Search results for: chick bean protein isolate

Authors: Ganesh K. Maurya, Reema Chaudhary, Neha Pandey, Hari S. Misra

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PprA, a pleiotropic protein participating in radioresistance, has been reported for its roles in DNA replication initiation, genome segregation, cell division and DNA repair in polyextremophile Deinococcus radiodurans. Interestingly, expression of deinococcal PprA in E. coli suppresses its growth by reducing the number of colony forming units and provide better resistance against γ-radiation than control. We employed different biochemical and cell biology studies using PprA and its DNA binding/polymerization mutants (K133E & W183R) in E. coli. Cells expressing wild type PprA or its K133E mutant showed reduction in the amount of genomic DNA as well as chromosome copy number in comparison to W183R mutant of PprA and control cells, which suggests the role of PprA protein in regulation of DNA replication initiation in E. coli. Further, E. coli cells expressing PprA or its mutants exhibited different impact on cell morphology than control. Expression of PprA or K133E mutant displayed a significant increase in cell length upto 5 folds while W183R mutant showed cell length similar to uninduced control cells. We checked the interaction of deinococcal PprA and its mutants with E. coli DnaA using Bacterial two-hybrid system and co-immunoprecipitation. We observed a functional interaction of EcDnaA with PprA and K133E mutant but not with W183R mutant of PprA. Further, PprA or K133E mutant has suppressed the ATPase activity of EcDnaA but W183R mutant of PprA failed to do so. These observations suggested that PprA protein regulates DNA replication initiation and cell morphology of surrogate E. coli.

Keywords: DNA replication, radioresistance, protein-protein interaction, cell morphology, ATPase activity

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Authors: Mario Pérez-Won, Anais Palma-Acevedo, Luis González-Cavieres, Roberto Lemus-Mondaca, Gipsy Tabilo-Munizaga

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The Chilean abalone (Concholepas Concholepas) is a gastropod mollusk; it has a high commercial value due to the qualities of its meat, especially hardness, as a critical acceptance parameter. However, its main problem is its short shelf-life which is usually extended using traditional technologies with high energy consumption. Therefore, applying different technologies for the pre-treatment and drying process is necessary. In this research, pulsed electric field (PEF) was used as a pre-treatment for vacuum microwave drying (VMD), freeze-drying (FD), and hot-air drying (HAD). Drying conditions and characteristics were set according to previous experiments. The Drying samples were analyzed in terms of physical quality (color, texture, microstructure, and rehydration capacity), protein quality (degree of hydrolysis and computer protein efficiency ratio), and energy parameters. Regarding quality, the treatment that obtained lower harness was PEF+FD (195 N ± 10), the lowest change of color was for treatment PEF+VMD (ΔE: 17 ± 1.5), and the best rehydration capacity was for treatment PEF+VMD (1.2 h for equilibrium). For protein quality, the highest Computer-Protein Efficiency Ratio was the sample 2.0 kV/ cm of PEF (index of 4.18 ± 0.26 at the end of the digestion). Moreover, about energetic consumption, results show that VMD decreases the drying process by 97% whether PEF was used or not. Consequently, it is possible to conclude that using PEF as a pre-treatment for VMD and FD treatments has advantages that must be used following the consumer’s needs or preferences.

Keywords: chilean abalone, freeze-drying, proteins, pulsed electric fields

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Authors: Abeer A. Q. Ahmed, Tracey McKay

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The increasing demand for renewable fuels and chemicals is pressuring manufacturing industry toward finding more sustainable cost-effective resources. Lignocellulose, such as agro-food wastes, is a suitable equivalent to petroleum for fine chemicals and fuels production. The complex structure of lignocellulose, however, requires a variety of enzymes in order to degrade its components into their respective building blocks that can be used further for the production of various value added products. This study aimed to isolate bacterial strain with the ability to produce a variety of lignocellulosic enzymes. One bacterial isolate was identified by 16S rRNA gene sequencing and phylogenetic analysis as Bacillus safensis LCX found to have CMCase, xylanase, manganese peroxidase, lignin peroxidase, and laccase activities. The enzymes production was induced by growing Bacillus safensis LCX in solid state fermentation using wheat straw, wheat bran, and corn stover. The activities of enzymes were determined by specific colorimetric assays. This study presents Bacillus safensis LCX as a promising source for lignocellulosic enzymes. These findings can extend the knowledge on agro-food wastes valorization strategies toward a sustainable production of fuels and chemicals.

Keywords: Bacillus safensis LCX, high valued chemicals, lignocellulosic enzymes, solid state fermentation

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Authors: Naureen Akhtar

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The stability of the protein in detergent-containing solution is the key for its successful crystallisation. Fluorescence-detection size-exclusion chromatography (FSEC) is a potential approach for screening monodispersity as well as the stability of protein in a detergent-containing-solution. In this present study, covalently linked Green Fluorescent Protein (GFP) to bacterial nitrate transporter, NarU from Escherichia coli was studied for pre-crystallisation trials by FSEC. Immobilised metal ion affinity chromatography (IMAC) and gel filtration were employed for their purification. The main objectives of this study were over-expression, detergent screening and crystallisation of nitrate transporter proteins. This study could not produce enough proteins that could realistically be taken forward to achieve the objectives set for this particular research. In future work, different combinations of variables like vectors, tags, creation of mutant proteins, host cells, position of GFP (N- or C-terminal) and/or membrane proteins would be tried to determine the best combination as the principle of technique is still promising.

Keywords: transporters, detergents, over-expression, crystallography

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Authors: M. Kowalski, M. Brodowski, K. Dziabowska, E. Czaczyk, W. Bialobrzeska, N. Malinowska, S. Zoledowska, R. Bogdanowicz, D. Nidzworski

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Boron-doped-carbon nanowall (BCNW) electrodes are recently in much interest among scientists. BCNWs are good candidates for biosensor purposes as they possess interesting electrochemical characteristics like a wide potential range and the low difference between redox peaks. Moreover, from technical parameters, they are mechanically resistant and very tough. The production process of the microwave plasma-enhanced chemical vapor deposition (MPECVD) allows boron to build into the structure of the diamond being formed. The effect is the formation of flat, long structures with sharp ends. The potential of these electrodes was checked in the biosensing field. The procedure of simple carbon electrodes modification by antibodies was adopted to BCNW for specific antigen recognition. Surface protein D deriving from H. influenzae pathogenic bacteria was chosen as a target analyte. The electrode was first modified with the aminobenzoic acid diazonium salt by electrografting (electrochemical reduction), next anti-protein D antibodies were linked via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry, and free sites were blocked by BSA. Cyclic voltammetry measurements confirmed the proper electrode modification. Electrochemical impedance spectroscopy records indicated protein detection. The sensor was proven to detect protein D in femtograms. This work was supported by the National Centre for Research and Development (NCBR) TECHMATSTRATEG 1/347324/12/NCBR/ 2017.

Keywords: anti-protein D antibodies, boron-doped carbon nanowall, impedance spectroscopy, Haemophilus influenzae.

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Authors: Shahram Naghizadeh Raeisi, Ali Alghooneh

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The loss of curd cohesiveness and syneresis are two common problems in the ultrafiltered cheese industry. In this study, by using membrane technology and protein modification, a modified cheese was developed and its properties were compared with a control sample. In order to decrease the lactose content and adjust the protein, acidity, dry matter and milk minerals, a combination of ultrafiltration, nanofiltration and reverse osmosis technologies was employed. For protein modification, a two-stage chemical and enzymatic reaction was employed before and after ultrafiltration. The physicochemical and microstructural properties of the modified ultrafiltered cheese were compared with the control one. Results showed that the modified protein enhanced the functional properties of the final cheese significantly (pvalue< 0.05), even if the protein content was 50% lower than the control one. The modified cheese showed 21 ± 0.70, 18 ± 1.10 & 25±1.65% higher hardness, cohesiveness and water-holding capacity values, respectively, than the control sample. This behavior could be explained by the developed microstructure of the gel network. Furthermore, chemical-enzymatic modification of milk protein induced a significant change in the network parameter of the final cheese. In this way, the indices of network linkage strength, network linkage density, and time scale of junctions were 10.34 ± 0.52, 68.50 ± 2.10 & 82.21 ± 3.85% higher than the control sample, whereas the distance between adjacent linkages was 16.77 ± 1.10% lower than the control sample. These results were supported by the results of the textural analysis. A non-linear viscoelastic study showed a triangle waveform stress of the modified protein contained cheese, while the control sample showed rectangular waveform stress, which suggested a better sliceability of the modified cheese. Moreover, to study the shelf life of the products, the acidity, as well as molds and yeast population, were determined in 120 days. It’s worth mentioning that the lactose content of modified cheese was adjusted at 2.5% before fermentation, while the lactose of the control one was at 4.5%. The control sample showed 8 weeks shelf life, while the shelf life of the modified cheese was 18 weeks in the refrigerator. During 18 weeks, the acidity of modified and control samples increased from 82 ± 1.50 to 94 ± 2.20 °D and 88 ± 1.64 to 194 ± 5.10 °D, respectively. The mold and yeast populations, with time, followed the semicircular shape model (R2 = 0.92, R2adj = 0.89, RMSE = 1.25). Furthermore, the mold and yeast counts and their growth rate in the modified cheese were lower than those for control one; Aforementioned result could be explained by the shortage of the source of energy for the microorganism in the modified cheese. The lactose content of the modified sample was less than 0.2 ± 0.05% at the end of fermentation, while this was 3.7 ± 0.68% in the control sample.

Keywords: non-linear viscoelastic, protein modification, semicircular shape model, ultrafiltered cheese

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Authors: Zi-Han Li, Lu Fang, Liang Wu, Dong-Yuan Chang, Manyuan Dong, Liang Ji, Qi Zhang, Ming-Hui Zhao, Sydney C. W. Tang, Lemin Zheng, Min Chen

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Uncoupling of mitochondrial respiration by chemical uncouplers has proven effective in ameliorating obesity, insulin resistance, and hyperglycemia, which were risk factors for diabetic nephropathy (DN). Recently, we found that ANXA1 could improve mitochondrial function to mitigate DN progression. However, the underlying mechanism is not fully clear yet. Here, we identified uncoupling protein 1 (UCP1), an inner membrane protein of mitochondria, as a key to mitochondrial homeostasis improved by ANXA1. Specifically, ANXA1 attenuated mitochondrial dysfunction via appropriately upregulating UCP1 by stabilizing its transcription factor GATA binding protein 3 (GATA3) by combining it with thioredoxin. Moreover, specific overexpression of UCP1 in the renal cortex rescued renal injuries in diabetic Anxa1-KO mice. UCP1 deletion aggravated renal injuries in HFD/STZ-induced diabetic mice. Mechanistically, UCP1 reduced mitochondrial fission through the aristaless-related homeobox (ARX)/cardiolipin synthase 1 (CRLS1) pathway. Therapeutically, CL316243, a UCP1 agonist, could attenuate established DN in db/db mice. This work established an alternative principle to harness the power of uncouplers for the treatment of DN.

Keywords: diabetic nephropathy, uncoupling protein 1, mitochondrial homeostasis, cardiolipin metabolism

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Authors: Belen Carrillo, Mauricio Mosquera

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Fatty acids are bioactive compounds widely used as nutritional supplements in the food and pharmaceutical industry. Bluefish such as sardines have a large variety of these fatty acids in their composition. The objective of this project is to extract these compounds from fishing wastes, to do this, heads of known species as Pinchagua (Opistonema libertate) were used. The conducted study represents a simplified alternative for obtaining and simultaneous saponification of oil through basic hydrolysis, which separates lipids from protein and saponifies sample all the same time to isolate the fatty acid accurately through salts formation. To do these different concentrations of sodium hydroxide were used, it was demonstrated at a concentration of 1 M the highest yield of saponified oil recovery corresponding a value of 3,64% was obtained. Subsequently, the saponified oil was subjected to an acid hydrolysis in which fatty acids were isolated. Different sulfuric acid concentrations and temperatures for the process were tested. Thus, it was shown that the great fatty acids variety were obtained at a 60 °C temperature and sulfuric acid concentration of 50% v/v. Among the obtained compounds the presence of acids such as palmitic, lauric, caproic and myristic are highlighted. Applications of this type of elements are varied and widely used in the nutritional supplements development. Thus, the described methodology proposes a simple mechanism in the revaluation of fishing industry wastes that allow directly generate high added value elements.

Keywords: fatty acids, hydrolysis, Pinchagua, saponification

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Authors: Sadegh Lotfieblisofla, Arash Khodabakhshi

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Tissue plasminogen activator (tPA) as a serine protease plays an important role in the fibrinolytic system and the dissolution of fibrin clots in human body. The production of this drug in plants such as tobacco could reduce its production costs. In this study, expression of tPA gene and protein targeting to different plant cell compartments, using various signal peptides has been investigated. For high level of expression, Kozak sequence was used after CaMV35S in the beginning of the gene. In order to design the final construction, Extensin, KDEL (amino acid sequence including Lys-Asp-Glu-Leu) and SP (γ-zein signal peptide coding sequence) were used as leader signals to conduct this protein into apoplast, endoplasmic reticulum and cytosol spaces, respectively. Cloned human tPA gene under the CaMV (Cauliflower mosaic virus) 35S promoter and NOS (Nopaline Synthase) terminator into pBI121 plasmid was transferred into tobacco explants by Agrobacterium tumefaciens strain LBA₄₄₀₄. The presence and copy number of genes in transgenic tobacco was proved by Southern blotting. Enzymatic activity of the rt-PA protein in transgenic plants compared to non-transgenic plants was confirmed by Zymography assay. The presence and amount of rt-PA recombinant protein in plants was estimated by ELISA analysis on crude protein extract of transgenic tobacco using a specific antibody. The yield of recombinant tPA in transgenic tobacco for SP, KDEL, Extensin signals were counted 0.50, 0.68, 0.69 microgram per milligram of total soluble proteins.

Keywords: tPA, recombinant, transgenic, tobacco

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Authors: Mohamad Ammar Ayass, Natalya Griko, Victor Pashkov, Wanying Cao, Kevin Zhu, Jin Zhang, Lina Abi Mosleh

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Respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent for coronavirus disease 2019 (COVID-19). Early evidence pointed at the angiotensin-converting enzyme 2 (ACE-2) expressed on the epithelial cells of the lung as the main entry point of SARS-CoV-2 into the cells. The viral entry is mediated by the binding of the Receptor Binding Domain (RBD) of the spike protein that is expressed on the surface of the virus to the ACE-2 receptor. As the number of SARS-CoV-2 variants continues to increase, mutations arising in the RBD of SARS-CoV-2 may lead to the ineffectiveness of RBD targeted neutralizing antibodies. To address this limitation, the objective of this study is to develop a combination of aptamers that target different regions of the RBD, preventing the binding of the spike protein to ACE-2 receptor and subsequent viral entry and replication. A safe and innovative biomedical tool was developed to inhibit viral infection and reduce the harms of COVID-19. In the present study, DNA aptamers were developed against a recombinant trimer S protein using the Systematic Evolution of Ligands by Exponential enrichment (SELEX). Negative selection was introduced at round number 7 to select for aptamers that bind specifically to the RBD domain. A series of 9 aptamers (ADI2010, ADI2011, ADI201L, ADI203L, ADI205L, ADIR68, ADIR74, ADIR80, ADIR83) were selected and characterized with high binding affinity and specificity to the RBD of the spike protein. Aptamers (ADI25, ADI2009, ADI203L) were able to bind and pull down endogenous spike protein expressed on the surface of SARS-CoV-2 virus in COVID-19 positive patient samples and determined by liquid chromatography- tandem mass spectrometry analysis (LC-MS/MS). LC-MS/MS data confirmed that aptamers can bind to the RBD of the spike protein. Furthermore, results indicated that the combination of the 9 best aptamers inhibited the binding of the purified trimer spike protein to the ACE-2 receptor found on the surface of Vero E6 cells. In the same experiment, the combined aptamers displayed a better neutralizing effect than antibodies. The data suggests that the selected aptamers could be used in therapy to neutralize the effect of the SARS-CoV-2 virus by inhibiting the interaction between the RBD and ACE-2 receptor, preventing viral entry into target cells and therefore blocking viral replication.

Keywords: aptamer, ACE-2 receptor, binding inhibitor, COVID-19, spike protein, SARS-CoV-2, treatment

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Authors: Ç. Gökçek-Saraç, Ş. Özen, N. Derin

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The N-methyl-D-aspartate (NMDA)-dependent pathway is the major intracellular signaling pathway implemented in both short- and long-term memory formation in the hippocampus which is the most studied brain structure because of its well documented role in learning and memory. However, little is known about the effects of RF-EMR exposure on NMDA receptor signaling pathway including activation of protein kinases, notably Ca²⁺/calmodulin-dependent protein kinase II alpha (CaMKIIα). The aim of the present study was to investigate the effects of acute and chronic 900 MHz RF-EMR exposure on both passive avoidance behaviour and hippocampal levels of CaMKIIα and its phosphorylated form (pCaMKIIα). Rats were divided into the following groups: Sham rats, and rats exposed to 900 MHz RF-EMR for 2 h/day for 1 week (acute group) or 10 weeks (chronic group), respectively. Passive avoidance task was used as a behavioural method. The hippocampal levels of selected kinases were measured using Western Blotting technique. The results of passive avoidance task showed that both acute and chronic exposure to 900 MHz RF-EMR can impair passive avoidance behaviour with minor effects on chronic group of rats. The analysis of western blot data of selected protein kinases demonstrated that hippocampal levels of CaMKIIα and pCaMKIIα were significantly higher in chronic group of rats as compared to acute groups. Taken together, these findings demonstrated that different duration times (1 week vs 10 weeks) of 900 MHz RF-EMR exposure have different effects on both passive avoidance behaviour of rats and hippocampal levels of selected protein kinases.

Keywords: hippocampus, protein kinase, rat, RF-EMR

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Authors: Prittesh Patel, Ramar Krishnamurthy

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In the present study, nine strains of C. falcatum obtained from different places and cultivars were characterized for sporulation, growth rate, and 18S rRNA gene sequence. All isolates had characteristic fast-growing sparse and fleecy aerial mycelia on potato dextrose agar with sickle shape conidia (length x width: varied from 20.0 X 3.89 to 25.52 X 5.34 μm) and blackish to orange acervuli with setae (length x width: varied from 112.37X 2.78 to 167.66 X 6.73 μm). They could be divided into two groups on the base of morphology; P1, dense mycelia with concentric growth and P2, sparse mycelia with uneven growth. Genomic DNA isolation followed by PCR amplification with ITS1 and ITS4 primer produced ~550bp amplicons for all isolates. Phylogeny generated by 18S rRNA gene sequence confirmed the variation in isolates and mainly grouped into two clusters; cluster 1 contained CoC671 isolates (cfNAV and cfPAR) and Co86002 isolate (cfTIM). Other isolates cfMAD, cfKAM, and cfMAR were grouped into cluster 2. Remaining isolates did not fall into any cluster. Isolate cfGAN, collected from Co86032 was found highly diverse of all the nine isolates. In a nutshell, we found considerable genetic divergence and morphological variation within C. falcatum accessions collected from different areas of south Gujarat, India and these can be used for the breeding program.

Keywords: Colletotrichum falcatum, ITS, morphology, red rot, sugarcane

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Authors: Nasser A. Al-Shabib

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Food allergens are most common non-native form when exposed to the immune system. Most food proteins undergo various treatments (e.g. thermal or proteolytic processing) during food manufacturing. Such treatments have the potential to impact the chemical structure of food allergens so as to convert them to more denatured or unfolded forms. The conformational changes in the proteins may affect the allergenicity of treated-allergens. However, most allergenic proteins possess high resistance against thermal modification or digestive enzymes. In the present study, ovomucoid (a major allergenic protein of egg white) was heated in phosphate-buffered saline (pH 7.4) at different temperatures, aqueous solutions and on different surfaces for various times. The results indicated that different antibody-based methods had different sensitivities in detecting the heated ovomucoid. When using one particular immunoassay‚ the immunoreactivity of ovomucoid increased rapidly after heating in water whereas immunoreactivity declined after heating in alkaline buffer (pH 10). Ovomucoid appeared more immunoreactive when dissolved in PBS (pH 7.4) and heated on a stainless steel surface. To the best of our knowledge‚ this is the first time that antibody-based methods have been applied for the detection of ovomucoid adsorbed onto different surfaces under various conditions. The results obtained suggest that use of antibodies to detect ovomucoid after food processing may be problematic. False assurance will be given with the use of inappropriate‚ non-validated immunoassays such as those available commercially as ‘Swab’ tests. A greater understanding of antibody-protein interaction after processing of a protein is required.

Keywords: ovomucoid, thermal treatment, solutions, surfaces

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Authors: Han-Bin Kim, Sooim Shin, Moonsung Choi

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There is a growing interest in introducing a desired functional group on enzymes in the field of protein engineering. In here, various redox centers were newly created using histidine tag, which is widely used for protein purification, plus cobalt in one of cupredoxins, amicyanin. The coordination of Cobalt-His tag and reactivity of the Co²⁺ loaded His-tag also were characterized. 3xHis-tag, 6xHis-tag, and 9xHis-tag were introduced on amicyanin by site-directed mutagenesis, and then Co²⁺ was loaded on each His-tagged amicyanin. The spectral changes at 330 nm corresponding to cobalt binding on His-tag site indicated the binding ratio of 3xHis-tag, 6xHis-tag, and 9xHis-tag to cobalt as 1:1, 1:2, 1:3 respectively. Based on kinetic studies of binding cobalt to 3xHis-tag, 6xHis-tag, and 9xHis-tagged amicyanin, the nature of the sites was elucidated. In addition, internal electron transfer properties between Cu¹⁺ site and engineered site of amicyanin were determined. These results provide insight into improvement of metal coordination and alternation of the redox properties of metal as a new catalytic site on proteins.

Keywords: amicyanin, cobalt, histidine, protein engineering

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Authors: Z. Mmango, U. Nwodo, L. V. Mabinya, A. I. Okoh

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Cellulose and hemicellulose account for a large portion of the world‘s plant biomass. In nature, these polysaccharides are intertwined forming complex materials that requires multiple and expensive treatment processes to free up the raw materials trapped in the matrix. Enzymatic degradation remains as the preferred technique as it is inexpensive and eco-friendly. However, the insufficiencies of enzyme battery systems in the degradation of lignocellulosic complex motivate the search for effective degrading enzymes from bacterial isolates from uncommon environment. The study aimed at the evaluation of actinomycetes isolated from saw dust samples collected from wood factory under bed. Cellulase and xylanase production was screened through organism culture on carboxyl methyl cellulose agar and Birchwood xylan. Halo zone indicating lignocellose utilization was shown by an isolate identified through 16S rRNA gene as Micrococcus luteus. The optimum condition for the production of cellulase and xylanase were incubation temperature of 25 °C, fermentation medium pH 5 and 10, agitation speed of 50 and 200 (rpm) and fermentation incubation time of 96 and 84 (h) respectively. The high cellulose and xylanase activity obtained from this isolate portends industrial relevance.

Keywords: carboxyl methyl cellulose, birchwood xylan, optimization, cellulase, xylanase, micrococcus, DNS method

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Authors: Ibinabo I. Ilaboya, Eustace A. Iyayi

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Phosphorus (P) is an indispensable mineral in broiler diets. Rice husk contains phytate-P and other nutrients like protein, carbohydrates, which are poorly digested in broiler chickens. Broiler chickens (BC) lacks sufficient phytase to help hydrolyse phytate-bound P. Hence excess of P is excreted by these chickens into the environment causing environmental pollution. Supplementation of such diets with microbial phytase helps to improve the digestibility of these nutrients. The study was conducted to determine the effect of phytase supplementation on the digestibility of crude protein (CP) and P of rice husk in BC. Six semi-purified diets of three levels of total P (3.46, 4.91 and 6.37g/kg) without and with 1,000 units of phytase per kg were formulated. Titanium dioxide was added to the diets at the rate of 5g/kg as an indigestible marker. At 20dposthatch, 288 broilers (Abor Acre) were weighed and allotted to the diets with 6 replicates of 8 birds each in a randomized complete block design. The birds had free access to the experimental diets until day 26 post-hatch. Phytase supplementation increased (p < 0.05) digestibility of P from 75-93%. Rice husk and its interaction with phytase had no significant (p > 0.05) effect on P digestibility, whereas there was significant (p < 0.01) effect on the interaction of rice husk with phytase on CP digestibility. There were linear increases (p < 0.01) in digested P and CP with phytase supplementation. The P and CP losses from the BC was reduced with the addition of phytase. Results suggest that supplementation of rice husk-based diets with microbial phytase improved pre-caecal digestibility of P and CP in broilers.

Keywords: crude protein, phosphorus, phytase, rice husk

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Authors: Akhmadjon Sultanov, Eun-Ho Lee, Hye-Jin Park, Young-Je Cho

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This study tested the anti-inflammatory effect of wild indigo (Baptisia tinctoria) root in Raw 264.7 cells. We prepared two extracts of B. tinctoria; one in water and the other in 50% ethanol. Then we evaluated the toxicities of the B. tinctoria root extracts at 10 to 100 mg mL-1 concentrations in raw 264.7 cells and observed 80% cell viability. The anti-inflammatory effect of B. tinctoria root extract in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells were observed with concentrations at 10, 30, and 50 μg mL-1. The results showed that 77.27-66.82% of nitric oxide (NO) production was inhibited by 50 μg mL-1 B. tinctoria root extract. The protein expression of Inducible NO synthase (iNOS) expression dramatically decreased by 93.14% and 52.65% in raw 264.7 cells treated with water and ethanol extracts of B. tinctoria root, respectively. Moreover, cyclooxygenase-2 (COX-2) protein expression decreased by 42.85% and 69.70% in raw 264.7 cells treated with water and ethanol extracts of B. tinctoria root, respectively. Furthermore, the mRNA expression of pro-inflammatory markers, such as tumor necrosis factor-alpha, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, and prostaglandin E synthase 2, was significantly suppressed in a concentration-dependent manner. Additionally, the B. tinctoria root extracts effectively inhibited enzymes involved in physiological activities. The B. tinctoria root extracts showed excellent anti-inflammatory effects and can be used as a functional material for biological activities.

Keywords: cytokine, macrophage, pro-inflammatory, protein expression, real-time PCR

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Authors: Zuzana Tatarkova, Ivana Pilchova, Michal Cibulka, Martin Kolisek, Peter Racay, Peter Kaplan

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Introduction: Normal functioning of mitochondria is crucial for cardiac performance. Mitochondria undergo mitophagy and biogenesis, and mitochondrial proteins are subject to extensive post-translational modifications. The state of mitochondrial homeostasis reflects overall cellular fitness and longevity. Perturbed mitochondria produce less ATP, release greater amounts of reactive molecules, and are more prone to apoptosis. Therefore mitochondrial turnover is an integral aspect of quality control in which dysfunctional mitochondria are selectively eliminated through mitophagy. Currently, the progressive deterioration of physiological functions is seen as accumulation of modified/damaged proteins with limiting regenerative ability and disturbance of such affected protein-protein communication throughout aging in myocardial cells. Methodologies: For our study was used immunohistochemistry, biochemical methods: spectrophotometry, western blotting, immunodetection as well as more sophisticated 2D electrophoresis and mass spectrometry for evaluation protein-protein interactions and specific post-translational modification. Results and Discussion: Mitochondrial stress response to reactive species was evaluated as electron transport chain (ETC) complexes, redox-active molecules, and their possible communication. Protein-protein interactions revealed a strong linkage between age and ETC protein subunits. Redox state was strongly affected in senescent mitochondria with shift in favor of more pro-oxidizing condition within cardiomyocytes. Acute myocardial ischemia and ischemia-reperfusion (IR) injury affected ETC complexes I, II and IV with no change in complex III. Ischemia induced decrease in total antioxidant capacity, MnSOD, GSH and catalase activity with recovery in some extent during reperfusion. While MnSOD protein content was higher in IR group, activity returned to 95% of control. Nitric oxide is one of the biological molecules that can out compete MnSOD for superoxide and produce peroxynitrite. This process is faster than dismutation and led to the 10-fold higher production of nitrotyrosine after IR injury in adult with higher protection in senescent ones. 2D protein profiling revealed 140 mitochondrial proteins, 12 of them with significant changes after IR injury and 36 individual nitrotyrosine-modified proteins further identified by mass spectrometry. Linking these two groups, 5 proteins were altered after IR as well as nitrated, but only one showed massive nitration per lowering content of protein after IR injury in adult. Conclusions: Senescent cells have greater proportion of protein content, which might be modulated by several post-translational modifications. If these protein modifications are connected to functional consequences and protein-protein interactions are revealed, link may lead to the solution. Assume all together, dysfunctional proteostasis can play a causative role and restoration of protein homeostasis machinery is protective against aging and possibly age-related disorders. This work was supported by the project VEGA 1/0018/18 and by project 'Competence Center for Research and Development in the field of Diagnostics and Therapy of Oncological diseases', ITMS: 26220220153, co-financed from EU sources.

Keywords: aging heart, mitochondria, proteomics, redox state

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Authors: Ammar Said Ahmed, M. Reissmann, R. Bortfeldt, G. A. Brockmann

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Milk protein genetic variants are of interest for characterizing domesticated mammalian species and breeds, and for studying associations with economic traits. The aim of this work was to analyze milk protein genetic variation in the Sudanese native cattle breeds, which have been gradually declining in numbers over the last years due to the breed substitution, and indiscriminate crossbreeding. The genetic variation at three milk protein genes αS1-casein (CSN1S1), αS2-casein (CSN1S2) and ƙ-casein (CSN3) was investigated in 250 animals belonging to five Bos indicus cattle breeds of Sudan (Butana, Kenana, White-nile, Erashy and Elgash). Allele specific primers were designed for five SNPs determine the CSN1S1 variants B and C, the CSN1S2 variants A and B, the CSN3 variants A, B and H. Allele, haplotype frequencies and genetic distances (D) were calculated and the phylogenetic tree was constructed. All breeds were found to be polymorphic for the studied genes. The CSN1S1*C variant was found very frequently (>0.63) in all analyzed breeds with highest frequency (0.82) in White-nile cattle. The CSN1S2*A variant (0.77) and CSN3*A variant (0.79) had highest frequency in Kenana cattle. Eleven haplotypes in casein gene cluster were inferred. Six of all haplotypes occurred in all breeds with remarkably deferent frequencies. The estimated D ranged from 0.004 to 0.049. The most distant breeds were White-nile and Kenana (D 0.0479). The results presented contribute to the genetic knowledge of indigenous cattle and can be used for proper definition and classification of the Sudanese cattle breeds as well as breeding, utilization, and potential development of conservation strategies for local breeds.

Keywords: milk protein, genetic variation, casein haplotype, Bos indicus

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Authors: Essam A. Makky, Siti H. Mohd Rasdi, J. B. Al-Dabbagh, G. F. Najmuldeen

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The synthesis of silver nano particles (SNPs) extensively studied by using chemical and physical methods. Here, the biological methods were used and give benefits in research field in the aspect of very low cost (from waste to wealth) and safe time as well. The study aims to isolate and exploit the microbial power in the production of industrially important by-products in nano-size with high economic value, to extract highly valuable materials from hazardous waste, to quantify nano particle size, and characterization of SNPs by X-Ray Diffraction (XRD) analysis. Disposal X-ray films were used as substrate because it consumes about 1000 tons of total silver chemically produced worldwide annually. This silver is being wasted when these films are used and disposed. Different bacterial isolates were obtained from various sources. Silver was extracted as nano particles by microbial power degradation from disposal X-ray film as the sole carbon source for ten days incubation period in darkness. The protein content was done and all the samples were analyzed using XRD, to characterize of silver (Ag) nano particles size in the form of silver nitrite. Bacterial isolates CL4C showed the average size of SNPs about 19.53 nm, GL7 showed average size about 52.35 nm and JF Outer 2A (PDA) showed 13.52 nm. All bacterial isolates partially identified using Gram’s reaction and the results obtained exhibited that belonging to Bacillus sp.

Keywords: nanotechnology, bioremediation, disposal X-ray film, nanoparticle, waste, XRD

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Authors: Yuexin Liu, Gordon B. Mills, Russell R. Broaddus, John N. Weinstein

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The lethality of endometrioid endometrial cancer (EEC) is primarily attributable to the high-stage diseases. However, there are no available biomarkers that predict EEC patient staging at the time of diagnosis. We aim to develop a predictive scheme to help in this regards. Using reverse-phase protein array expression profiles for 210 EEC cases from The Cancer Genome Atlas (TCGA), we constructed a Protein Scoring of EEC Staging (PSES) scheme for surgical stage prediction. We validated and evaluated its diagnostic potential in an independent cohort of 184 EEC cases obtained at MD Anderson Cancer Center (MDACC) using receiver operating characteristic curve analyses. Kaplan-Meier survival analysis was used to examine the association of PSES score with patient outcome, and Ingenuity pathway analysis was used to identify relevant signaling pathways. Two-sided statistical tests were used. PSES robustly distinguished high- from low-stage tumors in the TCGA cohort (area under the ROC curve [AUC]=0.74; 95% confidence interval [CI], 0.68 to 0.82) and in the validation cohort (AUC=0.67; 95% CI, 0.58 to 0.76). Even among grade 1 or 2 tumors, PSES was significantly higher in high- than in low-stage tumors in both the TCGA (P = 0.005) and MDACC (P = 0.006) cohorts. Patients with positive PSES score had significantly shorter progression-free survival than those with negative PSES in the TCGA (hazard ratio [HR], 2.033; 95% CI, 1.031 to 3.809; P = 0.04) and validation (HR, 3.306; 95% CI, 1.836 to 9.436; P = 0.0007) cohorts. The ErbB signaling pathway was most significantly enriched in the PSES proteins and downregulated in high-stage tumors. PSES may provide clinically useful prediction of high-risk tumors and offer new insights into tumor biology in EEC.

Keywords: endometrial carcinoma, protein, protein scoring of EEC staging (PSES), stage

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Authors: E. Oluwasola, E. Bamidele, E. Ogunbusola

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Enriched “aadun” was produced from maize with, supplemented with cray fish and beans. Sodium chloride (Nacl) was also added to the product which acts as preservatives. The produced enriched “aadun” was compared with commercial “aadun” organoleptically the result of the sensory evaluation carried out on the product showed that there is a statistical significant difference between the mouth feel of enriched and commercial “aadun” at 0.05 level of significance (t=5.499, P<0.05) Similarly, the mean difference between enriched and commercial “aadun” in terms of aroma (t=4.403, P<0.05), taste (t=4.592, P<0.05) colour (t=2.788, P<0.05) and general acceptability (t=3.894, P<0.05) is statistically significant at 95% confidence level in each case, therefore, it is clearly revealed that product 321 (Enriched “aadun”) is more acceptable and significant better than product 432 (commercial “aadun”) in all the attributes evaluated. The proximate analysis using standard methods of analysis was carried out which include the moisture content, ash and protein content for both the enriched aadun and commercial aadun the result showed moisture content 9%, ash 6.2%, protein 19.6% and 12.9% moisture content, 4%ash content, 8.75% protein for the commercial and improved aadun respectively.

Keywords: aadun, enriched, maize, supplemented

Procedia PDF Downloads 557

Authors: Abdelkarim M. Ertiame, D. W. Yu, D. L. Yu, J. B. Gomm

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In this paper, a new robust fault detection and isolation (FDI) scheme is developed to monitor a multivariable nonlinear chemical process called the Chylla-Haase polymerization reactor when it is under the cascade PI control. The scheme employs a radial basis function neural network (RBFNN) in an independent mode to model the process dynamics and using the weighted sum-squared prediction error as the residual. The recursive orthogonal Least Squares algorithm (ROLS) is employed to train the model to overcome the training difficulty of the independent mode of the network. Then, another RBFNN is used as a fault classifier to isolate faults from different features involved in the residual vector. The several actuator and sensor faults are simulated in a nonlinear simulation of the reactor in Simulink. The scheme is used to detect and isolate the faults on-line. The simulation results show the effectiveness of the scheme even the process is subjected to disturbances and uncertainties including significant changes in the monomer feed rate, fouling factor, impurity factor, ambient temperature and measurement noise. The simulation results are presented to illustrate the effectiveness and robustness of the proposed method.

Keywords: Robust fault detection, cascade control, independent RBF model, RBF neural networks, Chylla-Haase reactor, FDI under closed-loop control

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Authors: Hae-Jun Lee, Ji-Sun Shin, Kyung-Tae Lee

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Potentilla species (Rosasease) have been used in traditional medicine to treat different ailment, disease or malady. In this study, we investigated the anti-inflammatory effects of ethanol extracts of NUTT (EPP) in lipopolysaccharide (LPS)-induced Raw 264.7 macrophages and septic mice. EPP suppressed LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production in LPS-induced Raw 264.7 macrophages. Consistent with these observations, EPP reduced the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) by downregulation of their promoter activities. EPP inhibited tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) at production and mRNA levels. Molecularly, EPP attenuated the LPS-induced transcriptional activity, and DNA-binding activity of nuclear factor-κB (NF-κB), and this was associated with a decrease of translocation and phosphorylation of p65 NF-κB by inhibiting the inhibitory κB-α (IκB-α) degradation and IκB kinase-α/β (IKK-α/β) phosphorylation. Furthermore, EPP suppressed the LPS-induced activation of activator protein-1 (AP-1) by reducing the expression of c-Fos and c-Jun in nuclear. EPP also reduced the phosphorylation of mitogen-activated protein kinase (MAPK), such as p38 MAPK and c-Jun N-terminal kinase/stress-activated protein kinase (JNK). In a sepsis model, pretreatment with EPP reduced the LPS-induced lethality. Collectively, these results suggest that the anti-inflammatory effects of EPP were associated with the suppression of NF-κB and AP-1 activation, and support its possible therapeutic role for the treatment of sepsis.

Keywords: anti-inflammation, activator protein-1, nuclear factor κB, Potentilla paradoxa Nutt

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Authors: A. O Amosu, A. M Hammed, G. W. Maneveldt, D. V. Robertson-Andersson

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In aquaculture, feed utilization is an important factor affecting growth of the target species, and thus the success of the aquaculture operation. Growth of C. gariepinus fingerlings (weight 1.60 ± 0.05 g; length 4.50 ± 0.07cm) was monitored in a closed door hatchery for a period of 21 days in an experiment consisting of 4 treatments stocked at 20 fish/10 litre tanks, fed in triplicate twice daily (08:30, 17:30) at 4% body weight with weight changes recorded every 3 days. Treatments were: 1) FeedX; 2) 35% crude protein diet + non enriched Ulva spp (11.18% crude protein) (CD + NEU); 3) 35% crude protein diet + enriched Ulva spp (11.98% crude protein)(CD +EU) and 4) control diet of 35% crude protein (CD). The production of Ulva spp. biomass was cultivated for a period of 3 months. The result shows that the fish fed macroalgal enriched diet had good growth, though no significant difference (p > 0.05) was recorded amongst the weight gain, %weight gain, specific growth rates and nitrogen metabolism of diets CD + NEU, CD + EU and CD. Significant differences (p < 0.05), were, however, found in the food conversion ratio (FCR) and gross food conversion ratio (gFCR) among the fingerlings across all the different experimental diets. The best FCRs were recorded for control diet (0.79 ± 2.39) and the Ulva enriched (1.75 ± 1.34) diets. The results suggest that the fingerlings were able to utilize Ulva supplemented with control diet better than the FeedX. We have shown that Ulva supplemented diets are good substitutes for formulated and commercial feeds, with potential to be successful fish feed in aquaculture systems.

Keywords: aquaculture, clarias gariepinus, growth, macroalgae, nutrient, ulva

Procedia PDF Downloads 704

Authors: B. Makoudi, R. Ghanimi, A. Bargaz, M. Mouradi, M. Farissi, A. Kabbaj, J. J. Drevon, C. Ghoulam

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Legume species are able to establish a nitrogen fixing symbiosis with soil rhizobia that allows them, when it operates normally, to ensure their necessary nitrogen nutrition. This biological process needs high phosphorus (P) supply and consequently it is limited under low phosphorus availability. To overcome this constraint, legume-rhizobia symbiosis develops many mechanisms to increase P availability in the rhizosphere and also the efficiency of P fertilizers. The objectives of our research works are to understand the physiological and biochemical mechanisms implemented by legume-rhizobia symbiosis to increase its P use efficiency (PUE) in order to select legume genotypes-rhizobia strains combination more performing for BNF under P deficiency. Our studies were carried out on two grain legume species, common bean (Phaseolus vulgaris) and faba bean (Vicia faba) tested in farmers’ fields and in experimental station fewer than two soil phosphorus levels. Under field conditions, the P deficiency caused a significant decrease of Plant and nodule biomasses in all of the tested varieties with a difference between them. This P limitation increased the contents of available P in the rhizospheric soils that was positively correlated with the increase of phosphatases activities in the nodules and the rhizospheric soil. Some legume genotypes showed a significant increase of their P use efficiency under P deficiency. The P solubilization test showed that some rhizobia strains isolated from Haouz region presented an important capacity to grow on solid and liquid media with tricalcium phosphate as the only P source and their P solubilizing activity was confirmed by the assay of the released P in the liquid medium. Also, this P solubilizing activity was correlated with medium acidification and the excretion of acid phosphatases and phytases in the medium. Thus, we concluded that medium acidification and excretion of phosphatases in the rhizosphere are the prominent reactions for legume-rhizobia symbiosis to improve its P nutrition.

Keywords: legume, phosphorus deficiency, rhizobia, rhizospheric soil

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

Abstract:

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

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

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

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

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

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

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

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

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

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

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

Procedia PDF Downloads 66