3-D macro/microporous-nanofibrous bacterial cellulose scaffolds seeded with BMP-2 preconditioned mesenchymal stem cells exhibit remarkable potential for bone tissue engineering.

Bone repair using BMP-2 is a promising therapeutic approach in clinical practices, however, high dosages required to be effective pose issues of cost and safety. The present study explores the potential of low dose BMP-2 treatment via tissue engineering approach, which amalgamates 3-D macro/microporous-nanofibrous bacterial cellulose (mNBC) scaffolds and low dose BMP-2 primed murine mesenchymal stem cells (C3H10T1/2 cells). Initial studies on cell-scaffold interaction using unprimed C3H10T1/2 cells confirmed that scaffolds provided a propitious environment for cell adhesion, growth, and infiltration, owing to its ECM-mimicking nano-micro-macro architecture. Osteogenic studies were conducted by preconditioning the cells with 50 ng/mL BMP-2 for 15 min, followed by culturing on mNBC scaffolds for up to three weeks. The results showed an early onset and significantly enhanced bone matrix secretion and maturation in the scaffolds seeded with BMP-2 primed cells compared to the unprimed ones. Moreover, mNBC scaffolds alone were able to facilitate the mineralization of cells to some extent. These findings suggest that, with the aid of 'osteoinduction' from low dose BMP-2 priming of stem cells and 'osteoconduction' from nano-macro/micro topography of mNBC scaffolds, a cost-effective bone tissue engineering strategy can be designed for quick and excellent in vivo osseointegration.

Biotransformation of sweet lime pulp waste into high-quality nanocellulose with an excellent productivity using Komagataeibacter europaeus SGP37 under static intermittent fed-batch cultivation.

Herein, sweet lime pulp waste (SLPW) was utilized as a low- or no-cost feedstock for the production of bacterial nanocellulose (BNC) alone and in amalgamation with other nutritional supplements by the isolate K. europaeus SGP37 under static batch and static intermittent fed-batch cultivation. The highest yield (26.2±1.50gL-1) was obtained in the hot water extract of SLPW supplemented with the components of HS medium, which got further boosted to 38±0.85gL-1 as the cultivation strategy was shifted from static batch to static intermittent fed-batch. BNC obtained from various SLPW medium was similar or even superior to that obtained with standard HS medium in terms of its physicochemical properties. The production yields of BNC thus obtained are significantly higher and fit well in terms of industrial scale production.

Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.

Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded for a predicted protein of 42.3 kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.

Aspergillus niger PA2: a novel strain for extracellular biotransformation of L-tyrosine into L-DOPA.

L-DOPA (3,4-dihydroxyphenyl-L-alanine), an amino acid derivative is the most widely used drug of choice for the treatment of Parkinson's disease and other neurologic injuries. The present study deals with the elevated biochemical transformation of L-tyrosine to L-DOPA by Aspergillus niger PA2, a potent tyrosinase producer, isolated from decomposed food wastes. This appears to be the first report on A. niger as a notable extracellular tyrosinase producer. The extracellular tyrosinase activity produced remarkably higher levels of L-DOPA, i.e. 2.44 mg mL(-1) when the media was supplemented with 5 mg mL(-1) L-tyrosine. The optimum pH for tyrosinase production was 6.0, with the maximal L-DOPA production at the same pH. The product thus produced was analyzed by thin-layer chromatography, UV spectroscopy, high-performance liquid chromatography and Fourier transform infrared spectroscopy, that had denoted this to be L-DOPA. Kinetic parameters viz. Y p/s, Q s and Q p had further indicated the notable levels of production. Thus, Aspergillus niger PA2 could be a promising resource and may be further exploited for large-scale production of L-DOPA.

In vitro initiation of the acrosome reaction in the emu (Dromaius novaehollandiae).

1. An assessment of the efficiency of the acrosome reaction (AR) provides an important predictor of the fertilizing potential of semen and for diagnosis of the causes of infertility. A standardized protocol was therefore developed for initiation of the acrosome reaction in emu spermatozoa in vitro, and the role of CaCl2 or perivitelline membrane (PVM) proteins in determining the outcome of the reaction was investigated. 2. The acrosome reaction (assessed by FITC-PNA) was successfully induced in live spermatozoa by incubation for 2 min in NaCl-TES medium supplemented with 5 mM CaCl2. The maximum response was 32% live acrosome-reacted spermatozoa (LAR) achieved after 10 min incubation. 3. Compared to the outcome with 5 mM CaCl2 or PVM protein alone, the response was significantly better with a combination of PVM protein and CaCl2. 4. A significant variation in the percentage of LAR spermatozoa among individual males was observed. No treatment affected the percentage of dead acrosome-reacted spermatozoa. 5. The results emphasize the important role played by both PVM proteins and Ca(2+) in the in vitro initiation of the acrosome reaction.

Controlling bulk conductivity in topological insulators: key role of anti-site defects.

Intrinsic topological insulators are realized by alloying Bi(2)Te(3) with Bi(2)Se(3). Angle-resolved photoemission and bulk transport measurements reveal that the Fermi level is readily tuned into the bulk bandgap. First-principles calculations of the native defect landscape highlight the key role of anti-site defects for achieving this, and predict optimal growth conditions to realize maximally resistive topological insulators.

Spray drying of Tinospora cordifolia leaf and stem extract and evaluation of antioxidant activity.

Tinospora cordifolia (Guduchi) is widely used in folk medicine/ ayurvedic system of medicine, also in ayurvedic 'Rasayanas' to improve the immune system and used as general tonic, anti-periodic, anti-spasmodic, anti-inflammatory, anti-arthritic and anti-diabetic agent. Numerous studies have been reported on the health benefits of individual parts or whole Guduchi plant. However, most of the work has focused on the extracts of T. cordifolia. In this study, T. cordifolia leaf and stem extract powders were prepared using spray drying at 90 °C outlet temperature of the spray dryer. The powder morphology has also been studied by scanning electron microscopy. The antioxidant activity was followed by DPPH method. The leaf extract powder showed higher retention of antioxidant activity than stem extract powder.

Effect of Dietary L-ascorbic Acid (L-AA) on Production Performance, Egg Quality Traits and Fertility in Japanese Quail (Coturnix japonica) at Low Ambient Temperature.

Environmental stress boosts the levels of stress hormones and accelerates energy expenditure which subsequently imbalance the body's homeostasis. L-ascorbic acid (L-AA) has been recognized to mitigate the negative impact of environmental stress on production performances in birds. The present investigation was carried out to elucidate the effect of different dietary levels of L-AA on production performance, egg quality traits and fertility in Japanese quail at low ambient temperature. Sixty matured females (15 wks) were equally divided into three groups (20/group) based on the different dietary levels of L-AA (0, 250 and 500 ppm) and coupled with an equal number of males (1:1) obtained from the same hatch. They were managed in uniform husbandry conditions without restriction of feed and water at 14 h photo-schedule. Except for feed efficiency, body weight change, feed consumption and hen-day egg production were recorded highest in 500 ppm L-AA supplemented groups. Among the all egg quality traits studied, only specific gravity, shell weight and thickness differed significantly (p<0.05) in the present study. Fertility was improved significantly (p<0.01) to a dose dependent manner of L-AA. The findings of the present study concluded that dietary L-AA can be a caring management practice at least in part to alleviate the adverse effect of cold induced stress on production performance in Japanese quail.

Palm fatty acid biodiesel: process optimization and study of reaction kinetics.

The relatively high cost of refined oils render the resulting fuels unable to compete with petroleum derived fuel. In this study, biodiesel is prepared from palm fatty acid (PFA) which is a by-product of palm oil refinery. The process conditions were optimized for production of palm fatty acid methyl esters. A maximum conversion of 94.4% was obtained using two step trans-esterification with 1:10 molar ratio of oil to methanol at 65°C. Sulfuric acid and Sodium hydroxide were used as acid and base catalyst respectively. The composition of fatty acid methyl esters (FAME) obtained was similar to that of palm oil. The biodiesel produced met the established specifications of biodiesel of American Society for Testing and Materials (ASTM). The kinetics of the trans-esterification reaction was also studied and the data reveals that the reaction is of first order in fatty acid and methanol (MeOH) and over all the reaction is of second order.

Deacidification of high free fatty acid-containing rice bran oil by non-conventional reesterification process.

Rice bran (Oryza sativa) oil is known for containing higher amount of free fatty acid (FFA) generated by hydrolytic enzyme. This FFA causes heavy neutral oil losses due to saponification and emulsification since it produces large amount of soap in the conventional alkali refining process. In the present study the FFA of degummed rice bran oil (RBO) was significantly reduced by reesterifying it with glycerol. The reesterification process was carried out by varying the temperature and amount of excess glycerol using 0.2% catalyst (SnCl(2)). The maximum efficiency of the process could be attained at 200 degrees C using 70% excess glycerol and the maximum reduction in acid value (from 24.3 to 3.0) of RBO was observed within 4 h. The reesterified RBO was used to prepare stand oil and varnish and their performance characteristics were compared with those of the products obtained from dehydrated castor oil (DCO).

Hemolytic activity of aqueous extract of Livistona chinensis fruits.

Livistona chinensis is used as an anticancer agent in traditional Chinese medicine. In vitro, the extracts of fruits and seeds of L. chinensis are known to possess antiangiogenic and antiproliferative activities. Here we report the presence of phenolic compounds in L. chinensis fruits which show hemolytic activity. The hemolytic activity of phenolics is limited to an acid-precipitable fraction. Further, presence of proteins and lipids abrogated the hemolytic activity indicating astringent and membrane damaging activities as mechanisms of hemolysis. In conclusion, the hemolytic activity of phenolics in L. chinensis fruits is due to astringent and membrane damaging activities.

Discussion paper: The naming of Potato virus Y strains infecting potato.

Potato virus Y (PVY) strain groups are based on host response and resistance gene interactions. The strain groups PVY(O), PVY(C) and PVY(N) are well established for the isolates infecting potato in the field. A switch in the emphasis from host response to nucleotide sequence differences in the virus genomes, detection of isolates recombining sequences of different strains, and the need to recognize isolates that cause necrotic symptoms in potato tubers have led to the assignment of new acronyms, especially to isolates of the PVY(N) strain group. This discussion paper proposes that any newly found isolates should be described within the context of the original strain groups based on the original methods of distinguishing strains (i.e., tobacco and potato assays involving use of 'differential' potato cultivars). Additionally, sequence characterization of the complete genomes of isolates is highly recommended. However, it is acceptable to amend the names of PVY isolates with additional, specific codes to show that the isolate differs at the molecular, serological or phenotypic level from the typical strains within a strain group. The new isolates should preferably not be named using geographical, cultivar, or place-association designations. Since many new variants of PVY are being discovered, any new static classification system will be meaningless for the time being. A more systematic investigation and characterization of PVY from potato at the biological and molecular levels should eventually result in a biologically meaningful genetic strain concept.

Effects of sewage sludge amendment on heavy metal accumulation and consequent responses of Beta vulgaris plants.

Use of sewage sludge, a biological residue produced from sewage treatment processes in agriculture is an alternative disposal technique of waste. To study the usefulness of sewage sludge amendment for palak (Beta vulgaris var. Allgreen H-1), a leafy vegetable and consequent heavy metal contamination, a pot experiment was conducted by mixing sewage sludge at 20% and 40% (w/w) amendment ratios to the agricultural soil. Soil pH decreased whereas electrical conductance, organic carbon, total N, available P and exchangeable Na, K and Ca increased in soil amended with sewage sludge in comparison to unamended soil. Sewage sludge amendment led to significant increase in Pb, Cr, Cd, Cu, Zn and Ni concentrations of soil. Cd concentration in soil was found above the Indian permissible limit in soil at both the amendment ratios. The increased concentration of heavy metals in soil due to sewage sludge amendment led to increases in heavy metal uptake and shoot and root concentrations of Ni, Cd, Cu, Cr, Pb and Zn in plants as compared to those grown on unamended soil. Accumulation was more in roots than shoots for most of the heavy metals. Concentrations of Cd, Ni and Zn were more than the permissible limits of Indian standard in the edible portion of palak grown on different sewage sludge amendments ratios. Sewage sludge amendment in soil decreased root length, leaf area and root biomass of palak at both the amendment ratios, whereas shoot biomass and yield decreased significantly at 40% sludge amendment. Rate of photosynthesis, stomatal conductance and chlorophyll content decreased whereas lipid peroxidation, peroxidase activity and protein and proline contents, increased in plants grown in sewage sludge-amended soil as compared to those grown in unamended soil. The study clearly shows that increase in heavy metal concentration in foliage of plants grown in sewage sludge-amended soil caused unfavorable changes in physiological and biochemical characteristics of plants leading to reductions in morphological characteristics, biomass accumulation and yield. The study concludes that sewage sludge amendment in soil for growing palak may not be a good option due to risk of contamination of Cd, Ni and Zn and also due to lowering of yield at higher mixing ratio.

Bioconversion of grape must into modulated gluconic acid production by Aspergillus niger ORS-4.410.

AIMS: Analysis of regulators for modulated gluconic acid production under surface fermentation (SF) condition using grape must as the cheap carbohydrate source, by mutant Aspergillus niger ORS-4.410. Replacement of conventional fermentation condition by solid-state surface fermentation (SSF) for semi-continuous production of gluconic acid by pseudo-immobilization of A. niger ORS-4.410. METHODS AND RESULTS: Grape must after rectification was utilized for gluconic acid production in batch fermentation in SF and SSF processes using mutant strain of A. niger ORS-4.410. Use of rectified grape must led to the improved levels of gluconic acid production (80-85 g l(-1)) in the fermentation medium containing 0.075% (NH4)2HPO4; 0.1% KH2PO4 and 0.015% MgSO4.7H2O at an initial pH 6.6 (+/-0.1) under surface fermentation. Gluconic acid production was modulated by incorporating the 2% soybean oil, 2% starch and 1% H2O2 in fermentation medium at continuously high aeration rate (2.0 l min(-1)). Interestingly, 95.8% yield of gluconic acid was obtained when A. niger ORS-4.410 was pseudo-immobilized on cellulose fibres (bagasse) under SSF. Four consecutive fermentation cycles were achieved with a conversion rate of 0.752-0.804 g g(-1) of substrate into gluconic acid under SSF. CONCLUSIONS: Use of additives modulated the gluconic acid production under SF condition. Semi-continuous production of gluconic acid was achieved with pseudo-immobilized mycelia of A. niger ORS-4.410 having a promising yield (95.8%) under SSF condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioconversion of grape must into modulated gluconic acid production under SSF conditions can further be employed in fermentation industries by replacing the conventional carbohydrate sources and expensive, energy consuming fermentation processes.

Synthesis of biodiesel fuel from safflower oil using various reaction parameters.

Biodiesel fuel is gaining more and more importance because of the depletion and uncontrollable prices of fossil fuel resources. The use of vegetable oil and their derivatives as alternatives for diesel fuel is the best answer and as old as Diesel Engine. Chemically biodiesel fuel is the mono alkyl esters of fatty acids derived from renewable feed stocks like vegetable oils and animal fats. Safflower oil contains 75-80% of linoleic acid; the presence of this unsaturated fatty acid is useful in alleviating low temperature properties like pour point, cloud point and cold filter plugging point. In this paper we studied the effect of various parameters such as temperature, molar ratio (oil to alcohol), and concentration of catalyst on synthesis of biodiesel fuel from safflower oil. The better suitable conditions of 1:6 molar ratio (oil to alcohol), 60 degrees C temperature and catalyst concentration of 2% (by wt. of oil) were determined. The finally obtained biodiesel fuel was analyzed for fatty acid composition by GLC and some other properties such as flash point, specific gravity and acid value were also determined. From the results it was clear that the produced biodiesel fuel was with in the recommended standards of biodiesel fuel with 96.8% yield.

Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin.

Here, we assessed and compared the anticancer efficacy and associated mechanisms of silymarin and silibinin in human prostate cancer (PCA) PC3 cells; silymarin is comprised of silibinin and its other stereoisomers, including isosilybin A, isosilybin B, silydianin, silychristin and isosilychristin. Silymarin and silibinin (50-100 microg/ml) inhibited cell proliferation, induced cell death, and caused G1 and G2-M cell cycle arrest in a dose/time-dependent manner. Molecular studies showed that G1 arrest was associated with a decrease in cyclin D1, cyclin D3, cyclin E, cyclin-dependent kinase (CDK)4, CDK6 and CDK2 protein levels, and CDK2 and CDK4 kinase activity, together with an increase in CDK inhibitors (CDKIs) Kip1/p27 and Cip1/p21. Further, both agents caused cytoplasmic sequestration of cyclin D1 and CDK2, contributing to G1 arrest. The G2-M arrest by silibinin and silymarin was associated with decreased levels of cyclin B1, cyclin A, pCdc2 (Tyr15), Cdc2, and an inhibition of Cdc2 kinase activity. Both agents also decreased the levels of Cdc25B and cell division cycle 25C (Cdc25C) phosphatases with an increased phosphorylation of Cdc25C at Ser216 and its translocation from nucleus to the cytoplasm, which was accompanied by an increased binding with 14-3-3beta. Both agents also increased checkpoint kinase (Chk)2 phosphorylation at Thr68 and Ser19 sites, which is known to phosphorylate Cdc25C at Ser216 site. Chk2-specific small interfering RNA largely attenuated the silymarin and silibinin-induced G2-M arrest. An increase in the phosphorylation of histone 2AX and ataxia telangiectasia mutated was also observed. These findings indicate that silymarin and silibinin modulate G1 phase cyclins-CDKs-CDKIs for G1 arrest, and the Chk2-Cdc25C-Cdc2/cyclin B1 pathway for G2-M arrest, together with an altered subcellular localization of critical cell cycle regulators. Overall, we observed comparable effects for both silymarin and silibinin at equal concentrations by weight, suggesting that silibinin could be a major cell cycle-inhibitory component in silymarin. However, other silibinin stereoisomers present in silymarin also contribute to its efficacy, and could be of interest for future investigation.

Tinospora cordifolia induces enzymes of carcinogen/drug metabolism and antioxidant system, and inhibits lipid peroxidation in mice.

The present study is an effort to identify a potent chemopreventive agent against various diseases (including cancer) in which oxidative stress plays an important causative role. Here, we investigated the effect of a hydroalcoholic (80% ethanol: 20% distilled water) extract of aerial roots of Tinospora cordifolia (50 and 100mg/kg body wt./day for 2 weeks) on carcinogen/drug metabolizing phase-I and phase-II enzymes, antioxidant enzymes, glutathione (GSH) content, lactate dehydrogenase and lipid peroxidation in liver of 8-week-old Swiss albino mice. The modulatory effect of the extract was also examined on extrahepatic organs, i.e., lung, kidney and forestomach, for the activities of GSH S-transferase (GST), DT-diaphorase (DTD), superoxide dismutase (SOD) and catalase. Significant increases in the levels of acid-soluble sulfhydryl (-SH) and cytochrome P(450) contents, and enzyme activities of cytochrome P(450) reductase, cytochrome b(5) reductase, GST, DTD, SOD, catalase, GSH peroxidase (GPX) and GSH reductase (GR) were observed in the liver. Both treated groups showed decreased malondialdehyde (MDA) formation. In lung SOD, catalase and GST; in kidney SOD and catalase; and in forestomach SOD, DTD and GST showed significant increase at both dose levels of treatment. BHA (0.75%, w/w in diet), a pure antioxidant compound, was used as a positive control. This group showed increase in hepatic levels of GSH content, cytochrome b(5), DTD, GST, GR and catalase, whereas MDA formation was inhibited significantly. In the BHA-treated group, the lung and kidney showed increased levels of catalase, DTD and GST, whereas SOD was significantly increased in the kidney and forestomach; the latter also showed an increase in the activities of DTD and GST. The enhanced GSH level and enzyme activities involved in xenobiotic metabolism and maintaining antioxidant status of cells are suggestive of a chemopreventive efficacy of T. cordifolia against chemotoxicity, including carcinogenicity, which warrants further investigation of active principle (s) present in the extract responsible for the observed effects employing various carcinogenesis models.

Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models.

Antioxidant-rich fractions were extracted from pomegranate (Punica granatum) peels and seeds using ethyl acetate, methanol, and water. The extracts were screened for their potential as antioxidants using various in vitro models, such as beta-carotene-linoleate and 1,1-diphenyl-2-picryl hydrazyl (DPPH) model systems. The methanol extract of peels showed 83 and 81% antioxidant activity at 50 ppm using the beta-carotene-linoleate and DPPH model systems, respectively. Similarly, the methanol extract of seeds showed 22.6 and 23.2% antioxidant activity at 100 ppm using the beta-carotene-linoleate and DPPH model systems, respectively. As the methanol extract of pomegranate peel showed the highest antioxidant activity among all of the extracts, it was selected for testing of its effect on lipid peroxidation, hydroxyl radical scavenging activity, and human low-density lipoprotein (LDL) oxidation. The methanol extract showed 56, 58, and 93.7% inhibition using the thiobarbituric acid method, hydroxyl radical scavenging activity, and LDL oxidation, respectively, at 100 ppm. This is the first report on the antioxidant properties of the extracts from pomegranate peel and seeds. Owing to this property, the studies can be further extended to exploit them for their possible application for the preservation of food products as well as their use as health supplements and neutraceuticals.

Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.