Search results for: electrophoresis

Authors: Sandeep Kaur

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Phycoerythrin (PE) from the mesophilic filamentous cyanobacterium Nostoc piscinale PUPCCC 405.17, a good producer of phycobiliproteins, has been characterized in terms of their unit assembly and stability. The phycoerythrin was extracted by freeze-thawing the cells in water, concentrated by ammonium sulphate fractionation and purified by anion exchange chromatography. The purification process resulted in 2.90 fold increase in phycoerythrin purity reaching to 1.54. Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis of purified PE demonstrated three protein bands of 14.3, 27.54 and 39.81 kDa. The native PE also showed one band of 125.87 kDa, assumed to be a dimer (αβ)2γ based on results of non-denaturing PAGE. Lyophilized powder PE was more stable compared to phycoerythrin in the solution. The half-life of dry PE is 80 days when stored at 4 °C under dark. The phycoerythrin from this organism has potential applications in food as natural colour and as a fluorescent marker.

Keywords: characterization, Nostoc piscinale, phycoerythrin, purification

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Authors: Barun Chakrabarti, Dan Nir, Vladimir Yufit, P. V. Aravind, Nigel Brandon

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Fuel cell grade gas-diffusion layer carbon paper (CP) electrodes are subjected to electrophoresis in N,N’-dimethylformamide (DMF) consisting of reduced graphene oxide (rGO). The rGO modified electrodes are compared with CP in a single asymmetric all-vanadium redox battery system (employing a double serpentine flow channel for each half-cell). Peak power densities improved by 4% when the rGO deposits were facing the ion-exchange membrane (cell performance was poorer when the rGO was facing the flow field). Cycling of the cells showed least degradation of the CP electrodes that were coated with rGO in comparison to pristine samples.

Keywords: all-vanadium redox flow batteries, carbon paper electrodes, electrophoretic deposition, reduced graphene oxide

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Authors: P. Hashim, M. S. Mohd Ridzwan, J. Bakar

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Collagen was isolated from chicken feet by using papain and pepsin enzymes in acetic acid solution at 4°C for 24h with a yield of 18.16% and 22.94% by dry weight, respectively. Chemical composition and characteristics of chicken feet collagen such as amino acid composition, SDS-PAGE patterns, FTIR spectra and thermal properties were evaluated. The chicken feet collagen is rich in the amino acids glycine, glutamic acid, proline and hydroxyproline. Electrophoresis pattern demonstrated two distinct α-chains (α1 and α2) and β chain, indicating that type I collagen is a major component of chicken feet collagen. The thermal stability of collagen isolated by papain and pepsin revealed stable denaturation temperatures of 48.40 and 53.35°C, respectively. The FTIR spectra of both collagens were similar with amide regions in A, B, I, II, and III. The study demonstrated that chicken feet collagen using papain isolation method is possible as commercial alternative ingredient.

Keywords: chicken feet, collagen, papain, pepsin

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Authors: Ekaterina A. Savchenko, Elena N. Velichko, Evgenii T. Aksenov

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The development of pathological processes, such as cardiovascular and oncological diseases, are accompanied by changes in molecular parameters in cells, tissues, and serum. The study of the behavior of protein molecules in solutions is of primarily importance for diagnosis of such diseases. Various physical and chemical methods are used to study molecular systems. With the advent of the laser and advances in electronics, optical methods, such as scanning electron microscopy, sedimentation analysis, nephelometry, static and dynamic light scattering, have become the most universal, informative and accurate tools for estimating the parameters of nanoscale objects. The electrophoretic light scattering is the most effective technique. It has a high potential in the study of biological solutions and their properties. This technique allows one to investigate the processes of aggregation and dissociation of different macromolecules and obtain information on their shapes, sizes and molecular weights. Electrophoretic light scattering is an analytical method for registration of the motion of microscopic particles under the influence of an electric field by means of quasi-elastic light scattering in a homogeneous solution with a subsequent registration of the spectral or correlation characteristics of the light scattered from a moving object. We modified the technique by using the regime of total internal reflection with the aim of increasing its sensitivity and reducing the volume of the sample to be investigated, which opens the prospects of automating simultaneous multiparameter measurements. In addition, the method of total internal reflection allows one to study biological fluids on the level of single molecules, which also makes it possible to increase the sensitivity and the informativeness of the results because the data obtained from an individual molecule is not averaged over an ensemble, which is important in the study of bimolecular fluids. To our best knowledge the study of electrophoretic light scattering in the regime of total internal reflection is proposed for the first time, latex microspheres 1 μm in size were used as test objects. In this study, the total internal reflection regime was realized on a quartz prism where the free electrophoresis regime was set. A semiconductor laser with a wavelength of 655 nm was used as a radiation source, and the light scattering signal was registered by a pin-diode. Then the signal from a photodetector was transmitted to a digital oscilloscope and to a computer. The autocorrelation functions and the fast Fourier transform in the regime of Brownian motion and under the action of the field were calculated to obtain the parameters of the object investigated. The main result of the study was the dependence of the autocorrelation function on the concentration of microspheres and the applied field magnitude. The effect of heating became more pronounced with increasing sample concentrations and electric field. The results obtained in our study demonstrated the applicability of the method for the examination of liquid solutions, including biological fluids.

Keywords: light scattering, electrophoretic light scattering, electrophoresis, total internal reflection

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Authors: Alaa M. M. Badr Eldin, Marwa I. Ezzat, Mohammed S. Sedeek, Manal S. Afifi, Omar M. Sabry

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Cytotoxic activity of the total methanol extract against breast cancer cell line MCF-7 was amazing IC50 at 54 ug/ml. 130 polyphenolic compounds were tentatively identified in pomegranate peel (Punica granatum L.) methanol extract using HPLC-MS/MS technique. The antiestrogenic activity of the polyphenolic constituents found in pomegranate extract was confirmed experimentally in-vitro and by the in-silico molecular docking using gallagic acid, ellagic acid, and Punicalagin as these are considered model compounds confirmed in pomegranate peel extract. The methanolic extract was found to suppress ER, TGF-β, and NF-kB in-vitro gene expression strongly, and that was verified by qPCR and Western Blot gel electrophoresis techniques.

Keywords: HPLC-MS/MS, pomegranate, breast cancer, ovarian cancer, ER, TGF-β, NF-kB

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Authors: Hanaa A. S. Oraby, Amal A. M. Hassan, Mahmoud M. Sakr, Atef A. A. Haiba

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Applying the profiling approach for the assessment of proteomic variations due to genetic modification of the Egyptian tomato cultivar "Edkawy", three complementary approaches were used. These methods are amino acids analysis, gel electrophoresis, and Gas chromatography coupled with mass spectrometry (GC/MS). The results of the present study Show evidence of proteomic variations between both modified tomato and its non-modified counterpart. Amino acids concentrations, and the protein patterns separation on the 1D SDS-PAGE were not similar in the case of transformed tomato compared to that of the non-transformed counterpart. These detected differences are most likely derived from the process of transformation. Results also revealed that the efficiency of GC/MS approach to identify a mixture of unknown proteins is limited. GC/MS analysis was only able to identify few number of protein molecules. Therefore, more advanced and specific technologies like MALDI-TOF-MS are recommended to be employed.

Keywords: GMOs, unintended effects, proteomic variations, 1D SDS-PAGE, GC/MS

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Authors: Wang Yanping Karen, Mohd Rafeah Siti, Park MI Kyoung

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Formalin-fixed paraffin embedded (FFPE) tissue is important in the area of oncological diagnostics. This method of preserving tissues enabling them to be stored easily at ambient temperature for a long time. This decreases the risk of losing the DNA quantity and quality after extraction, reducing sample wastage, and making FFPE more cost effective. However, extracting DNA from FFPE tissue is a challenge as DNA purified is often highly cross-linked, fragmented, and degraded. In addition, this causes problems for many downstream processes. In this study, there will be a comparison of DNA extraction efficiency between One BioMed’s Xceler8 automated platform with commercial available extraction kits (Qiagen and Roche). The FFPE tissue slices were subjected to deparaffinization process, pretreatment and then DNA extraction using the three mentioned platforms. The DNA quantity were determined with real-time PCR (BioRad CFX ) and gel electrophoresis. The amount of DNA extracted with the One BioMed’s X8 platform was found to be comparable with the other two manual extraction kits.

Keywords: DNA extraction, FFPE tissue, qiagen, roche, one biomed X8

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Authors: Mari Jaakkola, Jasmiina Haverinen, Tiina Tolonen, Vesa Virtanen

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These process monitoring of biogas plant gives valuable information of the function of the process and help to maintain a stable process. The costs of basic monitoring are often much lower than the costs associated with re-establishing a biologically destabilised plant. Reactor acidification through reactor overload is one of the most common reasons for process deterioration in anaerobic digesters. This occurs because of a build-up of volatile fatty acids (VFAs) produced by acidogenic and acetogenic bacteria. VFAs cause pH values to decrease, and result in toxic conditions in the reactor. Ammonia ensures an adequate supply of nitrogen as a nutrient substance for anaerobic biomass and increases system's buffer capacity, counteracting acidification lead by VFA production. However, elevated ammonia concentration is detrimental to the process due to its toxic effect. VFAs are considered the most reliable analytes for process monitoring. To obtain accurate results, sample storage and transportation need to be carefully controlled. This may be a challenge for off-line laboratory analyses especially when the plant is located far away from the laboratory. The aim of this study was to investigate the correlation between fatty acids, ammonium, and bacteria in the anaerobic digestion samples obtained from an industrial biogas factory. The stability of the analytes was studied comparing the results of the on-site analyses performed in the factory site to the results of the samples stored at room temperature and -18°C (up to 30 days) after sampling. Samples were collected in the biogas plant consisting of three separate mesofilic AD reactors (4000 m³ each) where the main feedstock was swine slurry together with a complex mixture of agricultural plant and animal wastes. Individual VFAs, ammonium, and nutrients (K, Ca, Mg) were studied by capillary electrophoresis (CE). Longer chain fatty acids (oleic, hexadecanoic, and stearic acids) and bacterial profiles were studied by GC-MSD (Gas Chromatography-Mass Selective Detector) and 16S rDNA, respectively. On-site monitoring of the analytes was performed by CE. The main VFA in all samples was acetic acid. However, in one reactor sample elevated levels of several individual VFAs and long chain fatty acids were detected. Also bacterial profile of this sample differed from the profiles of other samples. Acetic acid decomposed fast when the sample was stored in a room temperature. All analytes were stable when stored in a freezer. Ammonium was stable even at a room temperature for the whole testing period. One reactor sample had higher concentration of VFAs and long chain fatty acids than other samples. CE was utilized successfully in the on-site analysis of separate VFAs and NH₄ in the biogas production site. Samples should be analysed in the sampling day if stored in RT or freezed for longer storage time. Fermentation reject can be stored (and transported) at ambient temperature at least for one month without loss of NH₄. This gives flexibility to the logistic solutions when reject is used as a fertilizer.

Keywords: anaerobic digestion, capillary electrophoresis, ammonium, bacteria

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Authors: Jian Peng, Wenhui Xiong, Zheng Lu

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An optimal recipe of amendment (nutrients and electron acceptors) was developed and dominant indigenous benzene-degrading microorganisms were characterized in this study. Lessons were learnt from the development of the optimal amendment recipe: (1) salinity and substantial initial concentration of benzene were detrimental for benzene biodegradation; (2) large dose of amendments can shorten the lag time for benzene biodegradation occurrence; (3) toluene was an essential co-substance for promoting benzene degradation activity. The stable isotope probing study identified incorporation 13C from 13C-benzene into microorganisms, which can be considered as a direct evidence of the occurrence of benzene biodegradation. The dominant mechanism for benzene removal was identified by quantitative polymerase chain reaction analysis to be nitrate reduction. Microbial analyses (denaturing gradient gel electrophoresis and 16S ribosomal RNA) demonstrated that members of genus Dokdonella spp., Pusillimonas spp., and Advenella spp. were predominant within the microbial community and involved in the anaerobic benzene bioremediation.

Keywords: benzene, enhanced anaerobic bioremediation, stable isotope probing, biosep biotrap

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Authors: Purva Nemavarkar, Badri Narain Pandey, Jitendra Kumar

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Introduction: The objective was to understand the effect of various radioprotectors on DNA damage repair enzyme and survival in gamma-irradiated wild and cdc mutants of S. pombe (fission yeast) cultured under permissive and restrictive conditions. DNA repair process, as influenced by radioprotectors, was measured by activity of DNA polymerase in the cells. The use of single cell gel electrophoresis assay (SCGE) or Comet Assay to follow gamma-irradiation induced DNA damage and effect of radioprotectors was employed. In addition, studying the effect of caffeine at different concentrations on S-phase of cell cycle was also delineated. Materials and Methods: S. pombe cells grown at permissive temperature (250C) and/or restrictive temperature (360C) were followed by gamma-radiation. Percentage survival and activity of DNA Polymerase (yPol II) were determined after post-irradiation incubation (5 h) with radioprotectors such as Caffeine, Curcumin, Disulphiram, and Ellagic acid (the dose depending on individual D 37 values). The gamma-irradiated yeast cells (with and without the radioprotectors) were spheroplasted by enzyme glusulase and subjected to electrophoresis. Radio-resistant cells were obtained by arresting cells in S-phase using transient treatment of hydroxyurea (HU) and studying the effect of caffeine at different concentrations on S-phase of cell cycle. Results: The mutants of S. pombe showed insignificant difference in survival when grown under permissive conditions. However, growth of these cells under restrictive temperature leads to arrest in specific phases of cell cycle in different cdc mutants (cdc10: G1 arrest, cdc22: early S arrest, cdc17: late S arrest, cdc25: G2 arrest). All the cdc mutants showed decrease in survival after gamma radiation when grown at permissive and restrictive temperatures. Inclusion of the radioprotectors at respective concentrations during post irradiation incubation showed increase in survival of cells. Activity of DNA polymerase enzyme (yPol II) was increased significantly in cdc mutant cells exposed to gamma-radiation. Following SCGE, a linear relationship was observed between doses of irradiation and the tail moments of comets. The radioprotection of the fission yeast by radioprotectors can be seen by the reduced tail moments of the yeast comets. Caffeine also exhibited its radio-protective ability in radio-resistant S-phase cells obtained after HU treatment. Conclusions: The radioprotectors offered notable radioprotection in cdc mutants when added during irradiation. The present study showed activation of DNA damage repair enzyme (yPol II) and an increase in survival after treatment of radioprotectors in gamma irradiated wild type and cdc mutants of S. pombe cells. Results presented here showed feasibility of applying SCGE in fission yeast to follow DNA damage and radioprotection at high doses, which are not feasible with other eukaryotes. Inclusion of caffeine at 1mM concentration to S phase cells offered protection and did not decrease the cell viability. It can be proved that at minimal concentration, caffeine offered marked radioprotection.

Keywords: radiation protection, cell cycle, fission yeast, comet assay, s-phase, DNA repair, radioprotectors, caffeine, curcumin, SCGE

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Authors: Hamed A. Keykha, Hadi Miri

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This paper describes a new method of soil stabilisation, electro-biogrouting (EBM), for improvement of soft soil with low hydraulic conductivity. This method uses an applied voltage gradient across the soil to induce the ions and bacteria cells through the soil matrix, resulting in CaCO3 precipitation and an increase of the soil shear strength in the process. The EBM were used effectively with two injection methods; bacteria injection and products of bacteria injection. The bacteria cells, calcium ions and urea were moved across the soil by electromigration and electro osmotic flow respectively. The products of bacteria (CO3-2) were moved by electromigration. The results showed that the undrained shear strength of the soil increased from 6 to 65 and 70 kPa for first and second injection method respectively. The injection of carbonate solution and calcium could be effectively flowed in the clay soil compare to injection of bacteria cells. The detection of CaCO3 percentage and its corresponding water content across the specimen showed that the increase of undrained shear strength relates to the deposit of calcite crystals between soil particles.

Keywords: Sporosarcina pasteurii, electrophoresis, electromigration, electroosmosis, biocement

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Authors: A. Jamsheera, F. Arjmand, D. K. Mohapatra

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Small molecules binding to specific sites along DNA molecule are considered as potential chemotherapeutic agents. Their role as mediators of key biological functions and their unique intrinsic properties make them particularly attractive therapeutic agents. Keeping in view, novel dipeptide complexes Cu(II)-Val-Pro (1), Zn(II)-Val-Pro (2), Cu(II)-Ala-Pro (3) and Zn(II)-Ala-Pro (4) were synthesized and thoroughly characterized using different spectroscopic techniques including elemental analyses, IR, NMR, ESI–MS and molar conductance measurements. The solution stability study carried out by UV–vis absorption titration over a broad range of pH proved the stability of the complexes in solution. In vitro DNA binding studies of complexes 1–4 carried out employing absorption, fluorescence, circular dichroism and viscometric studies revealed the binding of complexes to DNA via groove binding. UV–vis titrations of 1–4 with mononucleotides of interest viz., 5´-GMP and 5´-TMP were also carried out. The DNA cleavage activity of the complexes 1 and 2 were ascertained by gel electrophoresis assay which revealed that the complexes are good DNA cleavage agents and the cleavage mechanism involved a hydrolytic pathway. Furthermore, in vitro antitumor activity of complex 1 was screened against human cancer cell lines of different histological origin.

Keywords: dipeptide Cu(II) and Zn(II) complexes, DNA binding profile, pBR322 DNA cleavage, in vitro anticancer activity

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Authors: Luke Andrighetto, Paul G. Stevenson, Luke C. Henderson, Jim Pearson, Xavier A. Conlan

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As pseudoephedrine, a common ingredient in cold and flu medications is closely monitored and restricted in Australia, alternative methods of accessing it are of interest. The impurities and by-products of every reaction step of pseudoephedrine/ephedrine and methamphetamine synthesis have been mapped in order to develop a chemical fingerprint based on synthetic route. Likewise, seized methamphetamine contains a combination of different cutting agents and starting materials. Therefore, in-silico optimised two-dimensional HPLC with DryLab® and OpenMS® software has been used to efficiently separate complex seizure samples. An excellent match between simulated and real separations was observed. Targeted separation of model compounds was completed with significantly reduced method development time. This study produced a two-dimensional separation regime that offers unprecedented separation power (separation space) while maintaining a rapid analysis time that is faster than those previously reported for gas chromatography, single dimension high performance liquid chromatography or capillary electrophoresis.

Keywords: chemical fingerprint, ephedrine, methamphetamine, two-dimensional HPLC

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Authors: Mariedel Autriz, Angel Lambio, Renato Vega, Severino Capitan, Rita Laude

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The study was conducted to associate genetic polymorphism of the leptin gene T3469C with reproductive performance in purebred sows. DNA were isolated from hair follicles of 29 Landrace and 24 Large White sows. Amplification of the leptin gene was done followed by Hinf1digestion to determine the base at the T3469C site. Electrophoresis of the digestion products revealed that there were 25 Landrace and 15 Large White sows with the TT genotype while there were 3 Landrace and 6 Large White TC. There was 1 CC for Landrace and 3 for Large White. Significant genotype associations were observed for total litter size born and total born alive. Significant breed differences, on the other hand, was observed for gestation length and average birth weight. Significant breed by genotype interaction was observed in litter size total born and litter size born alive.

Keywords: genetic polymorphism, leptin, swine, T3469C

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Authors: Malik SS, Masood N, Mubarik S, Khadim TM

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Introduction: Xeroderma pigmentosum group G (XPG) gene plays a crucial role in the correction of UV-induced DNA damage through nucleotide excision repair pathway. Single nucleotide polymorphisms in XPG gene have been reported to be associated with different cancers. Current case-control study was designed to evaluate the relationship between one of the most frequently found XPG (rs1047768 T>C) polymorphism and breast cancer risk. Methodology: A total of 200 individuals were screened for this polymorphism including 100 pathologically confirmed breast cancer cases and age-matched 100 controls. Genotyping was carried out using Tetra amplification-refractory mutation system (ARMS) PCR and results were confirmed by gel electrophoresis. Results: Conditional logistic regression analysis showed significant association between TC genotype (OR: 8.9, CI: 2.0 – 38.7) and increased breast cancer risk. Although homozygous CC genotype was more frequent in patients as compared to controls, but it was statistically non-significant (OR: 3.9, CI: 0.4 – 35.7). Conclusion: In conclusion, XPG (rs1047768 T>C) polymorphism may contribute towards increased risk of breast cancer but other polymorphisms may also be evaluated to elucidate their role in breast cancer.

Keywords: XPG, breast cancer, NER, ARMS-PCR

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Authors: David D. Adams, Ibrahim B. Bello

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Spent oil contaminated Soil from ten selected mechanic workshops were investigated for their bacteria and bioremediation potentials. The bacterial isolates were morphologically and molecularly identified as Enterobacter hormaechei, Escherichia coli, Klebsiella pneumoniae, Shigella flexneri , Wesiella cibaria, Lactobacillus planetarium. The singles and a consortium of these bacteria incubated in the minimal salt medium incorporated with 1% engine oil exhibited various biodegradation rates, with the mixed consortium exhibiting the highest for this oil. The gene for the hydrocarbon enzyme Catechol 2, 3 dioxygenase (C2,30) was detected and amplified in Enterobacter hormaechei, Escherichia coli and Shigella flexneri using PCR and Agarose gel electrophoresis. The detection of the (C2,30) enzyme gene in, and the spent oil biodegradation activity exhibited by these bacteria suggest their possible possession of bioremediating potentials for the spent engine oil. It is therefore suggested that a pilot study on the field application of these bacteria for bioremediation and restoration of spent oil polluted environment should be done in mechanic workshops.

Keywords: spent engine oil, pollution, bacteria, enzyme, bioremediation, mechanic workshop

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Authors: C. Asha Poorna, N. S. Pradeep

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The biological function of lipase is to catalyze the hydrolysis of triacylglycerols to give free fatty acid, diacylglycerols, mono-acylglycerols and glycerol. They constitute the most important group of biocatalysts for biotechnological applications. The aim of the present study was to identify the lipolytic activity of Streptomyces sp. From soil sample collected from the sacred groves of southern Kerala. The culture conditions of the isolate were optimised and the enzyme was purified and characterised. The purification was attempted with acetone precipitation. The isolate observed to have high lipolytic activity and identified to be of Streptomyces strain. The purification was attempted with acetone precipitation. The purified enzyme observed to have an apparent molecular mass of ~60kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed maximum activity at 60oC and pH-8. The lipase showed tolerance towards different organic solvents like ethanol and methanol that are commonly used in transesterification reactions to displace alcohol from triglycerides contained in renewable resources to yield fatty acid alkyl esters known as biodiesel.

Keywords: lipase, Streptomyces, biodiesel, fatty acid, transesterification

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Authors: Masoud Alipanah, Sekineh Akbari, Gholamreza Dashab

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Myostatin genes or factor 8 affecting on growth and making differentiation works (GDF8) as a moderator in the development of skeletal muscle inhibitor. If mutations occurs in the coding region of myostatin, alter its inhibitory role and the muscle growth is increased. In this study, blood samples were collected randomly from 60 Kurdish sheep in northern Khorasan and DNA extraction was performed using a modified salt. A fragment 337 bp from exon 3 myostatin gene and-specific primers by using a polymerase chain reaction (PCR) were amplified. In order to detect different forms of an allele at this locus HaeΙΙΙ restriction enzymes and PCR-RFLP analysis were used. Band patterns clarification was performed using agarose gel electrophoresis. The frequency of genotypes mm, Mm, and MM, were respectively detected, 0, 0.15 and 0.85. The allele frequency for alleles m and M, were respectively, 0.07 and 0.93. The statistical analyses indicated that m allele was significantly associated with body weight. The results of this study suggest that the Myostatin gene possibly is a candidate gene that affects growth traits in Kurdish sheep.

Keywords: GDF8 gene, Kurdi Sheep of Northern Khorasan, polymorphism, weight traits

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

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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, antibiotics

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Authors: Chen Wang, Chun Liang

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Microsatellite instability (MSI) is characterized by high degree of polymorphism in microsatellite (MS) length due to a deficiency in mismatch repair (MMR) system. MSI is associated with several tumor types and its status can be considered as an important indicator for tumor prognostic. Conventional clinical diagnosis of MSI examines PCR products of a panel of MS markers using electrophoresis (MSI-PCR) which is laborious, time consuming, and less reliable. MSIpred, a python 2 package for automatic classification of MSI was released by this study. It computes important somatic mutation features from files in mutation annotation format (MAF) generated from paired tumor-normal exome sequencing data, subsequently using these to predict tumor MSI status with a support vector machine (SVM) classifier trained by MAF files of 1074 tumors belonging to four types. Evaluation of MSIpred on an independent 358-tumor test set achieved overall accuracy of over 98% and area under receiver operating characteristic (ROC) curve of 0.967. These results indicated that MSIpred is a robust pan-cancer MSI classification tool and can serve as a complementary diagnostic to MSI-PCR in MSI diagnosis.

Keywords: microsatellite instability, pan-cancer classification, somatic mutation, support vector machine

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Authors: H. Bouaziz, M. Sefi, J. de Lapuente, M. Borras, N. Zeghal

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Although arsenic trioxide has been the subject of toxicological research, in vitro cytotoxicity and genotoxicity studies using relevant cell models and uniform methodology are not well elucidated. Hence, the aim of the present study was to evaluate the cytotoxicity and genotoxicity induced by arsenic trioxide in human keratinocytes (HaCaT) using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and alkaline single cell gel electrophoresis (Comet) assays, respectively. Human keratinocytes were treated with different doses of arsenic trioxide for 4 h prior to cytogenetic assessment. Data obtained from the MTT assay indicated that arsenic trioxide significantly reduced the viability of HaCaT cells in a dose-dependent manner, showing a IC50 value of 34.18 ± 0.6 µM. Data generated from the comet assay also indicated a significant dose-dependent increase in DNA damage in HaCaT cells associated with arsenic trioxide exposure. We observed a significant increase in comet tail length and tail moment, showing an evidence of arsenic trioxide -induced genotoxic damage in HaCaT cells. This study confirms that the comet assay is a sensitive and effective method to detect DNA damage caused by arsenic.

Keywords: arsenic trioxide, cytotoxixity, genotoxicity, HaCaT

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Authors: Saleh H. Salmen, Sulaiman Ali Alharbi, Hany M. Yehia, Mohammad A. Khiyami, Milton Wainwright, Naiyf S. Alharbi, Arunachalam Chinnathambi

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An antifungal protein synthesized by Bacillus cereus has been partially purified by the use of ammonium sulfate precipitation and Sephadex-G-200 column chromatography. The protein was produced from Bacillus cereus grown in potato Dextrose Broth Medium (PDB) at 30 ºC for 3 days at 100 rpm. The protein showed antagonistic effect against some fungi and yeasts. Crude extract from medium and semi-purified protein were tested in vitro against both fungi and yeasts using the disc diffusion method in order to detect the inhibitory effect of the protein. Zones of inhibition of the following diameter were found (mm) were Alternaria alternate (28), Rhodotorula glutinis (20), Fusarium sp. (16), Rhizopus sp. (15), Penicillium digitatum (13), Mucor sp. (13) and Aspergillus niger (10). The isolated protein was found to have a molecular weight of ~35kDa by sodium deodecyl sulfate-poly acrylamide gel electrophoresis. The data showed that the protein of Bacillus cereus has antifungal activity, a fact which points to the possibility of using it as a bio-control agent against some fungi, findings which emphasize the potential role of B. cereus as an important bio-control agent.

Keywords: bacillus cereus, ~35kDa protein, molds, yeasts

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Authors: Dulanjali Ranasinghe, Isuru Supasan, Kaushalya Premachandra, Ranjan Dissanayake, Ajith Rajapaksha, Eustace Fernando

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Proteomics studies of organisms are considered to be significantly information-rich compared to their genomic counterparts because proteomes of organisms represent the expressed state of all proteins of an organism at a given time. In modern top-down and bottom-up proteomics workflows, the primary analysis methods employed are gel–based methods such as two-dimensional (2D) electrophoresis and mass spectrometry based methods. Machine learning (ML) and artificial intelligence (AI) have been used increasingly in modern biological data analyses. In particular, the fields of genomics, DNA sequencing, and bioinformatics have seen an incremental trend in the usage of ML and AI techniques in recent years. The use of aforesaid techniques in the field of proteomics studies is only beginning to be materialised now. Although there is a wealth of information available in the scientific literature pertaining to proteomics workflows, no comprehensive review addresses various aspects of the combined use of proteomics and machine learning. The objective of this review is to provide a comprehensive outlook on the application of machine learning into the known proteomics workflows in order to extract more meaningful information that could be useful in a plethora of applications such as medicine, agriculture, and biotechnology.

Keywords: proteomics, machine learning, gel-based proteomics, mass spectrometry

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Authors: Xueying Zhao, Ke Ma, Hui Li, Yu Cao, Fan Yang, Qingwen Xu, Wenbin Liu

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Massively parallel sequencing (MPS) technologies allow high-throughput sequencing analyses with a relatively affordable price and have gradually been applied to forensic casework. MPS technology identifies short tandem repeat (STR) loci based on sequence so that repeat motif variation within STRs can be detected, which may help one to infer the origin of the mutation in some cases. Here, we report on one case with one three-step mismatch (D18S51) in family trios based on both capillary electrophoresis (CE) and MPS typing. The alleles of the alleged father (AF) are [AGAA]₁₇AGAG[AGAA]₃ and [AGAA]₁₅. The mother’s alleles are [AGAA]₁₉ and [AGAA]₉AGGA[AGAA]₃. The questioned child’s (QC) alleles are [AGAA]₁₉ and [AGAA]₁₂. Given that the sequence variants in repeat regions of AF and mother are not observed in QC’s alleles, the QC’s allele [AGAA]₁₂ was likely inherited from the AF’s allele [AGAA]₁₅ by loss of three repeat [AGAA]. Besides, two new alleles of D18S51 in this study, [AGAA]₁₇AGAG[AGAA]₃ and [AGAA]₉AGGA[AGAA]₃, have not been reported before. All the results in this study were verified using Sanger-type sequencing. In summary, the MPS typing method can offer valuable information for forensic genetics research and play a promising role in paternity testing.

Keywords: family trios analysis, forensic casework, ion torrent personal genome machine (PGM), massively parallel sequencing (MPS)

Procedia PDF Downloads 302

Authors: Dina Helmy El-Ghonemy, Thanaa Hamed Ali

Abstract:

The aim of this study was to purify and characterize a keratinolytic enzyme produced by Aspergillus sp. DHE7 cultured in basal medium containing chicken feather as substrate. The enzyme was purified through ammonium sulfate saturation of 60%, followed by gel filtration chromatography in Sephadex G-100, with a 16.4-purification fold and recovery yield of 52.2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme is a monomeric enzyme with an apparent molecular mass of 30 kDa — the purified keratinase of Aspergillus sp. DHE7 exhibited activity in a broad range of pH (7- 9) and temperature (40℃-60℃) profiles with an optimal activity at pH eight and 50℃. The keratinolytic activity was inhibited by protease inhibitors such as phenylmethylsulfonyl fluoride and ethylenediaminetetraacetate, while no reduction of activity was detected by the addition of dimethyl sulfoxide (DMSO). Bivalent cations, Ca²⁺ and Mn²⁺, were able to greatly enhance the activity of keratinase by 125.7% and 194.8%, respectively, when used at one mM final concentration. On the other hand, Cu²⁺ and Hg²⁺ inhibited the enzyme activity, which might be indicative of essential vicinal sulfhydryl groups of the enzyme for productive catalysis. Furthermore, the purified keratinase showed significant stability and compatibility against the tested commercial detergents at 37ºC. Therefore, these results suggested that the purified keratinase from Aspergillus sp. DHE7 may have potential use in the detergent industry and should be of interest in the processing of poultry feather waste.

Keywords: Aspergillus sp. DHE7, biochemical characterization, keratinase, purification, waste management

Procedia PDF Downloads 124

Authors: Nutnicha Wongpadungkiat, Suwit Siriwatanayotin, Aluck Thipayarat, Punchira Vongsawasdi, Chotika Viriyarattanasak

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Chicken products are major export product of Thailand. With a dramatically increasing consumption of chicken product in the world, there are abundant wastes from chicken meat processing industry. Recently, much research in the development of value-added products from chicken meat industry has focused on the production of protein hydrolysate, utilized as food ingredients for human diet and animal feed. The present study aimed to determine the effect of thermal pre-treatment on functional properties of chicken protein hydrolysate. Chicken breasts were heated at 40, 60, 80 and 100ºC prior to hydrolysis by Alcalase at 60ºC, pH 8 for 4 hr. The hydrolysate was freeze-dried, and subsequently used for assessment of its functional properties molecular weight by gel electrophoresis (SDS-PAGE). The obtained results show that increasing the pre-treatment temperature increased oil holding capacity and emulsion stability while decreasing antioxidant activity and water holding capacity. The SDS-PAGE analysis showed the evidence of protein aggregation in the hydrolysate treated at the higher pre-treatment temperature. These results suggest the connection between molecular weight of the hydrolysate and its functional properties.

Keywords: chicken protein hydrolysate, enzymatic hydrolysis, thermal pretreatment, functional properties

Procedia PDF Downloads 270

Authors: A. Ameur Ameur, F. Chougrani, M. Halbouche

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In order to characterize the sheep's milk, we analyzed and compared, in a first stage of our work, the physical and chemical characteristics in two Algerian sheep breeds: Hamra race and race Ouled Djellal breeding at the station the experimental ITELV Ain Hadjar (Saïda Province). Analyses are performed by Ekomilk Ultra-analyzer (EON TRADING LLC, USA), they focused on the pH, density, freezing, fat, total protein, solids-the total dry extract. The results obtained for these parameters showed no significant differences between the two breeds studied. The second stage of this work was the isolation and characterization of milk proteins. For this, we used the precipitation of caseins phi [pH 4.6]. For this, we used the precipitation of caseins Phi (pH 4.6). After extraction, purification and assay, both casein and serum protein fractions were then assayed by the Bradford method and controlled by polyacrylamide gel electrophoresis (PAGE) in the different conditions (native, in the presence of urea and in the presence of SDS). The electrophoretic pattern of milk samples showed the presence similarities of four major caseins variants (αs1-, αs2-β-and k-casein) and two whey proteins (β-lactoglobulin, α-lactalbumin) of two races Hamra and Ouled Djellal. But compared to bovine milk, they have helped to highlight some peculiarities as related to serum proteins (α La β Lg) as caseins, including αs1-Cn.

Keywords: Hamra, Ouled Djellal, protein polymorphism, sheep breeds

Procedia PDF Downloads 557

Authors: Kalaumari Mayoral-Peña, Ana I. Montejano-Montelongo, Josué Reyes-Muñoz, Gonzalo A. Ortiz-Mancilla, Mayrin Rodríguez-Cruz, Víctor Hernández-Villalobos, Jesús A. Guzmán-López, Santiago García-Jacobo, Iván Licona-Vázquez, Grisel Fierros-Romero, Rosario Flores-Vallejo

Abstract:

The group B-lactamic antibiotics include some of the most frequently used small drug molecules against bacterial infections. Nevertheless, an alarming decrease in their efficacy has been reported due to the emergence of antibiotic-resistant bacteria. Infections caused by bacteria expressing extended Spectrum B-lactamases (ESBLs) are difficult to treat and account for higher morbidity and mortality rates, delayed recovery, and high economic burden. According to the Global Report on Antimicrobial Resistance Surveillance, it is estimated that mortality due to resistant bacteria will ascend to 10 million cases per year worldwide. These facts highlight the importance of developing low-cost and readily accessible detection methods of drug-resistant ESBLs bacteria to prevent their spread and promote accurate and fast diagnosis. Bacterial detection is commonly done using molecular diagnostic techniques, where PCR stands out for its high performance. However, this technique requires specialized equipment not available everywhere, is time-consuming, and has a high cost. Loop-Mediated Isothermal Amplification (LAMP) is an alternative technique that works at a constant temperature, significantly decreasing the equipment cost. It yields double-stranded DNA of several lengths with repetitions of the target DNA sequence as a product. Although positive and negative results from LAMP can be discriminated by colorimetry, fluorescence, and turbidity, there is still a large room for improvement in the point-of-care implementation. DNA origami is a technique that allows the formation of 3D nanometric structures by folding a large single-stranded DNA (scaffold) into a determined shape with the help of short DNA sequences (staples), which hybridize with the scaffold. This research aimed to generate DNA origami structures using LAMP products as scaffolds to improve the sensitivity to detect ESBLs in point-of-care diagnosis. For this study, the coding sequence of the CTM-X-15 ESBL of E. coli was used to generate the LAMP products. The set of LAMP primers were designed using PrimerExplorerV5. As a result, a target sequence of 200 nucleotides from CTM-X-15 ESBL was obtained. Afterward, eight different DNA origami structures were designed using the target sequence in the SDCadnano and analyzed with CanDo to evaluate the stability of the 3D structures. The designs were constructed minimizing the total number of staples to reduce costs and complexity for point-of-care applications. After analyzing the DNA origami designs, two structures were selected. The first one was a zig-zag flat structure, while the second one was a wall-like shape. Given the sequence repetitions in the scaffold sequence, both were able to be assembled with only 6 different staples each one, ranging between 18 to 80 nucleotides. Simulations of both structures were performed using scaffolds of different sizes yielding stable structures in all the cases. The generation of the LAMP products were tested by colorimetry and electrophoresis. The formation of the DNA structures was analyzed using electrophoresis and colorimetry. The modeling of novel detection methods through bioinformatics tools allows reliable control and prediction of results. To our knowledge, this is the first study that uses LAMP products and DNA-origami in combination to delect ESBL-producing bacterial strains, which represent a promising methodology for diagnosis in the point-of-care.

Keywords: beta-lactamases, antibiotic resistance, DNA origami, isothermal amplification, LAMP technique, molecular diagnosis

Procedia PDF Downloads 222

Authors: Kesavan Markkandan, Ha Young Park, Seung-Il Yoo, Sin-Gi Park, Junhyung Park

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For genetic identification of crop cultivars, insertions/deletions (InDel) markers have been preferred currently because they are easy to use, PCR based, co-dominant and relatively abundant. However, new InDels need to be developed for genetic studies of new varieties due to the difference of allele frequencies in InDels among the population groups. These new varieties are evolved with low levels of genetic diversity in specific genome loci with high recombination rate. In this study, we described soybean barcode system approach based on InDel makers, each of which is specific to a variation block (VB), where the genomes split by all assumed recombination sites. Firstly, VBs in crop cultivars were mined for transferability to VB-specific InDel markers. Secondly, putative InDels in the VB regions were identified for the development of barcode system by analyzing particular cultivar’s whole genome data. Thirdly, common VB-specific InDels from all cultivars were selected by gel electrophoresis, which were converted as 2D barcode types according to comparing amplicon polymorphisms in the five cultivars to the reference cultivar. Finally, the polymorphism of the selected markers was assessed with other cultivars, and the barcode system that allows a clear distinction among those cultivars is described. The same approach can be applicable for other commercial crops. Hence, VB-based genetic identification not only minimize the molecular markers but also useful for assessing cultivars and for marker-assisted breeding in other crop species.

Keywords: variation block, polymorphism, InDel marker, genetic identification

Procedia PDF Downloads 380

Authors: Anamarel Medina-Hernandez, Teresa Ponce-Noyola, Ileana Vera-Reyes, Ana C. Ramos-Valdivia

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Somatic embryos reproduce original seed characteristics and could be implemented in biotechnological studies. Jatropha curcas L. is an important plant for biodiesel production, but also is used in traditional medicine. Seeds from J. curcas are toxic because contain diterpenoids called phorbol esters, but in Mexico exist a non-toxic variety. Therefore, somatic embryos suspension cultures from non-toxic J. curcas variety were induced. In order to investigate the characteristics of somatic embryos, a differential proteomic analysis was made between pre-globular and globular stages by 2-D gel electrophoresis. 108 spots were differentially expressed (p<0.02), and 20 spots from globular somatic embryos were sequenced by MALDI-TOF-TOF mass spectrometry. A comparative analysis of terpenoids production between the two stages was made by RP-18 TLC plates. The sequenced proteins were related to energy production (68%), protein destination and storage (9%), secondary metabolism (9%), signal transduction (5%), cell structure (5%) and aminoacid metabolism (4%). Regarding terpenoid production, in pre-globular and globular somatic embryos were identified sterols and triterpenes of pharmacological interest (alpha-amyrin and betulinic acid) but also it was found compounds that were unique to each stage. The results of this work are the basis to characterize at different levels the J. curcas somatic embryos so that this system can be used efficiently in biotechnological processes.

Keywords: Jatropha curcas, proteomics, somatic embryo, terpenoids

Procedia PDF Downloads 254