Search results for: extracellular lignin- degrading enzymes

Authors: K. S. Zaytsev, Y. R. Nartsissov

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Glycine is one of the key inhibitory neurotransmitters in Central nervous system (CNS) meanwhile glycinergic transmission is highly dependable on its appropriate reuptake from synaptic cleft. Glycine transporters (GlyT) of types 1 and 2 are the enzymes providing glycine transport back to neuronal and glial cells along with Na⁺ and Cl⁻ co-transport. The distribution and stoichiometry of GlyT1 and GlyT2 differ in details, and GlyT2 is more interesting for the research as it reuptakes glycine to neuron cells, whereas GlyT1 is located in glial cells. In the process of GlyT2 activity, the translocation of the amino acid is accompanied with binding of both one chloride and three sodium ions consequently (two sodium ions for GlyT1). In the present study, we developed a computer simulator of GlyT2 and GlyT1 activity based on known experimental data for quantitative estimation of membrane glycine transport. The trait of a single protein functioning was described using the probability approach where each enzyme state was considered separately. Created scheme of transporter functioning realized as a consequence of elemental steps allowed to take into account each event of substrate association and dissociation. Computer experiments using up-to-date kinetic parameters allowed receiving the number of translocated glycine molecules, Na⁺ and Cl⁻ ions per time period. Flexibility of developed software makes it possible to evaluate glycine reuptake pattern in time under different internal characteristics of enzyme conformational transitions. We investigated the behavior of the system in a wide range of equilibrium constant (from 0.2 to 100), which is not determined experimentally. The significant influence of equilibrium constant in the range from 0.2 to 10 on the glycine transfer process is shown. The environmental conditions such as ion and glycine concentrations are decisive if the values of the constant are outside the specified range.

Keywords: glycine, inhibitory neurotransmitters, probability approach, single protein functioning

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Authors: V. M. Varagyan, G. A. Gevorgyan, K. V. Grigoryan, A. L. Varagyan

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Alaverdi region is situated in the northern part of the Republic of Armenia. Previous studies (1989) in Alaverdi region showed that due to soil irrigation with the highly polluted waters of the Debed and Shnogh rivers, the content of heavy metals in the brown forest steppe soils was significantly higher than the maximum permissible concentration as a result of which the fermentative activity in all the layers of the soils was stressed. Compared to the non-polluted soils, the activity of ferments in the plough layers of the highly polluted soils decreased by 44 - 68% (invertase – 60%, phosphatase – 44%, urease – 66%, catalase – 68%). In case of the soil irrigation with the polluted waters, a decrease in the intensity of fermentative reactions was conditioned by the high content of heavy metals in the soils and changes in chemical composition, physical and physicochemical properties. 20-year changes in the fermentative activity in the brown forest steppe soils in Alaverdi region were investigated. The activity of extracellular ferments in the soils was determined by the unification methods. The study has confirmed that self-recovery process occurs in soils previously polluted with heavy metals which can be revealed by fermentative activity. The investigations revealed that during 1989 – 2009, the activity of ferments in the plough layers of the medium and highly polluted soils increased by 31.2 – 52.6% (invertase – 31.2%, urease – 52.6%, phosphatase – 33.3%, catalase – 41.8%) and 24.1 – 87.0% (invertase – 40.4%, urease – 76.9%, phosphatase – 24.1%, catalase – 87.0%) respectively which indicated that the dynamic properties of the soils, which had been broken due to heavy metal pollution, were improved. In 1989, the activity of the Alaverdi copper smelting plant was temporarily stopped due to financial problems caused by the economic crisis and the absence of market, and the factory again started operation in 1997 and isn’t currently running at full capacity. As a result, the Debed river water has obtained a new chemical composition and comparatively good irrigation properties. Due to irrigation with this water, the gradually recovery of the soil dynamic properties, which had been broken due to irrigation with the waters polluted with heavy metals, was occurred. This is also explained by the fact that in case of irrigation with the partially cleaned water, the soil protective function against pollutants rose due to a content increase in humus and silt fractions. It is supposed that in case of the soil irrigation with the partially cleaned water, the intensity of fermentative reactions wasn’t directly affected by heavy metals.

Keywords: alaverdi region, heavy metal pollution, self-recovery, soil fermentative activity

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Authors: Jasna Čanadanović-Brunet, Gordana Ćetković, Sonja Djilas, Vesna Tumbas-Šaponjac, Jelena Vulić, Sladjana Stajčić

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Honey, produced by the honeybee, is a natural saturated sugar solution, which is mainly composed of a complex mixture of carbohydrates. Besides this, it also contains certain minor constituents, proteins, enzymes, amino and organic acids, lipids, vitamins, phenolic acids, flavonoids and carotenoids. Honey serves as a source of natural antioxidants, which are effective in reducing the risk of heart disease, cancer, immune-system decline, cataracts, and different inflammatory processes. Honey is consumed in its natural form alone, but also in combination with nuts and various kinds of dried fruits (plums, figs, cranberries, apricots etc.). The aim of this research was to investigate the contribution of dried apricot addition to polyphenols and flavonoids contents and antioxidant activities of honey. Some individual phenolic compounds in Serbian polyfloral honey (PH), linden honey (LH) and also in their mixtures with dried apricot, in 40% mass concentrations (PH40; LH40), were identified and quantified by HPLC. The most dominant phenolic compound was: gallic acid in LH (11.14 mg/100g), LH40 (42.65 mg/100g), PH (7.24 mg/100g) and catehin in PH40 (11.83 mg/100g). The antioxidant activity of PH, LH, PH40 and LH40 was tested by measuring their ability to scavenge hydroxyl radicals (OH) by electron spin resonance spectroscopy (ESR). Honey samples with 40% dried apricot exhibited better antioxidant activity measured by hydroxyl radical scavenging activity. The EC50 values, the amount of antioxidant necessary to decrease the initial concentration of OH radicals by 50%, were: EC50PH=3.36 mg/ml, EC50LH=13.36 mg/ml, EC50PH40=2.29 mg/ml, EC50 LH40=7.78 mg/ml. Our results indicate that supplementation of polyfloral honey and linden honey with dried apricots improves antioxidant activity of honey by enriching the phenolic composition.

Keywords: honey, dried apricot, HPLC, hydroxyl radical

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Authors: Boudjenah-Haroun Saliha, Isselnane Souad, Nouani Abdelwahab, Baaissa Babelhadj, Mati Abderrahmane

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Algeria is experiencing significant development of the dairy sector, where consumption of milk and milk products increased by 2.7 million tons in 2008 to 4,400,000 tons in 2013, and cheese production has reached 1640 tons in the year 2014 with average consumption of 0.7 kg/person/year. Although rennet is still the most used coagulating enzyme in cheese, its production has been growing worldwide shortage. This shortage is primarily due to a growing increase in the production and consumption of cheese, and the inability to increase in parallel the production of rennet. This shortage has caused very large fluctuations in its price). To overcome these obstacles, much research has been undertaken to find effective and competitive substitutes used industrially. For this, the selection of a local production of rennet substitute is desirable. It would allow a permanent supply with limited dependence on imports and price fluctuations. Investigations conducted by our research team showed that extracts coagulants from the stomachs of older camels are characterized by a coagulating power than those from younger camels. The objective of this work is to study the possibility of substituting commercial rennet coagulant by gastric enzymes from adult camels for coagulation bovine milk. Excerpts from the raw camel coagulants obtained are characterized through their teneures proteins and clotting and proteolytic activities. Milk clotting conditions by the action of these extracts were optimized. Milk clotting time all treated with enzyme preparations and under different conditions was calculated. Bovine rennet has been used for comparison. The results show that crude extracts from gastric adult camel can be good substituting bovine rennet.

Keywords: Algeria, camel, cheese, coagulation, gastric extracts, milk

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Authors: Felicia Orah Rein Moshe

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Soil erosion has become a priority global concern, impairing water quality and degrading ecosystem services. In Mediterranean climates, following a long dry period, the onset of rain occurs when agricultural soils are often bare and most vulnerable to erosion. Early storms transport sediments and sediment-bound pollutants into streams, along with dissolved chemicals. This results in loss of valuable topsoil, water quality degradation, and potentially expensive dredged-material disposal costs. Information on the provenance of fine sediment and priority sources of adsorbed pollutants represents a critical need for developing effective control strategies aimed at source reduction. Modifying sediment traps designed for marine systems, this study tested a cost-effective method to collect suspended sediments on a catchment scale to characterize stream water quality during first-flush storm events in a flashy Eastern Mediterranean coastal perennial stream. This study investigated the Kishon Basin, deploying sediment traps in 23 locations, including 4 in the mainstream and one downstream in each of 19 tributaries, enabling the characterization of sediment as a vehicle for transporting chemicals. Further, it enabled direct comparison of sediment-bound pollutants transported during the first-flush winter storms of 2020 from each of 19 tributaries, allowing subsequent ecotoxicity ranking. Sediment samples were successfully captured in 22 locations. Pesticides, pharmaceuticals, nutrients, and metal concentrations were quantified, identifying a total of 50 pesticides, 15 pharmaceuticals, and 22 metals, with 16 pesticides and 3 pharmaceuticals found in all 23 locations, demonstrating the importance of this transport pathway. Heavy metals were detected in only one tributary, identifying an important watershed pollution source with immediate potential influence on long-term dredging costs. Simultaneous sediment sampling at first flush storms enabled clear identification of priority tributaries and their chemical contributions, advancing a new national watershed monitoring approach, facilitating strategic plan development based on source reduction, and advancing the goal of improving the farm-stream interface, conserving soil resources, and protecting water quality.

Keywords: adsorbed pollution, dredged material, heavy metals, suspended sediment, water quality monitoring

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Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi

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Seed aging during storage is a complex biochemical and physiological processes that can lead to reduce seed germination. This phenomenon associated with increasing of total antioxidant activity during aging. To study the effects of hormones on seed aging, aged wheat seeds (control, 90 and 80% viabilities) were treated with GA3, Salicylic Acid, and paclobutrazol and antioxidant system were investigated as molecular biomarkers for seed vigor. The results showed that, seed priming treatment significantly affected germination percentage, normality seedling percentage, H2O2, MDA, CAT, APX, and GPX activates. Maximum germination percentage achieve in GA3 priming in control treatment. Germination percentage and normal seedling percentage increased in other GA3 priming treatment compared with other hormones. Also aging increased MDA, H2O2 content. MDA is considered sensitive marker commonly used for assessing membrane lipid peroxidation and H2O2result in toxicity to cellular membrane system and damages to plant cells. Amount of H2O2 and MDA declined in GA3 treatment. CAT, GPX and APX activities were reduced by increasing the aging time and at different levels of priming. The highest APX activity was observed in Salicylic Acid control treatment and the highest GPX and CAT activity was obtained in GA3 control treatment. The lowest MDA and H2O2 showed in GA3 control treatment, too. Hormone priming increased Antioxidant enzyme activity and decreased amount of reactive oxygen space and malondialdehyde (MDA) under aging treatment. Also, GA3 priming treatments have a significant effect on germination percentage and number of normal seedling. Generally aging seed, increase ROS and lipid peroxidation. Antioxidant enzymes activity of aged seeds increased after hormone priming.

Keywords: hormones priming, wheat, aging seed, antioxidant, lipid peroxidation

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Authors: Sasangka Prasetyawan, Anna Roosdiana, Diah Mardiana, Suratmo

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Pectinase are enzymes that hydrolyze pectin compounds. The use of this enzyme is primarily to reduce the viscosity of the beverage thus simplifying the purification process. Pectinase activity influenced by microbial sources . Exploration of two types of microbes that Aspergillus spp. and Bacillus spp. pectinase give different performance, but the use of local strain is still not widely studied. The aim of this research is exploration of pectinase from A. niger and B. firmus include production conditions and characterization. Bacillus firmus incubated and shaken at a speed of 200 rpm at pH variation (5, 6, 7, 8, 9, 10), temperature (30, 35, 40, 45, 50) °C and incubation time (6, 12, 18, 24, 30, 36 ) hours. Media was centrifuged at 3000 rpm, pectinase enzyme activity determined. Enzyme production by A. niger determined to variations in temperature and pH were similar to B. firmus, but the variation of the incubation time was 24, 48, 72, 96, 120 hours. Pectinase crude extract was further purified by precipitation using ammonium sulfate saturation in fraction 0-20 %, 20-40 %, 40-60 %, 60-80 %, then dialyzed. Determination of optimum conditions pectinase activity performed by measuring the variation of enzyme activity on pH (4, 6, 7, 8, 10), temperature (30, 35, 40, 45, 50) °C, and the incubation time (10, 20, 30, 40, 50) minutes . Determination of kinetic parameters of pectinase enzyme reaction carried out by measuring the rate of enzyme reactions at the optimum conditions, but the variation of the concentration of substrate (pectin 0.1 % , 0.2 % , 0.3 % , 0.4 % , 0.5 % ). The results showed that the optimum conditions of production of pectinase from B. firmus achieved at pH 7-8.0, 40-50 ⁰C temperature and fermentation time 18 hours. Purification of pectinase showed the highest purity in the 40-80 % ammonium sulfate fraction. Character pectinase obtained : the optimum working conditions of A. niger pectinase at pH 5 , while pectinase from B. firmus at pH 7, temperature and optimum incubation time showed the same value, namely the temperature of 50 ⁰C and incubation time of 30 minutes. The presence of metal ions can affect the activity of pectinase , the concentration of Zn 2 + , Pb 2 + , Ca 2 + and K + and 2 mM Mg 2 + above 6 mM inhibit the activity of pectinase .

Keywords: pectinase, Bacillus firmus, Aspergillus niger, green chemistry

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Authors: Naso Isaiah Thanavisuth

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Introduction: Medical cannabis can be used for treatment, including anorexia, pain, inflammation, multiple sclerosis, Parkinson's disease, epilepsy, cancer, and metabolic syndrome-related disorders. However, medical cannabis leads to adverse effects (AEs), which is delta-9-tetrahydrocannabinol (THC). In previous studies, the major of THC metabolism enzymes are CYP2C9. Especially, the variation of CYP2C9 gene consist of CYP2C9*2 on exon 3 (C430T) (Arg144Cys) and CYP2C9*3 on exon 7 (A1075C) (Ile359Leu) to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are a pharmacogenetics marker for prediction of THC-induced AEs in Thai patients. Objective: We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. Method: We enrolled 39 Thai patients with medical cannabis treatment consisting of men and women who were classified by clinical data. The quality of DNA extraction was assessed by using NanoDrop ND-1000. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay (ABI, Foster City, CA, USA). Results: All Thai patients who received the medical cannabis consist of twenty four (61.54%) patients who were female and fifteen (38.46%) were male, with age range 27- 87 years. Moreover, the most AEs in Thai patients who were treated with medical cannabis between cases and controls were tachycardia, arrhythmia, dry mouth, and nausea. Particularly, thirteen (72.22%) medical cannabis-induced AEs were female and age range 33 – 69 years. In this study, none of the medical cannabis groups carried CYP2C9*2 variants in Thai patients. The CYP2C9*3 variants (*1/*3, intermediate metabolizer, IM) and (*3/*3, poor metabolizer, PM) were found, three of thirty nine (7.69%) and one of thirty nine (2.56%) , respectively. Conclusion: This is the first study to confirm the genetic polymorphism of CYP2C9 and medical cannabis-induced AEs in the Thai population. Although, our results indicates that there is no found the CYP2C9*2. However, the variation of CYP2C9 allele might serve as a pharmacogenetics marker for screening before initiating the therapy with medical cannabis for prevention of medical cannabis-induced AEs.

Keywords: CYP2C9, medical cannabis, adverse effects, THC, P450

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Authors: Monisha Isaac

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Biotechnology is a discipline which explains the use of living organisms and systems to construct a product, or we can define it as an application or technology developed to use biological systems and organisms processes for a specific use. Particularly, it includes cells and its components use for new technologies and inventions. The tools developed can be further used in diverse fields such as agriculture, industry, research and hospitals etc. The 21^st century has seen a drastic development and advancement in biotechnology in India. Significant increase in Government of India’s outlays for biotechnology over the past decade has been observed. A sectoral break up of biotechnology-based companies in India shows that most of the companies are agriculture-based companies having interests ranging from tissue culture to biopesticides. Major attention has been given by the companies in health related activities and in environmental biotechnology. The biopharmaceutical, which comprises of vaccines, diagnostic, and recombinant products is the most reliable and largest segment of the Indian Biotech industry. India has developed its vaccine markets and supplies them to various countries. Then there are the bio-services, which mainly comprise of contract researches and manufacturing services. India has made noticeable developments in the field of bio industries including manufacturing of enzymes, biofuels and biopolymers. Biotechnology is also playing a crucial and significant role in the field of agriculture. Traditional methods have been replaced by new technologies that mainly focus on GM crops, marker assisted technologies and the use of biotechnological tools to improve the quality of fertilizers and soil. It may only be a small contributor but has shown to have huge potential for growth. Bioinformatics is a computational method which helps to store, manage, arrange and design tools to interpret the extensive data gathered through experimental trials, making it important in the design of drugs.

Keywords: biotechnology, advancement, agriculture, bio-services, bio-industries, bio-pharmaceuticals

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Authors: Muhammad Naveed Aslam, Rida Fatima, Anam Moosa, Muhammad Taimoor Shakeel

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Bacillus strains produce antifungal secondary metabolites making them potential candidates for suppressing Fusarium wilt of chickpea disease. In this study, eighteen Bacillus strains were evaluated for their antagonistic effect against Fusarium oxysporum f. sp. ciceris causing Fusarium wilt of chickpea disease. In a direct antifungal assay, thirteen strains showed significant inhibition zones while the remaining five strains did not produce inhibition zones of FOC. Bacillus thuringiensis CHGP12 was the most promising strain exhibiting the highest inhibition of FOC. Antifungal lipopeptides were extracted from CHGP12 strain which showed significant inhibition of the pathogen. Liquid chromatography mass spectrometry (LCMS) analysis revealed that CHGP12 was positive for the presence of iturin, fengycin, surfactin, bacillaene, bacillibactin, plantazolicin, and bacilysin. CHGP12 was tested for biochemical determinants in an in vitro qualitative test where it showed the ability to produce lipase, amylase, cellulase, protease, siderophores, and indole 3-acetic acid (IAA). Furthermore, in a greenhouse experiment CHGP12 also showed a significant decrease in the disease severity in treated plants compared to control. Moreover, CHGP12 also exhibited a significant increase in plant growth parameters viz, root and shoot growth parameters, stomatal conductance, and photosynthesis rate. Conclusively, our findings present the promising potential of Bacillus strain CHGP12 to suppress Fusarium wilt of chickpea and to promote plant growth.

Keywords: liquid chromatography mass spectrometry, growth promotion, antagonism, hydrolytic enzymes, inhibition, lipopeptides.

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Authors: Jos Olijve

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Introduction and Purpose: Endotoxins are found in the outer membrane of gram-negative bacteria and have profound in vitro and in vivo responses. They can trigger strong immune responses and negatively affect various cellar activities particular cells expressing toll-like receptors. They are therefore unwanted contaminants of biomaterials sourced from natural raw materials, and their activity must be as low as possible. Collagen and gelatin are natural extracellular matrix components and have, due to their low allergenic potential, suitable biological properties, and tunable physical characteristics, high potential in biomedical applications. The purpose of this study was to determine the influence of autoclave sterilization of gelatin on physical properties and endotoxin level compared to surfactant purified gelatin. Methods: Type A gelatin from Sigma-Aldrich (G1890) with endotoxin level of 35000 endotoxin units (EU) per gram gelatin and type A gelatins from Rousselot Gent with endotoxin activity of 30000 EU per gram were used. A 10 w/w% G1890 gelatin solution was autoclave sterilized during 30 minutes at 121°C and 1 bar over pressure. The physical properties and the endotoxin level of the sterilized G1890 gelatin were compared to a type A gelatin from Rousselot purified with Triton X100 surfactant. The Triton X100 was added to a concentration of 0.5 w/w% which is above the critical micellar concentration. The gelatin surfactant mixtures were kept for 30-45 minutes under constant stirring at 55-60°C. The Triton X100 was removed by active carbon filtration. The endotoxin levels of the gelatins were measured using the Endozyme recombinant factor C method from Hyglos GmbH (Germany). Results and Discussion: Autoclave sterilization significantly affect the physical properties of gelatin. Molecular weight of G1890 decreased from 140 to 50kDa, and gel strength decreased from 300 to 40g. The endotoxin level of the gelatin reduced after sterilization from 35000 EU/g to levels of 400-500 EU/g. These endotoxin levels are however still far above the upper endotoxin level of 0.05 EU/ml, which resembles 5 EU/g gelatin based on a 1% gelatin solution, to avoid cell proliferation alteration. Molecular weight and gel strength of Rousselot gelatin was not altered after Triton X100 purification and remained 150kDa and 300g respectively. The endotoxin levels of Triton X100 purified Rousselot gelatin was < 5EU/g gelatin. Conclusion: Autoclave sterilization of gelatin is, in comparison to Triton X100 purification, not efficient to inactivate endotoxin levels in gelatin to levels below the upper limit to avoid cell proliferation alteration. Autoclave sterilization gave a significant decrease in molecular weight and gel strength which makes autoclave sterilized gelatin, in comparison to Triton X100 purified gelatin, not suitable for 3D printing.

Keywords: endotoxin, gelatin, molecular weight, sterilization, Triton X100

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Authors: Dharmendra Pratap

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Large Cardamom (Amomum subulatum), family Zingiberaceae is an aromatic spice crop and has rich medicinal value. Large Cardamom is as synonymous to Sikkim as Tea is to Darjeeling. Since Sikkim alone contributes up to 88% of India's large cardamom production which is the world leader by producing over 50% of the global yield. However, the production of large cardamom has declined almost to half since last two decade. The economic losses have been attributed to two viral diseases namely, chirke and Foorkey. Chirke disease is characterized by light and dark green streaks on leaves. The affected leaves exhibit streak mosaic, which gradually coalesce, turn brown and eventually dry up. Excessive sprouting and formation of bushy dwarf clumps at the base of mother plants that gradually die characterize the foorkey disease. In our surveys in Sikkim–Darjeeling hill area during 2012-14, 40-45% of plants were found to be affected with foorkey disease and 10-15% with chirke. Mechanical and aphid transmission study showed banana as an alternate host for both the disease. For molecular identification, total genomic DNA and RNA was isolated from the infected leaf tissues and subjected to Rolling circle amplification (RCA) and RT-PCR respectively. The DNA concatamers produced in the RCA reaction were monomerized by different restriction enzymes and the bands corresponding to ~1 kb genomes were purified and cloned in the respective sites. The nucleotide sequencing results revealed the association of Nanovirus with the foorkey disease of large cardamom. DNA1 showed 74% identity with Replicase gene of FBNYV, DNA2 showed 77% identity with the NSP gene of BBTV and DNA3 showed 74% identity with CP gene of BBTV. The finding suggests the presence of a new species of nanovirus associated with foorkey disease of large cardamom in Sikkim and Darjeeling hills. The details of their epidemiology and other factors would be discussed.

Keywords: RCA, nanovirus, large cardamom, molecular virology and microbiology

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Authors: Farah Diab, Mohamad Khalil, Francesca Storace, Francesca Baldini, Piero Portincasaa, Giulio Lupidi, Laura Vergani

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Thymbra spicata is a thyme-like plant belonging to the Lamiaceae family that shows a global distribution, especially in the eastern Mediterranean region. Leaves of T. spicata contain large amounts of phenols such as phenolic acids (rosmarinic acid), phenolic monoterpenes (carvacrol), and flavonoids. In Lebanon, T. spicata is currently used as a culinary herb in salad and infusion, as well as for traditional medicinal purposes. Carvacrol (5-isopropyl-2-methyl phenol), the most abundant polyphenol in the organic extract and essential oils, has a great array of pharmacological properties. In fact, carvacrol is largely employed as a food additive and neutraceutical agent. Our aim is to investigate the beneficial effects of T. spicata ethanolic extract (TE) and its main component, carvacrol, using in vitro models of hepatic steatosis and endothelial dysfunction. As a further point, we focused on investigating if and how the binding of carvacrol to albumin, the physiological transporter for drugs in the blood, might be altered by the presence of high levels of fatty acids (FAs), thus impairing the carvacrol bio-distribution in vivo. For that reason, hepatic FaO cells treated with exogenous FAs such as oleate and palmitate mimic hepatosteatosis; endothelial HECV cells exposed to hydrogen peroxide are a model of endothelial dysfunction. In these models, we measured lipid accumulation, free radical production, lipoperoxidation, and nitric oxide release before and after treatment with carvacrol. The carvacrol binding to albumin with/without high levels of long-chain FAs was assessed by absorption and emission spectroscopies. Our findings show that both TE and carvacrol (i) counteracted lipid accumulation in hepatocytes by decreasing the intracellular and extracellular lipid contents in steatotic FaO cells; (ii) decreased oxidative stress in endothelial cells by significantly reducing lipoperoxidation and free radical production, as well as, attenuating the nitric oxide release; (ii) high levels of circulating FAs reduced the binding of carvacrol to albumin. The beneficial effects of TE and carvacrol on both hepatic and endothelial cells point to a nutraceutical potential. However, high levels of circulating FAs, such as those occurring in metabolic disorders, might hinder the carvacrol transport, bio-distribution, and pharmacodynamics.

Keywords: carvacrol, endothelial dysfunction, fatty acids, non-alcoholic fatty liver diseases, serum albumin

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Authors: A. Mulry, C. Kealey, D. B. Brady

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Planktonic bacteria can form biofilms which are microbial aggregates embedded within a matrix of extracellular polymeric substances (EPS). They can be found attached to abiotic or biotic surfaces. Biofilms are responsible for oral diseases such as dental caries, gingivitis and the progression of periodontal disease. Biofilms can resist 500 to 1000 times the concentration of biocides and antibiotics used to kill planktonic bacteria. Biofilm development on oral surfaces involves four stages, initial attachment, early development, maturation and dispersal of planktonic cells. The Minimum Inhibitory Concentration (MIC) was determined using a range of saturated and unsaturated fatty acids using the resazurin assay, followed by serial dilution and spot plating on BHI agar plates to establish the Minimum Bactericidal Concentration (MBC). Log reduction of bacteria was also evaluated for each fatty acid. The Minimum Biofilm Inhibition Concentration (MBIC) was determined using crystal violet assay in 96 well plates on forming and pre-formed S. mutans biofilms using BHI supplemented with 1% sucrose. Saturated medium-chain fatty acids Octanoic (C8.0), Decanoic (C10.0) and Undecanoic acid (C11.0) do not display strong antibiofilm properties; however, Lauric (C12.0) and Myristic (C14.0) display moderate antibiofilm properties with 97.83% and 97.5% biofilm inhibition with 1000 µM respectively. Monounsaturated, Oleic acid (C18.1) and polyunsaturated large chain fatty acids, Linoleic acid (C18.2) display potent antibiofilm properties with biofilm inhibition of 99.73% at 125 µM and 100% at 65.5 µM, respectively. Long-chain polyunsaturated Omega-3 fatty acids α-Linoleic (C18.3), Eicosapentaenoic Acid (EPA) (C20.5), Docosahexaenoic Acid (DHA) (C22.6) have displayed strong antibiofilm efficacy from concentrations ranging from 31.25-250µg/ml. DHA is the most promising antibiofilm agent with an MBIC of 99.73% with 15.625µg/ml. This may be due to the presence of six double bonds and the structural orientation of the fatty acid. To conclude, fatty acids displaying the most antimicrobial activity appear to be medium or long-chain unsaturated fatty acids containing one or more double bonds. Most promising agents include Omega-3-fatty acids Linoleic, α-Linoleic, EPA and DHA, as well as Omega-9 fatty acid Oleic acid. These results indicate that fatty acids have the potential to be used as antimicrobials and antibiofilm agents against S. mutans. Future work involves further screening of the most potent fatty acids against a range of bacteria, including Gram-positive and Gram-negative oral pathogens. Future work will involve incorporating the most effective fatty acids onto dental implant devices to prevent biofilm formation.

Keywords: antibiofilm, biofilm, fatty acids, S. mutans

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Authors: Osvaldo N. Oliveira Jr.

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The control of molecular architecture inherent in some experimental methods to produce nanostructured films has had great impact on devices of various types, including sensors and biosensors. The self-assembly monolayers (SAMs) and the electrostatic layer-by-layer (LbL) techniques, for example, are now routinely used to produce tailored architectures for biosensing where biomolecules are immobilized with long-lasting preserved activity. Enzymes, antigens, antibodies, peptides and many other molecules serve as the molecular recognition elements for detecting an equally wide variety of analytes. The principles of detection are also varied, including electrochemical methods, fluorescence spectroscopy and impedance spectroscopy. In this presentation an overview will be provided of biosensors made with nanostructured films to detect antibodies associated with tropical diseases and HIV, in addition to detection of analytes of medical interest such as cholesterol and triglycerides. Because large amounts of data are generated in the biosensing experiments, use has been made of computational and statistical methods to optimize performance. Multidimensional projection techniques such as Sammon´s mapping have been shown more efficient than traditional multivariate statistical analysis in identifying small concentrations of anti-HIV antibodies and for distinguishing between blood serum samples of animals infected with two tropical diseases, namely Chagas´ disease and Leishmaniasis. Optimization of biosensing may include a combination of another information visualization method, the Parallel Coordinate technique, with artificial intelligence methods in order to identify the most suitable frequencies for reaching higher sensitivity using impedance spectroscopy. Also discussed will be the possible convergence of technologies, through which machine learning and other computational methods may be used to treat data from biosensors within an expert system for clinical diagnosis.

Keywords: clinical diagnosis, information visualization, nanostructured films, layer-by-layer technique

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Authors: Bhojan Dhakal, Naba Raj Devkota, Chet Raj Upreti, Maheshwar Sapkota

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A study was conducted to find out the production potential, nutrient composition, and the variability of the most commonly available fodder trees along with the varying altitude to help optimize the dry matter requirement during winter lean period. The study was carried out from March to June, 2012 in Lho and Prok Village Development Committee of Manaslu Conservation Area (MCA), located in Gorkha district of Nepal. The other objective of the research was to learn the impact of climate change on livestock production linking it with feed availability. The study was conducted in two parts: social and biological. Accordingly, a households (HHs) survey was conducted to collect primary data from 70 HHs, focusing on the perception of respondents on impacts of climatic variability on the feeding management. The next part consisted of understanding yield potential and nutrient composition of the four most commonly available fodder trees (M. azedirach, M. alba, F. roxburghii, F. nemoralis), within two altitudes range: (1500-2000 masl and 2000-2500 masl) by using a RCB design in 2*4 factorial combination of treatments, each replicated four times. Results revealed that majority of the farmers perceived the change in climatic phenomenon more severely within the past five years. Farmers were using different adaptation technologies such as collection of forage from jungle, reducing unproductive animals, fodder trees utilization, and crop by product feeding at feed scarcity period. Ranking of the different fodder trees on the basis of indigenous knowledge and experiences revealed that F. roxburghii was the best-preferred fodder tree species (index value 0.72) in terms overall preferability whereas M. azedirach had highest growth and productivity (index value 0.77), F. roxburghii had highest adoptability (index value 0.69) and palatability (index value 0.69) as well. Similarly, fresh yield and dry matter yield of the each fodder trees was significant (P < 0.01) between the altitude and within species. Fodder trees yield analysis revealed that the highest dry matter (DM) yield (28 kg/tree) was obtained for F. roxburghii but that remained statistically similar (P > 0.05) to the other treatment. On the other hand, most of the parameters: ether extract (EE), acid detergent lignin (ADL), acid detergent fibre (ADF), cell wall digestibility (CWD), relative digestibility (RD), digestible nutrient (TDN), and Calcium (Ca) among the treatments were highly significant (P < 0.01). This indicates the scope of introducing productive and nutritive fodder trees species even at the high altitude to help reduce fodder scarcity problem during winter. The finding also revealed the scope of promoting all available local fodder trees species as crude protein content of these species were similar.

Keywords: fodder trees, yield potential, climate change, nutrient composition

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Authors: Areej K. Al-Jwaid, Dmitiry Berllio, Andrew Cundy, Irina Savina, Jonathan L. Caplin

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Phenol is a toxic compound that is widely distributed in the environment including the atmosphere, water and soil, due to the release of effluents from the petrochemical and pharmaceutical industries, coking plants and oil refineries. Moreover, a range of daily products, using phenol as a raw material, may find their way into the environment without prior treatment. The toxicity of phenol effects both human and environment health, and various physio-chemical methods to remediate phenol contamination have been used. While these techniques are effective, their complexity and high cost had led to search for alternative strategies to reduce and eliminate high concentrations of phenolic compounds in the environment. Biological treatments are preferable because they are environmentally friendly and cheaper than physico-chemical approaches. Some microorganisms such as Pseudomonas sp., Rhodococus sp., Acinetobacter sp. and Bacillus sp. have shown a high ability to degrade phenolic compounds to provide a sole source of energy. Immobilisation process utilising various materials have been used to protect and enhance the viability of cells, and to provide structural support for the bacterial cells. The aim of this study is to develop a new approach to the bioremediation of phenol based on an immobilisation strategy that can be used in wastewater. In this study, two bacterial species known to be phenol degrading bacteria (Pseudomonas mendocina and Rhodococus koreensis) were purchased from National Collection of Industrial, Food and Marine Bacteria (NCIMB). The two species and mixture of them were immobilised to produce macro porous crosslinked cell cryogels samples by using four types of cross-linker polymer solutions in a cryogelation process. The samples were used in a batch culture to degrade phenol at an initial concentration of 50mg/L at pH 7.5±0.3 and a temperature of 30°C. The four types of polymer solution - i. glutaraldehyde (GA), ii. Polyvinyl alcohol with glutaraldehyde (PVA+GA), iii. Polyvinyl alcohol–aldehyde (PVA-al) and iv. Polyetheleneimine–aldehyde (PEI-al), were used at different concentrations, ranging from 0.5 to 1.5% to crosslink the cells. The results of SEM and rheology analysis indicated that cell-cryogel samples crosslinked with the four cross-linker polymers formed monolithic macro porous cryogels. The samples were evaluated for their ability to degrade phenol. Macro porous cell–cryogels crosslinked with GA and PVA+GA showed an ability to degrade phenol for only one week, while the other samples crosslinked with a combination of PVA-al + PEI-al at two different concentrations have shown higher stability and viability to reuse to degrade phenol at concentration (50 mg/L) for five weeks. The initial results of using crosslinked cell cryogel samples to degrade phenol indicate that is a promising tool for bioremediation strategies especially to eliminate and remove the high concentration of phenol in wastewater.

Keywords: bioremediation, crosslinked cells, immobilisation, phenol degradation

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Authors: B. Dejanova, S. Petrovska, L. Todorovska, J. Pluncevic, S. Mancevska, V. Antevska, E. Sivevska, I. Karagjozova

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Introduction: Aging process is mainly related to endothelial function which may be impaired by oxidative stress (OS). Exercise is known to be beneficial to aging process, which may improve health and prevent appearance of chronic diseases in elderly. The aim of the study was to investigate the OS markers related to exercise. Methods: A number of 80 subjects (healthy volunteers) were examined (38 male and 32 female), divided in 3 age groups: group I ≤ 30 years (n=24); group II – 31-50 years (n=24); group III - ≥ 51 year (n=32). Each group was divided to subgroups of sedentary subjects (SS) and subjects who exercise (SE). Group I: SS (n=11), SE (n=13); group II: SS (n=13), SE (n=10); group III: SS (n=23) SE (n=9). Lipid peroxidation (LP) as a fluorimetric method with thiobarbituric acid was used to estimate OS. Antioxidative status was determined by cell antioxidants such as enzymes - superoxide dismutase (SOD), glutathione peroxidase (GPx) and glucose 6 phosphate (G-6-PD); and by extra cell antioxidants such as glutathione reductase (GR), nitric oxide (NO) and total antioxidant capacity (TAC). Results: Increased values of LP were noticed along the aging process: group I – 3.30±0.3 µmol/L; group II – 3.91±0.2 µmol/L; group III – 3.94±0.8 µmol/L (p<0.05), while no statistical significance was found between male and female subjects. Statistical significance for OS was not found between SS and SE in group I as it was found in group II (p<0.05) and in group III (p<0.01). No statistical significance was found for all cell antioxidants and GR within the groups, while NO and TAC showed lower values in SS compared to SE in II (p<0.05) and in group III (p<0.05). Discussion and conclusion: Aging process showed increased OS which may be either due to impaired function of scavengers of free radicals or due to their enormous production. Well balanced exercise might be one of the factors that keep the integrity of blood vessel endothelium which slows down the aging process. Possible mechanism of exercise beneficial influence is shear stress by upregulation of genes coding for nitric oxide bioavailability. Thus, due to obtained results we may conclude that OS is found to be diminished in the subject groups who perform exercise.

Keywords: oxidative stress, aging process, exercise, endothelial function

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Authors: Said Ghalem

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Honey is primarily composed of sugars: glucose and fructose. Depending honey, it's either fructose or glucose predominates. More the fructose concentration and the less the glycemic index (GI) is high. Thus, changes in the insulin response shows a decrease of the amount of insulin secreted at an increased fructose honey. Honey is also a compound that can reduce the lipid in the blood. Several studies on animals, but which remain to be checked in humans, have shown that the honey can have interesting effects when combined with other molecules: associated with Metformin (a medicine taken by diabetics), it shows the benefits and effects of diabetes preserves the tissue; associated ginger, it increases the antioxidant activity and thus avoids neurologic complications, neuropathic. Molecular modeling techniques are widely used in chemistry, biology, and the pharmaceutical industry. Most of the currently existing drugs target enzymes. Inhibition of DPP-4 is an important approach in the treatment of type 2 diabetes. We have chosen for the inhibition of DPP-4 the following molecules: Linagliptin (BI1356), Sitagliptin (Januvia), Vildagliptin, Saxagliptin, Alogliptin, and Metformin (Glucophage), that are involved in the disease management of type 2 diabetes and added to honey. For this, we used software Molecular Operating Environment. A Wistar rat study was initiated in our laboratory with a well-studied protocol; after sacrifice, according to international standards and respect for the animal This theoretical approach predicts the mode of interaction of a ligand with its target. The honey can have interesting effects when combined with other molecules, it shows the benefits and effects of honey preserves the tissue, it increases the antioxidant activity, and thus avoids neurologic complications, neuropathic or macrovascular. The organs, especially the kidneys of Wistar, shows that the parameters to renal function let us conclude that damages caused by diabetes are slightly perceptible than those observed without the addition of a high concentration of fructose honey.

Keywords: honey, molecular modeling, DPP4 enzyme, metformin

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Authors: Bellinda R. Chinowawa

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As part of a wider strategy to stifle civil society, Governments routinely resort to judicial harassment through the use of civil and criminal to impugn the integrity of human rights defenders and that of perceived political opponents. This phenomenon is rife in militarised or autocratic regimes where there is no tolerance for dissenting voices. Zimbabwe, ostensibly a presidential republic founded on the values of transparency, equality, freedom, is characterised by brutal suppression of perceived political opponents and those who assert their basic human rights. This is done through a wide range of tactics including unlawful arrests and detention, torture and other cruel, inhuman degrading treatment and enforced disappearances. Professionals including, journalists and doctors are similarly not spared from state attack. For human rights defenders, the most widely used tool of repression is that of judicial harassment where the judicial system is used to persecute them. This can include the levying of criminal charges, civil lawsuits and unnecessary administrative proceedings. Charges preferred against range from petty offences such as criminal nuisance to more serious charges of terrorism and subverting a constitutional government. Additionally, government sponsored individuals and organisations file strategic lawsuits with pecuniary implications order to intimidate and silence critics and engender self-censorship. Some HRDs are convicted and sentenced to prison terms, despite not being criminals in a true sense. While others are acquitted judicial harassment diverts energy and resources away from their human rights work. Through a consideration of statistical data reported by human rights organisations and face to face interviews with a cross section of human rights defenders, the article will map the incidence of judicial harassment in Zimbabwe. The article will consider the multi-level sociological and contextual factors which influence the Government of Zimbabwe to have easy recourse to criminal law and the debilitating effect of these actions on HRDs. These factors include the breakdown of the rule of law resulting in state capture of the judiciary, the proven efficacy of judicial harassment from colonial times to date, and the lack of an adequate redress mechanism at international level. By mapping the use of the judiciary as a tool of repression, from the inception of modern day Zimbabwe to date, it is hoped that HRDs will realise that they are part of a greater community of activists throughout the ages and should emboldened in the realisation that it is an age old tactic used by fallen regimes which should not deter them from calling for accountability.

Keywords: autocratic regime, colonial legacy, judicial harassment, human rights defenders

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Authors: A. K. Asekunowo, A O. T. Asafa, O. O. Okoh, O. T. Asekun, O. B. Familoni

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Background: Acalypha godseffiana is an important plant used both as an ornamental and herbs; its leaves are employed in management of diseases such as diabetics in Eastern Nigeria. Aim: The correlations of the polyphenolic compounds in the hypoglycemic potential of different extracts of leaves of A. godseffiana and their safety profile on cell lines were investigated. Materials and Methods: The phytochemical compositions and antioxidants potentials were determined using adopted methods. An in vitro approach was employed in determining the hypoglycemic potentials of the extracts on α-amylase and α-glucosidase. The Line weaver-Burke plot was used to evaluate the mechanisms of Inhibition mechanisms of the enzymes. Results and Conclusions: Antioxidants results revealed that total antioxidant capacity (TAC) of the acetone extract (IC50: 0.34 mg/mL) showed better activity compared to the standards (silymarine 0.52 mg/mL; gallic acid 0.51 mg/mL). In-vitro hypoglycemic activity of the extracts confirmed that acetone extract demonstrated strong and mild inhibitory potential against α-amylase and α-glucosidase respectively. The observed activity was concentration-dependent with IC50 values of 2.33 and 0.13 mg/mL. The observed hypoglycemic and anti-oxidant potentials of acetone extract A. godseffiana correlate to its high polyphenolic contents which include phenols (133.20 mg gallic acid g-1), flavonoid (350.60 mg quercetin g-1) and tannins (264.67 mg catechin g-1). The mechanisms of action exhibited by acetone extract of A. godseffiana were mixed non-competitive and uncompetitive; which can be attributed to its inhibitory properties on α-amylase and α-glucosidase respectively. This effect would cause reduction in the rate at which starch hydrolyse, boost palliated glucose levels; hence, making acetone extract of A. godseffiana a potential anti-hypoglycemic alternative.

Keywords: Acalypha godeseffiana, acetone extract, anti-hypoglycemia, antioxidant, phytochemicals

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Authors: Deepak Bhardwaj, Narendra Tuteja

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Global climate change is impacting large agrarian societies, especially those in countries located near the equator. Agriculture, and consequently, plant-based food, is the hardest hit in tropical and sub-tropical countries such as India due to an increased incidence of drought as well as an increase in soil salinity. One method that holds promise is AMF-rich biofertilizers which assist in activating proteins which in turn help alleviate abiotic stress in plants. In the present study, we identified two important species of (arbuscular mycorrhizal fungus) AMF belonging to Glomus and Gigaspora from the rhizosphere of the important medicinal plant Justicia adathoda. These two species have been found to be responsible for the abundance of Justicia adathoda in the semi-arid areas of the Jammu valley located in northern India, namely, the Union Territory of Jammu and Kashmir. We isolated the species of Glomus and Gigaspora from the rhizosphere of Justicia adathoda and used them as biofertilizers for the tomato plant. Significant improvements in the growth parameters were observed in the tomato plants inoculated with Glomus sp. and Gigaspora sp. in comparison with the tomato plants that were grown without AMF treatments. Tomato plants grown along with Glomus sp. and Gigaspora sp. have been observed to withstand 200 mM of salinity and 25% PEG stress. AMF also resulted in an increased concentration of proline and antioxidant enzymes in tomato plants. We also examined the expression levels of salinity and drought stress-inducible genes such as pea DNA helicase 45 (PDH 45) and genes of G-protein subunits of the tomato plants inoculated with and without AMF under stress and normal conditions. All the stress-inducible genes showed a significant increase in their gene expression under stress and AMF inoculation, while their levels were found to be normal under AMF inoculation without stress. We propose a model of abiotic stress alleviation in tomato plants with the help of external factors such as AMF and internally with the help of proteins like PDH 45 and G-proteins.

Keywords: AMF, abiotic stress, g-proteins, PDH-45

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Authors: Alan Ruiz-Padilla, Brando Villalobos-Villalobos, Yeniley Ruiz-Noa, Claudia Mendoza-Macías, Claudia Palafox-Sánchez, Miguel Marín-Rosales, Álvaro Cruz, Rubén Rangel-Salazar

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Introduction: In patients with rheumatoid arthritis, resistance or poor response to disease modifying antirheumatic drugs (DMARD) may be a reflection of the increase in g-P. The expression of g-P may be important in mediating the effluence of DMARD from the cell. In addition, P-glycoprotein is involved in the transport of cytokines, IL-1, IL-2 and IL-4, from normal lymphocytes activated to the surrounding extracellular matrix, thus influencing the activity of RA. The involvement of P-glycoprotein in the transmembrane transport of cytokines can serve as a modulator of the efficacy of DMARD. It was shown that a number of lymphocytes with glycoprotein P activity is increased in patients with RA; therefore, P-glycoprotein expression could be related to the activity of RA and could be a predictor of poor response to therapy. Objective: To evaluate in RA patients, if the G2677T/A MDR1 polymorphisms is associated with differences in the rate of therapeutic response to disease-modifying antirheumatic agents in patients with rheumatoid arthritis. Material and Methods: A prospective cohort study was conducted. Fifty seven patients with RA were included. They had an active disease according to DAS-28 (score >3.2). We excluded patients receiving biological agents. All the patients were followed during 6 months in order to identify the rate of therapeutic response according to the American College of Rheumatology (ACR) criteria. At the baseline peripheral blood samples were taken in order to identify the G2677T/A MDR1 polymorphisms using PCR- Specific allele. The fragment was identified by electrophoresis in polyacrylamide gels stained with ethidium bromide. For statistical analysis, the genotypic and allelic frequencies of MDR1 gene polymorphism between responders and non-responders were determined. Chi-square tests as well as, relative risks with 95% confidence intervals (95%CI) were computed to identify differences in the risk for achieving therapeutic response. Results: RA patients had a mean age of 47.33 ± 12.52 years, 87.7% were women with a mean for DAS-28 score of 6.45 ± 1.12. At the 6 months, the rate of therapeutic response was 68.7 %. The observed genotype frequencies were: for G/G 40%, T/T 32%, A/A 19%, G/T 7% and for A/A genotype 2%. Patients with G allele developed at 6 months of treatment, higher rate for therapeutic response assessed by ACR20 compared to patients with others alleles (p=0.039). Conclusions: Patients with G allele of the - G2677T/A MDR1 polymorphisms had a higher rate of therapeutic response at 6 months with DMARD. These preliminary data support the requirement for a deep evaluation of these and other genotypes as factors that may influence the therapeutic response in RA.

Keywords: pharmacogenetics, MDR1, P-glycoprotein, therapeutic response, rheumatoid arthritis

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Authors: Sidra Shaw, Sarenna Shaw, Chae Joon Lee, Irimpan Mathews, Eric Koehn

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One of the biggest public health concerns of our time is increasing antimicrobial resistance. As of 2019, the CDC has documented more than 2.8 million serious antibiotic resistant infections in the United States. Currently, antibiotic resistant infections are directly implicated in over 750,000 deaths per year globally. On our current trajectory, British economist Jim O’Neill predicts that by 2050, an additional 10 million people (about half the population of New York) will die annually due to drug resistant infections. As a result, new biochemical pathways must be targeted to generate next generation antibiotic drugs that will be effective against drug resistant bacteria. One enticing target is the biosynthesis of DNA within bacteria, as few drugs interrupt this essential life process. Thymidylate synthase enzymes are essential for life as they catalyze the synthesis of a DNA building block, 2′-deoxythymidine-5′-monophosphate (dTMP). In humans, the thymidylate synthase enzyme (TSase) has been shown to be distinct from the flavin-dependent thymidylate synthase (FDTS) produced by many pathogenic bacteria. TSase and FDTS have distinct structures and mechanisms of catalysis, which should allow selective inhibition of FDTS over human TSase. Currently, C. diff is one of the most antibiotic resistant bacteria, and no drugs that target thymine biosynthesis exist for C. diff. Here we present the initial biochemical characterization of FDTS from C. diff. Specifically, we examine enzyme kinetics and binding features of this enzyme to determine the nature of interaction with ligands/inhibitors and understand the molecular mechanism of catalysis. This research will provide more insight into the targetability of the C. diff FDTS enzyme for novel antibiotic drugs.

Keywords: flavin-dependent thymidylate synthase, FDTS, clostridioides difficile, C. diff, antibiotic resistance, DNA synthesis, enzyme kinetics, binding features

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Authors: Nayira A. Abd Elbaky, Amal J. Fatani, Hazar Yaqub, Nouf M. Al-Rasheed, Naglaa El-Orabi, Mai Osman

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The present work is aimed to evaluate the protective effect of N-acetyl cystiene (NAC), coenzyme Q10 (CoQ10), and their combination against carbon tetrachloride (CCl4)-induced cardiotoxicity in rats. CCl4 treatment significantly elevated the levels of cardiac oxidative stress bio markers including nitric oxide (NO) and malondialdehyde (MDA). A concomitant decrease in the level of reduced glutathione and the activity of membrane bound enzyme, calcium-adenosine triphosphatase were observed in the hearts of rats exposed to CCl4 compared to respective values in normal group. Quantitative analysis of myocardial energy metabolism revealed a significant decrease in the glucose content coupled with depletion in the activities of myocardial glycolytic enzymes as hexokinase (HK), phosphofructokinase (PFK) and lactate dehydrogenase (LDH) after CCl4 treatment. In addition, a significant elevation in myocardial hydroxyproline level was observed in CCl4 intoxicated rats indicating interstitial collagen accumulation. Pretreatment with either NAC, CoQ10 or their combination successively alleviated the alterations in myocardial oxidative stress and antioxidant markers, as well as effectively up-regulated the decrease in cardiac energetic biomarkers in CCl4 intoxicated rats. Moreover, these antioxidants markedly reduced myocardial hydroxyproline level versus that of CCl4-treated animals. In conclusion, the present results illustrated that the prophylactic use of the current antioxidant resulted in a remarkable cardioprotective effect against CCl4 induced myocardial damage, which suggest that they may candidates as prophylactic agents against different cardio-toxins.

Keywords: carbon tetrachloride, lipid peroxidation, antioxidant, energy metabolism, hydroxyproline

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Authors: Rashi Rajput, Manisha Singh

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Escitalopram oxalate (ETP), an FDA approved antidepressant drug from the category of SSRI (selective serotonin reuptake inhibitor) and is used in treatment of general anxiety disorder (GAD), major depressive disorder (MDD).When taken orally, it is metabolized to S-demethylcitalopram (S-DCT) and S-didemethylcitalopram (S-DDCT) in the liver with the help of enzymes CYP2C19, CYP3A4 and CYP2D6. Hence, causing side effects such as dizziness, fast or irregular heartbeat, headache, nausea etc. Therefore, targeted and sustained drug delivery will be a helpful tool for increasing its efficacy and reducing side effects. The present study is designed for formulating mucoadhesive nanoparticle formulation for the same Escitalopram loaded polymeric nanoparticles were prepared by ionic gelation method and characterization of the optimised formulation was done by zeta average particle size (93.63nm), zeta potential (-1.89mV), TEM (range of 60nm to 115nm) analysis also confirms nanometric size range of the drug loaded nanoparticles along with polydispersibility index of 0.117. In this research, we have studied the in vitro drug release profile for ETP nanoparticles, through a semi permeable dialysis membrane. The three important characteristics affecting the drug release behaviour were – particle size, ionic strength and morphology of the optimised nanoparticles. The data showed that on increasing the particle size of the drug loaded nanoparticles, the initial burst was reduced which was comparatively higher in drug. Whereas, the formulation with 1mg/ml chitosan in 1.5mg/ml tripolyphosphate solution showed steady release over the entire period of drug release. Then this data was further validated through mathematical modelling to establish the mechanism of drug release kinetics, which showed a typical linear diffusion profile in optimised ETP loaded nanoparticles.

Keywords: ionic gelation, mucoadhesive nanoparticle, semi-permeable dialysis membrane, zeta potential

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Authors: M. Umar Dahot, G. S. Mangrio

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In this study Agro-industrial waste was used as a carbon source, which is a low cost substrate. Along with this, various sugars and molasses of 2.5% and 5% were investigated as substrate/carbon source for the growth of A.niger and Pectinase production. Different nitrogen sources were also used. An overview of results obtained show that 5% sucrose, 5% molasses and 0.4% (NH4)2SO4 were found the best carbon and nitrogen sources for the production of pectinase by A. niger. The maximum production of pectinase (26.87units/ml) was observed at pH 6.0 after 72 hrs incubation. The optimum temperature for the maximum production of pectinase was achieved at 35ºC when maximum production of pectinase was obtained as 28.25Units/ml.Pectinase enzyme was purified with ammonium sulphate precipitation and dialyzed sample was finally applied on gel filtration chromatography (Sephadex G-100) and Ion Exchange DEAE A-50. The enzyme was purified 2.5 fold by gel chromatography on Sephadex G-100 and Four fractions were obtained, Fraction 1, 2, 4 showed single band while Fraction -3 showed multiple bands on SDS Page electrophoresis. Fraction -3 was pooled, dialyzed and separated on Sephdex A-50 and two fractions 3a and 3b showed single band. The molecular weights of the purified fractions were detected in the range of 33000 ± 2000 and 38000± 2000 Daltons. The purified enzyme was specifically most active with pure pectin, while pectin, Lemon pectin and orange peel given lower activity as compared to (control). The optimum pH and temperature for pectinase activity was found between pH 5.0 and 6.0 and 40°- 50°C, respectively. The enzyme was stable over the pH range 3.0-8.0. The thermostability of was determined and it was observed that the pectinase activity is heat stable and retains activity more than 40% when incubated at 90°C for 10 minutes. The pectinase activity of F3a and F3b was increased with different metal ions. The Pectinase activity was stimulated in the presence of CaCl2 up to 10-30%. ZnSO4, MnSO4 and Mg SO4 showed higher activity in fractions F3a and F3b, which indicates that the pectinase belongs to metalo-enzymes. It is concluded that A. niger is capable to produce pH stable and thermostable pectinase, which can be used for industrial purposes.

Keywords: pectinase, a. niger, production, purification, characterization

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Authors: E. Nakamachi, K. Matsumoto, K. Yamamoto, Y. Morita, H. Sakamoto

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In recently, electromagnetic and mechanical stimulations have been recognized as the effective extracellular environment stimulation technique to enhance the defected peripheral nerve tissue regeneration. In this study, we developed a new hybrid bioreactor by adopting 50 Hz uniform alternative current (AC) magnetic stimulation and 4% strain mechanical stimulation. The guide tube for nerve regeneration is mesh structured tube made of biodegradable polymer, such as polylatic acid (PLA). However, when neural damage is large, there is a possibility that peripheral nerve undergoes necrosis. So it is quite important to accelerate the nerve tissue regeneration by achieving enhancement of nerve axonal extension rate. Therefore, we try to design and fabricate the system that can simultaneously load the uniform AC magnetic field stimulation and the stretch stimulation to cells for enhancement of nerve axonal extension. Next, we evaluated systems performance and the effectiveness of each stimulation for rat adrenal pheochromocytoma cells (PC12). First, we designed and fabricated the uniform AC magnetic field system and the stretch stimulation system. For the AC magnetic stimulation system, we focused on the use of pole piece structure to carry out in-situ microscopic observation. We designed an optimum pole piece structure using the magnetic field finite element analyses and the response surface methodology. We fabricated the uniform AC magnetic field stimulation system as a bio-reactor by adopting analytically determined design specifications. We measured magnetic flux density that is generated by the uniform AC magnetic field stimulation system. We confirmed that measurement values show good agreement with analytical results, where the uniform magnetic field was observed. Second, we fabricated the cyclic stretch stimulation device under the conditions of particular strains, where the chamber was made of polyoxymethylene (POM). We measured strains in the PC12 cell culture region to confirm the uniform strain. We found slightly different values from the target strain. Finally, we concluded that these differences were allowable in this mechanical stimulation system. We evaluated the effectiveness of each stimulation to enhance the nerve axonal extension using PC12. We confirmed that the average axonal extension length of PC12 under the uniform AC magnetic stimulation was increased by 16 % at 96 h in our bio-reactor. We could not confirm that the axonal extension enhancement under the stretch stimulation condition, where we found the exfoliating of cells. Further, the hybrid stimulation enhanced the axonal extension. Because the magnetic stimulation inhibits the exfoliating of cells. Finally, we concluded that the enhancement of PC12 axonal extension is due to the magnetic stimulation rather than the mechanical stimulation. Finally, we confirmed that the effectiveness of the uniform AC magnetic field stimulation for the nerve axonal extension using PC12 cells.

Keywords: nerve cell PC12, axonal extension, nerve regeneration, electromagnetic-mechanical stimulation, bioreactor

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Authors: Vivek B. Ravindran, Aravind Surapaneni, Rebecca Traub, Sarvesh K. Soni, Andrew S. Ball

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Parasitic diseases have a devastating, long-term impact on human health and welfare. More than two billion people are infected with soil-transmitted helminths (STHs), including the roundworms (Ascaris), hookworms (Necator and Ancylostoma) and whipworm (Trichuris) with majority occurring in the tropical and subtropical regions of the world. Despite its low prevalence in developed countries, the removal of STHs from wastewater remains crucial to allow the safe use of sludge or recycled water in agriculture. Conventional methods such as incubation and optical microscopy are cumbersome; consequently, the results drastically vary from person-to-person observing the ova (eggs) under microscope. Although PCR-based methods are an alternative to conventional techniques, it lacks the ability to distinguish between viable and non-viable helminth ova. As a result, wastewater treatment industries are in major need for radically new and innovative tools to detect and quantify STHs eggs with precision, accuracy and being cost-effective. In our study, we focus on the following novel and innovative techniques: -Recombinase polymerase amplification and Surface enhanced Raman spectroscopy (RPA-SERS) based detection of helminth ova. -Use of metal nanoparticles and their relative nanozyme activity. -Colorimetric detection, differentiation and enumeration of genera of helminth ova using hydrolytic enzymes (chitinase and lipase). -Propidium monoazide (PMA)-qPCR to detect viable helminth ova. -Modified assay to recover and enumerate helminth eggs from fresh raw sewage. -Transcriptome analysis of ascaris ova in fresh raw sewage. The aforementioned techniques have the potential to replace current conventional and molecular methods thereby producing a standard protocol for the determination and enumeration of helminth ova in sewage sludge.

Keywords: colorimetry, helminth, PMA-QPCR, nanoparticles, RPA, viable

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Authors: Viktoriia Burkina, Vladimir Zlabek, Galia Zamaratskaia

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Cytochromes P450 (CYP) are important family of enzymes in Phase I metabolism. Environmental pollutants often act as inducers of the gene expression and activities CYP1A1 and CYP3A-like isoforms in fish. The activities are generally measured in the fish liver or gills, and less is known about tissue distribution of expression. In present study, the CYP1A1 and CYP3A-like activities were measured in rainbow trout liver, gill, intestine, heart, brain and gonads. The activities of CYP1A1 and CYP3A-like proteins were estimated as the rates of 7-ethoxyresorufin-O-deethylation (EROD) and benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation (BFCOD), respectively. The CYP1A1 and CYP3A-like activities were detectable in all investigated fish tissues, with the highest activity in hepatic tissue followed by heart > brain > gill > intestine > gonads. To confirm the presence of CYP1A1 in different tissues, EROD activity was measured in presence of the selective inhibitors ellipticine (CYP1A1), ketoconazole (CYP3A), 8-methoxypsoralen (human CYP2A) and diallyl sulphide (CYP2E1). It was found that ellipticine, ketoconazole and 8-methoxypsoralen inhibited hepatic EROD activity by 88-98%. Ellipticine inhibited gill, intestine, and gonad EROD activity by 50%. In conclusion, EROD and BFCOD activities were detected in rainbow trout liver, gill, intestine, heart, brain and gonads. Further studies are needed to fully identify all CYP450 isoforms responsible for these activities. Acknowledgement: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA “(No. CZ.1.05/2.1.00/01.0024), “CENAKVA Center Development “(No. CZ.1.05/2.1.00/19.0380), “CENAKVA II “(No. LO1205 under the NPU I program), and "Development of USB - International mobility (No. CZ.02.2.69/0.0/0.0/16_027/0008364).

Keywords: BFCOD, EROD, fish, phase I metabolism, selective inhibitors

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