Protective effect of Nelumbo nucifera (Gaertn.) against H2O2-induced oxidative stress on H9c2 cardiomyocytes.

Ischemic heart disease (IHD), a severe condition of myocardium facing impediment in the supply of basic needs for cellular metabolism is caused by atherosclerosis. Though statin drugs could control the use of surgery on IHD patients, the complete rehabilitation or prophylaxis can be achieved through herbal-based medicines viz. either in the form of crude extract or pure phytocompounds. In the present study, pretreatment with leaf extract of Nelumbo nucifera Gaertn. was investigated for cardioprotective activity-in vitro by mitigating H2O2-induced oxidative stress. Analysis such as estimation of antioxidants, lipid peroxidation, and DNA fragmentation assay revealed significant protective effect of plant extract on cardiomyocytes. Reactive oxygen species detection was done by using 2',7'-dichlorofluorescein diacetate, apoptosis detection with Acridine Orange/Ethidium Bromide and nuclear damage detection by diamidino-2-phenylindole which confirmed the protective effect of N. nucifera extract. In addition, gene expression studies of apoptotic regulatory genes (Bcl2 and Cas-9) resulted in significant protection of nucifera extract pretreated and maintained cells. To conclude, in vitro cardioprotective activity of N. nucifera against H2O2 induced oxidative stress was achieved at the concentration of 50 µg/ml. Therefore, major phytocompounds present in extract could be beneficial in managing cardiac complications in the future.

Biopolymer gel matrix as acellular scaffold for enhanced dermal tissue regeneration.

Biological grafts have drawbacks such as donor scarcity, disease transmission, tissue infection, while the scaffolds of either collagen or chitosan fabrics fail to become part of the tissue at the wound site, though they favor the formation of connective tissue matrix. This study developed a novel composite consisting of the combination of atelocollagen and chitosan in order to provide a biodegradable molecular matrix in gel form as a biomimetic surface for cell attachment, to promote the wound healing in excision wounds. We found that the topical application of biopolymer composite on the wound promoted cell proliferation, migration and collagen deposition overtime. The enhanced cellular activity in the collagen-chitosan treated wound tissue was also assed by increased levels of Platelet derived growth factor (PDGF) and Nerve growth factor (NGF) associated with elevated levels of antioxidants and decreased level of lipid peroxidation. The acellular matrix-like topical application material is designed to guide the eventual re-establishment of an anatomically normal skin. The results of this study demonstrate the feasibility of multi-cell regeneration on a molecular system that mimics tissue engineering in vivo.

Osmotic shock augments ethanol stress in Saccharomyces cerevisiae MTCC 2918.

Yeast cells sense and respond to hypertonicity. Saccharomyces cerevisiae MTCC 2918 was tested for its metabolic status in 1 M NaCl by cell viability analysis, intracellular glycerol content and total antioxidant capacity. Yeast cell viability was maximum in 1 M NaCl and 24 h addition of 1 M NaCl was effective in induction of hyperosmolarity. Increased glycerol contents in cells treated with salt indicated adaptation to osmotic stress with a maximum of 240.87 ± 0.38 mg/g dry weight (DW) at 72 h. The total antioxidant status with 1 M NaCl was 9.29 ± 0.39 mM/g DW at 96 h reflecting free radical quenching to overcome stress with increasing growth period. Considering that pre-adaptation to one type of stress evoked a protective response to other stress factors, we have attempted the cross adaptation of osmotic shock to high ethanol concentrations. In effect, we observed that osmotic shock lowered the cell survival by augmentation of cell toxicity by ethanol due to stress induction during exponential phase. Glycerol accumulation to an order of 470.27 ± 0.53 mg/g DW at 48 h in 1 M NaCl and 12% ethanol indicated that both stresses culminated in membrane disruption further leading to cell burst and contributed to the stress overload.

Hepatocyte growth factor incorporated chitosan nanoparticles augment the differentiation of stem cell into hepatocytes for the recovery of liver cirrhosis in mice.

BACKGROUND: Short half-life and low levels of growth factors in the niche of injured microenvironment necessitates the exogenous and sustainable delivery of growth factors along with stem cells to augment the regeneration of injured tissues. METHODS: Here, recombinant human hepatocyte growth factor (HGF) was incorporated into chitosan nanoparticles (CNP) by ionic gelation method and studied for its morphological and physiological characteristics. Cirrhotic mice received either hematopoietic stem cells (HSC) or mesenchymal stemcells (MSC) with or without HGF incorporated chitosan nanoparticles (HGF-CNP) and saline as control. Biochemical, histological, immunostaining and gene expression assays were carried out using serum and liver tissue samples. One way analysis of variance was used for statics application RESULTS: Serum levels of selected liver protein and enzymes were significantly increased in the combination of MSC and HGF-CNP (MSC+HGF-CNP) treated group. Immunopositive staining for albumin (Alb) and cytokeratin 18 (CK18), and reverse transcription-polymerase chain reaction (RT-PCR) for Alb, alpha fetoprotein (AFP), CK18, cytokeratin 19 (CK19) ascertained that MSC-HGF-CNP treatment could be an effective combination to repopulate liver parenchymal cells in the liver cirrhosis. Zymogram and western blotting for matrix metalloproteinases 2 and 9 (MMP2 and MMP9) revealed that MMP2 actively involved in the fibrolysis of cirrhotic tissue. Immunostaining for alpha smooth muscle actin (αSMA) and type I collagen showed decreased expression in the MSC+HGF-CNP treatment. These results indicated that HGF-CNP enhanced the differentiation of stem cells into hepatocytes and supported the reversal of fibrolysis of extracellular matrix (ECM). CONCLUSION: Bone marrow stem cells were isolated, characterized and transplanted in mice model. Biodegradable biopolymeric nanoparticles were prepared with the pleotrophic protein molecule and it worked well for the differentiation of stem cells, especially mesenchymal phenotypic cells. Transplantation of bone marrow MSC in combination with HGF-CNP could be an ideal approach for the treatment of liver cirrhosis.

Bacterial cellulose matrix with in situ impregnation of silver nanoparticles via catecholic redox chemistry for third degree burn wound healing.

In the present study, bacterial cellulose (BC) based nanocomposite dressing material was developed for third burn wound management by polydopamine (PD) coated BC with in situ reduction of silver nanoparticles (BC-PDAg). BC-PDAg nanocomposite was characterized to understand the morphological, physical and chemical properties. Antimicrobial activity of BC-PDAg against burn wound specific pathogens were significant. The in vitro cytotoxicity and proliferation studies revealed that BC-PDAg nanocomposite is biocompatible and it supports cell proliferation. Further, in vivo experiments on female albino Wistar rats confirmed that BC-PDAg was effective in wound healing by promoting re-epithelization, and collagen deposition as evidenced by histopathological analysis. Moreover, molecular gene expression study has revealed that BC-PDAg promotes healing process by regulating the expression of inflammatory, angiogenesis and growth factor genes. The overall performance of BC-PDAg nanocomposite suggests that it could be used as promising skin regenerative tool in modern medicine.

Thymol enriched bacterial cellulose hydrogel as effective material for third degree burn wound repair.

Bacterial cellulose is well known for its excellent contributions in biomedical applications due to its superior properties. However the lack of antimicrobial property restricts its use in wound healing. To address the complications in third degree burns, thymol enriched bacterial cellulose hydrogel (BCT) was developed in this study. The incorporation of thymol into bacterial cellulose along with its chemical and thermal changes were investigated by FTIR, TGA and DSC respectively. Antimicrobial studies revealed that BCT possess excellent biocidal activity against burn specific pathogens. The in vitro biocompatibility studies were carried out in mouse 3T3 fibroblast cells. The BCT hydrogel facilitated the growth of fibroblast cells, exhibiting low toxicity, and increased cell viability. The burn wound healing efficiency of the BCT hydrogel was examined in vivo using female albino Wistar rats. Histopathological studies reveal that the wound treated with BCT hydrogel showed faster wound closure than BC and control groups. All these findings, suggest that BCT hydrogel can be used as resourceful and natural burn wound dressing material.

Novel fibrous collagen-based cream accelerates fibroblast growth for wound healing applications: in vitro and in vivo evaluation.

The present study reports the development of a novel film-forming bovine collagenous cream (BCC) based on bovine collagen (BC). In this study, collagen was isolated from bovine forestomach tissue, a novel source, and a cream formulation was prepared using some other bioactive ingredients. The electrophoretic pattern of the BC was found to be similar to type I collagen. The purity of BC was examined by amino acid analysis, which confirmed the presence of atelocollagen. The physicochemical properties of BCC such as rheology, spreadability, and temperature stability were characterized. The antimicrobial activity was examined against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, and BCC displayed excellent inhibitory effect. In vitro biocompatibility studies using NIH 3T3 fibroblast cells showed enhanced cell viability. FACS analysis revealed the non-toxic nature of BCC toward cells. The cell morphology and proliferation on the BCC matrix was studied using SEM and fluorescence microscopy. The in vivo wound healing efficacy of the BCC as a topical wound dressing was demonstrated on full thickness excision wounds in rat models. The healing profile showed that the BCC significantly enhanced re-epithelialization, collagen deposition, and contraction in the wound healing process. The findings of this study provide a new opportunity for the utilization of the untapped byproducts of the meat industry for valorization. We expect that this kind of topical healing cream could be a potential candidate in wound management and future clinical needs.

Cleaner processing: a sulphide-free approach for depilation of skins.

The conventional unhairing process in leather making utilises large amount of lime and sodium sulphide which is hazardous and poses serious waste disposal concerns. Under acidic conditions, sodium sulphide liberates significant quantities of hydrogen sulphide which causes frequent fatal accidents. Further, the conventional unhairing process involves destruction of the hair leading to increased levels of biological oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS) and total suspended solids (TSS) in the effluent. A safe approach is needed to overcome such environmental and health problems through an eco-benign process. The present study deals with a clean technology in which the keratinous body is detached from the dermis using enzymes produced from Bacillus crolab MTCC 5468 by solid state fermentation (SSF) as an alternative to noxious chemicals. Complete unhairing of skin could be achieved with an enzyme concentration of 1.2 % (w/w). The bio-chemical parameters of the spent liquor of the enzymatic process were environmentally favourable when compared with conventional method. The study indicates that the enzymatic unhairing is a safe process which could be used effectively in leather processing to alleviate pollution and health problems.

Cr-induced cellular injury and necrosis in Glycine max L.: Biochemical mechanism of oxidative damage in chloroplast.

Chromium-induced toxicity and mechanisms of cell death involved in plants are yet to be fully elucidated. To understand the events of these processes, the stress response of the soybean plant using trivalent and hexavalent chromium compounds, namely, basic chromium sulphate (BCS) and potassium dichromate (K2Cr2O7) was investigated. The leaf surface morphology for stomatal aperture, wax deposition and presence of trichomes for chromium accumulation was examined by SEM-EDAX and light microscopy. The leaf mesophyll cell integrity was identified by trypan blue staining; chlorophyll autofluorescence, ROS generation and mitochondrial function were studied by fluorescence microscopy using different dyes. Isolated chloroplasts were analysed for micronutrients and total chromium content by AAS. Elevated Cr level and decreased Fe, Cu and Zn content in chloroplast revealed the active transportation of highly soluble Cr6+ species resulting in poor absorption of micronutrients. Cr accumulation as Cr(V) in chloroplast was noticed at g = 1.98 of electron paramagnetic resonance signal. Plants grown in Cr(VI) amended soil showed chemical modification of biological macromolecules in the chloroplast as observed from fourier transform infra-red (FTIR) spectra; the chloroplast DNA damage was confirmed by DAPI staining. Cr(VI)-treated plants showed significant reduction in the levels of various biochemical parameters. The results altogether clearly indicate that Cr(VI)-induced reactive oxygen species (ROS) production leads to oxidative stress-associated changes in the organelles, particularly in chloroplast, resulting in cell death.

In vitro secretion of zymogens by bovine pancreatic acini and ultra-structural analysis of exocytosis.

The aim of this study is to establish a bovine pancreatic acinar cell culture model with longer viability and functionality. The cells could be maintained in a functional state for upto 20 days with normal morphology. Cells were positive for amylase as observed by immunofluorescence staining. Acinar cells are spherical and range about 2-3 µm in diameter. The porosome formed by exocytosis and heterogenous enzyme granules of size ranging 100-300 nm were seen on the surface of cells by electron microscopy. The activity of the enzymes was high on day 15 and the activity profile of the enzymes is in the order: protease>lipase>amylase and the enzymes were identified by SDS-PAGE. Long-term culture of bovine pancreatic acini could be useful in studying the pathogenesis of pancreatitis. Since the bovine genome shares about 80% identity with the human genome, the cells derived from bovine pancreas can be engineered and used as a potential xenotransplant to treat conditions like pancreatitis as the tissue source is abundantly available.

The effect of nerve growth factor on the early responses during the process of wound healing.

In this study, we investigated the role of nerve growth factor (NGF)-incorporated collagen on wound healing in rats. Full-thickness excision wounds were made on the back of female rats weighing about 150-160 g. Topical application of NGF-incorporated collagen, at a concentration of 1 microg/1.2 mg collagen/cm(2), once a day, for 10 days resulted in complete healing of wounds on the 15th day. The concentrations of collagen, hexosamine and uronic acid in the granulation tissue were determined. The NGF-incorporated collagen-treated rats required shorter duration for the healing with an increased rate of wound contraction. Histological and electron microscopical evaluations were also performed, which reveal the activation of fibroblasts and endoplasmic reticulum and therefore increased level of collagen synthesis due to NGF application. These results clearly indicate that the topical application of NGF-incorporated collagen enhanced the rate of healing of excision wounds.

Inducible chromate reductase exhibiting extracellular activity in Bacillus methylotrophicus for chromium bioremediation.

Chromium toxicity is one of the major causes of environmental pollution due to its heavy discharge in industrial wastewaters. Chromate reduction is a viable method to detoxify hexavalent chromium to nontoxic trivalent species mediated by enzymes and metabolites. A new Bacillus methylotrophicus strain was isolated from tannery sludge and was an efficient candidate for chromate reduction. An initial chromate reductase activity of 212.84 U/mg protein was obtained at 48 h in a low-cost defined medium formulation with 0.25 mM chromate. The extracellular enzyme was inducible at 12h substrate addition with 312.99 U/mg at 48 h. Reduced glutathione was required for enhanced specific activity of 356.48 U/mg. Enzyme activity was optimum at pH 7.0 and at 30 °C, and was stable in the presence of EDTA, DTT and metal ions. The enzyme exhibited a Vmax of 59.89 µM/min/mg protein and a Km of 86.5 µM, suggesting feasibility of the reaction with K2Cr2O7 as substrate. Application of the crude reductase in tannery effluent resulted in 91.3% chromate reduction at 48 h. An enzyme-mediated chromate reduction process has therefore been developed for bioremediation of toxic chromium sp. in industrial effluents.

Facile production of ZnS quantum dot nanoparticles by Saccharomyces cerevisiae MTCC 2918.

Microbial synthesis of nanoparticles is a green route towards ecofriendly measures to overcome the toxicity and non-applicability of nanomaterials in clinical uses obtained by conventional physical and chemical approaches. Nanoparticles in the quantum regime have remarkable characteristics with excellent applicability in bioimaging. Yeasts have been commercially exploited for several industrial applications. ZnS nanoparticles as semiconductor quantum dots have mostly been synthesized by bacterial species. Here in, we have attempted to produce ZnS nanoparticles in quantum regime by Saccharomyces cerevisiae MTCC 2918 fungus and characterize its size and spectroscopic properties. Intracellular ZnS nanoparticles were produced by a facile procedure and freeze thaw extraction using 1mM zinc sulfate. The ZnS nanoparticles showed surface plasmon resonance band at 302.57nm. The ZnS nanoparticles were in low yield and in the size range of 30-40nm. Powder XRD analysis revealed that the nanoparticles were in the sphalerite phase. Photoluminescence spectra excited at 280nm and 325nm revealed quantum confinement effects. This suggests that yeasts have inherent sulfate metabolizing systems and are capable fungal sources to assimilate sulfate. Further insights are required to identify the transport/reducing processes that may have caused the synthesis of ZnS nanoparticles such as an oxidoreductase enzyme-mediated mechanism.

Preparation and characterization of aloe vera blended collagen-chitosan composite scaffold for tissue engineering applications.

Collagen-Chitosan (COL-CS) scaffolds supplemented with different concentrations (0.1-0.5%) of aloe vera (AV) were prepared and tested in vitro for their possible application in tissue engineering. After studying the microstructure and mechanical properties of all the composite preparations, a 0.2% AV blended COL-CS scaffold was chosen for further studies. Scaffolds were examined by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and thermogravimetry analysis (TGA) to understand the intermolecular interactions and their influence on the thermal property of the complex composite. Swelling property in phosphate buffered saline (pH 7.4) and in vitro biodegradability by collagenase digestion method were monitored to assess the stability of the scaffold in a physiological medium in a hydrated condition, and to assay its resistance against enzymatic forces. The scanning electron microscope (SEM) image of the scaffold samples showed porous architecture with gradual change in their morphology and reduced tensile properties with increasing aloe vera concentration. The FTIR spectrum revealed the overlap of the AV absorption peak with the absorption peak of COL-CS. The inclusion of AV to COL-CS increased the thermal stability as well as hydrophilicity of the scaffolds. Cell culture studies on the scaffold showed enhanced growth and proliferation of fibroblasts (3T3L1) without exhibiting any toxicity. Also, normal cell morphology and proliferation were observed by fluorescence microscopy and SEM. The rate of cell growth in the presence/absence of aloe vera in the scaffolds was in the order: COL-CS-AV > COL-CS > TCP (tissue culture polystyrene plate). These results suggested that the aloe vera gel-blended COL-CS scaffolds could be a promising candidate for tissue engineering applications.

Phytosynthesis of silver nanoparticles using Coccinia grandis leaf extract and its application in the photocatalytic degradation.

Silver nanoparticles were successfully synthesized from aqueous AgNO(3) through a simple green route using the leaf extract of Coccinia grandis as a reducing as well as capping agent. The results obtained from UV-vis spectrum, X-ray diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and Fourier-transform infra red spectroscopy (FTIR) and high-resolution transmission electron microscopy (HRTEM) revealed that the biosynthesis of silver nanoparticles were in the size range of 20-30 nm and is crystallized in face centered cubic symmetry. Further, the thermal stability of nanoparticles was studied using thermo gravimetric analyzer (TGA) and differential scanning calorimeter (DSC). Photocatalytic property of the Ag nanoparticles was investigated by degradation of Coomassie Brilliant Blue G-250 under UV light. The results show that Ag nanoparticles have suitable activity for the degradation of Coomassie Brilliant Blue G-250.

In vitro anti inflammatory activity of Aloe vera by down regulation of MMP-9 in peripheral blood mononuclear cells.

AIM OF THE STUDY: The anti-inflammatory activity of Aloe vera was investigated through MMP inhibition studies. The effect of Aloe vera on MMP-9 inhibition was tested on peripheral blood mononuclear cells (PBMC). MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from the heparinised venous blood by Ficoll Diatrizoate gradient centrifugation. The cell count and viability was determined using dye exclusion technique. Cytotoxicity was evaluated by MTT assay. Activation of MMP-9 was visualized by gelatin zymography. Inhibition of MMP-9 in the presence of aqueous extract of Aloe vera was detected by gelatin zymography and confirmed by RT-PCR. RESULTS: Peripheral blood mononuclear cells (PBMC) showed significant inhibition in the activity of MMP-9, indicating the in vitro inhibitory effect of Aloe vera on MMP-9. Zymographic analysis and RT-PCR showed that it caused a significant reduction in the production of MMP-9 in a concentration dependent manner. CONCLUSION: The inhibition of MMP-9 production in the cells was detected by gelatin zymography and was confirmed by RT-PCR.

Chitosan nanoparticles as a dual growth factor delivery system for tissue engineering applications.

Growth factors are essential in cellular signaling for migration, proliferation, differentiation and maturation. Sustainable delivery of therapeutic as well as functional proteins is largely required in the pharmacological and regenerative medicine. Here we have prepared chitosan nanoparticles (CNP) and incorporated growth factors such as epidermal growth factor (EGF) and fibroblast growth factor (FGF), either individually or in combination, which could ultimately be impregnated into engineered tissue construct. CNP was characterized by Fourier transform infrared (FTIR) spectroscopy, Zeta sizer and high resolution transmission electron microscope (HRTEM). The particles were in the size range of 50-100 nm with round and flat shape. The release kinetics of both EGF and FGF incorporated CNP showed the release of growth factors in a sustained manner. Growth factors incorporated nanoparticles did not show any toxicity against fibroblasts up to 4 mg/ml culture medium. Increased proliferation of fibroblasts in vitro evidenced the delivery of growth factors from CNP for cellular signaling. Western blotting results also revealed the poor inflammatory response showing less expression of proinflammatory cytokines such as IL-6 and TNFα in the macrophage cell line J774 A-1.

Differential anti-inflammatory and anti-fibrotic activity of transplanted mesenchymal vs. hematopoietic stem cells in carbon tetrachloride-induced liver injury in mice.

Bone marrow stem cells nullify acquired and non-acquired diseases of liver through multiple strategies including antiinflammation. However, little is known about the in vivo mechanism of immunomodulation by stem cells in mediating liver cirrhosis. Mesenchymal stem cells (MSC) or hematopoietic stem cells (HSC) isolated from bone marrow of male mice were transplanted into female mice with acute liver inflammation. Serum levels of liver proteins and aminotransferase as well as hepatic antioxidant enzymes were estimated. Immunostaining for the expression of tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), alpha smooth muscle actin (alpha-SMA) and type I collagen proteins was carried out and the expression of these mRNAs was also studied. After post-transplantation, the levels of serum albumin and aminotransferases became normal and the levels of antioxidants were significantly high in the MSC treated mice compared to HSC and control mice. Necrotic cells and invasion of neutrophils were not observed in histological sections of liver of MSC treated mice. Immunostaining showed that IL-6 and TNF-alpha were not expressed in the MSC treated mice when compared to the control and HSC treated mice. alpha-SMA representing activated myofibroblasts and type I collagen were not expressed in MSC treated group. These inflammatory and fibrogenic results were further confirmed by reverse transcription-polymerase chain reaction (RT-PCR). The acute inflammation ended with the formation of fibrosis in the HSC and control groups by the uncontrolled immunoreactions. Protection mechanism of MSC therapy against injury and fibrosis in the liver occurs by the suppression of inflammation. Our findings suggest that bone marrow MSC are capable of alleviating the immunoreactions leading to the fibrosis in the liver.