Search results for: recombinant protein purification

Authors: Demiraw Bikila, Tadesse Lejisa, Yosef Tolcha, Chala Bashea, Mehari Meles Tigist Getahun Genet Ashebir, Wossene Habtu, Feyissa Challa, Ousman Mohammed, Melkitu Kassaw, Adisu Kebede, Letebrhan G. Egzeabher, Endalkachew Befekadu, Mistire Wolde, Aster Tsegaye

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Background: Even though several factors affect reference intervals (RIs), the company-derived values are currently in use in many laboratories worldwide. However, little or no data is available regarding serum protein RIs, mainly in resource-limited setting countries like Ethiopia. Objective: To establish a reference interval for serum protein electrophoresis of apparently healthy adults in Addis Ababa, Ethiopia. Method: A cross-sectional study was conducted on a total of 297 apparently healthy adults from April-October 2019 in four selected sub-cities (Akaki, Kirkos, Arada, Yeka) of Addis Ababa, Ethiopia. Laboratory analysis of collected samples was performed using Capillarys 2 Flex Piercing analyzer, while statistical analysis was done using SPSS version 23 and med-cal software. Mann-Whitney test was used to check Partitions. Non-parametric method of reference range establishment was performed as per CLSI guideline EP28A3C. Result: The established RIs were: Albumin 53.83-64.59%, 52.24-63.55%; Alpha-1 globulin 3.04-5.40%, 3.44-5.60%; Alpha-2 globulin 8.0-12.67%, 8.44-12.87%; and Beta-1 globulin 5.01-7.38%, 5.14-7.86%. Moreover, Albumin to globulin ratio was 1.16-1.8, 1.09-1.74 for males and females, respectively. The combined RIs for Beta-2 globulin and Gamma globulin were 2.54-4.90% and 12.40-21.66%, respectively. Conclusion: The established reference interval for serum protein fractions revealed gender-specific differences except for Beta-2 globulin and Gamma globulin.

Keywords: serum protein electrophoresis, reference interval, Addis Ababa, Ethiopia

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Authors: Ketaki D. Belsare, Nicholas F. Polizzi, Lior Shtayer, William F. DeGrado

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Nature utilizes metalloproteins to perform chemical transformations with activities and selectivities that have long been the inspiration for design principles in synthetic and biological systems. The chemical reactivities of metalloproteins are directly linked to local environment effects produced by the protein matrix around the metal cofactor. A complete understanding of how the protein matrix provides these interactions would allow for the design of functional metalloproteins. The de novo computational design of proteins have been successfully used in design of active sites that bind metals like di-iron, zinc, copper containing cofactors; however, precisely designing active sites that can bind small molecule ligands (e.g., substrates) along with metal cofactors is still a challenge in the field. The de novo computational design of a functional metalloprotein that contains a purposefully designed substrate binding site would allow for precise control of chemical function and reactivity. Our research strategy seeks to elucidate the design features necessary to bind the cofactor protoporphyrin IX (hemin) in close proximity to a substrate binding pocket in a four helix bundle. First- and second-shell interactions are computationally designed to control orientation, electronic structure, and reaction pathway of the cofactor and substrate. The design began with a parameterized helical backbone that positioned a single histidine residue (as an axial ligand) to receive a second-shell H-bond from a Threonine on the neighboring helix. The metallo-cofactor, hemin was then manually placed in the binding site. A structural feature, pi-bulge was introduced to give substrate access to the protoporphyrin IX. These de novo metalloproteins are currently being tested for their activity towards hydroxylation and epoxidation. The de novo designed protein shows hydroxylation of aniline to 4-aminophenol. This study will help provide structural information of utmost importance in understanding de novo computational design variables impacting the functional activities of a protein.

Keywords: metalloproteins, protein design, de novo protein, biocatalysis

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Authors: Zelal Polat, Şebnem Harsa, Semra Ülkü

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Lactic acid exists in nature optically in two forms, L(+), D(-)-lactic acid, and has been used in food, leather, textile, pharmaceutical and cosmetic industries. Moreover, L(+)-lactic acid constitutes the raw material for the production of poly-L-lactic acid which is used in biomedical applications. Microbially produced lactic acid was aimed to be recovered from the fermentation media efficiently and economically. Among the various downstream operations, ion exchange chromatography is highly selective and yields a low cost product recovery within a short period of time. In this project, Lactobacillus casei NRRL B-441 was used for the production of L(+)-lactic acid from whey by fermentation at pH 5.5 and 37°C that took 12 hours. The product concentration was 50 g/l with 100% L(+)-lactic acid content. Next, the suitable resin was selected due to its high sorption capacity with rapid equilibrium behavior. Dowex marathon WBA, weakly basic anion exchanger in OH form reached the equilibrium in 15 minutes. The batch adsorption experiments were done approximately at pH 7.0 and 30°C and sampling was continued for 20 hours. Furthermore, the effect of temperature and pH was investigated and their influence was found to be unimportant. All the adsorption/desorption experiments were applied to both model lactic acid and biomass free fermentation broth. The ion exchange equilibria of lactic acid and L(+)-lactic acid in fermentation broth on Dowex marathon WBA was explained by Langmuir isotherm. The maximum exchange capacity (qm) for model lactic acid was 0.25 g La/g wet resin and for fermentation broth 0.04 g La/g wet resin. The equilibrium loading and exchange efficiency of L(+)-lactic acid in fermentation broth were reduced as a result of competition by other ionic species. The competing ions inhibit the binding of L(+)-lactic acid to the free sites of ion exchanger. Moreover, column operations were applied to recover adsorbed lactic acid from the ion exchanger. 2.0 M HCl was the suitable eluting agent to recover the bound L(+)-lactic acid with a flowrate of 1 ml/min at ambient temperature. About 95% of bound L(+)-lactic acid was recovered from Dowex marathon WBA. The equilibrium was reached within 15 minutes. The aim of this project was to investigate the purification of L(+)-lactic acid with ion exchange method from fermentation broth. The additional goals were to investigate the end product purity, to obtain new data on the adsorption/desorption behaviours of lactic acid and applicability of the system in industrial usage.

Keywords: fermentation, ion exchange, lactic acid, purification, whey

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Authors: Kshitij Bhardwaj, R.K. Trivedi, Shipra Dixit

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We used biological detoxification method that converts toxic residue waste of Jatropha curcas oil seeds (non edible oil seed) into industrial bio-products and animal feed material. Present study describes the complete degradation of phorbol esters by Aspergillus Niger strain during solid state fermentation (SSF) of deoiled Jatropha curcas seed cake. Phorbol esters were completely degraded in 15 days under the optimized SSF conditions viz deoiled cake 5.0 gm moistened with 5.0 ml distilled water; inoculum 2 ml of overnight grown Aspergillus niger; incubated at 30◦ C, pH 7.0. This method simultaneously induces the production of Protease enzyme by Aspergillus Niger which has high potential to be used in feedstuffs .The maximum Protease activities obtained were 709.16 mg/ml in Jatropha curcas oil seed cake. The protein isolate had small amounts of phorbol esters, phytic acid, and saponin without any lectin. Its minimum and maximum solubility were at pH 4.0&12.0. Water and oil binding capacities were 3.22 g water/g protein and 1.86 ml oil/g protein respectively.Emulsion activity showed high values in a range of basic pH. We concluded that Jatropha Curcas seed cake has a potential to be used as a novel source of functional protein for food or feed applications.

Keywords: solid state fermentation, Jatropha curcas, oil seed cake, phorbol ester

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Authors: W. P. Hu, J. V. Kumar, Jeffrey J. P. Tsai

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The inhibition of SH2 domain regulated protein-protein interactions is an attractive target for developing an effective chemotherapeutic approach in the treatment of disease. Molecular simulation is a useful tool for developing new drugs and for studying molecular recognition. In this study, we searched potential drug compounds for the inhibition of SH2 domain by performing structural similarity search in PubChem Compound Database. A total of 37 compounds were screened from the database, and then we used the LibDock docking program to evaluate the inhibition effect. The best three compounds (AP22408, CID 71463546 and CID 9917321) were chosen for MD simulations after the LibDock docking. Our results show that the compound CID 9917321 can produce a more stable protein-ligand complex compared to other two currently known inhibitors of Src SH2 domain. The compound CID 9917321 may be useful for the inhibition of SH2 domain based on these computational results. Subsequently experiments are needed to verify the effect of compound CID 9917321 on the SH2 domain in the future studies.

Keywords: nonpeptide inhibitor, Src SH2 domain, LibDock, molecular dynamics simulation

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Authors: Mateen A Khan, Taj Mohammad, Md. Imtaiyaz Hassan

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Alzheimer’s disease is characterized by the accumulation of the processing products of the amyloid beta peptide cleaved by amyloid precursor protein (APP). Iron increases the synthesis of amyloid beta peptides, which is why iron is present in Alzheimer's disease patients' amyloid plaques. Iron misregulation in the brain is linked to the overexpression of APP protein, which is directly related to amyloid-β aggregation in Alzheimer’s disease. The APP 5'-UTR region encodes a functional iron-responsive element (IRE) stem-loop that represents a potential target for modulating amyloid production. Targeted regulation of APP gene expression through the modulation of 5’-UTR sequence function represents a novel approach for the potential treatment of AD because altering APP translation can be used to improve both the protective brain iron balance and provide anti-amyloid efficacy. The molecular docking analysis of APP IRE RNA with eukaryotic translation initiation factors yields several models exhibiting substantial binding affinity. The finding revealed that the interaction involved a set of functionally active residues within the binding sites of eIF4F. Notably, APP IRE RNA and eIF4F interaction were stabilized by multiple hydrogen bonds with residues of APP IRE RNA and eIF4F. It was evident that APP IRE RNA exhibited a structural complementarity that tightly fit within binding pockets of eIF4F. The simulation studies further revealed the stability of the complexes formed between RNA and eIF4F, which is crucial for assessing the strength of these interactions and subsequent roles in the pathophysiology of Alzheimer’s disease. In addition, MD simulations would capture conformational changes in the IRE RNA and protein molecules during their interactions, illustrating the mechanism of interaction, conformational change, and unbinding events and how it may affect aggregation propensity and subsequent therapeutic implications. Our binding studies correlated well with the translation efficiency of APP mRNA. Overall, the outcome of this study suggests that the genomic modification and/or inhibiting the expression of amyloid protein by targeting APP IRE RNA can be a viable strategy to identify potential therapeutic targets for AD and subsequently be exploited for developing novel therapeutic approaches.

Keywords: Alzheimer's disease, Protein-RNA interaction analysis, molecular docking simulations, conformational dynamics, binding stability, binding kinetics, protein synthesis.

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Authors: H. Sakamoto, Y. Nakagawara, S. Oguri

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Drought stress is a critical environmental factor that adversely affects crop productivity and quality. Because of their immobile nature, plants have evolved mechanisms to sense and respond to drought stress. We identified a novel locus of Arabidopsis, designated DRA1 (drought responsive ankyrin 1), whose disruption leads to increased drought stress tolerance. DRA1 encodes a transmembrane protein with an ankyrin repeat motif that has been implicated in diverse cellular processes such as signal transduction. RT-PCR analysis revealed that there were at least two splicing variants of DRA1 transcripts in wild type plants. In response to drought stress, the levels of DRA1 transcripts retaining second and third introns were increased, whereas these introns were removed under unstressed conditions. These results suggest that DRA1 protein may negatively regulate plant drought tolerance and that the expression of DRA1 is regulated in response to drought stress by alternative splicing.

Keywords: alternative splicing, ankyrin repeat, Arabidopsis, drought tolerance

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Authors: Pawan Kumar Sharma, J. Stephan Sampath Kumar, S. Anand, Chandana B. L.

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Protein is the major component for the success in culture of shrimp and fishes. Apparently, excess dietary protein is undesirable, as it not only enhances the production cost but also leads to water quality deterioration. A field survey was conducted with aqua farmers of Kerala, India, a leading state in coastal aquaculture, to assess the role of protein component in feed that can be efficiently and effectively managed for sustainable aquaculture. The study showed an average feed amount of 13.55 ± 2.16 tonnes per hectare was being used by the farmers of Kerala. The average feed cost percentage of Rs. 57.76 ± 13.46 /kg was invested for an average protein level of 36.26 % ± 0.082 in the feed and Rs.78.95 ± 3.086 per kilogram of feed was being paid by the farmers. Study revealed that replacement of fish meal and fish oil within shrimp aquafeeds with alternative protein, and lipid sources can only be achieved if changes are made in the basic shrimp culturing practices, such as closed farming system through water recycling or zero-water exchange, and by maximizing in-situ, floc and natural food production within the culture system. The upshot of such production systems is that imports of high-quality feed ingredients and aqua feeds can eventually be eliminated, and the utilization of locally available feed ingredients from agricultural by-products can be greatly improved and maximized. The promotion of closed shrimp production systems would also greatly reduce water use and increase shrimp production per unit area but would necessitate the continuous provision of electricity for aeration during production. Alternative energy sources such as solar power might be used, and resource poor farming communities should also explore wind energy for use. The study concluded that farm made feed and closed farming systems are essential for the sustainability and profitability of the aquaculture industry.

Keywords: aqua feeds, floc, fish meal, protein, zero-water exchange

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Authors: Rosa Buxeda, Lorenzo Saliceti-Piazza, Rodolfo J. Romañach, Luis Ríos, Sandra L. Maldonado-Ramírez

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Biopharmaceuticals manufacturing is one of the major economic activities worldwide. Ninety-three percent of the workforce in a biomanufacturing environment concentrates in production-related areas. As a result, strategic collaborations between industry and academia are crucial to ensure the availability of knowledgeable workforce needed in an economic region to become competitive in biomanufacturing. In the past decade, our institution has been a key strategic partner with multinational biotechnology companies in supplying science and engineering graduates in the field of industrial biotechnology. Initiatives addressing all levels of the educational pipeline, from K-12 to college to continued education for company employees have been established along a ten-year span. The Amgen BioTalents Program was designed to provide undergraduate science and engineering students with training in biomanufacturing. The areas targeted by this educational program enhance their academic development, since these topics are not part of their traditional science and engineering curricula. The educational curriculum involved the process of producing a biomolecule from the genetic engineering of cells to the production of an especially targeted polypeptide, protein expression and purification, to quality control, and validation. This paper will report and describe the implementation details and outcomes of the first sessions of the program.

Keywords: biomanufacturing curriculum, interdisciplinary learning, workforce development, industry-academia partnering

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Authors: Jennifer Cuellar, Angie L. Parada, Kevin N. S. Chacon, Sol M. Mejia

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The purification of bio-ethanol through distillation methods is an unresolved issue at the biofuel industry because of the ethanol-water azeotrope formation, which increases the steps of the purification process and subsequently increases the production costs. Therefore, understanding the mixture nature at the molecular level could provide new insights for improving the current methods and/or designing new and more efficient purification methods. For that reason, the present study focuses on the evaluation and analysis of (ethanol)₉-water heterodecamers, as the systems with the minimum molecular proportion that represents the azeotropic concentration (96 %m/m in ethanol). The computational modelling was carried out with B3LYP-D3/6-311++G(d,p) in Gaussian 09. Initial explorations of the potential energy surface were done through two methods: annealing simulated runs and molecular dynamics trajectories besides intuitive structures obtained from smaller (ethanol)n-water heteroclusters, n = 7, 8 and 9. The energetic order of the seven stable heterodecamers determines the most stable heterodecamer (Hdec-1) as a structure forming a bicyclic geometry with the O-H---O hydrogen bonds (HBs) where the water is a double proton donor molecule. Hdec-1 combines 1 water molecule and the same quantity of every ethanol conformer; this is, 3 trans, 3 gauche 1 and 3 gauche 2; its abundance is 89%, its decamerization energy is -80.4 kcal/mol, i.e. 13 kcal/mol most stable than the less stable heterodecamer. Besides, a way to understand why methanol does not form an azeotropic mixture with water, analogous systems ((ethanol)10, (methanol)10, and (methanol)9-water)) were optimized. Topologic analysis of the electron density reveals that Hec-1 forms 33 weak interactions in total: 11 O-H---O, 8 C-H---O, 2 C-H---C hydrogen bonds and 12 H---H interactions. The strength and abundance of the most unconventional interactions (H---H, C-H---O and C-H---O) seem to explain the preference of the ethanol for forming heteroclusters instead of clusters. Besides, O-H---O HBs present a significant covalent character according to topologic parameters as the Laplacian of electron density and the relationship between potential and kinetic energy densities evaluated at the bond critical points; obtaining negatives values and values between 1 and 2, for those two topological parameters, respectively.

Keywords: ADMP, DFT, ethanol-water azeotrope, Grimme dispersion correction, simulated annealing, weak interactions

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Natalia Caldeira De Carvalho, Tassia Batista Pessato, Luis Gustavo R. Fernandes, Ricardo L. Zollner, Flavia Maria Netto

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Protein hydrolysates are ingredients of enteral diets and hypoallergenic formulas. Enzymatic hydrolysis is the most commonly used method for reducing the antigenicity of milk protein. The antigenicity and physicochemical characteristics of the protein hydrolysates depend on the reaction parameters. Among them, pH has been pointed out as of the major importance. Hydrolysis reaction in laboratory scale is commonly carried out under controlled pH (pH-stat). However, from the industrial point of view, controlling pH during hydrolysis reaction may be infeasible. This study evaluated the impact of pH control on the physicochemical properties and antigenicity of the hydrolysates of whey proteins with Alcalase. Whey protein isolate (WPI) solutions containing 3 and 7 % protein (w/v) were hydrolyzed with Alcalase 50 and 100 U g-1 protein at 60°C for 180 min. The reactions were carried out under controlled and uncontrolled pH conditions. Hydrolyses performed under controlled pH (pH-stat) were initially adjusted and maintained at pH 8.5. Hydrolyses carried out without pH control were initially adjusted to pH 8.5. Degree of hydrolysis (DH) was determined by OPA method, peptides profile was evaluated by HPLC-RP, and molecular mass distribution by SDS-PAGE/Tricine. The residual α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) concentrations were determined using commercial ELISA kits. The specific IgE and IgG binding capacity of hydrolysates was evaluated by ELISA technique, using polyclonal antibodies obtained by immunization of female BALB/c mice with α-La, β-Lg and BSA. In hydrolysis under uncontrolled pH, the pH dropped from 8.5 to 7.0 during the first 15 min, remaining constant throughout the process. No significant difference was observed between the DH of the hydrolysates obtained under controlled and uncontrolled pH conditions. Although all hydrolysates showed hydrophilic character and low molecular mass peptides, hydrolysates obtained with and without pH control exhibited different chromatographic profiles. Hydrolysis under uncontrolled pH released, predominantly, peptides between 3.5 and 6.5 kDa, while hydrolysis under controlled pH released peptides smaller than 3.5 kDa. Hydrolysis with Alcalase under all conditions studied decreased by 99.9% the α-La and β-Lg concentrations in the hydrolysates detected by commercial kits. In general, β-Lg concentrations detected in the hydrolysates obtained under uncontrolled pH were significantly higher (p<0.05) than those detected in hydrolysates produced with pH control. The anti-α-La and anti-β-Lg IgE and IgG responses to all hydrolysates decreased significantly compared to WPI. Levels of specific IgE and IgG to the hydrolysates were below 25 and 12 ng ml-1, respectively. Despite the differences in peptide composition and α-La and β-Lg concentrations, no significant difference was found between IgE and IgG binding capacity of hydrolysates obtained with or without pH control. These results highlight the impact of pH on the hydrolysates characteristics and their concentrations of antigenic protein. Divergence between the antigen detection by commercial ELISA kits and specific IgE and IgG binding response was found in this study. This result shows that lower protein detection does not imply in lower protein antigenicity. Thus, the use of commercial kits for allergen contamination analysis should be cautious.

Keywords: allergy, enzymatic hydrolysis, milk protein, pH conditions, physicochemical characteristics

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Authors: Mohammed Alasel, Michael Keusgen

Abstract:

Gold is a noble metal; in its nano-scale level (e.g. spherical nanoparticles), the conduction electrons are triggered to collectively oscillate with a resonant frequency when certain wavelengths of electromagnetic radiation interact with its surface; this phenomenon is known as surface plasmon resonance (SPR). SPR is responsible for giving the gold nanoparticles its intense red color depending mainly on its size, shape and distance between nanoparticles. A decreased distance between gold nanoparticles results in aggregation of them causing a change in color from red to blue. This aggregation enables gold nanoparticles to serve as a sensitive biosensoric indicator. In the proposed work, gold nanoparticles were modified with two proteins: i) Borrelia antigen, variable lipoprotein surface-exposed protein (VlsE), and ii) protein A. VlsE antigen induces a strong antibody response against Lyme disease and can be detected from early to late phase during the disease in humans infected with Borrelia. In addition, it shows low cross-reaction with the other non-pathogenic Borrelia strains. The high specificity of VlsE antigen to anti-Borrelia antibodies, combined simultaneously with the high specificity of protein A to the Fc region of all IgG human antibodies, was utilized to develop a rapid test for serological point of care diagnosis of borreliosis in human serum. Only in the presence of anti-Borrelia antibodies in the serum probe, an aggregation of gold nanoparticles can be observed, which is visible by a concentration-dependent colour shift from red (low IgG) to blue (high IgG). Experiments showed it is clearly possible to distinguish between positive and negative sera samples using a simple suspension of the two-protein modified gold nanoparticles in a very short time (30 minutes). The proposed work showed the potential of using such modified gold nanoparticles generally for serological diagnosis. Improved specificity and reduced assay time can be archived in applying increased salt concentrations combined with decreased pH values (pH 5).

Keywords: gold nanoparticles, gold aggregation, serological diagnosis, protein A, lyme borreliosis

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Authors: Kübra Aktaş, Nihat Akin

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Tarhana is a traditional Turkish fermented food. It is widely consumed as soup and includes many different ingredients such as wheat flour, various vegetables, and spices, yoghurt, bakery yeast. It can also be enriched by adding other ingredients. Thus, its nutritional properties can be enhanced. In this study, tarhana was supplemented with two different types of brans (rice bran and corn bran) and WPC (whey protein concentrate powder) to improve its nutritional and functional properties. Some chemical properties of tarhana containing two different brans and their levels (0, 5, 10 and 15%) and WPC (0, 5, 10%) were investigated. The results indicated that addition of WPC increased ash content in tarhanas which were fortified with rice and corn bran. The highest antioxidant and phenolic content values were obtained with addition of rice bran in tarhana formulation. Compared to tarhana with corn bran, rice bran addition gave higher oil content values. The cellulose content of tarhana samples was determined between 0.75% and 2.74% and corn bran showed an improving effect on cellulose contents of samples. In terms of protein content, addition of WPC into the tarhana raised protein content for the samples.

Keywords: corn, rice, tarhana, whey

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Authors: Muhammad Arifur Rahman, Talukdar M. Waliullah, Takashi Ushimaru

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Nucleophagy selectively degrades nuclear materials, especially nucleolus after nutrient starvation or inactivation of TORC1 kinase in budding yeast. Budding yeast phosphatidate (PA) phosphatase Pah1 that converts PA to diacylglycerol is essential for partitioning of lipid precursors between membrane and storage that is crucial for many aspects of cell growth and development. Pah1 is required for nuclear/ER membrane biogenesis and vacuole function, but whether Pah1 and its activator Nem1/Spo7 protein phosphatase complex are involved in autophagy is largely unknown. Loss of Pah1 causes expansion of the nucleus and fragmentation of the vacuole. Here we show that Pah1 is required for bulk autophagy and nucleophagy after TORC1 inactivation. Loss of Pah1 impaired nucleophagy severely and bulk autophagy to a lesser extent. Loss of the Pah1 activator Nem1-Spo7 protein phosphatase exhibited similar features.

Keywords: autophagy, Nem1/Spo7 phosphatase, Pah1, nucleophagy, TORC1

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Authors: Nijole Savickiene, Antonella Di Sotto, Gabriela Mazzanti, Rasa Starselskyte, Silvia Di Giacomo, Annabella Vitalone

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Plant lectins are non-enzymic and non-immune origin proteins that specifically recognize and bind to various sugar structures and possess the activity to agglutinate cells and/or precipitate polysaccharides and glycoconjugates. The emerging evidences showed that plant lectins contribute not only to tumour cell recognition but also to cell adhesion and localization, to signal transduction, to mitogenic cytotoxicity and apoptosis. Among chitin-binding lectins, the Urtica dioica agglutinin (UDA), which is a complex of different isoforms, has been poorly studied for its biological activity. In this context and according to the increasing interest for lectins as novel antitumor drugs, present paper aimed at evaluating the potential antimutagenic activity of a lectin-like glycoprotein-enriched fraction from aerial part of Urtica dioica L. Aim: to evaluate the potential chemopreventive properties of a protein - lectin fraction from the aerial part of Urtica dioica. Materials and methods: Protein – lectin fraction has been tested for the antimutagenic activity in bacteria (50–800 mg/plate; Ames test by the preincubation method) and for the cytotoxicity on human hepatoma HepG2 cells (0.06–2 mg/mL; 24 and 48 h incubation). Results: Protein – lectin fraction from stinging nettle was not cytotoxic on HepG2 cells up to 2 mg/mL; conversely, it exhibited a strong antimutagenic activity against the mutagen 2-aminoanthracene (2AA) in all strains tested (maximum inhibition of 56.78 and 61% in TA98, TA100, and WP2uvrA strains, respectively, at 800 mg/plate). Discussion and conclusions: Protein – lectin fraction from Urtica dioica L. possesses antimutagenic and radical scavenging properties. Being 2AA a pro-carcinogenic agent, we hypothesize that the antimutagenicity of it can be due to the inhibition of CYP450-isoenzymes, involved in the mutagen bioactivation.

Keywords: lectins, antimutagenicity, chemoprevention, Urtica dioica

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Authors: Kashif Ahmed

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Recent years researchers have been motivated towards extensive exploring of living organism, which could be utilized effectively in intense industrial conditions. The present study shows enhanced production, purification and characterization of industrial enzyme, invertase (Beta-D-fructofuranosidase) from Penicillium lilacinum. Various agricultural based by-products (cotton stalk, sunflower waste, rice husk, molasses and date syrup) were used as energy source. The highest amount of enzyme (13.05 Units/mL) was produced when the strain was cultured on growth medium containing date syrup as energy source. Yeast extract was used as nitrogen source after 96 h of incubation at incubation temperature of 40º C. Initial pH of medium was 8.0, inoculum size 6x10⁶ conidia and 200 rev/min agitation rate. The enzyme was also purified (7 folds than crude) and characterized. Molecular mass of purified enzyme (65 kDa) was determined by 10 % SDS-PAGE. Lineweaver-Burk Plot was used to determine Kinetic constants (Vmax 178.6 U/mL/min and Km 2.76 mM). Temperature and pH optima were 55º C and 5.5 respectively. MnCl₂ (52.9 %), MgSO₄ (48.9 %), BaCl₂ (24.6 %), MgCl₂ (9.6 %), CoCl₂ (5.7 %) and NaCl (4.2 %) enhanced the relative activity of enzyme and HgCl₂ (-92.8 %), CuSO₄ (-80.2 %) and CuCl₂ (-76.6 %) were proved inhibitors. The strain was showing enzyme activity even at extreme conditions of temperature (up to 60º C) and pH (up to 9), so it can be used in industries.

Keywords: invertase, Penicillium lilacinum, submerged fermentation, industrial enzyme

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Authors: Anurag Kumar Pandey, Tapan Kumar Nath, Santanu Dhara

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Polluted water by industrial effluents or dyes has become a major global concern, particularly in developing countries. Such environmental contaminants constitute a serious threat to biodiversity, ecosystems, and human health worldwide; thus, their treatment is critical. The usage of nanoparticles has been discovered to be a potential water treatment method with high efficiency, cheap manufacturing costs, and green synthesis. Carbon dots have attracted the interest of researchers due to their unique properties, such as high water solubility, ease of production, great electron-donating ability, and low toxicity. In this context, we synthesized iodine-doped clove buds-derived carbon dots (I-CCDs) for the Fenton-like degradation of environmental contaminants in water (such as methylene blue (MB) and rhodamine-B (Rh-B) dye). The formation of I-CCDs has been confirmed using various spectroscopy techniques. I-CCDs have demonstrated remarkable optical, cytocompatibility, and antibacterial capabilities. The C-dots that were synthesized were found to be an effective catalyst for the reduction of MB and Rh-B utilizing NaBH4 as a reducing agent. UV-visible spectroscopy was used to construct a detailed pathway for dye reduction step by step. As-prepared I-CCDs have the potential to be a promising solution for wastewater purification and treatment systems.

Keywords: iodine-doped carbon dots, wastewater treatment and purification, environmental friendly, antibacterial

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Authors: Tzan-Chain Lee

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Seeds are non-leaves green tissues. Photosynthesis begins with light absorption by chlorophyll and then the energy transfer between two pigment-protein complexes (PPC). Most studies of photosynthesis and PPC expression were focused on leaves; however, during seeds’ development were rare. Developed seeds from beginning pod (stage R3) to dried seed (stage R8), and the dried seed after sowing for 1-4 day, were analyzed for their chlorophyll contents. Thornber and MARS gel systems analysis compositions of PPC. Chlorophyll fluorescence was used to detect maximal photosynthetic efficiency (Fv/Fm). During soybean and black soybean seeds development (stages R3-R6), Fv/Fm up to 0.8, and then down-regulated after full seed (stage R7). In dried seed (stage R8), the two plant seeds lost photosynthetic activity (Fv/Fm=0), but chlorophyll degradation only occurred in soybean after full seed. After seeds sowing for 4 days, chlorophyll drastically increased in soybean seeds, and Fv/Fm recovered to 0.8 in the two seeds. In PPC, the two soybean seeds contained all PPC during seeds development (stages R3-R6), including CPI, CPII, A1, AB1, AB2, and AB3. However, many proteins A1, AB1, AB2, and CPI were totally missing in the two dried seeds (stage R8). The deficiency of these proteins in dried seeds might be caused by the incomplete photosynthetic activity. After seeds germination and seedling exposed to light for 4 days, all PPC were recovered, suggesting that completed PPC took place in the two soybean seeds. This study showed the reversibility of photosynthetic activity and pigment-protein complexes during soybean and black soybean seeds development.

Keywords: light-harvesting complex, pigment–protein complexes, soybean cotyledon, grana development

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Authors: Othman Elmahdy Othman

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The objectives of this study were to identify polymorphisms in the STAT5A using Restriction Fragment Length Polymorphism and DGAT1 using Single-Strand Conformation Polymorphism genes among three Egyptian goat breeds (Barki, Zaraibi, and Damascus) as well as investigate the effect of their genotypes on milk composition traits of Zaraibi goats. One hundred and fifty blood samples were collected for DNA extraction, 60 from Zaraibi, 40 from Damascus and 50 from Barki breeds. Fat, protein and lactose percentages were determined in Zaraibi goat milk using an automatic milk analyzer. Two genotypes, CC and CT (for STAT5A) and C-C- and C-C+ (for DGAT1), were identified in the three Egyptian goat breeds with different frequencies. The associations between these genotypes and milk fat, protein and lactose were determined in Zaraibi breed. The results showed that the STAT5A genotypes had significant effects on milk yield, protein, fat and lactose with the superiority of CT genotype over CC. Regarding DGAT1 polymorphism, the result showed the only association between it with milk fat where the animals with C-C+ genotype had greater milk fat than animals possess C-C- genotype. The association of combined genotypes with milk trait declared that the does with heterozygous genotypes for both genes are preferred than does with homozygous genotypes where the animals with CTC-C+ have more milk yield, fat and protein than those with CCC-C- genotype. In conclusion, the result showed that C/T and C-/C+ SNPs of STAT5A and DGAT1 genes respectively may be useful markers for assisted selection programs to improve goat milk composition

Keywords: DGAT1, genetic polymorphism, milk trait, STAT5A

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Authors: Hue Dinh, Binh Nguyen, Vivian Mendez, Phillip W. Taylor, Fleur Ponton

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Better understanding of how parental diet affects offspring traits is an important ecological and evolutionary question. In this study, we explored how maternal diet influences offspring physiology and resistance to infection using Bactrocera tryoni (Q-fly) as a system model. Female Q-flies were fed one of six single diets varying in their yeast-to-sugar ratio yielding six protein-to-carbohydrate ratios. As controls, we used females that were given a choice between yeast and sugar. Males were reared on a choice diet and allowed to mate with females 14 days post-emergence. Results showed that while maternal diet does not influence offspring developmental time, it has a strong effect on larval body weight. Mother fed either high-protein or high-sugar diet produced larger progeny. By challenging offspring with the bacterium Serratia marcescens, we found that female offspring from mothers fed high-sugar diet survived better the infection compared to those from mothers fed low-sugar diet. In contrast, male offspring produced by mother fed high-protein diet showed better resistance to the infection compared to those produced by mother fed low-protein diet. These results suggested sex-dependent transgenerational effects of maternal nutrition on offspring physiology and immunity.

Keywords: bacterial infection, Bactrocera tryoni, maternal diet, offspring, Serretia marcescens

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Authors: Tarana Siddika, Ilka U. Heinemann, Patrick O’Donoghue

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Protein kinase B (AKT1) is a serine/threonine kinase and central transducer of cell survival pathways. Typical approaches to study AKT1 biology in cells rely on growth factor or insulin stimulation that activates AKT1 via phosphorylation at two key regulatory sites (Threonine308, Serine473), yet cell stimulation also activates many other kinases and fails to differentiate the effect of the two main activating sites of AKT1 on downstream substrate phosphorylation and cell growth. While both AKT1 activating sites are associated with disease and used as clinical markers, in some cancers, high levels of Threonine308 phosphorylation are associated with poor prognosis while in others poor survival correlates with high Serine473 levels. To produce cells with specific AKT1 activity, a system was developed to deliver active AKT1 to human cells. AKT1 phospho-variants were produced from Escherichia coli with programmed phosphorylation by genetic code expansion. Tagging of AKT1 with an N-terminal cell penetrating peptide tag derived from the human immunodeficiency virus trans-activator of transcription (TAT) helped to enter AKT1 proteins in mammalian cells. The TAT-tag did not alter AKT1 kinase activity and was necessary and sufficient to rapidly deliver AKT1 protein variants that persisted in human cells for 24 h without the need to use transfection reagents. TAT-pAKT1T308, TAT-pAKT1S473 and TAT-pAKT1T308S473 proteins induced selective phosphorylation of the known AKT1 substrate GSK-3αβ, and downstream stimulation of the AKT1 pathway as evidenced by phosphorylation of ribosomal protein S6 at Serine240/244 in transfected cells. Increase in cell growth and proliferation was observed due to the transfection of different phosphorylated AKT1 protein variants compared to cells with TAT-AKT1 protein. The data demonstrate efficient delivery of AKT1 with programmed phosphorylation to human cells, thus establishing a cell-based model system to investigate signaling that is dependent on specific AKT1 activity and phosphorylation.

Keywords: cell penetrating peptide, cell signaling, protein kinase b (AKT1), phosphorylation

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Authors: Sergio D’Ambrosioa, Azza Dobousa, Chiara Schiraldia, Donatella Ciminib

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Probiotics are living microorganisms that give beneficial effects while consumed. Lactic acid bacteria and bifidobacteria are among the most representative strains assessed as probiotics and exploited as food supplements. Numerous studies demonstrated their potential as a therapeutic candidate for a variety of diseases (restoring gut flora, lowering cholesterol, immune response-enhancing, anti-inflammation and anti-oxidation activities). These beneficial actions are also due to biomolecules produced by probiotics, such as exopolysaccharides (EPSs), that demonstrate plenty of beneficial properties such as antimicrobial, antitumor, anti-biofilm, antiviral and immunomodulatory activities. Limosilactobacillus fermentum is a widely studied member of probiotics; however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications. However, few data are available on the development of fermentation processes for the large-scale production of probiotics biomass for industrial applications and for purification processes of EPSs at an industrial scale. For this purpose, L. fermentum strain was isolated from buffalo milk and used as a test example for biotechnological process development. The strain was able to produce up to 109 CFU/mL on a (glucose-based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media-rich of casein and beef extract. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover of about 5.7 × 1010CFU/gpowder of viable cells, indicating strain resistance to harsh processing conditions. Overall, these data demonstrate the possibility of obtaining and maintaining adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. A downstream EPS purification protocol based on ultrafiltration, precipitation and activated charcoal treatments showed a purity of the recovered polysaccharides of about 70-80%.

Keywords: probiotics, fermentation, exopolysaccharides (EPSs), purification

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Authors: Keri Alhadi Ighwela, Aziz Bin Ahmad, A. B. Abol-Munafi

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Three purified diets were formulated using fish meal, soya bean, wheat flour, palm oil, minerals and maltose. The carbohydrate in the diets was increased from 5 to 15% by changing the cellulose content to study the effect of dietary carbohydrate level on the growth parameters of Nile tilapia Oreochromis niloticus.The protein and the lipid contents were kept constant in all the diets. The results showed that, weight gain, protein efficiency ratio, net protein utilisation and hepatosomatic index of fish fed the diet containing 15% cellulose were the lowest among all groups. Addition, the fish fed the diet containing 5% cellulose had the best specific growth rate, and food conversion ratio. While, there was no effect of the dietary cellulose levels on condition factor and survival rate. These results indicate that Nile tilapia fingerlings are able to utilize dietary cellulose does not exceed 10% in their feed for optimum growth.

Keywords: dietary cellulose, growth parameters, oreochromis niloticus, purified diets

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Authors: Marwan Abdel Baset, Sally A. El Awdan, Marwa S. Khattab, Salma A. El-Marasy

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Rivaroxaban (RVX) id used to treat thrombosis however its effect on testicular IR hasnot been investigated yet. This study investigates the effect of RVX on testicular ischemia-reperfusion in rats. Rats were randomly allocated into 4 groups. The sham group, testicular ischemia-reperfusion (IR) group, the remaining 2 groups were treated with RVX in doses of7 and 14 mg/kg, respectively for a week prior IR. Then biochemical parameters were carried out in addition to western blot analysis. RVX-treated groups showed significant reduction in protein expression of hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) associated with elevation in testosterone level, reduction in malondialdehyde (MDA), elevation in glutathione peroxidase (GPX), reduction in nuclear factor kappa-B p65 (NF-ĸb p65), reduction in Bax (Bcl2-associated X protein) and elevation in BCL2 (B-cell lymphoma 2), content. Moreover, RVX reduced caspase-3 protein expression. It can be concluded that HIF-1α mediated RVX’s anti-oxidant, anti-inflammatory and, anti-apoptotic effect in rats subjected to testicular IR.

Keywords: HIF-1α, rats, rivaroxaban, testicular ischemia-reperfusion

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Authors: Muhammad Naeem, Amina Zubari, Abdus Salam, Syed Ali Ayub Bukhari, Naveed Ahmad Khan

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Seventy three wild Labeo bata of different body sizes, ranging from 8.20-16.00 cm total length and 7.4-86.19 g body weight, were studied for the analysis of body composition parameters (Water content, ash content, fat content, protein content) in relation to body size and condition factor. Mean percentage is found as for water 77.71 %, ash 3.42 %, fat 2.20 % and protein content 16.65 % in whole wet body weight. Highly significant positive correlations were observed between condition factor and body weight (r = 0.243). Protein contents, organic content and ash (% wet body weight) increase with increasing percent water contents for Labeo bata while these constituents (% dry body weight) and fat contents (% wet and dry body weight) have no influence on percent water. It was observed that variations in the body constituents have no association to body weight or length.

Keywords: Labeo bata, body size, body composition, condition factor

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Authors: Juan Jose Filgueira, Daniela Londono-Serna, Liliana Maria Hoyos

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Carnation (Dianthus caryophyllus) is one of the most important products of exportation in the floriculture industry worldwide. Fusariosis is the disease that causes the highest losses on farms, in particular the one produced by Fusarium oxysporum f.sp. dianthi, called vascular wilt. Gene identification and metabolic routes of the genes that participate in the building of the plant response to Fusarium are some of the current targets in the carnation breeding industry. The techniques for the identifying of resistant genes in the plants, is the analysis of the transcriptome obtained during the host-pathogen interaction. In this work, we report the cell transcriptome of different varieties of carnation that present differential response from Fusarium oxysporum f.sp. dianthi attack. The cells of the different hybrids produced in the outbreeding program were cultured in vitro and elicited with the parasite in a dual culture. The isolation and purification of mRNA was achieved by using affinity chromatography Oligo dT columns and the transcriptomes were obtained by using Illumina NGS techniques. A total of 85,669 unigenes were detected in all the transcriptomes analyzed and 31,000 annotations were found in databases, which correspond to 36.2%. The library construction of genic expression techniques used, allowed to recognize the variation in the expression of genes such as Germin-like protein, Glycosyl hydrolase family and Cinnamate 4-hydroxylase. These have been reported in this study for the first time as part of the response mechanism to the presence of Fusarium oxysporum.

Keywords: Carnation, Fusarium, vascular wilt, transcriptome

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Authors: Arfan Ali, Idrees Ahmad Nasir

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Potato (Solanum tuberosum) is an important cash crop and popular vegetable in Pakistan and throughout the world. Cold storage of potatoes accelerates the conversion of starch into reduced sugars (glucose and fructose). This process causes dry mass and bitter taste in the potatoes that are not acceptable to end consumers. In the current study, the phosphofructokinase B gene was cloned into the pET-30 vector for protein expression and the pCambia-1301 vector for plant expression. Amplification of a 930bp product from an E. coli strain determined the successful isolation of the phosphofructokinase B gene. Restriction digestion using NcoI and BglII along with the amplification of the 930bp product using gene specific primers confirmed the successful cloning of the PfkB gene in both vectors. The protein was expressed as a His-PfkB fusion protein. Western blot analysis confirmed the presence of the 35 Kda PfkB protein when hybridized with anti-His antibodies. The construct Fani-01 was evaluated transiently using a histochemical gus assay. The appearance of blue color in the agroinfiltrated area of potato leaves confirmed the successful expression of construct Fani-01. Further, the area displaying gus expression was evaluated for PfkB expression using ELISA. Moreover, PfkB gene expression evaluated through transient expression determined successful gene expression and highlighted its potential utilization for stable expression in potato to reduce sweetening due to long-term storage.

Keywords: potato, Solanum tuberosum, transformation, PfkB, anti-sweetening

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Authors: E. Obreque-Slier, F. Orellana-Rodríguez, R. López-Solís

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Phenolic compounds are secondary metabolites present in some foods, such as wine. Polyphenols comprise two main groups: flavonoids (anthocyanins, flavanols, and flavonols) and non-flavonoids (stilbenes and phenolic acids). Phenolic acids are low molecular weight non flavonoid compounds that are usually grouped into benzoic (gallic, vanillinic and protocatechuic acids) and cinnamic acids (ferulic, p-coumaric and caffeic acids). Likewise, tannic acid is an important polyphenol constituted mainly by gallic acid. Phenolic compounds are responsible for important properties in foods and drinks, such as color, aroma, bitterness, and astringency. Astringency is a drying, roughing, and sometimes puckering sensation that is experienced on the various oral surfaces during or immediately after tasting foods. Astringency perception has been associated with interactions between flavanols present in some foods and salivary proteins. Despite the quantitative relevance of phenolic acids in food and beverages, there is no information about its effect on salivary proteins and consequently on the sensation of astringency. The objective of this study was assessed the interaction of several phenolic acids (gallic, vanillinic, protocatechuic, ferulic, p-coumaric and caffeic acids) with saliva. Tannic acid was used as control. Thus, solutions of each phenolic acids (5 mg/mL) were mixed with human saliva (1:1 v/v). After incubation for 5 min at room temperature, 15-μL aliquots of the mixtures were dotted on a cellulose membrane and allowed to diffuse. The dry membrane was fixed in 50 g/L trichloroacetic acid, rinsed in 800 mL/L ethanol and stained for protein with Coomassie blue for 20 min, destained with several rinses of 73 g/L acetic acid and dried under a heat lamp. Both diffusion area and stain intensity of the protein spots were semiqualitative estimates for protein-tannin interaction (diffusion test). The rest of the whole saliva-phenol solution mixtures of the diffusion assay were centrifuged and fifteen-μL aliquots of each supernatant were dotted on a cellulose membrane, allowed to diffuse and processed for protein staining, as indicated above. In this latter assay, reduced protein staining was taken as indicative of protein precipitation (precipitation test). The diffusion of the salivary protein was restricted by the presence of each phenolic acids (anti-diffusive effect), while tannic acid did not alter diffusion of the salivary protein. By contrast, phenolic acids did not provoke precipitation of the salivary protein, while tannic acid produced precipitation of salivary proteins. In addition, binary mixtures (mixtures of two components) of various phenolic acids with gallic acid provoked a restriction of saliva. Similar effect was observed by the corresponding individual phenolic acids. Contrary, binary mixtures of phenolic acid with tannic acid, as well tannic acid alone, did not affect the diffusion of the saliva but they provoked an evident precipitation. In summary, phenolic acids showed a relevant interaction with the salivary proteins, thus suggesting that these wine compounds can also contribute to the sensation of astringency.

Keywords: astringency, polyphenols, tannins, tannin-protein interaction

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Authors: Priya Arora, Ashutosh Mishra

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Gene identification, towards the pursuit of mutated genes, leading to Parkinson’s disease, puts forward a challenge towards proactive cure of the disorder itself. Computational analysis is an effective technique for exploring genes in the form of protein sequences, as the theoretical and manual analysis is infeasible. The limitations and effectiveness of a particular computational method are entirely dependent on the previous data that is available for disease identification. The article presents a sequence-based classification method for the identification of genes responsible for Parkinson’s disease. During the initiation phase, the physicochemical properties of amino acids transform protein sequences into a feature vector. The second phase of the method employs Jaccard distances to select negative genes from the candidate population. The third phase involves artificial neural networks for making final predictions. The proposed approach is compared with the state of art methods on the basis of F-measure. The results confirm and estimate the efficiency of the method.

Keywords: disease gene identification, Parkinson’s disease, physicochemical properties of amino acid, protein sequences

Procedia PDF Downloads 141