High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays.

Algal biofuel production and mitigation potential in India

Energy and energy services are the backbone of growth and development in India and is increasingly dependent upon the use of fossil based fuels that lead to greenhouse gases (GHG) emissions and related concerns. Algal biofuels are being evolved as carbon (C)-neutral alternative biofuels. Algae are photosynthetic microorganisms that convert sunlight, water and carbon dioxide (CO2) to various sugars and lipids Tri-Acyl-Glycols (TAG) and show promise as an alternative, renewable and green fuel source for India. Compared to land based oilseed crops algae have potentially higher yields (5-12 g/m(2)/d) and can use locations and water resources not suited for agriculture. Within India, there is little additional land area for algal cultivation and therefore needs to be carried out in places that are already used for agriculture, e.g. flooded paddy lands (20 Mha) with village level technologies and on saline wastelands (3 Mha). Cultivating algae under such conditions requires novel multi-tier, multi-cyclic approaches of sharing land area without causing threats to food and water security as well as demand for additional fertilizer resources by adopting multi-tier cropping (algae-paddy) in decentralized open pond systems. A large part of the algal biofuel production is possible in flooded paddy crop land before the crop reaches dense canopies, in wastewaters (40 billion litres per day), in salt affected lands and in nutrient/diversity impoverished shallow coastline fishery. Mitigation will be achieved through avoidance of GHG, C-capture options and substitution of fossil fuels. Estimates made in this paper suggest that nearly half of the current transportation petro-fuels could be produced at such locations without disruption of food security, water security or overall sustainability. This shift can also provide significant mitigation avenues. The major adaptation needs are related to socio-technical acceptance for reuse of various wastelands, wastewaters and waste-derived energy and by-products through policy and attitude change efforts.

A computational study of heat transfer, soot production and transport in an acetylene-air laminar diffusion flame in the field of a line vortex

The present work involves a computational study of soot formation and transport in case of a laminar acetylene diffusion flame perturbed by a co nvecting line vortex. The topology of the soot contours (as in an earlier experimental work [4]) have been investigated. More soot was produced when vortex was introduced from the air si de in comparison to a fuel side vortex. Also the soot topography was more diffused in case of the air side vortex. The computational model was found to be in good agreement with the ex perimental work [4]. The computational simulation enabled a study of the various parameters affecting soot transport. Temperatures were found to be higher in case of air side vortex as compared to a fuel side vortex. In case of the fuel side vortex, abundance of fuel in the vort ex core resulted in stoichiometrically rich combustion in the vortex core, and more discrete so ot topography. Overall soot production too was low. In case of the air side vortex abundan ce of air in the core resulted in higher temperatures and more soot yield. Statistical techniques like probability density fun ction, correlation coefficient and conditional probability function were introduced to explain the transient dependence of soot yield and transport on various parameters like temperature, a cetylene concentration.

A computational study of flame dynamics on soot production and transport for an acetylene-air laminar diffusion flame in the field of a convecting line vortex

The present work involves a computational study of soot formation and transport in case of a laminar acetylene diffusion flame perturbed by a convecting line vortex. The topology of the soot contours (as in an earlier experimental work [4]) have been investigated. More soot was produced when vortex was introduced from the air side in comparison to a fuel side vortex. Also the soot topography was more diffused in case of the air side vortex. The computational model was found to be in good agreement with the experimental work [4]. The computational simulation enabled a study of the various parameters affecting soot transport. Temperatures were found to be higher in case of air side vortex as compared to a fuel side vortex. In case of the fuel side vortex, abundance of fuel in the vort ex core resulted in stoichiometrically rich combustion in the vortex core, and more discrete soot topography. Overall soot production too was low. In case of the air side vortex abundance of air in the core resulted in higher temperatures and more soot yield. Statistical techniques like probability density function, correlation coefficient and conditional probability function were introduced to explain the transient dependence of soot yield and transport on various parameters like temperature, a cetylene concentration.

Relative stabilities of condensed face sharing mono- and di-carboranes: CB20H18 and C2B19H18+

The relative energies of triangular face sharing condensed macro polyhedral carboranes: CB20H18 and C2B19H18+ derived from mono- and di-substitution of carbons in (4) B21H18- is calculated at B3LYP/6-31G* level. The relative energies, H center dot center dot center dot H non-bonding distances, NICS values, topological charge analysis and orbital overlap compatibility connotes the face sharing condensed macro polyhedral mono-carboranes, 8 (4-CB20H18) to be the lowest energy isomer. The di-carba- derivative, (36) 4,4'a-C2B19H18+ with carbons substituted in a different B-12 cage in (4) B21H18- in anti-fashion is the most stable isomer among 28 possibilities. This structure has less non-bonding H center dot center dot center dot H interaction and is in agreement with orbital-overlap compatibility, and these two have the pivotal role in deciding the stability of these clusters. An estimate of the inherent stability of these carboranes is made using near-isodesmic equations which show that CB20H18 (8) is in the realm of the possible. (C) 2015 Elsevier B.V. All rights reserved.

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H2O2, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl(+) cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl(+) CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl(+) cells. NAC enhances imatinib-induced apoptosis of Bcr-Abl(+) cells by endothelial nitric oxide synthase-mediated production of nitric oxide.

Immunization of male rabbits with sheep luteal receptor to LH results in production of antibodies exhibiting hormone-agonistic and -antagonistic activities

Antibodies to LH/chorionic gonadotrophin receptor (LH/CG-R; molecular weight 67 000), isolated in a homogenous state (established by SDS-PAGE and ligand blotting) from sheep luteal membrane using human CG (hCG)-Sepharose affinity chromatography, were raised in three adult male rabbits (R-I, R-II and R-III). Each of the rabbits received 20-30 mu g oi the purified receptor in Freund's complete adjuvant at a time. Primary immunization was followed by booster injection at intervals. Production of receptor antibodies was monitored by (1) determining the dilution of the serum (IgG fraction) that could specifically bind 50% of I-125-LH/CG-R added and (2) analysing sera for any chance in testosterone levels. Following primary immunization and the first booster, all three rabbits exhibited a 2.5- to 6.0-fold increase in serum testosterone over basal levels and this effect was spread over a period of time (similar to 40 days) coinciding with the rise and fall of receptor antibodies. The maximal antibody titre (ED(50)) produced at this time ranged from 1:350 to 1:100 to below detectable limits for R-I, R-II and R-III respectively. Subsequent immunizations followed by the second booster resulted in a substantial increase in antibody titre (ED(50) of 1:5000) in R-I, but this was not accompanied by any change in serum testosterone over preimmune levels, suggesting that with the progress of immunization the character of the antibody produced had also changed. Two pools of antisera from R-I collected 10 days following the booster (at day 70 (bleed I) and day 290 (bleed II)) were used in further experiments. IgG isolated from bleed I but not from bleed II antiserum showed a dose-dependent stimulation of testosterone production by mouse Leydig cells in vitro, thus confirming the in vivo hormone-mimicking activity antibodies generated during the early immunization phase. The IgG fractions from both bleeds were, however, capable of inhibiting (1) I-125-hCG binding to crude sheep luteal membrane (EC(50) of 1:70 and 1:350 for bleed I and II antisera respectively) and (2) ovine LH-stimulated testosterone production by mouse Leydig cells in vitro, indicating the presence oi antagonistic antibodies irrespective of the period of time during which the rabbits were immunized. The: fact that bleed I-stimulated testosterone production could be inhibited in a dose-dependent manner by the addition of IgG from bleed II to the mouse Leydig cell in vitro assay system showed that the agonistic activity is intrinsic to the bleed I antibody. The receptor antibody (bleed II) was also capable of blocking LH action in vivo, as rabbits passively (for 24 h with LH/CG-R antiserum) as well as actively (for 130 days) immunized against LH/CG-R failed to respond to a bolus injection of LH (50 mu g). At no time, however, was the serum testosterone reduced below the basal level. This study clearly shows that, unlike with LH antibody, attempts to achieve an LH deficiency effect in vivo by resorting to immunization with hole LH receptor is difficult, as receptor antibodies exhibit both hormone-mimicking (agonistic) as well as hormone-blocking (antagonistic) activities.

Defect production due to quenching through a multicritical point

We study the generation of defects when a quantum spin system is quenched through a multicritical point by changing a parameter of the Hamiltonian as t/tau, where tau is the characteristic timescale of quenching. We argue that when a quantum system is quenched across a multicritical point, the density of defects (n) in the final state is not necessarily given by the Kibble-Zurek scaling form n similar to 1/tau(d nu)/((z nu+1)), where d is the spatial dimension, and. and z are respectively the correlation length and dynamical exponent associated with the quantum critical point. We propose a generalized scaling form of the defect density given by n similar to 1/(tau d/(2z2)), where the exponent z(2) determines the behavior of the off-diagonal term of the 2 x 2 Landau-Zener matrix at the multicritical point. This scaling is valid not only at a multicritical point but also at an ordinary critical point.

On the cytotoxicity of carbon nanotubes

The cytotoxicity of carbon nanotubes (CNTs) is a major concern today well before its unusual physicochemical, mechanical, and electrical properties are fully exploited for commercial interests and subsequent mass production leading to greater possibilities for its exposure to humans and the environment. Contradictory reports on cytotoxicity of CNTs often appear in the literature and a mechanistic explanation of the reported toxicity remains obscure. We review here the conflicting results to focus categorically on an array of issues in CNT cytotoxicity. They include dispersion, aggregation status, coating or functionalization and immobilization, cellular uptake or internalization, purity in terms of metal catalyst contaminants, size and size distribution, surface area, surface chemistry and surface reactivity, cell types selected for experimentation as well as bioassay of nanotoxicity itself attesting as an issue in cytotoxicity. Recently a general agreement has emerged towards the potential toxicity of CNTs, although various paradigms explaining the mechanisms of CNT cytotoxicity continue to be elusive in the literature. A lack of synergy among various issues while studying cytotoxicity and most developed paradigms for the mechanism of CNT toxicity is highlighted.

Temperature characteristics of the billet and the dies in a small batch forging production

The work reported herein is part of an on-going programme to develop a computer code which, given the geometrical, process and material parameters of the forging operation, is able to predict the die and the billet cooling/heating characteristics in forging production. The code has been experimentally validated earlier for a single forging cycle and is now validated for a small batch production. To facilitate a step-by-step development of the code, the billet deformation has so far been limited to its surface layers, a situation akin to coining. The code has been used here to study the effects of die preheat-temperature, machine speed and rate of deformation the cooling/heating of the billet and the dies over a small batch of 150 forgings. The study shows: that there is a pre-heat temperature at which the billet temperature changes little from one forging to the next; that beyond a particular number of forgings, the machine speed ceases to have any pronounced influence on the temperature characteristics of the billet; and that increasing the rate of deformation reduces the heat loss from the billet and gives the billet a stable temperature profile with respect to the number of forgings. The code, which is simple to use, is being extended to bulk-deformation problems. Given a practical range of possible machine, billet and process specifics, the code should be able to arrive at a combination of these parameters which will give the best thermal characteristics of the die-billet system. The code is also envisaged as being useful in the design of isothermal dies and processes.

Axial and pseudoscalar current correlators and their couplings to eta and eta(') mesons

Correlators of singlet and octet axial currents, as well as anomaly and pseudoscalar densities have been studied using QCD sum rules. Several of these sum rules are used to determine the couplings f(eta)(8),f(eta)(0), f(eta)('8) and f(eta)('0). We find mutually consistent values which are also in agreement with phenomenological values obtained from data on various decay and production rates. While most of the sum rules studied by us are independent of the contributions of direct instantons and screening correction, the singlet-singlet current correlator and the anomaly-anomaly correlator improve by their inclusion.

Isolation and Culture of a Thermophilic Fungus, Melanocarpus albomyces, and Factors Influencing the Production and Activity of Xylanase

Summary: An uncommon thermophilic fungus, Melanocarpus albomyces, was isolated from soil and compost by incubating samples in a glucose/sorbose/asparagine liquid medium, followed by enrichment culture in medium containing sugarcane bagasse as carbon source. The culture filtrate protein of the fungus grown in the presence of bagasse or xylose hydrolysed xylan and some other polysaccharides but cellulose was not hydrolysed. High extracellular xylanase (EC 3.2.1.8) activity was produced by cultures grown on xylose or hemicellulosic materials. The enzyme was induced in glucose-grown washed mycelia in response to addition of xylose or xylan but not by alkyl or aryl β-D-xylosides. Cultures produced higher enzyme yields in shaken flasks than in a fermenter. Gel-filtration chromatography of culture filtrate protein showed the presence of two isoenzymes of xylanase, whose relative proportions varied with the carbon source used for growth. The extent of hydrolysis of heteroxylans or the hemicellulosic fraction of bagasse by culture filtrate protein preparations was greater when the cultures had been grown on bagasse rather than xylose as the inducing substrate. The activity of xylanase preparations was increased when an exogenous β-glucosidase was added.

An alternative approach to selective sea water oxidation for hydrogen production

Sea water electrolysis is one of the promising ways to produce hydrogen since it is available in plentiful supply on the earth. However, in sea water electrolysis toxic chlorine evolution is the preferred reaction over oxygen evolution at the anode. In this work, research has been focused on the development of electrode materials with a high selectivity for oxygen evolution over chlorine evolution. Selective oxidation in sea water electrolysis has been demonstrated by using a cation-selective polymer. We have used a perm-selective membrane (Nafion®), which electrostatically repels chloride ions (Cl−) to the electrode surface and thereby enhances oxygen evolution at the anode. The efficiency and behaviour of the electrode have been characterized by means of anode current efficiency and polarization studies. The surface morphology of the electrode has been characterized by using a scanning electron microscope (SEM). The results suggest that nearly 100% oxygen evolution efficiency could be achieved when using an IrO2/Ti electrode surface-modified by a perm-selective polymer.

Production of 2-phenethyl alcohol and 2-phenyllactic acid in Candida species

2-Phenethyl alcohol (2-PEA) and 2-phenyllactic acid (2-PLA) were isolated from the culture filtrates of Candida species grown in media containing peptone or phenylalanine as nitrogen source. These compounds were characterized by comparing their UV, IR, and NMR spectral properties with authentic samples. Candida species differed markedly in their production of 2-PEA and 2-PLA. Experiments using [14C]-phenylalanine indicated that both 2-PEA and 2-PLA are synthesised from L-phenylalanine. A pathway for the biosynthesis of 2-PEA from L-phenylalanine has been proposed.