Search results for: human epididymis protein 4

Authors: R. Charoen, S. Tipkanon, W. Savedboworn, N. Phonsatta, A. Panya

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Rice bran protein hydrolysates (RBPH) were prepared from defatted rice bran of two different Thai rice cultivars (Plai-Ngahm-Prachinburi; PNP and Khao Dok Mali 105; KDM105) using an enzymatic method. This research aimed to optimize enzyme-assisted protein extraction. In addition, the functional properties of RBPH and their stabilities to environmental stresses including pH (3 to 8), ionic strength (0 mM to 500 mM) and the thermal treatment (30 °C to 90 °C) were investigated. Results showed that enzymatic process for protein extraction of defatted rice bran was as follows: enzyme concentration 0.075 g/ 5 g of protein, extraction temperature 50 °C and extraction time 4 h. The obtained protein hydrolysate powders had a degree of hydrolysis (%) of 21.05% in PNP and 19.92% in KDM105. The solubility of protein hydrolysates at pH 4-6 was ranged from 27.28-38.57% and 27.60-43.00% in PNP and KDM105, respectively. In general, antioxidant activities indicated by total phenolic content, FRAP, ferrous ion-chelating (FIC), and 2,2’-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) of KDM105 had higher than PNP. In terms of functional properties, the emulsifying activity index (EAI) was was 8.78 m²/g protein in KDM105, whereas PNP was 5.05 m²/g protein. The foaming capacity at 5 minutes (%) was 47.33 and 52.98 in PNP and KDM105, respectively. Glutamine, Alanine, Valine, and Leucine are the major amino acid in protein hydrolysates where the total amino acid of KDM105 gave higher than PNP. Furthermore, we investigated environmental stresses on the stability of 5% oil in water emulsion (5% oil, 10 mM citrate buffer) stabilized by RBPH (3.5%). The droplet diameter of emulsion stabilized by KDM105 was smaller (d < 250 nm) than produced by PNP. For environmental stresses, RBPH stabilized emulsions were stable at pH around 3 and 5-6, at high salt (< 400 mM, pH 7) and at temperatures range between 30-50°C.

Keywords: functional properties, oil in water emulsion, protein hydrolysates, rice bran protein

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Authors: Ekin D. Horzum

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The aim of the proposal is to examine the relationship between human trafficking related corruption and human security. The proposal suggests that the human trafficking related corruption is about willingness of the states to turn a blind eye to the human trafficking cases. Therefore, it is important to approach human trafficking related corruption in terms of human security and human rights violation to find an effective way to fight against human trafficking. In this context, the purpose of this proposal is to examine the human trafficking related corruption as a safe haven in which trafficking thrives for perpetrators.

Keywords: human trafficking, human security, human rights, corruption, organized crime

Procedia PDF Downloads 439

Authors: Mahmoud M. Zakaria, Omnia F. Elmoursi, Mahmoud M. Gabr, Camelia A. AbdelMalak, Mohamed A. Ghoneim

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Many protocols were publicized for differentiation of human mesenchymal stem cells (MSCS) into insulin-producing cells (IPCs) in order to excrete insulin hormone ingoing to treat diabetes disease. Our aim is to evaluate relative gene expression for each independent protocol. Human bone marrow cells were derived from three volunteers that suffer diabetes disease. After expansion of mesenchymal stem cells, differentiation of these cells was done by three different protocols (the one-step protocol was used conophylline protein, the two steps protocol was depending on trichostatin-A, and the three-step protocol was started by beta-mercaptoethanol). Evaluation of gene expression was carried out by real-time PCR: Pancreatic endocrine genes, transcription factors, glucose transporter, precursor markers, pancreatic enzymes, proteolytic cleavage, extracellular matrix and cell surface protein. Quantitation of insulin secretion was detected by immunofluorescence technique in 24-well plate. Most of the genes studied were up-regulated in the in vitro differentiated cells, and also insulin production was observed in the three independent protocols. There were some slight increases in expression of endocrine mRNA of two-step protocol and its insulin production. So, the two-step protocol was showed a more efficient in expressing of pancreatic endocrine genes and its insulin production than the other two protocols.

Keywords: mesenchymal stem cells, insulin producing cells, conophylline protein, trichostatin-A, beta-mercaptoethanol, gene expression, immunofluorescence technique

Procedia PDF Downloads 186

Authors: Shiva Emami, Jenny Persson, Bengt Johansson Lindbom

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Group A streptococcus (GAS) is a prevalent human pathogen, causing a wide range of infections and diseases. One of the most well-known virulence factors in GAS is M protein, a surface protein that facilitates bacterial invasion. In this study, we used a recombinant GAS strain (GAS-2W) expressing M protein containing a hyper immunogenic peptide (2W). Mice were immunized three times with heat-killed-GAS subcutaneously at three weeks intervals. Three weeks post last immunization, mice were challenged intraperitoneally with a lethal dose of live GAS. In order to investigate the impact of IFN-ƴ and antibodies in protection against GAS infection, we used a mouse model knock-out for IFN-ƴ (IFN-ƴ KO). We observed immunization with GAS-2W strain can increase protection against GAS infection in mice compared with the original GAS strain. Higher levels of antibodies against M1 protein were measured in GAS-2W-immunized mice. There was also a significant increase in IgG2c response in mice immunized with GAS2W. By using IFN-ƴ KO mice, we showed that not a high level of total IgG, but IgG2c was correlated with protection through the i.p challenge. It also emphasizes the importance of IFN-ƴ cytokine to combat GAS by isotype switching to IgG2c (which is opsonic for phagocytosis). Our data indicate the crucial role of IFN-ƴ in the protective immune response that, together with IgG2c, can induce protection against GAS.

Keywords: Group A streptococcus, IgG2c, IFN-γ, protection

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Authors: El-Sohaimy Sobhy A., Androsova Natalia, Toshev Abuvali Djabarovec

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The conditions of the isolation of proteins from the hemp seeds were optimized in the current work. Moreover, the physicochemical and functional properties of hemp protein isolate were evaluated for its potential application in food manufacturing. The elastin protein is the most predominant protein in the protein profile with a molecular weight of 58.1 KDa, besides albumin, with a molecular weight of 31.5 KDa. The FTIR spectrum detected the absorption peaks of the amide I in 1750 and 1600 cm⁻¹, which pointed to C=O stretching while N-H was stretching at 1650-1580 cm⁻¹. The peak at 3250 was related to N-H stretching of primary aliphatic amine (3400-3300 cm⁻¹), and the N-H stretching for secondary (II) amine appeared at 3350-3310 cm⁻¹. Hemp protein isolate (HPI) was showed high content of arginine (15.52 g/100 g), phenylalanine+tyrosine (9.63 g/100 g), methionine + cysteine (5.49 g/100 g), leucine + isoleucine (5.21 g/100 g) and valine (4.53 g/100 g). It contains a moderate level of threonine (3.29 g/100 g) and lysine (2.50 g/100 g), with the limiting amino acid being a tryptophan (0.22 g/100 g HPI). HPI showed high water-holding capacity (4.5 ± 2.95 ml/g protein) and oil holding capacity (2.33 ± 1.88 ml/g) values. The foaming capacity of HPI was increased with increasing the pH values to reach the maximum value at pH 11 (67.23±3.20 %). The highest emulsion ability index of HPI was noted at pH 9 (91.3±2.57 m2/g) with low stability (19.15±2.03).

Keywords: Cannabis sativa ssp., protein isolate, isolation conditions, amino acid composition, chemical properties, functional properties

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Authors: Zhaojun Zheng, Yuanfa Liu, Jiaxin Li, Jinwei Li, Yong-jiang Xu, Chen Cao

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Black bean is an excellent protein source for preparing hydrolysates, which attract much attention due to their biological activity. The objective of this study was to characterize the physicochemical and antioxidant properties of black bean protein, hydrolyzed by ficin, bromelain or alcalase until 300 min of hydrolysis. Results showed that bromelain and alcalase hydrolysates possessed a higher degree of hydrolysis (DH) than that of ficin, thereby presenting different ultraviolet absorption, fluorescence intensity, and circular dichroism. Moreover, all hydrolysates possessed the capacity to scavenge DPPH radical with the lowest IC₅₀ of 21.11 µg/mL, as well as to chelate ferrous ion (Fe²⁺) with the IC₅₀ values ranging from 6.82 to 30.68 µg/mL. Intriguingly, the oxidation of linoleic acid, sunflower oil, and sunflower oil-in-water emulsion was remarkedly retarded by the three selected protein hydrolysates, especially by bromelain-treated protein hydrolysate, which might attribute to their high hydrophobicity and emulsifying properties. These findings can provide strong support for black bean protein hydrolysates to be employed in food products acting as natural antioxidant alternatives.

Keywords: antioxidant activity, black bean protein hydrolysate, emulsion physicochemical properties, sunflower oil

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Authors: Neslihan Taskale Karatug, Mustafa Akcelik

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Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

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Authors: Baigts-Allende Diana, Klojdová Iveta, Kozlu Ali, Metri-ojeda Jorge

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Emulsion gels are constituted by a colloidal system (emulsion) stabilized by a polymeric gel matrix. These systems are more homogeneous and stable than conventional emulsions and can behave as either gel-like or soft-solid. Protein-based emulsion gels (PEG) have been used as carrier systems of bioactive compounds and as food structuring to improve the texture and consistency, mainly in producing low-fat content products. This work studied the effect of protein: polysaccharide ratio 0.75:1.25, 1:1, and 1.25:0.75 (levels -1, 0, and +1) and pH values (2-9) on the stability of protein-based emulsion gels using soy protein isolate and sodium alginate. Protein emulsion capacity was enhaced with increased pH (6,7,8 and 9) compared to acid pH values. The smaller particle size for PEG was at pH 9 (~23µm); however, with increasing protein ratio (level +1), higher particle size was observed (~23µm). The same trend was observed for rheological measurements; the consistency index (K) increased at pH 9 for level -1 (1.17) in comparison to level +1 (0.45). The studied PEG showed good thermal stability at neutral and pH 9 (~98 %) for all biopolymer ratios. Optimal conditions in pH and biopolymer ratios were determined for PEG using soy protein and sodium alginate ingredients with potential use in elaborating stable systems for broad application in the food sector.

Keywords: emulsion gels, food structuring, biopolymers, food systems

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Authors: Y. Ruchi, A. Prerna, S. Deepshikha

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Amyotrophic lateral sclerosis (ALS), also known as “Lou Gehrig’s disease”. It is a neurodegenerative disease associated with degeneration of motor neurons in the cerebral cortex, brain stem, and spinal cord characterized by distal muscle weakness, atrophy, normal sensation, pyramidal signs and progressive muscular paralysis reflecting. ALS2 is a juvenile autosomal recessive disorder, slowly progressive, that maps to chromosome 2q33 and is associated with mutations in the alsin gene, a putative GTPase regulator. In this paper we have done homology modeling of alsin2 protein using multiple templates (3KCI_A, 4LIM_A, 402W_A, 4D9S_A, and 4DNV_A) designed using the Prime program in Schrödinger software. Further modeled structure is used to identify effective binding sites on the basis of structural and physical properties using sitemap program in Schrödinger software, structural and function analysis is done by using Prosite and ExPASy server that gives insight into conserved domains and motifs that can be used for protein classification. This paper summarizes the structural, functional and binding site property of alsin2 protein. These binding sites can be potential drug target sites and can be used for docking studies.

Keywords: ALS, binding site, homology modeling, neuronal degeneration

Procedia PDF Downloads 363

Authors: Suchao Donpudsa, Anchalee Tassanakajon, Vichien Rimphanitchayakit

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A crustin gene, LV-SWD1, previously found in the hemocyte cDNA library of Litopenaeus vannamei, contains the open reading frames of 288 bp encoding a putative protein of 96 amino acid residues. The putative signal peptides of the LV-SWD1 were identified using the online SignalP 3.0 with predicted cleavage sites between Ala24-Val25, resulting in 72 residue mature protein with calculated molecular mass of 7.4 kDa and predicted pI of 8.5. This crustin contains a Arg-Pro rich region at the amino-terminus and a single whey acidic protein (WAP) domain at the carboxyl-terminus. In order to characterize their properties and biological activities, the recombinant crustin protein was produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of Bacillus subtilis was inhibited by this recombinant crustin with MIC of about 25-50 µM.

Keywords: crustin, single whey acidic protein, Litopenaeus vannamei, antimicrobial activity

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Authors: Jiraporn Rojtinnakorn

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ICAM-2 (intercellular adhesion molecule 2) or CD102 (Cluster of Differentiation 102) is type I trans-membrane glycoproteins, composing 2-9 immunoglobulin-like C2-type domains. ICAM-2 plays the particular role in immune response and cell surveillance. It is concerned in innate and specific immunity, cell survival signal, apoptosis, and anticancer. EST clone of ICAM-2, from P. gigas blood cell EST libraries, showed high identity to human ICAM-2 (92%) with conserve region of ICAM N-terminal domain and part of Ig superfamily. Gene and protein of ICAM-2 has been founded in mammals. This is the first report of ICAM-2 in fish.

Keywords: ICAM-2, CD102, Pangasianodon gigas, antitumor

Procedia PDF Downloads 198

Authors: Natela Gerliani, Mohammed Aider

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Nowadays, the interest of using sustainable technologies for protein extraction from underutilized oilseeds is growing. Currently, a major disposal problem for the oil industry is by-products of plant food processing such as soybean meal. That is why valorization of soybean meal is important for the oil industry since it contains high-quality proteins and other valuable components. Generally, soybean meal is used in livestock and poultry feed but is rarely used in human feed. Though chemical composition of this meal compensate nutritional deficiency and can be used to balance protein in human food. Regarding the efficiency of soybean meal valorization, extraction is a key process for obtaining enriched protein ingredient, which can be incorporated into the food matrix. However, most of the food components such as proteins extracted from oilseeds by-products imply the utilization of organic and inorganic chemicals (e.g. acids, bases, TCA-acetone) having a significant environmental impact. In a context of sustainable production, the use of an electro-activation technology seems to be a good alternative. Indeed, the electro-activation technology requires only water, food grade salt and electricity as main materials. Moreover, this innovative technology helps to avoid special equipment and trainings for workers safety as well as transport and storage of hazardous materials. Electro-activation is a technology based on applied electrochemistry for the generation of acidic and alkaline solutions on the basis of the oxidation-reduction reactions that occur at the vicinity electrode/solution interfaces. It is an eco-friendly process that can be used to replace the conventional acidic and alkaline extraction. In this research, the electro-activation technology for protein extraction from soybean meal was carried out in the electro-activation reactor. This reactor consists of three compartments separated by cation and anion exchange membranes that allow creating non-contacting acidic and basic solutions. Different current intensities (150 mA, 300 mA and 450 mA) and treatment durations (10 min, 30 min and 50 min) were tested. The results showed that the extracts obtained by the electro-activation method have good quality in comparison to conventional extracts. For instance, extractability obtained with electro-activation method was 55% whereas with the conventional method it was only 36%. Moreover, a maximum protein quantity of 48 % in the extract was obtained with the electro-activation technology comparing to the maximum amount of protein obtained by conventional extraction of 41 %. Hence, the environmentally sustainable electro-activation technology seems to be a promising type of protein extraction that can replace conventional extraction technology.

Keywords: by-products, eco-friendly technology, electro-activation, soybean meal

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Authors: Patarasuda Chaisupa, R. Clay Wright

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The selection of appropriate biomolecular targets is a crucial aspect of biopharmaceutical development. The Auxin-Inducible Degron Degradation (AID) technology has demonstrated remarkable potential in efficiently and rapidly degrading target proteins, thereby enabling the identification and acquisition of drug targets. The AID system also offers a viable method to deplete specific proteins, particularly in cases where the degradation pathway has not been exploited or when the adaptation of proteins, including the cell environment, occurs to compensate for the mutation or gene knockout. In this study, we have engineered an improved AID system tailored to deplete proteins of interest. This AID construct combines the auxin-responsive E3 ubiquitin ligase binding domain, AFB2, and the substrate degron, IAA17, fused to the target genes. Essential genes of fungi with the lowest percent amino acid similarity to human and plant orthologs, according to the Basic Local Alignment Search Tool (BLAST), were cloned into the AID construct in S. cerevisiae (AID-tagged strains) using a modular yeast cloning toolkit for multipart assembly and direct genetic modification. Each E3 ubiquitin ligase and IAA17 degron was fused to a fluorescence protein, allowing for real-time monitoring of protein levels in response to different auxin doses via cytometry. Our AID system exhibited high sensitivity, with an EC50 value of 0.040 µM (SE = 0.016) for AFB2, enabling the specific promotion of IAA17::target protein degradation. Furthermore, we demonstrate how this improved AID system enhances quantitative functional studies of various proteins in fungi. The advancements made in auxin-inducible protein degradation in this study offer a powerful approach to investigating critical target protein viability in fungi, screening protein targets for drugs, and regulating intracellular protein abundance, thus revolutionizing the study of protein function underlying a diverse range of biological processes.

Keywords: synthetic biology, bioengineering, molecular biology, biotechnology

Procedia PDF Downloads 59

Authors: Amit Chaudhary, B. S. Yadav, P. K. Maurya, A. M., S. Srivastava, S. Singh, A. Mani

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Cold shock protein A (CspA) is major cold inducible protein present in Escherichia coli. The protein is involved in stabilizing secondary structure of RNA by working as chaperone during cold temperature. Two RNA binding motifs play key role in the stabilizing activity. This study aimed to investigate implications of low temperature on structure and RNA binding activity of E. coli CspA. Molecular dynamics simulations were performed to compare the stability of the protein at 37°C and 10 °C. The protein was mutated at RNA binding motifs and docked with RNA to assess the stability of both complexes. Results suggest that CspA as well as CspA-RNA complex is more stable at low temperature. It was also confirmed that RNP1 and RNP2 play key role in RNA binding.

Keywords: CspA, homology modelling, mutation, molecular dynamics simulation

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Authors: Naruemon Prapasuwannakul

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Soy milk residue is obtained as a byproduct from soy milk and tofu production with little economic value. It contains high protein and fiber as well as various minerals and phyto-chemical compounds. The objective of this research was to substitute soy milk residue for wheat flour in gyoza skin in order to enhance value of soy milk residue and increase protein and fiber content of gyoza skin. Wheat flour was replaced with soy milk residue from 0 to 40%. The soy milk residue prepared in this research contains 26.92% protein, 3.58% fiber, 2.88% lipid, 6.29% ash and 60.33% carbohydrate. The results showed that increasing soy milk residue decreased lightness (L*value), tensile strength and sensory attributes but increased redness (a*), yellowness (b*), protein and fiber contents of product. The result also showed that the gyoza skin substituted with 30% soy milk residue was the most acceptable (p≤0.05) and its protein and fiber content increased up to 45 % and 867 % respectively.

Keywords: Gyoza skin, sensory, soymilk residue, wheat flour

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Authors: Gyaltsen Dakpa, K. J. Senthil Kumar, Nai-Wen Tsao, Sheng-Yang Wang

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Context: Coronavirus disease 2019 (COVID-19) is a severe respiratory illness caused by the SARS-CoV-2 virus. One of the hallmarks of COVID-19 is a change in metabolism, which can lead to increased severity and mortality. The mechanism of SARS-CoV-2-mediated perturbations of metabolic pathways has yet to be fully understood. Research Aim: This study aimed to investigate the metabolic alteration caused by SARS-CoV-2 spike protein in Phorbol 12-myristate 13-acetate (PMA)-induced human monocytes (THP-1) and to examine the regulatory effect of natural compounds like Antcins A on SARS-CoV-2 spike protein-induced metabolic alteration. Methodology: The study used a combination of proton nuclear magnetic resonance (1H-NMR) and MetaboAnalyst 5.0 software. THP-1 cells were treated with SARS-CoV-2 spike protein or control, and the metabolomic profiles of the cells were compared. Antcin A was also added to the cells to assess its regulatory effect on SARS-CoV-2 spike protein-induced metabolic alteration. Findings: The study results showed that treatment with SARS-CoV-2 spike protein significantly altered the metabolomic profiles of THP-1 cells. Eight metabolites, including glycerol-phosphocholine, glycine, canadine, sarcosine, phosphoenolpyruvic acid, glutamine, glutamate, and N, N-dimethylglycine, were significantly different between control and spike-protein treatment groups. Antcin A significantly reversed the changes in these metabolites. In addition, treatment with antacid A significantly inhibited SARS-CoV-2 spike protein-mediated up-regulation of TLR-4 and ACE2 receptors. Theoretical Importance The findings of this study suggest that SARS-CoV-2 spike protein can cause significant metabolic alterations in THP-1 cells. Antcin A, a natural compound, has the potential to reverse these metabolic alterations and may be a potential candidate for developing preventive or therapeutic agents for COVID-19. Data Collection: The data for this study was collected from THP-1 cells that were treated with SARS-CoV-2 spike protein or a control. The metabolomic profiles of the cells were then compared using 1H-NMR and MetaboAnalyst 5.0 software. Analysis Procedures: The metabolomic profiles of the THP-1 cells were analyzed using 1H-NMR and MetaboAnalyst 5.0 software. The software was used to identify and quantify the cells' metabolites and compare the control and spike-protein treatment groups. Questions Addressed: The question addressed by this study was whether SARS-CoV-2 spike protein could cause metabolic alterations in THP-1 cells and whether Antcin A can reverse these alterations. Conclusion: The findings of this study suggest that SARS-CoV-2 spike protein can cause significant metabolic alterations in THP-1 cells. Antcin A, a natural compound, has the potential to reverse these metabolic alterations and may be a potential candidate for developing preventive or therapeutic agents for COVID-19.

Keywords: SARS-CoV-2-spike, ¹H-NMR, metabolomics, antcin-A, taiwanofungus camphoratus

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Authors: Jamaludin Sallim, Rozlina Mohamed, Roslina Abdul Hamid

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In this paper, we proposed an Ant Colony Optimization (ACO) algorithm together with Traveling Salesman Problem (TSP) approach to investigate the clustering problem in Protein Interaction Networks (PIN). We named this combination as ACOPIN. The purpose of this work is two-fold. First, to test the efficacy of ACO in clustering PIN and second, to propose the simple generalization of the ACO algorithm that might allow its application in clustering proteins in PIN. We split this paper to three main sections. First, we describe the PIN and clustering proteins in PIN. Second, we discuss the steps involved in each phase of ACO algorithm. Finally, we present some results of the investigation with the clustering patterns.

Keywords: ant colony optimization algorithm, searching algorithm, protein functional module, protein interaction network

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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: Surabhi Pandey, Pavuluri Srinivasa Rao

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Extrusion processing is a high-temperature short time (HTST) treatment which can improve protein quality and digestibility together with retaining active nutrients. In-vitro protein digestibility of plant protein-based foods is generally enhanced by extrusion. The current study aimed to investigate the effect of extrusion cooking on in-vitro protein digestibility (IVPD) and conformational modification of protein in green banana flour extrudates. Green banana flour was extruded through a co-rotating twin-screw extruder varying the moisture content, barrel temperature, screw speed in the range of 10-20 %, 60-80 °C, 200-300 rpm, respectively, at constant feed rate. Response surface methodology was used to optimise the result for IVPD. Fourier-transform infrared spectroscopy (FTIR) analysis provided a convenient and powerful means to monitor interactions and changes in functional and conformational properties of extrudates. Results showed that protein digestibility was highest in extrudate produced at 80°C, 250 rpm and 15% feed moisture. FTIR analysis was done for the optimised sample having highest IVPD. FTIR analysis showed that there were no changes in primary structure of protein while the secondary protein structure changed. In order to explain this behaviour, infrared spectroscopy analysis was carried out, mainly in the amide I and II regions. Moreover, curve fitting analysis showed the conformational changes produced in the flour due to protein denaturation. The quantitative analysis of the changes in the amide I and II regions provided information about the modifications produced in banana flour extrudates.

Keywords: extrusion, FTIR, protein conformation, raw banana flour, SDS-PAGE method

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Authors: Gulcin Yildiz, Hao Feng

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Soybean proteins are the most widely used and researched proteins in the food industry. Due to soy allergies among consumers, however, alternative legume proteins having similar functional properties have been studied in recent years. These alternative proteins are also expected to have a price advantage over soy proteins. One such protein that has shown good potential for food applications is pea protein. Besides the favorable functional properties of pea protein, it also contains fewer anti-nutritional substances than soy protein. However, a comparison of the physicochemical properties of pea protein isolate (PPI)-starch nanocomplexes and soy protein isolate (SPI)-starch nanocomplexes treated by ultrasound has not been well documented. This study was undertaken to investigate the effects of ultrasound treatment on the physicochemical properties of PPI-starch and SPI-starch nanocomplexes. Pea protein isolate (85% pea protein) provided by Roquette (Geneva, IL, USA) and soy protein isolate (SPI, Pro-Fam® 955) obtained from the Archer Daniels Midland Company were adjusted to different pH levels (2-12) and treated with 5 minutes of ultrasonication (100% amplitude) to form complexes with starch. The soluble protein content was determined by the Bradford method using BSA as the standard. The turbidity of the samples was measured using a spectrophotometer (Lambda 1050 UV/VIS/NIR Spectrometer, PerkinElmer, Waltham, MA, USA). The volume-weighted mean diameters (D4, 3) of the soluble proteins were determined by dynamic light scattering (DLS). The emulsifying properties of the proteins were evaluated by the emulsion stability index (ESI) and emulsion activity index (EAI). Both the soy and pea protein isolates showed a U-shaped solubility curve as a function of pH, with a high solubility above the isoelectric point and a low one below it. Increasing the pH from 2 to 12 resulted in increased solubility for both the SPI and PPI-starch complexes. The pea nanocomplexes showed greater solubility than the soy ones. The SPI-starch nanocomplexes showed better emulsifying properties determined by the emulsion stability index (ESI) and emulsion activity index (EAI) due to SPI’s high solubility and high protein content. The PPI had similar or better emulsifying properties at certain pH values than the SPI. The ultrasound treatment significantly decreased the particle sizes of both kinds of nanocomplex. For all pH levels with both proteins, the droplet sizes were found to be lower than 300 nm. The present study clearly demonstrated that applying ultrasonication under different pH conditions significantly improved the solubility and emulsify¬ing properties of the SPI and PPI. The PPI exhibited better solubility and emulsifying properties than the SPI at certain pH levels

Keywords: emulsifying properties, pea protein isolate, soy protein isolate, ultrasonication

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Authors: Zahra Yadollahi, Marjan Motiei, Natalia Kazantseva, Petr Saha

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Chitosan (CS)-whey protein isolate (WPI) core-shell nanoparticles were synthesized through self-assembly of whey protein isolated polyanions and chitosan polycations in the presence of tripolyphosphate (TPP) as a crosslinker. The formation of this type of nanostructures with narrow particle size distribution is crucial for developing delivery systems since the functional characteristics highly depend on their sizes. To achieve this goal, the nanostructure was optimized by varying the concentrations of WPI, CS, and TPP in the reaction mixture. The chemical characteristics, surface morphology, and particle size of the nanoparticles were evaluated.

Keywords: whey protein isolated, chitosan, nanoparticles, delivery system

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Authors: Makan Cheraghpour, Seyed Ahmad Hosseini, Damoon Ashtary-Larky, Saeed Shirali, Matin Ghanavati, Meysam Alipour

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Background: In addition to reducing body weight, the low-calorie diets can reduce the lean body mass. It is hypothesized that in addition to reducing the body weight, the low-calorie diets can maintain the lean body mass. So, the current study aimed at evaluating the effects of high-protein diet with calorie restriction on body composition in overweight and obese individuals. Methods: 36 obese and overweight subjects were divided randomly into two groups. The first group received a normal-protein, low-energy diet (RDA), and the second group received a high-protein, low-energy diet (2×RDA). The anthropometric indices including height, weight, body mass index, body fat mass, fat free mass, and body fat percentage were evaluated before and after the study. Results: A significant reduction was observed in anthropometric indices in both groups (high-protein, low-energy diets and normal-protein, low-energy diets). In addition, more reduction in fat free mass was observed in the normal-protein, low-energy diet group compared to the high -protein, low-energy diet group. In other the anthropometric indices, significant differences were not observed between the two groups. Conclusion: Independently of the type of diet, low-calorie diet can improve the anthropometric indices, but during a weight loss, high-protein diet can help the fat free mass to be maintained.

Keywords: diet, high-protein, body mass index, body fat percentage

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Authors: Sofia G. Meirinho, Luis G. Dias, António M. Peres, Lígia R. Rodrigues

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The emerging development of electrochemical aptasen sors has enabled the easy and fast detection of protein biomarkers in standard and real samples. Biomarkers are produced by body organs or tumours and provide a measure of antigens on cell surfaces. When detected in high amounts in blood, they can be suggestive of tumour activity. These biomarkers are more often used to evaluate treatment effects or to assess the potential for metastatic disease in patients with established disease. Osteopontin (OPN) is a protein found in all body fluids and constitutes a possible biomarker because its overexpression has been related with breast cancer evolution and metastasis. Currently, biomarkers are commonly used for the development of diagnostic methods, allowing the detection of the disease in its initial stages. A previously described RNA aptamer was used in the current work to develop a simple and sensitive electrochemical aptasensor with high affinity for human OPN. The RNA aptamer was biotinylated and immobilized on a gold electrode by avidin-biotin interaction. The electrochemical signal generated from the aptamer–target molecule interaction was monitored electrochemically using cyclic voltammetry in the presence of [Fe (CN) 6]−3/− as a redox probe. The signal observed showed a current decrease due to the binding of OPN. The preliminary results showed that this aptasensor enables the detection of OPN in standard solutions, showing good selectivity towards the target in the presence of others interfering proteins such as bovine OPN and bovine serum albumin. The results gathered in the current work suggest that the proposed electrochemical aptasensor is a simple and sensitive detection tool for human OPN and so, may have future applications in cancer disease monitoring.

Keywords: osteopontin, aptamer, aptasensor, screen-printed electrode, cyclic voltammetry

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Authors: Caspar S. Carson, Gabriel W. Prather, Nicholas E. Wong, Jeffery R. Anton, William H. McCoy

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Cutibacterium acnes is a commensal bacterium found on human skin that has been linked to acne. C. acnes can also be an opportunistic pathogen when it infiltrates the body during surgery. This pathogen can cause dangerous infections of medical implants, such as shoulder replacements, leading to life-threatening blood infections. Compounding this issue, C. acnes resistance to many antibiotics has become an increasing problem worldwide, creating a need for special forms of treatment. C. acnes expresses the protein RoxP, and it requires this protein to colonize human skin. Though this protein is required for C. acnes skin colonization, its function is not yet understood. Inhibition of RoxP function might be an effective treatment for C. acnes infections. To develop such reagents, the McCoy Laboratory generated four unique anti-RoxP antibodies. Preliminary studies in the McCoy Lab have established that each antibody binds a distinct site on RoxP. To assess the potential of these antibodies as therapeutics, it is necessary to specifically characterize these antibody epitopes and evaluate them in assays that assess their ability to inhibit RoxP-dependent C. acnes growth. To provide material for these studies, an antibody expression construct, Fv-clasp(v2), was adapted to encode anti-RoxP antibody sequences. The author hypothesizes that this expression strategy can produce sufficient amounts of >95% pure antibody fragments for further characterization of these antibodies. Four anti-RoxP Fv-clasp(v2) expression constructs (pET vector-based) were transformed into E. coli BL21-Gold(DE3) cells and a small-scale expression and purification trial was performed for each construct to evaluate anti-RoxP Fv-clasp(v2) yield and purity. Successful expression and purification of these antibody constructs will allow for their use in structural studies, such as protein crystallography and cryogenic electron microscopy. Such studies would help to define the antibody binding sites on RoxP, which could then be leveraged in the development of certain methods to treat C. acnes infection through RoxP inhibition.

Keywords: structural biology, protein expression, infectious disease, antibody, therapeutics, E. coli

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Authors: Dina Fatmawati, Sofia Mubarika, Mae Sri Wahyuningsih

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Chemotherapy for breast cancer is largely ineffective, but innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. In our previous study, the combination of Doxorubicin (Dox) and ethanolic extract of Dioscorea esculenta tuber ((EED) was found to have a synergistic effect on T47D human breast cancer cell line. In this study, we investigated the apoptotic effect of the combination on T47D human breast cancer cells and normal fibroblasts cell line and its effects on the expression of Caspase-3 and cleaved poly (ADP-Ribose) Polymerase-1 (cPARP-1) protein. T47D cell lines and fibroblasts cells were treated with the combination of Dox and EED. Apoptotic effect of the combination was determined using flow cytrometry assay. Protein expressions were determined by immunocytochemistry staining. The percentage of apoptotic cells were significantly higher in T47D cell lines (75%) than that of in fibroblast cells (23%). The expression of Caspase 3 (84.53%) and cPARP-1 (83.36%) were significantly higher in the cancer cell lines than those of normal cells. These results indicate that the combination of doxorubicin and Dioscorea esculenta is a promising candidate for the treatment of breast cancer cells.

Keywords: Dioscorea esculenta, Doxorubicin, apoptosis, immunocytochemistry, cancer cells

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Authors: Thanyaporn Senarai, Rapeepun Vanichviriyakit, Shinji Miyata, Chihiro Sato, Prapee Sretarugsa, Wattana Weerachatyanukul, Ken Kitajima

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In this study, we characterized a region-specific 250 kDa protein that was secreted of MSD fluid, which is believed to play dual functions in forming a spermatophoric wall for sperm physical protection, and in sperm membrane modification as part of sperm maturation process. The partial amino acid sequence and N-terminal sequencing revealed that the MSD-specific 250 kDa protein showed a high similarity with a plasma-rich protein, α-2 macroglobulin (α2M), so termed pp-α2M. This protein was a large glycoprotein contained predominantly mannose and GlcNAc. The expression of pp-α2M mRNA was detected in spermatic duct (SD), androgenic gland (AG) and hematopoietic tissue, while the protein expression was rather specific to the apical cytoplasm of MSD epithelium. The secretory pp-α2M in MSD fluid was acquired onto the MSD sperm membrane and was also found within the matrix of the acrosome. Distally, pp-α2M was removed from spermathecal sperm membrane, while its level kept constant in the sperm AC. Together the results indicate that pp-α2M is a 250 kDa region-specific secretory protein which plays roles in sperm physical protection and also acts as maturation factor in the P. pelagicus sperm.

Keywords: alpha-2 macroglobulin, blue swimming crab, sperm maturation, spermatic duct

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Authors: Mardiati Zain, Yetti Marlida, Elihasridas Elihasridas, Erpomen Erpomen, Andri Andri

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Background: Livestock productivity is greatly influenced by the energy and protein balance in diet. This study aimed to determine the energy and protein balance of male Simenthal cattle diet with protein and energy levels. The experimental design used was a randomized block design (RBD) 2x3x3 factorial design. There are two factors namely A level of energy diet that is 65% and 70% TDN. Factor B is a protein level of diet used were 10, 12 and 14% and each treatment is repeated three times. The weight of Simenthal cattle used ranged between 240 - 300 kg. Diet consisted of ammoniated rice straw and concentrated with ratio 40:60. Concentrate consisted of palm kernel cake, rice brain, cassava, mineral, and urea. The variables measured were digestibility of dry matter, organic matter and fiber, dry matter intake, daily gain, feed efficiency and blood characteristic. Results: There was no interaction between protein and energy level of diet on the nutrients intake (DM intake, OM intake, CP intake), weight gain and efficiency (P < 0.01). There was an interaction between protein and energy level of diet on digestibility (DM, OM, CP and allantoin urine (P > 0.01) Nutrients intake decreases with increasing levels of energy and protein diet, while nutrient digestibility, Avarage daily gain and feed efficiency increases with increasing levels of energy and protein diet. Conclusions: The result can be concluded that the best treatment was A2B1 which is energy level 70% TDN and protein 10%, where are dry matter intake 7.66 kg/d, daily gain 1.25 kg/d, feed efficiency 16.12%, and dry matter and organic matter digestibility 64.08 and 69.42% respectively.

Keywords: energy and protein ratio, simenthal cattle, rice straw ammoniated, digestibility

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Authors: Alireza Dantism

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Presently, describing the function of a protein sequence is one of the most common problems in biology. Usually, this problem can be facilitated by studying the three-dimensional structure of proteins. In the absence of a protein structure, comparative modeling often provides a useful three-dimensional model of the protein that is dependent on at least one known protein structure. Comparative modeling predicts the three-dimensional structure of a given protein sequence (target) mainly based on its alignment with one or more proteins of known structure (templates). Comparative modeling consists of four main steps 1. Similarity between the target sequence and at least one known template structure 2. Alignment of target sequence and template(s) 3. Build a model based on alignment with the selected template(s). 4. Prediction of model errors 5. Optimization of the built model There are many computer programs and web servers that automate the comparative modeling process. One of the most important advantages of these servers is that it makes comparative modeling available to both experts and non-experts, and they can easily do their own modeling without the need for programming knowledge, but some other experts prefer using programming knowledge and do their modeling manually because by doing this they can maximize the accuracy of their modeling. In this study, a web-based tool has been designed to predict the tertiary structure of proteins using PHP and Python programming languages. This tool is called EasyModel. EasyModel can receive, according to the user's inputs, the desired unknown sequence (which we know as the target) in this study, the protein sequence file (template), etc., which also has a percentage of similarity with the primary sequence, and its third structure Predict the unknown sequence and present the results in the form of graphs and constructed protein files.

Keywords: structural bioinformatics, protein tertiary structure prediction, modeling, comparative modeling, modeller

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Authors: Alexander A. Tokmakov

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Cell-free protein synthesis is widely used to synthesize recombinant proteins. It allows genome-scale expression of various polypeptides under strictly controlled uniform conditions. However, only a minor fraction of all proteins can be successfully expressed in the systems of protein synthesis that are currently used. The factors determining expression success are poorly understood. At present, the vast volume of data is accumulated in cell-free expression databases. It makes possible comprehensive bioinformatics analysis and identification of multiple features associated with successful cell-free expression. Here, we describe an approach aimed at identification of multiple physicochemical and structural properties of amino acid sequences associated with protein solubility and aggregation and highlight major correlations obtained using this approach. The developed method includes: categorical assessment of the protein expression data, calculation and prediction of multiple properties of expressed amino acid sequences, correlation of the individual properties with the expression scores, and evaluation of statistical significance of the observed correlations. Using this approach, we revealed a number of statistically significant correlations between calculated and predicted features of protein sequences and their amenability to cell-free expression. It was found that some of the features, such as protein pI, hydrophobicity, presence of signal sequences, etc., are mostly related to protein solubility, whereas the others, such as protein length, number of disulfide bonds, content of secondary structure, etc., affect mainly the expression propensity. We also demonstrated that amenability of polypeptide sequences to cell-free expression correlates with the presence of multiple sites of post-translational modifications. The correlations revealed in this study provide a plethora of important insights into protein folding and rationalization of protein production. The developed bioinformatics approach can be of practical use for predicting expression success and optimizing cell-free protein synthesis.

Keywords: bioinformatics analysis, cell-free protein synthesis, expression success, optimization, recombinant proteins

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Authors: Angela U. Makolo

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Protein-coding and Non-coding regions determine the biology of a sequenced transcriptome. Research advances have shown that Non-coding regions are important in disease progression and clinical diagnosis. Existing bioinformatics tools have been targeted towards Protein-coding regions alone. Therefore, there are challenges associated with gaining biological insights from transcriptome sequence data. These tools are also limited to computationally intensive sequence alignment, which is inadequate and less accurate to identify both Protein-coding and Non-coding regions. Alignment-free techniques can overcome the limitation of identifying both regions. Therefore, this study was designed to develop an efficient sequence alignment-free model for identifying both Protein-coding and Non-coding regions in sequenced transcriptomes. Feature grouping and randomization procedures were applied to the input transcriptomes (37,503 data points). Successive iterations were carried out to compute the gradient vector that converged the developed Protein-coding and Non-coding Region Identifier (PNRI) model to the approximate coefficient vector. The logistic regression algorithm was used with a sigmoid activation function. A parameter vector was estimated for every sample in 37,503 data points in a bid to reduce the generalization error and cost. Maximum Likelihood Estimation (MLE) was used for parameter estimation by taking the log-likelihood of six features and combining them into a summation function. Dynamic thresholding was used to classify the Protein-coding and Non-coding regions, and the Receiver Operating Characteristic (ROC) curve was determined. The generalization performance of PNRI was determined in terms of F1 score, accuracy, sensitivity, and specificity. The average generalization performance of PNRI was determined using a benchmark of multi-species organisms. The generalization error for identifying Protein-coding and Non-coding regions decreased from 0.514 to 0.508 and to 0.378, respectively, after three iterations. The cost (difference between the predicted and the actual outcome) also decreased from 1.446 to 0.842 and to 0.718, respectively, for the first, second and third iterations. The iterations terminated at the 390th epoch, having an error of 0.036 and a cost of 0.316. The computed elements of the parameter vector that maximized the objective function were 0.043, 0.519, 0.715, 0.878, 1.157, and 2.575. The PNRI gave an ROC of 0.97, indicating an improved predictive ability. The PNRI identified both Protein-coding and Non-coding regions with an F1 score of 0.970, accuracy (0.969), sensitivity (0.966), and specificity of 0.973. Using 13 non-human multi-species model organisms, the average generalization performance of the traditional method was 74.4%, while that of the developed model was 85.2%, thereby making the developed model better in the identification of Protein-coding and Non-coding regions in transcriptomes. The developed Protein-coding and Non-coding region identifier model efficiently identified the Protein-coding and Non-coding transcriptomic regions. It could be used in genome annotation and in the analysis of transcriptomes.

Keywords: sequence alignment-free model, dynamic thresholding classification, input randomization, genome annotation

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