Search results for: commet assay

Authors: Mohammad Shamim Hasan Mandal, Phu Minh, Do Tan Khang, Phung Thi Tuyen, Tran Dang Xuan

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Rice (Oryza sativa L.) is one of the major crops of Vietnam which has more than thousands of varieties. Many of the local varieties have greater potentiality but they are in danger of extinct. Rice plant contains many secondary metabolites that are allelopathic to other plants. Seven rice varieties were cultivated in the field condition at Hiroshima University, Japan; stems and leaves from each variety were collected later, they were extracted with methanol, hexane, ethyl acetate, butanol, and water. Total phenolic content and total flavonoid contents were high in ethyl acetate extracts. DPPH antioxidant assay results showed that the ethyl acetate extracts had the higher IC50 value. Therefore, the ethyl acetate extracts were selected for laboratory experimentation through petri dish assay. Results showed that the two-local variety Re nuoc and Nan chon completely inhibited the germination of radish seedlings. Further laboratory bioassay and field experimentation will be conducted to validate the laboratory bioassay findings.

Keywords: allelopathy, bioassay, Oryza sativa, Raphanus sativus

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Authors: Jacky Bhagat, Baban S Ingole

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In this study, in vivo experiments were carried out to investigate the effects of a toxic polycyclic aromatic hydrocarbon (PAH), benzo(k)fluoranthene (B[k]F), on marine gastropod, Morula granulata collected from Goa, west coast of India. Snails were exposed to different concentrations of B(k)F (1, 10, 25 and 50 µg/L) for 96 h. The genotoxic effects were evaluated by measuring DNA strand breaks using alkaline comet assay and oxidative stress were measured with the help of battery of biomarkers such as superoxide dismutase (SOD) catalase (CAT), glutathione-s-transferase (GST), and lipid peroxidation (LPO). Concentration-dependent increase in percentage tail DNA (TDNA) was observed in snails exposed to B(k)F. Exposure concentrations above 1 µg/L of B(k)F, showed significant increase in SOD activity and LPO value in snails. After 96 h, SOD activity were found to be doubled for 50 µg/L of B(k)F with reference to control. Significant increase in CAT and GST activity was observed at all exposure conditions at the end of the exposure time. Our study showed that B(k)F induces oxidative stress in snails which further lead to genotoxic damage.

Keywords: benzo(k)fluoranthene, comet assay, gastropod, oxidative stress

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Authors: Mehran Fadaeinasab, Hamed Karimian, Najihah Mohd Hashim, Hapipah Mohd Ali

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In this study, three indole alkaloids namely; rauvolfine B, macusine B, and isoreserpiline have been isolated from the dichloromethane crude extract of Rauvolfia reflexa bark (Apocynaceae). The structural elucidation of the isolated compounds has been performed using spectral methods such as UV, IR, MS, 1D, and 2D NMR. Rauvolfine B showed anti proliferation activity on HCT-116 cancer cell line, its cytotoxicity induction was observed using MTT assay in eight different cell lines. Annexin-V is serving as a marker for apoptotic cells and the Annexin-V-FITC assay was carried out to observe the detection of cell-surface Phosphatidylserine (PS). Apoptosis was confirmed by using caspase-8 and -9 assays. Cell cycle arrest was also investigated using flowcytometric analysis. rauvolfine B had exhibited significantly higher cytotoxicity against HCT-116 cell line. The treatment significantly arrested HCT-116 cells in the S phase. Together, the results presented in this study demonstrated that rauvolfine B inhibited the proliferation of HCT-116 cells and programmed cell death followed by cell cycle arrest.

Keywords: apocynacea, indole alkaloid, apoptosis, cell cycle arrest

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Authors: Sehrish Iftikhar, Ahmad Ali Shahid, Kiran Nawaz, Waheed Anwar

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Discovery and development of new compounds require intense studies in chemistry, biochemistry. Numerous experiments under laboratory-, greenhouse- and field conditions can be performed to select suitable candidates and to understand their full potential. Novel fungicides are fundamental to combat plant diseases. Fusarium solani is important plant pathogen. New broad spectrum foliar fungicides against complex II were designed in this study. Complex II, namely succinate dehydrogenase (SDH), or succinate quinone oxidoreductase (SQR) is a multi-subunit enzyme at the crossroads of TCA and ETC at the inner mitochondrial membrane. The need for new and innovative fungicides is driven by resistance management, regulatory hurdles and increasing customer expectations amongst others. Fungicidal activity was assessed for the effect on mycelial growth and spore germination of the fungi using fungicide amended media assay. In mycelial growth assay compounds C10 and C6 were highly active against all the isolates. The compounds C1 and C10 were found most potent in spore germination test. It fully proved that the SDHIs designed in this paper displayed as good inhibitory effects against Fusarium solani.

Keywords: Wilt, Fusarium, SDH, antifungal

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Authors: J. Carlos Kuri, Varun Vij, Raymond J. Turner, Orly Yadid-Pecht

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Celiac disease (CD) is an immune system disorder that is triggered by ingesting gluten. As a gluten-free (GF) diet has become a concern of many people for health reasons, a gold standard had to be nominated. Enzyme-linked immunosorbent assay (ELISA) has taken the seat of this role. However, multiple limitations were discovered, and with that, the desire for an alternative method now exists. Nucleic acid-based aptamers have become of great interest due to their selectivity, specificity, simplicity, and rapid-testing advantages. However, fluorescence-based aptasensors have been tagged as unstable, but lifespan details are rarely stated. In this work, the lifespan stability of a fluorescence-based aptasensor is shown over an 8-week-long study displaying the accuracy of the sensor and false negatives. This study follows 22 different samples, including GF and gluten-rich (GR) and soy sauce products, off-the-shelf products, and reference material from laboratories, giving a total of 836 tests. The analysis shows an accuracy of correctly classifying GF and GR products of 96.30% and 100%, respectively when the protocol is augmented with molecular sieves. The overall accuracy remains around 94% within the first four weeks and then decays to 63%.

Keywords: aptasensor, PEG, rGO, FAM, RM, ELISA

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Authors: Azimeh Rajaee, Lingyun Zhao, Shi Wang, Yaqiang Liu

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In recent years many studies have been focused on bismuth-based nanoparticles as radiosensitizer and contrast agent in radiation therapy and imaging due to the high atomic number (Z = 82), high photoelectric absorption, low cost, and low toxicity. This study aims to introduce a new multifunctional bismuth-based nanoparticle as a theranostic agent for radiotherapy, computed tomography (CT) and magnetic resonance imaging (MRI). We synthesized bismuth ferrite (BFO, BiFeO3) nanoparticles by sol-gel method and surface of the nanoparticles were modified by Polyethylene glycol (PEG). After proved biocompatibility of the nanoparticles, the ability of them as contract agent in Computed tomography (CT) and magnetic resonance imaging (MRI) was investigated. The relaxation time rate (R2) in MRI and Hounsfield unit (HU) in CT imaging were increased with the concentration of the nanoparticles. Moreover, the effect of nanoparticles on dose enhancement in low energy was investigated by clonogenic assay. According to clonogenic assay, sensitizer enhancement ratios (SERs) were obtained as 1.35 and 1.76 for nanoparticle concentrations of 0.05 mg/ml and 0.1 mg/ml, respectively. In conclusion, our experimental results demonstrate that the multifunctional nanoparticles have the ability to employ as multimodal imaging and therapy to enhance theranostic efficacy.

Keywords: molecular imaging, nanomedicine, radiotherapy, theranostics

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Authors: Eddy Neuhaus, Hanif Shirinzadeh, Cigdem Karaaslan, Elif Ince, Hande Gurer-Orhan, Sibel Suzen

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Reactive oxygen species (ROS) and oxidative stress can cause fatal damage to essential cell structures, including DNA. It is known that use of antioxidants could be advantageous in the prevention of various diseases such as cancer, cardiovascular diseases and neurodegenerative disorders. Since antioxidant properties of the indole ring-containing melatonin (MLT) has been described and evaluated, MLT-related compounds such as MLT metabolites and synthetic analogues are under investigation to determine which exhibit the highest activity with the lowest side-effects. Owing to indole and hydrazones appealing physiological properties and are mostly found in numerous biologically active compounds a series of indole-7-carbaldehyde hydrazone derivatives were synthesized, characterized and in vitro antioxidant activity was investigated by evaluating their reducing effect against oxidation of a redox-sensitive fluorescent probe. Cytotoxicity potential of all indole-based MLT analogues was investigated both by lactate dehydrogenase leakage assay and by MTT assay. This work was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) Research and Development Grant 112S599.

Keywords: melatonin, antioxidant activity, indole, hydrazone, oxidative stress

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Authors: S. Ibrahim, U. B. Abubakar, S. Danbirni, A. Usman, F. M. Ballah, C. A. Kudi, L. Lawson, G. H. Abdulrazak, I. A. Abdulkadir

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Performing culture and characterization of mycobacteria in low resource settings like Nigeria is a very difficult task to undertake because of the very few and limited laboratories carrying out such an experiment; this is a largely due to stringent and laborious nature of the tests. Hence, a rapid, simple and accurate test for characterization is needed. The “SD BIOLINE TB Ag MPT 64 Rapid ®” is a simple and rapid immunochromatographic test used in differentiating Mycobacteria into Mycobacterium tuberculosis (NTM). The 100 sputa were obtained from patients suspected to be infected with tuberculosis and presented themselves to hospitals for check-up and treatment were involved in the study. The samples were cultured in a class III Biosafety cabinet and level III biosafety practices were followed. Forty isolates were obtained from the cultured sputa, and there were identified as Acid-fast bacilli (AFB) using Zeihl-Neelsen acid-fast stain. All the isolates (AFB positive) were then subjected to the SD BIOLINE Analyses. A total of 31 (77.5%) were characterized as MTBC, while nine (22.5%) were NTM. The total turnaround time for the rapid assay was just 30 minutes as compared to a few days of phenotypic and genotypic method. It was simple, rapid and reliable test to differentiate MTBC from NTM.

Keywords: culture, mycobacteria, non tuberculous mycobacterium, SD Bioline

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Authors: Ibrahim Aly, Manal Ahmed, Mahmoud M. El-Shall

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Filariasis is a parasitic disease caused by small roundworms. The filarial worms are transmitted and spread by blood-feeding black flies and mosquitoes. Lymphatic filariasis (Elephantiasis) is caused by Wuchereriabancrofti, Brugiamalayi, and Brugiatimori. Elimination of Lymphatic filariasis necessitates an increasing demand for valid, reliable, and rapid diagnostic kits. Nanodiagnostics involve the use of nanotechnology in clinical diagnosis to meet the demands for increased sensitivity, specificity, and early detection in less time. The aim of this study was to evaluate the nano-based enzymelinked immunosorbent assay (ELISA) and novel nano-chip card as a rapid test for detection of filarial antigen in serum samples of human filariasis in comparison with traditional -ELISA. Serum samples were collected from an infected human with filarial gathered across Egypt's governorates. After receiving informed consenta total of 45 blood samples of infected individuals residing in different villages in Gharbea governorate, which isa nonendemic region for bancroftianfilariasis, healthy persons living in nonendemic locations (20 persons), as well as sera from 20 other parasites, affected patients were collected. The microfilaria was checked in thick smears of 20 µl night blood samples collected during 20-22 hrs. All of these individuals underwent the following procedures: history taking, clinical examination, and laboratory investigations, which included examination of blood samples for microfilaria using thick blood film and serological tests for detection of the circulating filarial antigen using polyclonal antibody- ELISA, nano-based ELISA, and nano-chip card. In the present study, a recently reported polyoclonal antibody specific to tegumental filarial antigen was used in developing nano-chip card and nano-ELISA compared to traditional ELISA for the detection of circulating filarial antigen in sera of patients with bancroftianfilariasis. The performance of the ELISA was evaluated using 45 serum samples. The ELISA was positive with sera from microfilaremicbancroftianfilariasis patients (n = 36) with a sensitivity of 80 %. Circulating filarial antigen was detected in 39/45 patients who were positive for circulating filarial antigen using nano-ELISA with a sensitivity of 86.6 %. On the other hand, 42 out of 45 patients were positive for circulating filarial antigen using nano-chip card with a sensitivity of 93.3%.In conclusion, using a novel nano-chip assay could potentially be a promising alternative antigen detection test for bancroftianfilariasis.

Keywords: lymphatic filariasis, nanotechnology, rapid diagnosis, elisa technique

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Authors: L. Bidyalakshmi, R. Ananthan, T. Longvah

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Antioxidants are substances that can prevent or delay oxidative damage of lipids, proteins and nucleic acids by reactive oxygen species. These help in lowering incidence of degenerative diseases such as cancer, arthritis, atherosclerosis, heart disease, inflammation, brain dysfunction and acceleration of the ageing process. The north eastern part of India falls among the global hotspots of biodiversity. Over the years, the local communities in the region have developed ingenious uses of many wild plants within their environment as food sources. Many of these less familiar foods form an integral part of the diet of these communities, and some are traditionally valued for its therapeutic effects. So the study was carried to estimate the antioxidant activity of some of these indigenous foods. Twenty-eight indigenous plant foods were studied for their antioxidant activity. Antioxidant activities were determined by using DPPH (2, 2-diphenyl-1-picrylhydrazyl) assay, FRAP (Ferric Reducing Antioxidant Power) assay and SOSA (Super Oxide Scavenging Assay). Out of the twenty-eight plant foods, there were thirteen leafy vegetables, four fruits, five roots and tubers, four spices and two mushrooms. Water extract and methanol extract of the samples were used for the analysis. The leafy vegetable samples exhibited antioxidant capacity with IC50 ranging from 8-1414 mg/ml for lipid extract and 34-37878 mg/ml for aqueous extract in DPPH assay. Total FRAP value ranging from 58-1005 mmol FeSO4 Eq/100g of the sample, which is comparatively higher than the antioxidant capacity of some commonly consumed leafy vegetables. In SOSA, water extract of leafy vegetables show a range of 0.05-193.68 µmol ascorbic acid equivalent/g of the samples. While the methanol extract of the samples show 0.20-21.94 µmol Trolox equivalent/g of the samples. Polygonum barbatum, Wendlandia glabrata and Polygonum posumbu have higher antioxidant activity among the leafy vegetables analysed. Among the fruits, Rhus hookerii showed the highest antioxidant activities in both FRAP and SOSA methods while Spondias magnifera exhibited higher antioxidant activity in DPPH method. Alocasia cucullata exhibited higher antioxidant activity in DPPH and FRAP assays while Alpinia galanga showed higher antioxidant activity in SOSA assay when compared to the other samples of roots and tubers. Elsholtzia communis showed high antioxidant activity in all the three parameters among the spices. For the mushrooms, Pleurotus ostreatus exhibited higher antioxidant activity than Auricularia delicate in DPPH and SOSA. The samples analysed exhibited antioxidant activity at varying levels and some exhibited higher antioxidant activity than the commonly consumed foods. So consumption of these less familiar foods may play a role in preventing human disease in which free radicals are involved. Further studies on these food samples on phytonutrients and its contribution to the antioxidant activities are required.

Keywords: antioxidant activity, DPPH, FRAP, SOSA

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Authors: Mahnoor Khan, Bashir Ahmad, Khizar Hayat, Saad Ahmad Khan, Laiba Ahmad, Shumaila Bashir, Abid Ali Khan

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The objective of this study was to synthesize the smallest possible size of ZnO NPs using a modified wet chemical synthesis method and to prepare core shell using polyethylene glycol (PEG) as shell material. Advanced and sophisticated techniques were used to confirm the synthesis, size, and shape of these NPs. Rounded, clustered NPs of size 5.343 nm were formed. Both the plain and core shell NPs were tested against MDR bacteria (E. cloacae, E. amnigenus, Shigella, S. odorifacae, Citrobacter, and E. coli). Both of the NPs showed excellent antibacterial properties, whereas E. cloacae showed maximum zone of inhibition of 16 mm, 27 mm, and 32 mm for 500 μg/ml, 1000 μg/ml, and 1500 μg/ml, respectively for plain ZnO NPs and 18 mm, 28 mm and 35 mm for 500 μg/ml, 1000 μg/ml and 1500 μg/ml for core shell NPs. These NPs were also biocompatible on human red blood cells showing little hemolysis of only 4% for 70 μg/ml for plain NPs and 1.5% for 70 μg/ml for core shell NPs. Core shell NPs were highly biocompatible because of the PEG. Their therapeutic effect as photosensitizers in photodynamic therapy (PDT) for cancer treatment was also monitored. The cytotoxicity of ZnO and PEG-ZnO was evaluated using MTT assay. Our results demonstrated that these NPs could generate ROS inside tumor cells after irradiation which in turn initiates an apoptotic pathway leading to cell death hence proving to be an effective candidate for PDT.

Keywords: ZnO, hemolysis, cytotoxiciy assay, photodynamic therapy, antibacterial

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Authors: Hanan Khojah, RuAngelie Edrada-Ebel

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Neurodegenerative disease including Alzheimer’s disease is a major cause of long-term disability. Oxidative stress is frequently implicated as one of the key contributing factors to neurodegenerative diseases. Protection against neuronal damage remains a great challenge for researchers. Ficus carica (commonly known as fig) is a species of great antioxidant nutritional value comprising a protective mechanism against innumerable health disorders related to oxidative stress as well as Alzheimer’s disease. The purpose of this work was to characterize the non-polar active metabolites in Ficus carica endocarp, mesocarp, and exocarp. Crude extracts were prepared using several extraction solvents, which included 1:1 water: ethylacetate, acetone and methanol. The dried extracts were then solvent partitioned between equivalent amounts of water and ethylacetate. Purification and fractionation were accomplished by high-throughput chromatography. The isolated metabolites were tested on their effect on human neuroblastoma cell line by cell viability test and cell cytotoxicity assay with acrolein. Molecular weights of the active metabolites were determined via LC–HRESIMS and GC-EIMS. Metabolomic profiling was performed to identify the active metabolites by using differential expression analysis software (Mzmine) and SIMCA for multivariate analysis. Structural elucidation and identification of the interested active metabolites were studied by 1-D and 2-D NMR. Significant differences in bioactivity against a concentration-dependent assay on acrolein radicals were observed between the three fruit parts. However, metabolites obtained from mesocarp and the endocarp demonstrated bioactivity to scavenge ROS radical. NMR profiling demonstrated that aliphatic compounds such as γ-sitosterol tend to induce neuronal bioactivity and exhibited bioactivity on the cell viability assay. γ-Sitosterol was found in higher concentrations in the mesocarp and was considered as one of the major phytosterol in Ficus carica.

Keywords: alzheimer, Ficus carica, γ-Sitosterol, metabolomics

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Authors: Donna M. Morrison, Lambert Chester, Coretta A. N. Samuels, David R. Ledoux

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A survey was conducted in the five rice-growing regions in Guyana to determine the presence of aflatoxins in multiple fractions of rice in June/October 2015 growing season. The fractions were paddy, steamed paddy, cargo rice, white rice and parboiled rice. Samples were analyzed by High Performance Liquid Chromatography. A subset of the samples was further analyzed by enzyme-linked immunosorbent assay (ELISA) for concurrence. All analyses were conducted at the University of Missouri, USA. Of the 186 samples tested, 16 had aflatoxin concentrations greater than 20 ppb the recommended limit for aflatoxins in food according to the United States Food and Drug Administration. An additional three samples had aflatoxin B₁ concentrations greater than the European Union Commission maximum levels for aflatoxin B₁ in rice at 5 µg/kg and total aflatoxins (B₁, B₂, G₁ and G₂) at 10 µg/kg. The survey indicates that there is no widespread aflatoxin problem in rice in Guyana. The incidence of aflatoxins appears to be localized.

Keywords: aflatoxin, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), rice fractions

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Authors: Subodh Kumar, Sanjeev Kumar Sharma, Gaurav Kaushik, Pramod Avti, Phulen Sarma, Bikash Medhi, Krishan Lal Khanduja

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Background: It is well known that cigarette smoke is one of the causative factors in various lung diseases especially cancer. Carcinogens and oxidant molecules present in cigarette smoke not only damage the cellular constituents (lipids, proteins, DNA) but may also regulate the molecular pathways involved in inflammation and cancer. Continuous oxidative stress caused by the constituents of cigarette smoke leads to higher PhospholipaseA₂ (PLA₂) activity, resulting in elevated levels of secondary metabolites whose role is well defined in cancer. To reduce the burden of chronic inflammation as well as oxidative stress, and higher levels of secondary metabolites, we checked the curative potential of PLA₂ inhibitor Bromoenol Lactone (BEL) during continuous exposure of cigarette smoke condensate (CSC). Aim: To check the therapeutic potential of Bromoenol Lactone (BEL), an inhibitor of PhospholipaseA₂s, in pathways of CSC-induced changes in type I and type II alveolar epithelial cells. Methods: Effect of BEL on CSC-induced PLA2 activity were checked using colorimetric assay, cellular toxicity using cell viability assay, membrane integrity using fluorescein di-acetate (FDA) uptake assay, reactive oxygen species (ROS) levels and apoptosis markers through flow cytometry, and cellular regulation using MAPKinases levels, in lung epithelium. Results: BEL significantly mimicked CSC-induced PLA₂ activity, ROS levels, apoptosis, and kinases level whereas improved cellular viability and membrane integrity. Conclusions: Current observations revealed that BEL may be a potential therapeutic agent during Cigarette smoke-induced anomalies in lung epithelium.

Keywords: cigarette smoke condensate, phospholipase A₂, oxidative stress, alveolar epithelium, bromoenol lactone

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Authors: Xiran Wang, Johanna Trinkl, Thomas Hoffmann, Wilfried Schwab

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Malonyltransferases (MATs) are enzymes that play a key role in the biosynthesis of secondary metabolites in plants, such as flavonoids and anthocyanins. As a kind of flavonoid-rich fruit, strawberries are an ideal model to study MATs. From Goodberry metabolome data, in the hybrid generation of 2 strawberries various, Fragaria × ananassa cv. 'Senga Sengana' and 'Candonga', we found the malonylated flavonoid concentration is significantly higher in 'Senga Sengana' compared with 'Candonga'. Therefore, we aimed to identify and characterize the malonyltransferases responsible for the different malonylated flavonoid concentrations in two different strawberry cultivars. In this study, we have found 6 MATs via genome mapping, metabolome analysis, gene cloning, and enzyme assay from strawberries, which catalyzed the malonylation of flavonoid substrates: quercetin-3-glucoside, kaempferol-3-glucoside, pelargonidin-3-glucoside, and cyanidin-3-glucoside. All four compounds reacted with FaMATs to varying degrees. These MATs have important implication into strawberries’ flavonoid biosynthesis, and also provide insights into insights into flavonoid biosynthesis, potential applications in agriculture, plant science, and pharmacy, and information on the regulation of secondary metabolism in plants.

Keywords: malonyltransferase, strawberry, flavonoid biosynthesis, enzyme assay

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Authors: Mmbulaheni Ramulondi, Sandy Van Vuuren, Helene De Wet

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The use of plant combinations to treat various medical conditions is not a new concept, and it is known that traditional people do not only rely on a single plant extract for efficacy but often combine various plant species for treatment. The knowledge of plant combinations is transferred from one generation to the other in the belief that combination therapy may enhance efficacy, reduce toxicity, decreases adverse effects, increase bioavailability and result in lower dosages. However, combination therapy may also be harmful when the interaction is antagonistic, since it may result in increasing toxicity. Although a fair amount of research has been done on the toxicity of medicinal plants, there is very little done on the toxicity of medicinal plants in combination. The aim of the study was to assess the toxicity potential of 19 plant combinations which have been documented as treatments of hypertension in northern KwaZulu-Natal by lay people. The aqueous extracts were assessed using two assays; the Brine shrimp assay (Artemia franciscana) and the Ames test (Mutagenicity). Only one plant combination (Aloe marlothii with Hypoxis hemerocallidea) in the current study has been previously assessed for toxicity. With the Brine shrimp assay, the plant combinations were tested in two concentrations (2 and 4 mg/ml), while for mutagenicity tests, they were tested at 5 mg/ml. The results showed that in the Brine shrimp assay, six combinations were toxic at 4 mg/ml. The combinations were Albertisia delagoensis with Senecio serratuloides (57%), Aloe marlothii with Catharanthus roseus (98%), Catharanthus roseus with Hypoxis hemerocallidea (66%), Catharanthus roseus with Musa acuminata (89%), Catharanthus roseus with Momordica balsamina (99%) and Aloe marlothii with Trichilia emetica and Hyphaene coriacea (50%). However when the concentration was reduced to 2 mg/ml, only three combinations were toxic which were Aloe marlothii with Catharanthus roseus (76%), Catharanthus roseus with Musa acuminata (66%) and Catharanthus roseus with Momordica balsamina (73%). For the mutagenicity assay, only the combinations between Catharanthus roseus with Hypoxis hemerocallidea and Catharanthus roseus with Momordica balsamina were mutagenic towards the Salmonella typhimurium strains TA98 and TA100. Most of the combinations which were toxic involve C. roseus which was also toxic when tested singularly. It is worth noting that C. roseus was one of the most frequently used plant species both to treat hypertension singularly and in combination and some of the individuals have been using this for the last 20 years. The mortality percentage of the Brine shrimp showed a significant correlation between dosage and toxicity thus toxicity was dosage dependant. A combination which is worth noting is the combination between A. delagoensis and S. serratuloides. Singularly these plants were non-toxic towards Brine shrimp, however their combination resulted in antagonism with the mortality rate of 57% at the total concentration of 4 mg/ml. Low toxicity was mostly observed, giving some validity to combined use, however the few combinations showing increased toxicity demonstrate the importance of analysing plant combinations.

Keywords: dosage, hypertension, plant combinations, toxicity

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Authors: Mondal Indranil, Bhakat Debjyoti, Mukhopadayay Asish K., Chatterjee Nabendu S.

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CS6 is the prevalent CF in our region and deciphering its molecular regulators would play a pivotal role in reducing the burden of ETEC pathogenesis. In prokaryotes, most of the genes are under the control of one operon and the promoter present upstream of the gene regulates the transcription of that gene. Here the promoter of CS6 was characterized by computational method and further analyzed by β-galactosidase assay and sequencing. Promoter constructs and deletions were prepared as required to analyze promoter activity. The effect of different additives on the CS6 promoter was analysed by the β-galactosidase assay. Bioinformatics analysis done by Softberry/BPROM predicted fur, lrp, and crp boxes, -10 and -35 region upstream of the CS6 gene. The promoter construction in no promoter plasmid pTL61T showed that region -573 to +1 is actually the promoter region as predicted. Sequential deletion of the region upstream of CS6 revealed that promoter activity remains the same when -573bp to -350bp is deleted. But after the deletion of the upstream region -350 bp to -255bp, promoter expression decreases drastically to 26%. Further deletion also decreases promoter activity up to a little range. So the region -355bp to -255bp holds the promoter sequence for the CS6 gene. Additives like iron, NaCl, etc., modulate promoter activity in a dose-dependent manner. From the promoter analysis, it can be said that the minimum region lies between -254 and +1. Important region(s) lies between -350 bp to -255 bp upstream in the promoter, which might have important elements needed to control CS6 gene expression.

Keywords: microbiology, promoter, colonization factor, ETEC

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Authors: Eva Tvrdá, Hana Greifová, Peter Ivanič, Norbert Lukáč

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The aim of this study was to evaluate the dose- and time-dependent in vitro effects of berberine (BER), a natural alkaloid with numerous biological properties on bovine spermatozoa during three time periods (0 h, 2 h, 24 h). Bovine semen samples were diluted and cultivated in physiological saline solution containing 0.5% DMSO together with 200, 100, 50, 10, 5, and 1 μmol/L BER. Spermatozoa motility was assessed using the computer assisted semen analyzer. The viability of spermatozoa was assessed by the metabolic (MTT) assay, production of superoxide radicals was quantified using the nitroblue tetrazolium (NBT) test, and chemiluminescence was used to evaluate the generation of reactive oxygen species (ROS). Cell lysates were prepared and the extent of lipid peroxidation (LPO) was evaluated using the TBARS assay. The results of the movement activity showed a significant increase in the motility during long term cultivation in case of concentrations ranging between 1 and 10 μmol/L BER (P < 0.01; P < 0.001; 24 h). At the same time, supplementation of 1, 5 and 10 μmol/L BER led to a significant preservation of the cell viability (P < 0.001; 24 h). BER addition at a range of 1-50 μmol/L also provided a significantly higher protection against superoxide (P < 0.05) and ROS (P < 0.001; P < 0.01) overgeneration as well as LPO (P < 0.01; P<0.05) after a 24 h cultivation. We may suggest that supplementation of BER to bovine spermatozoa, particularly at concentrations ranging between 1 and 50 μmol/L, may offer protection to the motility, viability and oxidative status of the spermatozoa, particularly notable at 24 h.

Keywords: berberine, bulls, motility, oxidative profile, spermatozoa, viability

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Authors: Muhammad Farooq Baig

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Objective: The purpose of this study was to compare results obtained using a single fecal specimen for O&P examination, direct immunofluorescence assay (DFA), and two conventional staining methods. Design: Hundred and fifty children fecal specimens were collected and examined by each method. The O&P and the DFA were used as the reference method. Setting: The study was performed at the laboratory in the Basic Medical Science Institute JPMC Karachi. Patients or Other Participants: The fecal specimens were collected from children with a suspected Giardia lamblia infection. Main Outcome Measures: The amount of agreement and disagreement between methods.1) Presence of giardiasis in our population. 2) The sensitivity and specificity of each method. Results: There was 45(30%) positive 105 (70%) negative on DFA, 41 (27.4%) positive 109 (72.6%) negative on iodine and 34 (22.6%) positive 116(77.4%) on saline method. The sensitivity and specificity of DFA in comparision to iodine were 92.2%, 92.7% respectively. The sensitivity and specificity of DFA in comparisoin to saline method were 91.2%, 87.9% respectively. The sensitivity of iodine method and saline method in compariosn to DFA were 82.2%, 68.8% respectively. There is mark diffrence in sensitivity of DFA to conventional method. Conclusion: The study supported findings of other investigators who concluded that DFA method have the greater sensitivity. The immunologic methods were more efficient and quicker than the conventional O&P method.

Keywords: direct immunofluorescence assay (DFA), ova and parasite (O&P), Giardia lamblia, children, medical science

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Authors: Giancarlo La Marca, Engy Shokry, Fabio Villanelli

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Epilepsy is one of the most common neurological disorders, with an estimated prevalence of 50 million people worldwide. Twenty five percent of the epilepsy population is represented in children under the age of 15 years. For antiepileptic drugs (AED), there is a poor correlation between plasma concentration and dose especially in children. This was attributed to greater pharmacokinetic variability than adults. Hence, therapeutic drug monitoring (TDM) is recommended in controlling toxicity while drug exposure is maintained. Carbamazepine (CBZ) is a first-line AED and the drug of first choice in trigeminal neuralgia. CBZ is metabolised in the liver into carbamazepine-10,11-epoxide (CBZE), its major metabolite which is equipotent. This develops the need for an assay able to monitor the levels of both CBZ and CBZE. The aim of the present study was to develop and validate a LC-MS/MS method for simultaneous quantification of CBZ and CBZE in dried blood spots (DBS). DBS technique overcomes many logistical problems, ethical issues and technical challenges faced by classical plasma sampling. LC-MS/MS has been regarded as superior technique over immunoassays and HPLC/UV methods owing to its better specificity and sensitivity, lack of interference or matrix effects. Our method combines advantages of DBS technique and LC-MS/MS in clinical practice. The extraction process was done using methanol-water-formic acid (80:20:0.1, v/v/v). The chromatographic elution was achieved by using a linear gradient with a mobile phase consisting of acetonitrile-water-0.1% formic acid at a flow rate of 0.50 mL/min. The method was linear over the range 1-40 mg/L and 0.25-20 mg/L for CBZ and CBZE respectively. The limit of quantification was 1.00 mg/L and 0.25 mg/L for CBZ and CBZE, respectively. Intra-day and inter-day assay precisions were found to be less than 6.5% and 11.8%. An evaluation of DBS technique was performed, including effect of extraction solvent, spot homogeneity and stability in DBS. Results from a comparison with the plasma assay are also presented. The novelty of the present work lies in being the first to quantify CBZ and its metabolite from only one 3.2 mm DBS disc finger-prick sample (3.3-3.4 µl blood) by LC-MS/MS in a 10 min. chromatographic run.

Keywords: carbamazepine, carbamazepine-10, 11-epoxide, dried blood spots, LC-MS/MS, therapeutic drug monitoring

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Authors: P. H. Mfengwana, S. S. Mashele, L. Verschaeve, R. Anthonissen, I. T. Manduna

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Gunnera perpensa is traditionally used mostly by women for the treatment of different gynaecological related conditions due to its proven uterine contractility effects. The uses of this plant include menstrual pain relief, treatment of infertility and promotion of easy labour. However, even though this plant species has been reported to possess numerous medicinal properties, to author’s best knowledge, its safety has not been investigated. Thus, this study was aimed at investigating the genotoxicity and antigenotoxicity of Gunnera perpensa aqueous, methanol and dichloromethane extracts. The in vitro toxicity of the plant extracts was assessed with the neutral red uptake (NRU) test. Genotoxic and antigenotoxic properties of Gunnera perpensa were investigated using high-throughput assays: bacterial Vitotox test and the alkaline comet assay with and without S9 activation on human C3A cells. Ethyl Methanesulfonate (EMS) and 4-nitroquinoline-oxide (4-NQO) were used as positive controls, respectively. All extracts showed toxicity in a dose-dependent manner; however, that does not mean they were all genotoxic. Methanol extract did show genotoxicity with S9 (metabolism) only at the highest concentration of 500 µg/ml due to increased DNA damage observed, however, no genotoxicity was observed from other concentrations. Therefore, the results show that Gunnera perpensa extracts are genotoxic and not safe for human use.

Keywords: antigenotoxicity, comet test, genotoxicity, Gunnera perpensa, vitotox assay

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Authors: Shabnam Abdi, Behzad Toosi

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Background: Melanoma is the most lethal type of malignant skin cancer in humans and dogs since it spreads rapidly throughout the body. Despite significant advances in treatment, cancer at an advanced stage has a poor prognosis. Hence, more effective treatments are needed to enhance outcomes with fewer side effects. Erythropoietin-producing hepatocellular receptors are the largest family of receptor tyrosine kinases and are divided into two subfamilies, EphA and EphB, both of which play a significant role in disease, especially cancer. Due to their association with proliferation and invasion in many aggressive types of cancer, Eph receptor tyrosine kinases (Eph RTKs) are promising cancer therapy molecules. Because these receptors have not been studied in canine melanoma, we investigated how EphA2 influences survival and tumorigenicity of melanoma cells. Methods: Expression of EphA2 protein in canine melanoma cell lines and human melanoma cell line was evaluated by Western blot. Melanoma cells were transduced with lentiviral particles encoding Eph-targeting shRNAs or non-silencing shRNAs (control) for silencing the expression of EphA2 receptor, and silencing was confirmed by Western blotting and immunofluorescence. The effect of siRNA treatment on cellular proliferation, colony formation, tumorsphere assay, invasion was analyzed by Resazurin assay Matrigel invasion assay, respectively. Results: Expression of EphA2 was detected in canine and human melanoma cell lines. Moreover, stably silencing EphA2 by specific shRNAs significantly and consistently decreased the expression of EphA2 protein in both human and canine melanoma cells. Proliferation, colony formation, tumorsphere and invasion of melanoma cells were significantly decreased in EphA2 siRNA-treated cells compared to control. Conclusion: Our data provide the first functional evidence that the EphA2 receptor plays a critical role in the malignant cellular behavior of melanoma in both human and dogs.

Keywords: ephA2, targeting, melanoma, human, canine

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Authors: Vasudha Devi, Arul Amuthan, K. Narayanan, Praveen KS, Venkata Rao J

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Background: Siddha Medicine is one of the Indian traditional medical systems, in which the cancer disease is mentioned as 'putrunoi' which literally means the disease of growth like termite mound. There are number of formulations available for the treatment of cancer disease. Neeradi muthu vallathymezugu (NMV) and thamira kattu chendooram (TKC) are two drugs commonly prescribed by Siddha physicians. These drugs have been clinically reported to be safe and effective when given orally. Though these formulations are in practice for centuries, no efforts have been made to standardize them and explore their anti-cancer potential systematically. Objective: To compare the cytotoxic activity of NMV and TKC with doxorubicin using cancer cell lines. Materials and methods: For this study, ethanol extract of NMV was taken, whereas TKC was used as such. In vitro cytotoxic activity was evaluated by sulphorhodamine (SRB) assay against human hepatic cancer cells (HepG2), human breast cancer cells (MCF-7) and human cervical cancer cells [KeLa]. Doxorubicin was used as the standard. The SRB assay is based on the ability of cellular proteins to bind with sulphorhodamine-B. The number of live cells in drug treated cell lines directly affects the color formation in the assay, which is estimated calorimetrically by measuring the absorbance at 540 nm to calculate the cytotoxicity (inhibitory concentration - IC50 value) of the drug. Results: The IC50values of NMV, TKC and doxorubicin against HepG2 were 3.08 µg/ml, 20.21 µg/ml and 1.21µg/ml respectively. In MCF-7, it was 11.75 µg/ml, 17.67 µg/ml and 2.8µg/ml. In HeLa, the values were 24.76 µg/ml, 73.35 µg/ml and 1.12µg/ml. Conclusions: The study proves the possible anti-cancer potential of these two formulations. Compared to TKC, NMV showed good cytotoxic effect even at low dose. Human hepatic cancer cells responded well even at very low dose, when compared to other cancer cells. Though, cytotoxic potential of these compounds was found to be less compared to doxorubicin, the isolated lead compound may have the potential to be used as an anticancer drug clinically.

Keywords: Neeradi muthu vallathymezugu (Hydnocarpus laurifolia), thamira kattu chendooram, cytotoxicity, in-vitro, Siddha Medicine

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Authors: Cheng-Cao Sun, Shu-Jun Li, Cuili Yang, Yongyong Xi, Liang Wang, Feng Zhang, De-Jia Li

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Hsa-miRNA-326 (miR-326) has recently been discovered having anticancer efficacy in different organs. However, the role of miR-326 on non-small cell lung cancer (NSCLC) is still ambiguous. In this study, we investigated the role of miR-326 on the development of NSCLC. The results indicated that miR-326 was significantly down-regulated in primary tumor tissues and very low levels were found in NSCLC cell lines. Ectopic expression of miR-326 in NSCLC cell lines significantly suppressed cell growth as evidenced by cell viability assay, colony formation assay and BrdU staining, through inhibition of cyclin D1, cyclin D2, CDK4, and up-regulation of p57(Kip2) and p21(Waf1/Cip1). In addition, miR-326 induced apoptosis, as indicated by concomitantly with up-regulation of key apoptosis protein cleaved caspase-3, and down-regulation of anti-apoptosis protein Bcl2. Moreover, miR-326 inhibited cellular migration and invasiveness through inhibition of matrix metalloproteinases (MMP)-7 and MMP-9. Further, oncogene CCND1 was revealed to be a putative target of miR-326, which was inversely correlated with miR-326 expression in NSCLC. Taken together, our results demonstrated that miR-326 played a pivotal role on NSCLC through inhibiting cell proliferation, migration, invasion, and promoting apoptosis by targeting oncogenic CCND1.

Keywords: hsa-miRNA-326 (miR-326), cyclin D1, non-small cell lung cancer (NSCLC), proliferation, apoptosis

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Authors: M. Pourshirazi, M. Esmaelifar, A. Aliahmadi, F. Yazdian, A. S. Hatamian Zarami, S. J. Ashrafi

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Medicinal fungi are the new potential source of drugs to improve the treatment of diseases with association to oxidative agents such as cancers. Monascus purpureus contains functional components potentially effective in improving human health. In the present work, ethanolic extract of Monascus purpureus (EEM) was evaluated for health improving potential mainly focusing on antioxidant and anticancer activities. Ferric ion reducing power (FRAP), scavenging of DPPH radicals and determining viability of breast carcinoma MCF-7 and cervical carcinoma HeLa cells with MTT assay were evaluated. Our data showed a significant antioxidant activity of EEM with 142.45 µg/ml inhibition concentration of 50% DPPH radicals and 2112.33 µg eq.Fe2+/mg extract of FRAP assay. These results might be caused by antioxidant components such as pigments and phenolic compounds. Further, the results demonstrated that EEM caused significant reduction in the viability of MCF-7 with IC50 of 7 µg/ml but not have good effect against viability of HeLa cells. Accordingly, Monascus purpureus is presented as a strong potential of breast cancer treatment. In further study, the mechanistic studies are needed to determine the mechanisms of anticancer activity of EEM.

Keywords: Monascus purpureus, antioxidant, cancer, ethanolic extract

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Authors: Y. Pilehvar-Soltanahmadi, F. Badrzadeh, N. Zarghami, S. Jalilzadeh-Tabrizi, R. Zamani

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Herbal compounds such as curcumin which decrease telomerase and gene expression have been considered as beneficial tools for lung cancer treatment. In this article, we compared the effects of pure curcumin and curcumin-loaded NIPAAm-MAA nanoparticles on telomerase and PinX1 gene expression in a lung cancer cell line. A tetrazolium-based assay was used for determination of cytotoxic effects of curcumin on the Calu-6 lung cancer cell line and telomerase and pinX1 gene expression was measured with real-time PCR. MTT assay showed that Curcumin-loaded NIPAAm-MAA inhibited the growth of the Calu-6 lung cancer cell line in a time and dose-dependent manner. Our q-PCR results showed that the expression of telomerase gene was effectively reduced as the concentration of curcumin-loaded NIPAAm-MAA increased while expression of the PinX1 gene became elevated. The results showed that curcumin loaded NIPAAm-MAA exerted cytotoxic effects on the Calu-6 cell line through down-regulation of telomerase and stimulation of pinX1 gene expression. NIPPAm-MAA could be the good carrier for such kinds of hydrophobic agent.

Keywords: curcumin, NIPAAm-MAA, PinX1, telomerase, lung cancer cells

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Authors: Maha Soltan, Amal Z. Hassan, Howaida I. Abd-Alla, Atef G. Hanna

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Two naturally known cardenolides; acovenoside A and acobioside A were isolated from the Egyptian cultivar; Acokanthera spectabilis leaves. It is an ornamental and poisonous plant that has been traditionally claimed for their medicinal properties against infectious microbes, killing worms and curing some inflammations at little amounts. We examined the growth inhibition effects of both cardenolides against four types of human cancer cell lines using Sulphorhodamine B assay. In addition, the clonogenic assay was also performed for testing the growth inhibiting power of the isolated compounds. An in vitro mechanistic investigation was further accomplished against hepatocellular carcinoma HepG2 cell line. Microscopic examination, colorimetric ELISA and flow cytometry techniques were our tools of proving at least part of the anticancer pathway of the tested compounds. Both compounds were able to inhibit the growth of 4 human cancer cell lines at less than 100 nM. In addition, they were able to activate the executioner Caspase-3 and apoptosis was then induced as a consequence of cell growth arrest at G2/M. An attention must be payed to those bioactive agents particularly when giving their activity against cancer cells at considerable small values while presenting safe therapeutic margins as indicated by literature.

Keywords: anticancer, cardenolides, Caspase-3, apoptosis

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Authors: Stela Papa, Klementina Puto, Migena Pllaha

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HCV is a hepatotropic RNA virus, transmitted primarily via the blood route, which causes progressive disease such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma. HCV nowadays is a global healthcare problem. A variety of immunoassays including old and new technologies are being applied to detect HCV in our country. These methods include Immunochromatography assays (ICA), Fluorescence immunoassay (FIA), Enzyme linked fluorescent assay (ELFA), and Enzyme linked immunosorbent assay (ELISA) to detect HCV antibodies in blood serum, which lately is being slowly replaced by more sensitive methods such as rapid automated analyzer chemiluminescence immunoassay (CLIA). The aim of this study is to estimate HCV infection in carriers and chronic acute patients and to evaluate the use of new diagnostic methods. This study was realized from September 2016 to May 2018. During this study period, 2913 patients were analyzed for the presence of HCV by taking samples from their blood serum. The immunoassays performed were ICA, FIA, ELFA, ELISA, and CLIA assays. Concluding, 82% of patients taken in this study, resulted infected with HCV. Diagnostic methods in clinical laboratories are crucial in the early stages of infection, in the management of chronic hepatitis and in the treatment of patients during their disease.

Keywords: CLIA, ELISA, Hepatitis C virus, immunoassay

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Authors: H. Alijani, M. Jabari, S. Matroodi, H. Zolqarnein, A. Sharafi, I. Zamani

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Actinomycetes, Gram-positive bacteria, are interesting as a main producer of secondary metabolites and are important industrially and pharmaceutically. The marine environment is a potential source for new actinomycetes, which can provide novel bioactive compounds and industrially important enzymes. The aims of this study were to isolate and identify novel actinomycetes from Persian Gulf sediments and screen these isolates for the production of secondary metabolites, especially antibiotics, Using phylogenetic (16S rRNA gene sequence), morphological and biochemical analyses. 15 different actinomycete strains from Persian Gulf sediments at a depth of 5-10 m were identified. DNA extraction was done using Cinnapure DNA Kit. PCR amplification of 16S rDNA gene was performed using F27 and R1492 primers. Phylogenetic tree analysis was performed using the MEGA 6 software. Most of the isolated strains belong to the genus namely Streptomyces (14), followed by Nocardiopsis (1). Antibacterial assay of the isolates supernatant was performed using a standard disc diffusion assay with replication (n=3). The results of disk diffusion assay showed that most active strain against Proteus volgaris and Bacillus cereus was AMJ1 (16.46±0.2mm and 13.78±0.2mm, respectively), against Salmonella sp. AMJ7 was the most effective strain (10.13±0.2mm), and AMJ1 and AHA5 showed more inhibitory activity against Escherichia coli (8.04±0.02 mm and 8.2±0.03 ). The AMJ6 strain showed best antibacterial activity against Klebsiella sp. (8.03±0.02mm). Antifungal activity of AMJ2 showed that it was most active strain against complex (16.05±0.02mm) and against Aspergillus flavus strain AMJ1 was most active strain (16.4±0.2mm) and highest antifungal activity against Trichophyton mentagrophytes, Microsporum gyp serum and Candida albicans, were shown by AHA1 (21.03±0.02mm), AHA3 and AHA7 (18±0.03mm), AMJ6 (21.03±0.2mm) respectively. Our results revealed that the marine actinomycetes of Persian Gulf sediments were potent source of novel antibiotics and bioactive compounds and indicated that the antimicrobial metabolites were extracellular. Most of the secondary metabolites and antibiotics are extracellular in nature and extracellular products of actinomycetes show potent antimicrobial activities.

Keywords: antibacterial activity, antifungal activity, marine actinomycetes, Persian Gulf

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Authors: Muzaffar Iqbal, Essam Ezzeldin, Khalid A. Al-Rashood

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Pomalidomide is a second generation oral immunomodulatory agent, being used for the treatment of multiple myeloma in patients with disease refractory to lenalidomide and bortezomib. In this study, a sensitive UPLC-MS/MS assay was developed and validated for high-throughput determination of pomalidomide in rat plasma using celecoxib as an internal standard (IS). Liquid liquid extraction using dichloromethane as extracting agent was employed to extract pomalidomide and IS from 200 µL of plasma. Chromatographic separation was carried on Acquity BEHTM C18 column (50 × 2.1 mm, 1.7 µm) using an isocratic mobile phase of acetonitrile:10 mM ammonium acetate (80:20, v/v), at a flow rate of 0.250 mL/min. Both pomalidomide and IS were eluted at 0.66 ± 0.03 and 0.80 ± 0.03 min, respectively with a total run time of 1.5 min only. Detection was performed on a triple quadrupole tandem mass spectrometer using electrospray ionization in negative mode. The precursor to product ion transitions of m/z 272.01 → 160.89 for pomalidomide and m/z 380.08 → 316.01 for IS were used to quantify them respectively, using multiple reaction monitoring mode. The developed method was validated according to regulatory guideline for bioanalytical method validation. The linearity in plasma sample was achieved in the concentration range of 0.47–400 ng/mL (r2 ≥ 0.997). The intra and inter-day precision values were ≤ 11.1% (RSD, %) whereas accuracy values ranged from - 6.8 – 8.5% (RE, %). In addition, other validation results were within the acceptance criteria and the method was successfully applied in a pharmacokinetic study of pomalidomide in rats.

Keywords: pomalidomide, pharmacokinetics, LC-MS/MS, celecoxib

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