Search results for: transmembrane protein

Authors: M. Sarraf, J. E. Moros, M. C. Sánchez

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Oleogels are self-standing systems that are able to trap edible liquid oil into a tridimensional network and also help to use less fat by forming crystallization oleogelators. There are different ways to generate oleogelation and oil structuring, including direct dispersion, structured biphasic systems, oil sorption, and indirect method (emulsion-template). The selection of processing conditions as well as the composition of the oleogels is essential to obtain a stable oleogel with characteristics suitable for its purpose. In this sense, one of the ingredients widely used in food products to produce oleogels and emulsions is polysaccharides. Basil seed gum (BSG), with the scientific name Ocimum basilicum, is a new native polysaccharide with high viscosity and pseudoplastic behavior because of its high molecular weight in the food industry. Also, proteins can stabilize oil in water due to the presence of amino and carboxyl moieties that result in surface activity. Whey proteins are widely used in the food industry due to available, cheap ingredients, nutritional and functional characteristics such as emulsifier and a gelling agent, thickening, and water-binding capacity. In general, the interaction of protein and polysaccharides has a significant effect on the food structures and their stability, like the texture of dairy products, by controlling the interactions in macromolecular systems. Using edible oleogels as oil structuring helps for targeted delivery of a component trapped in a structural network. Therefore, the development of efficient oleogel is essential in the food industry. A complete understanding of the important points, such as the ratio oil phase, processing conditions, and concentrations of biopolymers that affect the formation and stability of the emulsion, can result in crucial information in the production of a suitable oleogel. In this research, the effects of oil concentration and pressure used in the manufacture of the emulsion prior to obtaining the oleogel have been evaluated through the analysis of droplet size and rheological properties of obtained emulsions and oleogels. The results show that the emulsion prepared in the high-pressure homogenizer (HPH) at higher pressure values has smaller droplet sizes and a higher uniformity in the size distribution curve. On the other hand, in relation to the rheological characteristics of the emulsions and oleogels obtained, the predominantly elastic character of the systems must be noted, as they present values of the storage modulus higher than those of losses, also showing an important plateau zone, typical of structured systems. In the same way, if steady-state viscous flow tests have been analyzed on both emulsions and oleogels, the result is that, once again, the pressure used in the homogenizer is an important factor for obtaining emulsions with adequate droplet size and the subsequent oleogel. Thus, various routes for trapping oil inside a biopolymer matrix with adjustable mechanical properties could be applied for the creation of the three-dimensional network in order to the oil absorption and creating oleogel.

Keywords: basil seed gum, particle size, viscoelastic properties, whey protein

Procedia PDF Downloads 66

Authors: Adebola Ajayi, Olakunle M. Makanjuola

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Effect of sprouting period on proximate, functional and mineral properties of malted sorghum flour was evaluated. The study was carried out to determine the proximate, functional and mineral properties of sprouting period on malted sorghum flour produced. The malted sorghum flour was obtained by sorting, weighing, washing, steeping, draining, germination, drying, dry milling, sieving. Malted sorghum flour was evaluated for proximate composition, functional properties and mineral contents. Moisture, protein, fat content, crude fiber, ash contents and carbohydrate of 24 and 48 hours, were in the range of 10.50-11.0, 11.17-11.17, 1.50-4.00, 2.50-1.50, 1.50-1.54 and 73.15-70.79% respectively. Bulk density ranged between 0.64 and 0.59g/ml, water and oil absorption capacities ranged between 139.3 and 150.0 and 217.3 and 222.7g/g respectively. Calcium, Magnesium, Zinc, Iron and Manganese were also range of 12.5, 59.3-60.0, 3.22-3.25, 3.80-3.90 and 3.22-3.25 mg/100g respectively. The results indicate that the germination of red sorghum resulted in the enhancement of the nutritional quality and its functional properties.

Keywords: sprouting, sorghum, malted sorghum flour, cabinet dryer

Procedia PDF Downloads 208

Authors: Debora P. Arce, Flavia J. Krsticevic, Marco R. Bertolaccini, Joaquín Ezpeleta, Estela M. Valle, Sergio D. Ponce, Elizabeth Tapia

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Small Heat Shock Proteins (sHSPs) are low molecular weight chaperones that play an important role during stress response and development in all living organisms. Fruit maturation and oxidative stress can induce sHSP synthesis both in Arabidopsis and tomato plants. RNA-Seq technology is becoming widely used in various transcriptomics studies; however, analyzing and interpreting the RNA-Seq data face serious challenges. In the present work, we de novo assembled the Solanum lycopersicum transcriptome for three different maturation stages (mature green, breaker and red ripe). Differential gene expression analysis was carried out during tomato fruit development. We identified 12 sHSPs differentially expressed that might be involved in breaker and red ripe fruit maturation. Interestingly, these sHSPs have different subcellular localization and suggest a complex regulation of the fruit maturation network process.

Keywords: sHSPs, maturation, tomato, RNA-Seq, assembly

Procedia PDF Downloads 480

Authors: Fang Bo Yu, Li Bo Guan

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Biogas slurry is a low-cost source of crop nutrients and can offer extra benefits to soil fertility and fruit quality. However, its current utilization mode and low content of active ingredients limit its application scale. In this report, one growing season field research was conducted to assess the effects of concentrated biogas slurry on soil property, tomato fruit quality, and composition of the microflora in both non-rhizosphere and rhizosphere soils. The results showed that application of concentrated slurry could cause significant changes to tomato cultivation, including increases in organic matter, available N, P, and K, total N, and P, electrical conductivity, and fruit contents of amino acids, protein, soluble sugar, β-carotene, tannins, and vitamin C, together with the R/S ratios and the culturable counts of bacteria, actinomycetes, and fungi in soils. It could be concluded as the application is a practicable means in tomato production and might better service the sustainable agriculture in the near future.

Keywords: concentrated slurry, fruit quality, soil fertility, sustainable agriculture

Procedia PDF Downloads 458

Authors: Can Hu, Huixia Shi, Hongcheng Mei, Jun Zhu, Hongling Guo

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Textile fibers are important trace evidence and frequently encountered in criminal investigations. A significant aspect of fiber evidence examination is the determination of fiber dyes. Although several instrumental methods have been developed for dyes detection, the analysis speed is not fast enough yet. A rapid dye analysis method is still needed to further improve the efficiency of case handling. Capillary electrophoresis has the advantages of high separation speed and high separation efficiency and is an ideal method for the rapid analysis of fiber dyes. In this paper, acid dyes used for protein fiber dyeing were determined by a developed short-end injection capillary electrophoresis technique. Five acid red dyes with similar structures were successfully baseline separated within 5 min. The separation reproducibility is fairly good for the relative standard deviation of retention time is 0.51%. The established method is rapid and accurate which has great potential to be applied in forensic setting.

Keywords: acid dyes, capillary electrophoresis, fiber evidence, rapid determination

Procedia PDF Downloads 144

Authors: A. Natsir, M. Nadir, S. Syahrir, A. Mujnisa, N. Purnomo, A. R. Egan, B. J. Leury

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Rumen degradation characteristic of feedstuff is one of the prominent factors affecting microbial population in rumen of animal. High rumen degradation rate of faba bean protein may lead to inconstant rumen conditions that could have a prominent impact on rumen microbial diversity. Amplified Ribosomal DNA Restriction Analysis (ARDRA) is utilized to monitor diversity of rumen microbes on sheep fed low quality forage supplemented by faba beans. Four mature merino sheep with existing rumen cannula were used in this study according to 4 x 4 Latin square design. The results of study indicated that there were 37 different ARDRA types identified out of 136 clones examined. Among those clones, five main clone types existed across the treatments with different percentages. In conclusion, the ARDRA method is potential to be used as a routine tool to assess the temporary changes in the rumen community as a result of different feeding strategies.

Keywords: ARDRA method, cattle, genomic diversity, rumen microbes

Procedia PDF Downloads 361

Authors: E. Grochowska-Niedworok, K. Brukalo, B. Całyniuk, J. Piekorz, M. Kardas

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The aim of the study was to assess diets of residents of nursing homes. Provided by social welfare home, 10 day menus were introduced into the computer program Diet 5 and analyzed in respect of protein, fats, carbohydrates, energy, vitamin D and calcium. The resulting mean values of 10-day menus were compared with the existing Nutrition Standards for Polish population. The analysis menus showed that the average amount of energy supplied from food is not sufficient. Carbohydrates in food supply are too high and represent 257% of normal. The average value of fats and proteins supplied with food is adequate 85.2 g/day and 75.2 g/day. The calcium content of the diet is 513.9 mg/day. The amount of vitamin D supplied in the age group 51-65 years is 2.3 µg/day. Dietary errors that have been shown are due to the lack of detailed nutritional guidelines for nursing homes, as well as state-owned care facilities in general.

Keywords: assessment of diet, essential nutrients, social welfare home, nutrition

Procedia PDF Downloads 152

Authors: Andre Pastori D'Aurea, Lauriston Bertelli Feranades, Luis Eduardo Ferreira, Leandro Dias Pinto, Fabiana Ayumi Shiozaki

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The aim of this study was to improve the production of beef cattle on tropical pasture without harming this environment. On tropical pastures, cattle's live weight gain is lower than feedlot, and forage production is seasonable, changing from season to season. Thus, concerned with sustainable livestock production, the Premix Company has developed strategies to improve the production of beef cattle on tropical pasture to ensure sustainability of welfare and production. There are two important principles in this productivity system: 1) increase individual gains with use of better supplementation and 2) increase the productivity units with better forage quality like corn silage or other forms of forage conservations, actually used only in winter, and adding natural additives in the diet. This production system was applied from June 2017 to May 2018 in the Research Center of Premix Company, Patrocínio Paulista, São Paulo State, Brazil. The area used had 9 hectares of pasture of Brachiaria brizantha. 36 steers Nellore were evaluated for one year. The initial weight was 253 kg. The parameters used were daily average gain and gain per area. This indicated the corrections to be made and helped design future fertilization. In this case, we fertilized the pasture with 30 kg of nitrogen per animal divided into two parts. The diet was pasture and protein-energy supplements (0.4% of live weight). The supplement used was added with natural additive Fator P® – Premix Company). Fator P® is an additive composed by amino acids (lysine, methionine and tyrosine, 16400, 2980 and 3000 mg.kg-1 respectively), minerals, probiotics (Saccharomyces cerevisiae, 7 x 10E8 CFU.kg-1) and essential fatty acids (linoleic and oleic acids, 108.9 and 99g.kg-1 respectively). Due to seasonal changes, in the winter we supplemented the diet by increasing the offer of forage, supplementing with maize silage. It was offered 1% of live weight in silage corn and 0.4% of the live weight in protein-energetic supplements with additive Fator P ®. At the end of the period, the productivity was calculated by summing the individual gains for the area used. The average daily gain of the animals were 693 grams per day and was produced 1.005 kg /hectare/year. This production is about 8 times higher than the average of Brazilian meat national production. To succeed in this project, it is necessary to increase the gains per area, so it is necessary to increase the capacity per area. Pasture management is very important to the project's success because the dietary decisions were taken from the quantity and quality of the forage. We, therefore, recommend the use of animals in the growth phase because the response to supplementation is greater in that phase and we can allocate more animals per area. This system's carbon footprint reduces emissions by 61.2 percent compared to the Brazilian average. This beef cattle production system can be efficient and environmentally friendly to the natural. Another point is that bovines will benefit from their natural environment without competing or having an impact on human food production.

Keywords: cattle production, environment, pasture, sustainability

Procedia PDF Downloads 149

Authors: S. Najafi, E. Bertini, M. Pezzotti, G.B. Tornielli, S. Zenoni

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Grapevine is an important agricultural fruit crop plant consumed worldwide and with a key role in the global economy. Grapevine is strongly affected by both biotic and abiotic stresses, which impact grape growth at different stages, such as during plant and berry development and pre- and post-harvest, consequently causing significant economic losses. Recently global warming has propelled the anticipation of the onset of berry ripening, determining the reduction of a grape color and increased volatilization of aroma compounds. Climate change could negatively alter the physiological characteristics of the grape and affect the berry and wine quality. Modern plant breeding can provide tools such as genome editing for improving grape resilience traits while maintaining intact the viticultural and oenological quality characteristics of the genotype. This study aims at developing a platform for genome editing application in grapevine plants with the final goal to improve berry quality, biotic, and abiotic resilience traits. We chose to directly deliver ribonucleoproteins (RNP, preassembled Cas protein and guide RNA) into plant protoplasts, and, from these cell structures, regenerate grapevine plants edited in specific selected genes controlling traits of interest. Edited plants regenerated by somatic embryogenesis from protoplasts will then be sequenced and molecularly characterized. Embryogenic calli of Sultana and Shiraz cultivars were initiated from unopened leaves of in-vitro shoot tip cultures and from stamens, respectively. Leaves were placed on NB2 medium while stamens on callus initiation medium (PIV) medium and incubated in the dark at 28 °C for three months. Viable protoplasts, tested by FDA staining, isolated from embryogenic calli were cultured by disc method at 1*105 protoplasts/ml. Mature well-shaped somatic embryos developed directly in the protoplast culture medium two months later and were transferred in the light into to shooting medium for further growth. Regenerated plants were then transferred to the greenhouse; no phenotypic alterations were observed when compared to non in-vitro cultured plants. The performed experiments allowed to established an efficient protocol of embryogenic calli production, protoplast isolation, and regeneration of the whole plant through somatic embryogenesis in both Sultana and Shiraz. Regenerated plants, through direct somatic embryogenesis deriving from a single cell, avoid the risk of chimerism during the regeneration process, therefore improving the genome editing process. As pre-requisite of genome editing, an efficient method for transfection of protoplast by yellow fluorescent protein (YFP) marker genes was also established and experiments of direct delivery of CRISPR–Cas9 ribonucleoproteins (RNPs) in protoplasts to achieve efficient DNA-free targeted mutations are in progress.

Keywords: CRISPR-cas9, plant regeneration, protoplast isolation, Vitis vinifera

Procedia PDF Downloads 150

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

Procedia PDF Downloads 60

Authors: Iryna Atamaniuk, Hannah Boysen, Nils Wieczorek, Natalia Politaeva, Iuliia Bazarnova, Kerstin Kuchta

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Over the last decade due to climate change and a strategy of natural resources preservation, the interest for the aquatic biomass has dramatically increased. Along with mitigation of the environmental pressure and connection of waste streams (including CO₂ and heat emissions), microalgae bioeconomy can supply food, feed, as well as the pharmaceutical and power industry with number of value-added products. Furthermore, in comparison to conventional biomass, microalgae can be cultivated in wide range of conditions without compromising food and feed production, thus addressing issues associated with negative social and the environmental impacts. This paper presents the state-of-the art technology for microalgae bioeconomy from cultivation process to production of valuable components and by-streams. Microalgae Chlorella sorokiniana were cultivated in the pilot-scale innovation concept in Hamburg (Germany) using different systems such as race way pond (5000 L) and flat panel reactors (8 x 180 L). In order to achieve the optimum growth conditions along with suitable cellular composition for the further extraction of the value-added components, process parameters such as light intensity, temperature and pH are continuously being monitored. On the other hand, metabolic needs in nutrients were provided by addition of micro- and macro-nutrients into a medium to ensure autotrophic growth conditions of microalgae. The cultivation was further followed by downstream process and extraction of lipids, proteins and saccharides. Lipids extraction is conducted in repeated-batch semi-automatic mode using hot extraction method according to Randall. As solvents hexane and ethanol are used at different ratio of 9:1 and 1:9, respectively. Depending on cell disruption method along with solvents ratio, the total lipids content showed significant variations between 8.1% and 13.9 %. The highest percentage of extracted biomass was reached with a sample pretreated with microwave digestion using 90% of hexane and 10% of ethanol as solvents. Proteins content in microalgae was determined by two different methods, namely: Total Kejadahl Nitrogen (TKN), which further was converted to protein content, as well as Bradford method using Brilliant Blue G-250 dye. Obtained results, showed a good correlation between both methods with protein content being in the range of 39.8–47.1%. Characterization of neutral and acid saccharides from microalgae was conducted by phenol-sulfuric acid method at two wavelengths of 480 nm and 490 nm. The average concentration of neutral and acid saccharides under the optimal cultivation conditions was 19.5% and 26.1%, respectively. Subsequently, biomass residues are used as substrate for anaerobic digestion on the laboratory-scale. The methane concentration, which was measured on the daily bases, showed some variations for different samples after extraction steps but was in the range between 48% and 55%. CO₂ which is formed during the fermentation process and after the combustion in the Combined Heat and Power unit can potentially be used within the cultivation process as a carbon source for the photoautotrophic synthesis of biomass.

Keywords: bioeconomy, lipids, microalgae, proteins, saccharides

Procedia PDF Downloads 245

Authors: Kaomud Tyagi, Rajasree Chakraborty, Shantanu Kundu, Devkant Singha, Kailash Chandra, Vikas Kumar

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The melon thrips, Thrips palmi is a serious pest of a wide range of agriculture crops and also act as vectors for plant viruses (genus Tospovirus, family Bunyaviridae). More molecular data on this species is required to understand the cryptic speciation and evolutionary affiliations. Mitochondrial genomes have been widely used in phylogenetic and evolutionary studies in insect. So far, mitogenomes of five thrips species (Anaphothrips obscurus, Frankliniella intonsa, Frankliniella occidentalis, Scirtothrips dorsalis and Thrips imaginis) is available in the GenBank database. In this study, we sequenced the first complete mitogenome T. palmi and compared it with available thrips mitogenomes. We assembled the mitogenome from the whole genome sequencing data generated using Illumina Hiseq2500. Annotation was performed using MITOS web-server to estimate the location of protein coding genes (PCGs), transfer RNA (tRNAs), ribosomal RNAs (rRNAs) and their secondary structures. The boundaries of PCGs and rRNAs was confirmed manually in NCBI. Phylogenetic analyses were performed using the 13 PCGs data using maximum likelihood (ML) in PAUP, and Bayesian inference (BI) in MrBayes 3.2. The complete mitogenome of T. palmi was 15,333 base pairs (bp), which was greater than the genomes of A. obscurus (14,890bp), F. intonsa (15,215 bp), F. occidentalis (14,889 bp) and S. dorsalis South Asia strain (SA1) (14,283 bp), but smaller than the genomes of T. imaginis (15,407 bp) and S. dorsalis East Asia strain (EA1) (15,343bp). Like in other thrips species, the mitochondrial genome of T. palmi was represented by 37 genes, including 13 PCGs, large and small ribosomal RNA (rrnL and rrnS) genes, 22 transfer RNA (tRNAs) genes (with one extra gene for trn-Serine) and two A+T-rich control regions (CR1 and CR2). Thirty one genes were observed on heavy (H) strand and six genes on the light (L) strand. The six tRNA genes (trnG,trnK, trnY, trnW, trnF, and trnH) were found to be conserved in all thrips species mitogenomes in their locations relative to a protein-coding or rRNA gene upstream or downstream. The gene arrangements of T. palmi is very close to T. imaginis except the rearrangements in tRNAs genes: trnR (arginine), and trnE (glutamic acid) were found to be located between cox3 and CR2 in T. imaginis which were translocated between atp6 and CR1 in T. palmi; trnL1 (Leucine) and trnS1(Serine) were located between atp6 and CR1 in T. imaginis which were translocated between cox3 and CR2 in T. palmi. The location of CR1 upstream of nad5 gene was suggested to be ancestral condition of the thrips species in subfamily Thripinae, was also observed in T. palmi. Both the Maximum likelihood (ML) and Bayesian Inference (BI) phylogenetic trees generated resulted in similar topologies. The T. palmi was clustered with T. imaginis. We concluded that more molecular data on the diverse thrips species from different hierarchical level is needed, to understand the phylogenetic and evolutionary relationships among them.

Keywords: thrips, comparative mitogenomics, gene rearrangements, phylogenetic analysis

Procedia PDF Downloads 168

Authors: Hamad Al-Turaif, Usman Saeed

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The development of nanofibers with the help of electrospinning is being prioritized as a method of choice because of the simplicity and efficiency of the process. The parameters of the electrospinning process effectively convert the polymer solution into an electrospun final product made of the desired diameter of nanofiber. The aim of the study presented is to recognize and analyze the effect of proposed parameters on biodegradable and biocompatible polylactic acid (PLA)/polybutylene succinate (PBS) nanofiber developed by the electrospinning process. The morphology of the fiber is characterized by implementing Scanning Electron Microscope. Studies were conducted to characterize the result of using different electrospinning parameters on the final diameter and orientation of fiber. It was determined that varying polymer solution concentration, feed rate, and applied voltage show different outcomes. The best results were obtained at 6% polymer solution concentration, 20 kV, and 0.5 ml/h, which can be applicable for biomedical applications. Finally, protein adsorption and mechanical testing were conducted on the PLA/PBS fiber.

Keywords: electrospinning, polylactic acid, polybutylene succinate, morphology

Procedia PDF Downloads 132

Authors: Jiaojiao Zhang

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Boar sperm quality, as an important indicator of reproductive efficiency, directly affects the efficiency of livestock production. Here, this study was conducted to improve the boar sperm quality by using a non-thermal dielectric barrier discharge (DBD) plasma. Our results showed that DBD plasma exposure at 2.1 W for 15 s could improve boar sperm quality by increasing the exon methylation level of adenosine monophosphate-activated protein kinase (AMPK) and thus improving the glycolytic flux, mitochondrial function, and antioxidant capacity without damaging the integrity of sperm DNA and acrosome. In addition, DBD plasma could rescue DNA methyltransferase inhibitor decitabine-caused low sperm quality by reducing oxidative stress and mitochondrial damage. Therefore, the application of non-thermal plasma provides a new strategy for reducing sperm oxidative damage and improving sperm quality, which shows great potential in assisted reproduction to solve the problem of male infertility.

Keywords: non-thermal DBD plasma, sperm quality, AMPK methylation, energy metabolism, antioxidant capacity

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Authors: Dandan Shen, Hui-zhu Wang, Xi Yu, LiLi Gui, Xiao Wei, Xiao-yan Jiang, Da-wei Wang, Min Hong

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Yu-ping-feng-san (YPFS) is a clinical medicine for asthma and other allergic diseases, but the mechanism of YPFS on relapse of allergy is unclear. Currently, people come to realize the epithelial cells(EC) play a key role in stimulating and regulating local immune response. The study of thymic stromal lymphopoietin（TSLP derived from EC provides an important evidence that the EC can regulate immune response to stimulate allergic response. In this study, we observed the effect of YPFS on TSLP in vivo and in vitro. We established a method by using bronchial epithelial cells (16HBE) for screening potential bioactive components by HPLC-MS in YPFS and then analyzed the components in serum containing YPFS by UPLC-MS. The results showed that YPFS could decrease TSLP protein level in OVA-sensitized mice and 16HBE cells. Five components combing with the 16HBE cells were both detected in the serum.

Keywords: TSLP, bronchial epithelial cells, cell-binding, drug-containing serum

Procedia PDF Downloads 513

Authors: Anna Höfer, Johannes Herrmann, Patrick Meybohm, Christopher Lotz

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Mitochondria are pivotal for energy supply and regulation of cellular functions. Deficiencies of mitochondrial metabolism have been implicated in diverse stressful conditions including infections. Platelets are key mediators for thrombo-inflammation during development and resolution of acute respiratory distress syndrome (ARDS). Previous data point to an exhausted platelet phenotype in critically-ill patients with coronavirus 19 disease (COVID-19) impacting the course of disease. The objective of this work was to characterize platelet mitochondrial metabolism in patients suffering from COVID-19 ARDSA longitudinal analysis of platelet mitochondrial metabolism in 24 patients with COVID-19 induced ARDS compared to 35 healthy controls (ctrl) was performed. Blood samples were analyzed at two time points (t1=day 1; t2=day 5-7 after study inclusion). The activity of mitochondrial citrate synthase was photometrically measured. The impact of oxidative stress on mitochondrial permeability was assessed by a photometric calcium-induced swelling assay and the activity of superoxide dismutase (SOD) by a SOD assay kit. The amount of protein carbonylation and the activity of mitochondria complexes I-IV were photometrically determined. Levels of interleukins (IL)-1α, IL-1β and tumor necrosis factor (TNF-) α were measured by a Multiplex assay kit. Median age was 54 years, 63 % were male and BMI was 29.8 kg/m2. SOFA (12; IQR: 10-15) and APACHE II (27; IQR: 24-30) indicated critical illness. Median Murray Score was 3.4 (IQR: 2.8-3.4), 21/24 (88%) required mechanical ventilation and V-V ECMO support in 14/24 (58%). Platelet counts in ARDS did not change during ICU stay (t1: 212 vs. t2: 209 x109/L). However, mean platelet volume (MPV) significantly increased (t1: 10.6 vs. t2: 11.9 fL; p<0.0001). Citrate synthase activity showed no significant differences between ctrl and ARDS patients. Calcium induced swelling was more pronounced in patients at t1 compared to t2 and to ctrl (50µM; t1: 0.006 vs. ctrl: 0.016 ΔOD; p=0.001). The amount of protein carbonylation as marker for irreversible proteomic modification constantly increased during ICU stay and compared to ctrl., without reaching significance. In parallel, superoxid dismutase activity gradually declined during ICU treatment vs. ctrl (t2: - 29 vs. ctrl.: - 17 %; p=0.0464). Complex I analysis revealed significantly stronger activity in ARDS vs. ctrl. (t1: 0.633 vs. ctrl.: 0.415 ΔOD; p=0.0086). There were no significant differences in complex II, III or IV activity in platelets from ARDS patients compared to ctrl. IL-18 constantly increased during the observation period without reaching significance. IL-1α and TNF-α did not differ from ctrl. However, IL-1β levels were significantly elevated in ARDS (t1: 16.8; t2: 16.6 vs. ctrl.: 12.4 pg/mL; p1=0.0335, p2=0.0032). This study reveals new insights in platelet mitochondrial metabolism during COVID-19 caused ARDS. it data point towards enhanced platelet activity with a pronounced turnover rate. We found increased activity of mitochondria complex I and evidence for enhanced oxidative stress. In parallel, protective mechanisms against oxidative stress were narrowed with elevated levels of IL-1β likely causing a pro-apoptotic environment. These mechanisms may contribute to platelet exhaustion in ARDS.

Keywords: acute respiratory distress syndrome (ARDS), coronavirus 19 disease (COVID-19), oxidative stress, platelet mitochondrial metabolism

Procedia PDF Downloads 59

Authors: Nelson Rotimi Osungbemiro, Sanni Rafiu Olugbenga, Abayomi Olufemi Olajuyigbe

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Ninety Fingerlings of Clarias gariepinus were exposed to the pathogenic Flavobacterium columnare a Gram Negative bacteria responsible for high mortality in fish pond raised young fish (fries and fingerlings) of Clarias sp. in Southwestern Nigeria. After feeding with 40% crude protein pelletized fish feed for 5 days, the fishes were divided into two groups, one group was treated with extracts from Calendula officinalis flowers, while the second group was not treated (control). The results indicated that, at day 5, colony formation had been manifesting and at day 7, skin lesion occurred and at the 8th day, first mortality of fish occurred, and this continued steadily on the 9th-12th day when all the fishes were dead. Whereas, in the group that was treated with Calendula sp., no single mortality was recorded. This research shows that plant extract from Calendula flowers is an effective antimicrobial agent against the virulent pathogenic Flavobacterium columnare disease.

Keywords: antimicrobial, Flavobacterium columnare, Clarias gariepinus, fish

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Authors: Bouaziz H., Soudania N., Essafia M., Ben Amara I., Hakim A., Jamoussi K., Zeghal Km, Zeghal N.

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In this investigation, we have evaluated the effects of arsenic trioxide on hepatic function in pregnant and lactating Swiss albino mice and their suckling pups. Experiments were carried out on female mice given 175 ppm As2O3 in their drinking water from the 14th day of pregnancy until day 14 after delivery. Our results showed a significant decrease in plasma levels of total protein and albumin, cholesterol and triglyceride in As2O3 treated mice and their pups. The hyperbilirubinemia and the increased plasma total alkaline phosphatase activity suggested the presence of cholestasis. Transaminase activities as well as lactate deshydrogenase activity in plasma, known as biomarkers of hepatocellular injury, were elevated indicating hepatic cells’damage after treatment with As2O3. Exposure to arsenic led to an increase of liver thiobarbituric acid reactive substances level along with a concomitant decrease in the activities of superoxide dismutase, catalase and glutathione peroxidase and in glutathione.

Keywords: antioxidant status, arsenic trioxide, hepatotoxicity, mice, oxidative stress

Procedia PDF Downloads 255

Authors: R. Asmeda, A. Noorlaila, M. H. Norziah

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This research work was conducted to investigate the effects of different grinding techniques during the milling process of rice grains on physicochemical characteristics of rice flour produced. Dry grinding, semi-wet grinding, and wet grinding were employed to produce the rice flour. The results indicated that different grinding methods significantly (p ≤ 0.05) affected physicochemical and functional properties of starch except for the carbohydrate content, x-ray diffraction pattern and breakdown viscosity. Dry grinding technique caused highest percentage of starch damage compared to semi-wet and wet grinding. Protein, fat and ash content were highest in rice flour obtained by dry grinding. It was found that wet grinding produce flour with smallest average particle size (8.52 µm), resulting in highest process yield (73.14%). Pasting profiles revealed that dry grinding produce rice flour with significantly lowest pasting temperature and highest setback viscosity.

Keywords: average particle size, grinding techniques, physicochemical characteristics, rice flour

Procedia PDF Downloads 191

Authors: Y. Okumuş, A. Tuna, A. T. Seyhan, H. Çelebi

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Useful carbon fibers were derived from chicken feathers (PCFs) based on a two-step pyrolysis method. The collected PCFs were cleaned and categorized as black, white and brown. Differential scanning calorimeter (DSC) and thermo-gravimetric analyzer (TGA) were systemically used to design the pyrolysis steps. Depending on colors, feathers exhibit different glass transition (Tg) temperatures. Long-time heat treatment applied to the feathers emerged influential on the surface quality of the resulting carbon fibers. Fourier Transformation Infrared (FTIR) examination revealed that the extent of disulfide bond cleavage is highly associated with the feather melting stability. Scanning electron microscopy (SEM) examinations were employed to evaluate the morphological changes of feathers after pyrolysis. Of all, brown feathers were found to be the most promising to turn into useful carbon fibers without any trace of melting and shape distortion when pyrolysis was carried out at 230°C for 24 hours and at 450°C for 1 hour.

Keywords: poultry chicken feather, keratin protein fiber, pyrolysis, high carbonaceous fibers

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Authors: Cristiano Giordani, Marco Diociaiuti, Cecilia Bombelli, Laura Zanetti-Polzi, Marcello Belfiore, Raoul Fioravanti, Gianfranco Macchia

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We investigated induced functional effects by evaluating Ca2+-influx in liposomes and cell viability in HT22-DIFF neurons. Only solutions rich in unstructured Prefibrillar-Oligomers (PFOs) were able, in the presence of Monosialoganglioside-GM1 (GM1), to induce Ca2+-influx and were also neurotoxic, suggesting a correlation between the two phenomena. Thus, in the presence of GM1, we investigated the protein conformation and liposome modification due to the interaction. Circular Dichroism showed that GM1 fostered the formation of β-structures and Energy Filtered-Transmission Electron Microscopy that PFOs formed “amyloid-channels” as reported for Aβ. We speculate that electrostatic forces occurring between the positive PFOs and negative GM1 drive the initial binding, while the hydrophobic profile of the flexible PFO is responsible for the subsequent pore formation. Conversely, the rigid β-structured mature/fibers (MFs) and proto-fibers (PFs) were unable to induce membrane damage and Ca2+- influx.

Keywords: amyloid proteins, neurotoxicity, lipid-rafts, GM1

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Authors: Yeng Min Yi, Rosli Md Illias, Salehhuddin Hamdan

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Industrial biocatalysis is mainly based on the use of cell free or intracellular enzyme systems. However, the expensive cost and relatively lower operational stability of free enzymes limit practical use in industries. Cell surface display system can be used as a cost-efficient alternative to overcome the laborious purification and substrate transport limitation. In this research, TibA autotransporter from E. coli was used to display Aspergillus fumigatus xylanase (xyn). The amplified xyn was fused in between N-terminal signal peptide and C-terminal β-barrel of TibA. The cloned was transformed and expressed in E. coli BL21 (DE3). Outer membrane localization of TibA-xyn fusion protein was confirmed by SDS PAGE and western blot with expected size of 62.5 kDa. Functional display of xyn was examined by activity assay. Cell surface displayed xyn exhibited the highest activity at 37 °c, 0.3 mM IPTG. As a summary, TibA displaying system has the potential for further industrial applications. Moreover, this is the first report of the display of xylanase using TibA on the surface of E. coli.

Keywords: biocatalysis, cell surface display, Escherichia coli, TibA autotransporter

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Authors: Manab Bandhu Bera, Navdeep Singh, Paramjit Singh Panesar

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In view of exploiting the health benefits of probiotic yeast S.boulardii NCDC 363 and make it available in the form of non-dairy food products, study was undertaken. In this, probiotic yeast S.boulardii NCDC 363 was incorporated in the edible film made from sodium alginate (SA), whey protein concentrate (WPC) and glycerol (50%). Response surface methodology was used to optimize process variables such as; concentration of SA (0.25-0.75%), WPC (1-2%) and temperature (70-80°C) and also to investigate effect of these process variables on viability of probiotic yeast and hardness when applied as an edible coat on extruded food products. Accelerated storage stability of optimized probiotic extruded food products samples was determined at 38 C and 90% RH. The optimized products were packed in high-density polyethylene (HDPE) and aluminum laminated polyethylene (ALP) pouches at 38°C and relative humidity maintained was 90%. It was observed that product stored in ALP had better stability in terms of moisture absorption, hardness and viability.

Keywords: probiotic yeast, extruded food product, WPC, RSM

Procedia PDF Downloads 276

Authors: Sandeep Kumar Agrawal, Aditi Bhattacharya, Janvie Manhas, Krushna Bhatt, Yatin Kholakiya, Nupur Khera, Ajoy Roychoudhury, Sudip Sen

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Autologous cultured explants of human oral mucosal epithelial cells (OMEC) are a potential therapeutic modality for limbal stem cell deficiency (LSCD). Injury or inflammation of the ocular surface in the form of burns, chemicals, Stevens Johnson syndrome, ocular cicatricial pemphigoid etc. can lead to destruction and deficiency of limbal stem cells. LSCD manifests in the form of severe ocular surface diseases (OSD) characterized by persistent and recurrent epithelial defects, conjuntivalisation and neovascularisation of the corneal surface, scarring and ultimately opacity and blindness. Most of the cases of OSD are associated with severe dry eye pertaining to diminished mucin and aqueous secretion. Mycophenolate mofetil (MMF) has been shown to upregulate the mucin expression in conjunctival goblet cells in vitro. The aim of this study was to evaluate the effects of MMF on mucin expression in primary cultures of oral mucosal epithelial cells. With institutional ethics committee approval and written informed consent, thirty oral mucosal epithelial tissue samples were obtained from patients undergoing oral surgery for non-malignant conditions. OMEC were grown on human amniotic membrane (HAM, obtained from expecting mothers undergoing elective caesarean section) scaffold for 2 weeks in growth media containing DMEM & Ham’s F12 (1:1) with 10% FBS and growth factors. In vitro dosage of MMF was standardised by MTT assay. Analysis of stem cell markers was done using RT-PCR while mucin mRNA expression was quantified using RT-PCR and q-PCR before and after treating cultured OMEC with graded concentrations of MMF for 24 hours. Protein expression was validated using immunocytochemistry. Morphological studies revealed a confluent sheet of proliferating, stratified oral mucosal epithelial cells growing over the surface of HAM scaffold. The presence of progenitor stem cell markers (p63, p75, β1-Integrin and ABCG2) and cell surface associated mucins (MUC1, MUC15 and MUC16) were elucidated by RT-PCR. The mucin mRNA expression was found to be upregulated in MMF treated primary cultures of OMEC, compared to untreated controls as quantified by q-PCR with β-actin as internal reference gene. Increased MUC1 protein expression was validated by immunocytochemistry on representative samples. Our findings conclude that OMEC have the ability to form a multi-layered confluent sheet on the surface of HAM similar to a cornea, which is important for the reconstruction of the damaged ocular surface. Cultured OMEC has stem cell properties as demonstrated by stem cell markers. MMF can be a novel enhancer of mucin production in OMEC. It has the potential to improve dry eye in patients undergoing OMEC transplantation for bilateral OSD. Further clinical trials are required to establish the role of MMF in patients undergoing OMEC transplantation.

Keywords: limbal stem cell deficiency, mycophenolate mofetil, mucin, ocular surface disease

Procedia PDF Downloads 331

Authors: Allison Herlene Du Plessis, Dalena (R.M.) Van Rooyen, Wilma Ten Ham-Baloyi, Sihaam Jardien-Baboo

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Introduction: Women die in pregnancy and childbirth for five main reasons—severe bleeding, infections, unsafe abortions, hypertensive disorders (pre-eclampsia and eclampsia), and medical complications including cardiac disease, diabetes, or HIV/AIDS complicated by pregnancy. In 2015, WHO classified sepsis as the third highest cause for maternal mortalities in the world. Chorioamnionitis is a clinical syndrome of intrauterine infection during any stage of the pregnancy and it refers to ascending bacteria from the vaginal canal up into the uterus, causing infection. While the incidence rates for chorioamnionitis are not well documented, complications related to chorioamnionitis are well documented and midwives still struggle to identify this condition in time due to its complex nature. Few diagnostic methods are available in public health services, due to escalated laboratory costs. Often the affordable biomarkers, such as C-reactive protein CRP, full blood count (FBC) and WBC, have low significance in diagnosing chorioamnionitis. A lack of screening impacts on effective and timeous management of chorioamnionitis, and early identification and management of risks could help to prevent neonatal complications and reduce the subsequent series of morbidities and healthcare costs of infants who are health foci of perinatal infections. Objective: This integrative literature review provides an overview of current best research evidence on the screening of women at risk for chorioamnionitis. Design: An integrative literature review was conducted using a systematic electronic literature search through EBSCOhost, Cochrane Online, Wiley Online, PubMed, Scopus and Google. Guidelines, research studies, and reports in English related to chorioamnionitis from 2008 up until 2020 were included in the study. Findings: After critical appraisal, 31 articles were included. More than one third (67%) of the literature included ranked on the three highest levels of evidence (Level I, II and III). Data extracted regarding screening for chorioamnionitis was synthesized into four themes, namely: screening by clinical signs and symptoms, screening by causative factors of chorioamnionitis, screening of obstetric history, and essential biomarkers to diagnose chorioamnionitis. Key conclusions: There are factors that can be used by midwives to identify women at risk for chorioamnionitis. However, there are a paucity of established sociological, epidemiological and behavioral factors to screen this population. Several biomarkers are available to diagnose chorioamnionitis. Increased Interleukin-6 in amniotic fluid is the better indicator and strongest predictor of histological chorioamnionitis, whereas the available rapid matrix-metalloproteinase-8 test requires further testing. Maternal white blood cells count (WBC) has shown poor selectivity and sensitivity, and C-reactive protein (CRP) thresholds varied among studies and are not ideal for conclusive diagnosis of subclinical chorioamnionitis. Implications for practice: Screening of women at risk for chorioamnionitis by health care providers providing care for pregnant women, including midwives, is important for diagnosis and management before complications arise, particularly in resource-constraint settings.

Keywords: chorioamnionitis, guidelines, best evidence, screening, diagnosis, pregnant women

Procedia PDF Downloads 123

Authors: R. S. Fisca, Y. R. Elox, L. Umi, U. Z. Luttfia, Kun Harismah

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Petai (Parkia speciosa) is a plant indigenous to Southeast Asia. It is consumed either raw or cooked. It has been used in folk medicine to treat diabetes, hypertension, and kidney problems. It contains minerals and vitamins. Petai contains a lot of chemical compounds that are beneficial for health, including antioxidants, Vitamin B6 0,9mg, energy 142 g. cal, 10.4 g protein. 2 g fat, 22 g carbohydrates, 95 mg calcium, phosphorus 115 mg, 1 mg iron, 200 IU of vitamin A, vitamin B1 0.17 mg, 36 mg of vitamin C that can resolve various health problems. These chips are the result of innovation from petai packaged in such a way becomes a tasty snack chips and can be enjoyed by many people to relax and also nutritious for health. In the manufacture of petai chips require several steps of them start by boiling, flating, drying and the last frying. In introducing the products widely we sell petai chips with several methods. Some of these methods include direct sales, delivery order, online/social media, and open some booth at a few places and the car free day in Solo every sunday. Opportunity in selling petai chips is very wide because there is no competitors with similar business. With the innovation of petai chips become healthy snacks can be introduced to the public and can even be exported out of the country as one of the extraordinary snacks from Indonesia.

Keywords: antioxidants, chips, healty, petai

Procedia PDF Downloads 564

Authors: Truong H. Le, Ngoc M. To, Quang N. Tran, Luu T. Cao, Chi V. Le

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Introduction: In Vietnam, the current monitoring and treatment for ordinary diabetic patient mostly based on glucose monitoring with HbA1c test for every three months (recommended goal is HbA1c < 6.5%~7%). For diabetes in pregnant women or Gestational diabetes mellitus (GDM), glycemic control until the time of delivery is extremly important because it could reduce significantly medical implications for both the mother and the child. Besides, GDM requires continuos glucose monitoring at least every two weeks and therefore an alternative marker of glycemia for short-term control is considering a potential tool for the healthcare providers. There are published studies have indicated that the glycosylated serum protein is a better indicator than glycosylated hemoglobin in GDM monitoring. Based on the actual practice in Vietnam, this study was designed to evaluate the role of serum fructosamine as a monitoring tool in GDM treament and its correlations with fasting blood glucose (G0), 2-hour postprandial glucose (G2) and glycosylated hemoglobin (HbA1c). Methods: A cohort study on pregnant women diagnosed with GDM by the 75-gram oralglucose tolerance test was conducted at Endocrinology Department, Cho Ray hospital, Vietnam from June 2014 to March 2015. Cho Ray hospital is the final destination for GDM patient in the southern of Vietnam, the study population has many sources from other pronvinces and therefore researchers belive that this demographic characteristic can help to provide the study result as a reflection for the whole area. In this study, diabetic patients received a continuos glucose monitoring method which consists of bi-weekly on-site visit every 2 weeks with glycosylated serum protein test, fasting blood glucose test and 2-hour postprandial glucose test; HbA1c test for every 3 months; and nutritious consultance for daily diet program. The subjects still received routine treatment at the hospital, with tight follow-up from their healthcare providers. Researchers recorded bi-weekly health conditions, serum fructosamine level and delivery outcome from the pregnant women, using Stata 13 programme for the analysis. Results: A total of 500 pregnant women was enrolled and follow-up in this study. Serum fructosamine level was found to have a light correlation with G0 ( r=0.3458, p < 0.001) and HbA1c ( r=0.3544, p < 0.001), and moderately correlated with G2 ( r=0.4379, p < 0.001). During study timeline, the delivery outcome of 287 women were recorded with the average age of 38.5 ± 1.5 weeks, 9% of them have macrosomia, 2.8% have premature birth before week 35th and 9.8% have premature birth before week 37th; 64.8% of cesarean section and none of them have perinatal or neonatal mortality. The study provides a reference interval of serum fructosamine for GDM patient was 112.9 ± 20.7 μmol/dL. Conclusion: The present results suggests that serum fructosamine is as effective as HbA1c as a reflection of blood glucose control in GDM patient, with a positive result in delivery outcome (0% perinatal or neonatal mortality). The reference value of serum fructosamine measurement provided a potential monitoring utility in GDM treatment for hospitals in Vietnam. Healthcare providers in Cho Ray hospital is considering to conduct more studies to test this reference as a target value in their GDM treatment and monitoring.

Keywords: gestational diabetes mellitus, monitoring tool, serum fructosamine, Vietnam

Procedia PDF Downloads 280

Authors: Srie Rezeki Nur Endah, Richa Mardianingrum

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Cancer is one of the most feared diseases and is considered the leading cause of death worldwide. Generally, cancer drugs are synthetic drugs with relatively more expensive prices and have harmful side effects, so many people turn to traditional medicine, for example by utilizing herbal medicine. Erythrina poeppigiana is one of the plants that can be used as a medicinal plant containing 10,11-dihidroerisodin compounds that are useful anticancer etnofarmakologi. The purpose of this study was to identify the target of 10,11 dihydroerisodin receptor compound as in silico anticancer candidate. The pure isolate was tested physicochemically by MS (Mass Spectrometry), UV-Vis (Ultraviolet – Visible), IR (Infra Red), 13C-NMR (Carbon-13 Nuclear Magnetic Resonance), 1H-NMR (Hydrogen-1 Nuclear Magnetic Resonance), to obtain the structure of 10,11-dihydroerisodin alkaloid compound then identified to target receptors in silico. From the results of the study, it was found that 10,11-dihydroerisodin compound can work on the Serine / threonine-protein kinase Chk1 receptor that serves as an anti-cancer candidate.

Keywords: anti-cancer, Erythrina poeppigiana, target receptor, 10, 11- dihidroerisodin

Procedia PDF Downloads 246

Authors: Antonela Matana, Dubravka Brdar, Vesela Torlak, Marijana Popovic, Ivana Gunjaca, Ozren Polasek, Vesna Boraska Perica, Maja Barbalic, Ante Punda, Caroline Hayward, Tatijana Zemunik

Abstract:

Parathyroid hormone (PTH) plays a critical role in the regulation of bone mineral metabolism and calcium homeostasis. Higher PTH levels are associated with heart failure, hypertension, coronary artery disease, cardiovascular mortality and poorer bone health. A twin study estimated that 60% of the variation in PTH concentrations is genetically determined. Only one GWAS of PTH concentration has been reported to date. Identified loci explained 4.5% of the variance in circulating PTH, suggesting that additional genetic variants remain undiscovered. Therefore, the aim of this study was to identify novel genetic variants associated with PTH levels in a general population. We have performed a GWAS meta-analysis on 2596 individuals originating from three Croatian cohorts: City of Split and the Islands of Korčula and Vis, within a large-scale project of “10,001 Dalmatians”. A total of 7 411 206 variants, imputed using the 1000 Genomes reference panel, with minor allele frequency ≥ 1% and Rsq ≥ 0.5 were analyzed for the association. GWAS within each data set was performed under an additive model, controlling for age, gender and relatedness. Meta-analysis was conducted using the inverse-variance fixed-effects method. Furthermore, to identify sex-specific effects, we have conducted GWAS meta-analyses analyzing males and females separately. In addition, we have performed biological pathway analysis. Four SNPs, representing one locus, reached genome-wide significance. The most significant SNP was rs11099476 on chromosome 4 (P=1.15x10-8), which explained 1.14 % of the variance in PTH. The SNP is located near the protein-coding gene RASGEF1B. Additionally, we detected suggestive association with SNPs, rs77178854 located on chromosome 2 in the DPP10 gene (P=2.46x10-7) and rs481121 located on chromosome 1 (P=3.58x10-7) near the GRIK1 gene. One of the top hits detected in the main meta-analysis, intron variant rs77178854 located within DPP10 gene, reached genome-wide significance in females (P=2.21x10-9). No single locus was identified in the meta-analysis in males. Fifteen biological pathways were functionally enriched at a P<0.01, including muscle contraction, ion homeostasis and cardiac conduction as the most significant pathways. RASGEF1B is the guanine nucleotide exchange factor, known to be associated with height, bone density, and hip. DPP10 encodes a membrane protein that is a member of the serine proteases family, which binds specific voltage-gated potassium channels and alters their expression and biophysical properties. In conclusion, we identified 2 novel loci associated with PTH levels in a general population, providing us with further insights into the genetics of this complex trait.

Keywords: general population, genome-wide association analysis, parathyroid hormone, single nucleotide polymorphisms.

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Authors: Sung-Hee Hwang, Michael Lee

Abstract:

Upregulated Src activity has been implicated in a variety of cancers. Thus, Src family tyrosine kinase (SFK) inhibitors are often effective cancer treatments. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas mediated genome editing. The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA–DNA complementarity to identify target sites for sequence specific double-stranded DNA (dsDNA) cleavage. Interestingly, ATG5 KO cells clearly showed a greater proliferation rate than WT NIH 3T3 cells, implying that autophagy induction is cytotoxic. Also, the clonogenic survival of ATG5 KO cells was greater than WT cells. The MTT assay revealed that the cytotoxic effect of PP2 was weaker on ATG5 knockout cells than that WT cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. Furthermore, Cyto-ID autophagy assay also revealed that PP2 failed to induce autophagy in ATG5 knockout cells. Together, our findings suggest that the resistance to PP2 in ATG5 knockout cells is associated with defective autophagy.

Keywords: ATG5 knockout, Autophagy, CRISPR/Cas9, PP2

Procedia PDF Downloads 347