Search results for: SNaPshot assay

Authors: Soni Yadav, Sitansu Kumar Verma, Jitendra Singh, Ajay Kumar

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This article comprises detail information about L-asparaginase, encompassing topic such as various sources of L-asparaginase, mechanism and properties of L-asparaginase. Also describe the production, cultivation and purification of L-asparaginase along with information about the application of L-asparaginase. L-asparaginase catalyzes the conversion reaction to convert asparagine to aspartic acid and ammonia. Asparagine is a nutritional requirement for both normal and tumor cell. Present scenario has found that L-asparaginase has been found to be a best anti tumor or antileukemic agent. In the recent years this enzyme gained application in the field of clinical research pharmacologic and food industry. It has been characterized based on the enzyme assay principle hydrolyzing L-asparagine into L-aspartic acid and ammonia. It has been observed that eukaryotic microorganisms such as yeast and filamentous fungi have a potential for L-asparaginase production. L-asparaginase has been and is still one of the most lengthily studied therapeutic enzymes by scientist and researchers worldwide.

Keywords: L-asparaginase, antitumor, solid state fermentation, chemotherapeutic.

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Authors: Xin Kai, Juan Li, Sexin Huang, Zengliang Bai

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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.

Keywords: Hepatocellular carcinoma, Ethanol, Proteomics, survivin and AFP

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Authors: Noor Muzamil Mohamad, Abdul Manaf Ali, Hamzah Mohd Salleh

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Phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8) catalyze the hydrolysis of phytic acid (myoinositol hexakisphosphate) to the mono-, di-, tri-, tetra-, and pentaphosphates of myo-inositol and inorganic phosphate. Therrmophilic bacteria isolated from water sampled from hot spring. About 120 isolates of bacteria were successfully isolated form hot spring water sample and tested for extracellular phytase producing. After 5 passages of the screening on the PSM media, 4 isolates were found stable in producing phytase enzyme. The 16s RDNA sequencing for identification of bacteria using molecular technique revealed that all isolates those positive in phytase producing are belong to Geobacillus spp. And Anoxybacillus spp. Anoxybacillus rupiensis UniSZA-7 were identified for their carbon source utilization using Phenotype Microarray Plate of Biolog and found they utilize several kind of carbon source provided.

Keywords: Phytase, Phytic Acid, Thermophilic Bacteria, PSM Media and Phytase Assay

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Authors: Hojat Heidari-Bafroui, Amer Charbaji, Constantine Anagnostopoulos, Mohammad Faghri

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Phosphorus is an essential nutrient for plant life which is most frequently found as phosphate in water. Once phosphate is found in abundance in surface water, a series of adverse effects on an ecosystem can be initiated. Therefore, a portable and reliable method is needed to monitor the phosphate concentrations in the field. In this paper, an inexpensive dip strip device with the ascorbic acid/antimony reagent dried on blotting paper along with wet chemistry is developed for the detection of low concentrations of phosphate in water. Ammonium molybdate and sulfuric acid are separately stored in liquid form so as to improve significantly the lifetime of the device and enhance the reproducibility of the device’s performance. The limit of detection and quantification for the optimized device are 0.134 ppm and 0.472 ppm for phosphate in water, respectively. The device’s shelf life, storage conditions, and limit of detection are superior to what has been previously reported for the paper-based phosphate detection devices.

Keywords: Phosphate detection, paper-based device, molybdenum blue method, colorimetric assay.

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Authors: Yahia M., Chaoui N., Chaouch A., Massinissa Yahia

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Our study was designed to determine the metabolic  changes of some biochemical parameters (cholesterol, triglyceride,  Iron, uric acid, Urea and folic acid) and highlight their changes in 57  women of the region Batna, during the first trimester of pregnancy.  This practical work was done with 27 women with missed  miscarriage, compared with 30 control subjects of normal pregnant  women. The assay results revealed a highly significant difference (P  = 0.0006) between the two groups in serum iron (64.00 vs 93.54) and  in the rate of folate (6.70 vs 9.22) (P <0.001) but no difference was  found regarding the rate of Ca (9.69 vs 10.20), urea (0.19 vs 0.17),  UA (33.96 vs 32.76), CH (1.283 vs 1.431), and TG (0.8852 vs  0.8290). The present study indicates that iron deficiency and folate  are associated with missed miscarriage, but no direct  pathophysiological link has been determined. Further in-depth studies  are needed to determine the exact mechanism by which these deficits  lead to a missed miscarriage.

Keywords: Biochemical parameters, pregnant women, missed miscarriage.

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Authors: Mohd Azman A. Nurul, Husni Shafik, Almajano P. Maria, Gallego G. Maria

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The potential of using herbal Betula alba (BA) and Convolvulus arvensis (CA) as a natural antioxidant for food applications were investigated. Each plant extract was prepared by using pure ethanol, different concentration of ethanol aqueous solutions, including 50% and 75%, 50% methanol aqueous and water. Total phenolic content (TPC) was determined using Folin–Ciocalteau method and antioxidant activity were analyzed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, trolox equivalent antioxidant capacity (TEAC), Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) respectively. Ethanol extract of CA exhibited the highest TPC and antioxidant activity; however BA showed varies of antioxidant activity value in each assay. The BA and CA exhibit the potential sources of natural antioxidant for food commodities.

Keywords: Solvent effect, Antioxidant activity, Betula Alba, Convolvulus arvensis, Total Phenolic Content.

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Authors: Javier Sánchez, Laura Elena De León Prado, Dora Alicia Cortés Hernández

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Magnetic spinel ferrites are materials that possess size, magnetic properties and heating ability adequate for their potential use in biomedical applications. The Mn_0.5Ga_0.5Fe₂O₄ magnetic nanoparticles (MNPs) were synthesized by sol-gel method using citric acid as chelating agent of metallic precursors. The synthesized samples were identified by X-Ray Diffraction (XRD) as an inverse spinel structure with no secondary phases. Saturation magnetization (Ms) of crystalline powders was 45.9 emu/g, which was higher than those corresponding to GaFe₂O₄ (14.2 emu/g) and MnFe₂O₄ (40.2 emu/g) synthesized under similar conditions, while the coercivity field (Hc) was 27.9 Oe. The average particle size was 18 ± 7 nm. The heating ability of the MNPs was enough to increase the surrounding temperature up to 43.5 °C in 7 min when a quantity of 4.5 mg of MNPs per mL of liquid medium was tested. Cytotoxic effect (hemolysis assay) of MNPs was determined and the results showed hemolytic values below 1% in all tested cases. According to the results obtained, these synthesized nanoparticles can be potentially used as thermoseeds for hyperthermia therapy.

Keywords: Cytotoxicity, heating ability, manganese-gallium ferrite, magnetic hyperthermia.

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Authors: Zeinep A. Berkimbayeva, Almagul T. Mansharipova, Elmira M. Khussainova, Leyla B. Djansugurova

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It is believed that DNA damaging toxic metabolites contributes to the development of different pathological conditions. To prevent harmful influence of toxic agents, cells developed number of protecting mechanisms, such as enzymatic reaction of detoxification of reactive metabolites and repair of DNA damage. The aim of the study was to examine the association between polymorphism of GSTT1/GSTM1 and XRCC1/3 genes and coronary artery disease (CAD) incidence. To examine a polymorphism of these genes in CAD susceptibility in patients and controls, PCR based genotyping assay was performed. For GST genes, frequency of GSTM1 null genotype among CAD affected group was significantly increased than in control group (P<0.001). Frequencies of the GSTT1 null and positive alleles are almost equal in both groups (P>0.1). We found that neither XRCC1 Arg399Gln nor XRCC3 Thr241Met were associated with CAD risk. Obtained data suggests that GSTM1 null genotype carriers are more susceptible to CAD development.

Keywords: Cardiovascular disease, DNA reparation, gene polymorphism, risk factors, xenobiotic detoxification.

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Authors: N. B. Dhayanithi, T. T. Ajith Kumar, T. Balasubramanian

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Present study summarizes the control of Vibrio alginolyticus infection in hatchery reared Clownfish, Amphiprion sebae with the extract of the mangrove plant, Avicennia marina. Fishes with visible symptoms of hemorrhagic spots were chosen and the genomic DNA of the causative bacterium was isolated and sequenced based on 16S rDNA gene. The in vitro assay revealed that a fraction of A. marina leaf extract elucidated with ethyl acetate: methanol (6:4) showed a high activity (28 mm) at 125 μg/ml concentrations. About 4 % of the fraction fed along with live V. alginolyticus was significantly decreased the cumulative mortality (P<0.05) in the experimental groups than the control group. The responsible fraction was investigated by gas chromatography - mass spectroscopy and found the presence of active compounds. This is the first research in India to control vibriosis infection in marine ornamental fish with mangrove leaf extract.

Keywords: Amphiprion seabe, Avicennia marina, Gas Chromatography - Mass Spectroscopy, Vibrio alginolyticus

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Authors: Forough Ataollahi, Belinda Pingguan-Murphy, Wan Abu Bakar Wan Abas, Noor Azuan Abu Osman

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Endothelium proliferation is an important process in cardiovascular homeostasis and can be regulated by extracellular environment, as cells can actively sense mechanical environment. In this study, we evaluated endothelial cell proliferation on PDMS/alumina (Al₂O₃) composites and pure PDMS. The substrates were prepared from pure PDMS and its composites with 5% and 10% Al₂O₃ at curing temperature 50˚C for 4h and then characterized by mechanical, structural and morphological analyses. Higher stiffness was found in the composites compared to the pure PDMS substrate. Cell proliferation of the cultured bovine aortic endothelial cells on substrate materials were evaluated via Resazurin assay and 1, 1’-Dioctadecyl-1, 3, 3, 3’, 3’-Tetramethylindocarbocyanine Perchlorate-Acetylated LDL (Dil-Ac-LDL) cell staining, respectively. The results revealed that stiffer substrates promote more endothelial cells proliferation to the less stiff substrates. Therefore, this study firmly hypothesizes that the stiffness elevates endothelial cells proliferation.

Keywords: Bovine aortic endothelial cells, extra cellular matrix, proliferation, stiffness.

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Authors: Siti Aishah Abu Bakar, Madihah Zawawi, Abdul Manaf Ali, Aini Ideris

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Newcastle Disease Virus (NDV), an avian paramyxovirus, is a highly contagious, generalised virus disease of domestic poultry and wild birds characterized by gastro-intestinal, respiratory and nervous signs. In this study, it was shown that NDV strain AF2240 and V4-UPM are cytolytic to Human Promyelocytic Leukemia, HL60 and Human T-lymphoblastic Leukemia, CEM-SS cells. Results from MTT cytolytic assay showed that CD50 for NDV AF2240 against HL60 was 130 HAU and NDV V4-UPM against HL60 and CEM-SS were 110.6 and 150.9 HAU respectively. Besides, both strains were found to inhibit the proliferation of cells in a dose dependent manner. The mode of cell death either by apoptosis or necrosis was further analyzed using acridine orange and propidium iodide (AO/PI) staining. Our results showed that both NDV strains induced primarily apoptosis in treated cells at CD50 concentration. In conclusion, both NDV strains caused cytolytic effects primarily via apoptosis in leukemia cells.

Keywords: Apoptosis, Cytolytic, Leukaemia, Newcastle DiseaseVirus

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Authors: Ahmad M. Khalil, Ahmad M. Alshamali, Marwan H. Gagaa

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We investigated oxidative DNA damage caused by radio frequency radiation using 8-oxo-7, 8-dihydro-2'- deoxyguanosine (8-oxodG) generated in mice tissues after exposure to 900 MHz mobile phone radio frequency in three independent experiments. The RF was generated by a Global System for Mobile Communication (GSM) signal generator. The radio frequency field was adjusted to 25 V/m. The whole body specific absorption rate (SAR) was 1.0 W/kg. Animals were exposed to this field for 30 min daily for 30 days. 24 h post-exposure, blood serum, brain and spleen were removed and DNA was isolated. Enzyme-linked immunosorbent assay (ELISA) was used to measure 8-oxodG concentration. All animals survived the whole experimental period. The body weight of animals did not change significantly at the end of the experiment. No statistically significant differences observed in the levels of oxidative stress. Our results are not in favor of the hypothesis that 900 MHz RF induces oxidative damage.

Keywords: Mice, Mobile phone radiation, oxidative stress, 8-oxo-7, 8-dihydro-2'-deoxyguanosine

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Authors: Folasade F. Awe, Tayo N. Fagbemi, Comfort F. Ajibola, Adebanjo A. Badejo

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The antioxidant capability of beverage blends made from cocoa, zobo and ginger with standard antioxidant assay procedures was investigated. The DPPH (2,2-diphenyl-1- picrylhydrazyl) scavenging capacity ranged from 21.2-25.8% in comparison with GSH of 37.1%. The ferric reducing ability was highest in the zobo drink and lowest in ginger. The superoxide scavenging capacity was also highest in the zobo drink followed by the drink with alkalized cocoa. The metal chelating power decreased as the level of zobo in the blends decreases. The chelating power of zobo and ginger were significantly lower than the natural and alkalized cocoa. The 100% zobo drink inhibited linoleic acid till the fifth day while natural and alkalized cocoa as well as the blend with 50% alkalized cocoa inhibited linoleic acid greatly till the sixth day. The finding describes the potential health benefit of the phytochemical antioxidants of cocoa:zobo:ginger beverage blends.

Keywords: Antioxidant, cocoa, ginger, health benefit, zobo blend.

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Authors: N. Aliasgharzad, A. Molaei, S. Oustan

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Soil microbial activity is adversely affected by pollutants such as heavy metals, antibiotics and pesticides. Organic amendments including sewage sludge, municipal compost and vermicompost are recently used to improve soil structure and fertility. But, these materials contain heavy metals including Pb, Cd, Zn, Ni and Cu that are toxic to soil microorganisms and may lead to occurrence of more tolerant microbes. Among these, Pb is the most abundant and has more negative effect on soil microbial ecology. In this study, Pb levels of 0, 100, 200, 300, 400 and 500 mg Pb [as Pb(NO3)2] per kg soil were added to the pots containing 2 kg of a loamy soil and incubated for 6 months at 25°C with soil moisture of - 0.3 MPa. Dehydrogenase activity of soil as a measure of microbial activity was determined on 15, 30, 90 and 180 days after incubation. Triphenyl tetrazolium chloride (TTC) was used as an electron acceptor in this assay. PICTs (€IC50 values) were calculated for each Pb level and incubation time. Soil microbial activity was decreased by increasing Pb level during 30 days of incubation but the induced tolerance appeared on day 90 and thereafter. During 90 to 180 days of incubation, the PICT was gradually developed by increasing Pb level up to 200 mg kg-1, but the rate of enhancement was steeper at higher concentrations.

Keywords: Induced tolerance, soil microorganisms, Pb, PICT, pollutants.

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Authors: S.H. Mirdamadian, S.M. Khayam-Nekoui, H. Ghanavati

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Composting is the process in which municipal solid waste (MSW) and other organic waste materials such as biosolids and manures are decomposed through the action of bacteria and other microorganisms into a stable granular material which, applied to land, as soil conditioner. Microorganisms, especially those that are able to degrade polymeric organic material have a key role in speed up this process. The aim of this study has been established to isolation of microorganisms with high ability to production extracellular enzymes for degradation of natural polymers that are exists in MSW for decreasing time of degradation phase. Our experimental study for isolation designed in two phases: in first phase we isolated degrading microorganism with selected media that consist a special natural polymer such as cellulose, starch, lipids and etc as sole source of carbon. In second phase we selected microorganism that had high degrading enzyme production with enzymatic assay for seed production. However, our findings in pilot scale have indicated that usage of this microbial consortium had high efficiency for decreasing degradation phase.

Keywords: Biodegradation, Compost, Municipal Solid Waste, Waste Management.

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Authors: Alan Crowle, Philpp Thies

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s the floating offshore wind turbine industry continues to develop and grow, the capabilities of established port facilities need to be assessed as to their ability to support the expanding construction and installation requirements. This paper assesses current infrastructure requirements and projected changes to port facilities that may be required to support the floating offshore wind industry. Understanding the infrastructure needs of the floating offshore renewable industry will help to identify the port-related requirements. Floating offshore wind turbines can be installed further out to sea and in deeper waters than traditional fixed offshore wind arrays, meaning it can take advantage of stronger winds. Separate ports are required for substructure construction, fit-out of the turbines, moorings, subsea cables and maintenance. Large areas are required for the laydown of mooring equipment, inter array cables, turbine blades and nacelles. The capabilities of established port facilities to support floating wind farms are assessed by evaluation of size of substructures, height of wind turbine with regards to the cranes for fitting of blades, distance to offshore site and offshore installation vessel characteristics. The paper will discuss the advantages and disadvantages of using large land based cranes, inshore floating crane vessels or offshore crane vessels at the fit-out port for the installation of the turbine. Water depths requirements for import of materials and export of the completed structures will be considered. There are additional costs associated with any emerging technology. However, part of the popularity of Floating Offshore Wind Turbines stems from the cost savings against permanent structures like fixed wind turbines. Floating Offshore Wind Turbine developers can benefit from lighter, more cost effective equipment which can be assembled in port and towed to site rather than relying on large, expensive installation vessels to transport and erect fixed bottom turbines. The ability to assemble Floating Offshore Wind Turbines equipment on shore means minimising highly weather dependent operations like offshore heavy lifts and assembly, saving time and costs and reducing safety risks for offshore workers. Maintenance might take place in safer onshore conditions for barges and semi submersibles. Offshore renewables, such as floating wind, can take advantage of this wealth of experience, while oil and gas operators can deploy this experience at the same time as entering the renewables space. The floating offshore wind industry is in the early stages of development and port facilities are required for substructure fabrication, turbine manufacture, turbine construction and maintenance support. The paper discusses the potential floating wind substructures as this provides a snapshot of the requirements at the present time, and potential technological developments required for commercial development. Scaling effects of demonstration-scale projects will be addressed; however the primary focus will be on commercial-scale (30+ units) device floating wind energy farms.

Keywords: Floating offshore wind turbine, port logistics, installation, construction.

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Authors: Feng Chia Hsieh, Sheng Kuo Hsieh, Tzyy Rong Jinn

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Characterization and evaluation of the activity of Vespa basalis DPP-IV, which expressed in Spodoptera frugiperda 21 cells. The expression of rDPP-IV was confirmed by SDS–PAGE, Western blot analyses, LC-MS/MS and measurement of its peptidase specificity. One-step purification by Ni-NTA affinity chromatography and the total amount of rDPP-IV recovered was approximately 6.4mg per liter from infected culture medium; an equivalent amount would be produced by 1x109 infected Sf21 insect cells. Through the affinity purification led to highly stable rDPP-IV enzyme was recovered and with significant peptidase activity. The rDPP-IV exhibited classical Michaelis–Menten kinetics, with kcat/Km in the range of 10-500 mM-1×S-1 for the five synthetic substrates and optimum substrate is Ala-Pro-pNA. As expected in inhibition assay, the enzymatic activity of rDPP-IV was significantly reduced by 80 or 60% in the presence of sitagliptin (a DPP-IV inhibitor) or PMSF (a serine protease inhibitor), but was not apparently affected by iodoacetamide (a cysteine protease inhibitor).

Keywords: Dipeptidyl-Peptidase IV, Phenylmethylsulfonyl fluoride; Serine protease, Sitagliptin, Vespa basalis

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Authors: Hanina M. N., Hairul Shahril M., Ismatul Nurul Asyikin I., Abdul Jalil A. K., Salina M. R., Maryam M. R., Rosfarizan M.

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The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.

Keywords: Bacillus subtilis ATCC21332, Streptomycin Sulfate, extracellular proteins.

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcus lactis, recombinant, phytase, poultry.

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Authors: H. Rasouli Pirouzian, J. Hesari, S. Farajnia, M. Moghaddam, S. Ghiassifar, M. Manafi

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Lighvan cheese is basically made from sheep milk in the area of Sahand mountainside which is located in the North West of Iran. The main objective of this study was to investigate the effect of enterococci isolated from traditional Lighvan cheese on the quality of Iranian UF white during ripening. The experimental design was split plot based on randomized complete blocks, main plots were four types of starters and subplots were different ripening durations. Addition of Enterococcus spp. did not significantly (P<0.01) affect the pH and gross composition of cheeses. In the cheeses produced with Ent. faecalis and Ent. faecium strains, lipolysis rates were higher and flavor were improved. Moreover, proteolysis assay by measuring percentage of soluble nitrogen at pH 4.6 and urea polyacrylamide gel electrophoresis indicated the increase in proteolysis rate in the cheese containing Ent. faecalis and Ent. faecium strains compared to the control cheeses. Furthermore, the highest percentage of non- protein nitrogen was observed in the cheese containing Ent. faecium. In conclusion, the results showed the positive effect of the Ent. faecalis and Ent. faecium on secondary proteolysis, lipolysis and sensorial characteristics development of UF white cheeses.

Keywords: Enterococcus faecalis, Enterococcus faecium, Lighvan cheese, Lipolysis, Proteolysis, UF cheese

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Authors: Natasha Hussain, Maleeha Aslam, Robina Farooq

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Hepatitis B and hepatitis C are among the most significant hepatic infections all around the world that may lead to hepatocellular carcinoma. This study is first time performed at the blood transfussion centre of Omar hospital, Lahore. It aims to determine the sero-prevalence of these diseases by screening the apparently healthy blood donors who might be the carriers of HBV or HCV and pose a high risk in the transmission. It also aims the comparison between the sensitivity of two diagnostic tests; chromatographic immunoassay – one step test device and Enzyme Linked Immuno Sorbant Assay (ELISA). Blood serum of 855 apparently healthy blood donors was screened for Hepatitis B surface antigen (HBsAg) and for anti HCV antibodies. SPSS version 12.0 and X2 (Chi-square) test were used for statistical analysis. The seroprevalence of HCV was 8.07% by the device method and by ELISA 9.12% and that of HBV was 5.6% by the device and 6.43% by ELISA. The unavailability of vaccination against HCV makes it more prevalent. Comparing the two diagnostic methods, ELISA proved to be more sensitive.

Keywords: ELISA, Sensitivity comparison of diagnostic tests, seroprevalence of Hepatitis B and C

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Authors: N. Tsvetkova, R. Harizanov A. Ivanova, I. Rainova, N. Yancheva-Petrova, D. Strashimirov, R. Enikova, M. Videnova, E. Kaneva, I. Kaftandjiev, V. Levterova, I. Simeonovski, N. Yanev, G. Hinkov

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The Pneumocystis jirovecii (P. jirovecii) and protozoan of the genera Acanthamoeba, Cryptosporidium, and Toxoplasma gondii are opportunistic pathogens that can cause life-threatening infections in immunocompromised patients. Aim of the study was to evaluate the coinfection rate with opportunistic protozoal agents among Bulgarian patients diagnosed with P. jirovecii pneumonia. 38 pulmonary samples were collected from 38 patients (28 HIV-infected) with P. jirovecii infection. P. jirovecii DNA was detected by real-time PCR targeting the large mitochondrial subunit ribosomal RNA gene. Acanthamoeba was determined by genus-specific conventional PCR assay. Real-time PCR for the detection of a Toxoplasma gondii and Cryptosporidium DNA fragment was used. Pneumocystis DNA was detected in all 38 specimens; 28 (73.7%) were from HIV-infected patients. Three (10,7%) of them were coinfected with T. gondii and 1 (3.6%) with Cryptosporidium. In the group of non-HIV-infected (n = 10), Cryptosporidium DNA was detected in an infant (10%). Acanthamoeba DNA was not found in the tested samples. The current study showed a relatively low rate of coinfections of Cryptosporidium spp./T. gondii and P. jirovecii in the Bulgarian patients studied.

Keywords: Coinfection, opportunistic protozoal agents, Pneumocystis jirovecii, pulmonary infections.

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Authors: Dehpour A. A., Eslami B., Rezaie S., Hashemian S. F., Shafie F., Kiaie M.

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The aims of study were investigation on chemical composition essential oil and the effect of extract of Coronilla varia on antimicrobial and cytotoxicity activity. The essential oils of Coronilla varia is obtained by hydrodistillation and analyzed by (GC/MS) for determining their chemical composition and identification of their components. Antibacterial activity of plant extract was determined by disc diffusion method and anticancer activity measured by MTT assay. The major components in essential oil were Caryophyllene Oxide (60.19%), Alphacadinol (4.13%) and Homoadantaneca Robexylic Acid (3.31%). The extracts from Coronilla varia had interesting activity against Proteus mirabilis in the concentration of 700 μg/disc and did not show any activity against Staphylococus aureus, Bacillus subtillis, Klebsiella pneumonia and Entrobacter cloacae. The positive control, Ampicillin, Chloramphenicol and Cenphalothin had shown zone of inhibition resistant all bacteria. The ethanol extract of Corohilla varia inhibited on MCF7 cell lines. IC50 0.6(mg/ml) was the optimum concentration of extract from Coronilla varia inhibition of cell line growth. The MCF7 cancer cell line and Proteus mirabilis were more sensitive to Coronilla varia ethanol extract.

Keywords: Coronilla varia, Essential oil, Antibacterial, Anticancer, HeLa cell line.

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Authors: Marek Halenár, Eva Tvrdá, Simona Baldovská, Ľubomír Ondruška, Peter Massányi, Adriana Kolesárová

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This study aimed to assess the in vitro effects of different concentrations of the Viscum album extract on the motility, viability, and reactive oxygen species (ROS) production by rabbit spermatozoa during different time periods (0, 2, and 8h). Spermatozoa motility was assessed by using the CASA (Computer aided sperm analysis) system. Cell viability was evaluated by using the metabolic activity MTT assay, and the luminol-based luminometry was applied to quantify the ROS formation. The CASA analysis revealed that low Viscum concentrations were able to prevent a rapid decline of spermatozoa motility, especially in the case of concentrations ranging between 1 and 5 µg/mL (P<0.05 with respect to time 8h). At the same time, concentrations ranging between 1 and 100 µg/mL of the extract led to a significant preservation of the cell viability (P<0.05 in case of 5, 50 and 100 µg/mL; P<0.01 with respect to 1 and 10 µg/mL, time 8h). 1 and 5 µg/mL of the extract exhibited antioxidant characteristics, translated into a significant reduction of the ROS production, particularly notable at time 8h (P<0.01). The results indicate that the Viscum extract is capable of delaying the damage inflicted to the spermatozoon by the in vitro environment.

Keywords: CASA, mistletoe, mitochondrial activity, motility, reactive oxygen species, rabbits, spermatozoa, Viscum album.

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Authors: A. Panacek, M. Kilianova, R. Prucek, V. Husickova, R. Vecerova, M. Kolar, L. Kvitek, R. Zboril

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In this work we describe the preparation of NanoSilver/methylcellulose hydrogel containing silver nanoparticles (NPs) for topical bactericidal applications. Highly concentrated dispersion of silver NPs as high as of 5g/L of silver with diameter of 10nm was prepared by reduction of AgNO₃ via strong reducing agent NaBH₄. Silver NPs were stabilized by addition of sodium polyacrylate in order to prevent their aggregation at such high concentration. This way synthesized silver NPs were subsequently incorporated into methylcellulose suspension at elevated temperature resulting in formation of NanoSilver/methylcellulose hydrogel when temperature cooled down to laboratory conditions. In vitro antibacterial activity assay proved high bactericidal and fungicidal efficiency of silver NPs alone in the form of dispersion as well as in the form of hydrogel against broad spectrum of bacteria and yeasts including highly multiresistant strains such as methicillin-resistant Staphylococcus aureus. A very low concentrations of silver as low as 0.84mg/L Ag in as-prepared dispersion gave antibacterial performance. NanoSilver/methylcellulose hydrogel showed antibacterial action at the lowest used silver concentration equal to 25mg/L. Such prepared NanoSilver/methylcellulose hydrogel represent promising topical antimicrobial formulation for treatment of burns and wounds.

Keywords: Antimicrobial, burn, hydrogel, silver NPs.

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Authors: Titin Andri Wihastuti, Djanggan Sargowo

Abstract:

NFκB is a transcription factor regulating many function of the vessel wall. In the normal condition , NFκB is revealed diffuse cytoplasmic expressionsuggesting that the system is inactive. The presence of activation NFκB provide a potential pathway for the rapid transcriptional of a variety of genes encoding cytokines, growth factors, adhesion molecules and procoagulatory factors. It is likely to play an important role in chronic inflamatory disease involved atherosclerosis. There are many stimuli with the potential to active NFκB, including hyperlipidemia. We used 24 mice which was divided in 6 groups. The HFD given by et libitum procedure during 2, 4, and 6 months. The parameters in this study were the amount of NFKB activation ,H2O2 as ROS and VCAM-1 as a product of NFKB activation. H2O2 colorimetryc assay performed directly using Anti Rat H2O2 ELISA Kit. The NFKB and VCAM-1 detection obtained from aorta mice, measured by ELISA kit and imunohistochemistry. There was a significant difference activation of H2O2, NFKB and VCAM-1 level at induce HFD after 2, 4 and 6 months. It suggest that HFD induce ROS formation and increase the activation of NFKB as one of atherosclerosis marker that caused by hyperlipidemia as classical atheroschlerosis risk factor.

Keywords: High Fat Diet, NFKB, H2O2, atherosclerosis

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Authors: Hai Xu, Yiwei Wang, Xi Bao, Bihua Deng, Pengcheng Li, Yu Lu

Abstract:

T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 10¹⁰ pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac^®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.

Keywords: Gonadotrophin releasing hormone, GnRH, immunocastration, T7 phage, phage vaccine.

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Authors: Mahmood Ahmad, Ghulam Murtaza, Sonia Khiljee, Muhammad Asadullah Madni

Abstract:

A new reverse phase-high performance liquid chromatography (RP-HPLC) method with fluorescent detector (FLD) was developed and optimized for Norfloxacin determination in human plasma. Mobile phase specifications, extraction method and excitation and emission wavelengths were varied for optimization. HPLC system contained a reverse phase C18 (5 μm, 4.6 mm×150 mm) column with FLD operated at excitation 330 nm and emission 440 nm. The optimized mobile phase consisted of 14% acetonitrile in buffer solution. The aqueous phase was prepared by mixing 2g of citric acid, 2g sodium acetate and 1 ml of triethylamine in 1 L of Milli-Q water was run at a flow rate of 1.2 mL/min. The standard curve was linear for the range tested (0.156–20 μg/mL) and the coefficient of determination was 0.9978. Aceclofenac sodium was used as internal standard. A detection limit of 0.078 μg/mL was achieved. Run time was set at 10 minutes because retention time of norfloxacin was 0.99 min. which shows the rapidness of this method of analysis. The present assay showed good accuracy, precision and sensitivity for Norfloxacin determination in human plasma with a new internal standard and can be applied pharmacokinetic evaluation of Norfloxacin tablets after oral administration in human.

Keywords: Norfloxacin, Aceclofenac sodium, Methodoptimization, RP-HPLC method, Fluorescent detection, Calibrationcurve.

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Authors: S. Yodyingyong, C. Chaichana, C. Nuchsuk, S. Roytrakul, N. P. T-Thienprasert, S. Ratanapo

Abstract:

Protein hydrolysates prepared from a number of medicinal plants are promising sources of various bioactive peptides. In this work, proteins from dried whole plant of Euphorbia hirta Linn. were extracted and digested with pepsin for 12h. The hydrolysates of lesser than 3 KDa were fractionated by a cut-off membrane. The peptide hydrolysate was then purified by an anion-exchange chromatography on DEAE-Sephacel™ column and reverse-phase chromatography on Sep-pak C₁₈ column, respectively. The cytotoxic effect of each peptide fraction against a gastric carcinoma cell line (KATO-III, ATCC No. HTB103) was investigated using colorimetric MTT viability assay. A human liver cell line (Chang Liver, CLS No. 300139) was used as a control normal cell line. Two purified peptide peaks, peak l and peak ll at 100µg peptides mL^-1 affected cell viability of the gastric cancer cell lines to 63.85±4.94 and 66.92±6.46%, respectively. Our result showed for the first time that the peptide fractions derived from protein hydrolysate of Euphorbia hirta Linn. have anti-gastric cancer activity, which offers a potential novel and natural anti-gastric cancer remedy.

Keywords: Cytotoxic, peptides, Euphorbia hirta Linn., gastric carcinoma.

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Authors: Ikram Mohamed Eltayeb Elsiddig, Abdel Khalig Muddather, Hiba Abdel Rahman Ali, Saad Mohamed Hussein Ayoub

Abstract:

Anogeissus leiocarpus (Combretaceae) is well known for its medicinal uses in African traditional medicine, for treating many human diseases mainly skin diseases and infections. Mycetoma disease is a fungal and/ or bacterial skininfection, mainly cause by Madurella mycetomatis fungus. This study was carried out in vitro to investigate the antifungal activity of Anogeissus leiocarpus leaf extracts against the isolated pathogenic Madurella mycetomatis, by using the NCCLS modified method compared to Ketoconazole standard drug, and MTT assay. The bioactive fraction was subjected to chemical analysis implementing different chromatographic analytical methods (TLC, HPLC, and LC-MS/MS). The results showed significance antifungal activity of A. leiocarpus leaf extracts against the isolated pathogenic M. mycetomatis, compared to negative and positive controls. The chloroform fraction showed the highest antifungal activity. The chromatographic analysis of the chloroform fraction with the highest activity showed the presence of important bioactive compounds such as ellagic and flavellagic acids derivatives, flavonoids and stilbenoid, which are well known for their antifungal activity.

Keywords: Anogeissus leiocarpus, crude extracts and fractions of Anogeissus leiocarpus, in vitro susceptibility of Madurella mycetomatis, Madurella mycetomatis.

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