The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

ENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCH

The delicate balance between the production and disposal of proteins is vital for the changes required in the cell to respond to given stimulus. Ubiquitination is a protein modification with a range of signaling outcomes when ubiquitin is attached to a protein through a highly ordered enzymatic cascade process. Understanding ubiquitination is a growing field and nowadays the application of chemical reactions allows the isolation of quantitative materials for structural studies. Therefore, in this dissertation it is described some of these suitable chemical methodologies to produce an isopeptide bond toward the polymerization of ubiquitin bypassing the enzymatic control with the purpose of showing if these chemical modifications have a direct impact on the structure of ubiquitin. First, the possibility of incorporating non-natural lysine analogs known as mercaptolysines into the polypeptide chain of Ubiquitin was explored when they were attached to ubiquitin by native chemical ligation at its C terminus. The sulfhydryl group was used for the attachment of a paramagnetic label to map the surface of ubiquitin. Second, the condensation catalyzed by silver nitrate was used for the dimer assembly. In particular, the main focus was on examining whether orthogonal protection and deprotection of each monomer have an impact on the reaction yield, since the synthetic strategy has been previously attempted successfully. Third, the formation of ubiquitin dimers was approached by building an inter-ubiquitin linkage mimicking the isopeptide bond with two approaches, the classic disulfide exchange as well as the thiol-ene click reaction by thermal initiation in aqueous conditions. After assembling the dimeric units, they were studied by Nuclear Magnetic Resonance, in order to establish a conformational state profile which depends on the pH conditions. The latter is a very important concept since some ligands have a preferred affinity when the protein-protein hydrophobic patches are in close proximity.

Enzyme-free glucose biosensor based on low density CNT forest grown directly on a Si/SiO2 substrate

A highly sensitive nonenzymatic amperometric glucose sensor was fabricated by using Ni nanoparticles homogeneously dispersed within and on the top of a vertically aligned CNT forest (CNT/Ni nanocomposite sensor), which was directly grown on a Si/SiO2 substrate. The surface morphology and elemental analysis were characterized using scanning electron microscopy and energy dispersive spectroscopy, respectively. Cyclic voltammetry and chronoamperometry were used to evaluate the catalytic activities of CNT/Ni electrode. The CNT/Ni nanocomposite sensor exhibited a great enhancement of anodic peak current after adding 5 mM glucose in alkaline solution. The sensor can also be applied to the quantification of glucose content with a linear range covering from 5 μM to 7 mM, a high sensitivity of 1433 μA mM-1 cm-2, and a low detection limit of 2 μM. The CNT/Ni nanocomposite sensor exhibits good reproducibility and long-term stability, moreover, it was also relatively insensitive to commonly interfering species, such as uric acid, ascorbic acid, acetaminophen, sucrose and d-fructose. © 2013 Elsevier B.V.

A Chloro-Bridged Linear Chain Imine-Copper(II) Complex and Its Application as an Enzyme-Free Amperometric Biosensor for Hydrogen Peroxide

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Serum-free process development:improving the yield and consistency of human mesenchymal stromal cell production

Background aims: The cost-effective production of human mesenchymal stromal cells (hMSCs) for off-the-shelf and patient specific therapies will require an increasing focus on improving product yield and driving manufacturing consistency. Methods: Bone marrow-derived hMSCs (BM-hMSCs) from two donors were expanded for 36 days in monolayer with medium supplemented with either fetal bovine serum (FBS) or PRIME-XV serum-free medium (SFM). Cells were assessed throughout culture for proliferation, mean cell diameter, colony-forming potential, osteogenic potential, gene expression and metabolites. Results: Expansion of BM-hMSCs in PRIME-XV SFM resulted in a significantly higher growth rate (P < 0.001) and increased consistency between donors compared with FBS-based culture. FBS-based culture showed an inter-batch production range of 0.9 and 5 days per dose compared with 0.5 and 0.6 days in SFM for each BM-hMSC donor line. The consistency between donors was also improved by the use of PRIME-XV SFM, with a production range of 0.9 days compared with 19.4 days in FBS-based culture. Mean cell diameter has also been demonstrated as a process metric for BM-hMSC growth rate and senescence through a correlation (R² = 0.8705) across all conditions. PRIME-XV SFM has also shown increased consistency in BM-hMSC characteristics such as per cell metabolite utilization, in vitro colony-forming potential and osteogenic potential despite the higher number of population doublings. Conclusions: We have increased the yield and consistency of BM-hMSC expansion between donors, demonstrating a level of control over the product, which has the potential to increase the cost-effectiveness and reduce the risk in these manufacturing processes.

Transfer free suspended graphene devices on silicon using electrodeposited copper

Transfer free processes using Cu films greatly simplify the fabrication of reliable suspended graphene devices. In this paper, the authors report on the use of electrodeposited Cu films on Si for transfer free fabrication of suspended graphene devices. The quality of graphene layers on optimized electrodeposited Cu and Cu foil are found to be the same. By selectively etching the underlying Cu, the authors have realized by a transfer free process metal contacted, suspended graphene beams up to 50 mu m in length directly on Si. The suspended graphene beams do not show any increase in defect levels over the as grown state indicating the efficiency of the transfer free process. Measured room temperature electronic mobilities of up to 5200 cm(2)/V.s show that this simpler and CMOS compatible route has the potential to replace the foil based route for such suspended nano and micro electromechanical device arrays. (C) 2014 American Vacuum Society.

Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

A heterofunctional support for enzyme immobilization may be defined as that which possesses several distinct functionalities on its surface able to interact with a protein. We will focus on those supports in which a final covalent attachment between the enzyme and the support is achieved. Heterofunctionality sometimes has been featured in very old immobilization techniques, even though in many instances it has been overlooked, giving rise to some misunderstandings. In this respect, glutaraldehyde-activated supports are the oldest multifunctional supports. Their matrix has primary amino groups, the hydrophobic glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. Thus, immobilization may start (first event of the immobilization) via different causes and may involve different positions of the enzyme surface depending on the activation degree and immobilization conditions. Other “classical” heterofunctional supports are epoxy commercial supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization is performed at high ionic strength to permit protein adsorption, so that covalent attachment may take place at a later stage. Starting from these old immobilization techniques, tailor-made heterofunctional supports have been designed to permit a stricter control of the enzyme immobilization process. The requirement is to find conditions where the main covalent reactive moieties may have very low reactivity toward the enzyme. In this Review we will discuss the suitable properties of the groups able to give the covalent attachment (intending a multipoint covalent attachment), and the groups able to produce the first enzyme adsorption on the support. Prospects, limitations, and likely pathways for the evolution (e.g., coupling of site-directed mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional supports) will be discussed in this Review.

Pyrimidine-Derived Prolinamides as Recoverable Bifunctional Organocatalysts for Enantioselective Inter- and Intramolecular Aldol Reactions under Solvent-Free Conditions

Chiral L-prolinamides 2 containing the (R,R)- and (S,S)-trans-cyclohexane-1,2-diamine scaffold and a 2-pyrimidinyl unit are synthesized and used as general organocatalysts for intermolecular and intramolecular aldol reactions with 1,6-hexanedioic acid as a co-catalyst under solvent-free conditions. The intermolecular reaction between ketone–aldehyde and aldehyde–aldehyde must be performed under wet conditions with catalyst (S,S)-2b at 10 °C, which affords anti-aldols with high regio-, diastereo-, and enantioselectivities. For the Hajos–Parrish–Eder–Sauer–Wiechert reaction, both diastereomers of catalyst 2 give similar results at room temperature in the absence of water to give the corresponding Wieland–Miescher ketone and derivatives. Both types of reactions were scaled up to 1 g, and the organocatalysts were recovered by extractive workup and reused without any appreciable loss in activity. DFT calculations support the stereochemical results of the intermolecular process and the bifunctional role played by the organocatalyst by providing a computational comparison of the H-bonding networks occurring with catalysts 2a and 2b.

Enzymatic production of oligosaccharides in centrifugal fields

The aims of this work have been to identify an enzymatic reaction system suitable to investigate and develop the high-speed centrifuge as a novel reaction system for performing such reactions. The production of galacto-oligosaccharides by the trans-galactosyl activity of the enzyme β-galactosidase on lactose monohydrate was identified as a model enzymatic system to elucidate the principles of this type of process. Galacto-oligosaccharides have attracted considerable commercial interest as food additives which have been shown to be beneficial to the health of the human gastrointestinal tract. The development of a single unit operation capable of controlling the biosynthesis of galacto-oligosaccharides whilst simultaneously separating the enzyme from the reaction products would reduce downstream processing costs. This thesis shows for the first time that by using a combination of (a) immobilised or insolubilised β-galactosidase , (b) a rate-zonal centrifugation technique, and (c) various applied centrifugal fields, that a high-speed centrifuge could be used to control the formation of galacto-oligosaccharides whilst removing the enzyme from the reaction products. By layering a suspension of insolubilised β-galactosidase on top of a lactose monohydrate density gradient and centrifuging, the applied centrifugal fields generated produced sedimentation of the enzyme particles through the substrate. The higher sedimentation rate of the enzyme compared to those of the reaction products allowed for separation to take place. Complete sedimentation, or pelleting of the enzyme permits the possible recovery and re-use. Insolubilisation of the enzyme allowed it to be sedimented through the substrate gradient using much lower applied centrifugal fields than that required to sediment free soluble enzyme and this allowed for less expensive centrifugation equipment to be used. Using free soluble and insolubilised β-galactosidase stirred-batch reactions were performed to investigate the kinetics of lactose monohydrate hydrolysis and galacto-oligosaccharide formation. Based on these results a preliminary mathematical model based on Michaelis-Menten kinetics was produced. It was found that the enzyme insolubilisation process using a chemical cross-linking agent did not affect the process of galacto-oligosaccharide formation. Centrifugation experiments were performed and it was found that by varying the applied centrifugal fields that the yield of galacto-oligosaccharides could be controlled. The higher the applied centrifugal fields the lower the yield of galacto-oligosaccharides. By increasing the applied centrifugal fields the 'contact time' between the sedimenting enzyme and the substrate was reduced, which produced lower yields. A novel technique involving pulsing the insolubilised enzyme through the substrate gradient was developed and this was found to produce higher yields of galacto-oligosaccharide compared to using a single enzyme loading equivalent to the total combined activity of the pulses. Comparison of the galacto-oligosaccharide yields between stirred-batch and centrifugation reactions showed that the applied centrifugal fields did not adversely affect the transgalactosyl activity of the insolubilised enzyme.

A systematic review of treatments for atopic dermatitis

Atopic dermatitis is a very common inflammatory skin disease, particularly in children. A systematic review of randomised controlled trials of treatments for atopic dermatitis (AD) was carried out to assess how many trials exist, what they cover, what they do not cover, the research gaps, provide a 'blue print' for future Cochrane Reviews and assist those making treatment recommendations by summarising the available RCT evidence, using descriptive statistics. The Cochrane Collaboration systematic review process formed the basis of the methodology, from which over 4000 studies were located via electronic database searches and hand searching of journals. A total of 292 trials were finally included covering 9 treatment groups and over 48 individual treatments. There are lots of trials covering lots of interventions but gaps are evident. However, there is evidence of a benefit in the treatment of atopic dermatitis with topical corticosteroids, psychological approaches, UV light, ascomycin derivatives, topical tacrolimus and oral cyclosporin. Treatments that show limited evidence of a benefit include non-sedatory antihistamines, topical doxepin, the oral antibiotic Cefadroxil on clinically infected AD, the topical antibacterial Mupirocin on clinically uninfected AD, Chinese herbs, hypnotherapy and biofeedback, massage therapy, dietary manipulation, house dust mite reduction, patient education, emollients, allergen antibody complexes of house dust mite and thymic extracts. Treatments that show no evidence of benefit include sedatory antihistamines, oral sodium cromoglycate, oral antibiotics on clinically uninfected AD, topical antibacterials, topical antifungals, aromatherapy essential oils, borage oil, fish oil, evening primrose oil, enzyme-free clothes detergent, cotton clothing, house dust mite hyposensitisation, salt baths, topical coal tar, topical cyclosporin and platelet-activating-factor antagonist. When interpreting the conclusions of this thesis it is important to understand that lack of evidence does not equal lack of efficacy, particularly considering the interventions that are commonly in use today to treat atopic dermatitis that have not been subjected to RCTs, such as occlusive dressings, water softening devices and stress management among many others.

Plasma enables edge-to-center-oriented graphene nanoarrays on Si nanograss

The formation of clearly separated vertical graphenenanosheets on silicon nanograss support is demonstrated. The plasma-enabled, two-stage mask-free process produced self-organized vertical graphenes of a few carbon layers (as confirmed by advanced microanalysis), prominently oriented in the substrate center–substrate edge direction. It is shown that the width of the alignment zone depends on the substrate conductivity, and thus the electric field in the vicinity of the growth surface is responsible for the graphene alignment. This finding is confirmed by the Monte Carlo simulations of the ion flux distribution in the silicon nanograss pattern.

Plasma-enabled growth of single-crystalline SiC/AlSiC Core–Shell nanowires on porous alumina templates

We report the catalyst-free synthesis of the arrays of core–shell, ultrathin, size-uniform SiC/AlSiC nanowires on the top of a periodic anodic aluminum oxide template. The nanowires were grown using an environmentally friendly, silane-free process by exposing the silicon supported porous alumina template to CH4 + H2 plasmas. High-resolution scanning and transmission electron microscopy studies revealed that the nanowires have a single-crystalline core with a diameter of about 10 nm and a thin (1–2 nm) amorphous AlSiC shell. Because of their remarkable length, high aspect ratio, and very high surface area-to-volume ratio, these unique structures are promising for nanoelectronic and nanophotonic applications that require efficient electron emission, light scattering, etc. A mechanism for nanowire growth is proposed based upon the reduction of the alumina template to nanosized metallic aluminum droplets forming between nanopores. The subsequent incorporation of silicon and carbon atoms from the plasma leads to nucleation and growth from the top of the alumina template.

A bottom-up approach for optimization of friction stir processing parameters; a study on aluminium 2024-T3 alloy

Friction stir processing (FSP) is emerging as one of the most competent severe plastic deformation (SPD) method for producing bulk ultra-fine grained materials with improved properties. Optimizing the process parameters for a defect free process is one of the challenging aspects of FSP to mark its commercial use. For the commercial aluminium alloy 2024-T3 plate of 6 mm thickness, a bottom-up approach has been attempted to optimize major independent parameters of the process such as plunge depth, tool rotation speed and traverse speed. Tensile properties of the optimum friction stir processed sample were correlated with the microstructural characterization done using Scanning Electron Microscope (SEM) and Electron Back-Scattered Diffraction (EBSD). Optimum parameters from the bottom-up approach have led to a defect free FSP having a maximum strength of 93% the base material strength. Micro tensile testing of the samples taken from the center of processed zone has shown an increased strength of 1.3 times the base material. Measured maximum longitudinal residual stress on the processed surface was only 30 MPa which was attributed to the solid state nature of FSP. Microstructural observation reveals significant grain refinement with less variation in the grain size across the thickness and a large amount of grain boundary precipitation compared to the base metal. The proposed experimental bottom-up approach can be applied as an effective method for optimizing parameters during FSP of aluminium alloys, which is otherwise difficult through analytical methods due to the complex interactions between work-piece, tool and process parameters. Precipitation mechanisms during FSP were responsible for the fine grained microstructure in the nugget zone that provided better mechanical properties than the base metal. (C) 2014 Elsevier Ltd. All rights reserved.