Search results for: fluorescent proteins

Authors: Mario Pérez-Won, Vilbett Briones L., Guido Trautmann, María José Bugueño, Gipsy Tabilo-Munizaga, Luis Gonzalez-Cavieres

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The fishing industry generates a significant amount of shrimp byproducts, which often result in environmental contamination. Protein extraction from these by-products is a potential solution to minimize waste and revalue the by-products. To improve the extraction of proteins (by chemical method) from shrimp (Pleuroncodes monodon) by-products, the emerging technologies of ohmic heating (OH), microwaves (MW) and pulsed electric fields (PEF) were used. The results show that microwaves, electrical pulses, and ohmic heating improved performance by 28.19%, 19.25%, and 3.65%, respectively. Furthermore, conformational changes were studied by DSC and FTIR. Subsequently, the use of these proteins in electrospinning technology was evaluated. In conclusion, this study demonstrates that the application of emerging technologies, can significantly improve the extraction yield of proteins from shrimp by-products.

Keywords: electrospinning, emerging technologies, improving extraction, shrimp by-products

Procedia PDF Downloads 43

Authors: Majid Hejazian, Nam-Trung Nguyen

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Over the past few years, microfluidic devices have generated significant attention from industry and academia due to advantages such as small sample volume, low cost and high efficiency. Microfluidic devices have applications in chemical, biological and industry analysis and can facilitate assay of bio-materials and chemical reactions, separation, and sensing. Micromixers are one of the important microfluidic concepts. Micromixers can work as stand-alone devices or be integrated in a more complex microfluidic system such as a lab on a chip (LOC). Micromixers are categorized as passive and active types. Passive micromixers rely only on the arrangement of the phases to be mixed and contain no moving parts and require no energy. Active micromixers require external fields such as pressure, temperature, electric and acoustic fields. Rapid and efficient mixing is important for many applications such as biological, chemical and biochemical analysis. Achieving fast and homogenous mixing of multiple samples in the microfluidic devices has been studied and discussed in the literature recently. Improvement in mixing rely on effective mass transport in microscale, but are currently limited to molecular diffusion due to the predominant laminar flow in this size scale. Using magnetic field to elevate mass transport is an effective solution for mixing enhancement in microfluidics. The use of a non-uniform magnetic field to improve mass transfer performance in a microfluidic device is demonstrated in this work. The phenomenon of mixing ferrofluid and DI-water streams has been reported before, but mass transfer enhancement for other non-magnetic species through magnetic field have not been studied and evaluated extensively. In the present work, permanent magnets were used in a simple microfluidic device to create a non-uniform magnetic field. Two streams are introduced into the microchannel: one contains fluorescent dye mixed with diluted ferrofluid to induce enhanced mass transport of the dye, and the other one is a non-magnetic DI-water stream. Mass transport enhancement of fluorescent dye is evaluated using fluorescent measurement techniques. The concentration field is measured for different flow rates. Due to effect of magnetic field, a body force is exerted on the paramagnetic stream and expands the ferrofluid stream into non-magnetic DI-water flow. The experimental results demonstrate that without a magnetic field, both magnetic nanoparticles of the ferrofluid and the fluorescent dye solely rely on molecular diffusion to spread. The non-uniform magnetic field, created by the permanent magnets around the microchannel, and diluted ferrofluid can improve mass transport of non-magnetic solutes in a microfluidic device. The susceptibility mismatch between the fluids results in a magnetoconvective secondary flow towards the magnets and subsequently the mass transport of the non-magnetic fluorescent dye. A significant enhancement in mass transport of the fluorescent dye was observed. The platform presented here could be used as a microfluidics-based micromixer for chemical and biological applications.

Keywords: ferrofluid, mass transfer, micromixer, microfluidics, magnetic

Procedia PDF Downloads 194

Authors: P. Behera, K. K. Singh, D. K. Saini, M. De

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Implementation of 2D materials in the detection of bio analytes is highly advantageous in the field of sensing because of its high surface to volume ratio. We have designed our sensor array with different cationic two-dimensional MoS₂, where surface modification was achieved by cationic thiol ligands with different functionality. Green fluorescent protein (GFP) was chosen as signal transducers for its biocompatibility and anionic nature, which can bind to the cationic MoS₂ surface easily, followed by fluorescence quenching. The addition of bio-analyte to the sensor can decomplex the cationic MoS₂ and GFP conjugates, followed by the regeneration of GFP fluorescence. The fluorescence response pattern belongs to various analytes collected and transformed to linear discriminant analysis (LDA) for classification. At first, 15 different proteins having wide range of molecular weight and isoelectric points were successfully discriminated at 50 nM with detection limit of 1 nM. The sensor system was also executed in biofluids such as serum, where 10 different proteins at 2.5 μM were well separated. After successful discrimination of protein analytes, the sensor array was implemented for bacteria sensing. Six different bacteria were successfully classified at OD = 0.05 with a detection limit corresponding to OD = 0.005. The optimized sensor array was able to classify uropathogens from non-uropathogens in urine medium. Further, the technique was applied for discrimination of bacteria possessing resistance to different types and amounts of drugs. We found out the mechanism of sensing through optical and electrodynamic studies, which indicates the interaction between bacteria with the sensor system was mainly due to electrostatic force of interactions, but the separation of native bacteria from their drug resistant variant was due to Van der Waals forces. There are two ways bacteria can be detected, i.e., through bacterial cells and lysates. The bacterial lysates contain intracellular information and also safe to analysis as it does not contain live cells. Lysates of different drug resistant bacteria were patterned effectively from the native strain. From unknown sample analysis, we found that discrimination of bacterial cells is more sensitive than that of lysates. But the analyst can prefer bacterial lysates over live cells for safer analysis.

Keywords: array-based sensing, drug resistant bacteria, linear discriminant analysis, two-dimensional MoS₂

Procedia PDF Downloads 113

Authors: Eva Slaninova, Diana Cernayova, Zuzana Sedrlova, Katerina Mrazova, Petr Sedlacek, Jana Nebesarova, Stanislav Obruca

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Cyanobacteria are gram-negative prokaryotes belonging to a group of photosynthetic bacteria. In comparison with heterotrophic microorganisms, cyanobacteria utilize atmospheric nitrogen and carbon dioxide without any additional substrates. This ability of these microorganisms could be employed in biotechnology for the production of bioplastics, concretely polyhydroxyalkanoates (PHAs) which are primarily accumulated as a storage material in cells in the form of intracellular granules. In this study, there two cyanobacterial cultures from genera Synechocystis were used, namely Synechocystic sp. PCC 6803 and Synechocystis salina CCALA 192. There were optimized and used several various approaches, including microscopic techniques such as cryo-scanning electron microscopy (Cryo-SEM) and transmission electron microscopy (TEM), and fluorescence lifetime imaging microscopy using Nile red as a fluorescent probe (FLIM). Due to these instrumental techniques, the morphology of intracellular space and surface of cells were characterized. The next group of methods which were employed was spectroscopic techniques such as UV-Vis spectroscopy measured in two modes (turbidimetry and integration sphere) and Fourier transform infrared spectroscopy (FTIR). All these diverse techniques were used for the detection and characterization of pigments (chlorophylls, carotenoids, phycocyanin, etc.) and PHAs, in our case poly (3-hydroxybutyrate) (P3HB). To verify results, gas chromatography (GC) was employed concretely for the determination of the amount of P3HB in biomass. Cyanobacteria were also characterized as polyhydroxybutyrate producers by flow cytometer, which could count cells and at the same time distinguish cells including P3HB and without due to fluorescent probe called BODIPY and live/dead fluorescent probe SYTO Blue. Based on results, P3HB content in cyanobacteria cells was determined, as also the overall fitness of the cells. Acknowledgment: Funding: This study was partly funded by the projectGA19-29651L of the Czech Science Foundation (GACR) and partly funded by the Austrian Science Fund (FWF), project I 4082-B25.

Keywords: cyanobacteria, fluorescent probe, microscopic techniques, poly(3hydroxybutyrate), spectroscopy, chromatography

Procedia PDF Downloads 196

Authors: Yakup Ulusu, Numan Eczacıoğlu, İsa Gökçe, Helen Waller, Jeremy H. Lakey

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Besides having the appropriate amino acid sequence to perform the function of proteins, it is important to have correct conformation after this sequence to process. To consist of this conformation depends on the amino acid sequence at the primary structure, hydrophobic interaction, chaperones and enzymes in charge of folding etc. Misfolded proteins are not functional and tend to be aggregated. Cysteine originating disulfide cross-links make stable this conformation of functional proteins. When two of the cysteine amino acids come side by side, disulfide bond is established that forms a cystine bridge. Due to this feature cysteine plays an important role on the formation of three-dimensional structure of many proteins. There are two cysteine amino acids (C44, C69) in the Tol-A-III protein. Unlike protein disulfide bonds from within his own, any non-specific cystine bridge causes a change in the three dimensional structure of the protein. Proteins can be expressed in various host cells as directly or fusion (chimeric). As a result of overproduction of the recombinant proteins, aggregation of insoluble proteins in the host cell can occur by forming a crystal structure called inclusion body. In general fusion proteins are produced for provide affinity tags to make proteins more soluble and production of some toxic proteins via fusion protein expression system like pTolT. Proteins can be modified by using a site-directed mutagenesis. By this way, creation of non-specific disulfide crosslinks can be prevented at fusion protein expression system via the present cysteine replaced by another amino acid such as serine, glycine or etc. To do this, we need; a DNA molecule that contains the gene that encodes for the target protein, required primers for mutation to be designed according to site directed mutagenesis reaction. This study was aimed to be replaced cysteine encoding codon TGT with serine encoding codon AGT. For this sense and reverse primers designed (given below) and used site-directed mutagenesis reaction. Several new copy of the template plasmid DNA has been formed with above mentioned mutagenic primers via polymerase chain reaction (PCR). PCR product consists of both the master template DNA (wild type) and the new DNA sequences containing mutations. Dpn-l endonuclease restriction enzyme which is specific for methylated DNA and cuts them to the elimination of the master template DNA. E. coli cells obtained after transformation were incubated LB medium with antibiotic. After purification of plasmid DNA from E. coli, the presence of the mutation was determined by DNA sequence analysis. Developed this new plasmid is called PtolT-δ.

Keywords: site directed mutagenesis, Escherichia coli, pTolT, protein expression

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Authors: Manju Kanu, Subrata Sinha, Surabhi Johari

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Viral proteins of Ebola viruses belong to one of the best studied viruses; however no effective prevention against EBOV has been developed. Epitope-based vaccines provide a new strategy for prophylactic and therapeutic application of pathogen-specific immunity. A critical requirement of this strategy is the identification and selection of T-cell epitopes that act as vaccine targets. This study describes current methodologies for the selection process, with Ebola virus as a model system. Hence great challenge in the field of ebola virus research is to design universal vaccine. A combination of publicly available bioinformatics algorithms and computational tools are used to screen and select antigen sequences as potential T-cell epitopes of supertypes Human Leukocyte Antigen (HLA) alleles. MUSCLE and MOTIF tools were used to find out most conserved peptide sequences of viral proteins. Immunoinformatics tools were used for prediction of immunogenic peptides of viral proteins in zaire strains of Ebola virus. Putative epitopes for viral proteins (VP) were predicted from conserved peptide sequences of VP. Three tools NetCTL 1.2, BIMAS and Syfpeithi were used to predict the Class I putative epitopes while three tools, ProPred, IEDB-SMM-align and NetMHCII 2.2 were used to predict the Class II putative epitopes. B cell epitopes were predicted by BCPREDS 1.0. Immunogenic peptides were identified and selected manually by putative epitopes predicted from online tools individually for both MHC classes. Finally sequences of predicted peptides for both MHC classes were looked for common region which was selected as common immunogenic peptide. The immunogenic peptides were found for viral proteins of Ebola virus: epitopes FLESGAVKY, SSLAKHGEY. These predicted peptides could be promising candidates to be used as target for vaccine design.

Keywords: epitope, b cell, immunogenicity, ebola

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Authors: Himani Sharma, Amit Agrawal

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Stability of the Cassie and Wenzel wetting states depends on intrinsic contact angle and geometric features on a surface that was exploited in capturing biofluids in microwells. However, the mechanism of imbibition of biofluids in the microwells is not well implied in terms of wettability of a substrate. In this work, we experimentally demonstrated filling dynamics in hydrophilic and hydrophobic microwells by protein solutions. Towards this, we utilized lotus leaf as a mold to fabricate microwells on a Polydimethylsiloxane (PDMS) surface. Lotus leaf containing micrometer-sized blunt-conical shaped pillars with a height of 8-15 µm and diameter of 3-8 µm were transferred on to PDMS. Furthermore, PDMS surface was treated with oxygen plasma to render the hydrophilic nature. A 10µL droplets containing fluorescein isothiocyanate (FITC) - labelled bovine serum albumin (BSA) were rested on both hydrophobic (θa = 108o, where θa is the apparent contact angle) and hydrophilic (θa = 60o) PDMS surfaces. A time-dependent fluorescence microscopy was conducted on these modified PDMS surfaces by recording the fluorescent intensity over a 5 minute period. It was observed that, initially (at t=1 min) FITC-BSA was accumulated on the periphery of both hydrophilic and hydrophobic microwells due to incomplete penetration of liquid-gas meniscus. This deposition of FITC-BSA on periphery of microwell was not changed with time for hydrophobic surfaces, whereas, a complete filling was occurred in hydrophilic microwells (at t=5 mins). This attributes to a gradual movement of three-phase contact line along the vertical surface of the hydrophilic microwells as compared to stable pinning in the hydrophobic microwells as confirmed by Surface Evolver simulations. In addition, if the cavities are presented on hydrophobic surfaces, air bubbles will be trapped inside the cavities once the aqueous solution is placed over these surfaces, resulting in the Cassie-Baxter wetting state. This condition hinders trapping of proteins inside the microwells. Thus, it is necessary to impart hydrophilicity to the microwell surfaces so as to induce the Wenzel state, such that, an entire solution will be fully in contact with the walls of microwells. Imbibition of microwells by protein solutions was analyzed in terms fluorescent intensity versus time. The present work underlines the importance of geometry of microwells and surface wettability of substrate in wetting and effective capturing of solid sub-phases in biofluids.

Keywords: BSA, microwells, surface evolver, wettability

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Authors: Emine G. Cansu Ergun

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Conjugated system based sensors for selective detection of metal ions have been taking attention during last two decades. Fluorescent sensors are the promising candidates for ion detection due to their high selectivity towards metal ions, and rapid response times. Detection of mercury in an environmenet is important since mercury is a toxic element for human. Beyond the maximum allowable limit, mercury may cause serious problems in human health by spreading into the atmosphere, water and the food chain. In this study, a quinoxaline and 3,4-ethylenedioxy thiophene based donor-acceptor-donor type conjugated molecule used as a fluorescent sensor for detecting the mercury ion in aqueous medium. Among other various cations, existence of mercury resulted in a full quenching of the fluorescence signal. Then, a paper based sensor is constructed and used for mercury detection. As a result it is concluded that the offering sensor is a good candidate for selective mercury detection in aqueous media both in solution and paper based forms.

Keywords: Conjugated molecules , fluorescence quenching, metal ion detection , sensors

Procedia PDF Downloads 129

Authors: Shayli Varasteh Moradi, Wayne A. Johnston, Dejan Gagoski, Kirill Alexandrov

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The demand for a rapid and more efficient technique to identify protein-protein interaction particularly in the areas of therapeutics and diagnostics development is growing. The method described here is a rapid in vitro protein-protein interaction analysis approach based on AlphaLISA technology combined with Leishmania tarentolae cell-free protein production (LTE) system. Cell-free protein synthesis allows the rapid production of recombinant proteins in a multiplexed format. Among available in vitro expression systems, LTE offers several advantages over other eukaryotic cell-free systems. It is based on a fast growing fermentable organism that is inexpensive in cultivation and lysate production. High integrity of proteins produced in this system and the ability to co-express multiple proteins makes it a desirable method for screening protein interactions. Following the translation of protein pairs in LTE system, the physical interaction between proteins of interests is analysed by AlphaLISA assay. The assay is performed using unpurified in vitro translation reaction and therefore can be readily multiplexed. This approach can be used in various research applications such as epitope mapping, antigen-antibody analysis and protein interaction network mapping. The intra-viral protein interaction network of Zika virus was studied using the developed technique. The viral proteins were co-expressed pair-wise in LTE and all possible interactions among viral proteins were tested using AlphaLISA. The assay resulted to the identification of 54 intra-viral protein-protein interactions from which 19 binary interactions were found to be novel. The presented technique provides a powerful tool for rapid analysis of protein-protein interaction with high sensitivity and throughput.

Keywords: AlphaLISA technology, cell-free protein expression, epitope mapping, Leishmania tarentolae, protein-protein interaction

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Authors: Wael El-Deeb

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The present study aimed to investigate the diagnostic importance of acute phase proteins in urinary tract infection (UTI) in cattle. We describe the clinical, bacteriological and biochemical findings in 99 lactating cows. Blood and urine samples from diseased (n=84) and control healthy cows (n=15) were submitted to laboratory investigations. The urine analysis revealed hematuria and pyuria in UTI group. The isolated bacteria were E.coli (43/84) Corynebacterium spp, (31/84), Proteus spp. (6/84) and Streptococcus spp (4/84). The concentrations of Haptoglobin (Hp), serum amyloid A (SAA), α1-Acid glycoprotein (AGP), fibrinogen (Fb), total protein, albumen, and globulin were higher in cows with UTI when compared to healthy ones. Fifty-one of 84 cows with UTI were successfully treated. The levels of Hp, SAA, AGP, total protein, and globulin were associated with the odds of treatment failure. Conclusively, acute phase proteins could be used as diagnostic and prognostic biomarkers in cows with UTI.

Keywords: cows, urinary, infections, haptoglobin, serum Amyloid A

Procedia PDF Downloads 697

Authors: Win Nee Phong, Tau Chuan Ling, Pau Loke Show

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From an industrial perspective, the exploitation of microalgae for protein source is of great economical and commercial interest due to numerous attractive characteristics. Nonetheless, the release of protein from microalgae is limited by the multiple layers of the rigid thick cell wall that generally contain a large proportion of cellulose. Thus an efficient cell disruption process is required to rupture the cell wall. The conventional downstream processing methods which typically involve several unit operational steps such as disruption, isolation, extraction, concentration and purification are energy-intensive and costly. To reduce the overall cost and establish a feasible technology for the success of the large-scale production, microalgal industry today demands a more cost-effective and eco-friendly technique in downstream processing. One of the main challenges to extract the proteins from microalgae is the presence of rigid cell wall. This study aims to provide some guidance on the selection of the efficient solvent to facilitate the proteins released during the cell disruption process. The effects of solvent types such as methanol, ethanol, 1-propanol and water in rupturing the microalgae cell wall were studied. It is interesting to know that water is the most effective solvent to recover proteins from microalgae and the cost is cheapest among all other solvents.

Keywords: green, microalgae, protein, solvents

Procedia PDF Downloads 227

Authors: Narin Salehiyan, Ramin Ghasemi Shayan

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The largest genus of plant viruses, the potyvirus, is responsible for significant crop losses. Potyviruses are aphid sent in a nonpersistent way, and some of them are likewise seed communicated. As significant microorganisms, potyviruses are substantially more examined than other plant infections having a place with different genera, and their review covers numerous parts of plant virology, like utilitarian portrayal of viral proteins, sub-atomic communication with hosts and vectors, structure, scientific classification, development, the study of disease transmission, and determination. Biotechnological utilizations of potyviruses are likewise being investigated. During this last ten years, significant advances have been made in the comprehension of the sub-atomic science of these infections and the elements of their different proteins. Potyvirus multiplication, movement, and transmission, as well as potyvirus/plant compatible interactions, including pathogenicity and symptom determinants, are updated following a general overview of the family Potyviridae and the potyviral proteins. it end the survey giving data on biotechnological uses of potyviruses.

Keywords: virology, poty, virus, genome, genetic

Procedia PDF Downloads 41

Authors: G. Carrero, C. Contreras, M. J. Hendzel

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Linker histones or histones H1 are highly mobile nuclear proteins that regulate the organization of chromatin and limit DNA accessibility by binding to the chromatin structure (DNA and associated proteins). It is known that this binding process is driven by both slow (strong binding) and rapid (weak binding) interactions. However, the exact binding mechanism has not been fully described. Moreover, the existing models only account for one type of bound population that does not distinguish explicitly between the weakly and strongly bound proteins. Thus, we propose different systems of reaction-diffusion equations to describe explicitly the rapid and slow interactions during a FRAP (Fluorescence Recovery After Photobleaching) experiment. We perform a model comparison analysis to characterize the binding mechanism of histone H1 and provide new meaningful biophysical information on the kinetics of histone H1.

Keywords: FRAP (Fluorescence Recovery After Photobleaching), histone H1, histone H1 binding kinetics, linker histone, reaction-diffusion equation

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Authors: Mahsa Jalili, Trude Johansen, Signe Dille Lovmo, Turid Rustad, Rolf Erik Olsen, Atle M. Bones

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The quality of fish muscle is a key factor in fish industry, and dietary ingredients can influence fish quality. The aim of this study was to examine the impact of krill meal, soybean meal, Bactocell® and butyrate fortified feeds and control diet on characteristics of salmon fillet. Thirty Atlantic salmon (6 per each group) were farmed for 12 weeks. All the fish were killed and frozen immediately. The white muscle from top posterior part of dorsal fin was dissected to analyze fat content, carotenoid content, content of water-soluble and salt-soluble proteins, cathepsin B and cathepsin B-L activities. ANOVA test was used to analyze mean and standard error of mean values at 0.05 significance level. There were significant difference in cathepsin B activity, water-soluble proteins and salt-soluble proteins (p-value= 0.005, 0.009 and 0.002). The mean values of other factors were not significantly different among the groups. Cathepsin B activity was higher in soymeal group. Water-soluble proteins were reported higher in soy meal and krill groups and salt-soluble proteins were significantly higher in soy meal and butyrate rich diets. Although soy meal has proven effect on enteritis, it results in higher percentage of protein in fillets. On the other hand, this feeding may have role in textural deterioration of fillets owing to higher values of endogenous cathepsin B in soymeal group.

Keywords: aquaculture, food quality, Krill protein extract, prebiotics, probiotics, Salmo salar, soy

Procedia PDF Downloads 177

Authors: R. Gounina-Allouane, N. Ouali, F. Z. Berrabah, A. Bentaleb

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For a long time, the Gram-positive spore-forming bacteria Bacillus thuringiensis (Bt) has been widely used in biological control against devastating and disease vectors insects. This is due to the insecticidal activity of its crystalline parasporal inclusion (crystals) predominantly comprised of one or more proteins (Cry and Cyt proteins) also called δ-endotoxins, produced during sporulation. The shape and composition of Bt crystals vary among strains and crystalline proteins are extremely varied (more than 475 cry gene were discovered). The insecticidal activity of Bt crystals is very well studied, thus their insecticidal mode of action is well established, however, their antimicrobial effect is largely unknown. The lack of data on the antimicrobial effect of crystalline proteins of Bt and the need for searching new antimicrobial molecules encouraged us to carried out this study. The antibacterial effect of δ-endotoxines produced by two Bt stains; a strain isolated from soil at northern of Algeria (Bt 7.2.B), and a strain isolated from a bioinsecticide (Bacillus thuringiensis var aizawai), activated by proteolysis, was assayed on clinical bacterial strains and ATCC collection ones respectively. Gram positive and negative clinical bacterial strains (Escherichia coli, Klebsiella pneumonaie, Pseudomonas aeruginosa, Staphylococcus aureus) were sensitive to activated Bt 72B endotoxins. Similarly, bacterial strains from ATCC collection (Escherichia coli ATCC 25922, Pseudomonas aerugenosa ATCC 27853, Staphylococcus aureus ATCC 25923) were sensitive to activated B. thuringiensis var aizawai δ-endotoxines. The activated δ-endotoxins were separated by SDS-PAGE.

Keywords: Bacillus thuringiensis, crystals, cry proteins, δ-endotoxins, antibacterial activity

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Authors: C. Busuioc, S. I. Jinga, E. Pavel

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The continuous need of more non-volatile memories with a higher storage capacity, smaller dimensions and weight, as well as lower costs, has led to the exploration of optical lithography on active media, as well as patterned magnetic composites. In this context, optical lithography is a technique that can provide a significant decrease of the information bit size to the nanometric scale. However, there are some restrictions that arise from the need of breaking the optical diffraction limit. Major achievements have been obtained by employing a vitoceramic material as active medium and a laser beam operated at low power for the direct writing procedure. Thus, optical discs with ultra-high density were fabricated by a conventional melt-quenching method starting from analytical purity reagents. They were subsequently used for 3D recording based on their photosensitive features. Naturally, the next step consists in the elucidation of the composition and structure of the active centers, in correlation with the use of silver and rare-earth compounds for the synthesis of the optical supports. This has been accomplished by modern characterization methods, namely transmission electron microscopy coupled with selected area electron diffraction, scanning transmission electron microscopy and electron energy loss spectroscopy. The influence of laser diode parameters, silver concentration and fluorescent compounds formation on the writing process and final material properties was investigated. The results indicate performances in terms of capacity with two order of magnitude higher than other reported information storage systems. Moreover, the fluorescent photosensitive vitroceramics may be integrated in other applications which appeal to nanofabrication as the driving force in electronics and photonics fields.

Keywords: data storage, fluorescent compounds, laser writing, vitroceramics

Procedia PDF Downloads 206

Authors: Laura M. Hovsepyan, Gayane S. Ghazaryan, Hasmik V. Zanginyan

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Hypertension (HD) is the most common cardiovascular pathology that causes disability and mortality in the working population. Most often, heart failure (HF), which is based on myocardial remodeling, leads to death in hypertension. Recently, endothelial dysfunction (EDF) or a violation of the functional state of the vascular endothelium has been assigned a significant role in the structural changes in the myocardium and the occurrence of heart failure in patients with hypertension. It has now been established that tissues affected by inflammation form increased amounts of superoxide radical and NO, which play a significant role in the development and pathogenesis of various pathologies. They mediate inflammation, modify proteins and damage nucleic acids. The aim of this work was to study the processes of oxidative modification of proteins (OMP) and the production of nitric oxide in hypertension. In the experimental work, the blood of 30 donors and 33 patients with hypertension was used. For the quantitative determination of OMP products, the based on the reaction of the interaction of oxidized amino acid residues of proteins and 2,4-dinitrophenylhydrazine (DNPH) with the formation of 2,4-dinitrophenylhydrazones, the amount of which was determined spectrophotometrically. The optical density of the formed carbonyl derivatives of dinitrophenylhydrazones was recorded at different wavelengths: 356 nm - aliphatic ketone dinitrophenylhydrazones (KDNPH) of neutral character; 370 nm - aliphatic aldehyde dinirophenylhydrazones (ADNPH) of neutral character; 430 nm - aliphatic KDNFG of the main character; 530 nm - basic aliphatic ADNPH. Nitric oxide was determined by photometry using Grace's solution. Adsorption was measured on a Thermo Scientific Evolution 201 SF at a wavelength of 546 nm. Thus, the results of the studies showed that in patients with arterial hypertension, an increased level of nitric oxide in the blood serum is observed, and there is also a tendency to an increase in the intensity of oxidative modification of proteins at a wavelength of 270 nm and 363 nm, which indicates a statistically significant increase in aliphatic aldehyde and ketone dinitrophenylhydrazones. The increase in the intensity of oxidative modification of blood plasma proteins in the studied patients, revealed by us, actually reflects the general direction of free radical processes and, in particular, the oxidation of proteins throughout the body. A decrease in the activity of the antioxidant system also leads to a violation of protein metabolism. The most important consequence of the oxidative modification of proteins is the inactivation of enzymes.

Keywords: hypertension (HD), oxidative modification of proteins (OMP), nitric oxide (NO), oxidative stress

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Authors: R. Sulakshana, R. Lakshmi

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Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR associate (CRISPR/Cas) is an adaptive immunity system found in bacteria and archaea. It has been modified to serve as a potent gene editing tool. Moreover, it has found widespread use in the field of genome research because of its accessibility and low cost. Several bioinformatics methods have been created to aid in the construction of specific single guide RNA (sgRNA), which is highly active and crucial to CRISPR/Cas performance. Various Cas proteins, including Cas1, Cas2, Cas9, and Cas12, have been used to create genome engineering tools because of their programmable sequence specificity. Class 1 and 2 CRISPR/Cas systems, as well as the processes of all known Cas proteins (including Cas9 and Cas12), are discussed in this review paper. In addition, the various CRISPR methodologies and their tools so far discovered are discussed. Finally, the challenges and issues in the CRISPR system along with future works, are presented.

Keywords: gene editing tool, Cas proteins, CRISPR, guideRNA, programmable sequence

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Authors: Irina Veselova, Maria Makedonskaya, Olga Eremina, Alexandr Sidorov, Eugene Goodilin, Tatyana Shekhovtsova

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Such catecholamines as dopamine, norepinephrine, and epinephrine are the principal neurotransmitters in the sympathetic nervous system. Catecholamines and their metabolites are considered to be important markers of socially significant diseases such as atherosclerosis, diabetes, coronary heart disease, carcinogenesis, Alzheimer's and Parkinson's diseases. Currently, neurotransmitters can be studied via electrochemical and chromatographic techniques that allow their characterizing and quantification, although these techniques can only provide crude spatial information. Besides, the difficulty of catecholamine determination in biological materials is associated with their low normal concentrations (~ 1 nM) in biomaterials, which may become even one more order lower because of some disorders. In addition, in blood they are rapidly oxidized by monoaminooxidases from thrombocytes and, for this reason, the determination of neurotransmitter metabolism indicators in an organism should be very rapid (15—30 min), especially in critical states. Unfortunately, modern instrumental analysis does not offer a complex solution of this problem: despite its high sensitivity and selectivity, HPLC-MS cannot provide sufficiently rapid analysis, while enzymatic biosensors and immunoassays for the determination of the considered analytes lack sufficient sensitivity and reproducibility. Fluorescent and SERS-sensors remain a compelling technology for approaching the general problem of selective neurotransmitter detection. In recent years, a number of catecholamine sensors have been reported including RNA aptamers, fluorescent ribonucleopeptide (RNP) complexes, and boronic acid based synthetic receptors and the sensor operated in a turn-off mode. In this work we present the fluorescent and SERS turn-on sensor systems based on the bio- or chemorecognizing nanostructured films {chitosan/collagen-Tb/Eu/Cu-nanoparticles-indicator reagents} that provide the selective recognition, visualization, and sensing of the above mentioned catecholamines on the level of nanomolar concentrations in biomaterials (cell cultures, tissue etc.). We have (1) developed optically transparent porous films and gels of chitosan/collagen; (2) ensured functionalization of the surface by molecules-'recognizers' (by impregnation and immobilization of components of the indicator systems: biorecognizing and auxiliary reagents); (3) performed computer simulation for theoretical prediction and interpretation of some properties of the developed materials and obtained analytical signals in biomaterials. We are grateful for the financial support of this research from Russian Foundation for Basic Research (grants no. 15-03-05064 a, and 15-29-01330 ofi_m).

Keywords: biomaterials, fluorescent and SERS-recognition, neurotransmitters, solid-phase turn-on sensor system

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Authors: Parisa Farzaneh, Azade Harati

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Mushrooms, or macro-fungi, as an important superfood, contain many bioactive compounds, particularly bio-peptides. In this research, mushroom proteins were extracted by buffer or buffer plus salt (0.15 M), along with an ultrasound bath to extract the intercellular protein. As a result, the highest amount of proteins in mushrooms were categorized into albumin. Proteins were also hydrolyzed and changed into peptides through endogenous and exogenous proteases, including gastrointestinal enzymes. The potency of endogenous proteases was also higher in Agaricus bisporus than Terfezia claveryi, as their activity ended at 75 for 15 min. The blanching process, endogenous enzymes, the mixture of gastrointestinal enzymes (pepsin-trypsin-α-chymotrypsin or trypsin- α-chymotrypsin) produced the different antioxidant and antibacterial hydrolysates. The peptide fractions produced with different cut-off ultrafilters also had various levels of radical scavenging, lipid peroxidation inhibition, and antibacterial activities. The bio-peptides with superior bioactivities (less than 3 kD of T. claveryi) were resistant to various environmental conditions (pH and temperatures). Therefore, they are good options to be added to nutraceutical and pharmaceutical preparations or functional foods, even during processing.

Keywords: bio-peptide, mushrooms, gastrointestinal enzymes, bioactivity

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Authors: Mayuva Youngsabanant, Suphaphorn Rabiab, Hatairuk Tungkasen, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The porcine follicular fluid proteins (pFF) of healthy small size ovarian follicles (1-3 mm in diameters) of Large White pig ovaries were collected by sterile technique. They were used for testing the effect on cell viability and characterization of Vero cell lines using MTT assay. Two hundred microliter of round shape Vero cell lines were culture in 96 well plates with DMEM for 24 h. After that, they were attachment to substrate and some changed into fibroblast shape and spread over the surface after culture for 48 h. Then, Vero cell lines were treated with pFF at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins/mL for 24 h. Yields of the best results were analyzed by using one-way ANOVA. MTT assay reviewed an increasing in percentage of viability of Vero cell lines indicated that at concentration of 400-600 µg proteins/mL showed higher percentage of viability (115.64 ± 6.95, 106.91 ± 5.27 and 116.73 ± 20.15) than control group. They were significantly different from the control group (p < 0.05) but lower than the positive control group (DMEM with 10% heat treated fetal bovine serum). Cell lines showed normal character in fibroblast elongate shape after treated with pFF except in high concentration of pFF. This result implies that pFF of small size ovarian follicle at concentration of 400-600 µg proteins/mL could be optimized concentration for using as a supplement in Vero cell line culture medium to promote cell viability instead of growth hormone from fetal bovine serum. This merit could be applied in other cell biotechnology researches. Acknowledgements: This work was funded by a grant from Silpakorn University and Faculty of Science, Silpakorn University, Thailand.

Keywords: cell viability, porcine follicular fluid, MTT assay, Vero cell line

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Authors: Ahmed S. Eissa

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Whey represents about 85-95% of the milk volume and about 55% of milk nutrients. Whey proteins are of special importance in formulated foods due to their rich nutritional and functional benefits. Whey proteins form large polymers upon heating to a temperature greater than the denaturation temperature. Hydrophobic interactions play an important role in building whey protein polymers. In this study, dissociation of hydrophobic interactions of whey protein polymers was done by adding Sodium Dodecyl Sulphonate (SDS). At low SDS concentrations, protein polymers were dissociated to smaller chains, as revealed by dilution solution viscometry (DSV). Interestingly, at higher SDS concentrations, polymer molecules got larger in size. Intrinsic viscosity was increased to many folds when raising the SDS concentration from 0.5% to 2%. Complex molecular arrangement leads to the formation of larger macromolecules, due to micelle formation. The study opens a venue for manipulating and enhancing whey protein functional properties by manipulating the hydrophobic interactions.

Keywords: whey proteins, hydrophobic interactions, SDS

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Authors: Shveta Bathla, Preeti Rawat, Sudarshan Kumar, Rubina Baithalu, Jogender Singh Rana, Tushar Kumar Mohanty, Ashok Kumar Mohanty

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Pregnancy is a complex process which includes series of events such as fertilization, formation of blastocyst, implantation of embryo, placental formation and development of fetus. The success of these events depends on various interactions which are synchronized by endocrine interaction between a receptive dam and competent embryo. These interactions lead to change in expression of hormones and proteins. But till date no protein biomarker is available which can be used to detect successful completion of these events. We employed quantitative proteomics approach to develop putative serological biomarker which has diagnostic applicability for early detection of pregnancy in cows. For this study, sera were collected from control (non-pregnant, n=6) and pregnant animals on successive days of pregnancy (7, 19, 45, n=6). The sera were subjected to depletion for removal of albumin using Norgen depletion kit. The tryptic peptides were labeled with iTRAQ. The peptides were pooled and fractionated using bRPLC over 80 min gradient. Then 12 fractions were injected to nLC for identification and quantitation in DDA mode using ESI. Identification using Mascot search revealed 2056 proteins out of which 352 proteins were differentially expressed. Twenty proteins were upregulated and twelve proteins were down-regulated with fold change > 1.5 and < 0.6 respectively (p < 0.05). The gene ontology studies of DEPs using Panther software revealed that majority of proteins are actively involved in catalytic activities, binding and enzyme regulatory activities. The DEP'S such as NF2, MAPK, GRIPI, UGT1A1, PARP, CD68 were further subjected to pathway analysis using KEGG and Cytoscape plugin Cluego that showed involvement of proteins in successful implantation, maintenance of pluripotency, regulation of luteal function, differentiation of endometrial macrophages, protection from oxidative stress and developmental pathways such as Hippo. Further efforts are continuing for targeted proteomics, western blot to validate potential biomarkers and development of diagnostic kit for early pregnancy diagnosis in cows.

Keywords: bRPLC, Cluego, ESI, iTRAQ, KEGG, Panther

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Authors: Fengmei Li, Li Xu, Guoliang Xia

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

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

Procedia PDF Downloads 326

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

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

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

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

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

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

Procedia PDF Downloads 346

Authors: Mayuva Youngsabanant-Areekijseree, Chanikarn Srinark, S. Sengsai, Mayuree Pumipaiboon

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Our project was aimed at morphology of oocytes, follicle cells and follicular fluid proteins study of Large White pig (at local slaughter house in Nakhon Pathom Province). The porcine oocytes and follicular fluid of healthy small follicles (1-2 mm), medium follicles (3-6 mm in diameters) and large follicles (7-8 mm and 10 mm in diameter) were aspirated and collected from the ovary by sterile technique. Then, the oocytes and the follicle cells were separated from the fluid. The oocytes were round shape and surrounded by zona pellucida with numerous layers of cumulus cells. Based on the number of cumulus cell layers surrounding oocytes, the oocytes were classified into 5 types, which were intact-, multi-, partial-cumulus layer oocyte, completely denuded oocyte and degenerative oocyte. The collected oocytes showed high percentages of intact- and multi- cumulus cell layers in the small follicles (53.48%) medium follicles (56.94%) and large follicles (56.52%) which have high potential to develop into mature oocytes in vitro. Proteins from follicular fluid of 3 size follicles were separated by SDS-PAGE and LC/MS/MS. The molecular weight of follicular fluid proteins from the small follicles were 24, 60-65, 79, 110, 140, 160, and > 220 kDa. Meanwhile, the follicular fluid protein from medium and large follicle contained 52, 65, 79, 90, 110, 120, 160, 190 and > 220 kDa. Almost all proteins played important roles in promoting and regulating growth and development of oocytes and ovulation. This finding was an initial tool for in vitro testing and applied biotechnology research. Acknowledgements: The project was funded by a grant from Silpakorn University Research & Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: follicular fluid protein, LC/MS/MS, porcine oocyte, SDS-PAGE, reproductive biology

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Authors: Waqas Shafqat Chattha, Muhammad Iqbal, Amir Shakeel

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Transcriptional factors are proteins that play a vital role in regulating the transcription of target genes in different biological processes and are being widely studied in different plant species. In the current era of genomics, plant genomes sequencing has directed to the genome-wide identification, analyses and categorization of diverse transcription factor families and hence provide key insights into their structural as well as functional diversity. The DNA-binding with One Finger (DOF) proteins belongs to C2-C2-type zinc finger protein family. DOF proteins are plant-specific transcription factors implicated in diverse functions including seed maturation and germination, phytohormone signalling, light-mediated gene regulation, cotton-fiber elongation and responses of the plant to biotic as well as abiotic stresses. In this context, a genome-wide in-silico analysis of DOF TF family in diploid cotton species i.e. Gossypium raimondii has enabled us to identify 55 non-redundant genes encoding DOF proteins renamed as GrDofs (Gossypium raimondii Dof). Gene distribution studies have shown that all of the GrDof genes are unevenly distributed across 12 out of 13 G. raimondii chromosomes. The gene structure analysis illustrated that 34 out of 55 GrDof genes are intron-less while remaining 21 genes have a single intron. Protein sequence-based phylogenetic analysis of putative 55 GrDOFs has divided these proteins into 5 major groups with various paralogous gene pairs. Molecular evolutionary studies aided with the conserved domain as well as gene structure analysis suggested that segmental duplications were the principal contributors for the expansion of Dof genes in G. raimondii.

Keywords: diploid cotton , G. raimondii, phylogenetic analysis, transcription factor

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Authors: Salwa Karboune, Amanda Waglay

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Recent studies indicate that health-promoting functional ingredients and nutraceuticals can help support and improve the overall public health, which is timely given the aging of the population and the increasing cost of health care. The development of novel ‘natural’ functional ingredients is increasingly challenging. Biocatalysis offers powerful approaches to achieve this goal. Our recent research has been focusing on the development of innovative biocatalytic approaches towards the isolation of protein isolates from potato by-products and the generation of peptides. Potato is a vegetable whose high-quality proteins are underestimated. In addition to their high proportion in the essential amino acids, potato proteins possess angiotensin-converting enzyme-inhibitory potency, an ability to reduce plasma triglycerides associated with a reduced risk of atherosclerosis, and stimulate the release of the appetite regulating hormone CCK. Potato proteins have long been considered not economically feasible due to the low protein content (27% dry matter) found in tuber (Solanum tuberosum). However, potatoes rank the second largest protein supplying crop grown per hectare following wheat. Potato proteins include patatin (40-45 kDa), protease inhibitors (5-25 kDa), and various high MW proteins. Non-destructive techniques for the extraction of proteins from potato pulp and for the generation of peptides are needed in order to minimize functional losses and enhance quality. A promising approach for isolating the potato proteins was developed, which involves the use of multi-enzymatic systems containing selected glycosyl hydrolase enzymes that synergistically work to open the plant cell wall network. This enzymatic approach is advantageous due to: (1) the use of milder reaction conditions, (2) the high selectivity and specificity of enzymes, (3) the low cost and (4) the ability to market natural ingredients. Another major benefit to this enzymatic approach is the elimination of a costly purification step; indeed, these multi-enzymatic systems have the ability to isolate proteins, while fractionating them due to their specificity and selectivity with minimal proteolytic activities. The isolated proteins were used for the enzymatic generation of active peptides. In addition, they were applied into a reduced gluten cookie formulation as consumers are putting a high demand for easy ready to eat snack foods, with high nutritional quality and limited to no gluten incorporation. The addition of potato protein significantly improved the textural hardness of reduced gluten cookies, more comparable to wheat flour alone. The presentation will focus on our recent ‘proof-of principle’ results illustrating the feasibility and the efficiency of new biocatalytic processes for the production of innovative functional food ingredients, from potato by-products, whose potential health benefits are increasingly being recognized.

Keywords: biocatalytic approaches, functional ingredients, potato proteins, peptides

Procedia PDF Downloads 353

Authors: Arredondo Valdés Roberto, Hernández Castillo Francisco Daniel, Laredo Alcalá Elan Iñaky, Gonzalez Gallegos Esmeralda, Castro Del Angel Epifanio

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The common bean or Phaseolus vulgaris L. is the most important food legume for direct consumption in the world. Fusarium dry rot in the major fungus disease affects Phaseolus vulgaris L, after planting. In another hand, Rhizoctonia can be found on all underground parts of the plant and various times during the growing season. In recent years, the world has conducted studies about the use of natural products as substitutes for herbicides and pesticides, because of possible ecological and economic benefits. Plants respond to fungal invasion by activating defense responses associated with accumulation of several enzymes and inhibitors, which prevent pathogen infection. This study focused on the role of proteins, peroxidase (POD), phenylalanine ammonia lyase (PAL), in imparting resistance to soft rot pathogens by applied different bacterial consortium, formulated and provided by Biofertilizantes de Méxicanos industries, analyzing the enzyme activity at different times of application (6 h, 12 h and 24 h). The resistance of these treatments was correlated with high POD and PAL enzyme activity as well as increased concentrations of proteins. These findings show that PAL, POD and synthesis of proteins play a role in imparting resistance to Phaseolus vulgaris L. soft rot infection by Fusarium and Rhizoctonia.

Keywords: fusarium, peroxidase, phenylalanine ammonia lyase, rhizoctonia

Procedia PDF Downloads 319