Search results for: amino%20acids

Authors: Salman Akrokayan

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Synthetic cDNA (ScDNA) of granulocyte colony-stimulating factor (G-CSF) was constructed using a DNA synthesizer with the aim to increase its expression level. 5' end of the ScDNA of G-CSF coding region was modified by decreasing the GC content without altering the predicted amino acids sequence. The identity of the resulting protein from ScDNA was confirmed by the highly specific enzyme-linked immunosorbent assay. In conclusion, a synthetic G-CSF cDNA in combination with the recombinant DNA protocol offers a rapid and reliable strategy for synthesizing the target protein. However, the commercial utilization of this methodology requires rigorous validation and quality control.

Keywords: synthetic cDNA, recombinant G-CSF, cloning, gene expression

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Authors: Hamza Zidoum

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This paper investigates the impact of human genetic variation on the function of human proteins using machine-learning algorithms. Single-Nucleotide Polymorphism represents the most common form of human genome variation. We focus on the single amino-acid polymorphism located in the coding region as they can affect the protein function leading to pathologic phenotypic change. We use several supervised Machine Learning methods to identify structural properties correlated with increased risk of the missense mutation being damaging. SVM associated with Principal Component Analysis give the best performance.

Keywords: single-nucleotide polymorphism, machine learning, feature selection, SVM

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Authors: Restu Misrianti

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The amino acid variation of Asn (allele A) at position 631 in Mx gene was specific to positive antiviral to avian viral desease. This research was aimed at identifying polymorphism of Mx gene in duck using molecular technique. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique was used to select the genotype of AA, AG and GG. There were thirteen duck from Indragiri Hulu regency (Riau Province) used in this experiment. DNA amplification results showed that the Mx gene in duck is found in a 73 bp fragment. Mx gene in duck did not show any polymorphism. The frequency of the resistant allele (AA) was 0%, while the frequency of the susceptible allele (GG) was 100%.

Keywords: duck, Mx gene, PCR, RFLP

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Authors: Sze Wing Ho, Nagendra P. Shah

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Lactic acid bacteria (LAB) have shown the beneficial effects on human gastrointestinal tract, such as protects diarrhea induced by lactose intolerance or enteric pathogens. Citrulline is a non-protein amino acid and also the precursors of arginine and nitric oxide, it has shown to enhance intestinal barrier function. Citrulline has shown to improve the growth of some strains of LAB, it is important for LAB to have a sufficient cell concentration to contribute the effects. Therefore, the aims of this study were to investigate the effect of combining citrulline with LAB on the anti-adhesion effect against pathogens and the effect on the cell integrity. The effect of citrulline on selected LAB was determined by incubating in 0%, 0.1% or 0.2% citrulline enriched MRS broth for 18 h. The adhesion ability of LAB and the anti-adhesion effect of LAB and citrulline against pathogens were performed on IPEC-J2 cell line. Transepithelial electrical resistance (TEER) assay was used to measure the tight junction (TJ) integrity. TJ proteins (claudin-1, occludin and zonula occluden-1 (ZO-1)) were determined by western blot analysis. It found that the growth of Lactobacillus helveticus ASCC 511 was significantly stimulated by 0.2% citrulline compared with control during 18 h fermentation. The adhesion of L. helveticus ASCC 511 and Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) ASCC 756 was increased when supplemented with citrulline. Citrulline has shown significant inhibitory effect on the adhesion of Escherichia coli PELI0480 (O157:H7), Shigella sonnei ATCC 25931, Staphyloccocus aureus CMCC26003 and Cronobacter sakazakii ATCC 29544. The anti-adhesion effect of L. helveticus ASCC 511, L. bulgaricus ASCC 756 and Lactobacillus paracasei ASCC 276 against Cronobacter sakazakii ATCC 29544 was significantly enhanced with citrulline supplementation. Treatments with citrulline and LAB were able to maintain the TEER of IPEC-J2 cell and shown the positive effect on the TJ proteins. In conclusion, citrulline had stimulating effect on some strains of LAB and determined to improve the adhesion of LAB on intestinal epithelial cell, to enhance the inhibitory effect on enteric pathogens adhesion as well as had beneficial effects on maintaining cell integrity. It implied LAB supplemented with citrulline might have advantageous effects on gastrointestinal tracts.

Keywords: citrulline, lactic acid bacteria, amino acid, anti-adhesion effect, cell integrity

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Authors: A. Kouloumpis, P. Zygouri, G. Potsi, K. Spyrou, D. Gournis

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Much of the research effort on graphene focuses on its use as building block for the development of new hybrid nanostructures with well-defined dimensions and behavior suitable for applications among else in gas storage, heterogeneous catalysis, gas/liquid separations, nanosensing and biology. Towards this aim, here we describe a new bottom-up approach, which combines the self-assembly with the Langmuir Schaefer technique, for the production of fullerene-intercalated graphene hybrid materials. This new method uses graphene nanosheets as a template for the grafting of various fullerene C60 molecules (pure C60, bromo-fullerenes, C60Br24, and fullerols, C60(OH)24) in a bi-dimensional array, and allows for perfect layer-by-layer growth with control at the molecular level. Our film preparation approach involves a bottom-up layer-by-layer process that includes the formation of a hybrid organo-graphene Langmuir film hosting fullerene molecules within its interlayer spacing. A dilute water solution of chemically oxidized graphene (GO) was used as subphase on the Langmuir-Blodgett deposition system while an appropriate amino surfactant (that binds covalently with the GO) was applied for the formation of hybridized organo-GO. After the horizontal lift of a hydrophobic substrate, a surface modification of the GO platelets was performed by bringing the surface of the transferred Langmuir film in contact with a second amino surfactant solution (capable to interact strongly with the fullerene derivatives). In the final step, the hybrid organo-graphene film was lowered in the solution of the appropriate fullerene derivative. Multilayer films were constructed by repeating this procedure. Hybrid fullerene-based thin films deposited on various hydrophobic substrates were characterized by X-ray diffraction (XRD) and X-ray reflectivity (XRR), FTIR, and Raman spectroscopies, Atomic Force Microscopy, and optical measurements. Acknowledgments. This research has been co‐financed by the European Union (European Social Fund – ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF)‐Research Funding Program: THALES. Investing in knowledge society through the European Social Fund (no. 377285).

Keywords: hybrids, graphene oxide, fullerenes, langmuir-blodgett, intercalated structures

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Authors: Mohamed S. Sasi

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The research project aim is to study the hydrolysis of 8-diethylphosphate-1-naphthalenol with hydroxylamine in water. 8-diethylphosphate-1-naphthalenol, 1 was successfully synthesized and its rate of reaction with hydroxylamine was studied at 60°C. Pseudo first order behavior was observed. The rate of P-O cleavage of 1 at 60°C (7.43 x 10-3 M-1s-1) was found to be 178 fold and 7 fold slower than diethyl 8-dimethylamino-1-naphthyl phosphate, 3 at 60°C (1.32 M-1s-1) and diethyl 8-amino-1-naphthyl phosphate, 2 at 90 °C (5.5 x 10-2 M-1s-1) respectively. The rate of P-O cleavage of 1 with hydroxylamine was found to be faster than that of 4-chlorophenyl-1-cyclopropylphosphate triester, 5 where the reaction was too slow to observe at 60°C.

Keywords: phosphate esters, intramolecular hydrogen bonding

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Authors: Olga V. Chervyakova, Elmira T. Tailakova, Vitaliy M. Strochkov, Kulyaisan T. Sultankulova, Nurlan T. Sandybayev, Lev G. Nemchinov, Rosemarie W. Hammond

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Sheep pox is a highly contagious infection that OIE regards to be one of the most dangerous animal diseases. It causes enormous economic losses because of death and slaughter of infected animals, lower productivity, cost of veterinary and sanitary as well as quarantine measures. To control spread of sheep pox infection the attenuated vaccines are widely used in the Republic of Kazakhstan and other Former Soviet Union countries. In spite of high efficiency of live vaccines, the possible presence of the residual virulence, potential genetic instability restricts their use in disease-free areas that leads to necessity to exploit new approaches in vaccine development involving recombinant DNA technology. Vaccines on the basis of recombinant proteins are the newest generation of prophylactic preparations. The main advantage of these vaccines is their low reactogenicity and this fact makes them widely used in medical and veterinary practice for vaccination of humans and farm animals. The objective of the study is to produce recombinant immunogenic proteins for development of the high-performance means for sheep pox prophylaxis. The SPV proteins were chosen for their homology with the known immunogenic vaccinia virus proteins. Assay of nucleotide and amino acid sequences of the target SPV protein genes. It has been shown that four proteins SPPV060 (ortholog L1), SPPV074 (ortholog H3), SPPV122 (ortholog A33) and SPPV141 (ortholog B5) possess transmembrane domains at N- or C-terminus while in amino acid sequences of SPPV095 (ortholog А 4) and SPPV117 (ortholog А 27) proteins these domains were absent. On the basis of these findings the primers were constructed. Target genes were amplified and subsequently cloned into the expression vector рЕТ26b(+) or рЕТ28b(+). Six constructions (pSPPV060ΔТМ, pSPPV074ΔТМ, pSPPV095, pSPPV117, pSPPV122ΔТМ and pSPPV141ΔТМ) were obtained for expression of the SPV genes under control of T7 promoter in Escherichia coli. To purify and detect recombinant proteins the amino acid sequences were modified by adding six histidine molecules at C-terminus. Induction of gene expression by IPTG was resulted in production of the proteins with molecular weights corresponding to the estimated values for SPPV060, SPPV074, SPPV095, SPPV117, SPPV122 and SPPV141, i.e. 22, 30, 20, 19, 17 and 22 kDa respectively. Optimal protocol of expression for each gene that ensures high yield of the recombinant protein was identified. Assay of cellular lysates by western blotting confirmed expression of the target proteins. Recombinant proteins bind specifically with antibodies to polyhistidine. Moreover all produced proteins are specifically recognized by the serum from experimentally SPV-infected sheep. The recombinant proteins SPPV060, SPPV074, SPPV117, SPPV122 and SPPV141 were also shown to induce formation of antibodies with virus-neutralizing activity. The results of the research will help to develop a new-generation high-performance means for specific sheep pox prophylaxis that is one of key moments in animal health protection. The research was conducted under the International project ISTC # K-1704 “Development of methods to construct recombinant prophylactic means for sheep pox with use of transgenic plants” and under the Grant Project RK MES G.2015/0115RK01983 "Recombinant vaccine for sheep pox prophylaxis".

Keywords: prophylactic preparation, recombinant protein, sheep pox virus, subunit vaccine

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Authors: D. J. Madhu Kumar, D. Jagadeesh Prasad, Sana Sheik, E. P. Rejeesh

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A series of Mannich bases derived from 1,2,4-triazole(3a-k and 4a-k) are synthesized by treating a Schiff base with various substituted primary/secondary amines and formaldehyde. The Schiff base is prepared by treating 3-methyl-4-amino-5-mercapto-1,2,4-triazole with 3,4-dimethoxybenzaldehyde in the presence of acid catalyst. The triazole is prepared by treating acetic acid with thiocarbohydrazide at reflux temperature. All the synthesized samples are characterised by FT-IR, 1H-NMR, and LC-MASS spectral studies and screened for their anti-oxidant activity.

Keywords: mannich bases, anti-oxidant activity, schiff base, triazole

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Authors: Thanthrige Thiunuwan Priyathilaka, Don Anushka Sandaruwan Elvitigala, Bong-Soo Lim, Hyung-Bok Jeong, Jehee Lee

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Toll like receptors (TLRs) are a phylogeneticaly conserved family of pattern recognition receptors, which participates in the host immune responses against various pathogens and pathogen derived mitogen. TLR21, a non-mammalian type, is almost restricted to the fish species even though those can be identified rarely in avians and amphibians. Herein, this study was carried out to identify and characterize TLR21 from rock bream (Oplegnathus fasciatus) designated as RbTLR21, at transcriptional and genomic level. In this study, the full length cDNA and genomic sequence of RbTLR21 was identified using previously constructed cDNA sequence database and BAC library, respectively. Identified RbTLR21 sequence was characterized using several bioinformatics tools. The quantitative real time PCR (qPCR) experiment was conducted to determine tissue specific expressional distribution of RbTLR21. Further, transcriptional modulation of RbTLR21 upon the stimulation with Streptococcus iniae (S. iniae), rock bream iridovirus (RBIV) and Edwardsiella tarda (E. tarda) was analyzed in spleen tissues. The complete coding sequence of RbTLR21 was 2919 bp in length which can encode a protein consisting of 973 amino acid residues with molecular mass of 112 kDa and theoretical isoelectric point of 8.6. The anticipated protein sequence resembled a typical TLR domain architecture including C-terminal ectodomain with 16 leucine rich repeats, a transmembrane domain, cytoplasmic TIR domain and signal peptide with 23 amino acid residues. Moreover, protein folding pattern prediction of RbTLR21 exhibited well-structured and folded ectodomain, transmembrane domain and cytoplasmc TIR domain. According to the pair wise sequence analysis data, RbTLR21 showed closest homology with orange-spotted grouper (Epinephelus coioides) TLR21with 76.9% amino acid identity. Furthermore, our phylogenetic analysis revealed that RbTLR21 shows a close evolutionary relationship with its ortholog from Danio rerio. Genomic structure of RbTLR21 consisted of single exon similar to its ortholog of zebra fish. Sevaral putative transcription factor binding sites were also identified in 5ʹ flanking region of RbTLR21. The RBTLR 21 was ubiquitously expressed in all the tissues we tested. Relatively, high expression levels were found in spleen, liver and blood tissues. Upon induction with rock bream iridovirus, RbTLR21 expression was upregulated at the early phase of post induction period even though RbTLR21 expression level was fluctuated at the latter phase of post induction period. Post Edwardsiella tarda injection, RbTLR transcripts were upregulated throughout the experiment. Similarly, Streptococcus iniae induction exhibited significant upregulations of RbTLR21 mRNA expression in the spleen tissues. Collectively, our findings suggest that RbTLR21 is indeed a homolog of TLR21 family members and RbTLR21 may be involved in host immune responses against bacterial and DNA viral infections.

Keywords: rock bream, toll like receptor 21 (TLR21), pattern recognition receptor, genomic characterization

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Authors: Hamzeh Alipour, Masoumeh Bagheri, Abbasali Raz, Javad Dadgar Pakdel, Kourosh Azizi, Aboozar Soltani, Mohammad Djaefar Moemenbellah-Fard

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Background: Maggot debridement therapy is an appropriate, effective, and controlled method using sterilized larvae of Luciliasericata (L.sericata) to treat wounds. Netrin-A is an enzyme in the Laminins family which secreted from salivary gland of L.sericata with a central role in neural regeneration and angiogenesis. This study aimed to production of new recombinant Netrin-A protein of Luciliasericata larvae by baculovirus expression vector system (BEVS) in SF9. Material and methods: In the first step, gene structure was subjected to the in silico studies, which were include determination of Antibacterial activity, Prion formation risk, homology modeling, Molecular docking analysis, and Optimization of recombinant protein. In the second step, the Netrin-A gene was cloned and amplified in pTG19 vector. After digestion with BamH1 and EcoR1 restriction enzymes, it was cloned in pFastBac HTA vector. It was then transformed into DH10Bac competent cells, and the recombinant Bacmid was subsequently transfected into insect Sf9 cells. The expressed recombinant Netrin-A was thus purified in the Ni-NTA agarose. This protein evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay method. Results: The Bacmid vector structure with Netrin-A was successfully constructed and then expressed as Netrin-A protein in the Sf9 cell lane. The molecular weight of this protein was 52 kDa with 404 amino acids. In the in silico studies, fortunately, we predicted that recombinant LSNetrin-A have Antibacterial activity and without any prion formation risk.This molecule hasa high binding affinity to the Neogenin and a lower affinity to the DCC-specific receptors. Signal peptide located between amino acids 24 and 25. The concentration of Netrin-A recombinant protein was calculated to be 48.8 μg/ml. it was confirmed that the characterized gene in our previous study codes L. sericata Netrin-A enzyme. Conclusions: Successful generation of the recombinant Netrin-A, a secreted protein in L.sericata salivary glands, and because Luciliasericata larvae are used in larval therapy. Therefore, the findings of the present study could be useful to researchers in future studies on wound healing.

Keywords: blowfly, BEVS, gene, immature insect, recombinant protein, Sf9

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Authors: Elif Tugce Aksun Tumerkan, Chris Lowe, Hannah Krupa

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Fluorescent proteins are self-sufficient in forming chromophores with a visible wavelength from 3 amino acids sequence within their own polypeptide structure. This chromophore – a molecule that absorbs a photon of light and exhibits an energy transition equal to the energy of the absorbed photon. Fluorescent proteins (FPs) consisted of a chain of 238 amino acid residues and composed of 11 beta strands shaped in a cylinder surrounding an alpha helix structure. A better understanding of the system of the chromospheres and the increasing advance in protein engineering in recent years, the properties of FPs offers the potential for new applications. They have used sensors and probes in molecular biology and cell-based research that giving a chance to observe these FPs tagged cell localization, structural variation and movement. For clarifying functional uses of fluorescent proteins, electrophoretic properties of these proteins are one of the most important parameters. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is used for determining electrophoretic properties commonly. While there are many techniques are used for determining the functionality of protein-based research, SDS-PAGE analysis can only provide a molecular level assessment of the proteolytic fragments. Before SDS-PAGE analysis, fluorescent proteins need to successfully purified. Due to directly purification of the target, FPs is difficult from the animal, gene expression is commonly used which must be done by transformation with the plasmid. Furthermore, used gel within electrophoresis and staining agents properties have a key role. In this review, the different factors that have the impact on the electrophoretic properties of fluorescent proteins explored. Fluorescent protein separation and purification are the essential steps before electrophoresis that should be done very carefully. For protein purification, gene expression process and following steps have a significant function. For successful gene expression, the properties of selected bacteria for expression, used plasmid are essential. Each bacteria has own characteristics which are very sensitive to gene expression, also used procedure is the important factor for fluorescent protein expression. Another important factors are gel formula and used staining agents. Gel formula has an effect on the specific proteins mobilization and staining with correct agents is a key step for visualization of electrophoretic bands of protein. Visuality of proteins can be changed depending on staining reagents. Apparently, this review has emphasized that gene expression and purification have a stronger effect than electrophoresis protocol and staining agents.

Keywords: cell biology, gene expression, staining agents, SDS-page

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Authors: Komala Arsi, Ann M. Donoghue, Dan J. Donoghue

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Methionine is an essential dietary amino acid necessary to promote growth and health of poultry. Synthetic methionine is commonly used as a supplement in conventional poultry diets and is temporarily allowed in organic poultry feed for lack of natural and organically approved sources of methionine. It has been a challenge to find a natural, sustainable and cost-effective source for methionine which reiterates the pressing need to explore potential alternatives of methionine for organic poultry production. Fish have high concentrations of methionine, but wild-caught fish are expensive and adversely impact wild fish populations. Asian carp (AC) is an invasive species and its utilization has the potential to be used as a natural methionine source. However, to our best knowledge, there is no proven technology to utilize this fish as a methionine source. In this study, we co-extruded Asian carp and soybean meal to form a dry-extruded, methionine-rich AC meal. In order to formulate rations with the novel extruded carp meal, the product was tested on cecectomized roosters for its amino acid digestibility and total metabolizable energy (TMEn). Excreta was collected and the gross energy, protein content of the feces was determined to calculate Total Metabolizable Energy (TME). The methionine content, digestibility and TME values were greater for the extruded AC meal than control diets. Carp meal was subsequently tested as a methionine source in feeds formulated for broilers, and production performance (body weight gain and feed conversion ratio) was assessed in comparison with broilers fed standard commercial diets supplemented with synthetic methionine. In this study, broiler chickens were fed either a control diet with synthetic methionine or a treatment diet with extruded AC meal (8 replicates/treatment; n=30 birds/replicate) from day 1 to 42 days of age. At the end of the trial, data for body weights, feed intake and feed conversion ratio (FCR) was analyzed using one-way ANOVA with Fisher LSD test for multiple comparisons. Results revealed that birds on AC diet had body weight gains and feed intake comparable to diets containing synthetic methionine (P > 0.05). Results from the study suggest that invasive AC-derived fish meal could potentially be an effective and inexpensive source of sustainable natural methionine for organic poultry farmers.

Keywords: Asian carp, methionine, organic, poultry

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Authors: Mahboobeh Mohadeszadeh, Majid Saghi

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Polyoxometalates (POMs) are important inorganic compounds that have been considered specifically in recent years due to abundant attributes and applications. Those POMs that have one central tetrahedral atom called keggin. The binding Amino-acid groups to keggin structure give the antivirus effect to these compounds. A new organic-inorganic hybrid structure, with formula (Gly)2H4SiW12O40 was synthesized. Investigation on Anti-viral effect of this compound showed the (Gly)2H4SiW12O40 prevents infection of Tobacco Mosaic Virus (TMV) on the Nicotianatabacum plants.

Keywords: Polyoxometalate, Keggin, Organic-inorganic salt, TMV

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Authors: Mahboobeh Mohadeszadeh, Majid Saghi

Abstract:

Polyoxometalates (POMs) are important inorganic compounds that have been considered specifically in recent years due to abundant attributes and applications. Those POMs that have one central tetrahedral atom called keggin. The binding Amino-acid groups to keggin structure give the antivirus effect to these compounds. A new organic-inorganic hybrid structure, with formula (Gly)2H4SiW12O40 was synthesized. Investigation on Anti-viral effect of this compound showed the (Gly)2H4SiW12O40 prevents infection of Tobacco Mosaic Virus (TMV) on the Nicotianatabacum plants.

Keywords: polyoxometalate, keggin, organic-inorganic salt, TMV

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Authors: Ali Bahri Lubis

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Tryptophan oxidation by Heme-dioxygenase enzyme is initial important stepTryptophan oxidation by Heme-dioxygenase enzyme is initial important step in kynurenine pathway implicating to several severe diseases such as Parkinson’s Disease, Huntington Disease, poliomyelitis and cataract. It is crucial to comprehend the oxidation mechanism with the hope to find decent treatment upon abovementioned diseases. The mechanism has been debatable since no one has been yet proved the mechanism obviously. In this research we have attempted to prove mechanistic steps of tryptophan oxidation via human indoleamine dioxygenase (h-IDO) using various substrates: L-tryptophan, L-tryptophan (indole-ring-2-13C), L-fully-labelled13C-tryptophan, L-N-methyl-tryptophan, L-tryptophan and 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid. All enzyme assay experiments were measured using a UV-Vis spectrophotometer, LC-MS, 1H-NMR, and HSQC. We also successfully synthesized enzyme products as our control in NMR measurements. The result exhibited that the distinct substrates produced N-formyl kynurenine (NFK) and hydroxypyrrolloindoleamine carboxylate acid (HPIC) in different concentrations and isomers, correlated to the proposal of considered mechanism reaction in kynurenine pathway implicating to several severe diseases such as Parkinson’s Disease, Huntington Disease, poliomyelitis and cataract. It is crucial to comprehend the oxidation mechanism with the hope to find decent treatment for the abovementioned diseases. The mechanism has been debatable since no one has yet proven the mechanism obviously. In this research we have attempted to prove mechanistic steps of tryptophan oxidation via human indoleamine dioxygenase (h-IDO) using various substrates: L-tryptophan, L-tryptophan (indole-ring-2-13C), L-fully-labelled13C-tryptophan, L-N-methyl-tryptophan, L-tryptophan and 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid. All enzyme assay experiments were measured using a UV-Vis spectrophotometer, LC-MS, 1H-NMR and HSQC. We also successfully synthesized enzyme products as our control in NMR measurements. The result exhibited that the distinct substrates produced N-formyl kynurenine (NFK) and hydroxypyrrolloindoleamine carboxylate acid (HPIC) in different concentrations and isomers, correlated to the proposal of considered mechanism reaction.

Keywords: heme-dioxygenase enzyme, tryptophan oxidation, kynurenine pathway, n-formyl kynurenine

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Authors: Hiral D. Trivedi, Dinesh S. Patel, Sachin P. Shukla

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We have immobilized alcohol dehydrogenase on k-carrageenan, which is a natural polysaccharide obtained from seaweeds by entrapment and on copolymer of acrylamide and 2-hydroxy ethylmethaacrylate by covalent coupling. We have optimized all the immobilization parameters and also carried the comparison studies of both. In case of copolymer of acrylamide and 2-hydroxy ethylmethaacrylate, we have activated both the amino and hydroxyl group individually and simultaneously using different activating agents and obtained some interesting results. We have found that covalently bound enzyme was found to be better under all tested conditions. The reaction on ethanol was carried out using these immobilized systems.

Keywords: alcohol dehydrogenase, acrylamide-co-2-hydroxy ethylmethaacrylate, ethanol, k-carrageenan

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Authors: Mahboobeh Mohadeszadeh, Majid Saghi

Abstract:

Polyoxometalates (POMs) are important inorganic compounds that have been considered specifically in recent years due to abundant attributes and applications. Those POMs that have one central tetrahedral atom called keggin. The binding Amino-acid groups to keggin structure give the antivirus effect to these compounds. A new organic-inorganic hybrid structure, with formula (Gly)2H4SiW12O40, was synthesized. Investigation on the anti-viral effect of this compound showed the (Gly)2H4SiW12O40 prevents infection of Tobacco Mosaic Virus (TMV) on the Nicotianatabacum plants.

Keywords: polyoxometalate, keggin, organic-inorganic salt, TMV

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Authors: Kittisak Jantanasakulwong, Toshiaki Ougizawa

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Thermoplastic starch (TPS) was prepared by melt-blending of cassava starch with glycerol (70/30 wt%/wt%) at 130 ◦C for 10 min. Chitosan (CTS) was used as a compatibilizer. TPS/CTS blend was melt-blended with maleic anhydride grafted ethylene-1-butene rubber (EB-MAH) in the composition of 80/20 respectively. Addition of CTS in TPS/EB-MAH blend decreased particles size of EB-MAH rubber to 1µm in TPS matrix. Mechanical properties, solubility, swelling property, morphology, and water contact angle of TPS/EB-MAH blend were improved by CTS incorporation. FTIR confirmed a reaction had occurred between amino groups (-NH2) of CTS and the MAH groups of EB-MAH. This reaction and the enhanced miscibility between TPS and CTS improved morphology and properties of the TPS/EB-MAH/CTS blend.

Keywords: thermoplastic starch, rubber, reactive blending, chitosan

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Authors: Balgabay Sadepovich Maikanov, Laura Tyulegenovna Auteleyeva, Seidenova Simbat Polatbekovna

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In this article reduced nutritional value of the rabbits’ meat at 1, 1 dimethylhydrazine experimental toxicosis is shown. The assay was performed on liquid chromatograph SHIMADZU LC-20 Prominence (Japan) with fluorometric and spectrophotometric detector. This research has revealed that samples of rabbit meat of the experimental group had significant differences from the control group:in amino acids concentration from 1.2% to 9.1%; vitamin concentration from 11.2% to 60.5%, macro – minerals concentration from 17.4% to 78.1% and saturated fatty acids concentration from 17,1% to 34.5%, respectively. The decrease in the chemical composition of rabbits’ meat at 1,1 dimethylhydrazine toxicosis may be due to changes in the internal processes associated with impaired metabolic homeostasis of animals.

Keywords: 1, 1-dimethylhydrazine, metabolic homeostasis, nutritional value, rabbit meat

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Authors: Yew Mun Yip, Dawei Zhang

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Proteins are the biological machinery that executes specific vital functions in every cell of the human body by folding into their 3D structures. When a protein misfolds from its native structure, the machinery will malfunction and lead to misfolding diseases. Although in vitro experiments are able to conclude that the mutations of the amino acid sequence lead to incorrectly folded protein structures, these experiments are unable to decipher the folding process. Therefore, molecular dynamic (MD) simulations are employed to simulate the folding process so that our improved understanding of the folding process will enable us to contemplate better treatments for misfolding diseases. MD simulations make use of force fields to simulate the folding process of peptides. Secondary structures are formed via the hydrogen bonds formed between the backbone atoms (C, O, N, H). It is important that the hydrogen bond energy computed during the MD simulation is accurate in order to direct the folding process to the native structure. Since the atoms involved in a hydrogen bond possess very dissimilar electronegativities, the more electronegative atom will attract greater electron density from the less electronegative atom towards itself. This is known as the polarization effect. Since the polarization effect changes the electron density of the two atoms in close proximity, the atomic charges of the two atoms should also vary based on the strength of the polarization effect. However, the fixed atomic charge scheme in force fields does not account for the polarization effect. In this study, we introduce the polarized structure-specific backbone charge (PSBC) model. The PSBC model accounts for the polarization effect in MD simulation by updating the atomic charges of the backbone hydrogen bond atoms according to equations derived between the amount of charge transferred to the atom and the length of the hydrogen bond, which are calculated from quantum-mechanical calculations. Compared to other polarizable models, the PSBC model does not require quantum-mechanical calculations of the peptide simulated at every time-step of the simulation and maintains the dynamic update of atomic charges, thereby reducing the computational cost and time while accounting for the polarization effect dynamically at the same time. The PSBC model is applied to two different β-peptides, namely the Beta3s/GS peptide, a de novo designed three-stranded β-sheet whose structure is folded in vitro and studied by NMR, and the trpzip peptides, a double-stranded β-sheet where a correlation is found between the type of amino acids that constitute the β-turn and the β-propensity.

Keywords: hydrogen bond, polarization effect, protein folding, PSBC

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Authors: Aylin W Sahin, Alice Jaeger, Laura Nyhan, Gregory Belt, Steffen Münch, Elke K. Arendt

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Our current food systems cause an increase in malnutrition resulting in more people being overweight or obese in the Western World. Additionally, our natural resources are under enormous pressure and the greenhouse gas emission increases yearly with a significant contribution to climate change. Hence, transforming our food systems is of highest priority. Plant-based food products have a lower environmental impact compared to their animal-based counterpart, representing a more sustainable protein source. However, most plant-based protein ingredients, such as soy and pea, are lacking indispensable amino acids and extremely limited in their functionality and, thus, in their food application potential. They are known to have a low solubility in water and change their properties during processing. The low solubility displays the biggest challenge in the development of milk alternatives leading to inferior protein content and protein quality in dairy alternatives on the market. Moreover, plant-based protein ingredients often possess an off-flavour, which makes them less attractive to consumers. EverPro, a plant-protein isolate originated from Brewer’s Spent Grain, the most abundant by-product in the brewing industry, represents the missing piece in the plant protein portfolio. With a protein content of >85%, it is of high nutritional value, including all indispensable amino acids which allows closing the protein quality gap of plant proteins. Moreover, it possesses high techno-functional properties. It is fully soluble in water (101.7 ± 2.9%), has a high fat absorption capacity (182.4 ± 1.9%), and a foaming capacity which is superior to soy protein or pea protein. This makes EverPro suitable for a vast range of food applications. Furthermore, it does not cause changes in viscosity during heating and cooling of dispersions, such as beverages. Besides its outstanding nutritional and functional characteristics, the production of EverPro has a much lower environmental impact compared to dairy or other plant protein ingredients. Life cycle assessment analysis showed that EverPro has the lowest impact on global warming compared to soy protein isolate, pea protein isolate, whey protein isolate, and egg white powder. It also contributes significantly less to freshwater eutrophication, marine eutrophication and land use compared the protein sources mentioned above. EverPro is the prime example of sustainable ingredients, and the type of plant protein the food industry was waiting for: nutritious, multi-functional, and environmentally friendly.

Keywords: plant-based protein, upcycled, brewers' spent grain, low environmental impact, highly functional ingredient

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Authors: Tetsuo Okutsu

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We have developed a crystallization plate with the function of promoting protein crystallization. A gold thin film is deposited on the crystallization plate. A protein solution is dropped thereon, and crystallization is promoted when the protein is irradiated with light of a wavelength that protein does not absorb. Protein is densely adsorbed on the gold thin film surface. The light excites the surface plasmon resonance of the gold thin film, the protein is excited by the generated enhanced electric field induced by surface plasmon resonance, and the amino acid residues are radicalized to produce protein dimers. The dimers function as templates for protein crystals, crystallization is promoted.

Keywords: lysozyme, plasmon, protein, crystallization, RNaseA

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Authors: Ahmed A. Al-Amiery, Yasmin K. Al-Majedy, Abdul Amir H. Kadhum, Abu Bakar Mohamad

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The efficiency of synthesized thiosemicarbazone namely 2-(1,5-dimethyl-4-(2-methylbenzylidene)amino)-2-phenyl-1H-pyrazol-3(2H)-ylidene) hydrazinecarbothioamide investigated as corrosion inhibitor of mild steel in 1N H2SO4 using electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization (PD) in addition of scanning electron microscopy (SEM). The results showed that this inhibitor behaved as a good corrosion inhibitor even at low concentration with a mean efficiency of 93%. Polarization technique and EIS were tested in different concentrations reveal that this compound is adsorbed on the mild steel, therefore blocking the active sites and the adsorption follows the Langmuir adsorption isotherm model. SEM shows that mild steel surface is nearly perfect for mild steel which was immersed in a solution of H2SO4 with corrosion inhibitor.

Keywords: corrosion inhibitor, thiosemicarbazide, electrochemical impedance, electrochemical impedance spectroscopy

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Authors: Hateem Zafar Kayani, Nageen Hussain

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The main aim is to analyze the mutations of DNASE I gene in diabetic patients. A total of 120 diabetes patients and 120 controls were sampled. The total number of male diabetic patients included in the study was 79 (66%) while female patients were 41 (34%) in number. Exon 8 of the DNASE I gene was amplified by using thermo cycler. The possible band of interest was located at 165 base pairs. Two samples showed similar missense mutations at 127th position of exon 8 which replaced amino acid Arginine (Arg) to Glutamine (Gln). All controls showed no mutations. The association of diabetes with different levels of blood pressure and body mass index (BMI) were found to be significant.

Keywords: deoxyribonuclease I, polymerase chain reaction, insulin-dependent diabetes mellitus, non-insulin dependent diabetes mellitus

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Authors: Sophio Kobauri, Temur Kantaria, Nina Kulikova, David Tugushi, Ramaz Katsarava

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The elaboration of effective drug delivery vehicles is still topical nowadays since targeted drug delivery is one of the most important challenges of the modern nanomedicine. The last decade has witnessed enormous research focused on synthetic cationic polymers (CPs) due to their flexible properties, in particular as non-viral gene delivery systems, facile synthesis, robustness, not oncogenic and proven gene delivery efficiency. However, the toxicity is still an obstacle to the application in pharmacotherapy. For overcoming the problem, creation of new cationic compounds including the polymeric nano-size particles – nano-containers (NCs) loading with different pharmaceuticals and biologicals is still relevant. In this regard, a variety of NCs-based drug delivery systems have been developed. We have found that amino acid-based biodegradable polymers called as pseudo-proteins (PPs), which can be cleared from the body after the fulfillment of their function are highly suitable for designing pharmaceutical NCs. Among them, one of the most promising are NCs made of biodegradable Cationic PPs (CPPs). For preparing new cationic NCs (CNCs), we used CPPs composed of positively charged amino acid L-arginine (R). The CNCs were fabricated by two approaches using: (1) R-based homo-CPPs; (2) Blends of R-based CPPs with regular (neutral) PPs. According to the first approach NCs we prepared from CPPs 8R3 (composed of R, sebacic acid and 1,3-propanediol) and 8R6 (composed of R, sebacic acid and 1,6-hexanediol). The NCs prepared from these CPPs were 72-101 nm in size with zeta potential within +30 ÷ +35 mV at a concentration 6 mg/mL. According to the second approach, CPPs 8R6 was blended in organic phase with neutral PPs 8L6 (composed of leucine, sebacic acid and 1,6-hexanediol). The NCs prepared from the blends were 130-140 nm in size with zeta potential within +20 ÷ +28 mV depending on 8R6/8L6 ratio. The stability studies of fabricated NCs showed that no substantial change of the particle size and distribution and no big particles’ formation is observed after three months storage. In vitro biocompatibility study of the obtained NPs with four different stable cell lines: A549 (human), U-937 (human), RAW264.7 (murine), Hepa 1-6 (murine) showed both type cathionic NCs are biocompatible. The obtained data allow concluding that the obtained CNCs are promising for the application as biodegradable drug delivery vehicles. This work was supported by the joint grant from the Science and Technology Center in Ukraine and Shota Rustaveli National Science Foundation of Georgia #6298 'New biodegradable cationic polymers composed of arginine and spermine-versatile biomaterials for various biomedical applications'.

Keywords: biodegradable polymers, cationic pseudo-proteins, nano-containers, drug delivery vehicles

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Authors: Kuheli Chakrabarty, Animesh Ghosh, Atanu Roy, Gourab Kanti Das

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Aldol reaction is an important class of carbon-carbon bond forming reactions. One of the popular ways to impose asymmetry in aldol reaction is the introduction of chiral auxiliary that binds the approaching reactants and create dissymmetry in the reaction environment, which finally evolves to enantiomeric excess in the aldol products. The last decade witnesses the usage of natural amino acids as chiral auxiliary to control the stereoselectivity in various carbon-carbon bond forming processes. In this context, L-proline was found to be an effective organocatalyst in asymmetric aldol additions. In last few decades the use of water as solvent or co-solvent in asymmetric organocatalytic reaction is increased sharply. Simple amino acids like L-proline does not catalyze asymmetric aldol reaction in aqueous medium not only that, In organic solvent medium high catalytic loading (~30 mol%) is required to achieve moderate to high asymmetric induction. In this context, huge efforts have been made to modify L-proline and 4-hydroxy-L-proline to prepare organocatalyst for aqueous medium asymmetric aldol reaction. Here, we report the result of our DFT calculations on asymmetric aldol reaction of benzaldehyde, p-NO2 benzaldehyde and t-butyraldehyde with a number of ketones using L-proline hydrazide as organocatalyst in wet solvent free condition. Gaussian 09 program package and Gauss View program were used for the present work. Geometry optimizations were performed using B3LYP hybrid functional and 6-31G(d,p) basis set. Transition structures were confirmed by hessian calculation and IRC calculation. As the reactions were carried out in solvent free condition, No solvent effect were studied theoretically. Present study has revealed for the first time, the direct involvement of two water molecules in the aldol transition structures. In the TS, the enamine and the aldehyde is connected through hydrogen bonding by the assistance of two intervening water molecules forming a supramolecular network. Formation of this type of supramolecular assembly is possible due to the presence of protonated -NH2 group in the L-proline hydrazide moiety, which is responsible for the favorable entropy contribution to the aldol reaction. It is also revealed from the present study that, water assisted TS is energetically more favorable than the TS without involving any water molecule. It can be concluded from this study that, insertion of polar group capable of hydrogen bond formation in the L-proline skeleton can lead to a favorable aldol reaction with significantly high enantiomeric excess in wet solvent free condition by reducing the activation barrier of this reaction.

Keywords: aldol reaction, DFT, organocatalysis, transition structure

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Authors: Darshna Sharma, Suban K. Sahoo

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The detection of intracellular fluoride in human cancer cell HeLa was achieved by chemosensors derived from vitamin B6 cofactors using fluorescence imaging technique. These sensors were first synthesized by condensation of pyridoxal/pyridoxal phosphate with 2-amino(thio)phenol. The anion recognition ability was explored by experimental (UV-VIS, fluorescence and 1H NMR) and theoretical DFT [(B3LYP/6-31G(d,p)] methods in DMSO and mixed DMSO-H2O system. All the developed sensors showed both naked-eye detectable color change and remarkable fluorescence enhancement in the presence of F- and AcO-. The anion recognition was occurred through the formation of hydrogen bonded complexes between these anions and sensor, followed by the partial deprotonation of sensor. The detection limit of these sensors were down to micro(nano) molar level of F- and AcO-.

Keywords: chemosensors, fluoride, acetate, turn-on, live cells imaging, DFT

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Authors: Abdoulie K. Ceesay

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Within the sequence of the whole genome, it is known that 99.9% of the human genome is similar, whilst our difference lies in just 0.1%. Among these minor dissimilarities, the most common type of genetic variations that occurs in a population is SNP, which arises due to nucleotide substitution in a protein sequence that leads to protein destabilization, alteration in dynamics, and other physio-chemical properties’ distortions. While causing variations, they are equally responsible for our difference in the way we respond to a treatment or a disease, including various cancer types. There are two types of SNPs; synonymous single nucleotide polymorphism (sSNP) and non-synonymous single nucleotide polymorphism (nsSNP). sSNP occur in the gene coding region without causing a change in the encoded amino acid, while nsSNP is deleterious due to its replacement of a nucleotide residue in the gene sequence that results in a change in the encoded amino acid. Predicting the effects of cancer related nsSNPs on protein stability, function, and dynamics is important due to the significance of phenotype-genotype association of cancer. In this thesis, Data of 5 oncogenes (ONGs) (AKT1, ALK, ERBB2, KRAS, BRAF) and 5 tumor suppressor genes (TSGs) (ESR1, CASP8, TET2, PALB2, PTEN) were retrieved from ClinVar. Five common in silico tools; Polyphen, Provean, Mutation Assessor, Suspect, and FATHMM, were used to predict and categorize nsSNPs as deleterious, benign, or neutral. To understand the impact of each variation on the phenotype, Maestro, PremPS, Cupsat, and mCSM-NA in silico structural prediction tools were used. This study comprises of in-depth analysis of 10 cancer gene variants downloaded from Clinvar. Various analysis of the genes was conducted to derive a meaningful conclusion from the data. Research done indicated that pathogenic variants are more common among ONGs. Our research also shows that pathogenic and destabilizing variants are more common among ONGs than TSGs. Moreover, our data indicated that ALK(409) and BRAF(86) has higher benign count among ONGs; whilst among TSGs, PALB2(1308) and PTEN(318) genes have higher benign counts. Looking at the individual cancer genes predisposition or frequencies of causing cancer according to our research data, KRAS(76%), BRAF(55%), and ERBB2(36%) among ONGs; and PTEN(29%) and ESR1(17%) among TSGs have higher tendencies of causing cancer. Obtained results can shed light to the future research in order to pave new frontiers in cancer therapies.

Keywords: tumor suppressor genes (TSGs), oncogenes (ONGs), non synonymous single nucleotide polymorphism (nsSNP), single nucleotide polymorphism (SNP)

Procedia PDF Downloads 61

Authors: S. D. N. K. Bathige, G. I. Godahewa, Navaneethaiyer Umasuthan, Jehee Lee

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Kunitz-type serine protease inhibitors (KTIs) are identified in various organisms including animals, plants and microbes. These proteins shared single or multiple Kunitz inhibitory domains link together or associated with other types of domains. Characteristic Kunitz type domain composed of around 60 amino acid residues with six conserved cysteine residues to stabilize by three disulfide bridges. KTIs are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis and inflammation. In this study, two Kunitz-type domain containing protein was identified from rock bream database and designated as RbKunitz. The coding sequence of RbKunitz encoded for 507 amino acids with 56.2 kDa theoretical molecular mass and 5.7 isoelectric point (pI). There are several functional domains including MANEC superfamily domain, PKD superfamily domain, and LDLa domain were predicted in addition to the two characteristic Kunitz domain. Moreover, trypsin interaction sites were also identified in Kunitz domain. Homology analysis revealed that RbKunitz shared highest identity (77.6%) with Takifugu rubripes. Completely conserved 28 cysteine residues were recognized, when comparison of RbKunitz with other orthologs from different taxonomical groups. These structural evidences indicate the rigidity of RbKunitz folding structure to achieve the proper function. The phylogenetic tree was constructed using neighbor-joining method and exhibited that the KTIs from fish and non-fish has been evolved in separately. Rock bream was clustered with Takifugu rubripes. The SYBR Green qPCR was performed to quantify the RbKunitz transcripts in different tissues and challenged tissues. The mRNA transcripts of RbKunitz were detected in all tissues (muscle, spleen, head kidney, blood, heart, skin, liver, intestine, kidney and gills) analyzed and highest transcripts level was detected in gill tissues. Temporal transcription profile of RbKunitz in rock bream blood tissues was analyzed upon LPS (lipopolysaccharide), Poly I:C (Polyinosinic:polycytidylic acid) and Edwardsiella tarda challenge to understand the immune responses of this gene. Compare to the unchallenged control RbKunitz exhibited strong up-regulation at 24 h post injection (p.i.) after LPS and E. tarda injection. Comparatively robust expression of RbKunits was observed at 3 h p.i. upon Poly I:C challenge. Taken together all these data indicate that RbKunitz may involve into to immune responses upon pathogenic stress, in order to protect the rock bream.

Keywords: Kunitz-type, rock bream, immune response, serine protease inhibitor

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Authors: Gulmira Zhakupova, Tamara Tultabayeva, Aknur Muldasheva, Assem Sagandyk

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The development of technology and production of products with increased biological value based on the use of natural food raw materials are important tasks in the policy of the food market of the Republic of Kazakhstan. For Kazakhstan, livestock farming, in particular sheep farming, is the most ancient and developed industry and way of life. The history of the Kazakh people is largely connected with this type of agricultural production, with established traditions using dairy products from sheep's milk. Therefore, the development of new technologies from sheep’s milk remains relevant. In addition, one of the most promising areas for the development of food technology for therapeutic and prophylactic purposes is sheep milk products as a source of protein, immunoglobulins, minerals, vitamins, and other biologically active compounds. This article presents the results of research on the study of milk processing technology. The objective of the study is to study the possibilities of processing sheep milk and its role in human nutrition, as well as the results of research to improve the technology of sheep milk products. The studies were carried out on the basis of sanitary and hygienic requirements for dairy products in accordance with the following test methods. To perform microbiological analysis, we used the method for identifying Salmonella bacteria (Horizontal method for identifying, counting, and serotyping Salmonella) in a certain mass or volume of product. Nutritional value is a complex of properties of food products that meet human physiological needs for energy and basic nutrients. The protein mass fraction was determined by the Kjeldahl method. This method is based on the mineralization of a milk sample with concentrated sulfuric acid in the presence of an oxidizing agent, an inert salt - potassium sulfate, and a catalyst - copper sulfate. In this case, the amino groups of the protein are converted into ammonium sulfate dissolved in sulfuric acid. The vitamin composition was determined by HPLC. To determine the content of mineral substances in the studied samples, the method of atomic absorption spectrophotometry was used. The study identified the technological parameters of sheep milk products and determined the prospects for researching sheep milk products. Microbiological studies were used to determine the safety of the study product. According to the results of the microbiological analysis, no deviations from the norm were identified. This means high safety of the products under study. In terms of nutritional value, the resulting products are high in protein. Data on the positive content of amino acids were also obtained. The results obtained will be used in the food industry and will serve as recommendations for manufacturers.

Keywords: dairy, milk processing, nutrition, colostrum

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