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ABSTRACT This work studied the amplification, cloning and determination of the GmDREB2 gene from the soybean cultivar DT2008 and five Vietnamese local soybean cultivars (DT26, DT51, DVN5, CB, CBD) and designed the vector carrying the structure containing GmDREB2 gene from cultivar DT2008 (best drought tolerant). The coding region of GmDREB2 gene isolated from six soybean cultivars was 480 nucleotides in length, encoding 159 amino acids. The recombinant structure was designed as 35S- GmDREB2- c-myc and its expression was analysed in transgenic tobacco plants. Recombinant DREB2 protein was expressed in five transgenic tobacco lines with molecular weights close to 20 kDa. During the drought conditions, the proline accumulation of the transgenic tobacco lines was higher than on wild-type (WT) plants, with the rate from 211.17 to 332.44% after five days of drought stress, and from 262.79 to 466.04% after nine days of drought stress. The two lines, TG2 and TG12 had the highest increase rate. These results provided the basis to generate drought-tolerant soybean plants by GmDREB2 overexpression.

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ABSTRACT This work studied the acid phosphatase (APase) activity from culture medium (extracellular, eAPase) and mycelial extract (intracellular, iAPase) of Aspergillus niger F111. The influence of fungus growth and phosphate concentration of the media on the synthesis and secretion of phosphatase was demonstrated. The effects of pH, substrate concentration and inorganic and organic compounds added to the reaction mixture on APase activity were also studied. Both enzymes were repressed by high concentrations of phosphate. Overexpression of iAPase in relation to eAPase was detected; iAPase activity was 46.1 times higher than eAPase. The maximal activity of eAPase was after 24h of fungus growth and for iAPase was after 96h. Optimal pH and substrate concentrations were 4.5 and 8.0 mM, respectively. Michaelis-Menten constant (Km) for the hydrolysis of p-nitrophenyl phosphate was 0.57 mM with Vmax = 14,285.71 U mg-1 mycelium for the iAPase and 0.31 mM with V max = 147.06 U mg-1 mycelium for eAPase. Organic substances had little effect on acid phosphatases when compared with the salts. Both the APases were inhibited by 10 mM KH 2PO4 and 5 mM (NH4)2MoO4; eAPase was also inhibited by 1 mM CoCl2.

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ABSTRACT The present investigation was aimed to detect the specific polypeptide(s) appeared during the sequential stages of differentiation. Among different explants, only nodal explants showed good results for callusing. Depending on the fresh and dry weight, best callus growth was observed on MS medium supplemented with NAA (2.5 mg/L) in Dioscorea alata and 2, 4-D (2.0 mg/L) in D. deltoidea, respectively. This callus was used for the regeneration. Roots differentiation was observed on MS medium + NAA (2.0 mg/L) + IBA (0.5 mg/L) and shoots on MS medium + BAP (2.0 mg/L) + NAA (0.5 mg/L) in D. alata while in D. deltoidea, roots on RT medium + IAA (1.0 mg/L) and shoots on RT medium + BAP (1.0 mg/L) + NAA (0.5 mg/L). Continuous decrease was seen in the total soluble protein during the differentiation in D. alatawhereas in D. deltoidea, the protein content decreased upto initiation stage. Four root specific polypeptides (MW 25.56, 24.35, 19.13 and 18.2 kDa) and three shoot specific polypeptides (MW 53.7, 25.12 and 19.13 kDa) were synthesized during the differentiation in D. alata. Similarly, two root specific (MW 33.9 and 31.69 kDa) and one shoot specific (MW 16.98 kDa) polypeptide band were appeared during differentiation in D. deltoidea.

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ABSTRACT Oxytetracycline (OTC) production by Streptomyces rimosus was studied in batch and fed-batch cultures in shake flask and bioreactor levels using semi-defined medium. First, the effect of glucose concentration on OTC production and growth kinetics was studied intensively. The optimal glucose concentration in the medium was 15 g/L. Higher glucose concentrations supported higher biomass production by less volumetric and specific antibiotic production. Based on these data, cultivations were carried out at semi-industrial scale 15 L bioreactor in batch culture. At bioreactor level, cell growth and OTC production were higher compared to the shake flask culture by about 18 and 38%, respectively. During the bioreactor cultivation, glucose was totally consumed after only 48 h. Thus, the fed-batch experiment was designed for mono-glucose feeding and complete medium feeding to increase the OTC production by overcoming carbon limitations. The results showed that the fed-batch culture using constant glucose feeding strategy with rate of 0.33 g/L/h produced 1072 mg/L. On the other hand, feeding with complete medium resulted in 45% higher biomass but less OTC production by about 26% compared to mono-glucose fed culture. A further improvement in this process was achieved in by keeping the dissolved oxygen (DO) value at 60% saturation by cascading the glucose feeding pump with the DO controller. The later feeding strategy resulted in higher antibiotic production, reaching 1414 mg/L after 108 h.

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ABSTRACT An effective and improved plant regeneration system was successfully developed using shoot tip explants taken from a two years old mature plant of Cassia angustifolia. The effect of different cytokinins, [6-benzyladenine (BA), Kinetin (Kin) and thidiazuron (TDZ)] at different concentrations (0.5-10 µM) were evaluated as augmented with Murashige and Skoog (MS 1962) medium. Among all the cytokinins tested, TDZ (5.0 µM) was optimum in inducing multiple shoots as compared to BA and Kin. The rate of shoot multiplication was increased when optimal concentration (5.0 µM) of BA and Kin was tested with different concentration (0.1-1.0 µM) of Indole-3- acetic acid (IAA). Among all the combinations tested, the maximum rate of shoot multiplication was obtained on MS medium supplemented with 5.0 µM BA and 0.5 µM IAA. The number of the shoots and shoot length developed in TDZ was increased when transferred to MS medium devoid of TDZ. After every subculture, rate of the shoot multiplication and shoot length showed increment and continued even after fifth subculture without any decline rate. In vitro rooting in regenerated shoots were best obtained in half-strength MS medium supplemented with 2.0 µM indole-3- butyric acid (IBA). Plantlets with well-developed shoot and roots were successfully hardened off in earthen pots containing garden soil and grown in greenhouse with 80% survival rate.

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ABSTRACT In this study, a potential novel cellulolytic bacteriumArthrobacter sp. HPG166 was isolated from the hindgut of root-feeding larvae Holotrichia parallela. Optimization of fermentation factors for endoglucanase production byArthrobacter sp. HPG166 was carried out via response surface methodology. Sodium carboxymethylcellulose 1.19% (w/v) and beef extract 0.35% (w/v) were the ideal combination of carbon and nitrogen sources for enzyme production; the optimum temperature and pH for cellulase production were 34°C and pH 8.0 respectively. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.411 U mL-1 was obtained. The crude endoglucanase was thermotolerant as it retained 50.31% of its activity after incubation at 70°C for an hour. Metal profile of the enzyme indicated that Mg2+ and Na+ were strong stimulators while Mn2+ and Co+ drastically inhibited its activity. Due to its particular characteristics, this enzyme could have potential for industrial applications.

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ABSTRACT The aim of this study was to optimize the biosynthesis of silver nanoparticles using leaves of Aegle marmelos as the primary source. The optimal reaction medium comprised 2:1 concentration of leaf extract and 6mM concentration of silver nitrate solution (pH 7. The biosynthesized silver nanoparticles were confirmed by UV-Vis spectroscopy at 420 nm, XRD and FTIR analysis. The antimicrobial properties of silver nanoparticles were confirmed with Bacillus subtilis andPseudomonas aeruginosa.

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ABSTRACT In this study, 154 isolates capable of producing extracellular phytate-degrading activity were isolated from four soil samples from volcanic areas in Central Java, Indonesia. Six strains with high phytate-degrading activity were selected for strain identification and characterization of the corresponding phytate-degrading enzyme. Blast analysis of 16S rRNA gene sequences revealed high similarities for all the six isolates to reference sequences belonging to the genus Bacillus. Isolates MS5, MC6, D10 and D16 showed 99% sequence identity to B. cereus, while isolate MC8 exhibited 99% sequence identity to B. aryabhatti and D6 99% sequence identity to B. psychrotolerans. The crude extracellular phytase preparations from the isolates showed following optimal conditions for phytate dephosphorylation: pH 4.0 and 50°C (isolate D10), pH 5.0 and 60°C (isolate MC6, and isolate MS5), pH 6.0 and 50°C (isolate D16) and pH 6.0 and 60°C (isolate D6) and pH 6.0 and 40°C (isolate MC8). Zn2+ and Fe3+ strongly inhibited phytate dephosphorylation with all phytase preparations studied. In the presence of Ca2+, an increase in phytase activity of 10-15% was obtained.

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ABSTRACT The aim of this work was to study the co-production of nattokinase and poly (γ-glutamic acid) by Bacillus subtilis natto with soybean and rice husk under solid-state fermentation (SSF). The results showed that the size of soybean particle and rice husk significantly improved the co-production of nattokinase and poly (γ-glutamic acid), yielding 2503.4 IU/gs and 320 mg/gs, respectively in the improved culture medium composed of 16.7% soybean flour and 13.3% rice husk with 70% water content. The yields increased by approximate 7- and 2-fold factor relative to their original ones. Thus, the co-production of nattokinase and poly (γ-glutamic acid) under SSF could be considered as an efficient method to exploit agro-residues for economical production of some higher-value products.

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ABSTRACT The aim of this work was to develop and evaluate an indirect enzyme-linked immunosorbent assay (ELISA) and made a serological screening for specific antibodies to BoHV-4. Bovine serum samples were collected from different Brazilian states and evaluated for the presence of antibodies for BoHV-4 and BoHV-1. The serological results obtained showed that the indirect ELISA assay could be applied for the detection of specific antibodies for BoHV-4. The ELISA test allowed concluding that BoHV-4 is present in bovines in every Brazilian state from which serum samples were collected. The ELISA assay here standardized proved to be useful for the epidemiological studies and showed a positivity range from 1.8 to 66% in Brazil.

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ABSTRACT The aim of this study was to investigate the effect of the bark extract Bathysa cuspidata on chemically induced preneoplastic colorectal lesions in Wistar rats. Forty male rats were randomly divided into four groups (n = 10 each): saline (control group, oral administration of saline solution 0.9%); dimethylsulfoxide (DMSO, vehicle control), B200 (treated with 200 mg/kg bark extract of B. cuspidata), and B400 (treated with 400 mg/kg bark extract of B. cuspidata). Administration of treatments was carried out by the gavage. The animals received four subcutaneous injections of 1,2-dimethylhydrazine (DMH, 40 mg/kg) in the initial two weeks of the experiment to induce preneoplastic colorectal lesions. After 15 weeks, the animals were euthanized and the presence of aberrant crypt foci (ACF), body weight, biochemical analyses, and oxidative stress markers were measured. The extract of B. cuspidata decreased the levels of superoxide dismutase (SOD), but did not influence the levels of catalase (CAT), malondialdehyde (MDA), nitric oxide or protein carbonyl, compared with the saline group. The animals supplemented with a more concentrated B. cuspidata extract (B400) showed a significant reduction in the number of ACF in all the portions of the intestinal mucosa. The study demonstrated that the bark extract of B. cuspidata at 400 mg/kg reduced the preneoplastic colorectal lesions in an animal model of colon cancer and that the effect could be dose-dependent.

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ABSTRACT The aim of this work was to investigate whether the free radical scavengers, L-arginine (L-arg) and/or carnosine, either alone, or in combination would modulate tissue injury induced by hypoxia by measuring Fischer's ratio [concentrations of branched chain amino acids (BCAAs)/aromatic amino acids]. Decreased Fischer's ratios and increased malondialdehyde (MDA) led to pathogeneses of many diseases. Rats were injected with sodium nitrite (60 mg/kg) to establish hypoxia. They were treated with L-arg, (200 mg/ kg) and/or carnosine (200 mg/ kg) and their combination 24 and 1 h prior to sodium nitrite intoxication. The results revealed that hypoxia significantly decreased hemoglobin, arginine, citrulline and proline and increased sLDH, MDA , ammonia , urea, BCAAs (valine, leucine and isoleucine) and aromatic amino acids (phenylalanine and tyrosine ). The Fischer's ratio was decreased compared with the control; the administration of the aforementioned antioxidants ameliorated most of the previously altered parameters. It was concluded that Fischer's ratio was a valuable tool for understanding the pathology of hemic hypoxia, evaluating the degree of the modulatory effect of various natural antioxidants and the synergy between L-arg and carnosine in ameliorating the effect of sodium nitrite on amino acids pattern. Thus, it could be recommended to administer the combination of L-arg and carnosine in the areas of high altitudes to combat the hazard effect of hypoxia on hemoglobin concentration and MDA level.

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ABSTRACT Destabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA). Crabs collected in Narta Lagoon, Vlora (Albania) during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT) and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05), indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05) than in control group, demonstrating the presence of stress on the animals. These results showed that C. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.

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ABSTRACT The present study aimed to investigate the histological alterations in common carp gills caused by a fosetyl-Al and fenamidone based fungicide tested in laboratory conditions at 30, 38 and 50 mg/L concentration. In general, all the tested concentrations activated compensatory-adaptive mechanisms, which caused pathological changes in the fish gills. Results showed different histological alterations in the gill structure, which included lamellar lifting, edema, proliferation of the glandular cells and epithelium, covering the gill filament, fusion and degenerative alterations. Blood circulatory system showed vasodilatation of the secondary lamellae and aneurysms. Overall, there was enhancement of the gill histological changes, which was dose-dependent, i.e., proportional to the increasing fungicide concentrations. Thus, based on the results, it was concluded that the histological alterations in common carp gills could be applied as possible biomarkers in risk assessment and monitoring programs for pesticide contamination of aquatic ecosystems.

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ABSTRACT In this work, the genetic relationship among twelve Garcinia cambogia (Gaertn.) Desr. accessions were evaluated using Random Amplified Polymorphic DNA markers. The samples were part of the germplasm collected and maintained at NBPGR Regional station, Thrissur, India. Out of thirty RAPD primers used for screening, seven primers produced a total of 128 polymorphic markers in twelve accessions. The Polymorphic Information Content (PIC) ranged from 0.28 (OPA18) to 0.37 (OPA9) and Marker Index (MI) ranged between 3.61 (OPA12) and 5.93 (OPA3) among the primers used. Jaccard's coefficient of genetic similarity ranged between 0.07 and 0.64. The dendrogram constructed based on the similarity matrix generated from the molecular and morphological data showed the genetic relationship among the sampled accessions. Mantel matrix test showed a positive correlation (r = 0.49) between the cluster analysis of RAPD data and morphological data. The clustering pattern in the molecular dendrogram and Principle Coordinate Analysis (PCoA) showed that the genotypes were diverse, which was in congruence with the similarity index values and morphological dendrogram. High frequency of similarity values in the range of 0.11 to 0.17 suggested the existence of high genetic diversity among the accessions. The high level of genetic diversity among the studied accessions of G.cambogia was also supported by the large variation in the morphological characters observed in the flowers, leaves, fruits and seeds of these sampled accessions. This is the first report for the molecular based genetic diversity studies for these accessions.

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ABSTRACT The aim of this work was to assess the damage of DNA in human blood cell and sperm in vitro under the influence of furan. These cells were administered 0-600 μM of furan at 37 and 32oC for 30 and 60 min, respectively. A significant increase in tail DNA%, tail length and moment indicating DNA damage was observed at increasing doses when compared to the controls. The treatment with 300 and 600 μM of furan showed a maximum increase of 86.74 ± 2.43 and 93.29 ± 8.68 compared to the control tail DNA% in lymphocytes. However, only 600 μM of furan showed a maximum increase of 94.71 ± 6.24 compared to the control tail DNA% in sperm. The results suggested that furan caused DNA damage in lymphocytes at increasing doses, but appeared to have not the same effect on human sperm at the low doses. Genotoxic activity had an impact on the risk assessment of furan formed on the food for human cells. Therefore, it would be important to further investigate these properties of furan as the food mutagen.

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ABSTRACT Polyhydroxybutyrate (PHB) is a renowned biodegradable plastic that do not release any toxins or residues in the environment like petroleum based plastics. In the present study, 50 bacteria isolated from mangrove niche, Saudi Arabia, were screened for maximum PHB production. All the 50 strains showed positive for PHB production, of which one strain showed maximum of 137 mgL-1. The most PHB accumulated bacterium was selected and identified as Bacillus thuringiensis KSADL127, based on phenotypic characterization and 16S rRNA sequence analysis. Characterization of extracted PHB was carried out by FT-IR, NMR, UV spectroscopy, DSC, TGA, and LC-MS, which later confirmed the presence of intracellular accumulated polymer and substantiated as PHB.

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ABSTRACT A lipase producing bacterium was isolated from oil contaminated effluents of various industries from Sheikhupura Road, Pakistan, and, on the basis of biochemical and 16S rRNA ribotyping, was identified as Bacillus subtilis. The optimum temperature and pH for the growth of the culture were 37ºC and 7.0, respectively. B. subtilis I-4 had a lag phase of 4 h in LB medium while this phase prolonged to 6 h in oil containing medium. The optimum temperature and pH for the enzyme activity were 50ºC and 7.0, respectively. Maximum lipase activity was found in the presence of Ca ions. Olive oil and Tween 80 induced lipase gene in the bacterium while concentration of oil greater than 2% retarded the growth of the organism. In addition to lipase B. subtilis I-4 also produced alkane hydroxylase and biosurfactant which could make this bacterium potential candidate for lipase production as well as bioremediation of oil-contaminated wastewater.

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Undaria pinnatifida (U. pinnatifida) is a highly invasive species and has caused concern all over the world because it has invaded coastal environments, has the potential to displace native species, significantly alters habitat for associated fauna, and disturbs navigation. Any attempt to eradicate it would be futile, owing to the elusive, microscopic gametophyte, and because the alga thrives in sites rich in anthropic activities. Venice Lagoon is the largest Mediterranean transitional environment and the spot of the highest introduction of non-indigenous species, including U. pinnatifida, which is removed as a waste. We demonstrated that polysaccharide extracts from U. pinnatifida have an anticoagulant effect on human blood in vitro and are not cytotoxic. The results obtained by PT (normal values 70-120%) and APTT (normal values 28-40s) assays were significantly prolonged by the polysaccharide extracts of U. pinnatifida, therefore algal extracts are ideal candidates as antithrombotic agents.

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ABSTRACT In this work, the mixture of alginate and soy protein isolate used as a wall material was developed to encapsulate Enterococcus faecalis HZNU P2 (E. faecalis HZNU P2). The survival ability in the simulated gastric fluid (SGF) and bile salt solution, storage stability at different temperatures and release properties in the simulated intestinal fluid (SIF) of encapsulated cells were assessed. The results showed that encapsulation could offer sufficient protection toE. faecalis HZNU P2. The viability of encapsulated E. faecalis HZNU P2 did not decrease in SGF at pH 2.5 or 2.0 after 2 h incubation, while free cells were reduced from 11 to 9.85 log CFU/mL in SGF (pH 2.5) at the same exposure time. Only minor viability of encapsulatedE. faecalis HZNU P2 lost in 1.0 or 2.0% bile salt solution for 1 or 2 h exposure, compared with no survival of free E. faecalis HZNU P2 under the same conditions. Encapsulated E. faecalis HZNU P2 was completely released from the microspheres in SIF within 1 h. The viability of encapsulated E. faecalisHZNU P2 stored for two weeks at 4°C was fully retained. Viabilities of encapsulated E. faecalis HZNU P2, 9.6 and 9.0 Log CFU/g were obtained at 25 and 37°C after 21 days storage, respectively. However, around 1.0 log CFU/mL of free cells was reduced after two weeks storage at 4°C. Encapsulated E. faecalis HZNU P2 using soy protein isolate and alginate as wall materials could play an important role in food applications.