with bulk Pt/Au atomic ratios (Pt surface coverages) corresponding to 0.19 (half monolayer), 0.39 (monolayer), 0.58 (1.5 monolayer) and 0.88 (2 monolayers). The colloidal particles were coated with octadecanethiol and phase-transferred into chlroform-hexane mixture to facilitate sample preparation for structural characterization. The structure of the resultant nanoparticles were determined to be Au@Pt using HRTEM, SAED, XPS, UV-vis and confirmed by cyclic voltammetry (CV) studies. Monolayers of octadecanethiol coated Au@Pt nanoparticles were self-assembled at an air-water interface and transfer printed twice onto a gold substrate to form bilayer films for electrochemical characterization. Electrochemical activity on such films was observed only after the removal of the octadecanethiol ligand coating the nanoparticles, using a RF plasma etching process. The electrochemical activity (HOR, MOR studies) of Au@Pt nanoparticles was found to be highest for particles having a two atom thick platinum overlayer. These nanoparticles can significantly enhance platinum utilization in electrocatalytic applications as their platinum content based activity was three times higher than pure platinum nanoparticles.