Search results for: detoxification enzymes

Authors: Soufiane Fal, Abderahim Aasfar, Ali Ouhssain, Hasnae Choukri, Abelaziz Smouni, Hicham El Arroussi

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Heavy metals contamination is a major environmental concern around the world. It has a harmful impact on plant productivity and poses a serious risk to humans and animals health. In the present study, the effect of Microalgae Crude Extract (MCE) on tomato growth and nutrients uptake exposed to 2 mM Pb2+ and Cd2+ was investigated. In results, 2 mM Pb2+ and Cd2+ showed a significant reduction of tomatobiomass and perturbation in nutrients absorption. Moreover, MCE application in tomato plant exposed to Pb2+ and Cd2+ showed a significant enhancement of biomass compared to tomato plants under Pb2+ and Cd2+. On the other hand, MCE application favoured heavy metals accumulation in root and inhibited their translocation to shoot as phytostabilisation mechanism. Tomato plants showed biochemical responses to Pb2+ and Cd2+ stress with elevation of scavenging enzymes and molecules such as POD, CAT, SOD, Proline, and polyphenols, etc. In addition, the treatment by MCE showed a significant reduction level of the majority of these parameters. Furthermore, the metabolomic analysis revealed a significant change in important metabolites. Pb2+ and Cd2+ showed decrease in SFA and increase of UFA, VLFA, alkanes, alkenes, sterols, which known accumulated as tolerance and resistance mechanism to heavy metal (H.M) stress. However, MCE treatment showed the inverse of these response to return tomato plants to normal state and enhanced tolerance and resistance to heavy metal stress. In the present study, we emphasized that MCE can alleviate H.M stress, enhance tomato plant growth nutrients absorption and improve biochemical responses.

Keywords: microalgae crude extract, heavy metal stress, nutrient uptake, metabolomic analysis, solanum lycopersicum (Tomato), phytostabilisation

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Authors: Jinyoung Park, Eun Joo Song

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Introduction: Deubiquitinating enzymes (DUBs) are proteases that cleave ubiquitin or ubiquitin-like modifications on substrates. Deubiquitination could regulate cellular physiology, such as signal transduction, DNA damage and repair, and cell cycle progression. Although more than 100 DUBs are encoded in the human and the importance of DUBs has been realized, the functions of most DUBs are unknown. This study aims to identify the molecular mechanism by which deubiquitinating enzyme USP35 regulates cell cycle progression for the first time. Methods: USP35 RNAi was mainly used to identify the function of USP35 in cell cycle progression. To find substrates of USP35, we analyzed protein-protein interaction using LC-MS. Several biological methods, such as ubiquitination assay, cell synchronization, immunofluorescence, and immunoprecipitation assay were used to investigate the exact mechanism by which USP35 affects successful completion of mitosis. Results: USP35 knockdown caused not only reduction of mitotic cell number but also induction of mitotic cells with abnormal spindle formation. Actually, cell proliferation was decreased by USP35 knockdown. Interestingly, we found that loss of USP35 decreased the stability and expression of Aurora B, a member of chromosomal passenger complex (CPC), and the phosphorylation of its substrate. Indeed, USP35 interacted with Aurora B and deubiquitinated it. In addition, USP35 knockdown induced abnormal localization of Aurora B in mitotic cells. Finally, CDH1-mediated ubiquitination of Aurora B level was rescued by USP35 overexpression, but not inactive form of USP35, USP35 C450A. Discussion: Our findings suggest that USP35 regulates Aurora B-mediated mitotic spindle assembly and G2-M transition by blocking CDH1-induced degradation of Aurora B.

Keywords: USP35, HSP90, Aurora B, cell cycle progression

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Authors: Hanen Bouaziz, Françoise Croute, Najiba Zeghal

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In order to investigate the toxic effects of fluoride on cerebrum maturity of suckling mice, we treated adult female mice of Swiss Albinos strain by 500 ppm NaF in their drinking water from the 15th day of pregnancy until the day 14 after delivery. All mice were sacrificed on day 14 after parturition. During treatment, levels of thiobarbituric acid reactive substances, the marker of lipid peroxidation extend, increased, while the activities of the antioxidant enzymes such as glutathione peroxidase, superoxide dismutase and catalase and the level of glutathione decreased significantly in cerebellum compared with those of the control group. These results suggested that fluoride enhanced oxidative stress, thereby disturbing the antioxidant defense of nursing pups. In addition, acetylcholinesterase activity in cerebellum was inhibited after treatment with fluoride. In cerebellum of mice, migration of neurons from the external granular layer to the internal granular layer occurred postnatally. Key guidance signals to these migrating neurons were provided by laminin, an extracellular matrix protein fixed to the surface of astrocytes. In the present study, we examined the expression and distribution of laminin in cerebellum of 14-day-old mice. Immunoreactive laminin was disappeared by postnatal day 14 in cerebellum parenchyma of control pups and was restricted to vasculature despite the continued presence of granular cells in the external granular layer. In contrast, in cerebellum of NaF treated pups, laminin was deposited in organised punctuate clusters in the molecular layer. These data indicated that the disruption of laminin distribution might play a major role in the profound derangement of neuronal migration observed in cerebellum of NaF treated pups.

Keywords: acetylcholinesterase activity, cerebellum, laminin, oxidative stress, suckling mice

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Authors: Idan Chiyanzu, Maruping Mangena

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Crude glycerol, a major by-product from the transesterification of triacylglycerides with alcohol to biodiesel, is known to have a broad range of applications. For example, its bioconversion can afford a wide range of chemicals including alcohols, organic acids, hydrogen, solvents and intermediate compounds. In bacteria, the 2-oxoglutarate dioxygenase (2-OGD) enzymes are widely found among the Pseudomonas syringae species and have been recognized with an emerging importance in ethylene formation. However, the use of optimized enzyme function in recombinant systems for crude glycerol conversion to ethylene is still not been reported. The present study investigated the production of ethylene from crude glycerol using engineered E. coli MG1655 and JM109 strains. Ethylene production with an optimized expression system for 2-OGD in E. coli using a codon optimized construct of the ethylene-forming gene was studied. The codon-optimization resulted in a 20-fold increase of protein production and thus an enhanced production of the ethylene gas. For a reliable bioreactor performance, the effect of temperature, fermentation time, pH, substrate concentration, the concentration of methanol, concentration of potassium hydroxide and media supplements on ethylene yield was investigated. The results demonstrate that the recombinant enzyme can be used for future studies to exploit the conversion of low-priced crude glycerol into advanced value products like light olefins, and tools including recombineering techniques for DNA, molecular biology, and bioengineering can be used to allowing unlimited the production of ethylene directly from the fermentation of crude glycerol. It can be concluded that recombinant E.coli production systems represent significantly secure, renewable and environmentally safe alternative to thermochemical approach to ethylene production.

Keywords: crude glycerol, bioethylene, recombinant E. coli, optimization

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Authors: Alessio Giovannelli, Claudia Cocozza, Enrico Marchi, Valerio Giorgio Muzzini, Eleftherios Touloupakis, Raffaella Margherita Zampieri

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In Mediterranean countries, forest fires are recurrent events that need to be considered as a central component of regional and global forest management strategies and biodiversity restoration programmes. The response of tree function to fire damage can vary widely, also taking into account species, season, age of the tree, etc. Trees that survive fire may have different levels of physiological functionality, which may result in reduced growth or increased susceptibility to delayed mortality. An approach to assessing irreversible physiological injury in trees could help to inform management decisions at burned sites for biodiversity restoration, environmental safety and understanding of ecosystem functional adaptations. Physiological proxies for latent tree mortality, such as cambial cell death, reduced or absent starch and soluble sugar content in C sinks, and ethanol accumulation in the phloem, are considered proxies for cell death. However, their determination requires time-consuming laboratory protocols, making the approach unfeasible as a practical option in the field, but recent findings have shown that biosensors could be usefully applied to overcome these limitations. The study will focus on the development of amperometric biosensors capable of detecting a few target molecules in the phloem and xylem (such as ethanol and glucose) that have recently been identified as proxies for latent tree mortality. The results of a specific experiment on a stand of Pinus pinaster subjected to prescribed fire are reported.

Keywords: enzymes, glucose, ethanol, prescribed fires

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Authors: Bryony Davies

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Many cell and tissue culture practices ignore the effects of gravity on cell biology, and little is known about how cell components may move in response to gravitational forces. Starfish oocytes provide an excellent model for interrogating the movement of cell components due to their unusually large size, ease of handling, and high transparency. Chromosomes from starfish oocytes can be visualised by microinjection of the histone-H2B-mCherry plasmid into the oocytes. The movement of the chromosomes can then be tracked by live-cell fluorescence microscopy. The results from experiments using these methods suggest that there is a replicable downward movement of centrally located chromosomes at a median velocity of 0.39 μm/min. Chromosomes nearer the nuclear boundary showed more restricted movement. Chromosome density and shape could also be altered by microinjection of restriction enzymes, primarily Alu1, before imaging. This was found to alter the speed of chromosome movement, with chromosomes from Alu1-injected nuclei showing a median downward velocity of 0.60 μm/min. Overall, these results suggest that there is a non-negligible movement of chromosomes in response to gravitational forces and that this movement can be altered by enzyme activity. Future directions based on these results could interrogate if this observed downward movement extends to other cell components and to other cell types. Additionally, it may be important to understand whether gravitational orientation and vertical positioning of cell components alter cell behaviour. The findings here may have implications for current cell culture practices, which do not replicate cell orientations or external forces experienced in vivo. It is possible that a failure to account for gravitational forces in 2D cell culture alters experimental results and the accuracy of conclusions drawn from them. Understanding possible behavioural changes in cells due to the effects of gravity would therefore be beneficial.

Keywords: starfish, oocytes, live-cell imaging, microinjection, chromosome dynamics

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Authors: Ronald Villamar-Torres, Michael Staudt, Christopher Viot

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Cotton Gossypium hirsutum L. is one of the most important industrial crops, producing the world leading natural textile fiber, but is very prone to arthropod attacks that reduce crop yield and quality. Cotton cultivation, therefore, makes an outstanding use of chemical pesticides. In reaction to herbivorous arthropods, cotton plants nevertheless show natural defense reactions, in particular through volatile organic compounds (VOCs) emissions. These natural defense mechanisms are nowadays underutilized but have a very high potential for cotton cultivation, and elucidating their genetic bases will help to improve their use. Simulating herbivory attacks by mechanical wounding of cotton plants in greenhouse, we studied by qPCR the changes in gene expression for genes of the terpenoids biosynthesis pathway. Differentially expressed genes corresponded to higher levels of the terpenoids biosynthesis pathway and not to enzymes synthesizing particular terpenoids. The genes were mapped on the G. hirsutum L. reference genome; their global relationships inside the general metabolic pathways and the biosynthesis of secondary metabolites were visualized with iPath2. The chromatographic profiles of VOCs emissions indicated first monoterpenes and sesquiterpenes emissions, dominantly four molecules known to be involved in plant reactions to arthropod attacks. As a result, the study permitted to identify potential key genes for the emission of volatile terpenoids by cotton plants in reaction to an arthropod attack, opening possibilities for molecular-assisted cotton breeding in benefit of smallholder cotton growers.

Keywords: biosynthesis pathways, cotton, mechanisms of plant defense, terpenoids, volatile organic compounds

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Authors: Olga Shapovalova, Vladimir Vinogradov

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Among many different sol – gel matrices only alumina can be successfully parenteral injected in the human body. And this is not surprising, because boehmite (aluminium oxyhydroxide) is the metal oxide approved by FDA and EMA for intravenous and intramuscular administrations, and also has been using for a longtime as adjuvant for producing of many modern vaccines. In our earlier study, it has been shown, that denaturation temperature of enzymes entrapped in sol-gel boehmite matrix increases for 30 – 60 °С with preserving of initial activity. It makes such matrices more attractive for long-term storage of non-stable drugs. In current work we present ultrasound-assisted sol-gel synthesis of nano-boehmite. This method provides bio-friendly, very stable, highly homogeneous alumina sol with using only water and aluminium isopropoxide as a precursor. Many parameters of the synthesis were studied in details: time of ultrasound treatment, US frequency, surface area, pore and nanoparticle size, zeta potential and others. Here we investigated the dependence of stability of colloidal sols and textural properties of the final composites as a function of the time of ultrasonic treatment. Chosen ultrasonic treatment time was between 30 and 180 minutes. Surface area, average pore diameter and total pore volume of the final composites were measured by surface and pore size analyzer Nova 1200 Quntachrome. It was shown that the matrices with ultrasonic treatment time equal to 90 minutes have the biggest surface area 431 ± 24 m2/g. On the other had such matrices have a smaller stability in comparison with the samples with ultrasonic treatment time equal to 120 minutes that have the surface area 390 ± 21 m2/g. It was shown that the stable sols could be formed only after 120 minutes of ultrasonic treatment, otherwise the white precipitate of boehmite is formed. We conclude that the optimal ultrasonic treatment time is 120 minutes.

Keywords: boehmite matrix, stabilisation, ultrasound-assisted sol-gel synthesis

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Authors: Kidane workelul, Li xu, Xiaoyang Pang, Jiaping Lv

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Dairy products are exceptionally ideal media for the growth of microorganisms because of their high nutritional content. There are several ways that milk might get contaminated throughout the milking process, including how the raw milk is transported and stored, as well as how long it is kept before being processed. Psychrotrophic bacteria are among the one which can deteriorate the quality of milk mainly their heat resistance proteas and lipase enzyme. For this research purpose 8 selected strains of Psychrotrophic bacteria (Entrococcus hirae, Pseudomonas fluorescens, Pseudomonas azotoformans, Pseudomonas putida, Exiguobacterium indicum, Pseudomonas paralactice, Acinetobacter indicum, Serratia liquefacients)are chosen and try to determine their characteristics based on the research methodology protocol. Thus, the 8 selected strains are cultured, plated incubate, extracted their genomic DNA and genome DNA was amplified, the purpose of the study was to identify their Psychrotrophic properties, lipase hydrolysis positive test, their optimal incubation temperature, designed primer using the noble strain P,flourescens conserved region area in target with lipA gene, optimized primer specificity as well as sensitivity and PCR detection for lipase positive strains using the design primers. Based on the findings both the selected 8 strains isolated from stored raw milk are Psychrotrophic bacteria, 6 of the selected strains except the 2 strains are positive for lipase hydrolysis, their optimal temperature is 20 to 30 OC, the designed primer specificity is very accurate and amplifies for those strains only with lipase positive but could not amplify for the others. Thus, the result is promising and could help in detecting the Psychrotrophic bacteria producing heat resistance enzymes (lipase) at early stage before the milk is processed and this will safe production loss for the dairy industry.

Keywords: dairy industry, heat-resistant, lipA, milk, primer and psychrotrophic

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Authors: Tarek Elnimr, Rabab El-kelany

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Heavy metals are environmental contaminants that may pose long-term health risks. Unfortunately, the consequent implementation of preventive measures was generally delayed, causing important negative effects to the exposed populations. The objective of this study was to determine whether co-consumption of nutritional supplements as selenium and vitamin E would treat the hazardous effects of exposure to mercury, lead and cadmium. 108 workers (60 males and 48 females) were the subject of this study, their ages ranged from 19-63 years, (M = 29.5±10.12). They were working in lamp factory for an average of 0.5-40 years (M= 5.3±8.8). Twenty control subjects matched for age and gender were used for comparison. All workers were subjected to neuropsychiatric evaluation. General Health Questionnaire (GHQ-28) revealed that 44.4% were complaining of anxiety, 52.7% of depression, 41.6% of social dysfunction and 22.2% of somatic symptoms. Cognitive tests revealed that long-term memory was not affected significantly when compared with controls, while short term memory and perceptual ability were affected significantly. Blood metal levels were measured by Inductively Coupled Plasma – optical emission spectrometry(ICP-OES), and revealed that the mean blood mercury, lead and cadmium concentrations before treatment were 1.6 mg/l, 0.39 mg/l and 1.7 µg/l, while they decreased significantly after treatment to 1.2 mg/l, 0.29 mg/l and 1.3 µg/l respectively. Anti-oxidative enzymes (paraoxonase and catalase) and lipid peroxidation product (malondialdehyde) were measured before and after treatment with selenium and vitamin E, and showed significant improvement. It could be concluded that co-consumption of selenium and vitamin E produces significant decrease in mercury, lead and cadmium levels in blood.

Keywords: mercury, lead, cadmium, neuropsychiatric impairment, selenium, vitamin E

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Authors: T. M. Clark, S. J. Coggins, R. Malik, J. King, R. Korman

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Objectives: This observational study investigated the use of molnupiravir for treatment of cats with naturally occurring feline infectious peritonitis. Methods: From September 2022 to February 2024, 66 cats diagnosed with FIP across 32 veterinary practices, mainly in Australia, were enrolled. Of these, 54 cats met the inclusion criteria. Complete remission was defined by the resolution of clinical signs and normalisation of A:G ratio (to ≥0.6). Presumptive remission was defined as sustained resolution of FIP-related clinical signs for at least 100 days post cessation of antiviral therapy. Results: In Cohort 1, 18 cats were treated with molnupiravir monotherapy. Thirteen achieved complete remission and three attained presumptive remission, resulting in an overall remission rate of 89% and a provisional cure rate of 72%, with three relapses. Cohort 2 included 29 cats treated with a short induction course of GS-441524 and/or remdesivir before switching to molnupiravir; 23 attained complete remission, and two achieved presumptive remission. The overall cure rate was 86% with no relapses. Seven cats in cohort 3 were initially treated with extended courses of GS-441524, remdesivir, and/or mefloquine and experienced treatment failure or relapse. Molnupiravir was introduced as a rescue therapy; 6 achieved complete remission and 1 achieved presumed remission, resulting in a 100% cure rate with no relapses. Few adverse effects were reported, with the most notable including neutropenia, transient elevations in hepatic enzymes, and polydipsia/polyuria. Conclusion and Relevance: Molnupiravir as a monotherapy, or in combination with other antivirals, represents an accessible, effective treatment for FIP when given at a dosage of 10-15 mg/kg BID. Success occurred across various presentations of FIP, including cases with ocular and neurological involvement.

Keywords: feline infectious peritonitis, FIP, molnupiravir, nucleoside analogue, antiviral

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Authors: Peng Hou

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Natural products produced from marine bacteria serve as an immense reservoir for anti-infective drugs and therapeutic agents. Nowadays, heterologous expression of gene clusters of interests has been widely adopted as an effective strategy for natural product discovery. Briefly, the heterologous expression flowchart would be: biosynthetic gene cluster identification, pathway construction and expression, and product detection. However, gene cluster capture using traditional Transformation-associated recombination (TAR) protocol is low-efficient (0.5% positive colony rate). To make things worse, most of these putative new natural products are only predicted by bioinformatics analysis such as antiSMASH, and their corresponding natural products biosynthetic pathways are either not expressed or expressed at very low levels under laboratory conditions. Those setbacks have inspired us to focus on seeking new technologies to efficiently edit and refractor of biosynthetic gene clusters. Recently, two cutting-edge techniques have attracted our attention - the CRISPR-Cas9 and Gibson Assembly. By now, we have tried to pretreat Brevibacillus laterosporus strain genomic DNA with CRISPR-Cas9 nucleases that specifically generated breaks near the gene cluster of interest. This trial resulted in an increase in the efficiency of gene cluster capture (9%). Moreover, using Gibson Assembly by adding/deleting certain operon and tailoring enzymes regardless of end compatibility, the silent construct (~80kb) has been successfully refactored into an active one, yielded a series of analogs expected. With the appearances of the novel molecular tools, we are confident to believe that development of a high throughput mature pipeline for DNA assembly, transformation, product isolation and identification would no longer be a daydream for marine natural product discovery.

Keywords: biosynthesis, CRISPR-Cas9, DNA assembly, refactor, TAR cloning

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Authors: Eduzor Esther, Uhiara Ngozi, Ya’u Abubakar Umar, Anayo Jacob Gabriel, Umar Ahmed

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The study investigated the characteristic effect of treating okra seed and soybeans seed oil with crude enzymes extract from malted rice. The oils from okra seeds and soybeans were obtained by solvent extraction method using N-hexane solvent. Soybeans seeds had higher percentage oil yield than okra seed. 250ml of each oil was thoroughly mixed with 5ml of the malted rice extract at 400C for 5mins and then filtered and regarded as treated oil while another batch of 250ml of each oil was not mixed with the malted rice extract and regarded as untreated oil. All the oils were analyzed for specific gravity, refractive index, emulsification capacity, absortivity, TSS and viscosity. Treated okra seed and soybeans oil gave higher values for specific gravity, than the untreated oil for okra seed and soybeans oil respectively. The emulsification capacity values were also higher for treated oils, when compared to the untreated oil, for okra seed and soybeans oil respectively. Treated okra seed and soybeans oil also had higher range of values for absorptivity, than the untreated oil for okra seed and soybeans respectively. The ranges of T.S.S values of the treated oil were also higher, than those of the untreated oil for okra seed and soybeans respectively. The results of viscosity showed that the treated oil had higher values, than the untreated oil for okra seed and soybeans oil respectively. However, the results of refractive index showed that the untreated oils had higher values ranges of than the treated oils for okra seed and soybeans respectively. Treated oil show better quality in respect to the parameters analyst, except the refractive index which is slightly less but also is within the rangiest of standard, the oils are high in unsaturation especially okra oil when compared with soya beans oil. It is recommended that, treated oil of okra seeds and soya beans can serve better than many oils that presently in use such as ground nut oil, palm oil and cotton seeds oil.

Keywords: extract, malted, oil, okra, rice, seed, soybeans

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Authors: Amamra Ryma, Djebar Mohamed Reda, Moumeni Ouissem, Otmani Hadjer, Berrebbah Houria

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The problem of environmental contamination by the excessive use of organics cannot be neglected. Extensive application is usually companied with serious problems and health risk. It is established that many chemicals, in common use, can produce some toxic effects on biological systems through their mode of action or by production of free radicals that damage all cell compounds. Deltamethrin, a widely used type II pyrethroid pesticide, is one of the most common contaminants in freshwater aquatic system. In this study, we investigate the effects of deltamethrin exposure on the induction of oxidative stress to the freshwater ciliate Paramecium tetraurelia. After the treatment of paramecium cells with increasing concentrations of insecticide, we followed up the growth kinetics, generation time and the percentage response. Also, we studied the variation in biomarkers of stress such as: GSH content, GST, GPX and CAT activities. Our results showed a significant decrease in the proliferation of cells correlated by the decrease in generation number and the increase in generation time. Also, we noted an inhibition in the percentage response. The monitoring of biomarkers revealed depletion in GSH content in a proportional and dose dependent manner accompanied by an increase in the GST activity. In parallel, a strong induction in the CAT and GPX activities was noted specially for the highest dose. In summary, under the current experimental conditions, deltamethrin is highly toxic to the freshwater ciliate Paramecium tetraurelia. Exposure to low concentrations showed significant adverse on growth accompanied with the induction of oxidative damage supported by the decrease in GSH content and the intensification of the antioxidant enzymes.

Keywords: deltamethrin, Paramecium tetraurelia, growth, oxidative stress, biomarkers, antioxidant

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Authors: Moustafa Elhamouly, Masayuki Shimada

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The production of competent oocytes is essential for reproductivity in mammals. Maintenance of mitochondrial efficiency is required to supply the ATP necessary for granulosa cell proliferation during the follicular development process. Treatment with Pyrroloquinoline quinone (PQQ) has been reported to increase the number of ovulated oocytes and pups per delivery in mice by maintaining healthy mitochondrial function. This study aimed to elucidate how PQQ maintains mitochondrial function during ovarian follicle growth. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ. The effects of PQQ on beta-oxidation, mitochondrial function, mitophagy, and mitochondrial biogenesis were examined. PQQ increased beta-oxidation-related genes and CPT1 protein content in granulosa cells and this was associated with a decreased phosphorylation of P38 signaling protein. Using the fatty acid oxidation assay on the flux analyzer, PQQ increased the reliance of beta-oxidation on the endogenous fatty acids and was associated with a mild UCP-dependant mitochondrial uncoupling, ATP production, mitophagy, and mitochondrial biogenesis. PQQ also increased the expression of endogenous antioxidant enzymes. Thus, PQQ induced beta-oxidation in growing granulosa cells relying on endogenous fatty acids. And reduced the Reactive oxygen species (ROS) production by inducing a mild mitochondrial uncoupling with keeping high mitochondrial function. Damaged mitochondria were recycled by the induced mitophagy and replaced by the increased mitochondrial biogenesis. Collectively, PQQ may enhance reproductivity by maintaining the efficiency of mitochondria to produce enough ATP required for normal folliculogenesis.

Keywords: granulosa cells, mitochondrial uncoupling, mitophagy, pyrroloquinoline quinone (PQQ), reactive oxygen species (ROS).

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Authors: Sara Arafeh, Kovriguine Evguine

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Cytochromes P450 are enzymes that require a supply of electrons to catalyze the synthesis of steroid hormones, fatty acids, and prostaglandin hormone. Cytochrome P450 Oxidoreductase (CYPOR), a membrane bound enzyme, provides these electrons in its open conformation. CYPOR has two cytosolic domains (FAD domain and FMN domain) and an N-terminal in the membrane. In its open conformation, electrons flow from NADPH, FAD, and finally to FMN where cytochrome P450 picks up these electrons. In the closed conformation, cytochrome P450 does not bind to the FMN domain to take the electrons. It was found that when the cytosolic domains are isolated, CYPOR could not bind to cytochrome P450. This suggested that the membrane environment is important for CYPOR function. This project takes the initiative to better understand the dynamics of CYPOR in its full length. Here, we determine the distance between specific sites in the FAD and FMN binding domains in CYPOR by Forster Resonance Energy Transfer (FRET) and Ultrafast TA spectroscopy with and without NADPH. The approach to determine these distances will rely on labeling these sites with red and infrared fluorophores. Mimic membrane attachment is done by inserting CYPOR in lipid nanodiscs. By determining the distances between the donor-acceptor sites in these domains, we can observe the open/closed conformations upon reducing CYPOR in the presence and absence of cytochrome P450. Such study is important to better understand CYPOR mechanism of action in various endosomal membranes including hepatic CYPOR which is vital in plasma cholesterol homeostasis. By investigating the conformational cycles of CYPOR, we can synthesize drugs that would be more efficient in affecting the steroid hormonal levels and metabolism of toxins catalyzed by Cytochrome P450.

Keywords: conformational cycle of CYPOR, cytochrome P450, cytochrome P450 oxidoreductase, FAD domain, FMN domain, FRET, Ultrafast TA Spectroscopy

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Authors: Martynova Alina, Lyubov Buzoleva

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Streptococcus pneumoniae is the causative agent of pneumonias and meningitids throught all the world. Having high genetic diversity, this microorganism can cause different clinical forms of pneumococcal infections and microbiologically it is really difficult diagnosed by routine methods. Also, epidemiological surveillance requires more developed methods of molecular typing because the recent method of serotyping doesn't allow to distinguish invasive and non-invasive isolates properly. Non-coding genome of bacteria seems to be the interesting source for seeking of highly distinguishable markers to discriminate the subspecies of such a variable bacteria as Streptococcus pneumoniae. Technically, we proposed scheme of discrimination of S.pneumoniae strains with amplification of non-coding region (SP_1932) with the following restriction with 2 types of enzymes of Alu1 and Mn1. Aim: This research aimed to compare different methods of typing and their application for molecular epidemiology purposes. Methods: we analyzed population of 100 strains of S.pneumoniae isolated from different patients by different molecular epidemiology methods such as pulse-field gel electophoresis (PFGE), restriction polymorphism analysis (RFLP) and multilolocus sequence typing (MLST), and all of them were compared with classic typing method as serotyping. The discriminative power was estimated with Simpson Index (SI). Results: We revealed that the most discriminative typing method is RFLP (SI=0,97, there were distinguished 42 genotypes).PFGE was slightly less discriminative (SI=0,95, we identified 35 genotypes). MLST is still the best reference method (SI=1.0). Classic method of serotyping showed quite weak discriminative power (SI=0,93, 24 genotypes). In addition, sensivity of RFLP was 100%, specificity was 97,09%. Conclusion: the most appropriate method for routine epidemiology surveillance is RFLP with non-coding region of Streptococcsu pneumoniae, then PFGE, though in some cases these results should be obligatory confirmed by MLST.

Keywords: molecular epidemiology typing, non-coding genome, Streptococcus pneumoniae, MLST

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Authors: Debajyoti Bose

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Chitosan has received much attention as a functional biopolymer for diverse applications, especially in pharmaceutics and medicine. Chitosan is a positively charged natural biodegradable and biocompatible polymer. It is a linear polysaccharide consisting of β-1,4 linked monomers of glucosamine and N-acetylglucosamine. Chitosan can be mainly obtained from fungal sources during large fermentation process. In this study,three different fungal strains Aspergillus niger NCIM 1045, Aspergillus oryzae NCIM 645 and Mucor indicus MTCC 3318 were used for the production of chitosan. The growth mediums were optimized for maximum fungal production. The produced chitosan was characterized by determining degree of deacetylation. Chitosan possesses one reactive amino at the C-2 position of the glucosamine residue, and these amines confer important functional properties to chitosan which can be exploited for biofabrication to generate various chemically modified derivatives and explore their potential for pharmaceutical field. Chitosan nanoparticles were prepared by ionic cross-linking with tripolyphosphate (TPP). The major effect on encapsulation and release of protein (e.g. enzyme diastase) in chitosan-TPP nanoparticles was investigated in order to control the loading and release efficiency. It was noted that the chitosan loading and releasing efficiency as a nanocapsule, obtained from different fungal sources was almost near to initial enzyme activity(12026 U/ml) with a negligible loss. This signify, chitosan can be used as a polymeric drug as well as active component or protein carrier material in dosage by design due to its appealing properties such as biocompatibility, biodegradability, low toxicity and relatively low production cost from abundant natural sources. Based upon these initial experiments, studies were also carried out on modification of chitosan based nanocapsules incorporated with physiologically important enzymes and nutraceuticals for target delivery.

Keywords: fungi, chitosan, enzyme, nanocapsule

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Authors: Iman Kamranfar, Gang-Ping Xue, Salma Balazadeh, Bernd Mueller-Roeber

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Adverse environmental conditions such as salinity stress, high temperature and drought limit plant growth and typically lead to precocious tissue degeneration and leaf senescence, a process by which nutrients from photosynthetic organs are recycled for the formation of flowers and seeds to secure reaching the next generation under such harmful conditions. In addition, abiotic stress affects developmental patterns that help the plant to withstand unfavourable environmental conditions. We discovered an NAC (for NAM, ATAF1, 2, and CUC2) transcription factor (TF), called RAF in the following, which plays a central role in abiotic drought stress-triggered senescence and the control of developmental adaptations to stressful environments. RAF is an ABA-responsive TF; RAF overexpressors are hypersensitive to abscisic acid (ABA) and exhibit precocious senescence while knock-out mutants show delayed senescence. To explore the RAF gene regulatory network (GRN), we determined its preferred DNA binding sites by binding site selection assay (BSSA) and performed microarray-based expression profiling using inducible RAF overexpression lines and chromatin immunoprecipitation (ChIP)-PCR. Our studies identified several direct target genes, including those encoding for catabolic enzymes acting during stress-induced senescence. Furthermore, we identified various genes controlling drought stress-related developmental changes. Based on our results, we conclude that RAF functions as a central transcriptional regulator that coordinates developmental programs with stress-related inputs from the environment. To explore the potential agricultural applications of our findings, we are currently extending our studies towards crop species.

Keywords: abiotic stress, Arabidopsis, development, transcription factor

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Authors: G. K. Sruthi, Sila Bhattacharya

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Sorghum is an important food crop in the dry tropical areas of the world, and possesses significant levels of phytochemicals and dietary fiber to offer health benefits. However, the absence of gluten is a limitation for converting the sorghum dough into sheeted/flattened/rolled products. Chapathi/roti (flat unleavened bread prepared conventionally from whole wheat flour dough) was attempted from sorghum as wheat gluten causes allergic reactions leading to celiac disease. Dynamic oscillatory rheology of sorghum flour dough (control sample) and enzyme treated sorghum doughs were studied and linked to the attributes of the finished ready-to-eat product. Enzymes like amylase, xylanase, and a mix of amylase and xylanase treated dough affected drastically the rheological behaviour causing a lowering of dough consistency. In the case of amylase treated dough, marked decrease of the storage modulus (G') values from 85513 Pa to 23041 Pa and loss modulus (G") values from 8304 Pa to 7370 Pa was noticed while the phase angle (δ) increased from 5.6 to 10.1o for treated doughs. There was a 2 and 3 fold increase in the total sugar content after α-amylase and xylanase treatment, respectively, with simultaneous changes in the structure of the dough and finished product. Scanning electron microscopy exhibited enhanced extent of changes in starch granules. Amylase and mixed enzyme treatment produced a sticky dough which was difficult to roll/flatten. The dough handling properties were improved by the use of xylanase and quality attributes of the chapath/roti. It is concluded that enzyme treatment can offer improved rheological status of gluten free doughs and products.

Keywords: sorghum dough, amylase, xylanase, dynamic oscillatory rheology, sensory assessment

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Authors: Samaneh Nazemi, Ayla Bahramian, Marzieh Aghazadeh

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Saliva is a clear liquid composed of water, electrolytes, glucose, amylase, glycoproteins, and antimicrobial enzymes. The presence of a wide range of molecules and proteins in saliva has made this fluid valuable in screening for some diseases as well as epidemiological studies. Saliva is easier than serum to collect in large populations. Due to the importance of sodium and glucose levels in many biological processes, this study investigates the relationship between sodium and glucose levels in salivary and serum samples of healthy individuals referring to Tabriz Dental School. This descriptive-analytical study was performed on 40 healthy individuals referred to the Oral Diseases Department of Tabriz Dental School. Serum and saliva samples were taken from these patients according to standard protocols. Data were presented as mean (standard deviation) and frequency (percentage) for quantitative and qualitative variables. Pearson test, paired-samples T-test and SPSS 24 software were used to determine the correlation between serum and salivary levels of these biomarkers. In this study, P less than 0.05% is considered significant. Out of 40 participants in this study, 14 (35%) were male, and 26 (65%) were female. According to the results of this study, the mean salivary sodium (127.53 ml/dl) was lower than the mean serum sodium (141.2725 ml/dl). In contrast, the mean salivary glucose (4.55 ml/dl) was lower than the mean serum glucose (89.7575 ml/dl). The result of paired samples T-test (p-value<0.05) showed that there is a statistically significant difference between the mean of serum sodium and salivary sodium, as well as between the serum glucose and salivary glucose. Pearson correlation test results showed that there is no significant correlation between serum sodium and salivary sodium (p-value >0.05), but here is a positive correlation between serum glucose and salivary glucose (p-value<0.001). Both serum sodium and glucose were higher than salivary sodium and glucose.In conclusion, this study found that there was not a statistical relationship between salivary glucose and serum glucose and also salivary sodium and serum sodium of healthy individuals. Perhaps salivary samples can’t be used to measure glucose and sodium in these individuals.

Keywords: glucose, saliva, serum, sodium

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Authors: Muhammad Iqbal, Hafiz Muhammad Tauqeer, Hamza Rafaqat, Muhammad Naveed, Muhammad Awais Irshad

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Soil pollution with chromium (Cr) has become one of the most important concerns due to its toxicity for humans. To date, various remediation approaches have been employed for the remediation and management of Cr contaminated soils. Phytoextraction is an eco-friendly and emerging remediation approach which has gained attention due to several advantages over conventional remediation approach. The use of energy crops for phytoremediation is an emerging trend worldwide. These energy crops have high tolerance against various environmental stresses, the potential to grow in diverse ecosystems and high biomass production make them a suitable candidate for phytoremediation of contaminated soils. The removal efficiency of plants in phytoextraction depends upon several soil and plant factors including solubility, bioavailability and metal speciation in soils. A pot scale experiment was conducted to evaluate the phytoextraction potential of Arundo donax L. with the application of acetic acid (A.A) in Cr contaminated soils. Plants were grown in pots filled with 5 kg soils for 90 days. After 30 days plants acclimatization in pot conditions, plants were treated with various levels of Cr (2.5 mM, 5 mM, 7.5 mM, 10 mM) and A.A (Cr 2.5 mM + A.A 2.5 mM, Cr 5 mM + A.A 2.5 mM, Cr 7.5 mM + A.A 2.5 mM, Cr 10 mM + A.A 2.5 mM). The application of A.A significantly increased metal uptake and in roots and shoots of A. donax. This increase was observed at Cr 7.5 mM + A.A 2.5 mM but at high concentrations, visual symptoms of Cr toxicity were observed on leaves. Similarly, A.A applications also affect the activities of key enzymes including catalase (CAT), superoxidase dismutase (SOD), and ascorbate peroxidase (APX) in leaves of A. donax. Based on results it is concluded that the applications of A.A acid for phytoextraction is an alternative approach for the management of Cr affected soils and synthetic chelators should be replaced with organic acids.

Keywords: acetic acid, A. donax, chromium, energy crop, phytoextraction

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Authors: Jyoti Kumari, Pavuluri Srinivasa Rao

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Jackfruit (ArtocarpusheterophyllusL.) is an underutilized yet highly nutritious fruit with unique flavour, known for its therapeutic and culinary properties. Fresh jackfruit bulb has a very short shelf life due to high moisture and sugar content leading to microbial and enzymatic browning, hindering its consumer acceptability and marketability. An attempt has been made for the preservation of the ripe jackfruit bulbs, by the application of high pressure (HP) over a range of 200-500 MPa at ambient temperature for dwell times ranging from 5 to 20 min. The physicochemical properties of jackfruit bulbs such as the pH, TSS, and titrable acidity were not affected by the pressurization process. The ripening index of the fruit bulb also decreased following HP treatment. While the ascorbic acid and antioxidant activity of jackfruit bulb were well retained by high pressure processing (HPP), the total phenols and carotenoids showed a slight increase. The HPP significantly affected the colour and textural properties of jackfruit bulb. High pressure processing was highly effective in reducing the browning index of jackfruit bulbs in comparison to untreated bulbs. The firmness of the bulbs improved upon the pressure treatment with longer dwelling time. The polyphenol oxidase has been identified as the most prominent oxidative enzyme in the jackfruit bulb. The enzymatic activity of polyphenol oxidase and peroxidase were significantly reduced by up to 40% following treatment at 400 MPa/15 min. HPP of jackfruit bulbs at ambient temperatures is shown to be highly beneficial in improving the shelf stability, retaining its nutrient profile, color, and appearance while ensuring the maximum inactivation of the spoilage enzymes.

Keywords: antioxidant capacity, ascorbic acid, carotenoids, color, HPP-high pressure processing, jackfruit bulbs, polyphenol oxidase, peroxidase, total phenolic content

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Authors: Jiang Xu, Daoping Wang, Yinghong Pan

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The jointing-booting stage is a critical period of both vegetative growth and reproductive growth in rice. Therefore, the proteomic analysis of the mutant Osbri1, whose corresponding gene OsBRI1 encodes the putative BRs receptor OsBRI1, at jointing-booting stage is very important for understanding the effects of BRs on vegetative and reproductive growth. In this study, the proteomes of leaves from an allelic mutant of the DWARF 61 (D61, OsBRI1) gene, Fn189 (dwarf54, d54) and its wild-type variety T65 (Taichung 65) at jointing-booting stage were analysed by using a Q Exactive plus orbitrap mass spectrometer, and more than 3,100 proteins were identified in each sample. Ontology analysis showed that these proteins distribute in various space of the cells, such as the chloroplast, mitochondrion, and nucleus, they functioned as structural components and/or catalytic enzymes and involved in many physiological processes. Moreover, quantitative analysis displayed that 266 proteins were differentially expressed in two samples, among them, 77 proteins decreased and 189 increased more than two times in Fn189 compared with T65, the proteins whose content decreased in Fn189 including b5-like Heme/Steroid binding domain containing protein, putative retrotransposon protein, putative glutaminyl-tRNA synthetase, and higher content proteins such as mTERF, putative Oligopeptidase homologue, zinc knuckle protein, and so on. A former study founded that the transcription level of a mTERF was up-regulated in the leaves of maize seedling after EBR treatment. In our experiments, it was interesting that one mTERF protein increased, but another mTERF decreased in leaves of Fn189 at jointing-booting stage, which suggested that BRs may have differential regulation mechanisms on the expression of various mTERF proteins. The relationship between other differential proteins with BRs is still unclear, and the effects of BRs on rice protein contents and its regulation mechanisms still need further research.

Keywords: bri1 mutant, jointing-booting stage, proteomic analysis, rice

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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: Narin Ozturk, Xiao-Mei Li, Sylvie Giachetti, Francis Levi, Alper Okyar

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P-glycoprotein (P-gp, MDR1, ABCB1) is a transmembrane protein acting as an ATP-dependent efflux pump and functions as a biological barrier by extruding drugs and xenobiotics out of cells in healthy tissues especially in intestines, liver and brain as well as in tumor cells. The circadian timing system controls a variety of biological functions in mammals including xenobiotic metabolism and detoxification, proliferation and cell cycle events, and may affect pharmacokinetics, toxicity and efficacy of drugs. Selective mTOR (mammalian target of rapamycin) inhibitor everolimus is an immunosuppressant and anticancer drug that is active against many cancers, and its pharmacokinetics depend on P-gp. The aim of this study was to investigate the dosing time-dependent toxicity of everolimus with respect to the intestinal P-gp expression rhythms in mdr1a::Luc mice using Real Time-Biolumicorder (RT-BIO) System. Mdr1a::Luc male mice were synchronized with 12 h of Light and 12 h of Dark (LD12:12, with Zeitgeber Time 0 – ZT0 – corresponding Light onset). After 1-week baseline recordings, everolimus (5 mg/kg/day x 14 days) was administered orally at ZT1-resting period- and ZT13-activity period- to mdr1a::Luc mice singly housed in an innovative monitoring device, Real Time-Biolumicorder units which let us monitor real-time and long-term gene expression in freely moving mice. D-luciferin (1.5 mg/mL) was dissolved in drinking water. Mouse intestinal mdr1a::Luc oscillation profile reflecting P-gp gene expression and locomotor activity pattern were recorded every minute with the photomultiplier tube and infrared sensor respectively. General behavior and clinical signs were monitored, and body weight was measured every day as an index of toxicity. Drug-induced body weight change was expressed relative to body weight on the initial treatment day. Statistical significance of differences between groups was validated with ANOVA. Circadian rhythms were validated with Cosinor Analysis. Everolimus toxicity changed as a function of drug timing, which was least following dosing at ZT13, near the onset of the activity span in male mice. Mean body weight loss was nearly twice as large in mice treated with 5 mg/kg everolimus at ZT1 as compared to ZT13 (8.9% vs. 5.4%; ANOVA, p < 0.001). Based on the body weight loss and clinical signs upon everolimus treatment, tolerability for the drug was best following dosing at ZT13. Both rest-activity and mdr1a::Luc expression displayed stable 24-h periodic rhythms before everolimus and in both vehicle-treated controls. Real-time bioluminescence pattern of mdr1a revealed a circadian rhythm with a 24-h period with an acrophase at ZT16 (Cosinor, p < 0.001). Mdr1a expression remained rhythmic in everolimus-treated mice, whereas down-regulation was observed in P-gp expression in 2 of 4 mice. The study identified the circadian pattern of intestinal P-gp expression with an unprecedented precision. The circadian timing depending on the P-gp expression rhythms may play a crucial role in the tolerability/toxicity of everolimus. The circadian changes in mdr1a genes deserve further studies regarding their relevance for in vitro and in vivo chronotolerance of mdr1a-transported anticancer drugs. Chronotherapy with P-gp-effluxed anticancer drugs could then be applied according to their rhythmic patterns in host and tumor to jointly maximize treatment efficacy and minimize toxicity.

Keywords: circadian rhythm, chronotoxicity, everolimus, mdr1a::Luc mice, p-glycoprotein

Procedia PDF Downloads 342

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

Procedia PDF Downloads 356

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

Procedia PDF Downloads 441

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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