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International Science Index

Commenced in January 1999 Frequency: Monthly Edition: International Paper Count: 22

Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering

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    Molecular Identification of ESBL Genesbla GES-1, blaVEB-1, blaCTX-M blaOXA-1, blaOXA-4,blaOXA-10 and blaPER-1 in Pseudomonas aeruginosa Strains Isolated from Burn Patientsby PCR, RFLP and Sequencing Techniques
    Fourty one strains of ESBL producing P.aeruginosa which were previously isolated from burn patients in Kerman University general hospital, Iran were subjected to PCR, RFLP and sequencing in order to determine the type of extended spectrum β- lactamases (ESBL), the restriction digestion pattern and possibility of mutation among detected genes. DNA extraction was carried out by phenol chloroform method. PCR for detection of bla genes was performed using specific primer for each gene. Restriction Fragment Length Polymorphism (RFLP) for ESBL genes was carried out using EcoRI, NheI, PVUII, EcoRV, DdeI, and PstI restriction enzymes. The PCR products were subjected to direct sequencing of both the strands for identification of the ESBL genes.The blaCTX-M, blaVEB-1, blaPER-1, blaGES-1, blaOXA-1, blaOXA-4 and blaOXA-10 genes were detected in the (n=1) 2.43%, (n=41)100%, (n=28) 68.3%, (n=10) 24.4%, (n=29) 70.7%, (n=7)17.1% and (n=38) 92.7% of the ESBL producing isolates respectively. The RFLP analysis showed that each ESBL gene has identical pattern of digestion among the isolated strains. Sequencing of the ESBL genes confirmed the genuinety of PCR products and revealed no mutation in the restriction sites of the above genes. From results of the present investigation it can be concluded that blaVEB-1 and blaCTX-M were the most and the least frequently isolated ESBL genes among the P.aeruginosa strains isolated from burn patients. The RFLP and sequencing analysis revealed that same clone of the bla genes were indeed existed among the antibiotic resistant strains.
    Development of All-male Fingerlings by Heat Treatment and the Genetic Mechanism of Heat Induced Sex Determination in Nile Tilapia(Oreochromis niloticus L.)
    Juvenile Nile tilapia subjected to heat treatment at temperatures ranging from 260C to 370C showed positive correlation (P
    Induction of Alternative Oxidase Activity in Candida albicans by Oxidising Conditions

    Candida albicans ATCC 10231 had low endogenous activity of the alternative oxidase compared with that of C. albicans ATCC 10261. In C. albicans ATCC 10231 the endogenous activity declined as the cultures aged. Alternative oxidase activity could be induced in C. albicans ATCC 10231 by treatment with cyanide, but the induction of this activity required the presence of oxygen which could be replaced, at least in part, with high concentrations of potassium ferricyanide. We infer from this that the expression of the gene encoding the alternative oxidase is under the control of a redoxsensitive transcription factor.

    In Vitro Antibacterial and Antifungal Effects of a 30 kDa D-Galactoside-Specific Lectin from the Demosponge, Halichondria okadai
    The present study has been taken to explore the screening of in vitro antimicrobial activities of D-galactose-binding sponge lectin (HOL-30). HOL-30 was purified from the marine demosponge Halichondria okadai by affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa with a single polypeptide by SDS-PAGE under non-reducing and reducing conditions. HOL-30 agglutinated trypsinized and glutaraldehydefixed rabbit and human erythrocytes with preference for type O erythrocytes. The lectin was subjected to evaluation for inhibition of microbial growth by the disc diffusion method against eleven human pathogenic gram-positive and gram-negative bacteria. The lectin exhibited strong antibacterial activity against gram-positive bacteria, such as Bacillus megaterium and Bacillus subtilis. However, it did not affect against gram-negative bacteria such as Salmonella typhi and Escherichia coli. The largest zone of inhibition was recorded of Bacillus megaterium (12 in diameter) and Bacillus subtilis (10 mm in diameter) at a concentration of the lectin (250 μg/disc). On the other hand, the antifungal activity of the lectin was investigated against six phytopathogenic fungi based on food poisoning technique. The lectin has shown maximum inhibition (22.83%) of mycelial growth of Botrydiplodia theobromae at a concentration of 100 μg/mL media. These findings indicate that the lectin may be of importance to clinical microbiology and have therapeutic applications.
    Biological Effects of a Carbohydrate-Binding Protein from an Annelid, Perinereis nuntia Against Human and Phytopathogenic Microorganisms
    Lectins have a good scope in current clinical microbiology research. In the present study evaluated the antimicrobial activities of a D-galactose binding lectin (PnL) was purified from the annelid, Perinereis nuntia (polychaeta) by affinity chromatography. The molecular mass of the lectin was determined to be 32 kDa as a single polypeptide by SDS-PAGE under both reducing and non-reducing conditions. The hemagglutinating activity of the PnL showed against trypsinized and glutaraldehyde-fixed human erythrocytes was specifically inhibited by D-Gal, GalNAc, Galβ1-4Glc and Galα1-6Glc. PnL was evaluated for in vitro antibacterial screening studies against 11 gram-positive and gram-negative microorganisms. From the screening results, it was revealed that PnL exhibited significant antibacterial activity against gram-positive bacteria. Bacillus megaterium showed the highest growth inhibition by the lectin (250 μg/disc). However, PnL did not inhibit the growth of gram-negative bacteria such as Vibrio cholerae and Pseudomonas sp. PnL was also examined for in vitro antifungal activity against six fungal phytopathogens. PnL (100 μg/mL) inhibited the mycelial growth of Alternaria alternata (24.4%). These results indicate that future findings of lectin applications obtained from annelids may be of importance to life sciences.
    A New Method for Rapid DNA Extraction from Artemia (Branchiopoda, Crustacea)
    Artemia is one of the most conspicuous invertebrates associated with aquaculture. It can be considered as a model organism, offering numerous advantages for comprehensive and multidisciplinary studies using morphologic or molecular methods. Since DNA extraction is an important step of any molecular experiment, a new and a rapid method of DNA extraction from adult Artemia was described in this study. Besides, the efficiency of this technique was compared with two widely used alternative techniques, namely Chelex® 100 resin and SDS-chloroform methods. Data analysis revealed that the new method is the easiest and the most cost effective method among the other methods which allows a quick and efficient extraction of DNA from the adult animal.
    Biological Characterization of the New Invasive Brine Shrimp Artemia franciscana in Tunisia: Sabkhet Halk El-Menzel

    Endemic Artemia franciscana populations can be found throughout the American continent and also as an introduced specie in several country all over the world, such as in the Mediterranean region where Artemia franciscana was identified as an invasive specie replacing native Artemia parthenogenetica and Artemia salina. In the present study, the characterization of the new invasive Artemia franciscana reported from Sabkhet Halk El-Menzel (Tunisia) was done based on the cysts biometry, nauplii instar-I length, Adult sexual dimorphism and fatty acid profile. The mean value of the diameter of non-decapsulated and decapsulated cysts, chorion thickness and naupliar length is 235.8, 226.3, 4.75 and 426.8 μm, respectively. Sexual dimorphism for adults specimen showed that maximal distance between compound eyes, diameter for compound eyes, length of first antenna and the abdomen length compared to the total body length ratio, are the most important variables for males and females discrimination with a total contribution of 62.39 %. The analysis of fatty acid methyl esters profile of decapsulated cysts resulted in low levels of linolenic acid (LLA, C18:3n-3) and high levels of eicosapentaenoic acid (EPA, C20:5n-3) with 3.11 and 11.10 %, respectively. Low quantity of docosahexaenoic acid (DHA, 22:6n-3) was also observed with 0.17 mg.g-1 dry weight.

    Supercritical Fluid Extraction of Lutein Esters from Marigold Flowers and their Hydrolysis by Improved Saponification and Enzyme Biocatalysis
    Lutein is a dietary oxycarotenoid which is found to reduce the risks of Age-related Macular Degeneration (AMD). Supercritical fluid extraction of lutein esters from marigold petals was carried out and was found to be much effective than conventional solvent extraction. The saponification of pre-concentrated lutein esters to produce free lutein was studied which showed a composition of about 88% total carotenoids (UV-VIS spectrophotometry) and 90.7% lutein (HPLC). The lipase catalyzed hydrolysis of lutein esters in conventional medium was investigated. The optimal temperature, pH, enzyme concentration and water activity were found to be 50°C, 7, 15% and 0.33 respectively and the activity loss of lipase was about 25% after 8 times re-use in at 50°C for 12 days. However, the lipase catalyzed hydrolysis of lutein esters in conventional media resulted in poor conversions (16.4%).
    Biochemical Characteristics of Sorghum Flour Fermented and/or Supplemented with Chickpea Flour
    Sorghum flour was supplemented with 15 and 30% chickpea flour. Sorghum flour and the supplement were fermented at 35 oC for 0, 8, 16, and 24 h. Changes in pH, titrable acidity, total soluble solids, protein content, in vitro protein digestibility and amino acid composition were investigated during fermentation and/or after supplementation of sorghum flour with chickpea. The pH of the fermenting material decreased sharply with a concomitant increase in the titrable acidity. The total soluble solids remained unchanged with progressive fermentation time. The protein content of sorghum cultivar was found to be 9.27 and that of chickpea was 22.47%. The protein content of sorghum cultivar after supplementation with15 and 30% chickpea was significantly (P ≤ 0.05) increased to 11.78 and 14.55%, respectively. The protein digestibility also increased after fermentation from 13.35 to 30.59 and 40.56% for the supplements, respectively. Further increment in protein content and digestibility was observed when supplemented and unsupplemented samples were fermented for different periods of time. Cooking of fermented samples was found to increase the protein content slightly and decreased digestibility for both supplements. Amino acid content of fermented and fermented and cooked supplements was determined. Supplementation was found to increase the lysine and therionine content. Cooking following fermentation decreased lysine, isoleucine, valine and sulfur containg amino acids.
    Cloning of a β-Glucosidase Gene (BGL1) from Traditional Starter Yeast Saccharomycopsis fibuligera BMQ 908 and Expression in Pichia pastoris
    β-Glucosidase is an important enzyme for production of ethanol from lignocellulose. With hydrolytic activity on cellooligosaccharides, especially cellobiose, β-glucosidase removes product inhibitory effect on cellulases and forms fermentable sugars. In this study, β-glucosidase encoding gene (BGL1) from traditional starter yeast Saccharomycosis fibuligera BMQ908 was cloned and expressed in Pichia pastoris. BGL1 of S. fibuligera BMQ 908 shared 98% nucleotide homology with the closest GenBank sequence (M22475) but identity in amino-acid sequences of catalytic domains. Recombinant plasmid pPICZαA/BGL1 containing the sequence encoding BGL1 mature protein and α-factor secretion signal was constructed and transformed into methylotrophic yeast P. pastoris by electroporation. The recombinant strain produced single extracellular protein with molecular weight of 120 kDa and cellobiase activity of 60 IU/ml. The optimum pH of the recombinant β-glucosidase was 5.0 and the optimum temperature was 50°C.
    Biodiversity and Phytosociological Analysis of Plants around the Municipal Drains in Jaunpur
    The habitat where the present study has been carried out is productive in relation to nutrient quality and they may perform several useful functions, but are also threatened for their existence. Hence, the proposed work, will add much new information about biodiversity of macrophytes in drains and their embankment. All the species were identified with their different stages of growth which encountered on the three selected sites (I, II and III). The number of species occurring at each site is grouped seasonally, i.e. summer, rainy and winter season and the species were further recorded for the study of phytosociology. Phytosociological characters such as frequency, density and abundance were influenced by the climatic, anthropogenic and biotic stresses prevailing at the three study sites. All the species present at the study sites have shown maximum values of frequency, density and abundance in rainy season in comparison to that of summer and winter seasons.
    Apoptosis Induced by Low-concentration Ethanol in Hepatocellular Carcinoma Cell Strains and Down-regulated AFP and Survivin Analysis by Proteomic Technology

    Ethanol is generally used as a therapeutic reagent against Hepatocellular carcinoma (HCC or hepatoma) worldwide, as it can induce Hepatocellular carcinoma cell apoptosis at low concentration through a multifactorial process regulated by several unknown proteins. This paper provides a simple and available proteomic strategy for exploring differentially expressed proteins in the apoptotic pathway. The appropriate concentrations of ethanol required to induce HepG2 cell apoptosis were first assessed by MTT assay, Gisma and fluorescence staining. Next, the central proteins involved in the apoptosis pathway processs were determined using 2D-PAGE, SDS-PAGE, and bio-software analysis. Finally the downregulation of two proteins, AFP and survivin, were determined by immunocytochemistry and reverse transcriptase PCR (RT-PCR) technology. The simple, useful method demonstrated here provides a new approach to proteomic analysis in key bio-regulating process including proliferation, differentiation, apoptosis, immunity and metastasis.

    A Simple Affymetrix Ratio-transformation Method Yields Comparable Expression Level Quantifications with cDNA Data
    Gene expression profiling is rapidly evolving into a powerful technique for investigating tumor malignancies. The researchers are overwhelmed with the microarray-based platforms and methods that confer them the freedom to conduct large-scale gene expression profiling measurements. Simultaneously, investigations into cross-platform integration methods have started gaining momentum due to their underlying potential to help comprehend a myriad of broad biological issues in tumor diagnosis, prognosis, and therapy. However, comparing results from different platforms remains to be a challenging task as various inherent technical differences exist between the microarray platforms. In this paper, we explain a simple ratio-transformation method, which can provide some common ground for cDNA and Affymetrix platform towards cross-platform integration. The method is based on the characteristic data attributes of Affymetrix- and cDNA- platform. In the work, we considered seven childhood leukemia patients and their gene expression levels in either platform. With a dataset of 822 differentially expressed genes from both these platforms, we carried out a specific ratio-treatment to Affymetrix data, which subsequently showed an improvement in the relationship with the cDNA data.
    Screening Wheat Parents of Mapping Population for Heat and Drought Tolerance, Detection of Wheat Genetic Variation

    To evaluate genetic variation of wheat (Triticum aestivum) affected by heat and drought stress on eight Australian wheat genotypes that are parents of Doubled Haploid (HD) mapping populations at the vegetative stage, the water stress experiment was conducted at 65% field capacity in growth room. Heat stress experiment was conducted in the research field under irrigation over summer. Result show that water stress decreased dry shoot weight and RWC but increased osmolarity and means of Fv/Fm values in all varieties except for Krichauff. Krichauff and Kukri had the maximum RWC under drought stress. Trident variety was shown maximum WUE, osmolarity (610 mM/Kg), dry mater, quantum yield and Fv/Fm 0.815 under water stress condition. However, the recovery of quantum yield was apparent between 4 to 7 days after stress in all varieties. Nevertheless, increase in water stress after that lead to strong decrease in quantum yield. There was a genetic variation for leaf pigments content among varieties under heat stress. Heat stress decreased significantly the total chlorophyll content that measured by SPAD. Krichauff had maximum value of Anthocyanin content (2.978 A/g FW), chlorophyll a+b (2.001 mg/g FW) and chlorophyll a (1.502 mg/g FW). Maximum value of chlorophyll b (0.515 mg/g FW) and Carotenoids (0.234 mg/g FW) content belonged to Kukri. The quantum yield of all varieties decreased significantly, when the weather temperature increased from 28 ÔùªC to 36 ÔùªC during the 6 days. However, the recovery of quantum yield was apparent after 8th day in all varieties. The maximum decrease and recovery in quantum yield was observed in Krichauff. Drought and heat tolerant and moderately tolerant wheat genotypes were included Trident, Krichauff, Kukri and RAC875. Molineux, Berkut and Excalibur were clustered into most sensitive and moderately sensitive genotypes. Finally, the results show that there was a significantly genetic variation among the eight varieties that were studied under heat and water stress.

    Biometrical Comparison of Artemia urmiana Günther, 1899 (Crustacea: Anostraca) Cysts between Rainy and Drought Years (1994-2003/4) from Urmia Lake, Iran
    Nowadays, biometrical characterizations of Artemia cysts are used as one of the most important factors in the study of Artemia populations and intraspecific particularity; meanwhile these characters can be used as economical indices. For example, typically high hatching efficiency is possible due to the small diameter of cysts (high number per gram); therefore small diameter of cysts show someway high quality of cysts. This study was performed during a ten year period, including two different ecological conditions: rainy and drought. It is important from two different aspects because it covers alteration of A. urmiana during ten years also its variation in the best and worst environmental situations in which salinity increased from 173.8 ppt in 1994 to 280.8 ppt in 2003/4. In this study the biometrical raw data of Artemia urmiana cysts at seven stations from the Urmia Lake in 1994 and their seven identical locations at 26 studied stations in 2003/4 were reanalyzed again and compared together. Biometrical comparison of untreated and decapsulated cysts in each of the seven similar stations showed a highly significant variation between 1994 and 2003/4. Based on this study, in whole stations the untreated and decapsulated cysts from 1994 were larger than cysts of 2003/4 without any exception. But there was no logical relationship between salinity and chorion thickness in the Urmia Lake. With regard to PCA analyses the stations of two different studied years certainly have been separated with factor 1 from each other. In conclusion, the interaction between genetic and environmental factors can determine and explain variation in the range of cysts diameter in Artemia.
    Effect of Addition of Separan at Different Concentrations as a Flocculants on Quality of Sugar Cane Juice
    The study was designed to evaluate the use of low concentrations of separan flocculent (Less than 3 ppm) on physicochemical properties of sugar cane juice. Colour, pH, purity, turbidity, pol, brix, reducing sugars tannins and polyphenols of crushed cane (green and burned) juice, mixed juice and clarified juice were studied. The results showed that pol, brix, reducing sugar and turbidity are higher in crushed cane juice. Clarified burned juice had low turbidity, reducing sugars, pol and brix but had significantly lower pH, purity and colour when compared to crushed juice. Polyphenols of the crushed juice (1.19%) decreased significantly in the clarified juice to 0.006%. Addition of separan at a concentration of 0.015 ppm reduced significantly colour, polyphenols and tannins and reducing sugar compared to the control.
    Statistical Optimization of Enzymatic Hydrolysis of Potato (Solanum tuberosum) Starch by Immobilized α-amylase
    Enzymatic hydrolysis of starch from natural sources finds potential application in commercial production of alcoholic beverage and bioethanol. In this study the effect of starch concentration, temperature, time and enzyme concentration were studied and optimized for hydrolysis of Potato starch powder (of mesh 80/120) into glucose syrup by immobilized (using Sodium arginate) α-amylase using central composite design. The experimental result on enzymatic hydrolysis of Potato starch was subjected to multiple linear regression analysis using MINITAB 14 software. Positive linear effect of starch concentration, enzyme concentration and time was observed on hydrolysis of Potato starch by α-amylase. The statistical significance of the model was validated by F-test for analysis of variance (p ≤ 0.01). The optimum value of starch concentration, enzyme concentration, temperature, time and were found to be 6% (w/v), 2% (w/v), 40°C and 80min respectively. The maximum glucose yield at optimum condition was 2.34 mg/mL.
    Inhibition of the Growth of Pathogenic Candida spp. by Salicylhydroxamic Acid

    Candida spp. are common and aggressive pathogens. Because of the growing resistance of Candida spp. to current antifungals, novel targets, found in Candida spp. but not in humans or other flora, have to be identified. The alternative oxidase (AOX) is one such possibility. This enzyme is insensitive to cyanide, but is sensitive to compounds such as salicylhydroxamic acid (SHAM), disulfiram and n-alkyl gallates. The growth each of six Candida spp. was inhibited significantly by ~13 mM SHAM or 2 mM cyanide, albeit to differing extents. In C. dubliniensis, C. krusei and C. tropicalis the rate of O2 uptake was inhibited by 18-36% by 25 mM SHAM, but this had little or no effect on C. glabrata, C. guilliermondii or C. parapsilosis. Although SHAM substantially inhibited the growth of Candida spp., it is unlikely that the inhibition of AOX was the cause. Salicylhydroxamic acid is used therapeutically in the treatment of urinary tract infections and urolithiasis, but it also has some potential in the treatment of Candida spp. infection.

    Application of Central Composite Design Based Response Surface Methodology in Parameter Optimization and on Cellulase Production Using Agricultural Waste
    Response Surface Methodology (RSM) is a powerful and efficient mathematical approach widely applied in the optimization of cultivation process. Cellulase enzyme production by Trichoderma reesei RutC30 using agricultural waste rice straw and banana fiber as carbon source were investigated. In this work, sequential optimization strategy based statistical design was employed to enhance the production of cellulase enzyme through submerged cultivation. A fractional factorial design (26-2) was applied to elucidate the process parameters that significantly affect cellulase production. Temperature, Substrate concentration, Inducer concentration, pH, inoculum age and agitation speed were identified as important process parameters effecting cellulase enzyme synthesis. The concentration of lignocelluloses and lactose (inducer) in the cultivation medium were found to be most significant factors. The steepest ascent method was used to locate the optimal domain and a Central Composite Design (CCD) was used to estimate the quadratic response surface from which the factor levels for maximum production of cellulase were determined.
    Bioprocessing of Proximally Analyzed Wheat Straw for Enhanced Cellulase Production through Process Optimization with Trichodermaviride under SSF
    The purpose of the present work was to study the production and process parameters optimization for the synthesis of cellulase from Trichoderma viride in solid state fermentation (SSF) using an agricultural wheat straw as substrates; as fungal conversion of lignocellulosic biomass for cellulase production is one among the major increasing demand for various biotechnological applications. An optimization of process parameters is a necessary step to get higher yield of product. Several kinetic parameters like pretreatment, extraction solvent, substrate concentration, initial moisture content, pH, incubation temperature and inoculum size were optimized for enhanced production of third most demanded industrially important cellulase. The maximum cellulase enzyme activity 398.10±2.43 μM/mL/min was achieved when proximally analyzed lignocellulosic substrate wheat straw inocubated at 2% HCl as pretreatment tool along with distilled water as extraction solvent, 3% substrate concentration 40% moisture content with optimum pH 5.5 at 45°C incubation temperature and 10% inoculum size.
    Effect of Transglutaminase Cross Linking on the Functional Properties as a Function of NaCl Concentration of Legumes Protein Isolate
    The effect of cross linking of the protein isolates of three legumes with the microbial enzyme transglutaminase (EC on the functional properties at different NaCl concentration was studied. The reduction in the total free amino groups (OD340) of the polymerized protein showed that TGase treatment cross-linking the protein subunit of each legume. The solubility of the protein polymer of each legume was greatly improved at high concentration of NaCl. At 1.2 M NaCl the solubility of the native legumes protein was significantly decreased but after polymerization slightly improved. Cross linked proteins were less turbid on heating to higher temperature as compared to native proteins and the temperature at which the protein turns turbid also increased in the polymerized proteins. The emulsifying and foaming properties of the protein polymer were greatly improved at all concentrations of NaCl for all legumes.
    Human Elastin-derived Biomimetic Coating Surface to Support Cell Growth
    A new sythetic gene coding for a Human Elastin-Like Polypeptide was constructed and expressed. The recombinant product was tested as coating agent to realize a surface suitable for cell growth. Coatings showed peculiar features and different human cell lines were seeded and cultured. All cell lines tested showed to adhere and proliferate on this substrate that has been shown also to exert a specific effect on cells, depending on cell type.