HspB5 protects mouse neural stem/progenitor cells from paraquat toxicity.

INTRODUCTION: HspB5 (αB-crystallin) is known to be involved in a variety of cellular functions, including, protection of cells from oxidative damage and inhibiting apoptosis. Neural stem/progenitor cells (NSPCs) have significant therapeutic value, especially in the NSC/NPC transplantation therapy. However, the viability of the transplanted NSPCs remains low because of various factors, including oxidative stress. OBJECTIVE: The current investigation explored the possible role of HspB5 in the protection of mouse NSPCs (mNSPCs) against paraquat-induced toxicity. METHODS: The recombinant human HspB5 was expressed in E.coli and was purified using gel filtration and Ion-exchange chromatography. The biophysical characterization of HspB5 was carried out using DLS, CD, and Analytical Ultracentrifugation (SV); the chaperone activity of HspB5 was determined by alcohol dehydrogenase aggregation assay. We have subjected the mNSPCs to paraquat-induced oxidative stress and monitored the protective ability of HspB5 by MTT assay and Hoechst-PI staining. Furthermore, increase in the expression of the anti-apoptotic protein, procaspase-3 was monitored using western blotting. RESULTS: The recombinant HspB5 was purified to its homogeneity and was characterized using various biophysical techniques. The externally added FITC-labeled HspB5 was found to be localized within the cytoplasm of mNSPCs. Our Immunocytochemistry results showed that the externally added FITC-labeled HspB5 not only entered the cells but also conferred cytoprotection against paraquat-induced toxicity. The protective events were monitored by a decrease in the PI-positive cells and an increase in the procaspase-3 expression through Immunocytochemistry and Western blotting respectively. CONCLUSION: Our results clearly demonstrate that exogenously added recombinant human HspB5 enters the mNSPCs and confers protection against paraquat toxicity.

Mechanistic role of perfusion culture on bone regeneration.

Bone tissue engineering (BTE) aims to develop engineered bone tissue to substitute conventional bone grafts. To achieve this, culturing the cells on the biocompatible three-dimensional (3D) scaffold is one alternative approach. The new functional bone tissue regeneration could be feasible by the synergetic combinations of cells, biomaterials and bioreactors. Although the field of biomaterial design/development for BTE applications attained reasonable success, development of suitable bioreactor remains still a major challenge. Tissue engineering bioreactors provide the microenvironment required for neo-tissue regeneration, and also can be used to study the physio-chemical cues effect on cell proliferation and differentiation in order to produce functional tissue. In this direction, various bioreactors have been developed and evaluated for the successful development of engineered bone tissue. Continues assessment of tissue development and limitations of the bioreactors lead to the progression of perfusion flow bioreactor system. Improvements in perfusion reactor system were able to yield multiple tissue engineered constructs with uniform cell distribution, easy to operate protocols and also effectively handled for the functional tissue development to meet the adequate supply of engineered graft for clinical application.

Improved osteogenic differentiation of umbilical cord blood MSCs using custom made perfusion bioreactor.

BACKGROUND: 3D cell culture is an appropriate method to develop engineered bone tissue, where different bioreactors have been designed to mitigate the challenges in 3D culture. Currently, we tailored a perfusion reactor to witness human mesenchymal stem cells (MSCs) proliferation and differentiation over polylactic acid-polyethylene glycol (PLA/PEG) composite scaffolds. METHODS: The composite scaffolds with different weight ratios of PLA and PEG were prepared using solvent casting-particulate leaching technique. Human umbilcal card blood MSCs were cultured under dynamic and static conditions to elucidate the role of dynamic fluid flow in osteogenesis of MSCs. RESULTS: The human MSCs distribution over the scaffolds was confirmed with fluorescent microscopy. Alkaline phosphatase (ALP), calcium mineralization, and collagen formation were found to be higher in PLA90 scaffolds than PLA100 and PLA75. PLA90 scaffolds with better cell adhesion/proliferartion were considered for bioreactor studies and they exhibited enhanced ALP, Ca+2 mineralization and collagen formation under dynamic perfusion than static culture. We further confirmed our observation by looking at expression levels of osteogenic marker (Runx2 and osteonectin) in differentiated MSCs subjected to perfusion culture compared to static culture. CONCLUSION: The results of the current investigation once again proves that dynamic perfusion cultures improve the osteogenic differentiation of MSCs over hybrid polymer scaffolds (PLA90) for effective bone regeneration.

Role of signaling pathways in mesenchymal stem cell differentiation.

Mesenchymal Stem Cells (MSCs) are self-renewing cells with ability to differentiate into organized, functional network of cells. In recent past research in developing clinical applications for MSCs has increased significantly. MSCs exhibit multi potential proliferation, and are capable of differentiating into cartilage, bone, neuronal cells and adipocytes, etc. Signaling pathways, transcription factors and growth factors modulate the differentiation of MSCs into different cell lineages. Besides, physical factors may regulate the molecular differentiation of stem cells. The main theme of this paper is to review the signaling pathways related to bone morphogenetic proteins (BMPs), epidermal growth factors (EGF), transforming growth factors (TGF), wingless type MMTV integration site (wnt) proteins, fibroblastic growth factor (FGF), and transcriptional regulating factors significance in the MSCs differentiation.

Foil assisted replica molding for fabrication of microfluidic devices and their application in vitro.

We present a simple, rapid, benchtop, Foil Assisted Rapid Molding (FARM) method for the fabrication of microfluidic devices. This novel technique involves the use of aluminium foil, pen and an X-Y plotter to create semi-circular or plano-concave, shallow microchannels. It is an easy do-it-yourself (DIY) technique for creating a microfluidic device in three simple steps: (1) create a channel design using the CAD software, (2) plot the patterns on aluminium foil and (3) use the reverse of the engraved foil as a mold to create microfluidic devices. In this report, we present a detailed study of the proposed method by varying a range of parameters such as foil thickness, tip material, and tip sizes and by investigating their effect on the creation of channels with varying geometry. Furthermore, we demonstrated the cytocompatibility of these devices in vitro.

Significance of biotic factors in mesenchymal stem cell fate in regenerative medicine.

Stem and progenitor cell research is a complex and exciting field which promises curative discoveries in numerous areas including cancer, diabetes, and regenerative medicine. Use of biotic factors or growth factors has played an essential role in the development of stem cell research. These biologically active components have been administered into stem cells either to improve or maintain the stem cell proliferation, or to encourage controlled differentiation into more defined cell types. Small molecules such as 6-Bromoindirubin-3´-oxime (BIO), cardiogenol-C, etc can help stem cell research by controlling or influencing the regulatory changes in a controlled manner and to help understand the mechanisms during stem cell differentiation. Extra cellular matrix (ECM) is another significant biotic factor, which mediates cell and tissue behavior by influencing cell-matrix interactions. Thus, in this review we would like to emphasize significance of various growth factors in stem cell research.

Influence of dilute acid and alkali pretreatment on reducing sugar production from corncobs by crude enzymatic method: a comparative study.

In the present study, two commonly used catalysts in chemical pretreatment, sulfuric acid and sodium hydroxide, were tested to evaluate the effect of solid-to-liquid ratio on pretreatment and enzymatic hydrolysis. Solid to liquid ratio (S/L) was influential on sugars released with an increase in the S/L ratio between 0.03 and 0.2. Enzymatic digestibility of 0.25 M H2SO4 pretreated corncobs were released more sugars (415.12 mg/mL); whereas, corncobs pretreated with NaOH released 350.12 mg/mL of reducing sugars at S/L 0.05. Further, in comparison with NaOH pretreated corncobs, acid treated material substantially increased the accessibility and digestibility of cellulose during crude enzymatic hydrolysis (28.96 FPU) and released 398.95 mg/mL reducing sugars.

Optimization of amorphadiene production in engineered yeast by response surface methodology.

Isoprenoids are among the most diverse bioactive compounds synthesized by biological systems. The superiority of these compounds has expanded their utility from pharmaceutical to fragrances, including biofuel industries. In the present study, an engineered yeast strain Saccharomyces cerevisiae (YCF-AD1) was optimized for production of Amorpha-4, 11-diene, a precursor of anti-malarial drug using response surface methodology. The effect of four critical parameters such as KH2PO4, methionine, pH and temperature were evaluated both qualitatively and quantitatively and further optimized for enhanced amorphadiene production by using a central composite design and model validation. The "goodness of fit" of the regression equation and model fit (R2) of 0.9896 demonstrate this study to be an effective model. Further, this model will be used to validate theoretically and experimentally at the higher level of amorphadiene production with the combination of the optimized values of KH2PO4 (4.0), methionine (1.49), pH (5.4) and temperature (33 °C).

Combination of ERG9 Repression and Enzyme Fusion Technology for Improved Production of Amorphadiene in Saccharomyces cerevisiae.

The yeast strain (Saccharomyces cerevisiae) MTCC 3157 was selected for combinatorial biosynthesis of plant sesquiterpene amorpha-4,11-diene. Our main objective was to overproduce amorpha 4-11-diene, which is a key precursor molecule of artemisinin (antimalarial drug) produced naturally in plant Artemisia annua through mevalonate pathway. Farnesyl diphosphate (FPP) is a common intermediate metabolite of a variety of compounds in the mevalonate pathway of yeast and leads to the production of ergosterols, dolichol and ubiquinone, and so forth. In our studies, FPP converted to amorphadiene (AD) by expressing heterologous amorphadiene synthase (ADS) in yeast. First, ERG9 (squalane synthase) promoter of yeast was replaced with repressible methionine (MET3) promoter by using bipartite gene fusion method. Further to overcome the loss of the intermediate FPP through competitive pathways in yeast, fusion protein technology was adopted and farnesyldiphosphate synthase (FPPS) of yeast has been coupled with amorphadiene synthase (ADS) of plant origin (Artemisia annua L.) where amorphadiene production was improved by 2-fold (11.2 mg/L) and 4-fold (25.02 mg/L) in yeast strains YCF-002 and YCF-005 compared with control strain YCF-AD (5.5 mg/L), respectively.

Physical and degradation properties of PLGA scaffolds fabricated by salt fusion technique.

Tissue engineering scaffolds require a controlled pore size and interconnected pore structures to support the host tissue growth. In the present study, three dimensional (3D) hybrid scaffolds of poly lactic acid (PLA) and poly glycolic acid (PGA) were fabricated using solvent casting/particulate leaching. In this case, partially fused NaCl particles were used as porogen (200-300µ) to improve the overall porosity (≥90%) and internal texture of scaffolds. Differential scanning calorimeter (DSC) analysis of these porous scaffolds revealed a gradual reduction in glass transition temperature (Tg) (from 48°C to 42.5°C) with increase in hydrophilic PGA content. The potential applications of these scaffolds as implants were further tested for their biocompatibility and biodegradability in four simulated body fluid (SBF) types in vitro. Whereas, simulated body fluid (SBF) Type1 with the optimal amount of HCO3 (-) ions was found to be more appropriate and sensible for testing the bioactivity of scaffolds. Among three combinations of polymer scaffolds, sample B with a ratio of 75:25 of PLA: PGA showed greater stability in body fluids (pH 7.2) with an optimum degradation rate (9% to 12% approx). X-ray diffractogram also confirmed a thin layer of hydroxyapatite deposition over sample B with all SBF types in vitro.

Study on osteoblast like behavior of umbilical cord blood cells on various combinations of PLGA scaffolds prepared by salt fusion.

The osteogenic potential of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) on porous poly lactide-co-glycolide (PLGA) scaffolds have been reported to differentially support osteogenic differentiation based on polymer composition (80:20, 75:25 and 70:30 percent of PLA: PGA, respectively). Along with polymer composition; fused NaCl crystal matrix prior to solvent casting improves the porosity and pore interconnectivity in 3D scaffolds, which has significant impact on cell proliferation. FTIR and XRD studies of PLGA scaffolds also verified the intermolecular interactions, phase distribution and crystallinity in scaffolds. Among three scaffold combinations, sample B (75:25) has showed maximum porosity with optimum water uptake/retention abilities. Impact of polymer composition and porosity on cell proliferation was investigated through MTT assay, where sample B was observed to be supporting better cell proliferation,due to its internal structure. The above results were further confirmed by ALP and Col-I gene expression studies using RT-PCR. Immuno fluorescent studies also revealed the extracellular filamentous actin organization over the scaffolds, where cell adhesion and proliferation was found to be higher with increase in PGA content, which is a hydrophilic polymer.

Enhanced proliferation and osteogenic differentiation of human umbilical cord blood stem cells by L-ascorbic acid, in vitro.

The multilineage potentiality of cord blood stem cells has been experimentally proven in a number of cell based therapies. Umbilical cord blood (UCB) derived mesenchymal stem cells (MSCs), on prolonged exposure with Lascorbic acid have been successfully differentiated in to osteoblasts (bone forming cells) without altering the phenotype of the cells. In this case study, the role of L-ascorbic acid on collagen biosynthesis and mineral deposition in MSCs has been assessed, which are ultimately matured in to an insoluble extra cellular matrix (ECM), giving mechanical strength to the bone cells. Moreover, up to specific concentration of L-ascorbic acid (250µM), proliferation as well as differentiation potential of the cells remains unaltered. Further increase in concentrations of L-ascorbic acid (500 µM) reduced the cell proliferation and subsequently leads to morphological changes in the cultures. This may be due to an immature antioxidant defense system, which can be overcome by treating the cell cultures with antioxidants. Our final results conclude that Lascorbic acid has positive effect on the ostogenic differentiation of cord blood stem cells, and the concentration of ascorbic acid is vital in cell proliferation and differentiation.

Development of petri net-based dynamic model for improved production of farnesyl pyrophosphate by integrating mevalonate and methylerythritol phosphate pathways in yeast.

In this case study, we designed a farnesyl pyrophosphate (FPP) biosynthetic network using hybrid functional Petri net with extension (HFPNe) which is derived from traditional Petri net theory and allows easy modeling with graphical approach of various types of entities in the networks together. Our main objective is to improve the production of FPP in yeast, which is further converted to amorphadiene (AD), a precursor of artemisinin (antimalarial drug). Natively, mevalonate (MEV) pathway is present in yeast. Methyl erythritol phosphate pathways (MEP) are present only in higher plant plastids and eubacteria, but not present in yeast. IPP and DAMP are common isomeric intermediate in these two pathways, which immediately yields FPP. By integrating these two pathways in yeast, we augmented the FPP synthesis approximately two folds higher (431.16 U/pt) than in MEV pathway alone (259.91 U/pt) by using HFPNe technique. Further enhanced FPP levels converted to AD by amorphadiene synthase gene yielding 436.5 U/pt of AD which approximately two folds higher compared to the AD (258.5 U/pt) synthesized by MEV pathway exclusively. Simulation and validation processes performed using these models are reliable with identified biological information and data.

Cellulase production under solid-state fermentation by Trichoderma reesei RUT C30: statistical optimization of process parameters.

Sugar cane bagasse was used as substrate for cellulase production using Trichoderma reesei RUT C30, and the culture parameters were optimized for enhancing cellulase yield. The culture parameters, such as incubation temperature, duration of incubation, and inducer concentration, were optimized for enhancing cellulase yield using a Box-Behnken experimental design. The optimal level of each parameter for maximum cellulase production by the fungus was determined. Predicted results showed that cellulase production was highest (25.6 FPAase units per gram dry substrate) when the inducer concentration was 0.331 ml/gds, and the incubation temperature and time were 33 degrees C and 67 h, respectively. Crude inducer generated by cellulase action was found to be very effective in inducing cellulases. Validation of predicted results was done, and the experimental values correlated well with that of the predicted.