Collagen Promotes Higher Adhesion,Survival and Proliferation of Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) can differentiate into several cell types and are desirable candidates for cell therapy and tissue engineering. However, due to poor cell survival, proliferation and differentiation in the patient, the therapy outcomes have not been satisfactory. Although several studies have been done to understand the conditions that promote proliferation, differentiation and migration of MSC in vitro and in vivo, still there is no clear understanding on the effect of non-cellular bio molecules. Of the many factors that influence the cell behavior, the immediate cell microenvironment plays a major role. In this context, we studied the effect of extracellular matrix (ECM) proteins in controlling cell survival, proliferation, migration and directed MSC differentiation. We found that collagen promoted cell proliferation, cell survival under stress and promoted high cell adhesion to the cell culture surface. Increased osteogenic differentiation accompanied by high active RHOA (Ras homology gene family member A) levels was exhibited by MSC cultured on collagen. In conclusion, our study shows that collagen will be a suitable matrix for large scale production of MSC with high survival rate and to obtain high osteogenic differentiation for therapy.     

Artificial Intelligence versus Statistical Modeling and Optimization of Cholesterol Oxidase Production by using Streptomyces Sp.

Cholesterol oxidase (COD) is a bi-functional FAD-containing oxidoreductase which catalyzes the oxidation of cholesterol into 4-cholesten-3-one. The wider biological functions and clinical applications of COD have urged the screening, isolation and characterization of newer microbes from diverse habitats as a source of COD and optimization and over-production of COD for various uses. The practicability of statistical/ artificial intelligence techniques, such as response surface methodology (RSM), artificial neural network (ANN) and genetic algorithm (GA) have been tested to optimize the medium composition for the production of COD from novel strain Streptomyces sp. NCIM 5500. All experiments were performed according to the five factor central composite design (CCD) and the generated data was analysed using RSM and ANN. GA was employed to optimize the models generated by RSM and ANN. Based upon the predicted COD concentration, the model developed with ANN was found to be superior to the model developed with RSM. The RSM-GA approach predicted maximum of 6.283 U/mL COD production, whereas the ANN-GA approach predicted a maximum of 9.93 U/mL COD concentration. The optimum concentrations of the medium variables predicted through ANN-GA approach were: 1.431 g/50 mL soybean, 1.389 g/50 mL maltose, 0.029 g/50 mL MgSO₄, 0.45 g/50 mL NaCl and 2.235 ml/50 mL glycerol. The experimental COD concentration was concurrent with the GA predicted yield and led to 9.75 U/mL COD production, which was nearly two times higher than the yield (4.2 U/mL) obtained with the un-optimized medium. This is the very first time we are reporting the statistical versus artificial intelligence based modeling and optimization of COD production by Streptomyces sp. NCIM 5500.     

Phytochemical analysis of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> extract and its antimicrobial activity

The present study describes the phytochemical profile and antimicrobial activity of Andrographis paniculata. For the present investigation, two samples of A. paniculata extracts, obtained by extraction in chloroform and chloroform + HCl, respectively, were compared for their antimicrobial activity and further subjected to GC-MS analysis to find out the nature of the compounds responsible for the antimicrobial activity. The antibacterial activities were assessed by measuring the diameter of the inhibition zones, MIC and MBC values. Compared to the chloroform + HCl extract, the chloroform extract showed better antimicrobial activity against all the nine pathogenic bacterial strains tested. The chloroform extract was observed to be active against the opportunistic and pathogenic gram-negative bacteria, indicating its potential application related to noscomial infections. GC-MS results revealed phenols, aromatic carboxylic acids and esters in the chloroform extract to be the molecules responsible for the antimicrobial activity of A. paniculata. This is the first report on analysis of antimicrobial components from A. paniculata, and our results confer the utility of this plant extract in developing a novel broad spectrum antimicrobial agent.     

Evaluation of diverse soybean germplasm for root growth and architecture

Root characteristics of soybean (Glycine max (L.) Merr.) improve drought avoidance by increasing water uptake from the soil profile. Screening genotypes for improved root architecture without breaking the taproots or losing lateral roots is a challenge. Due to difficulty in separating roots from field or potting soil, a rapid and effective screening method with a suitable growth medium to assess root characteristics under controlled conditions needs to be established. We describe two screening techniques “the cone system” and “the tube system” using turface:sand medium. In the cone system thirty four soybean lines including cultivars and exotic plant introduction (PI) lines were evaluated for tap root length and root biomass, 12 days after sowing. Eight replications per line were grown in a growth chamber. Significant differences among genotypes for tap root length were detected by the cone system. Validity of results from the cone system was tested by evaluating root growth 21 days after planting for eight lines in the tube system. A coefficient of determination of 0.72 indicated good agreement between the two screening systems for evaluating genotypes for rooting depth. The cone system will be a useful method to easily and rapidly assess soybean genotypes for root growth.     

Hzf Determines cell survival upon genotoxic stress by modulating p53 transactivation

A critical unresolved issue about the genotoxic stress response is how the resulting activation of the p53 tumor suppressor can lead either to cell-cycle arrest and DNA repair or to apoptosis. We show here that hematopoietic zinc finger (Hzf), a zinc-finger-containing p53 target gene, modulates p53 transactivation functions in an autoregulatory feedback loop. Hzf is induced by p53 and binds to its DNA-binding domain, resulting in preferential transactivation of proarrest p53 target genes over its proapoptotic target genes. Thus, p53 activation results in cell-cycle arrest in Hzf wild-type MEFs, while in Hzf(-/-) MEFs, apoptosis is induced. Exposure of Hzf null mice to ionizing radiation resulted in enhanced apoptosis in several organs, as compared to in wild-type mice. These findings provide novel insights into the regulation of p53 transactivation function and suggest that Hzf functions as a key player in regulating cell fate decisions in response to genotoxic stress.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

Evidence for an interaction between the SH3 domain and the N-terminal extension of the essential light chain in class II myosins

The function of the src-homology 3 (SH3) domain in class II myosins, a distinct beta-barrel structure, remains unknown. Here, we provide evidence, using electron cryomicroscopy, in conjunction with light-scattering, fluorescence and kinetic analyses, that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues followed by a repeating sequence of seven Pro/Ala residues. It is widely believed that the highly charged region interacts with actin, while the Pro/Ala-rich sequence forms a rigid tether that bridges the approximately 9 nm distance between the myosin lever arm and the thin filament. In order to localize the N terminus of ELC in the actomyosin complex, an engineered Cys was reacted with undecagold-maleimide, and the labeled ELC was exchanged into myosin subfragment-1 (S1). Electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, we suggest that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.     

Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

Single exposure gamma-irradiation amplifies xanthine oxidase activity and induces endothelial dysfunction in rat aorta

Irradiation of the heart and vasculature can cause a spectrum of cardiovascular complications, including increased risk of myocardial infarction or coronary heart disease. Although irradiation is implicated in oxidant stress and chronic inflammation, the underlying molecular mechanisms have not been elucidated. We tested the hypothesis that irradiation-initiated upregulation of xanthine oxidase (XO), a primary source of cardiovascular reactive oxygen species, contributes to endothelial dysfunction and increased vascular stiffness. Twenty-two, 3-month-old Sprague–Dawley male rats were gamma-irradiated at the following doses: 0, 50, 160, and 500 cGy. Rats exposed to 500 cGy showed a significant increase in endothelial XO expression and a twofold increase in XO activity, compared to the 0 cGy controls. Endothelial function was investigated ex vivo through vascular tension dose–responses to the endothelial dependent vasodilator, acetylcholine. Endothelial-dependent relaxation in aorta of the 500 cGy exposed rats was significantly attenuated from the control group. Remarkably, specific inhibition of XO with oxypurinol restored the relaxation response to that of the control. Furthermore, these ex vivo results are reflected in vivo through alterations in vascular stiffness, as measured by pulse wave velocity (PWV). As early as 1-day post-exposure, rats exhibited a significant increase in PWV from pre-exposure. The PWV of irradiated rats (50, 160, and 500 cGy) were greater than those of 0 cGy control rats at 1 day, 1 and 2 weeks. The sham and irradiated rats possessed equivalent pre-exposure PWV, with sham showing no change over 2 weeks. Thus, these findings suggest that early upregulation of XO contributes to oxidative stress and endothelial nitro-redox imbalance with resultant endothelial dysfunction and altered vascular mechanics. Furthermore, these data identify XO as a potential molecular target for attenuating irradiation-induced cardiovascular injury.     

Development of injectable thermogelling chitosan-inorganic phosphate solutions for biomedical applications

Thermosetting polymers are attractive candidates for biomedical applications as noninvasive therapeutic delivery vehicles. In the present study, the feasibility of developing a neutral physiological temperature setting injectable formulation based on chitosan and an inorganic phosphate salt have been demonstrated. The in situ gelling system was developed by adding different concentrations of ammonium hydrogen phosphate (AHP) to chitosan solution. The resulting solutions have pH in the range of approximately 7-7.2. The gelling time of the chitosan-AHP solution was determined by incubating the solutions at 37 degrees C. Depending on the concentrations of AHP added, the gelling time varied from 5 min to 30 h at 37 degrees C. Addition of various diluents to chitosan-AHP solution did not significantly change the gelling time of the solutions. The gels were found to be cytocompatible as evidenced from in vitro cytocompatibility evaluation using MC3T3-E1 mouse osteoblast like cells. The feasibility of using the gels as a stem cell carrier vehicle as well as a macromolecular delivery vehicle has been demonstrated.