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.

Growth kinetics and immune response of chimeric foot-and-mouth disease virus serotype `O' produced through replication competent mini genome of serotype Asia 1, 63/72, in BHK cell lines

Regular vaccinations with potent vaccine, in endemic countries and vaccination to live in non-endemic countries are the methods available to control foot-and-mouth disease. Selection of candidate vaccine strain is not only cumbersome but the candidate should grow well for high potency vaccine preparation. Alternative strategy is to generate an infectious cDNA of a cell culture-adapted virus and use the replicon for development of tailor-made vaccines. We produced a chimeric `O' virus in the backbone of Asia 1 and studied its characteristics. The chimeric virus showed high infectivity titre (>10(10)) in BHK 21 cell lines, revealed small plague morphology and there was no cross reactivity with antiserum against Asia I. The virus multiplies rapidly and reaches peak at 12 h post infection. The vaccine prepared with this virus elicited high antibody titres.

Pulsed Electrical Stimulation and Surface Charge Induced Cell Growth on Multistage Spark Plasma Sintered Hydroxyapatite-Barium Titanate Piezobiocomposite

The primary purpose of the present work was to illustrate whether cell proliferation can be enhanced on electroactive bioceramic composite, when the cells are cultured in the presence of external electrical stimulation. The two different aspects of the influence of electric field (E-field) application toward stimulating the growth/proliferation of bone/connective tissue cells in vitro, (a) intermittent delivery of extremely low strength pulsed electrical stimulation (0.5-4V/cm, 400s DC pulse) and (b) surface charge generated by electrical poling (10kV/cm) of hydroxyapatite (HA)-BaTiO3 piezobiocomposite have been demonstrated. The experimental results establish that the cell growth can be enhanced using the new culture protocol of the intermittent delivery of electrical pulses within a narrow range of stimulation parameters. The optimal E-field strength for enhanced cellular response for mouse fibroblast L929 and osteogenic cells is in the range of 0.5-1V/cm. The MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay results suggested the increased viability of E-field treated cells over 7d in culture, implicating the positive impact of electrical pulses on proliferation behavior. The alizarin red assay results showed noticeable increase in Ca-deposition on the E-field treated samples in comparison to their untreated counterparts. The negatively charged surfaces of developed piezocomposite stimulated the cell growth in a statistically noticeable manner as compared with the uncharged or positively charged surfaces of similar composition.

Characterisation and bioactivity of oosporein produced by endophytic fungus Cochliobolus kusanoi isolated from Nerium oleander L.

Bioactive compounds comprising secondary metabolites produced by endophytic fungi have wide applications in pharmacology and agriculture. Isolation, characterisation and evaluation of biological activities of secondary metabolites were carried out from Cochliobolus kusanoi an endophytic fungus of Nerium oleander L. The fungus was identified based on 18S rDNA sequence analysis. There are no reports available on the compounds of C. kusanoi hence, antimicrobial metabolite produced by this fungus was extracted and purified by fractionation using hexane, diethyl ether, dichloromethane, ethyl acetate and methanol. Out of all the solvent fractions, the methanol fraction exhibited better antimicrobial activity which was further purified and characterised as oosporein. Oosporein from C. kusanoi exhibited broad spectrum in vitro antimicrobial, antioxidant and cytotoxic activities. The characterisation and antioxidant activity of oosporein from C. kusanoi are reported for the first time.

Influence of melt pouring temperature and plate inclination on solidification and microstructure of A356 aluminum alloy produced using oblique plate

The preparation of semisolid slurry of A356 aluminum alloy using an oblique plate was investigated. A356 alloy melt undergoes partial solidification when it flows down on an oblique plate cooled from underneath by counter flowing water. It results in continuous formation of columnar dendrites on plate wall. Due to forced convection, these dendrites are sheared off into equiaxed/fragmented grains and then washed away continuously to produce semisolid slurry at plate exit. Melt pouring temperature provides required condition of solidification whereas plate inclination enables necessary shear for producing semisolid slurry of desired quality. Slurry obtained was solidified in metal mould to produce semisolid-cast billets of desired microstructure. Furthermore, semisolid-cast billets were heat treated to improve surface quality. Microstructures of both semisolid-cast and heat-treated billets were analyzed. Effects of melt pouring temperature and plate inclination on solidification and microstructure of billets produced using oblique plate were described. The investigations involved four different melt pouring temperatures (620, 625, 630 and 635 degrees C) associated with four different plate inclinations (30 degrees, 45 degrees, 60 degrees and 75 degrees). Melt pouring temperature of 625 degrees C with plate inclination of 60 degrees shows fine and globular microstructures and it is the optimum.

Origin of 1/f noise in graphene produced for large-scale applications in electronics

The authors report a detailed investigation of the flicker noise (1/f noise) in graphene films obtained from chemical vapour deposition (CVD) and chemical reduction of graphene oxide. The authors find that in the case of polycrystalline graphene films grown by CVD, the grain boundaries and other structural defects are the dominant source of noise by acting as charged trap centres resulting in huge increase in noise as compared with that of exfoliated graphene. A study of the kinetics of defects in hydrazine-reduced graphene oxide (RGO) films as a function of the extent of reduction showed that for longer hydrazine treatment time strong localised crystal defects are introduced in RGO, whereas the RGO with shorter hydrazine treatment showed the presence of large number of mobile defects leading to higher noise amplitude.

Influence of plate length and plate cooling rate on solidification and microstructure of A356 alloy produced by oblique plate

A356 alloy melt solidifies partially when it flows down on an oblique plate cooled from bottom by counter flowing water. Columnar dendrites are continuously formed on the plate wall. Because of the forced convection, these dendrites are sheared off into equiaxed/fragmented grains and then washed away continuously by producing semisolid slurry at plate exit. Plate cooling rate provides required extent/amount of solidification whereas plate length enables necessary shear for producing semisolid slurry of desired quality. Slurry obtained is solidified in metal mould to produce semisolid-cast billets of desired microstructure. Furthermore, semisolid-cast billets are also heat-treated to improve surface quality. Microstructures of both semisolid-cast and heat-treated billets are compared. The effects of plate length and plate cooling rate on solidification and microstructure of billets produced by using oblique plate are illustrated. Three different plate lengths (200 mm, 250 mm, 300 mm) associated with three different heat transfer coefficients (1000, 2000 and 2500 W/(m(2).K)) are involved. Plate length of 250 mm with heat transfer coefficient of 2000 W/(m(2).K) gives fine and globular microstructures and is the optimum as there is absolutely no possibility of sticking of slurry to plate wall.

Interplay of Substrate Conductivity, Cellular Microenvironment, and Pulsatile Electrical Stimulation toward Osteogenesis of Human Mesenchymal Stem Cells in Vitro

The influences of physical stimuli such as surface elasticity, topography, and chemistry over mesenchymal stem cell proliferation and differentiation are well investigated. In this context, a fundamentally different approach was adopted, and we have demonstrated the interplay of inherent substrate conductivity, defined chemical composition of cellular microenvironment, and intermittent delivery of electric pulses to drive mesenchymal stem cell differentiation toward osteogenesis. For this, conducting polyaniline (PANI) substrates were coated with collagen type 1 (Coll) alone or in association with sulfated hyaluronan (sHya) to form artificial extracellular matrix (aECM), which mimics the native microenvironment of bone tissue. Further, bone marrow derived human mesenchymal stem cells (hMSCs) were cultured on these moderately conductive (10(-4)10(-3) S/cm) aECM coated PANI substrates and exposed intermittently to pulsed electric field (PEF) generated through transformer-like coupling (TLC) approach over 28 days. On the basis of critical analysis over an array of end points, it was inferred that Coll/sHya coated PANI (PANI/Coll/sHya) substrates had enhanced proliferative capacity of hMSCs up to 28 days in culture, even in the absence of PEF stimulation. On the contrary, the adopted PEF stimulation protocol (7 ms rectangular pulses, 3.6 mV/cm, 10 Hz) is shown to enhance osteogenic differentiation potential of hMSCs. Additionally, PEF stimulated hMSCs had also displayed different morphological characteristics as their nonstimulated counterparts. Concomitantly, earlier onset of ALP activity was also observed on PANI/Coll/sHya substrates and resulted in more calcium deposition. Moreover, real-time polymerase chain reaction results indicated higher mRNA levels of alkaline phosphatase and osteocalcin, whereas the expression of other osteogenic markers such as Runt-related transcription factor 2, Col1A, and osteopontin exhibited a dynamic pattern similar to control cells that are cultured in osteogenic medium. Taken together, our experimental results illustrate the interplay of multiple parameters such as substrate conductivity, electric field stimulation, and aECM coating on the modulation of hMSC proliferation and differentiation in vitro.

Synergistic effect of polymorphism, substrate conductivity and electric field stimulation towards enhancing muscle cell growth in vitro

Poly(vinylidene difluoride), a well-known candidate for artificial muscle patch applications is a semi-crystalline polymer with a host of attributes such as piezo- and pyroelectricity, polymorphism along with low dielectric constant and stiffness. The present work explores the unique interplay among the factors (conductivity, polymorphism and electrical stimulation) towards cell proliferation on poly(vinylidene difluoride) (PVDF)-based composites. In this regard, multi-walled carbon nanotubes (MWNTs) are introduced in the PVDF matrix (limited to 2%) through melt mixing to increase the conductivity of PVDF. The addition of MWNTs also led to an increase in the fraction of piezoelectric beta-phase, tensile strength and modulus. The melting and crystallization behaviour of PVDF-MWNT together with FT-IR confirms that the crystallization is found to be aided by the presence of MWNT. The conducting PVDF-MWNTs are used as substrates for the growth of C2C12 mouse myoblast cells and electrical stimulation with a range of field strengths (0-2 V cm(-1)) is intermittently delivered to the cells in culture. The cell viability results suggest that metabolically active cell numbers can statistically increase with electric stimulation up to 1 V cm(-1), only on the PVDF + 2% MWNT. Summarising, the current study highlights the importance of biophysical cues on cellular function at the cell-substrate interface. This study further opens up new avenues in designing conducting substrates, that can be utilized for enhancing cell viability and proliferation and also reconfirms the lack of toxicity of MWNTs, when added in a tailored manner.

Theoretical and Experimental Evaluation of Pulsating Torque Produced by Induction Motor Drives Controlled With Advanced Bus-Clamping Pulsewidth Modulation

This paper investigates possible reduction of pulsating torque in open-loop and vector-controlled induction motor drives through deployment of certain advanced bus-clamping pulsewidth modulation (ABCPWM) method. Toward this goal, a simple and machine-independent method is proposed to analyze the torque harmonic spectrum of a voltage source inverter fed induction motor, operated with any real-time pulsewidth modulation (PWM) method. The analytically evaluated torque harmonic spectra, pertaining to conventional space vector PWM (CSVPWM), bus-clamping PWM (BCPWM), and ABCPWM, are validated through simulation and experimental results. Theoretical and experimental studies bring out the superiority of the ABCPWM in terms of torque harmonics over CSVPWM and BCPWM. The magnitude of the dominant torque harmonic with the ABCPWM scheme is shown to be significantly lower than that with CSVPWM, over a wide range of speed. The rms torque ripple (i.e., total rms value of all harmonic torques) is lower with ABCPWM than with BCPWM over the entire range of speed.

Effect of current density on the microstructure and corrosion properties of plasma electrolytic oxidation (PEO) coatings on AM50 Mg alloy produced in an electrolyte containing clay additives

Plasma electrolytic oxidation coatings were produced on AM50 Mg alloy in alkaline phosphate based electrolyte with montmorillonite clay additives employing current densities of 30, 60, and 120 mA/cm(2). The effect of current density on the microstructure and corrosion properties of the coating was investigated. The clay additives got melted and reactively incorporated into the coating forming an amorphous phase, at all the current densities. However, the coating was predominantly amorphous only at 30 mA/cm(2) and with increasing current density, increasing fractions of crystalline phases were formed. Higher current densities resulted in increased thickness of the coating, but reduced the compactness of the coatings. Electrochemical impedance spectroscopy tests in 0.5 wt.% (0.08 M) and 3.5 wt.% (0.6 M) NaCl solution revealed that the coatings processed at 30 mA/cm(2) exhibited a relatively better initial corrosion resistance owing to its relatively defect-free barrier layer and compactness of the coating. However, the presence of amorphous phases in significant amounts and lack of MgO in the coating resulted in increased rate of dissolution of the coatings and degradation of corrosion resistance. Coatings produced at higher current densities exhibited initial inferior corrosion resistance due to a more defective barrier layer and increased porosity in pore band and outer porous layer. However, the increased amount of crystalline phases and an increased amount of MgO, which resisted dissolution, counterbalanced the negative effects of defective barrier and increased porosity resulting in a relatively lower rate of the degradation of the corrosion resistance. Thus, the corrosion resistance of all the coatings continuously decreased with time and became similar after prolonged immersion in NaCl solution. Increasing current density, therefore, did not prove to be beneficial for the improvement of the corrosion performance of the PEO coatings. (C) 2016 Elsevier B.V. All rights reserved.

Influence of the Processing Conditions on the Characteristics of the Clad Layers Produced with Laminar Plasma Technology

Laminar plasma technology was used to produce ceramic hardened layers of Al2O3-40% mass Ni composite powders on stainless steel substrates. In order to investigate the influences of processing conditions on the morphologies of the surface modified layers, two different powder-feeding methods were tested, one with carrier gas called the powder injection method, and the other without carrier gas called powder transfers method. The microscopic investigations demonstrate that the cross-section of the clad layers consists of two distinct microstructural regions, in which the Al2O3 phases exhibit different growth mechanisms. When the powder transfers method is adopted, the number density and volume fraction of the Al2O3 particles increase considerably and their distributions exhibit zonal periodical characteristics. When the powder-feeding rate increases, the microstructure of the Al2O3 phases changes from a small globular to a long needle shape. Finite element simulations show that the transient thermo-physical features of the pool substances, such as solidification rate and cooling rate, influence strongly the mechanisms of the nucleation and the directional growth of the Al2O3 phases in the thermal processing.

The effect of carbon encapsulation on the magnetic properties of Ni nanoparticles produced by arc discharge in de-ionized water

Despite intensive research on optimizing the methods for depositing carbon encapsulated ferromagnetic nanoparticles, the effect of the carbon cages remains unclear. In the present work, the effect of the graphitic cages on the magnetization of the ferromagnetic core has been studied by comparing the magnetic properties of pure and carbon encapsulated Ni particles of the same size. The carbon encapsulated Ni particles were formed using an electric arc discharge in de-ionized water between a solid graphite cathode and an anode consisting of Ni and C in a mass ratio of Ni:C = 7:3. This method is shown to have potential for low cost production of carbon encapsulated Ni nanoparticle samples with narrow particle size distributions. X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM) analysis were used to study the crystallography, morphology, and size distribution of the encapsulated and pure Ni nanoparticle samples. The availability of encapsulated particles with various sizes allowed us to elucidate the role of carbon cages in size-dependent properties. Our data suggest that even though encapsulation is beneficial for protection against hostile chemical environments and for avoiding low proximity phenomena, it suppresses the saturation magnetization of the Ni cores.

Investigation of High-Speed Combustible Gas Ignited by a Hot Gas Jet Produced in the Shock Tube

The high-speed combustible gas ignited by a hot gas jet, which is induced by shock focusing, was experimentally investigated. By use of the separation mode of shock tube, the test section of a single shock tube is split into two parts, which provide the high-speed flow of combustible gas and pilot flame of hot gas jet, respectively. In the interface of two parts of test sections the flame of jet was formed and spread to the high-speed combustible gas. Two kinds of the ignitions, 3-D “line-flame ignition” and 2-D “plane-flame ignition”, were investigated. In the condition of 3-D “lineflame ignition” of combustion, thicker hot gas jet than pure air jet, was observed in schlieren photos. In the condition of 2-D “plane-flame ignition” of combustion, the delay time of ignition and the angle of flame front in schlieren photos were measured, from which the velocity of flame propagation in the high-speed combustible gas is estimated in the range of 30–90 m/s and the delay time of ignition is estimated in the range of 0.12–0.29 ms.