Search results for: recombinant enzymes

Authors: Milda Nainyte, Viktoras Masevicius

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Biological methylation is a methyl group transfer from S-adenosyl-L-methionine (AdoMet) onto N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules. The reaction is catalyzed by enzymes called AdoMet-dependent methyltransferases (MTases), which represent more than 3 % of the proteins in the cell. As a general mechanism, the methyl group from AdoMet replaces a hydrogen atom of nucleophilic center producing methylated DNA and S-adenosyl-L-homocysteine (AdoHcy). Recently, DNA methyltransferases have been used for the sequence-specific, covalent labeling of biopolymers. Two types of MTase catalyzed labeling of biopolymers are known, referred as two-step and one-step. During two-step labeling, an alkylating fragment is transferred onto DNA in a sequence-specific manner and then the reporter group, such as biotin, is attached for selective visualization using suitable chemistries of coupling. This approach of labeling is quite difficult and the chemical hitching does not always proceed at 100 %, but in the second step the variety of reporter groups can be selected and that gives the flexibility for this labeling method. In the one-step labeling, AdoMet analog is designed with the reporter group already attached to the functional group. Thus, the one-step labeling method would be more comfortable tool for labeling of biopolymers in order to prevent additional chemical reactions and selection of reaction conditions. Also, time costs would be reduced. However, effective AdoMet analog appropriate for one-step labeling of biopolymers and containing cleavable bond, required for reduction of PCR interferation, is still not known. To expand the practical utility of this important enzymatic reaction, cofactors with activated sulfonium-bound side-chains have been produced and can serve as surrogate cofactors for a variety of wild-type and mutant DNA and RNA MTases enabling covalent attachment of these chains to their target sites in DNA, RNA or proteins (the approach named methyltransferase-directed Transfer of Activated Groups, mTAG). Compounds containing hex-2-yn-1-yl moiety has proved to be efficient alkylating agents for labeling of DNA. Herein we describe synthetic procedures for the preparation of N-biotinoyl-N’-(pent-4-ynoyl)cystamine starting from the coupling of cystamine with pentynoic acid and finally attaching the biotin as a reporter group. The synthesis of the first AdoMet based cofactor containing a cleavable reporter group and appropriate for one-step labeling was developed.

Keywords: adoMet analogs, DNA alkylation, cofactor, methyltransferases

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Authors: Siti Aisya Gany, Swee Ching Tan, Sook Yee Gan

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Diminished antioxidant defense or increased production of reactive oxygen species in the biological system can result in oxidative stress which may lead to various neurodegenerative diseases including Alzheimer’s disease (AD). Microglial activation also contributes to the progression of AD by producing several pro-inflammatory cytokines, nitric oxide (NO), and prostaglandin E2 (PGE2). Oxidative stress and inflammation have been reported to be possible pathophysiological mechanisms underlying AD. In addition, the cholinergic hypothesis postulates that memory impairment in patient with AD is also associated with the deficit of cholinergic function in the brain. Although a number of drugs have been approved for the treatment of AD, most of these synthetic drugs have diverse side effects and yield relatively modest benefits. Marine algae have great potential in pharmaceutical and biomedical applications as they are valuable sources of bioactive properties such as anti-coagulation, anti-microbial, anti-oxidative, anti-cancer and anti-inflammatory. Hence, this study aimed to provide an overview of the properties of Malaysian seaweeds (Padina australis, Sargassum polycystum and Caulerpa racemosa) in inhibiting oxidative stress, neuroinflammation and cholinesterase enzymes. All tested samples significantly exhibit potent DPPH and moderate Superoxide anion radical scavenging ability (P<0.05). Hexane and methanol extracts of S. polycystum exhibited the most potent radical scavenging ability with IC50 values of 0.1572 ± 0.004 mg/ml and 0.8493 ± 0.02 for DPPH and ABTS assays, respectively. Hexane extract of C. racemosa gave the strongest superoxide radical inhibitory effect (IC50 of 0.3862± 0.01 mg/ml). Most seaweed extracts significantly inhibited the production of cytokine (IL-6, IL-1 β, TNFα) and NO in a concentration-dependent manner without causing significant cytotoxicity to the lipopolysaccharide (LPS)-stimulated microglia cells (P<0.05). All extracts suppressed cytokine and NO level by more than 80% at the concentration of 0.4mg/ml. In addition, C. racemosa and S. polycystum also showed anti-acetylcholinesterase activities with the IC50 values ranging from 0.086-0.115 mg/ml. Moreover, C. racemosa and P. australis were also found to be active against butyrylcholinesterase with IC50 values ranging from 0.118-0.287 mg/ml.

Keywords: anti-cholinesterase, anti-oxidative, neuroinflammation, seaweeds

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Authors: Debamitra Chakravorty, Pratap K. Parida

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Cold-adapted proteases enhance wash performance in low-temperature laundry resulting in a reduction in energy consumption and wear of textiles and are also used in the dehairing process in leather industries. Unfortunately, the possible drawbacks of using cold-adapted proteases are their instability at higher temperatures. Therefore, proteases with broad temperature stability are required. Unfortunately, wild-type cold-adapted proteases exhibit instability at higher temperatures and thus have low shelf lives. Therefore, attempts to engineer cold-adapted proteases by protein engineering were made previously by directed evolution and random mutagenesis. The lacuna is the time, capital, and labour involved to obtain these variants are very demanding and challenging. Therefore, rational engineering for cold stability without compromising an enzyme's optimum pH and temperature for activity is the current requirement. In this work, mutations were rationally designed with the aid of high throughput computational methodology of network analysis, evolutionary conservation scores, and molecular dynamics simulations for Savinase from Bacillus lentus with the intention of rendering the mutants cold stable without affecting their temperature and pH optimum for activity. Further, an attempt was made to incorporate a mutation in the most stable mutant rationally obtained by this method to introduce oxidative stability in the mutant. Such enzymes are desired in detergents with bleaching agents. In silico analysis by performing 300 ns molecular dynamics simulations at 5 different temperatures revealed that these three mutants were found to be better in cold stability compared to the wild type Savinase from Bacillus lentus. Conclusively, this work shows that cold adaptation without losing optimum temperature and pH stability and additionally stability from oxidative damage can be rationally designed by in silico enzyme engineering. The key findings of this work were first, the in silico data of H5 (cold stable savinase) used as a control in this work, corroborated with its reported wet lab temperature stability data. Secondly, three cold stable mutants of Savinase from Bacillus lentus were rationally identified. Lastly, a mutation which will stabilize savinase against oxidative damage was additionally identified.

Keywords: cold stability, molecular dynamics simulations, protein engineering, rational design

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Authors: Tai Chen, Caihuan Tian, Xiuxia Ren, Jingqi Xue, Xiuxin Zhang

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The herbaceous peony has become increasingly popular worldwide in recent years, especially as a cut flower with great economic value. However, peony has a very short vase life, only 3-5 d usually, which seriously affects its commodity value. In this study, we used the cut peony (Paeonia lactiflora cv. Sarah) as a material and found that melatonin treatment significantly improved its postharvest performance. In the control group, its vase life was 4.8 d, accompanied by petal dropping at last; melatonin treatment (40 μM) increased this time to 6.9 d without petal dropping at the end. Further study showed that melatonin treatment significantly increased the activity of antioxidant enzymes as well as reduced sugar content in petals, whereas the starch content in petals decreased. These results indicated that melatonin treatment may delay the oxidation reaction caused by aging, which also provides extra energy for maintaining flowering. Through full-length transcriptome sequencing, a total of 2819 differentially expressed genes (DEGs) between control and melatonin treatment groups were identified. KEGG enrichment analysis showed that these DEGs were mainly involved in three pathways, including melatonin synthesis, starch and sucrose conversion, and plant disease resistance. After the RT-qPCR verification, we identified three DEGs, named PlBAM3, PlWRKY22 and PlTIP1, and they should play major roles in melatonin-improved postharvest performance. One possible reason is that PlBAM3 caused maltose production (by starch degradation), maintained the proline biosynthesis, and then alleviated oxidative stress. Another reason is that both PlBAM3 and PlWRKY22 are key drought resistance regulators, which have the ability to alleviate osmotic stress and improve water absorption, which may also help to improve the postharvest quality of cut peony. In addition, PlTIP1 is involved in the sugar signal pathway, indicating sugar may also as a signal substance during this process. Our work may give new ideas for developing new ways to prolong the vase life of cut peony and improve its commodity value eventually.

Keywords: cut peony, melatonin, vase life, oxidation reaction, energy supply, differentially expressed genes

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Authors: Bharat Rawat, Shekhar Rajbhandari, Yadav Bhatta, Jay Prakash Jaiswal, Shivaji Bikram Silwal, Rajiv Shrestha, Shova Sunuwar

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Introduction: The honey produced by the bees fed on Rhobdodendron species containing grayanotoxin is known as mad honey. Grayanotoxin is found in honey obtained from the nectar of Rhododendron species growing on the mountains of the Black Sea region of Turkey and also in Japan, Nepal, Brazil, parts of North America, and Europe. Although the incidence of grayanotoxin poisoning is rare, there is concern that the number of cases per year will rise with the increasing demand for organic products. Mad honey intoxication might present with mild symptoms of cardiovascular, gastrointestinal and neurological systems or might also present with a life-threatening form with AV block and cardiovascular collapse. In this article, we describe the summary of five cases, which came to our hospital with mad honey related cardiac complications. Findings: In last one year, five cases presented in the emergency department with sudden onset of Loss of consciousness, dizziness, shortness of breath. They felt difficulty after the consumption of 1-3 teaspoonful of wild honey. The honey was brought from most of the rural parts of Nepal like khotang. Some of them also came with vomiting, dizziness, and loose stool. On examination, most of them had severe bradycardia and low blood pressure. No abnormalities were detected on systemic examinations. In one patient, ECG and cardiac enzymes showed features of the acute coronary syndrome, but his treadmill test done few days later was normal. All patients were managed with inj. Atropine, I/V normal saline, and other supportive measures and discharged in a stable condition within one or two days. Conclusions: Rhododendrons is the national flower of Nepal. The specific species of rhododendron found in Nepal which contains the toxin is not known. Bees feeding on these rhododendrons are known to transfer the grayanotoxin to the honey they produce. Most symptoms are mild and resolve themselves without medical intervention. Signs and symptoms of grayanotoxin poisoning rarely last more than 24 hours and are usually not fatal. Some signs of mad honey poisoning include Bradycardia, Cardiac arrhythmia, Hypotension, Nausea and Vomiting. They respond to close monitoring and appropriate supportive treatment. Normally, patients recover completely with no residual damage to the heart or its conduction system.

Keywords: rhobdodendron, honey, grayanotoxin, bradycardia

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Authors: Susmita Goswami, Joyeeta Mitra, Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Prabir K. Paul

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'Malbhog’, an indigenous banana variety, much prized for its flavour and delicacy suffers production losses due to Fusarium oxysporum f.sp. cubense. The pathogen enters young plants through feeder roots causing wilting of plants ultimately leading to death of plants. The pathogen spreads rapidly to other plants in the field. In eastern part of India, this variety escapes the onslaught of the pathogen when either co-cultivated or rotated with Amorphophallus campanulatus (yam). The present study provides an insight into the physiological aspect of the biocontrol by yam. In vitro application of sterile aqueous extract of yam tuber (100gm/100ml distilled water and its 1:10 and 1:100 dilutions) were mixed with PDA media which was substantially inoculated with spores of Fusarium oxysporum f.sp. cubense. The extract could significantly reduce germination of pathogen spores. Banana variety susceptible to Fusarium sp was raised in soil rite under aseptic conditions. Spores of the pathogen (106 spores/ml) were inoculated into the soil rite. The plants were spread with aqueous extract of yam. The control plants were treated with sterilized distilled water. The activity of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POX) were estimated in leaves and roots at interval of 24 hours for 5 days after treatment. The incidence of wilt disease was recorded after two weeks. The results demonstrated that yam extract could induce significant activity of PAL, PPO and POX along with accumulation of phenols in both roots and leaves of banana plants. However, significantly high activity of enzymes and phenol accumulation was observed in roots. The disease incidence was significantly low in yam treated plants. The results clearly demonstrated the control of the pathogen due to induction of defense mechanism in the host by the extract. The observed control of the pathogen in the field could possibly be due to induction of such defense responses in host by exudates leached into the soil from yam tubers. Yam extract could be a potential source of environment-friendly biocide against Panama wilt of banana.

Keywords: Amorphophallus campanulatus, banana, Fusarium oxysporum f.sp. cubense, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), peroxidase (POX)

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Authors: Varsha Salian, Shama Rao, N. Narendra, B. Mohana Kumar

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Evolving evidence proposes the existence of a highly tumorigenic subpopulation of undifferentiated, self-renewing cancer stem cells, responsible for exhibiting resistance to conventional anti-cancer therapy, recurrence, metastasis and heterogeneous tumor formation. Importantly, the mechanisms exploited by cancer stem cells to resist chemotherapy are very less understood. Oral squamous cell carcinoma (OSCC) is one of the most regularly diagnosed cancer types in India and is associated commonly with alcohol and tobacco use. Therefore, the isolation and in vitro characterization of cancer stem-like cells from patients with OSCC is a critical step to advance the understanding of the chemoresistance processes and for designing therapeutic strategies. With this, the present study aimed to establish and characterize cancer stem-like cells in vitro from OSCC. The primary cultures of cancer stem-like cell lines were established from the tissue biopsies of patients with clinical evidence of an ulceroproliferative lesion and histopathological confirmation of OSCC. The viability of cells assessed by trypan blue exclusion assay showed more than 95% at passage 1 (P1), P2 and P3. Replication rate was performed by plating cells in 12-well plate and counting them at various time points of culture. Cells had a more marked proliferative activity and the average doubling time was less than 20 hrs. After being cultured for 10 to 14 days, cancer stem-like cells gradually aggregated and formed sphere-like bodies. More spheroid bodies were observed when cultured in DMEM/F-12 under low serum conditions. Interestingly, cells with higher proliferative activity had a tendency to form more sphere-like bodies. Expression of specific markers, including membrane proteins or cell enzymes, such as CD24, CD29, CD44, CD133, and aldehyde dehydrogenase 1 (ALDH1) is being explored for further characterization of cancer stem-like cells. To summarize the findings, the establishment of OSCC derived cancer stem-like cells may provide scope for better understanding the cause for recurrence and metastasis in oral epithelial malignancies. Particularly, identification and characterization studies on cancer stem-like cells in Indian population seem to be lacking thus provoking the need for such studies in a population where alcohol consumption and tobacco chewing are major risk habits.

Keywords: cancer stem-like cells, characterization, in vitro, oral squamous cell carcinoma

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Authors: Huzaifa Bilal

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Russian wheat aphid (Diuraphis noxia, Kurdjumov) is considered an economically important wheat (Triticum aestivum L.) pest worldwide and in South Africa. The RWA damages wheat plants and reduces annual yields by more than 10%. Even though pest management by pesticides and resistance breeding is an attractive option, chemicals can cause harm to the environment. Furthermore, the evolution of resistance-breaking aphid biotypes has out-paced the release of resistant cultivars. An alternative strategy to reduce the impact of aphid damage on plants, such as priming, which sensitizes plants to respond effectively to subsequent attacks, is necessary. In this study, wheat plants at the seedling and flag leaf stages were primed by salicylic acid and isolate representative of two races of the leaf rust pathogen Puccinia triticina Eriks. (Pt), before RWA (South African RWA biotypes 1 and 4) infestation. Randomized complete block design experiments were conducted in the greenhouse to study plant-pest interaction in primed and non-primed plants. Analysis of induced aphid damage indicated salicylic acid differentially primed wheat cultivars for increased resistance to the RWASA biotypes. At the seedling stage, all cultivars were primed for enhanced resistance to RWASA1, while at the flag leaf stage, only PAN 3111, SST 356 and Makalote were primed for increased resistance. The Puccinia triticina efficaciously primed wheat cultivars for excellent resistance to RWASA1 at the seedling and flag leaf stages. However, Pt failed to enhance the four Lesotho cultivars' resistance to RWASA4 at the seedling stage and PAN 3118 at the flag leaf stage. The induced responses at the seedling and flag leaf stages were positively correlated in all the treatments. Primed plants induced high activity of antioxidant enzymes like peroxidase, ascorbate peroxidase and superoxide dismutase. High antioxidant activity indicates activation of resistant responses in primed plants (primed by salicylic acid and Puccina triticina). Isolates of avirulent Pt races can be a worthy priming agent for improved resistance to RWA infestation. Further confirmation of the priming effects needs to be evaluated at the field trials to investigate its application efficiency.

Keywords: Russian wheat aphis, salicylic acid, puccina triticina, priming

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

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

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

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Authors: Natan C. G. Silva, Carlos A. C. Girao Neto, Marcele M. S. Vasconcelos, Luciana R. B. Goncalves, Maria Valderez P. Rocha

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Galactooligosaccharides (GOS) are sugars with prebiotic function that can be synthesized chemically or enzymatically, and this last one can be promoted by the action of β-galactosidases. In addition to favoring the transgalactosylation reaction to form GOS, these enzymes can also catalyze the hydrolysis of lactose. A highly studied type of GOS is lactulose because it presents therapeutic properties and is a health promoter. Among the different raw materials that can be used to produce lactulose, whey stands out as the main by-product of cheese manufacturing, and its discarded is harmful to the environment due to the residual lactose present. Therefore, its use is a promising alternative to solve this environmental problem. Thus, lactose from whey is hydrolyzed into glucose and galactose by β-galactosidases. However, in order to favor the transgalactosylation reaction, the medium must contain fructose, due this sugar reacts with galactose to produce lactulose. Then, the glucose-isomerase enzyme can be used for this purpose, since it promotes the isomerization of glucose into fructose. In this scenario, the aim of the present work was first to develop β-galactosidase biocatalysts of Kluyveromyces lactis and to apply it in the integrated reactions of hydrolysis, isomerization (with the glucose-isomerase from Streptomyces murinus) and transgalactosylation reaction, using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% glutaraldehyde was evaluated using different enzymatic loads (2, 5, 7, 10, and 12 mg/g). Subsequently, the hydrolysis and transgalactosylation reactions were studied and conducted at 50°C, 120 RPM for 20 minutes. In parallel, the isomerization of glucose into fructose was evaluated under conditions of 70°C, 750 RPM for 90 min. After, the integration of the three processes for the production of lactulose was investigated. Among the evaluated loads, 7 mg/g was chosen because the best activity of the derivative (44.3 U/g) was obtained, being this parameter determinant for the reaction stages. The other parameters of immobilization yield (87.58%) and recovered activity (46.47%) were also satisfactory compared to the other conditions. Regarding the integrated process, 94.96% of lactose was converted, achieving 37.56 g/L and 37.97 g/L of glucose and galactose, respectively. In the isomerization step, conversion of 38.40% of glucose was observed, obtaining a concentration of 12.47 g/L fructose. In the transgalactosylation reaction was produced 13.15 g/L lactulose after 5 min. However, in the integrated process, there was no formation of lactulose, but it was produced other GOS at the same time. The high galactose concentration in the medium probably favored the reaction of synthesis of these other GOS. Therefore, the integrated process proved feasible for possible production of prebiotics. In addition, this process can be economically viable due to the use of an industrial residue as a substrate, but it is necessary a more detailed investigation of the transgalactosilation reaction.

Keywords: beta-galactosidase, glucose-isomerase, galactooligosaccharides, lactulose, whey

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Authors: Saddam Husain Dhobi, Bikrant Karki

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The main objective of this paper is save money, balance ecosystem of the terrestrial organism, control global warming, and enhancing the storage capacity of the battery with requiring weight and thinness by using Cyanobacteria in the battery. To fulfill this purpose of paper we can use different methods: Analysis, Biological, Chemistry, theoretical and Physics with some engineering design. Using this different method, we can produce the special type of battery that has the long life, high storage capacity, and clean environment, save money so on and by using the byproduct of Cyanobacteria i.e. glucose. Cyanobacteria are a special type of bacteria that produces different types of extracellular glucoses and oxygen with the help of little sunlight, water, and carbon dioxide and can survive in freshwater, marine and in the land as well. In this process, O₂ is more in the comparison to plant due to rapid growth rate of Cyanobacteria. The required materials are easily available in this process to produce glucose with the help of Cyanobacteria. Since CO₂, is greenhouse gas that causes the global warming? We can utilize this gas and save our ecological balance and the byproduct (glucose) C₆H₁₂O₆ can be utilized for raw material for the battery where as O₂ escape is utilized by living organism. The glucose produce by Cyanobateria goes on Krebs's Cycle or Citric Acid Cycle, in which glucose is complete, oxidizes and all the available energy from glucose molecule has been release in the form of electron and proton as energy. If we use a suitable anodes and cathodes, we can capture these electrons and protons to produce require electricity current with the help of byproduct of Cyanobacteria. According to "Virginia Tech Bio-battery" and "Sony" 13 enzymes and the air is used to produce nearly 24 electrons from a single glucose unit. In this output power of 0.8 mW/cm, current density of 6 mA/cm, and energy storage density of 596 Ah/kg. This last figure is impressive, at roughly 10 times the energy density of the lithium-ion batteries in your mobile devices. When we use Cyanobacteria in battery, we are able to reduce Carbon dioxide, Stop global warming, and enhancing the storage capacity of battery more than 10 times that of lithium battery, saving money, balancing ecology. In this way, we can produce energy from the Cyanobacteria and use it in battery for different benefits. In addition, due to the mass, size and easy cultivation, they are better to maintain the size of battery. Hence, we can use Cyanobacteria for the battery having suitable size, enhancing the storing capacity of battery, helping the environment, portability and so on.

Keywords: anode, byproduct, cathode, cyanobacteri, glucose, storage capacity

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Authors: Virag Vass, Ilona Bereczki, Erzsebet Szabo, Nora Debreczeni, Aniko Borbas, Pal Herczegh, Arpad Tosaki

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Hydrogen sulfide (H₂S) is a toxic gas, but it is produced by certain tissues in a small quantity. According to earlier studies, ibuprofen and H₂S has a protective effect against damaging heart tissue caused by ischemia-reperfusion. Recently, we have been investigating the effect of a new water-soluble H₂S releasing ibuprofen molecule administered after artificially generated ischemia-reperfusion on isolated rat hearts. The H₂S releasing property of the new ibuprofen derivative was investigated in vitro in medium derived from heart endothelial cell isolation at two concentrations. The ex vivo examinations were carried out on rat hearts. Rats were anesthetized with an intraperitoneal injection of ketamine, xylazine, and heparin. After thoracotomy, hearts were excised and placed into ice-cold perfusion buffer. Perfusion of hearts was conducted in Langendorff mode via the cannulated aorta. In our experiments, we studied the dose-effect of the H₂S releasing molecule in Langendorff-perfused hearts with the application of gradually increasing concentration of the compound (0- 20 µM). The H₂S releasing ibuprofen derivative was applied before the ischemia for 10 minutes. H₂S concentration was measured with an H₂S detecting electrochemical sensor from the coronary effluent solution. The 10 µM concentration was chosen for further experiments when the treatment with this solution was occurred after the ischemia. The release of H₂S is occurred by the hydrolyzing enzymes that are present in the heart endothelial cells. The protective effect of the new H₂S releasing ibuprofen molecule can be confirmed by the infarct sizes of hearts using the Triphenyl-tetrazolium chloride (TTC) staining method. Furthermore, we aimed to define the effect of the H₂S releasing ibuprofen derivative on autophagic and apoptotic processes in damaged hearts after investigating the molecular markers of these events by western blotting and immunohistochemistry techniques. Our further studies will include the examination of LC3I/II, p62, Beclin1, caspase-3, and other apoptotic molecules. We hope that confirming the protective effect of new H₂S releasing ibuprofen molecule will open a new possibility for the development of more effective cardioprotective agents with exerting fewer side effects. Acknowledgment: This study was supported by the grants of NKFIH- K-124719 and the European Union and the State of Hungary co- financed by the European Social Fund in the framework of GINOP- 2.3.2-15-2016-00043.

Keywords: autophagy, hydrogen sulfide, ibuprofen, ischemia, reperfusion

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Authors: Jia-Ling Ho, Xiu-Ru Zhang, Zwe-Ling Kong

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Diabetes mellitus (DM) is a major health problem that affects patients’ life quality throughout the world due to its many complications. It characterized by chronic hyperglycemia with oxidative stress, which impaired male reproductive function. Fibroblast growth factor 21 (FGF21) is a metabolic regulator that is required for normal spermatogenesis and protects against diabetes-induced germ cell apoptosis. Echinacea purpurea ethanol extract (EE), which contain phenolic acid and isobutylamide, had been proven to have antidiabetic property. Silica-chitosan nanoparticles (Nano-CS) has drug delivery and controlled release properties. This study aims to investigate whether silica-chitosan nanoparticles encapsulated EE (Nano-EE) had more ameliorating male infertility by analyzing the effect of testicular FGF21. The Nano-EE was characterized before used to treatment the diabetic rat model. Male Sprague-Dawley (SD) rats were obtained and divided into seven groups. A group was no induced Streptozotocin (STZ), marked as normal group. Diabetic rats were induced into diabetes by STZ (33 mg/kg). A diabetic group was no treatment with sample (diabetic control group), and other groups were treatment by Nano-CS (465 mg/kg), Nano-EE (93, 279, 465 mg/kg), and metformin (Met) (200 mg/kg) used as reference drug for 7 weeks. Our results indicated that the average nanoparticle size and zeta potential of Nano-EE were 2630 nm and -21.3 mV, respectively. The encapsulation ratio of Nano-EE was about 70%. It also confirmed the antioxidative activity was unchanged by comparing the DPPH and ABTS scavenging of Nano-EE and EE. In vivo test, Nano-EE can improve the STZ induced hyperglycemia, insulin resistance, and plasma FGF21 levels. Nano-EE has increased sperm motility, mitochondria membrane potential (MMP), plasma testosterone level, and reduction of abnormal sperm, nitric oxide (NO), superoxide production as well as reactive oxygen species (ROS). In addition, in plasma antioxidant enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) was increased whereas pro-inflammatory cytokines TNF-α, and IL-1β were decreased. Further, in testis, protein content of FGF21, PGC-1α, and SIRT1 were improved. Nano-EE might improve diabetes-induced down-regulation of testicular FGF21 and SIRT1/PGC-1α signaling hence maintain spermatogenesis.

Keywords: diabetes mellitus, Echinacea purpurea, reproductive dysfunction, silica-chitosan nanoparticles

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Authors: Roya Bakhtiar, Alireza Abdolmohammadi, Hadi Hajarian, Zahra Nikousefat, Davood, Kalantar-Neyestanaki

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The objective of the present study was to determine the polymorphism in the leptin (332G>A) and its association with biometric traits in Sanjabi sheep. For this purpose, blood samples from 96 rams were taken, and tail length, width tail, circumference tail, body length, body width, and height were simultaneously recorded. PCR was performed using specific primer to amplify 463 bp fragment including exon 3 of leptin gene, and PCR products were digested by Cail restriction enzymes. The 332G>A (at 332th nucleotide of exon 3 leptin gene) that caused an amino acid change from Arg to Gln was detected by Cail (CAGNNNCTG) endonuclease, as the endonuclease cannot cut this region if G nucleotide is located in this position. Three genotypes including GG (463), GA (463, 360and 103 bp) and GG (360 bp and 103 bp) were identified after digestion by enzyme. The estimated frequencies of three genotypes including GG, GA, and AA for 332G>A locus were 0.68, 0.29 and 0.03 and those were 0.18 and 0.82 for A and G alleles, respectively. In the current study, chi-square test indicated that 332G>A positions did not deviate from the Hardy–Weinberg (HW) equilibrium. The most important reason to show HW equation was that samples used in this study belong to three large local herds with a traditional breeding system having random mating and without selection. Shannon index amount was calculated which represent an average genetic variation in Sanjabi rams. Also, heterozygosity estimated by Nei index indicated that genetic diversity of mutation in the leptin gene is moderate. Leptin gene polymorphism in the 332G>A had significant effect on body length (P<0.05) trait, and individuals with GA genotype had significantly the higher body length compared to other individuals. Although animals with GA genotype had higher body width, this difference was not statistically significant (P>0.05). This non-synonymous SNP resulted in different amino acid changes at codon positions111(R/Q). As leptin activity is localized, at least in part, in domains between amino acid residues 106-1406, it is speculated that the detected SNP at position 332 may affect the activity of leptin and may lead to different biological functions. Based to our results, due to significant effect of leptin gene polymorphism on body size traits, this gene may be used a candidate gene for improving these traits.

Keywords: body size, Leptin gene, PCR-RFLP, Sanjabi sheep

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Authors: Omar Pillaca-Pullo, Arturo Alejandro-Paredes, Carol Flores-Fernandez, Marijuly Sayuri Kina, Amparo Iris Zavaleta

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Aqueous two-phase system (ATPS) is an interesting alternative for separating industrial enzymes due to it is easy to scale-up and low cost. Polyethylene glycol (PEG) mixed with potassium phosphate or magnesium sulfate is one of the most frequently polymer/salt ATPS used, but the consequences of its use is a high concentration of phosphates and sulfates in wastewater causing environmental issues. Citrate could replace these inorganic salts due to it is biodegradable and does not produce toxic compounds. On the other hand, statistical design of experiments is widely used for ATPS optimization and it allows to study the effects of the involved variables in the purification, and to estimate their significant effects on selected responses and interactions. The 24 factorial design with four central points (20 experiments) was employed to study the partition and purification of proteases produced by Pseudomonas sp. in PEG/citrate ATPS system. ATPS was prepared with different sodium citrate concentrations [14, 16 and 18% (w/w)], pH values (7, 8 and 9), PEG molecular weight (2,000; 4,000 and 6,000 g/mol) and PEG concentrations [18, 20 and 22 % (w/w)]. All system components were mixed with 15% (w/w) of the fermented broth and deionized water was added to a final weight of 12.5 g. Then, the systems were mixed and kept at room temperature until to reach two-phases separation. Volumes of the top and bottom phases were measured, and aliquots from both phases were collected for subsequent proteolytic activity and total protein determination. Influence of variables such as PEG molar mass (MPEG), PEG concentration (CPEG), citrate concentration (CSal) and pH were evaluated on the following responses: purification factor (PF), activity yield (Y), partition coefficient (K) and selectivity (S). STATISTICA program version 10 was used for the analysis. According to the obtained results, higher levels of CPEG and MPEG had a positive effect on extraction, while pH did not influence on the process. On the other hand, the CSal could be related with low values of Y because of the citrate ions have a negative effect on solubility and enzymatic structure. The optimum values of Y (66.4 %), PF (1.8), K (5.5) and S (4.3) were obtained at CSal (18%), MPEG (6,000 g/mol), CPEG (22%) and pH 9. These results indicated that the PEG/citrate system is accurate to purify these Pseudomonas sp. proteases from fermented broth as a first purification step.

Keywords: citrate, polyethylene glycol, protease, Pseudomonas sp

Procedia PDF Downloads 194

Authors: D. Nagalakshmi, S. Parashuramulu K. Sadasiva Rao, G. Aruna, L. Vikram

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The zinc (Zn) is the second most abundant trace element in mammals and birds, forming structural component of over 300 enzymes, playing an important role in anti-oxidant defense, immune response and reproduction. Organic trace minerals are more readily absorbed from the digestive tract and more biologically available compared with its inorganic salt. Thus, the present study was undertaken on 60 adult female Sprague Dawley rats (275±2.04 g) for experimental duration of 12 weeks to investigate the effect of dietary Zn supplementation from various organic sources on immunity, reproduction, oxidative defense mechanism and blood biochemical profile. The rats were randomly allotted to 30 replicates (2 per replicate) which were in turn randomly allotted to 5 dietary treatments varying in Zn source i.e., one inorganic source (Zn carbonate) and 4 organic sources (Zn-proteinate, Zn-propionate, Zn-amino acid complex and Zn-methionine) so as to supply NRC recommended Zn concentration (12 ppm Zn). Supplementation of organic Zn had no effect on various haematological and serum biochemical constituents compared to inorganic Zn fed rats. The TBARS and protein carbonyls concentration in liver indicative of oxidative stress was comparable between various organic and inorganic groups. The glutathione reductase activity in haemolysate (P<0.05) and reduced glutathione concentration in liver (P<0.01) was higher when fed organic Zn and RBC catalase activity was higher (P<0.01) on Zn methionine compared to other organic sources tested and the inorganic source. The humoral immune response assessed as antibody titres against sheep RBC was higher (P<0.05) when fed organic sources of zinc compared to inorganic source. The cell mediated immune response expressed as delayed type hypersensitivity reaction was higher (P<0.05) in rats fed Zn propionate with no effect of other organic Zn sources. The serum progesterone concentration was higher (P<0.05) in rats fed organic Zn sources compared to inorganic zinc. The data on ovarian folliculogenesis indicated that organic Zn supplementation increased (P<0.05) the number of graafian follicles and corpus luteum with no effect on primary, secondary and tertiary follicle number. The study indicated that rats fed organic sources of Zn had higher antioxidant enzyme activities, immune response and serum progesterone concentration with higher number of mature follicles. Though the effect of feeding various organic sources were comparable, rats fed zinc methionine had higher antioxidant activity and cell mediated immune response was higher in rats on Zn propionate.

Keywords: organic zinc, immune, rats, reproductive

Procedia PDF Downloads 286

Authors: Ayda Maadani Mallak, Amir lakzian, Elham Khodaverdi, Gholam Hossein Haghnia

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The use of pharmaceuticals and personal care products such as antibiotics and antibacterials has been increased in recent years. Since the major part of consumed compounds remains unchanged in the wastewater treatment plant, they will easily find their way into the human food chain following the land use of sewage sludge (SS). Biological treatment of SS is one the most effective methods for expunging contaminants. White rot fungi, due to their ligninolytic enzymes, are extensively used to degrade organic compounds. Among all three different morphological forms and growth patterns of filamentous fungi (mycelia, clumps, and pellets), fungal pellet formation has been the subject of interest in industrial bioprocesses. Therefore this study was aimed to investigate the uptake of tetracycline (TC) and triclosan (TCS) by radish plant (Raphanus sativus) from soil amended with untreated and pretreated SS by P. ostreatus fungal pellets under greenhouse conditions. The experimental soil was amended with 1) Contaminated SS with TC at a concentration of 100 mgkg-1 and pretreated by fungal pellets, 2) Contaminated SS with TC at 100 mgkg-1 and untreated with fungal pellets, 3) Contaminated SS with TCS at a concentration of 50 mgkg-1 and pretreated by fungal pellets, 4) contaminated SS with TCS at 50 mgkg-1 and untreated with fungal pellets. An uncontaminated and untreated SS-amended soil also was considered as control treatment. An AB SCIEX 3200 QTRAP LC-MS/MS system was used in order to analyze the concentration of TC and TCS in plant tissues and soil medium. Results of this study revealed that the presence of TC and TCS in SS-amended soil decreased the radish biomass significantly. The reduction effect of TCS on dry biomass of shoot and root was 39 and 45% compared to controls, whereas for TC, the reduction percentage for shoot and root was 27 and 40.6%, respectively. However, fungal treatment of SS by P. ostreatus pellets reduced the negative effect of both compounds on plant biomass remarkably, as no significant difference was observed compared to control treatments. Pretreatment of SS with P. ostreatus also caused a significant reduction in translocation factor (concentration in shoot/root), especially for TC compound up to 32.3%, whereas this reduction for TCS was less (8%) compared to untreated SS. Generally, the results of this study confirmed the positive effect of using fungal pellets in SS amendment to decrease TC and TCS uptake by radish plants. In conclusion, P. ostreatus fungal pellets might provide future insights into bioaugmentation to remove antibiotics from environmental matrices.

Keywords: antibiotic, fungal pellet, sewage sludge, white-rot fungi

Procedia PDF Downloads 157

Authors: N. S. Risqina, G. Edijanti, P. S. Nurita, L. Endang, R. A. Siti, R. Tri

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Skin is an important and vital organs and also as a mirror of health and life. Facial skin care is one of the main emphasis to get the beautiful, healthy, and fresh skin. Potentially antioxidant phenolic compounds shows, antimutagen, antitumor, anti-inflammatory, and anti-cancer. Flavonoids are a group of polyphenolic compounds that have the nature of free radicals, inhibiting the oxidative and hydrolytic enzymes as well as anti-inflammatory. Bitter melon (Momordica charantia Linn) is a plant that contains flavonoids, and phenolic antioxidant activity. Bitter melon has strong antioxidant activity that can counteract the free radicals.These compounds can prevent free radicals that cause premature aging. Gel masks including depth cleansing is the cosmetics which work in depth and could raise the dead skin cells. Measurement of antioxidant activity of the extract and gel mask is done by using the immersion method of DPPH. IC50 value of ethanol extract of bitter melon fruit of 287.932 ppm. The preparation of gel mask bitter melon fruit extract, necessary to test the effectiveness of antioxidants using DPPH method is done by measuring the inhibition of DPPH and using UV spectrophotometer at the wavelength of maximum DPPH solution. Tests conducted at the beginning and end of the evaluation (day 0 and day 28). The purpose of this study is to determine the antioxidant activity of the bitter melon's extract and to determine the antioxidant activity of ethanol extract gel mask pare in varying concentrations, ie 1xIC100 (0.295%), 2xIC100 (0.590%) and 4xIC100 (1.180%). Evaluation of physical properties of the preparation on (Day-0,7,14,21, and 28) and evaluation of antioxidant activity (day 0 and 28). Data were analyzed using One Way ANOVA to determine differences in the physical properties of each formula. The statistical results showed that differences in the formula and storage time affects the adhesion, dispersive power, dry time and pH it is shown on a significant value of p <0.05, but longer storage does not affect the pH because the significance value p> 0,05. The antioxidant test showed that there are differences in antioxidant activity in all formulas. Measurement of antioxidant activity of bitter melon fruit extract gel mask on day 0 with a concentration of 0.295%, 0.590%, and 1.180%, respectively, are 124,209.277 ppm, ppm 83819.223 and 47323.592 ppm, whereas day 28 consecutive 130 411, 495 ppm, and 53239.806 95561.645 ppm ppm. The Conclusions drawn that there are antioxidant activity in preparation gel mask of bitter melon fruit extract. The antioxidant activity of bitter melon fruit extract gel mask on the day 0 with a concentration of 0.295%, 0.590%, and 1.180%, respectively, are 124,209.277 ppm, ppm 83819.223 and 47323.592 ppm, whereas on day 28 of antioxidant activity gel mask bitter melon fruit extract with a concentration of 0.295%, 0.590%, and 1.180% in succession, namely: 130,411.495 ppm, ppm 95561.645 and 53239.806 ppm.

Keywords: antioxdant, bitter melon, gel mask, IC50

Procedia PDF Downloads 470

Authors: Selisha A. Sooklal, Phelelani Mpangase, Shaun Aron, Karl Rumbold

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Organically bound fluorine (C-F bond) is extremely rare in nature. Despite this, the first fluorinated secondary metabolite, fluoroacetate, was isolated from the plant Dichapetalum cymosum (commonly known as Gifblaar). However, the enzyme responsible for fluorination (fluorinase) in Gifblaar was never isolated and very little progress has been achieved in understanding this process in higher plants. Fluorinated compounds have vast applications in the pharmaceutical, agrochemical and fine chemicals industries. Consequently, an enzyme capable of catalysing a C-F bond has great potential as a biocatalyst in the industry considering that the field of fluorination is virtually synthetic. As with any biocatalyst, a range of these enzymes are required. Therefore, it is imperative to expand the exploration for novel fluorinases. This study aimed to gain molecular insights into secondary metabolite biosynthesis in Gifblaar using a high-throughput sequencing-based approach. Mechanical wounding studies were performed using Gifblaar leaf tissue in order to induce expression of the fluorinase. The transcriptome of the wounded and unwounded plant was then sequenced on the Illumina HiSeq platform. A total of 26.4 million short sequence reads were assembled into 77 845 transcripts using Trinity. Overall, 68.6 % of transcripts were annotated with gene identities using public databases (SwissProt, TrEMBL, GO, COG, Pfam, EC) with an E-value threshold of 1E-05. Sequences exhibited the greatest homology to the model plant, Arabidopsis thaliana (27 %). A total of 244 annotated transcripts were found to be differentially expressed between the wounded and unwounded plant. In addition, secondary metabolic pathways present in Gifblaar were successfully reconstructed using Pathway tools. Due to lack of genetic information for plant fluorinases, a transcript failed to be annotated as a fluorinating enzyme. Thus, a local database containing the 5 existing bacterial fluorinases was created. Fifteen transcripts having homology to partial regions of existing fluorinases were found. In efforts to obtain the full coding sequence of the Gifblaar fluorinase, primers were designed targeting the regions of homology and genome walking will be performed to amplify the unknown regions. This is the first genetic data available for Gifblaar. It has provided novel insights into the mechanisms of metabolite biosynthesis and will allow for the discovery of the first eukaryotic fluorinase.

Keywords: biocatalyst, fluorinase, gifblaar, transcriptome

Procedia PDF Downloads 273

Authors: Gianmaria Califano, Júlio Augusto Lucena Maciel, Olfa Zarrouk, Miguel Damasio, Jose Silvestre, Ana Margarida Fortes

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Global warming, with rapid and sudden changes in meteorological conditions, is one of the major constraints to ensuring agricultural and crop resilience in the Mediterranean regions. Several strategies are being adopted to reduce the pressure of drought stress on grapevines at regional and local scales: improvements in the irrigation systems, adoption of interline cover crops, and adaptation of pruning techniques. However, still, more can be achieved if also microbial compartments associated with plants are considered in crop management. It is known that the microbial community change according to several factors such as latitude, plant variety, age, rootstock, soil composition and agricultural management system. Considering the increasing pressure of the biotic and abiotic stresses, it is of utmost necessity to also evaluate the effects of drought on the microbiome associated with the grapevine, which is a commercially important crop worldwide. In this study, we characterize the diversity and the structure of the microbial community under three long-term irrigation levels (100% ETc, 50% ETc and rain-fed) in a drought-tolerant grapevine cultivar present worldwide, Syrah. To avoid the limitations of culture-dependent methods, amplicon sequencing with target primers for bacteria and fungi was applied to the same soil samples. The use of the DNeasy PowerSoil (Qiagen) extraction kit required further optimization with the use of lytic enzymes and heating steps to improve DNA yield and quality systematically across biological treatments. Target regions (16S rRNA and ITS genes) of our samples are being sequenced with Illumina technology. With bioinformatic pipelines, it will be possible to obtain a characterization of the bacterial and fungal diversity, structure and composition. Further, the microbial communities will be assessed for their functional activity, which remains an important metric considering the strong inter-kingdom interactions existing between plants and their associated microbiome. The results of this study will lay the basis for biotechnological applications: in combination with the establishment of a bacterial library, it will be possible to explore the possibility of testing synthetic microbial communities to support plant resistance to water scarcity.

Keywords: microbiome, metabarcoding, soil, vinegrape, syrah, global warming, crop sustainability

Procedia PDF Downloads 123

Authors: Yemane Tadesse Gebreslassie, Fisseha Guesh Gebremeskel

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Nanotechnology has made remarkable advancements in recent years, revolutionizing various scientific fields, industries, and research institutions through the utilization of metal and metal oxide nanoparticles. Among these nanoparticles, copper oxide nanoparticles (CuO NPs) have garnered significant attention due to their versatile properties and wide-range applications, particularly, as effective antimicrobial and anticancer agents. CuO NPs can be synthesized using different methods, including physical, chemical, and biological approaches. However, conventional chemical and physical approaches are expensive, resource-intensive, and involve the use of hazardous chemicals, which can pose risks to human health and the environment. In contrast, biological synthesis provides a sustainable and cost-effective alternative by eliminating chemical pollutants and allowing for the production of CuO NPs of tailored sizes and shapes. This comprehensive review focused on the green synthesis of CuO NPs using various biological resources, such as plants, microorganisms, and other biological derivatives. Current knowledge and recent trends in green synthesis methods for CuO NPs are discussed, with a specific emphasis on their biomedical applications, particularly in combating cancer and microbial infections. This review highlights the significant potential of CuO NPs in addressing these diseases. By capitalizing on the advantages of biological synthesis, such as environmental safety and the ability to customize nanoparticle characteristics, CuO NPs have emerged as promising therapeutic agents for a wide range of conditions. This review presents compelling findings, demonstrating the remarkable achievements of biologically synthesized CuO NPs as therapeutic agents. Their unique properties and mechanisms enable effective combating against cancer cells and various harmful microbial infections. CuO NPs exhibit potent anticancer activity through diverse mechanisms, including induction of apoptosis, inhibition of angiogenesis, and modulation of signaling pathways. Additionally, their antimicrobial activity manifests through various mechanisms, such as disrupting microbial membranes, generating reactive oxygen species, and interfering with microbial enzymes. This review offers valuable insights into the substantial potential of biologically synthesized CuO NPs as an alternative approach for future therapeutic interventions against cancer and microbial infections.

Keywords: copper oxide nanoparticles, green synthesis, nanotechnology, microbial infection

Procedia PDF Downloads 63

Authors: Yemane Tadesse Gebreslassie, Fisseha Guesh Gebremeskel

Abstract:

Nanotechnology has made remarkable advancements in recent years, revolutionizing various scientific fields, industries, and research institutions through the utilization of metal and metal oxide nanoparticles. Among these nanoparticles, copper oxide nanoparticles (CuO NPs) have garnered significant attention due to their versatile properties and wide-range applications, particularly, as effective antimicrobial and anticancer agents. CuO NPs can be synthesized using different methods, including physical, chemical, and biological approaches. However, conventional chemical and physical approaches are expensive, resource-intensive, and involve the use of hazardous chemicals, which can pose risks to human health and the environment. In contrast, biological synthesis provides a sustainable and cost-effective alternative by eliminating chemical pollutants and allowing for the production of CuO NPs of tailored sizes and shapes. This comprehensive review focused on the green synthesis of CuO NPs using various biological resources, such as plants, microorganisms, and other biological derivatives. Current knowledge and recent trends in green synthesis methods for CuO NPs are discussed, with a specific emphasis on their biomedical applications, particularly in combating cancer and microbial infections. This review highlights the significant potential of CuO NPs in addressing these diseases. By capitalizing on the advantages of biological synthesis, such as environmental safety and the ability to customize nanoparticle characteristics, CuO NPs have emerged as promising therapeutic agents for a wide range of conditions. This review presents compelling findings, demonstrating the remarkable achievements of biologically synthesized CuO NPs as therapeutic agents. Their unique properties and mechanisms enable effective combating against cancer cells and various harmful microbial infections. CuO NPs exhibit potent anticancer activity through diverse mechanisms, including induction of apoptosis, inhibition of angiogenesis, and modulation of signaling pathways. Additionally, their antimicrobial activity manifests through various mechanisms, such as disrupting microbial membranes, generating reactive oxygen species, and interfering with microbial enzymes. This review offers valuable insights into the substantial potential of biologically synthesized CuO NPs as an alternative approach for future therapeutic interventions against cancer and microbial infections.

Keywords: biological synthesis, copper oxide nanoparticles, microbial infection, nanotechnology

Procedia PDF Downloads 62

Authors: Eun-Young Kwon, Myung-Sook Choi, Su-Jung Cho, Ji-Young Choi, So Young Kim, Youngji Han

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Overweight and obesity have been linked to a low-grade chronic inflammatory response and an increased risk of developing metabolic syndrome including insulin resistance, type 2 diabetes mellitus and certain types of cancers. Luteolin is a dietary flavonoid with anti-inflammatory, anti-oxidant, anti-cancer and anti-diabetic properties. However, little is known about the detailed mechanism associated with the effect of luteolin on inflammation-related obesity and its complications. The aim of the present study was to reveal the anti-diabetic effect of luteolin in diet-induced obesity mice using “transcriptomics” tool. Thirty-nine male C57BL/6J mice (4-week-old) were randomly divided into 3 groups and were fed normal diet, high-fat diet (HFD, 20% fat) and HFD+0.005% (w/w) luteolin for 16 weeks. Luteolin improved insulin resistance, as measured by HOMA-IR and glucose tolerance, along with preservation action of pancreatic β-cells, compared to the HFD group. Luteoiln was significantly decreased the levels of leptin and ghrelin that play a pivotal role in energy balance, and the macrophage low-grade inflammation marker sCD163 (soluble Cd antigen 163) in plasma. Activities of hepatic anti-oxidant enzymes (catalase and glutathione peroxidase) were increased, while the levels of plasma transaminase (GOT and GPT) and oxidative damage markers (hepatic mitochondria H2O2 and TBARS) were markedly decreased by luteolin supplementation. In addition, luteolin increased oxidative capacity and fatty acid utilization by presenting decrease in enzyme activities of citrate synthase, cytochrome C oxidase and β-hydroxyacyl CoA dehydrogenase and UCP3 gene expression compared to high-fat diet. Moreover, our microarray results of muscle also revealed down-regulated gene expressions associated with TCA cycle by HFD were reversed to normal level by luteolin treatment. Taken together, our results indicate that luteolin is one of bioactive components for improving insulin resistance by increasing oxidative capacity, modulating anti-oxidant metabolism and suppressing inflammatory signaling cascades in diet-induced obese mice. These results provide possible therapeutic targets for prevention and treatment of diet-induced obesity and its complications.

Keywords: anti-oxidant metabolism, diabetes, luteolin, oxidative capacity

Procedia PDF Downloads 337

Authors: Wei-Ju Huang, Hung-Pin Hsu

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[Background] In congestive heart failure (CHF), it has always been the principle of clinical treatment to control the water retention mechanism in the body to prevent excessive fluid retention. Early control of sympathetic nerves, Renin-Angiotensin-Aldosterone system (RAA system, RAAS), or strengthening of Atrial Natriuretic Peptide (ANP) was the point. In RAA system, related hormones, such as angiotensin, or enzymes in the pathway, such as ACE-I, can be used with corresponding inhibitors to reduce water content.[Aim] In recent years, clinical studies have pointed out that if different mechanisms are combined, the control effect seems to be better. For example, recent studies showed that ENTRESTO, a combination of Sacubitril and Valsartan, is a good new drug for CHF. Sacubitril is a prodrug. After activation, it can inhibit neprilysin and act as a neprilysin inhibitor (ARNI) to reduce the breakdown of natriuretic peptides(ANP). Valsartan is a kind of angiotensin receptor blocker (ARB), both of which are used to treat heart failure at the same time, have excellent curative effects.[Materials and Methods] Considering the side effects of this drug, coughing and a few cases of diarrhea were observed. However, the effect of this drug on the patient's intestinal tract has not been confirmed. On the other hand, studies have pointed out that ANP supplement can improve the CHF and increase the inhibitory effect on cancer cells. Therefore, the purpose of this study is to use a special microbial detection method to prove that whether oral drugs have an effect on microorganisms.The experimental method uses Nissui Compact Dry to observe the situation in different types of microorganisms. After the drug is dissolved in water, it is implanted in a petri dish, and the presence of different microorganisms is detected through different antibody reactions to confirm whether the drug has some toxicology in the gut.[Results and Discussion]From the above experimental results, it can be known that among the effects of Sacubitril and Valsartan on the basic microbial flora of the human body, low doses had no significant effect on Escherichia coli or intestinal bacteria. If Sacubitril or Valsartan with a high concentration of 3mg/ml is used alone or under the stimulation of a high concentration of the two drugs, it has a significant inhibitory effect on Escherichia coli. However, in terms of the effect on intestinal bacteria, high concentration of Sacubitril has a more significant inhibitory effect on intestinal bacteria, while high concentration of Valsartan has a less significant inhibitory effect on intestinal bacteria. The inhibitory effect of the combination of the two drugs on intestinal bacteria is also less significant.[Conclusion]The results of this study can be used as a further reference for the possible side effects of the clinical use of Sacubitril and Valsartan on the intestinal tract of patients,

Keywords: sacubitril, valsartan, entresto, congestive heart failure (CHF)

Procedia PDF Downloads 67

Authors: Ayodele A. Otaiku

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Purpose: Nigeria has become the largest importer of rice in Africa and second in the world, 2015. Investigate interactions of organic rice farming on soil quality and health from bio-waste converted to biofertilizer and its environmental impact on rice crop. Methodology: Bio-wastes, poultry waste, organic agriculture wastes, wood ash mixed with microbial inoculant organisms called OBD-Plus microbes (broad spectrum) composted in anaerobic digester to OBD-biofertilizer (2010 - 2012) uses microbes to build humus and other stable carbons. Two field experiments were carried out at Badeggi, Niger state in 2011 and 2012 to evaluate the response of lowland rice production using biofertilizer. The experimental field was laid out in a strip-plot design with five treatments and three replications and at twenty-one day old seedlings of FARO 44 and FARO 52 rice varieties were transplanted. Plots without fertiliser application served as control. Findings: The highest rice grain yield increase of 4.4 t/ha over the control in 2012 against the Nigeria average of lowland rice grain yields of 1.5 t/ha. The utilization of OBD-Biofertilizer can decrease the use of chemical nitrogen fertilizer, prevent the depletion of soil organic matter and reduce environmental pollution. Increasing the floodwater productivity and optimizing the recycling of nutrients cum grazer populations and disease by biocontrols microbes present in the OBD-Biofertilizer. Organic matter in the soil improves by 58% and C/N 15 (2011) and 13.35 (2012). Implications: OBD- Biofertilizer produce plant growth hormones such as indole acetic acid (IAA), glomalin related soil protein and extracellular enzymes as phosphatases that promote soil health and quality. Conclusion: Microorganisms can enhance nutrients use efficiency by increasing root surface area e.g., mycorrhizal, fungi, promoting other beneficial symbioses of the host plant and microbial interactions resulting to increase in soil organic matter. By 2030, climate change is projected to depress cereal production in Africa by 2 to 3 percent. Improved seeds and increased fertilizer use should more than compensate, but this factor will still weigh heavily on efforts to make progress.

Keywords: OBD-plus microbial consortia, OBD-biofertilizer, rice production, soil quality, sustainable agriculture

Procedia PDF Downloads 269

Authors: Siti Nur Syazni Mohd Zuki, Tan Ling Ling, Nina Suhaity Azmi, Chong Kwok Feng, Lee Yook Heng

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Nitrite (NO2-) ion is used prevalently as a preservative in processed meat. Elevated levels of nitrite also found in edible bird’s nests (EBNs). Consumption of NO2- ion at levels above the health-based risk may cause cancer in humans. Spectrophotometric Griess test is the simplest established standard method for NO2- ion detection, however, it requires careful control of pH of each reaction step and susceptible to strong oxidants and dyeing interferences. Other traditional methods rely on the use of laboratory-scale instruments such as GC-MS, HPLC and ion chromatography, which cannot give real-time response. Therefore, it is of significant need for devices capable of measuring nitrite concentration in-situ, rapidly and without reagents, sample pretreatment or extraction step. Herein, we constructed a microspheres-based microbial optode for visual quantitation of NO2- ion. Raoutella planticola, the bacterium expressing NAD(P)H nitrite reductase (NiR) enzyme has been successfully extracted by microbial technique from EBN collected from local birdhouse. The whole cells and the lipophilic Nile Blue chromoionophore were physically absorbed on the photocurable poly(n-butyl acrylate-N-acryloxysuccinimide) [poly (nBA-NAS)] microspheres, whilst the reduced coenzyme NAD(P)H was covalently immobilized on the succinimide-functionalized acrylic microspheres to produce a reagentless biosensing system. Upon the NiR enzyme catalyzes the oxidation of NAD(P)H to NAD(P)+, NO2- ion is reduced to ammonium hydroxide, and that a colour change from blue to pink of the immobilized Nile Blue chromoionophore is perceived as a result of deprotonation reaction increasing the local pH in the microspheres membrane. The microspheres-based optosensor was optimized with a reflectance spectrophotometer at 639 nm and pH 8. The resulting microbial bio-optode membrane could quantify NO2- ion at 0.1 ppm and had a linear response up to 400 ppm. Due to the large surface area to mass ratio of the acrylic microspheres, it allows efficient solid state diffusional mass transfer of the substrate to the bio-recognition phase, and achieve the steady state response as fast as 5 min. The proposed optical microbial biosensor requires no sample pre-treatment step and possesses high stability as the whole cell biocatalyst provides protection to the enzymes from interfering substances, hence it is suitable for measurements in contaminated samples.

Keywords: acrylic microspheres, microbial bio-optode, nitrite ion, reflectometric

Procedia PDF Downloads 448

Authors: Amnah M. A. Alsuhaibani, Amal N. Al-Kuraieef

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Introduction: In the recent decades, production of low-calorie jams is needed for diabetics that comprise low calorie fruits and low calorie sweeteners. Object: the research aimed to prepare low calorie formulated pumpkin jams (fructose, stevia and aspartame) incorporated with soy bean and evaluate the jams through chemical analysis and sensory evaluation after storage for six month. Moreover, the possible effect of consumption of low calorie jams on diabetic rats was investigated. Methods: Five formulas of pumpkin jam with different sucrose, fructose, stevia and aspartame sweeteners and soy bean were prepared and stored at 10 oC for six month compared to ordinary pumpkin jam. Chemical composition and sensory evaluation of formulated jams were evaluated at zero time, 3 month and 6 month of storage. The best three acceptable pumpkin jams were taken for biological study on diabetic rats. Rats divided into group (1) served as negative control and streptozotocin induce diabetes four rat groups that were positive diabetic control (group2), rats fed on standard diet with 10% sucrose soybean jam, fructose soybean jam and stevia soybean jam (group 3, 4&5), respectively. Results: The content of protein, fat, ash and fiber were increased but carbohydrate was decreased in low calorie formulated pumpkin jams compared to ordinary jam. Production of aspartame soybean pumpkin jam had lower score of all sensory attributes compared to other jam then followed by stevia soybean Pumpkin jam. Using non nutritive sweeteners (stevia & aspartame) with soybean in processing jam could lower the score of the sensory attributes after storage for 3 and 6 months. The highest score was recorded for sucrose and fructose soybean jams followed by stevia soybean jam while aspartame soybean jam recorded the lowest score significantly. The biological evaluation showed a significant improvement in body weight and FER of rats after six weeks of consumption of standard diet with jams (Group 3,4&5) compared to Group1. Rats consumed 10% low calorie jam with nutrient sweetener (fructose) and non nutrient sweetener (stevia) soybean jam (group 4& 5) showed significant decrease in glucose level, liver function enzymes activity, and liver cholesterol & total lipids in addition of significant increase of insulin and glycogen compared to the levels of group 2. Conclusion: low calorie pumpkin jams can be prepared by low calorie sweeteners and soybean and also storage for 3 months at 10oC without change sensory attributes. Consumption of stevia pumpkin jam fortified with soybean had positive health effects on streptozoticin induced diabetes in rats.

Keywords: pumpkin jam, HFCS, aspartame, stevia, storage

Procedia PDF Downloads 183

Authors: Muhammad Muzamal, Anders Rasmuson

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Wood material is extensively considered as a raw material for the production of bio-polymers, bio-fuels and value-added chemicals. However, the shortcoming in using wood as raw material is that the enzymatic hydrolysis of wood material is difficult because the accessibility of enzymes to hemicelluloses and cellulose is hindered by complex chemical and physical structure of the wood. The steam explosion (SE) pre-treatment improves the digestion of wood material by creating both chemical and physical modifications in wood. In this process, first, wood chips are treated with steam at high pressure and temperature for a certain time in a steam treatment vessel. During this time, the chemical linkages between lignin and polysaccharides are cleaved and stiffness of material decreases. Then the steam discharge valve is rapidly opened and the steam and wood chips exit the vessel at very high speed. These fast moving wood chips collide with each other and with walls of the equipment and disintegrate to small pieces. More damaged and disintegrated wood have larger surface area and increased accessibility to hemicelluloses and cellulose. The energy required for an increase in specific surface area by same value is 70 % more in conventional mechanical technique, i.e. attrition mill as compared to steam explosion process. The mechanism of wood disintegration during the SE pre-treatment is very little studied. In this study, we have simulated collision and impact of wood chips (dimension 20 mm x 20 mm x 4 mm) with each other and with walls of the vessel. The wood chips are simulated as a 3D orthotropic material. Damage and fracture in the wood material have been modelled using 3D Hashin’s damage model. This has been accomplished by developing a user-defined subroutine and implementing it in the FE software ABAQUS. The elastic and strength properties used for simulation are of spruce wood at 12% and 30 % moisture content and at 20 and 160 OC because the impacted wood chips are pre-treated with steam at high temperature and pressure. We have simulated several cases to study the effects of elastic and strength properties of wood, velocity of moving chip and orientation of wood chip at the time of impact on the damage in the wood chips. The disintegration patterns captured by simulations are very similar to those observed in experimentally obtained steam exploded wood. Simulation results show that the wood chips moving with higher velocity disintegrate more. Moisture contents and temperature decreases elastic properties and increases damage. Impact and collision in specific directions cause easy disintegration. This model can be used to efficiently design the steam explosion equipment.

Keywords: dynamic simulation, disintegration of wood, impact, steam explosion pretreatment

Procedia PDF Downloads 400

Authors: Shohei Matsuo, Saki Mikai, Hiroshi Morita

Abstract:

Objectives: Shochu is a popular Japanese distilled spirits. In the production of shochu, the filamentous fungus Aspergillus kawachii has traditionally been used. A. kawachii produces two types of starch hydrolytic enzymes, α-amylase (enzymatic liquefaction) and glucoamylase (enzymatic saccharification). Liquid culture system is a relatively easy microorganism to ferment with relatively low cost of production compared for solid culture. In liquid culture system, acid-unstable α-amylase (α-A) was produced abundantly, but, acid-stable α-amylase (Aα-A) was not produced. Since there is high enzyme productivity, most in shochu brewing have been adopted by a solid culture method. In this study, therefore, we investigated production of Aα-A in liquid culture system. Materials and methods: Microorganism Aspergillus kawachii NBRC 4308 was used. The mold was cultured at 30 °C for 7~14 d to allow formation of conidiospores on slant agar medium. Liquid Culture System: A. kawachii was cultured in a 100 ml of following altered SLS medium: 1.0 g of rice flour, 0.1 g of K2HPO4, 0.1 g of KCl, 0.6 g of tryptone, 0.05 g of MgSO4・7H2O, 0.001 g of FeSO4・7H2O, 0.0003 g of ZnSO4・7H2O, 0.021 g of CaCl2, 0.33 of citric acid (pH 3.0). The pH of the medium was adjusted to the designated value with 10 % HCl solution. The cultivation was shaking at 30 °C and 200 rpm for 72 h. It was filtered to obtain a crude enzyme solution. Aα-A assay: The crude enzyme solution was analyzed. An acid-stable α-amylase activity was carried out using an α-amylase assay kit (Kikkoman Corporation, Noda, Japan). It was conducted after adding 9 ml of 100 mM acetate buffer (pH 3.0) to 1 ml of the culture product supernatant and acid treatment at 37°C for 1 h. One unit of a-amylase activity was defined as the amount of enzyme that yielded 1 mmol of 2-chloro-4-nitrophenyl 6-azide-6-deoxy-b-maltopentaoside (CNP) per minute. Results and Conclusion: We experimented with co-culture of A. kawachii and lactobacillus in order to get control of pH in altered SLS medium. However, high production of acid-stable α-amylase was not obtained. We experimented with yoghurt or milk made an addition to liquid culture. The result indicated that high production of acid-stable α-amylase (964 U/g-substrate) was obtained when milk made an addition to liquid culture. Phosphate concentration in the liquid medium was a major cause of increased acid-stable α-amylase activity. In liquid culture, acid-stable α-amylase activity was enhanced by milk, but Fats and oils in the milk were oxidized. In addition, Tryptone is not approved as a food additive in Japan. Thus, alter SLS medium added to skim milk excepting for the fats and oils in the milk instead of tryptone. The result indicated that high production of acid-stable α-amylase was obtained with the same effect as milk.

Keywords: acid-stable α-amylase, liquid culture, milk, shochu

Procedia PDF Downloads 284

Authors: Manohar Salla, Mark S. Butler, Ruby Pelingon, Geraldine Kaeslin, Daniel E. Croker, Janet C. Reid, Jong Min Baek, Paul V. Bernhardt, Elizabeth M. J. Gillam, Matthew A. Cooper, Avril A. B. Robertson

Abstract:

MCC950 is a potent and selective inhibitor of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome that shows early promise for treatment of inflammatory diseases. The identification of major metabolites of lead molecule is an important step during drug development process. It provides an information about the metabolically labile sites in the molecule and thereby helping medicinal chemists to design metabolically stable molecules. To identify major metabolites of MCC950, the compound was incubated with human liver microsomes and subsequent analysis by (+)- and (−)-QTOF-ESI-MS/MS revealed a major metabolite formed due to hydroxylation on 1,2,3,5,6,7-hexahydro-s-indacene moiety of MCC950. This major metabolite can lose two water molecules and three possible regioisomers were synthesized. Co-elution of major metabolite with each of the synthesized compounds using HPLC-ESI-SRM-MS/MS revealed the structure of the metabolite (±) N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. Subsequent synthesis of individual enantiomers and coelution in HPLC-ESI-SRM-MS/MS using a chiral column revealed the metabolite was R-(+)- N-((1-hydroxy-1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-4-(2-hydroxypropan-2-yl)furan-2-sulfonamide. To study the possible cytochrome P450 enzyme(s) responsible for the formation of major metabolite, MCC950 was incubated with a panel of cytochrome P450 enzymes. The result indicated that CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C18, CYP2C19, CYP2J2 and CYP3A4 are most likely responsible for the formation of the major metabolite. The biological activity of the major metabolite and the other synthesized regioisomers was also investigated by screening for for NLRP3 inflammasome inhibitory activity and cytotoxicity. The major metabolite had 170-fold less inhibitory activity (IC50-1238 nM) than MCC950 (IC50-7.5 nM). Interestingly, one regioisomer had shown nanomolar inhibitory activity (IC50-232 nM). However, no evidence of cytotoxicity was observed with any of these synthesized compounds when tested in human embryonic kidney 293 cells (HEK293) and human liver hepatocellular carcinoma G2 cells (HepG2). These key findings give an insight into the SAR of the hexahydroindacene moiety of MCC950 and reveal a metabolic soft spot which could be blocked by chemical modification.

Keywords: Cytochrome P450, inflammasome, MCC950, metabolite, microsome, NLRP3

Procedia PDF Downloads 252