Search results for: single cell protein (SCP)
9068 In vitro Modeling of Aniridia-Related Keratopathy by the Use of Crispr/Cas9 on Limbal Epithelial Cells and Rescue
Authors: Daniel Aberdam
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Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by reduced visual acuity. Patients have also progressive disorders including cataract, glaucoma and corneal abnormalities making their condition very challenging to manage. Aniridia-related keratopathy (ARK), caused by a combination of factors including limbal stem-cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface, affects up to 95% of patients. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub-epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. Although animal models partially recapitulate this disease, there is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9 technology) to introduce a nonsense mutation found in patients into one allele of the PAX6 gene into limbal stem cells. Resulting mutated clones, expressing half of the amount of PAX6 protein and thus representative of haploinsufficiency were further characterized. Sequencing analysis showed that no off-target mutations were induced. The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also reduced. Remarkably, addition of soluble recombinant PAX6 protein into the culture medium was sufficient to activate endogenous PAX6 gene and, as a consequence, rescue the phenotype. It strongly suggests that our in vitro model recapitulates well the epithelial defect and becomes a powerful tool to identify drugs that could rescue the corneal defect in patients. Furthermore, we demonstrate that the homeotic transcription factor Pax6 is able to be uptake naturally by recipient cells to function into the nucleus.Keywords: Pax6, crispr/cas9, limbal stem cells, aniridia, gene therapy
Procedia PDF Downloads 1989067 Physicochemical and Functional Characteristics of Hemp Protein Isolate
Authors: El-Sohaimy Sobhy A., Androsova Natalia, Toshev Abuvali Djabarovec
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The conditions of the isolation of proteins from the hemp seeds were optimized in the current work. Moreover, the physicochemical and functional properties of hemp protein isolate were evaluated for its potential application in food manufacturing. The elastin protein is the most predominant protein in the protein profile with a molecular weight of 58.1 KDa, besides albumin, with a molecular weight of 31.5 KDa. The FTIR spectrum detected the absorption peaks of the amide I in 1750 and 1600 cm⁻¹, which pointed to C=O stretching while N-H was stretching at 1650-1580 cm⁻¹. The peak at 3250 was related to N-H stretching of primary aliphatic amine (3400-3300 cm⁻¹), and the N-H stretching for secondary (II) amine appeared at 3350-3310 cm⁻¹. Hemp protein isolate (HPI) was showed high content of arginine (15.52 g/100 g), phenylalanine+tyrosine (9.63 g/100 g), methionine + cysteine (5.49 g/100 g), leucine + isoleucine (5.21 g/100 g) and valine (4.53 g/100 g). It contains a moderate level of threonine (3.29 g/100 g) and lysine (2.50 g/100 g), with the limiting amino acid being a tryptophan (0.22 g/100 g HPI). HPI showed high water-holding capacity (4.5 ± 2.95 ml/g protein) and oil holding capacity (2.33 ± 1.88 ml/g) values. The foaming capacity of HPI was increased with increasing the pH values to reach the maximum value at pH 11 (67.23±3.20 %). The highest emulsion ability index of HPI was noted at pH 9 (91.3±2.57 m2/g) with low stability (19.15±2.03).Keywords: Cannabis sativa ssp., protein isolate, isolation conditions, amino acid composition, chemical properties, functional properties
Procedia PDF Downloads 1689066 Physicochemical and Antioxidative Characteristics of Black Bean Protein Hydrolysates Obtained from Different Enzymes
Authors: Zhaojun Zheng, Yuanfa Liu, Jiaxin Li, Jinwei Li, Yong-jiang Xu, Chen Cao
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Black bean is an excellent protein source for preparing hydrolysates, which attract much attention due to their biological activity. The objective of this study was to characterize the physicochemical and antioxidant properties of black bean protein, hydrolyzed by ficin, bromelain or alcalase until 300 min of hydrolysis. Results showed that bromelain and alcalase hydrolysates possessed a higher degree of hydrolysis (DH) than that of ficin, thereby presenting different ultraviolet absorption, fluorescence intensity, and circular dichroism. Moreover, all hydrolysates possessed the capacity to scavenge DPPH radical with the lowest IC₅₀ of 21.11 µg/mL, as well as to chelate ferrous ion (Fe²⁺) with the IC₅₀ values ranging from 6.82 to 30.68 µg/mL. Intriguingly, the oxidation of linoleic acid, sunflower oil, and sunflower oil-in-water emulsion was remarkedly retarded by the three selected protein hydrolysates, especially by bromelain-treated protein hydrolysate, which might attribute to their high hydrophobicity and emulsifying properties. These findings can provide strong support for black bean protein hydrolysates to be employed in food products acting as natural antioxidant alternatives.Keywords: antioxidant activity, black bean protein hydrolysate, emulsion physicochemical properties, sunflower oil
Procedia PDF Downloads 1329065 Experimental Investigation of Performance Anode Side of PEM Fuel Cell with Spin Method Coated with YSZ+SDC
Authors: Gürol Önal, Kevser Dinçer, Salih Yayla
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In this study, performance of proton exchange membrane PEM fuel cell was experimentally investigated. Coating on the anode side of the PEM fuel cell was accomplished with the spin method by using YSZ+SDC. A solution having 0,1 gr YttriaStabilized Zirconia (YSZ) + 0,1 Samarium-Doped Ceria (SDC) + 10 mL methanol was prepared. This solution was taken out and filled into a micro-pipette. Then the anode side of PEM fuel cell was coated with YSZ+ SDC by using spin method. In the experimental study, current, voltage and power performances before and after coating were recorded and then compared to each other. It was found that the efficiency of PEM fuel cell increases after the coating with YSZ+SDC.Keywords: fuel cell, Polymer Electrolyte Membrane (PEM), membrane, spin method
Procedia PDF Downloads 5479064 Protein Stabilized Foam Structures as Protective Carrier Systems during Microwave Drying of Probiotics
Authors: Jannika Dombrowski, Sabine Ambros, Ulrich Kulozik
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Due to the increasing popularity of healthy products, probiotics are still of rising importance in food manufacturing. With the aim to amplify the field of probiotic application to non-chilled products, the cultures have to be preserved by drying. Microwave drying has proved to be a suitable technique to achieve relatively high survival rates, resulting from drying at gentle temperatures, among others. However, diffusion limitation due to compaction of cell suspension during drying can prolong drying times as well as deteriorate product properties (grindability, rehydration performance). Therefore, we aimed to embed probiotics in an aerated matrix of whey proteins (surfactants) and di-/polysaccharides (foam stabilization, probiotic protection) during drying. As a result of the manifold increased inner surface of the cell suspension, drying performance was enhanced significantly as compared to non-foamed suspensions. This work comprises investigations on suitable foam matrices, being stable under vacuum (variation of protein concentration, type and concentration of di-/polysaccharide) as well as development of an applicable microwave drying process in terms of microwave power, chamber pressure and maximum product temperatures. Performed analyses included foam characteristics (overrun, drainage, firmness, bubble sizes), and properties of the dried cultures (survival, activity). In addition, efficiency of the drying process was evaluated.Keywords: foam structure, microwave drying, polysaccharides, probiotics
Procedia PDF Downloads 2529063 Development of Protein-based Emulsion Gels For Food Structuring
Authors: Baigts-Allende Diana, Klojdová Iveta, Kozlu Ali, Metri-ojeda Jorge
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Emulsion gels are constituted by a colloidal system (emulsion) stabilized by a polymeric gel matrix. These systems are more homogeneous and stable than conventional emulsions and can behave as either gel-like or soft-solid. Protein-based emulsion gels (PEG) have been used as carrier systems of bioactive compounds and as food structuring to improve the texture and consistency, mainly in producing low-fat content products. This work studied the effect of protein: polysaccharide ratio 0.75:1.25, 1:1, and 1.25:0.75 (levels -1, 0, and +1) and pH values (2-9) on the stability of protein-based emulsion gels using soy protein isolate and sodium alginate. Protein emulsion capacity was enhaced with increased pH (6,7,8 and 9) compared to acid pH values. The smaller particle size for PEG was at pH 9 (~23µm); however, with increasing protein ratio (level +1), higher particle size was observed (~23µm). The same trend was observed for rheological measurements; the consistency index (K) increased at pH 9 for level -1 (1.17) in comparison to level +1 (0.45). The studied PEG showed good thermal stability at neutral and pH 9 (~98 %) for all biopolymer ratios. Optimal conditions in pH and biopolymer ratios were determined for PEG using soy protein and sodium alginate ingredients with potential use in elaborating stable systems for broad application in the food sector.Keywords: emulsion gels, food structuring, biopolymers, food systems
Procedia PDF Downloads 689062 Harnessing of Electricity from Distillery Effluent and Simultaneous Effluent Treatment by Microbial Fuel Cell
Authors: Hanish Mohammed, C. H. Muthukumar Muthuchamy
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The advancement in the science and technology has made it possible to convert electrical energy into any desired form. It has given electrical energy a place of pride in the modern world. The survival of industrial undertakings and our social structure depends primarily upon low cost and uninterrupted supply of electrical energy. Microbial fuel cell (MFC) is a promising and emerging technique for sustainable bioelectricity generation and wastewater treatment. MFCs are devices which are capable of converting organic matter to electricity/hydrogen with help of microorganisms. Different kinds of wastewater could be used in this technique, distillery effluent is one of the most troublesome and complex and strong organic effluent with high chemical oxygen demand of 1,53,846 mg/L. A single cell MFC unit was designed and fabricated for the distillery effluent treatment and to generate electricity. Due to the high COD value of the distillery effluent helped in the production of energy for 74 days. The highest voltage got from the fuel cell is 206 mV on the 30th day. A maximum power density obtained from the MFC was 9.8 mW, treatment efficiency was evaluated in terms of COD removal and other parameters. COD removal efficiencies were around 68.5 % and other parameters such as Total Hardness (81.5%), turbidity (70 %), chloride (66%), phosphate (79.5%), Nitrate (77%) and sulphate (71%). MFC using distillery effluent is a promising new unexplored substrate for the power generation and sustainable treatment technique through harnessing of bioelectricity.Keywords: microbial fuel cell (MFC), bioelectricity, distillery effluent, wastewater treatment
Procedia PDF Downloads 2039061 The Current Use of Cell Phone in Education
Authors: Elham A. Alsadoon, Hamadah B. Alsadoon
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Educators try to design learning environments that are preferred by their students. With the wide-spread adoption of cell phones surpassing any other technology, educators should not fail to invest in the power of such technology. This study aimed to explore the current use of cell phones in education among Saudi students in Saudi universities and how students perceive such use. Data was collected from 237 students at King Saud University. Descriptive analysis was used to analyze the data. A T-test for independent groups was used to examine whether there was a significant difference between males and females in their perception of using cell phones in education. Findings suggested that students have a positive attitude toward the use of cell phones in education. The most accepted use was for sending notification to students, which has already been experienced through the Twasel system provided by King Saud University. This electronic system allows instructors to easily send any SMS or email to their students. The use of cell phone applications came in the second rank of using cell phones in education. Students have already experienced the benefits of having these applications handy wherever they go. On the other hand, they did not perceive using cell phones for assessment as practical educational usage. No gender difference was detected in terms of students’ perceptions toward using cell phones in education.Keywords: cell phone, mobile learning, educational sciences, education
Procedia PDF Downloads 4079060 Detection of Alzheimer's Protein on Nano Designed Polymer Surfaces in Water and Artificial Saliva
Authors: Sevde Altuntas, Fatih Buyukserin
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Alzheimer’s disease is responsible for irreversible neural damage of brain parts. One of the disease markers is Amyloid-β 1-42 protein that accumulates in the brain in the form plaques. The basic problem for detection of the protein is the low amount of protein that cannot be detected properly in body liquids such as blood, saliva or urine. To solve this problem, tests like ELISA or PCR are proposed which are expensive, require specialized personnel and can contain complex protocols. Therefore, Surface-enhanced Raman Spectroscopy (SERS) a good candidate for detection of Amyloid-β 1-42 protein. Because the spectroscopic technique can potentially allow even single molecule detection from liquid and solid surfaces. Besides SERS signal can be improved by using nanopattern surface and also is specific to molecules. In this context, our study proposes to fabricate diagnostic test models that utilize Au-coated nanopatterned polycarbonate (PC) surfaces modified with Thioflavin - T to detect low concentrations of Amyloid-β 1-42 protein in water and artificial saliva medium by the enhancement of protein SERS signal. The nanopatterned PC surface that was used to enhance SERS signal was fabricated by using Anodic Alumina Membranes (AAM) as a template. It is possible to produce AAMs with different column structures and varying thicknesses depending on voltage and anodization time. After fabrication process, the pore diameter of AAMs can be arranged with dilute acid solution treatment. In this study, two different columns structures were prepared. After a surface modification to decrease their surface energy, AAMs were treated with PC solution. Following the solvent evaporation, nanopatterned PC films with tunable pillared structures were peeled off from the membrane surface. The PC film was then modified with Au and Thioflavin-T for the detection of Amyloid-β 1-42 protein. The protein detection studies were conducted first in water via this biosensor platform. Same measurements were conducted in artificial saliva to detect the presence of Amyloid Amyloid-β 1-42 protein. SEM, SERS and contact angle measurements were carried out for the characterization of different surfaces and further demonstration of the protein attachment. SERS enhancement factor calculations were also completed via experimental results. As a result, our research group fabricated diagnostic test models that utilize Au-coated nanopatterned polycarbonate (PC) surfaces modified with Thioflavin-T to detect low concentrations of Alzheimer’s Amiloid – β protein in water and artificial saliva medium. This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) Grant No: 214Z167.Keywords: alzheimer, anodic aluminum oxide, nanotopography, surface enhanced Raman spectroscopy
Procedia PDF Downloads 2839059 The Second Generation of Tyrosine Kinase Inhibitor Afatinib Controls Inflammation by Regulating NLRP3 Inflammasome Activation
Authors: Shujun Xie, Shirong Zhang, Shenglin Ma
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Background: Chronic inflammation might lead to many malignancies, and inadequate resolution could play a crucial role in tumor invasion, progression, and metastases. A randomised, double-blind, placebo-controlled trial shows that IL-1β inhibition with canakinumab could reduce incident lung cancer and lung cancer mortality in patients with atherosclerosis. The process and secretion of proinflammatory cytokine IL-1β are controlled by the inflammasome. Here we showed the correlation of the innate immune system and afatinib, a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR) in non-small cell lung cancer. Methods: Murine Bone marrow derived macrophages (BMDMs), peritoneal macrophages (PMs) and THP-1 were used to check the effect of afatinib on the activation of NLRP3 inflammasome. The assembly of NLRP3 inflammasome was check by co-immunoprecipitation of NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC), disuccinimidyl suberate (DSS)-cross link of ASC. Lipopolysaccharide (LPS)-induced sepsis and Alum-induced peritonitis were conducted to confirm that afatinib could inhibit the activation of NLRP3 in vivo. Peripheral blood mononuclear cells (PBMCs) from non-small cell lung cancer (NSCLC) patients before or after taking afatinib were used to check that afatinib inhibits inflammation in NSCLC therapy. Results: Our data showed that afatinib could inhibit the secretion of IL-1β in a dose-dependent manner in macrophage. Moreover, afatinib could inhibit the maturation of IL-1β and caspase-1 without affecting the precursors of IL-1β and caspase-1. Next, we found that afatinib could block the assembly of NLRP3 inflammasome and the ASC speck by blocking the interaction of the sensor protein NLRP3 and the adaptor protein ASC. We also found that afatinib was able to alleviate the LPS-induced sepsis in vivo. Conclusion: Our study found that afatinib could inhibit the activation of NLRP3 inflammasome in macrophage, providing new evidence that afatinib could target the innate immune system to control chronic inflammation. These investigations will provide significant experimental evidence in afatinib as therapeutic drug for non-small cell lung cancer or other tumors and NLRP3-related diseases and will explore new targets for afatinib.Keywords: inflammasome, afatinib, inflammation, tyrosine kinase inhibitor
Procedia PDF Downloads 1129058 ROCK Signaling and Radio Resistance: The Association and the Effect
Authors: P. Annapurna, Cecil Ross, Sudhir Krishna, Sweta Srivastava
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Irradiation plays a pivotal role in cervical cancer treatment, however some tumors exhibit resistance to therapy while some exhibit relapse, due to better repair and enhanced resistance mechanisms operational in their cells. The present study aims to understand the signaling mechanism operational in resistance phenotype and in the present study we report the role of Rho GTPase associated protein kinase (ROCK) signaling in cervical carcinoma radio-resistance. ROCK signaling has been implicated in several tumor progressions and is important for DNA repair. Irradiation of spheroid cultures of SiHa cervical carcinoma derived cell line at 6Gy resulted in generation of resistant cells in vitro which had better clonogenic abilities and formed larger and more colonies, in soft agar colony formation assay, as compared to the non-irradiated cells. These cells also exhibited an enhanced motility phenotype. Cell cycle profiling showed the cells to be blocked in G2M phase with enhanced pCDC2 levels indicating onset of possible DNA repair mechanism. Notably, 3 days post-irradiation, irradiated cells showed increased ROCK2 translocation to the nucleus with enhanced protein expression as compared to the non-irradiated cells. Radio-sensitization of the resistant cells was enhanced using Y27632, an inhibitor to ROCK signaling. The treatment of resistant cells with Y27632 resulted in increased cell death upon further irradiation. This observation has been confirmed using inhibitory antibodies to ROCK1/2. Result show that both ROCK1/2 have a functional contribution in radiation resistance of cervical cancer cells derived from cell lines. Interestingly enrichment of stem like cells (Hoechst negative cells) was also observed upon irradiation and these cells were markedly sensitive to Y27632 treatment. Our results thus suggest the role of ROCK signaling in radio-resistance in cervical carcinoma. Further studies with human biopsies, mice models and mechanistic of ROCK signaling in the context of radio-resistance will clarify the role of this molecule further and allow for therapeutics development.Keywords: cervical carcinoma, radio-resistance, ROCK signaling, cancer treatment
Procedia PDF Downloads 3149057 A Structure-Switching Electrochemical Aptasensor for Rapid, Reagentless and Single-Step, Nanomolar Detection of C-Reactive Protein
Authors: William L. Whitehouse, Louisa H. Y. Lo, Andrew B. Kinghorn, Simon C. C. Shiu, Julian. A. Tanner
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C-reactive protein (CRP) is an acute-phase reactant and sensitive indicator for sepsis and other life-threatening pathologies, including systemic inflammatory response syndrome (SIRS). Currently, clinical turn-around times for established CRP detection methods take between 30 minutes to hours or even days from centralized laboratories. Here, we report the development of an electrochemical biosensor using redox probe-tagged DNA aptamers functionalized onto cheap, commercially available screen-printed electrodes. Binding-induced conformational switching of the CRP-targeting aptamer induces a specific and selective signal-ON event, which enables single-step and reagentless detection of CRP in as little as 1 minute. The aptasensor dynamic range spans 5-1000nM (R=0.97) or 5-500nM (R=0.99) in 50% diluted human serum, with a LOD of 3nM, corresponding to 2-orders of magnitude sensitivity under the clinically relevant cut-off for CRP. The sensor is stable for up to one week and can be reused numerous times, as judged from repeated real-time dosing and dose-response assays. By decoupling binding events from the signal induction mechanism, structure-switching electrochemical aptamer-based sensors (SS-EABs) provide considerable advantages over their adsorption-based counterparts. Our work expands on the retinue of such sensors reported in the literature and is the first instance of an SS-EAB for reagentless CRP detection. We hope this study can inspire further investigations into the suitability of SS-EABs for diagnostics, which will aid translational R&D toward fully realized devices aimed at point-of-care applications or for use more broadly by the public.Keywords: structure-switching, C-reactive protein, electrochemical, biosensor, aptasensor.
Procedia PDF Downloads 539056 High-Throughput Screening and Selection of Electrogenic Microbial Communities Using Single Chamber Microbial Fuel Cells Based on 96-Well Plate Array
Authors: Lukasz Szydlowski, Jiri Ehlich, Igor Goryanin
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We demonstrate a single chamber, 96-well-plated based Microbial Fuel Cell (MFC) with printed, electronic components. This invention is aimed at robust selection of electrogenic microbial community under specific conditions, e.g., electrode potential, pH, nutrient concentration, salt concentration that can be altered within the 96 well plate array. This invention enables robust selection of electrogenic microbial community under the homogeneous reactor, with multiple conditions that can be altered to allow comparative analysis. It can be used as a standalone technique or in conjunction with other selective processes, e.g., flow cytometry, microfluidic-based dielectrophoretic trapping. Mobile conductive elements, like carbon paper, carbon sponge, activated charcoal granules, metal mesh, can be inserted inside to increase the anode surface area in order to collect electrogenic microorganisms and to transfer them into new reactors or for other analytical works. An array of 96-well plate allows this device to be operated by automated pipetting stations.Keywords: bioengineering, electrochemistry, electromicrobiology, microbial fuel cell
Procedia PDF Downloads 1339055 Polymorphisms of STAT5A and DGAT1 Genes and Their Associations with Milk Trait in Egyptian Goats
Authors: Othman Elmahdy Othman
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The objectives of this study were to identify polymorphisms in the STAT5A using Restriction Fragment Length Polymorphism and DGAT1 using Single-Strand Conformation Polymorphism genes among three Egyptian goat breeds (Barki, Zaraibi, and Damascus) as well as investigate the effect of their genotypes on milk composition traits of Zaraibi goats. One hundred and fifty blood samples were collected for DNA extraction, 60 from Zaraibi, 40 from Damascus and 50 from Barki breeds. Fat, protein and lactose percentages were determined in Zaraibi goat milk using an automatic milk analyzer. Two genotypes, CC and CT (for STAT5A) and C-C- and C-C+ (for DGAT1), were identified in the three Egyptian goat breeds with different frequencies. The associations between these genotypes and milk fat, protein and lactose were determined in Zaraibi breed. The results showed that the STAT5A genotypes had significant effects on milk yield, protein, fat and lactose with the superiority of CT genotype over CC. Regarding DGAT1 polymorphism, the result showed the only association between it with milk fat where the animals with C-C+ genotype had greater milk fat than animals possess C-C- genotype. The association of combined genotypes with milk trait declared that the does with heterozygous genotypes for both genes are preferred than does with homozygous genotypes where the animals with CTC-C+ have more milk yield, fat and protein than those with CCC-C- genotype. In conclusion, the result showed that C/T and C-/C+ SNPs of STAT5A and DGAT1 genes respectively may be useful markers for assisted selection programs to improve goat milk compositionKeywords: DGAT1, genetic polymorphism, milk trait, STAT5A
Procedia PDF Downloads 1469054 Parental Diet Effects on Offspring Body Size and Pathogen Resistance in Bactrocera tryoni
Authors: Hue Dinh, Binh Nguyen, Vivian Mendez, Phillip W. Taylor, Fleur Ponton
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Better understanding of how parental diet affects offspring traits is an important ecological and evolutionary question. In this study, we explored how maternal diet influences offspring physiology and resistance to infection using Bactrocera tryoni (Q-fly) as a system model. Female Q-flies were fed one of six single diets varying in their yeast-to-sugar ratio yielding six protein-to-carbohydrate ratios. As controls, we used females that were given a choice between yeast and sugar. Males were reared on a choice diet and allowed to mate with females 14 days post-emergence. Results showed that while maternal diet does not influence offspring developmental time, it has a strong effect on larval body weight. Mother fed either high-protein or high-sugar diet produced larger progeny. By challenging offspring with the bacterium Serratia marcescens, we found that female offspring from mothers fed high-sugar diet survived better the infection compared to those from mothers fed low-sugar diet. In contrast, male offspring produced by mother fed high-protein diet showed better resistance to the infection compared to those produced by mother fed low-protein diet. These results suggested sex-dependent transgenerational effects of maternal nutrition on offspring physiology and immunity.Keywords: bacterial infection, Bactrocera tryoni, maternal diet, offspring, Serretia marcescens
Procedia PDF Downloads 1339053 Development and Characterisation of a Microbioreactor 'Cassette' for Cell Culture Applications
Authors: Nelson Barrientos, Matthew J. Davies, Marco C. Marques, Darren N. Nesbeth, Gary J. Lye, Nicolas Szita
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Microbioreactor technology is making important advances towards its application in cell culture and bioprocess development. In particular, the technology promises flexible and controllable devices capable to perform parallelised experimentation at low cost. Currently, state of the art methods (e.g. optical sensors) allow the accurate monitoring of the microbioreactor operation. In addition, the laminar flow regime encountered in these devices allows more predictive fluid dynamics modelling, improving the control over the soluble, physical and mechanical environment of the cells. This work describes the development and characterisation of a novel microbioreactor cassette system (microbioreactor volume is 150 μL. The volumetric oxygen transfer coefficient (KLa) and mixing time have been characterised to be between 25 to 113 h-1 and 0.5 and 0.1 s, respectively. In addition, the Residence time distribution (RTD) analysis confirms that the reactor operates at well mixed conditions. Finally, Staphylococcus carnosus TM300 growth is demonstrated via batch culture experiments. Future work consists in expanding the optics of the microbioreactor design to include the monitoring of variables such as fluorescent protein expression, among others.Keywords: microbioreactor, cell-culture, fermentation, microfluidics
Procedia PDF Downloads 3999052 Anti-Nutritional Factors, In-Vitro Trypsin, Chymotrypsin and Peptidase Multi Enzyme Protein Digestibility of Some Melon (Egusi) Seeds and Their Protein Isolates
Authors: Joan O. Ogundele, Aladesanmi A. Oshodi, Adekunle I. Amoo
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Abstract In-vitro multi-enzyme protein digestibility (IVMPD) and some anti-nutritional factors (ANF) of five melon (egusi) seed flours (MSF) and their protein isolates (PI) were carried out. Their PI have potentials comparable to that of soya beans. It is important to know the IVMPD and ANF of these protein sources as to ensure their safety when adapted for use as alternate protein sources to substitute for cow milk, which is relatively expensive in Nigeria. Standard methods were used to produce PI of Citrullus colocynthis, Citrullus vulgaris, African Wine Kettle gourd (Lageneria siceraria I), Basket Ball gourd (Lagenaria siceraria II) and Bushel Giant Gourd (Lageneria siceraria III) seeds and to determine the ANF and IVMPD of the MSF and PI unheated and at 37oC. Multi-enzymes used were trypsin, chymotrypsin and peptidase. IVMPD of MSF ranged from (70.67±0.70) % (C. vulgaris) to (72.07± 1.79) % (L.siceraria I) while for their PI ranged from 74.33% (C.vulgaris) to 77.55% (L.siceraria III). IVMPD of the PI were higher than those of MSF. Heating increased IVMPD of MSF with average value of 79.40% and those of PI with average of 84.14%. ANF average in MSF are tannin (0.11mg/g), phytate (0.23%). Differences in IVMPD of MSF and their PI at different temperatures may arise from processing conditions that alter the release of amino acids from proteins by enzymatic processes. ANF in MSF were relatively low, but were found to be lower in the PI, therefor making the PI safer for human consumption as an alternate source of protein.Keywords: Anti-nutrients, Enzymatic protein digestibility, Melon (egusi)., Protein Isolates.
Procedia PDF Downloads 1079051 Structural Evaluation of Cell-Filled Pavement
Authors: Subrat Roy
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This paper describes the findings of a study carried out for evaluating the performance of cell-filled pavement for low volume roads. Details of laboratory investigations and the methodology adopted for construction of cell-filled pavement are presented. The aim of this study is to evaluate the structural behaviour of cement concrete filled cell pavement laid over three different types of subbases (water bound macadam, soil-cement and moorum). A formwork of cells of a thin plastic sheet was used to construct the cell-filled pavements to form flexible, interlocked block pavements. Surface deflections were measured using falling weight deflectometer and benkelman beam methods. Resilient moduli of pavement layers were estimated from the measured deflections. A comparison of deflections obtained from both the methodology is also presented.Keywords: cell-filled pavement, WBM, FWD, Moorum
Procedia PDF Downloads 2919050 Effect of Low Temperature on Structure and RNA Binding of E.coli CspA: A Molecular Dynamics Based Study
Authors: Amit Chaudhary, B. S. Yadav, P. K. Maurya, A. M., S. Srivastava, S. Singh, A. Mani
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Cold shock protein A (CspA) is major cold inducible protein present in Escherichia coli. The protein is involved in stabilizing secondary structure of RNA by working as chaperone during cold temperature. Two RNA binding motifs play key role in the stabilizing activity. This study aimed to investigate implications of low temperature on structure and RNA binding activity of E. coli CspA. Molecular dynamics simulations were performed to compare the stability of the protein at 37°C and 10 °C. The protein was mutated at RNA binding motifs and docked with RNA to assess the stability of both complexes. Results suggest that CspA as well as CspA-RNA complex is more stable at low temperature. It was also confirmed that RNP1 and RNP2 play key role in RNA binding.Keywords: CspA, homology modelling, mutation, molecular dynamics simulation
Procedia PDF Downloads 3669049 Comparison of Inexpensive Cell Disruption Techniques for an Oleaginous Yeast
Authors: Scott Nielsen, Luca Longanesi, Chris Chuck
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Palm oil is obtained from the flesh and kernel of the fruit of oil palms and is the most productive and inexpensive oil crop. The global demand for palm oil is approximately 75 million metric tonnes, a 29% increase in global production of palm oil since 2016. This expansion of oil palm cultivation has resulted in mass deforestation, vast biodiversity destruction and increasing net greenhouse gas emissions. One possible alternative is to produce a saturated oil, similar to palm, from microbes such as oleaginous yeast. The yeasts can be cultured on sugars derived from second-generation sources and do not compete with tropical forests for land. One highly promising oleaginous yeast for this application is Metschnikowia pulcherrima. However, recent techno-economic modeling has shown that cell lysis and standard lipid extraction are major contributors to the cost of the oil. Typical cell disruption techniques to extract either single cell oils or proteins have been based around bead-beating, homogenization and acid lysis. However, these can have a detrimental effect on lipid quality and are energy-intensive. In this study, a vortex separator, which produces high sheer with minimal energy input, was investigated as a potential low energy method of lysing cells. This was compared to four more traditional methods (thermal lysis, acid lysis, alkaline lysis, and osmotic lysis). For each method, the yeast loading was also examined at 1 g/L, 10 g/L and 100 g/L. The quality of the cell disruption was measured by optical cell density, cell counting and the particle size distribution profile comparison over a 2-hour period. This study demonstrates that the vortex separator is highly effective at lysing the cells and could potentially be used as a simple apparatus for lipid recovery in an oleaginous yeast process. The further development of this technology could potentially reduce the overall cost of microbial lipids in the future.Keywords: palm oil substitute, metschnikowia pulcherrima, cell disruption, cell lysis
Procedia PDF Downloads 1919048 SIPTOX: Spider Toxin Database Information Repository System of Protein Toxins from Spiders by Using MySQL Method
Authors: Iftikhar Tayubi, Tabrej Khan, Rayan Alsulmi, Abdulrahman Labban
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Spider produces a special kind of substance. This special kind of substance is called a toxin. The toxin is composed of many types of protein, which differs from species to species. Spider toxin consists of several proteins and non-proteins that include various categories of toxins like myotoxin, neurotoxin, cardiotoxin, dendrotoxin, haemorrhagins, and fibrinolytic enzyme. Protein Sequence information with references of toxins was derived from literature and public databases. From the previous findings, the Spider toxin would be the best choice to treat different types of tumors and cancer. There are many therapeutic regimes, which causes more side effects than treatment hence a different approach must be adopted for the treatment of cancer. The combinations of drugs are being encouraged, and dramatic outcomes are reported. Spider toxin is one of the natural cytotoxic compounds. Hence, it is being used to treat different types of tumors; especially its positive effect on breast cancer is being reported during the last few decades. The efficacy of this database is that it can provide a user-friendly interface for users to retrieve the information about Spiders, toxin and toxin protein of different Spiders species. SPIDTOXD provides a single source information about spider toxins, which will be useful for pharmacologists, neuroscientists, toxicologists, medicinal chemists. The well-ordered and accessible web interface allows users to explore the detail information of Spider and toxin proteins. It includes common name, scientific name, entry id, entry name, protein name and length of the protein sequence. The utility of this database is that it can provide a user-friendly interface for users to retrieve the information about Spider, toxin and toxin protein of different Spider species. The database interfaces will satisfy the demands of the scientific community by providing in-depth knowledge about Spider and its toxin. We have adopted the methodology by using A MySQL and PHP and for designing, we used the Smart Draw. The users can thus navigate from one section to another, depending on the field of interest of the user. This database contains a wealth of information on species, toxins, and clinical data, etc. This database will be useful for the scientific community, basic researchers and those interested in potential pharmaceutical Industry.Keywords: siptoxd, php, mysql, toxin
Procedia PDF Downloads 1689047 Parental Monitoring of Learners’ Cell Phone Use in the Eastern Cape, South Africa
Authors: Melikhaya Skhephe, Robert Mawuli Kwasi Boadzo, Zanoxolo Berington Gobingca
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This research study sought to examine parental monitoring of learners’ cell phone use in the Eastern Cape, South Africa. To this end, the researchers employed a quantitative approach. Data were obtained through questionnaires, with a sample of 15 parents having been purposively selected. The findings revealed that parents are unaware that they have to monitor the learner’s cell phone. Another finding was that parents in the 21-century did not support the use of mobile phones in education. The researchers recommend that parent’s discussion forums be created to educate parents on how a cell phone can be used in education. Cellphone companies need to be encouraged to educate parents on how they monitor cell phones used by learners. Another recommendation was that network providers need to restrict access to searching on the internet according to age.Keywords: parental monitoring, app blocking services, learner’s cell phone use, cell phone
Procedia PDF Downloads 1489046 WT1 Exprassion in Malignant Surface Epithelial Ovarian Tumors
Authors: Mahmoodreza Tahamtan
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Background: Malignant surface epithelial ovarian tumors (SEOT) account for approximately 90% of primary ovarian cancer. Wilms tumor gene (WT1) product was defined as a tumor suppressor gene, but today it is considered capable of performing oncogenic functions. There seems to be differences in WT1 expression patterns among SEOT subtypes. We evaluate the immunohistochemical expression of WT1 protein among different histologic subtypes of SEOT. Materials and Methods: Immunohistochemistry for WT1 was done on 35 serous cystadenocarcinomas, 9 borderline serous tumors, 3 mucinous cystadenocarcinomas, 10 borderline mucinous tumors, 7 endometrioid ovarian carcinomas, 3 clear cell carcinomas, 1 malignant Brenner tumor, 2 metastatic adenocarcinomas, and 6 endometrial adenocarcinomas. A tumor was considered negative if < 1% of tumor cells were stained.Positive reactions were graded as follows:1+,1%-24%; 2+,25%-49%; 3+,50%-74%; 4+,75%-100%. Results: Of the 35 cases of ovarian serous cystadenocarcinoma, 30(85.7%) were diffusely positive (3+,4+),4 showed reactivity of < 50% of the tumor cells (1+,2+), and one were negative. All 9 borderline serous tumors showed immunoreactivity with WT1. All the mucinous tumors(n:13), endometrioid carcinomas (n: 7), clear cell carcinomas (n: 3), metastatic adenocarcinomas (n: 2) and primary endometrial carcinomas (n:6) were negative. The single malignant Brenner tumor showed a positive reaction for WT1(4+) Conclusion: WT1 is a good marker to distinguish primary ovarian serous carcinomas from other surface epithelial tumors (especially endometrioid subtype) and metastatic carcinomas (especially endometrial serous carcinoma), other than malignant mesothelioma. We cannot rely to the degree of expression inorder to separate high grade borderline serous tumors from low grade ones.Keywords: WT1, ovary, epithelial tumors, malignant
Procedia PDF Downloads 879045 Utilization of Soymilk Residue for Wheat Flour Substitution in Gyoza skin
Authors: Naruemon Prapasuwannakul
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Soy milk residue is obtained as a byproduct from soy milk and tofu production with little economic value. It contains high protein and fiber as well as various minerals and phyto-chemical compounds. The objective of this research was to substitute soy milk residue for wheat flour in gyoza skin in order to enhance value of soy milk residue and increase protein and fiber content of gyoza skin. Wheat flour was replaced with soy milk residue from 0 to 40%. The soy milk residue prepared in this research contains 26.92% protein, 3.58% fiber, 2.88% lipid, 6.29% ash and 60.33% carbohydrate. The results showed that increasing soy milk residue decreased lightness (L*value), tensile strength and sensory attributes but increased redness (a*), yellowness (b*), protein and fiber contents of product. The result also showed that the gyoza skin substituted with 30% soy milk residue was the most acceptable (p≤0.05) and its protein and fiber content increased up to 45 % and 867 % respectively.Keywords: Gyoza skin, sensory, soymilk residue, wheat flour
Procedia PDF Downloads 3919044 Investigation of Astrocyte Physiology on Stiffness-Controlled Cellulose Acetate Nanofiber as a Tissue Scaffold
Authors: Sun Il Yu, Jung Hyun Joo, Hwa Sung Shin
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Astrocytes are known as dominant cells in CNS and play a role as a supporter of CNS activity and regeneration. Recently, three-dimensional culture of astrocytes were actively applied to understand in vivo astrocyte works. Electrospun nanofibers are attractive for 3D cell culture system because they have a high surface to volume ratio and porous structure, and have already been used for 3D astrocyte cultures. In this research, the stiffness of cellulose acetate (CA) nanofiber was controlled by heat treatment. As stiffness increased, astrocyte cell viability and adhesion increased. Reactivity of astrocyte was also upregulated in stiffer CA nanofiber in terms of GFAP, an intermediate filament protein. Finally, we demonstrated that stiffness-controllable CA is attractive for astrocyte tissue engineering.Keywords: astrocyte, cellulose acetate, nanofiber, tissue scaffold
Procedia PDF Downloads 3479043 Mathematical Modeling of Cell Volume Alterations under Different Osmotic Conditions
Authors: Juliana A. Knocikova, Yann Bouret, Médéric Argentina, Laurent Counillon
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Cell volume, together with membrane potential and intracellular hydrogen ion concentration, is an essential biophysical parameter for normal cellular activity. Cell volumes can be altered by osmotically active compounds and extracellular tonicity. In this study, a simple mathematical model of osmotically induced cell swelling and shrinking is presented. Emphasis is given to water diffusion across the membrane. The mathematical description of the cellular behavior consists in a system of coupled ordinary differential equations. We compare experimental data of cell volume alterations driven by differences in osmotic pressure with mathematical simulations under hypotonic and hypertonic conditions. Implications for a future model are also discussed.Keywords: eukaryotic cell, mathematical modeling, osmosis, volume alterations
Procedia PDF Downloads 4549042 The Effect of Particulate Matter on Cardiomyocyte Apoptosis Through Mitochondrial Fission
Authors: Tsai-chun Lai, Szu-ju Fu, Tzu-lin Lee, Yuh-Lien Chen
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There is much evidence that exposure to fine particulate matter (PM) from air pollution increases the risk of cardiovascular morbidity and mortality. According to previous reports, PM in the air enters the respiratory tract, contacts the alveoli, and enters the blood circulation, leading to the progression of cardiovascular disease. PM pollution may also lead to cardiometabolic disturbances, increasing the risk of cardiovascular disease. The effects of PM on cardiac function and mitochondrial damage are currently unknown. We used mice and rat cardiomyocytes (H9c2) as animal and in vitro cell models, respectively, to simulate an air pollution environment using PM. These results indicate that the apoptosis-related factor PUMA, a regulator of apoptosis upregulated by p53, is increased in mice treated with PM. Apoptosis was aggravated in cardiomyocytes treated with PM, as measured by TUNEL assay and Annexin V/PI. Western blot results showed that CASPASE3 was significantly increased and BCL2 (B-cell lymphoid 2) was significantly decreased under PM treatment. Concurrent exposure to PM increases mitochondrial reactive oxygen species (ROS) production by MitoSOX Red staining. Furthermore, using Mitotracker staining, PM treatment significantly shortened mitochondrial length, indicating mitochondrial fission. The expression of mitochondrial fission-related proteins p-DRP1 (phosphodynamics-related protein 1) and FIS1 (mitochondrial fission 1 protein) was significantly increased. Based on these results, the exposure to PM worsens mitochondrial function and leads to cardiomyocyte apoptosis.Keywords: particulate matter, cardiomyocyte, apoptosis, mitochondria
Procedia PDF Downloads 939041 Distinct Antiviral Pathway for ZFP36-Like Family Members Against Flavivirus Infection
Authors: Ren-Jye Lin, Li-Hsiung Lin, Bing-Cheng Liu, Ching-Len Liao
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The human zinc finger protein 36-like protein family, containing zinc finger protein 36-like 1 (ZFP36L1) and zinc finger protein 36-like 2 (ZFP36L2), belongs to CCCH-type zinc-finger protein identified as an RNA-binding protein that participates in controlling posttranscriptional regulation via RNA decay pathways. Recently, we demonstrated that human ZFP36L1 showed potent antiviral activity against flavivirus Infection by both 5´-3´ XRN1 and 3´-5´RNA-exosome RNA decay pathways (Journal of Virology 2022 Jan 12;96(1): e0166521). However, another zinc finger protein 36-like protein member, ZFP36L2, in the host defense response against flaviviruses has yet to be addressed. Here, we also demonstrate that ZFP36L2 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L2 reduced JEV and DENV infection, and ZFP36L2 knockdown significantly promoted viral replication. Distinct from the antiviral mechanism of ZFP36L1, ZFP36L2 inhibits flavivirus infection by only a 5´-3´ XRN1-mediated RNA decay pathway but not the 3´-5´RNA-exosome RNA decay pathway. Human ZFP36L1 and ZFP36L2 can restrict flavivirus replication by directly binding and destabilizing viral RNA. Thus, for the first time, human zinc finger protein 36-like family members, ZFP36L1 and ZFP36L2, are identified as host antiviral factors that can bind and degrade flavivirus viral RNA by diverse antiviral mechanisms.Keywords: ZFP36L1, ZFP36L2, 5'-3' exonuclease XRN1, antiviral mechansim
Procedia PDF Downloads 659040 Hippocampus Proteomic of Major Depression and Antidepressant Treatment: Involvement of Cell Proliferation, Differentiation, and Connectivity
Authors: Dhruv J. Limaye, Hanga Galfalvy, Cheick A. Sissoko, Yung-yu Huang, Chunanning Tang, Ying Liu, Shu-Chi Hsiung, Andrew J. Dwork, Gorazd B. Rosoklija, Victoria Arango, Lewis Brown, J. John Mann, Maura Boldrini
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Memory and emotion require hippocampal cell viability and connectivity and are disrupted in major depressive disorder (MDD). Applying shotgun proteomics and stereological quantification of neural progenitor cells (NPCs), intermediate neural progenitors (INPs), and mature granule neurons (GNs), to postmortem human hippocampus, identified differentially expressed proteins (DEPs), and fewer NPCs, INPs and GNs, in untreated MDD (uMDD) compared with non-psychiatric controls (CTRL) and antidepressant-treated MDD (MDDT). DEPs lower in uMDD vs. CTRL promote mitosis, differentiation, and prevent apoptosis. DEPs higher in uMDD vs. CTRL inhibit the cell cycle, and regulate cell adhesion, neurite outgrowth, and DNA repair. DEPs lower in MDDT vs. uMDD block cell proliferation. We observe group-specific correlations between numbers of NPCs, INPs, and GNs and an abundance of proteins regulating mitosis, differentiation, and apoptosis. Altered protein expression underlies hippocampus cellular and volume loss in uMDD, supports a trophic effect of antidepressants, and offers new treatment targets.Keywords: proteomics, hippocampus, depression, mitosis, migration, differentiation, mitochondria, apoptosis, antidepressants, human brain
Procedia PDF Downloads 949039 RACK1 Integrates Light and Brassinosteroid Signaling to Coordinate Cell Division During Root Soil Penetration
Authors: Liang Jiansheng, Zhu Wei
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Light and brassinosteroids are essential external and internal cues for plant survival. Although the coordination of light with phytohormone signals is crucial for plant growth and development, the molecular connection between light and brassinosteroid signaling during root soil penetration remains elusive. Here, we reveal that light-stabilized RACK1 couples a brassinosteroid signaling cascade to drive cell division in root meristems. RACK1 family scaffold proteins positively regulate light-induced the promotion of root elongation during soil penetration. Under the light condition, RACK1A interacts with both phyB and SPA1, then reinforces the phyB-SPA1 association to accumulate its abundance in roots. In response to brassinosteroid signals, RACK1A competes with BKI1 to attenuate the BRI1-BKI1 interaction, thereby leading to activating BRI1 actions in root development. Furthermore, RACK1A binds to BES1 to repress its DNA binding activity toward the target gene CYCD3;1. This ultimately allows to release the inhibition of CYCD3;1 transcription, and promotes cell division during root growth. Our study illustrates a new mechanistic model of how plants engage scaffold proteins in transducing light information to facilitate brassinosteroid signaling for root growth in the soil.Keywords: root growth, cell division, light signaling, brassinosteroid signaling, soil penetration, scaffold protein, RACK1
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