Topological constraints in nucleic acid hybridization kinetics

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of ‘helix-driven wrapping’. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary ‘kissing hairpins’ demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex.     

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.     

Effects of cryopreservation on the mononuclear cells of patients with lung cancer and normal controls

Studies were carried out to determine the effects of cryopreservation on the mitogen-induced proliferative and immunoglobulin-producing abilities of peripheral blood mononuclear cells (MNC) of patients with lung cancer and normal controls. One-half of each sample of cells was tested fresh, while the other half was frozen, thawed immediately, and cultured at the same time. The responses of each sample of cryopreserved cells were compared to the responses of fresh cells from the same individual in simultaneous assays. The cryopreserved mononuclear cells of most of the lung cancer patients showed significantly enhanced plaque-forming cell (PFC) responses after stimulation with pokeweed mitogen (PWM). No such significant differences were observed between the proliferative responses of cryopreserved and fresh cells against phytohemagglutinin (PHA) or PWM stimulation. The cryopreserved MNC of some of the normal controls also showed a similar increase in the PFC responses, although to a lesser extent. Coculture of cryopreserved B cell-enriched populations of cells with fresh T cell-enriched fractions obtained from these patients also resulted in the generation of a higher number of PFCs as compared to the number of PFCs observed after coculture of fresh B and T cell-enriched populations. The results indicate that the suppressor activities of monocytes/macrophages and other non-T cells (NK cells) are sensitive to cryopreservation. The results also show that the MNC of patients with lung cancer can be cryopreserved and used for subsequent B and T cell assays.     

Process simulation for recombinant protein production: cost estimation and sensitivity analysis for heparinase I expressed in Escherichia coli

Heparinase I from flavobacterium heparinum has several potential clinical applications; the resulting high demands on protein purity and quantity can be met by recombinant expression in Escherichia coli. Based on laboratory scale experiments with insoluble heparinase I expression followed by renaturation, a process for production of 3 kg/year of heparinase I was designed. We present a comparative analysis of the production costs of soluble and insoluble heparinase I expression, as well as a generalized approach to sensitivity analysis, based on perturbation around a base case design scenario. This may assist focusing further development on process steps for which improvements both are feasible and result in significant cost saving. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 575-582, 1997.     

Anthocyanin production in callus cultures ofDaucus carota as influenced by nutrient stress and osmoticum

Summary Daucus carota callus developed red pigments under the influence of indole-3 acetic acid and kinetin. Maximum yield of anthocyanin at the end of 3 weeks was 5.4% on dry weight basis. The callus subjected to phosphate and nitrate stress produced 7.2% and 8.5% anthocyanin respectively. Feeding of sucrose at 7.5% level resulted in production of 15% anthocyanin. Mannitol as osmoticum had positive influence on anthocyanin production.     

Bacterial community dynamics involved in Reactive Orange M2R dye degradation using a real time quantitative PCR and scale up studies using sequence batch reactor

Bacterial consortium volatile suspended soil (VSS) (Vatva soil sample) with a capability of azo dye Reactive Orange M2R (ROM2R) decolorization and degradation (shown in our earlier studies using Fourier transform infrared spectroscopy (FTIR) and phytotoxicity studies) was isolated from industrial wastewaters by enrichment culture technique. The present study was carried out to study bacterial population dynamics in consortium Vatva soil sample (VSS) during azo dye ROM2R degradation and to identify the consortium members that were actively involved in the degradation process. To achieve this goal, a real-time Polymerase chain reaction (PCR) assay targeting species-specific region of 16S rDNA of each consortial bacteria was developed to provide quantitative information about the bacterial abundance during azo dye degradation. The real-time PCR assay indicated that Pseudomonas aeruginosa (VSS-6) dominated consortium bacterial community during the active continuous bioremediation process. Attempt has been made to scale up from 100 ml volume to 10 L operation volume with intermittent additions (batch fed loadings) in a Sequence batch reactor (SBR). The development of VSS consortium biomass (MLVSS), changes in COD and biochemical oxygen demand, and the dye degradation were studied under conditions simulating the operations of biological effluent treatment in an attempt to develop a commercially applicable dye effluent treatment process unit.     

Point of Care Tuberculosis Sero-Diagnosis Kit for Wild Animals: Combination of Proteins for Improving the Diagnostic Sensitivity and Specificity

Tuberculosis is a significant problem globally for domestic animals as well as captive and free ranging wild life. Rapid point of care (POC) serology kits are well suited for the diagnosis of TB in wild animals. However, wild animals are invariably exposed to environmental non-pathogenic mycobacterium species with the development of cross reacting antibodies. In the present study, POC TB diagnosis kit was developed using a combination of pathogenic Mycobacteria specific recombinant antigens and purified protein derivatives of pathogenic and non-pathogenic Mycobacteria. To benchmark the TB antibody detection kit, particularly in respect to specificity which could not be determined in wildlife due to the lack of samples from confirmed uninfected animals, we first tested well-characterized sera from 100 M. bovis infected and 100 uninfected cattle. Then we investigated the kit’s performance using sera samples from wildlife, namely Sloth Bears (n = 74), Elephants (n = 9), Cervidae (n = 14), Felidae (n = 21), Cape buffalo (n = 2), Wild bear (n = 1) and Wild dog (n = 1).In cattle, a sensitivity of 81% and a specificity of 90% were obtained. The diagnostic sensitivity of the kit was 94% when the kit was tested using known TB positive sloth bear sera samples. 47.4% of the in-contact sloth bears turned seropositive using the rapid POC TB diagnostic kit. Seropositivity in other wild animals was 25% when the sera samples were tested using the kit. A point of care TB sero-diagnostic kit with the combination of proteins was developed and the kit was validated using the sera samples of wild animals.