Search results for: recombinant protein processing

Authors: Evgeniya Y. Yuzbasheva, Tigran V. Yuzbashev, Natalia I. Perkovskaya, Elizaveta B. Mostova

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Cell-surface display of lipase is of great interest as it has many applications in the field of biotechnology owing to its unique advantages: simplified product purification, and cost-effective downstream processing. One promising area of application for whole-cell biocatalysts with surface displayed lipase is biodiesel synthesis. Biodiesel is biodegradable, renewable, and nontoxic alternative fuel for diesel engines. Although the alkaline catalysis method has been widely used for biodiesel production, it has a number of limitations, such as rigorous feedstock specifications, complicated downstream processes, including removal of inorganic salts from the product, recovery of the salt-containing by-product glycerol, and treatment of alkaline wastewater. Enzymatic synthesis of biodiesel can overcome these drawbacks. In this study, Lip2p lipase was displayed on Yarrowia lipolytica cells via C- and N-terminal fusion variant. The active site of lipase is located near the C-terminus, therefore to prevent the activity loosing the insertion of glycine-serine linker between Lip2p and C-domains was performed. The hydrolytic activity of the displayed lipase reached 12,000–18,000 U/g of dry weight. However, leakage of enzyme from the cell wall was observed. In case of C-terminal fusion variant, the leakage was occurred due to the proteolytic cleavage within the linker peptide. In case of N-terminal fusion variant, the leaking enzyme was presented as three proteins, one of which corresponded to the whole hybrid protein. The calculated number of recombinant enzyme displayed on the cell surface is approximately 6–9 × 105 molecules per cell, which is close to the theoretical maximum (2 × 106 molecules/cell). Thus, we attribute the enzyme leakage to the limited space available on the cell surface. Nevertheless, cell-bound lipase exhibited greater stability to short-term and long-term temperature treatment than the native enzyme. It retained 74% of original activity at 60°C for 5 min of incubation, and 83% of original activity after incubation at 50°C during 5 h. Cell-bound lipase had also higher stability in organic solvents and detergents. The developed whole-cell biocatalyst was used for recycling biodiesel synthesis. Two repeated cycles of methanolysis yielded 84.1–% and 71.0–% methyl esters after 33–h and 45–h reactions, respectively.

Keywords: biodiesel, cell-surface display, lipase, whole-cell biocatalyst

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Authors: Oscar Rubiano, Oscar Ortiz, Natalia Morales, Lida Alfonso, Johana Alvarado, Adriana Gutierrez, Daniel Botero

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Athletes who usually use protein supplements, are those who practice strength and power sports, whose goal is to achieve a large muscle mass. However, it has also been explored in sports or endurance activities such as cycling, and where despite requiring high power, prominent muscle development can impede good competitive performance due to the determinant of body mass for good performance of the athlete body. This research shows, the effect with protein supplements establishes a protein - muscle mass ratio, although in a lesser proportion the relationship between protein types and muscle power. Thus, we intend to explore as a first approximation, the behavior of muscle power in lower limbs after the intake of two protein supplements from different sources. The aim of the study was to describe the behavior of muscle power in lower limbs after the consumption of animal protein (AP) and vegetable protein (VP) in four route cyclists from 18 to 20 years of the Bogota cycling league. The methodological design of this study is quantitative, with a non-probabilistic sampling, based on a pre-experimental model. The jumping power was evaluated before and after the intervention by means of the squat jump test (SJ), Counter movement jump (CMJ) and Abalacov (AB). Cyclists consumed a drink with whey protein and a soy isolate after training four times a week for three months. The amount of protein in each cyclist, was calculated according to body weight (0.5 g / kg of muscle mass). The results show that subjects who consumed PV improved muscle strength and landing strength. In contrast, the power and landing force decreased for subjects who consumed PA. For the group that consumed PV, the increase was positive at 164.26 watts, 135.70 watts and 33.96 watts for the AB, SJ and CMJ jumps respectively. While for PA, the differences of the medians were negative at -32.29 watts, -82.79 watts and -143.86 watts for the AB, SJ and CMJ jumps respectively. The differences of the medians in the AB jump were positive for both the PV (121.61 Newton) and PA (454.34 Newton) cases, however, the difference was greater for PA. For the SJ jump, the difference for the PA cases was 371.52 Newton, while for the PV cases the difference was negative -448.56 Newton, so the difference was greater in the SJ jump for PA. In jump CMJ, the differences of the medians were negative for the cases of PA and PV, being -7.05 for PA and - 958.2 for PV. So the difference was greater for PA. The conclusion of this study shows that serum protein supplementation showed no improvement in muscle power in the lower limbs of the cyclists studied, which could suggest that whey protein does not have a beneficial effect on performance in terms of power, either, showed an impact on body composition. In contrast, supplementation with soy isolate showed positive effects on muscle power, body.

Keywords: animal protein (AP), muscle power, supplements, vegetable protein (VP)

Procedia PDF Downloads 152

Authors: Hamid Hossein Khezri, Afsaneh Javdani-Mallak

Abstract:

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Cannabidiol (CBD) is one of the members of cannabinoid compounds found in cannabis. CBD and Cannabinol (CBN), as the other extract of plant Cannabis were able to reduce myofascial pain in rats with immunosuppressive and anti-inflammatory activities. In this study, we accomplished protein-protein BLAST, and the sequence was found to be for Gamma-aminobutyric acid receptor subunit alpha-1 (GBRA1) chain A and its 3D structure was subsequently downloaded from Protein Data Bank. The structures of the ligands, cannabinol, and cannabidiol, were obtained from PubChem. After the necessary process of the obtained files, AutoDock Vina was used to perform molecular docking. Docking between the ligands and GBRA1 chain A revealed that cannabinol has a higher affinity to GBRA1 (binding energy = -7.5 kcal/mol) compared to cannabidiol (binding energy = -6.5 kcal/mol). Furthermore, cannabinol seems to be able to interact with 10 residues of the protein, out of which 3 are in the neurotransmitter-gated ion-channel transmembrane domain of GBRA1, whereas cannabidiol interacts with two other residues. Although the results of this project do not indicate the activating /or inhibitory capability of the studied compounds, it suggests that cannabinol can act as a relatively strong ligand for GBRA1.

Keywords: protein-ligand docking, cannabinol, cannabidiol, GBRA1

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Authors: Aida Kalantari, Boyang Ji, Tao Chen, Ivan Mijakovic

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3-hydroxypropanoic acid (3-HP) is one of the most important biomass-derivable platform chemicals that can be converted into a number of industrially important compounds. There have been several attempts at production of 3-HP from renewable sources in cell factories, focusing mainly on Escherichia coli, Klebsiella pneumoniae, and Saccharomyces cerevisiae. Despite the significant progress made in this field, commercially exploitable large-scale production of 3-HP in microbial strains has still not been achieved. In this study, we investigated the potential of Bacillus subtilis to be used as a microbial platform for bioconversion of glycerol into 3-HP. Our recombinant B. subtilis strains overexpress the two-step heterologous pathway containing glycerol dehydratase and aldehyde dehydrogenase from various backgrounds. The recombinant strains harboring the codon-optimized synthetic pathway from K. pneumoniae produced low levels of 3-HP. Since the enzymes in the heterologous pathway are sensitive to oxygen, we had to perform our experiments in micro-aerobic conditions. Under these conditions, the cell produces lactate in order to regenerate NAD+, and we found the lactate production to be in competition with the production of 3-HP. Therefore, based on the in silico predictions, we knocked out the glycerol kinase (glpk), which in combination with growth on glucose, resulted in improving the 3-HP titer to 1 g/L and the removal of lactate. Cultivation of the same strain in an enriched medium improved the 3-HP titer up to 7.6 g/L. Our findings provide the first report of successful introduction of the biosynthetic pathway for conversion of glycerol into 3-HP in B. subtilis.

Keywords: bacillus subtilis, glycerol, 3-hydroxypropanoic acid, metabolic engineering

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Authors: Zahra Akbari, Farzin Zokaee, Talat Ghomashchi

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Whey is a by-product of the dairy industry whose major components are lactose (44–52 g/L), proteins (6–8 g/L) and mineral salts (4–9 g/L). Approximately 50% of 121 million tons of whey produced in the world in 1993 were disposed into rivers, lakes or other water bodies, treated in wastewater treatment plants or loaded onto land. This represents a significant loss of resources and causes serious pollution problems since whey is a heavy organic pollutant with high COD and BOD values, 40–60 g/L and 50–80 g/L, respectively. The removal of cheese whey proteins and minerals represent an important task both in environmental and in food sciences. The most important treatments which are considered in this study, have been done by using lime, Al2O3, FeCl3 and AlCl3 along with heating and also acidic-alkaline method. Results show that the best way for removal of protein is accomplished with adding HCl to decrease pH from 6 to 4, boiling for 20 min, and filtering protein aggregates. Also partial demineralization in whey solution for reducing ash is accomplished by adding NaOH to increase pH to 7.2 and heating solution for 20 min.

Keywords: whey treatment, dairy industry, precipitation, protein, mineral

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Authors: Sulhee Lee, Geon Kim, Young-Seo Park

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Unlike the traditional milk sterilization methods (LTLT, HTST, or UHT), pulsed electric field (PEF) technology is a non-thermal pasteurization process. This technology minimizes energy required for heat treatment in food processing, changes in sensory properties, and physical losses. In this study, structural changes of bovine milk proteins, the amount of immunoproteins such as IgG, and their storability by PEF treatment were examined. When the changes of protein content in PEF-treated milk were examined using HPLC, the amounts of α-casein and β-lactoglobulin were reduced over 40% each, whereas those of κ-casein and β-casein did not change. The amount of α-casein in HTST milk was reduced to 50%. When PEF was applied to milk at the energy level of 250 kJ, the amounts of IgG, IgA, β-lactoglobulin (β-LG), lactoferrin, and α-lactalbumin (α-LA) decreased by 43, 41, 35, 63, and 45%, respectively. When milk was sterilized by LTLT process followed by PEF process at the level of 150 kJ, the concentrations of IgG, IgA, β-LG, lactoferrin, and α-LA were 56.6, 10.6, 554, 2.8 and 660.1 μg/mL, respectively. When the bovine milk was sterilized by LTLT process followed by PEF process at the energy level of 180 kJ, storability of immunoproteins of milk was the highest and the concentrations of IgG, IgA, and β-LG decreased by 79.5, 6.5, and 134.5 μg/mL, respectively, when compared with the initial concentrations of those proteins. When bovine milk was stored at 4℃ after sterilization through HTST sterilizer followed by PEF process at the energy level of 200 kJ, the amount of lactoferrin decreased 7.3% after 36 days of storage, whereas that of lactoferrin of raw milk decreased 16.4%. Our results showed that PEF treatment did not change the protein structure nor induce protein denaturation in milk significantly when compared with LTLT or HTST sterilization. Also, LTLT or HTST process in combination with PEF were more effective than LTLT only or HTST only process in the conservation of immunoproteins in bovine milk.

Keywords: pulsed electric field, bovine milk, immunoproteins, sterilization

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Authors: Shahram Naghizadeh Raeisi, Ali Alghooneh

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The application of whey protein in drink industry to enhance the nutritional value of the products is important. Furthermore, the gelification of protein during thermal treatment and shelf life makes some limitations in its application. So, the main goal of this research is manufacturing of high concentrate whey protein orange drink with appropriate shelf life. In this way, whey protein was 5 to 30% hydrolyzed ( in 5 percent intervals at six stages), then thermal stability of samples with 10% concentration of protein was tested in acidic condition (T= 90 °C, pH=4.2, 5 minutes ) and neutral condition (T=120° C, pH:6.7, 20 minutes.) Furthermore, to study the shelf life of heat treated samples in 4 months at 4 and 24 °C, the time sweep rheological test were done. At neutral conditions, 5 to 20% hydrolyzed sample showed gelling during thermal treatment, whereas at acidic condition, was happened only in 5 to 10 percent hydrolyzed samples. This phenomenon could be related to the difference in hydrodynamic radius and zeta potential of samples with different level of hydrolyzation at acidic and neutral conditions. To study the gelification of heat resistant protein solutions during shelf life, for 4 months with 7 days intervals, the time sweep analysis were performed. Cross over was observed for all heat resistant neutral samples at both storage temperature, while in heat resistant acidic samples with degree of hydrolysis, 25 and 30 percentage at 4 and 20 °C, it was not seen. It could be concluded that the former sample was stable during heat treatment and 4 months storage, which made them a good choice for manufacturing high protein drinks. The Scheffe polynomial model and numerical optimization were employed for modeling and high protein orange drink formula optimization. Scheffe model significantly predicted the overal acceptance index (Pvalue<0.05) of sensorial analysis. The coefficient of determination (R2) of 0.94, the adjusted coefficient of determination (R2Adj) of 0.90, insignificance of the lack-of-fit test and F value of 64.21 showed the accuracy of the model. Moreover, the coefficient of variable (C.V) was 6.8% which suggested the replicability of the experimental data. The desirability function had been achieved to be 0.89, which indicates the high accuracy of optimization. The optimum formulation was found as following: Modified whey protein solution (65.30%), natural orange juice (33.50%), stevia sweetener (0.05%), orange peel oil (0.15%) and citric acid (1 %), respectively. Its worth mentioning that this study made an appropriate model for application of whey protein in drink industry without bitter flavor and gelification during heat treatment and shelf life.

Keywords: croos over, orange beverage, protein modification, optimization

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Authors: Berhan Fikru

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The objective of this study was to develop new complementary food from maize, soybean and moringa for young children. The complementary foods were formulated with linear programming (LP Nutri-survey software) and Faffa (corn soya blend) use as control. Analysis were made for formulated blends and compared with the control and recommended daily intake (RDI). Three complementary foods composed of maize, soya bean, moringa and sugar with ratio of 65:20:15:0, 55:25:15:5 and 65:20:10:5 for blend 1, 2 and 3, respectively. The blends were formulated based on the protein, energy, mineral (iron, zinc an calcium) and vitamin (vitamin A and C) content of foods. The overall results indicated that nutrient content of faffa (control) was 16.32 % protein, 422.31 kcal energy, 64.47 mg calcium, 3.8 mg iron, 1.87mg zinc, 0.19 mg vitamin A and 1.19 vitamin C; blend 1 had 17.16 % protein, 429.84 kcal energy, 330.40 mg calcium, 6.19 mg iron, 1.62 mg zinc, 6.33 mg vitamin A and 4.05 mg vitamin C; blend 2 had 20.26 % protein, 418.79 kcal energy, 417.44 mg calcium, 9.26 mg iron, 2.16 mg zinc, 8.43 mg vitamin A and 4.19 mg vitamin C whereas blend 3 exhibited 16.44 % protein, 417.42 kcal energy, 242.4 mg calcium, 7.09 mg iron, 2.22 mg zinc, 3.69 mg vitamin A and 4.72 mg vitamin C, respectively. The difference was found between all means statically significance (P < 0.05). Sensory evaluation showed that the faffa control and blend 3 were preferred by semi-trained panelists. Blend 3 had better in terms of its mineral and vitamin content than FAFFA corn soya blend and comparable with WFP proprietary products CSB+, CSB++ and fulfills the WHO recommendation for protein, energy and calcium. The suggested formulation with Moringa powder can therefore be used as a complementary food to improve the nutritional status and also help solve problems associated with protein energy and micronutrient malnutrition for young children in developing countries, particularly in Ethiopia.

Keywords: corn soya blend, proximate composition, micronutrient, mineral chelating agents, complementary foods

Procedia PDF Downloads 266

Authors: Jean Paul Hategekimana, Bampire Claudine, Niyonsenga Nadia, Irakoze Josiane

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Peas, soybeans and rice are crops which are grown in Rwanda and are available in rural and urban local markets and they give contribution in reduction of health problems especially in fighting malnutrition and food insecurity in Rwanda. Several research activities have been conducted on how cereals flour can be mixed with legumes flour for developing baked products which are rich in protein, fiber, minerals as they are found in legumes. However, such activity was not yet well studied in Rwanda. The aim of the present study was to develop bread and biscuit products from peas, soybeans and rice as functional ingredients combined with wheat flour and then analyze the nutritional content and consumer acceptability of new developed products. The malnutrition problem can be reduced by producing bread and biscuits which are rich in protein and are very accessible for every individual. The processing of bread and biscuit were made by taking peas flour, soybeans flour and rice flour mixed with wheat flour and other ingredients then a dough was made followed by baking. For bread, two kind of products were processed, for each product one control and three experimental samples in different three ratios of peas and rice were prepared. These ratios were 95:5, 90:10 and 80:20 for bread from peas and 85:5:10, 80:10:10 and 70:10:20 for bread from peas and rice. For biscuit, two kind of products were also processed, for each product one control sample and three experimental samples in three different ratios were prepared. These ratios are 90:5:5,80:10:10 and 70:10:20 for biscuit from peas and rice and 90:5:5,80:10:10 and 70:10:20 for biscuit from soybean and rice. All samples including the control sample were analyzed for the consumer acceptability (sensory attributes) and nutritional composition. For sensory analysis, bread from of peas and rice flour with wheat flour at ratio 85:5:10 and bread from peas only as functional ingredient with wheat flour at ratio 95:5 and biscuits made from a of soybeans and rice at a ratio 90:5:5 and biscuit made from peas and rice at ratio 90:5:5 were most acceptable compared to control sample and other samples in different ratio. The moisture, protein, fat, fiber and minerals (Sodium and iron.) content were analyzed where bread from peas in all ratios was found to be rich in protein and fiber compare to control sample and biscuit from soybean and rice in all ratios was found to be rich in protein and fiber compare to control sample.

Keywords: bakery products, peas and rice flour, wheat flour, sensory evaluation, proximate composition

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Authors: Asnate Kirse, Daina Karklina, Sandra Muizniece-Brasava, Ruta Galoburda

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Pulses are high in plant protein and dietary fiber, and contain slowly digestible starches. Innovative products from pulses could increase their consumption and benefit consumer health. This study was conducted to evaluate physicochemical stability of processed cowpea (Vigna unguiculata (L.) Walp. cv. Fradel) and maple pea (Pisum sativum var. arvense L. cv. Bruno) spreads at 5 °C temperature during 62-day storage. Physicochemical stability of pulse spreads was compared after sous vide treatment (80 °C/15 min) and high pressure processing (700 MPa/10 min/20 °C). Pulse spreads were made by homogenizing cooked pulses in a food processor together with salt, citric acid, oil, and bruschetta seasoning. A total of four different pulse spreads were studied: Cowpea spread without and with seasoning, maple pea spread without and with seasoning. Transparent PA/PE and light proof PET/ALU/PA/PP film pouches were used for packaging of pulse spreads under vacuum. The parameters investigated were pH, water activity and mass losses. Pulse spreads were tested on days 0, 15, 29, 42, 50, 57 and 62. The results showed that sous-vide treatment and high pressure processing had an insignificant influence on pH, water activity and mass losses after processing, irrespective of packaging material did not change (p>0.1). pH and water activity of sous-vide treated and high pressure processed pulse spreads in different packaging materials proved to be stable throughout the storage. Mass losses during storage accounted to 0.1% losses. Chosen sous-vide treatment and high pressure processing regimes and packaging materials are suitable to maintain consistent physicochemical quality of the new products during 62-day storage.

Keywords: cowpea, flexible packaging, maple pea, water activity

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Authors: Fengmei Li, Li Xu, Guoliang Xia

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Whey acidic proteins (WAP) domain-containing proteins in crustacean are involved in innate immune response against microbial invasion. In the present study, a novel double WAP domain (DWD)-containing protein gene 3 was identified from Chinese mitten crab Eriocheir sinensis (designated EsDWD3) by expressed sequence tag (EST) analysis and PCR techniques. The full-length cDNA of EsDWD3 was of 1223 bp, consisting of a 5′-terminal untranslated region (UTR) of 74 bp, a 3′ UTR of 727 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 423 bp. The ORF encoded a polypeptide of 140 amino acids with a signal peptide of 22 amino acids. The deduced protein sequence EsDWD3 showed 96.4 % amino acid similar to other reported EsDWD1 from E. sinensis, and phylogenetic tree analysis revealed that EsDWD3 had closer relationships with the reported two double WAP domain-containing proteins of E. sinensis species.

Keywords: Chinese mitten crab, Eriocheir sinensis, cloning, double WAP domain-containing protein

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Authors: Hamid Hossein Khezri, Afsaneh Javdani-Mallak

Abstract:

Fast inhibitory neurotransmission in the mammalian nervous system is largely mediated by GABAA receptors, chloride-selective members of the superfamily of pentameric Cys-loop receptors. Cannabidiol (CBD) is one of the members of cannabinoid compounds found in cannabis. CBD and Cannabinol (CBN), as the other extract of plant Cannabis, were able to reduce myofascial pain in rats with immunosuppressive and anti-inflammatory activities. In this study, we accomplished protein-protein BLAST and the sequence was found to be for Gamma-aminobutyric acid receptor subunit alpha-1 (GBRA1) chain A and its 3D structure was subsequently downloaded from Protein Data Bank. The structures of the ligands cannabinol and cannabidiol were obtained from PubChem. After a necessary process of the obtained files, AutoDock Vina was used to performing molecular docking. Docking between the ligands and GBRA1 chain A revealed that cannabinol has a higher affinity to GBRA1 (binding energy = -7.5 kcal/mol) compared to cannabidiol (binding energy = -6.5 kcal/mol). Furthermore, cannabinol seems to be able to interact with 10 residues of the protein, out of which 3 are in the neurotransmitter-gated ion-channel transmembrane domain of GBRA1, whereas cannabidiol interacts with two other residues. Although the results of this project do not indicate the activating /or inhibitory capability of the studied compounds, it suggests that cannabinol can act as a relatively strong ligand for GBRA1.

Keywords: protein-ligand docking, cannabinol, cannabidiol, GBRA1

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Authors: Keaghan Brown

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The prioritization of drug resistance mutations impacting protein folding or protein-drug and protein-DNA interactions within macromolecular systems is critical to the success of treatment regimens. With a continual increase in computational tools to assess these impacts, the need for scalability and reproducibility became an essential component of computational analysis and experimental research. Here it introduce a bioinformatics pipeline that combines several structural analysis tools in a simplified workflow, by optimizing the present computational hardware and software to automatically ease the flow of data transformations. Utilizing preestablished software tools, it was possible to develop a pipeline with a set of pre-defined functions that will automate mutation introduction into the HIV-1 Integrase protein structure, calculate the gain and loss of polar interactions and calculate the change in energy of protein fold. Additionally, an automated molecular dynamics analysis was implemented which reduces the constant need for user input and output management. The resulting pipeline, Automated Mutation Introduction and Analysis (AMIA) is an open source set of scripts designed to introduce and analyse the effects of mutations on the static protein structure as well as the results of the multi-conformational states from molecular dynamic simulations. The workflow allows the user to visualize all outputs in a user friendly manner thereby successfully enabling the prioritization of variant systems for experimental validation.

Keywords: automated workflow, variant prioritization, drug resistance, HIV Integrase

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Authors: Zyrah Lou R. Samar, Genecarlo Liwanag

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Type 2 Diabetes Mellitus is the more prevalent type, caused by a combination of insulin resistance and inadequate insulin response to hyperglycemia1. Aside from pharmacologic interventions, medical nutrition therapy is an integral part of the management of patients with Type 2 Diabetes Mellitus. Whey protein, which is one of the best protein sources, has been investigated for its applicability in improving glycemic control in patients with Type 2 Diabetes Mellitus. This systematic review and meta-analysis was conducted to measure the magnitude of the effect of whey protein on glycemic control in type 2 diabetes mellitus. The aim of this review is to evaluate the efficacy and safety of whey protein in patients with type 2 diabetes mellitus. Methods: A systematic electronic search for studies in the PubMed and Cochrane Collaboration database was done. Included in this review were randomized controlled trials of whey protein enrolling patients with type 2 diabetes mellitus. Three reviewers independently searched, assessed, and extracted data from the individual studies. Results: A systematic literature search on online databases such as Cochrane Central Registry, PubMed, and Herdin Plus was conducted in April to September 2021 to identify eligible studies. The search yielded 21 randomized controlled trials after removing duplicates. Only 5 articles were included after reviewing the full text, which met the criteria for selection. Conclusion: Whey protein supplementation significantly reduced fasting blood glucose. However, it did not reduce post-prandial blood glucose, HbA1c level, and weight when compared with the placebo. There has been a considerate heterogeneity across all studies, which may have contributed/confounded its effects. A larger sample size and better inclusion, and a more specific study may be included in the future reviews.

Keywords: whey protein, diabetes, nutrition, fasting blood sugar, postprandial glucose, HbA1c, weight reduction

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Authors: Jiun-Nan Hou, Tien-Huang Chen, Wei-June Chen

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Dengue viruses (DENVs) are naturally transmitted between humans by mosquito vectors. Mosquito cells usually survive DENV infection, allowing infected mosquitoes to retain an active status for virus transmission. In this study, we found that DENV2 virus infection in mosquito cells causes the unfolded protein response (UPR) that activates the protein kinase RNA-like endoplasmic reticulum kinase (PERK) signal pathway, leading to shutdown of global protein translation in infected cells which was apparently regulated by the PERK signal pathway. According to observation in this study, the PERK signal pathway in DENV2-infected C6/36 cells alleviates ER stress, and reduces initiator and effector caspases, as well as the apoptosis rate via shutdown of cellular proteins. In fact, phosphorylation of eukaryotic initiation factor 2ɑ (eIF2ɑ) by the PERK signal pathway may impair recruitment of ribosomes that bind to the mRNA 5’-cap structure, resulting in an inhibitory effect on canonical cap-dependent cellular protein translation. The resultant pro-survival “byproduct” of infected mosquito cells is undoubtedly advantageous for viral replication. This finding provides insights into elucidating the PERK-mediated modulating web that is actively involved in dynamic protein synthesis, cell survival, and viral replication in mosquito cells.

Keywords: cap-dependent protein translation, dengue virus, endoplasmic reticulum stress, mosquito cells, PERK signal pathway

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Authors: Michelangelo Liban, Debbie Liquete

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A 70-year-old right-handed Filipina was seen by the Neurology service due to a new onset headache, bi-occipital in location, dull squeezing in character with a pain score of 8/10 with associated nausea and one episode of non-projectile, which provided no relief. Due to the alarming features of the headache despite the absence of risk factors and an essentially normal neurologic examination, a cranial CTA+CTV was done, which revealed a small left frontal and small right pontine hyper density with minimal perilesional edema. Findings also revealed filling defects in the straight and right transverse sinus and a consideration of hypoplastic left transverse sinus with no definite evidence of aneurysm nor A-V malformation. She had normal levels of D-Dimer, Protein C, ANA and Anti-DS DNA but had a low Protein S of 56% (N.V is 70-120%). Antithrombin, homocysteine and Factor V Leiden were not done due to unavailability of the tests. She was then treated as a case of Cerebral Venous Thrombosis with multiple hemorrhage from venous infraction and was given anticoagulants which provided relief of the headache. She did not manifest with any further cortical, bulbar or sensorimotor deficits hence was discharged improved after 15 hospital days. To our knowledge, there are no case reports of patients with CVT from Protein S deficiency and venous anomaly that presented with multiple hemorrhage from venous infarction, more so affecting the brainstem. In this paper, a rare location of CVT in a newly diagnosed Protein S deficient patient is presented together with an uneventful course and favorable outcome.

Keywords: protein S deficiency, cerebral venous thrombosis, pontine hemorrhage from venous infarction, elderly

Procedia PDF Downloads 39

Authors: Donatella Cimini, Sergio D’ambrosio, Saba Sadiq, Chiara Schiraldi

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Chondroitin sulfate (CS), as well as modified CS, and unsulfated chondroitin, are largely applied in research today. CS is a linear glycosaminoglycan normally present in cartilage-rich tissues and bones in the form of proteoglycans decorated with sulfate groups in different positions. CS is used as an effective non-pharmacological alternative for the treatment of osteoarthritis, and other potential applications in the biomedical field are being investigated. Some bacteria, such as E. coli K4, produce a polysaccharide that is a precursor of CS (unsulfated chondroitin). This work focused on the construction of integrative E. coli K4 recombinant strains overexpressing genes (kfoA, kfoF, pgm and galU in different combinations) involved in the biosynthesis of the nucleotide sugars necessary for polysaccharide synthesis. Strain growth and polymer production were evaluated using renewable waste materials as substrates in shake flasks and small-scale batch fermentation processes. Results demonstrated the potential to replace pure sugars with cheaper medium components to establish environmentally sustainable and cost-effective production routes for potential industrial development. In fact, although excellent fermentation results have been described so far by employing strains that naturally produce chondroitin-like polysaccharides on semi-defined media, there is still the need to reduce manufacturing costs by providing a cost-effective biotechnological alternative to currently used animal-based extraction procedures.

Keywords: E. coli K4, chondroitin, microbial cell factories, glycosaminoglycans, renewable resources

Procedia PDF Downloads 46

Authors: P. Paengkoum, S. C. Chen, S. Paengkoum

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Six male growing Thai-indigenous beef cattle with body weight (BW) of 154±13.2 kg were randomly assigned in replicated 3×3 Latin square design, and fed with different levels of crude protein (CP) in total mixed ration (TMR) diets. CP levels in diets were 4.3%, 7.3% and 10.3% base on dry matter (DM). Ruminal ammonia nitrogen (NH3-N) and blood urea nitrogen (BUN) concentrations increased (P<0.01) with increasing CP levels. Moreover, there is a positive relationship between BUN and ruminal NH3-N. Rumen pH, total volatile fatty acid (VFA), molar proportions of acetate, propionate and butyrate were not affected by CP levels (P>0.05).

Keywords: Thai-indigenous beef cattle, crude protein, volatile fatty acid (VFA), total mixed ration (TMR) diets

Procedia PDF Downloads 249

Authors: Geevika J. Ganegama Arachchi, Steve H. Flint, Lynn McIntyre, Cristina D. Cruz, Beatrice M. Dias-Wanigasekera, Craig Billington, J. Andrew Hudson, Anthony N. Mutukumira

Abstract:

In seafood processing plants, Listeriamonocytogenes(L. monocytogenes)likely exists in a metabolically stressed state due to the nutrient-deficient environment, processing treatments such as heating, curing, drying, and freezing, and exposure to detergents and disinfectants. Stressed L. monocytogenes cells have been shown to be as pathogenic as unstressed cells. This study investigated lytic efficacy of (LiMN4L, LiMN4p, and LiMN17) which were previouslycharacterized as virulent against physiologically stressed cells of three seafood borne L. monocytogenesstrains (19CO9, 19DO3, and 19EO3).Physiologically compromised cells ofL. monocytogenesstrains were prepared by aging cultures in TrypticaseSoy Broth at 15±1°C for 72 h; heat injuringcultures at 54±1 - 55±1°C for 40 - 60 min;salt-stressing cultures in Milli-Q water were incubated at 25±1°C in darkness for three weeks; and incubating cultures in 9% (w/v) NaCl at 15±1°C for 72 h. Low concentrations of physiologically compromised cells of three L. monocytogenesstrainswere challenged in vitrowith high titre of three phages in separate experiments using Fish Broth medium (aqueous fish extract) at 15 °C in order to mimic the environment of seafood processing plant. Each phage, when present at ≈9 log10 PFU/ml, reduced late exponential phase cells of L. monocytogenes suspended in fish protein broth at ≈2-3 log10 CFU/ml to a non-detectable level (< 10 CFU/ml). Each phage, when present at ≈8.5 log10 PFU/ml, reduced both heat-injured cells present at 2.5-3.6 log10 CFU/ml and starved cells that were showed coccoid shape, present at ≈2-3 log10 CFU/ml to < 10 CFU/ml after 30 min. Phages also reduced salt-stressed cellspresent at ≈3 log10 CFU/ml by > 2 log10. L. monocytogenes (≈8 log10 CFU/ml) were reduced to below the detection limit (1 CFU/ml) by the three successive phage infections over 16 h, indicating that emergence of spontaneous phage resistance was infrequent. The three virulent phages showed high decontamination potential for physiologically stressed L. monocytogenes strains from seafood processing environments.

Keywords: physiologically stressed L. monocytogenes, heat injured, seafood processing environment, virulent phage

Procedia PDF Downloads 110

Authors: Rabab Makki, Deputy Chief Dietitian

Abstract:

Malnutrition is commonly seen in Liver Disease patients. Prevalence of malnutrition in cirrhosis, is as high as 65-90%. Protein depletion and reduced muscle function are common. There are many mechanisms of malnutrition in liver cirrhosis e.g. insulin resistance, low respiratory quotient, increased glucogenesis etc. Nutrition support improves outcome in patients unable to maintain an intake of 35-40 Kcal/kg and 1.2-1.5 gm/kg/day. Simple methods of assessment such as subjective global assessment, calorie counting, MMC are useful. The value of BCAAs remains uncertain despite a considerable number of studies. Normal protein diets have been given safely to patients with hepatic encephalopathy. Restriction of protein not more than 48 hours pre- and pro-biotic, glutamine, fish oil etc are all part of the latest advanced techniques used.

Keywords: liver cirrhosis, omega 3 for liver disease, nutrition management, malnutrition

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Authors: K. Tejasri, K. Suvarna Vani, S. Prathyusha, S. Ramya

Abstract:

Proteins are chained sequences of amino acids which are brought together by the peptide bonds. Many varying formations of the chains are possible due to multiple combinations of amino acids and rotation in numerous positions along the chain. Protein structure prediction is one of the crucial goals worked towards by the members of bioinformatics and theoretical chemistry backgrounds. Among the four different structure levels in proteins, we emphasize mainly the secondary level structure. Generally, the secondary protein basically comprises alpha-helix and beta-sheets. Multi-class classification problem of data with disparity is truly a challenge to overcome and has to be addressed for the beta strands. Imbalanced data distribution constitutes a couple of the classes of data having very limited training samples collated with other classes. The secondary structure data is extracted from the protein primary sequence, and the beta-strands are predicted using suitable machine learning algorithms.

Keywords: proteins, secondary structure elements, beta-sheets, beta-strands, alpha-helices, machine learning algorithms

Procedia PDF Downloads 68

Authors: Christina Roman, Deepak Koirala, Joseph A. Piccirilli

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Phage displaying synthetic Fab libraries have been used to obtain Fabs that bind to specific RNA targets with high affinity and specificity. These Fabs have been demonstrated to facilitate RNA crystallization. However, the antibody framework used in the construction of these phage display libraries contains numerous bulky, flexible, and charged residues, which facilitate solubility and hinder aggregation. These residues can interfere with crystallization due to the entropic cost associated with burying them within crystal contacts. To systematically reduce the surface entropy of the Fabs and improve their crystallization properties, a protein engineering strategy termed surface entropy reduction (SER) is being applied to the Fab framework. In this approach, high entropy residues are mutated to smaller ones such as alanine or serine. Focusing initially on Fab BL3-6, which binds an RNA AAACA pentaloop with 20nM affinity, the SER P server (http://services.mbi.ucla.edu/SER/) was used and analysis was performed on existing RNA-Fab BL3-6 co-crystal structures. From this analysis twelve surface entropy reduced mutants were designed. These SER mutants were expressed and are now being measured for their crystallization and diffraction performance with various RNA targets. So far, one mutant has generated 3.02 angstrom diffraction with the yjdF riboswitch RNA. Ultimately, the most productive mutations will be combined into a new Fab framework to be used in a optimized phage displayed Fab library.

Keywords: antibody fragment, crystallography, RNA, surface entropy reduction

Procedia PDF Downloads 154

Authors: Sodamade Abiodun, Adeboye Olubunmi Omolara

Abstract:

Problems associated with protein malnutrition are still prevalent in third-world countries, leading to the constant search for plants that can serve as nutrients and medicinal purposes. Huckleberry is one of the plants that has been proven useful locally in the treatment of numerous ailments and diseases. A fresh sample of Huckleberry was collected from a vegetable garden situated near the Erelu dam of the Emmanuel Alayande College of Education campus, Oyo. The sample was authenticated at the forestry research institute of Nigeria (FRIN) Ibadan. The leaves of the plant were plucked and processed for leaf protein concentrates before proximate composition; mineral analysis phytochemical and antimicrobial properties of the leaf protein concentrates were determined using a standard method of analysis. The results of proximate constituents showed; moisture content; 9.89±0.051g/100g, Ash; 3.23±0.12g/100g, crude fat; 3.96±0.11g/100g and 61.27±0.56g/100g of Nitrogen free extractive results of the mineral analysis showed that the sample contains Mg; 0.081±0.00mg/100g, Ca; 42.30±0.05mg/100g, Na; 27.57±0.09mg/100g, K; 6.81±0.01mg/100g, P; 8.90±0.03mg/100g Fe; 0.51±0.00mg/100g, Zn; 0.021±0.00mg/100g, Cd; 0.04±0.04mg/100g, Pb; 0.002±0.00mg/100g, Cr; 0.041±0.00mg/100g while cadmium was not detected in the sample. The result of phytochemical analysis of leaf protein concentrates of the Huckleberry showed the presence of Alkaloid, Saponin, Flavonoid, Tanin, Coumarin, steroid, Terpenoid, cordial glycosides, Glycosides, Quinones, Anthocyanin, phytosterols, and phenols. Ethanolic extracts of the Huckleberry leaf protein concentrates showed that it contains bioactive compounds that are capable of eradicating some tested microorganisms; Staphylococcus aureus, Streptococcus pyogenes, Streptococcus faecalis, Pseudomonas aeruginosa, Klebisidlae pneumonia and Proteus merabilis. The results of the analysis of leaf protein concentrates of Huckleberry showed that the sample contains high nutrient and mineral constituents and phytochemical compounds that could make the sample useful for medicinal activities.

Keywords: huckleberry, mentha piperita, phytochemical, leaf protein concentrates, nutritional characteristics

Procedia PDF Downloads 54

Authors: H. Sakamoto, S. Kurosawa, M. Suzuki, S. Oguri

Abstract:

In Arabidopsis, several genes encoding proteins with ankyrin repeats and trans-membrane domains (AtANKTM) have been identified as mediators of biotic and abiotic stress responses. It has been known that the expression of an AtANKTM gene, At2g24600, is induced in response to abiotic stress and that there are four splicing variants derived from this locus. In this study, by RT-PCR and sequencing analysis, an unknown splicing variant of the At2g24600 transcript was identified. Based on differences in the predicted amino acid sequences, the five splicing variants are divided into three groups. The three predicted proteins are highly homologous, yet have different numbers of ankyrin repeats and trans-membrane domains. It is generally considered that ankyrin repeats mediate protein-protein interaction and that the number of trans-membrane domains affects membrane topology of proteins. The protein variants derived from the At2g24600 locus may have different molecular functions each other.

Keywords: alternative splicing, ankyrin repeats, trans-membrane domains, arabidopsis

Procedia PDF Downloads 346

Authors: Sumi Manjipparambil Surendran, Subrata Majumdar

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Aim: The aim of the study is to find out if low PAPP-A (Pregnancy-Associated Plasma Protein A) levels in the first trimester are associated with adverse obstetric outcome. Methods: A retrospective study was carried out on 114 singleton pregnancies having undergone combined test screening. Results: There is statistically significant increased incidence of low birth weight infants in the low PAPP-A group. However, significant association was not found in the incidence of pre-eclampsia, miscarriage, and placental abruption. Conclusion: Low PAPP-A in the first trimester is associated with fetal growth restriction. Recommendation: Women with low PAPP-A levels in first trimester pregnancy screening require consultant-led care and serial growth scans.

Keywords: pregnancy, pregnancy-associated plasma protein A, PAPP-A, fetal growth restriction, trimester

Procedia PDF Downloads 111

Authors: Lucretia Ramnath

Abstract:

Fishmeal is extensively used for fish farming but is an expensive fish feed ingredient. A cheaper alternate to fishmeal is single cell protein (SCP) which can be cultivated on fermentable sugars recovered from organic waste streams such as pulp and paper mill sludge (PPMS). PPMS has a high cellulose content, thus is suitable for glucose recovery through enzymatic hydrolysis but is hampered by lignin and ash. To render PPMS amenable for enzymatic hydrolysis, the PPMS waspre-treated to produce a glucose-rich hydrolysate which served as a feed stock for the production of fungal SCP. The PPMS used in this study had the following composition: 72.77% carbohydrates, 8.6% lignin, and 18.63% ash. The pre-treatments had no significant effect on lignin composition but had a substantial effect on carbohydrate and ash content. Enzymatic hydrolysis of screened PPMS was previously optimized through response surface methodology (RSM) and 2-factorial design. The optimized protocol resulted in a hydrolysate containing 46.1 g/L of glucose, of which 86% was recovered after downstream processing by passing through a 100-mesh sieve (38 µm pore size). Vogel’s medium supplemented with 10 g/L hydrolysate successfully supported the growth of Fusarium venenatum, conducted using standard growth conditions; pH 6, 200 rpm, 2.88 g/L ammonium phosphate, 25°C. A maximum F. venenatum biomass of 45 g/L was produced with a yield coefficient of 4.67. Pulp and paper mill sludge hydrolysate contained approximately five times more glucose than what was needed for SCP production and served as a suitable carbon source. We have shown that PPMS can be successfully beneficiated for SCP production.

Keywords: pulp and paper waste, fungi, single cell protein, hydrolysate

Procedia PDF Downloads 167

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

Abstract:

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

Procedia PDF Downloads 43

Authors: Kieren Luellman, Makenzi Rockwell, Eduardo Callegari, Nichole Haag, Chun Wu

Abstract:

Multiple sclerosis (MS) is an autoimmune disorder that damages the myelin sheath of neurons in the central nervous system. The presence of Acinetobacter bacteria and anti-Acinetobacter antibodies in MS patients has led to the hypothesis that the bacteria may contribute to MS pathogenesis. In this study, the protein sequences of Acinetobacter baumannii were compared to five peptides from three mammalian myelin proteins, i.e., Proteolipid Protein (PLP): PLP 139-151, PLP 178-191, Myelin Basic Protein (MBP): MBP 84-104 and Myelin Oligodendrocyte Glycoprotein (MOG): MOG 35-55 and MOG 92-106 respectively, known to induce experimental autoimmune encephalomyelitis (EAE), a condition similar to MS. We found 11 hits (i.e., with five or more amino acid sequence similarity) in Acinetobacter baumannii, which are identical or similar to PLP139-151, 32 hits to PLP178-191, 35 to MBP 84-104, 41 hits to MOG 35-55 and 26 hits to MOG92-106. In addition, Western blotting was used to assess possible interaction between the bacterial proteins and human anti-MBP, anti-MOG, and anti-PLP antibodies produced in rabbits, corresponding to MBP 84-104, MOG 35-55, and PLP 139-151, respectively. We found that both human Polyclonal anti-MOG antibody and anti-PLP antibody recognized a protein or more proteins of the same molecular mass of around 25 kDa. in Acinetobacter baumannii. The results suggested that this/these protein(s) might potentially serve as antigen(s) to induce anti-MOG antibody and anti-PLP antibody production in mammalian B cells. The proteomic study identified 433 hits, among which the sequence of Acinetobacter baumannii protein 491 subunit A matches a previously published enzyme Acinetobacter 3-Oxoadipate CoA-Transferase, in which a fragment of its peptide was observed to recognize MS patient serum via ELISA method. Our findings might pave the road to understanding one of the pathogenesis mechanisms of MS.

Keywords: multiple sclerosis, pathogenesis, Acinetobacter baumannii, antibody recognition

Procedia PDF Downloads 80

Authors: Harika Atmaca, Emir Bozkurt, Latife Merve Oktay, Selim Uzunoglu, Ruchan Uslu, Burçak Karaca

Abstract:

Microarrays have been developed for highly parallel enzyme-linked immunosorbent assay (ELISA) applications. The most common protein arrays are produced by using multiple monoclonal antibodies, since they are robust molecules which can be easily handled and immobilized by standard procedures without loss of activity. Protein expression profiling with protein array technology allows simultaneous analysis of the protein expression pattern of a large number of proteins. Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate, Ecteinascidia turbinata, has been shown to have antitumor effects. Here, we used a novel proteomic approach to explore the mechanism of action of trabectedin in prostate cancer cell line PC-3 by apoptosis antibody microarray. XTT cell proliferation kit and Cell Death Detection Elisa Plus Kit (Roche) was used for measuring cytotoxicity and apoptosis. Human Apoptosis Protein Array (R&D Systems) which consists of 35 apoptosis related proteins was used to assess the omic protein expression pattern. Trabectedin induced cytotoxicity and apoptosis in prostate cancer cells in a time and concentration-dependent manner. The expression levels of the death receptor pathway molecules, TRAIL-R1/DR4, TRAIL R2/DR5, TNF R1/TNFRSF1A, FADD were significantly increased by 4.0-, 21.0-, 4.20- and 11.5-fold by trabectedin treatment in PC-3 cells. Moreover, mitochondrial pathway related pro-apoptotic proteins Bax, Bad, Cytochrome c, and Cleaved Caspase-3 expressions were induced by 2.68-, 2.07-, 2.8-, and 4.5-fold and the expression levels of anti-apoptotic proteins Bcl-2 and Bcl-XL were reduced by 3.5- and 5.2-fold in PC-3 cells. Proteomic (antibody microarray) analysis suggests that the mechanism of action of trabectedin may be exerted via the induction of both intrinsic and extrinsic apoptotic pathways. The antibody microarray platform can be utilised to explore the molecular mechanism of action of novel anticancer agents.

Keywords: trabectedin, prostate cancer, omic protein expression profile, apoptosis

Procedia PDF Downloads 416

Authors: S. Mowafi, A. Abou El-Kheir, M. Abou Taleb, H. El-Sayed

Abstract:

Two proteinic biopolymers; namely keratin and sericin, were extracted from their respective natural resources by simple appropriate methods. The said proteins were dissolved in the appropriate solvents followed by regeneration in a form of film polyvinyl alcohol. Protein-starch-potassium iodide (PSPI) composite was prepared by anchoring starch and potassium iodide mixture onto the film surface using appropriate polymeric material. The possibility of using PSPI composite for determination of the concentration of chlorine ions in domestic as well as industrial water was examined. The concentration of chlorine in water was determined spectrophotometrically by measuring the intensity of blue colour of formed between starch and the released iodine obtained by interaction of potassium iodide chlorine in the tested water sample.

Keywords: chlorine, protein, potassium iodide, water

Procedia PDF Downloads 344